TLR4促进肿瘤细胞免疫逃逸和凋亡抵抗的分子机制研究
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摘要
临床与流行病学调查研究发现慢性感染和炎症紊乱与肿瘤发生有关,其中慢性炎症促进肿瘤免疫逃逸,被认为是导致肿瘤发生和发展的重要原因之一。近年来肿瘤细胞表面表达的Toll样受体(Toll-like receptors,TLRs)与肿瘤免疫逃逸的关系倍受关注。研究证明,在慢性感染或炎症条件下,TLR4诱导肿瘤细胞生成大量的免疫抑制性因子抑制免疫细胞启动免疫应答,同时局部感染细胞大量增殖并抵抗凋亡促进细胞存活。因此打破肿瘤免疫逃逸可能成为预防和治疗肿瘤的重要手段。雷帕霉素(Rapamycin)是一种广泛用于治疗自身免疫和移植排斥疾病的免疫抑制剂,最近几年用于治疗肿瘤、抑制血管形成并促进肿瘤细胞凋亡。因此,我们提出Rapamycin这种抗炎药物是否能够阻断TLR4介导肿瘤免疫逃逸,从而起到抗肿瘤的效应。
研究结果发现,人HT29和鼠CT26结肠癌细胞组成性地表达较高水平的TLR4,提示TLR4信号可能促进结肠癌细胞免疫逃逸。首先我们用RT-PCR方法检测TLR4信号对免疫抑制因子表达谱的影响,结果显示,LPS刺激后,LPS上调CT26细胞中IL-6和COX-2的表达,随后证明LPS刺激能够促进IL-6和PGE2的合成,这些因子不仅抑制免疫细胞功能而且能够促进肿瘤转移、浸润和存活。然后,我们研究了Rapamycin这种抗炎药物是否能够抑制TLR4介导结肠癌细胞IL-6和PGE2的生成。RT-PCR和ELISA检测都证实,在Rapamycin存在情况下,TLR4-介导的IL-6和COX-2表达以及IL-6和PGE2合成均显著降低,提示Rapamycin能够显著抑制TLR4介导免疫抑制介质的生成。
由于IL-6和PGE2是非常重要的调节多种细胞增殖、凋亡、迁移和浸润的分子,因此,我们进一步研究是否LPS能够通过IL-6和PGE2自分泌促进细胞转移和浸润,以及是否Rapamycin抑制了LPS诱导IL-6和PGE2的产生,从而降低CT26细胞的活力。为了证实这一推断,我们在下层小室分别加入重组鼠IL-6或PGE2,发现外源性的IL-6和PGE2能够逆转Rapamycin对CT26细胞迁移和浸润的抑制效应,而且,同时加入IL-6和PGE2时,几乎全部恢复Rapamycin的上述抑制效应。结果表明,Rapamycin能够抑制LPS诱导结肠癌细胞的迁移和浸润,因而,在临床治疗时,Rapamycin可能通过抑制结肠癌细胞分泌IL-6和PGE2而发挥抑制结肠癌转移的抗肿瘤效应。
我们先前的实验发现,TLR4能够诱导人肺癌细胞的凋亡抵抗,那么Rapamycin是否能够逆转LPS诱导结肠癌的凋亡抵抗效应呢?Annixin V/PI分析表明,LPS能够降低OXL或DXR所诱导人HT29和鼠CT26结肠癌细胞的凋亡效应,进一步研究发现,Rapamycin能够逆转TLR4所介导的这一凋亡抵抗,表明Rapamycin能够增强肿瘤细胞对抗肿瘤药物的敏感性,表现出抗肿瘤效应。
Rapamycin抑制TLR4信号促进CT26结肠癌细胞IL-6、PGE2分泌并降低LPS诱导的凋亡抵抗。那么,其可能的分子信号机制如何呢?首先我们利用FACS对TLR4受体膜表面的表达进行分析,发现Rapamycin能够抑制TLR4在细胞膜上的表达,但对TLR4的转录水平没有影响,提示Rapamycin可能通过转录后调节如抑制TLR4的糖基化或向胞膜的转运过程而降低膜表面的表达,Rapamycin介导TLR4的下调可能削弱了LPS所诱导肿瘤的免疫逃逸。随后我们对TLR4介导的MAPKs、Akt和NF-κB信号途径进行了研究。结果发现,在CT26细胞中,Rapamycin对LPS诱导的MAPKs激酶p38、ERK1/2、JNK1/2的活化没有影响,但显著抑制了Akt的磷酸化及NF-κB信号途径。提示Rapamycin可能通过抑制这两条信号通路从而抑制IL-6和PGE2的合成及凋亡抵抗逆转。为了证实这一点,Akt和NF-κB信号分子特异性的抑制剂LY209400及PDTC处理阻断信号后,ELISA检测免疫抑制性因子的生成,结果显示与Rapamycin相似,LY209400及PDTC均能有效抑制IL-6和PGE2的分泌;同样地,LY209400及PDTC均能有效逆转LPS对肿瘤药物OXL和DXR的抵抗作用。可推断Rapamycin作用于Akt和NF-κB信号分子抑制TLR4介导的CT26分泌IL-6和PGE2及OXL和DXR诱导的凋亡抵抗。
以上研究结果显示,Rapamycin作用于Akt和NF-κB信号分子打破肿瘤细胞的免疫逃逸,Rapamycin是PI3K/Akt通路有效的抑制剂,但对于NF-κB抑制机制还不清楚。众所周知,I-κB的磷酸化及降解促进NF-κB的核转位。I-κB上游的激酶为IKKα/β,IKKα/β是Akt的调节靶分子,这些通路参与对外界的炎症应答。我们发现LY209400(Akt的抑制剂)与Rapamycin一样均能够抑制IKKα/β和NF-κB的活化,说明Rapamycin通过抑制LPS诱导的Akt/IKKα/β/NF-κB的级联信号抑制结肠癌细胞免疫逃逸和转移。
综上所述,Rapamycin能够降低结肠癌TLR4膜表面的表达和抑制Akt/IKKα/β/NF-κB级联通路,从而抑制了TLR4介导的结肠癌细胞自分泌IL-6和PGE2及其所诱导的肿瘤迁移和浸润,并逆转了TLR4所介导结肠癌细胞凋亡抵抗。本实验结果从新的角度解释了Rapamycin通过抑制参与免疫逃逸的因子的形成及增强抗肿瘤药物敏感性从而打破免疫逃逸以发挥抗肿瘤效应,进一步提示了Rapamycin可用于结肠癌的治疗。
目前,肿瘤治疗主要依赖于手术、放疗和化疗,然而,临床观察发现,在某些情况下,放化疗可以促进肝癌发展,然而其内在机制依然不明。因此,为了提高治疗效果,探究在治疗过程中HCC的生物学功能及肿瘤恶化机制尤为重要。本文所研究旨在探讨在放疗或生物治疗过程中,肝癌的免疫逃逸和发展机制。
诸多研究表明,肿瘤微环境中,持续的炎症刺激和慢性感染导致或促进肿瘤发生、发展。近年来发现,表达于肿瘤中的TLR4受体通过诱导肿瘤细胞释放免疫抑制性的因子和凋亡抵抗从而介导免疫逃逸。
高频率重组蛋白-1(High-mobility group box-1,HMGB1)最初被鉴定为DNA体细胞转录调控协同因子。免疫刺激时,炎症细胞和免疫细胞被活化导致HMGB1被包装到溶酶体然后释放到胞外环境;坏死或受损的体细胞以被动的方式释放HMGB1。一旦释放到胞外,HMGB1发挥致炎因子作用活化内皮细胞,促进血管形成,炎症细胞和干细胞向血管外的迁移,通过活化一系列关键信号途径从而启动炎症反应,调节细胞的活化、生长及死亡。因此,HMGB1涉及到多种疾病,包括帕会森、脓血症、缺血再灌注及自身免疫疾病。HMGB1到胞外环境且与肿瘤增殖和转移有关。最近研究表明,HMGB1与肿瘤增殖和转移有关,而且,HMGB1是TLR4的内源性配体,因此,我们研究了在放疗和生物治疗过程中由坏死细胞释放的HMGB1是否能够作为一个旁分泌因子促进肝癌的发展。
我们研究发现,在放疗和TRAIL治疗过程中,源于死亡的肝癌细胞株SMMC-7721上清能够诱导SMMC-7721细胞表达免疫抑制性细胞因子VEGF和趋化因子MIP-3α、IP-10和MCP-1。为了确定是否死亡细胞释放的HMGB1诱导了这种炎症效应,我们干扰了SMMC-7221细胞HMGB1,结果发现,HMGB1干扰的坏死细胞的上清对MIP-3α的诱导能力显著减弱,证实了死亡细胞释放HMGB1并将信号传递于其周边细胞。重组的HMGB1也能促进高水平MIP-3α的表达,进一步表明了上清中的HMGB1促进了免疫抑制性细胞因子和趋化因子的表达。为了明确HMGB1是由凋亡细胞还是坏死细胞释放HMGB1,利用放射和TRAIL处理SMMC-7721,Annixin V/PI凋亡检测表明,是死亡细胞而非凋亡细胞释放的HMGB1促进肿瘤细胞的免疫逃逸,因为源于死亡细胞的上清能够促进SMMC-7721表达,但凋亡细胞上清不能诱导其表达。这些研究结果表明,在放疗和TRAIL治疗过程中,死亡肝癌细胞上清中所含的HMGB1促进了免疫抑制过程。
由于HMGB1能够介导神经节瘤的生长和扩散,所以我们研究了HMGB1和死亡细胞上清是否能够促进肝癌细胞增殖。MTT检测发现,HMGB1能够促进SMMC-7721增殖,BrdU参入法检测细胞增殖进一步证实死亡细胞上清和HMGB1能够促进肝癌细胞生长。然后,我们研究了是否HMGB1通过加速细胞周期进程从而促进肝癌细胞增殖。细胞周期分析表明,重组HMGB1加速G1向S期的转化,从而促进肝癌细胞的生长。这些实验结果提示,由于放疗和化疗诱导死亡细胞可能通过释放高水平HMGB1加速细胞周期进程从而促进肿瘤的生长和恶化。
由于细胞周期素A,D和E是G1向S相转化所必需分子,然后利用蛋白印记方法检测是否HMGB1影响细胞周期素A,D,E的表达,我们发现,HMGB1刺激SMMC-7721后,细胞周期素cyclinD的表达显著增加,但对细胞周期素A和E的表达没有影响。另外,因为p53、p21和p27都是负调节细胞周期进程的分子,因此我们推测可能这些分子在这一过程中发挥重要作用。Western blot显示HMGB1显著下调p27的表达,但不影响p21和p53的表达。因为只有p27在T187位点的磷酸化能被泛素化并被蛋白酶体识别,因此我们检测了是否HMGB1影响p27的磷酸化。Western blot表明,HMGB1刺激后,SMMC-7721细胞中p27—T187磷酸化显著增加。综合以上结果,提示下调p27及增强cyclinD可能是HMGB1加速肝癌细胞周期进程从而促进肝癌细胞增殖和生长的机制之一。
在治疗肿瘤的过程中,最主要的瓶颈之一是对抗肿瘤药物的耐受从而不能杀伤肿瘤。我们推测HMGB1可能参与肿瘤的抗凋亡效应。SMMC-7721细胞用HMGB1预处理,然后TRAIL再行行刺激,Annixin V/PI分析发现,HMGB1降低了TRAIL所诱导SKMC-7721的凋亡。为了进一步阐明HMGB1导致凋亡抵抗效应的机制,用蛋白印迹检测了促调亡和抗凋亡蛋白,我们发现,HMGB1刺激SMMC-7721后,抗凋亡蛋白Bcl-xL显著上调,但Bcl-2、Bax或Bak没有变化。这些结果提示,HMGB1诱导Bcl-xL表达很可能是其促进肝癌细胞逃避TRAIL诱导的凋亡从而促进肝癌细胞存活的机制之一。
如前面所述,重组HMGB1和源于死亡细胞的上清能够促进肝癌SMMC-7721细胞免疫抑制细胞因子VEGF和趋化因子MIP-3α,IP-10和MCP-1的表达。另外,HMGB1是TLR4内源性配体,因而,为了进一步证实HMGB1是否通过TLR4/MyD88信号途径而介导免疫逃逸的诱导,我们构建了针对于SMMC-7721细胞TLR4和MyD88的干扰载体。RT-PCR检测表明,HMGB1或死亡细胞上清处理TLR4或MyD88缺陷的SMMC-7721细胞后,对MIP-3αmRNA诱导表达能力显著降低,说明重组的HMGB1和死亡细胞的上清介导MIP-3α表达依赖于TLR4/MyD88信号途径。
另外,与LPS刺激相似,HMGB1能够活化SMMC-7721细胞p38MAPK、ERK1/2、JNK和NF-κB信号通路。因此,我们研究了HMGB1是否通过MAPK或NF-κB参与细胞周期和凋亡抵抗的调节。Western blot检测表明MEK1/2(PD98059)和ERK1/2(U0126)抑制剂能够显著抑制HMGB1诱导的SMMC-7721细胞p27磷酸化及其降解以及周期素D1的表达。而NF-κB抑制剂PDTC下调HMGB1所诱导的抗凋亡蛋白Bcl-xL的表达。因此,HMGB1/TLR4诱导的肝癌细胞周期转化是由MEK1/2/ERK1/2信号通路介导,而N-κB是促进HMGB1诱导肝癌细胞凋亡抵抗的关键信号分子。因此,HMGB1/TLR4信号介导肝癌细胞凋亡抵抗和加速细胞周期转化过程,从而促进肝癌免疫逃逸和进一步发展。
综上所述,由30 Gy-co-ray放疗或TRAIL处理所导致死亡的肝癌细胞的分泌上清能够反馈性地诱导肝癌细胞表达免疫抑制细胞因子VEGF和趋化因子MIP-3α、IP-10和MCP-1,死亡的人肝细胞癌细胞释放的HMGB1介导了此种效应。HMGB1通过增强cyclinD表达及p27-T187磷酸化和降解而促进了肝癌细胞G1—S期的进程。另外,HMGB1可通过上调抗凋亡蛋白Bcl-xL而增强肝癌细胞对TRAIL诱导凋亡的抵抗,提示,放化疗导致死亡的肝癌细胞释放的HMGB1参与了肿瘤的逃避免疫和凋亡抵抗。干扰HMGB1或TLR4/MyD88之后,死亡的肝癌细胞以及重组HMGB1均不能有效诱导肝癌细胞表达MIP-3α,进一步证实了HMGB1/TLR4/MyD88途径依赖的放化疗促进肝癌的免疫逃逸作用。因此,我们研究结果提供的实验结果直接证明了在放化疗过程中坏死的肝癌细胞能够通过释放HMGB1并通过TLR4/MyD88依赖途径介导了肝癌免疫逃逸和凋亡抵抗,提示通过中和或干扰肝癌细胞中HMGB1并与化疗联合应用可能有助于肝癌的治疗。
Toll-like receptors(TLRs)are expressed in many kinds of immune cells,including antigen-presenting cells dendritic cells(DC),macrophages and B cells.Also,recent studies show that TLRs can be expressed by tumor cells,and TLR signaling contributes to tumor immune esacpe and promotes tumor metastasis.However,the mechanisms by which TLR-triggered tumor immune esacpe and tumor progression remain to be fully understood.Inspired by the data that the chronic inflammation may induce tumorigenesis of colon cancer,in this study,we investigated the TLR expression in colon cancer cells and then what's the function of TLR signaling in the immune esacape and apoptosis resistance of colon cancer cells.Our findings demonstrate that both human HT29 and mouse CT26 colon cancer cells express high level of TLR4,suggesting TLR4 signaling might contribute to immune escape.Firstly, we screened production of the immune suppressive cytokines by TLR4-ligated colon cancer cells through RT-PCR and ELISA.The data showed that the expression of IL-6, COX-2 and production of IL-6,PGE2 were significantly upregulated by CT26 colon cancer cells stimulated with LPS.It's well known that IL-6 and PGE2 can suppress the function of immune cells and promote tumor cell migration,invasion and survival. Then,we investigated whether Rapamycin,anti-inflammatory regent,affected TLR4-mediated IL-6 and PGE2 production by colon cancer cells.The data of RT-PCR, real-time PCR and ELSIA demonstrated that Rapamycin inhibited IL-6 and COX-2 expression,and IL-6 and PGE2 production by CT26 cells stimulated with LPS.These data indicated that Rapamycin could effectively suppress TLR4-mediated production of immunosuppressive mediators by colon cancer cells.
As IL-6 and PGE2 are both the important mediators that regulate cell proliferation, apoptosis,migration and invasion of several cell types,so we wondered whether LPS could promote migration and invasion of CT26 cells,and whether inhibition of LPS-induced IL-6 and PGE2 production by Rapamycin was responsible for the deceased mobility of CT26 cells.We added recombinant mIL-6 or PGE2 in the lower chamber and observed both exogenous IL-6 and PGE2 could reverse the inhibitory effect of Rapamycin on CT26 cell migration and invasion,and simultaneous addition of IL-6 and PGE2 almost totally reversed the inhibitory effect of Rapamycin.These results indicated that LPS-induced IL-6 and PGE2 can promote invasion of colon cancer cells,and Rapamycin may inhibit TLR4-promoted invasion of colon cancer cells by suppressing cancer cell autocrine production of IL-6 and PGE2.
Our previous study demonstrated that TLR4 could induce apoptosis resistance of lung cancer cells.We wondered whether Rapamycin could reduce TLR-induced apoptosis resistance of cancer cells.Annixin V/PI analysis demonstrated LPS stimulation could significantly reduce the apoptosis of both human HT29 and murine CT26 colon cancer cells induced by OXL or DXR.In addition,Rapamycin could increase apoptosis of LPS-induced CT26 cancer cells to OXL or DXR treatment,indicating that Rapamycin could reduce TLR4-evoked apoptotic resistance in colon cancer cells, and enhance the sensitivity of cancer cells to anti-tumor drugs,and thus exhibiting anti-tumor effect.
Then we explored the mechanism for Rapamycin to inhibite TLR4-triggered IL-6 and PGE2 production and apoptosis resistance of CT26 cells.Firstly,Western blot and FACS analysis demonstrated that Rapamycin could inhibit significantly surface expression of TLR4 in CT26 cells stimulated by LPS without alternation TLR4 mRNA expression,suggesting that TLR4 distribution might be modulated via post-transcriptional regulation such as TLR4 membrane transport or its glycosylation modification.These data suggested that Rapamycin-mediated downregulation of TLR4 expression may contribute to the suppressive effect of Rapamycin on tumor immune escape.Then we investigated which TLR4 signaling pathway involved in the inhibitory effect of Rapamycin on immune escape.Western blot analysis showed that LPS-mediated phosphorylation of p38,JNK,ERK1/2 MAP kinase remained unchanged in the presence of Rapamycin,but the activation of Akt and NF-κB were significantly disrupted by Rapamycin,indicating that Akt and NF-κB inactivation were involved in the process.To confirm the inhibitory effect of Rapamycin,CT26 cells were incubated with the PI3K/Akt(LY294002)and NF-κB specific inhibitor (PDTC)in presence LPS.Similar to Rapamycin,both LY294002 and PDTC significantly inhibited LPS-induced IL-6 and PGE2 secretion,and reverse the apoptosis resistance to OXL and DXR treatment for colon cancer cells.These results demonstrated that inactivation of Akt and NF-κB pathways is involved in the suppressive effect of Rapamycin on the LPS-increased IL-6,PGE2 secretion and apoptosis resistance.
As above demonstrated,Rapamycin inhibited Akt and NF-κB activation triggered by LPS.Rapamycin is the potent inhibitor of PI3K/Akt pathway.Up to now,how Rapamycin inhibits LPS-induced NF-κB activity remains to be fully understood. NF-κB activation is regulated by I-κBαphosphorylation and subsequent I-κBαdegradation,allowing NF-κB to translocate into the nucleus.I-κBαphosphorylation is regulated by I-κB kinase(IKKα/β).It is generally accepted that activation of IKKα/βis the regulatory target for Akt,which involved in cellular signaling in response to pro-inflammatory stimuli.We found that Rapamycin suppresses the LPS-induced Akt/IKKα/β/NF-κB cascade pathway,thus blocking TLR-triggered the immune escape and apoptosis resistance of tumor cells.
In summary,Rapamycin could suppress the TLR4-induced IL-6,PGE2 secretion and inhibit autocrine IL-6,PGE2-mediated migration and invasion,and reverse TLR4-induced apoptosis resistance of colon cancer cells through downregulation of surface TLR4 expreesion of colon cancer cells and inhibition of Akt/IKKα/β/NF-κB pathways.Our results have provided the new insight into the mechansim for the antitumor effect of Rapamycin,and indicate the potential application of Rapamycin in colon cancer treatment.
Surgery,radiotherapy and chemotherapy are all conventional approaches for cancer treatments.However,clinical observations suggest that sometimes radiotherapy and chemotherapy may promote progression of hepatocellular carcinoma(HCC).However, up to now,the underlying mechanisms remain unknown.Therefore,to improve treatment outcomes of HCC,a better understanding of mechanisms that underlie the behavior of HCC and contribute to HCC progression induced by radiotherapy and chemotherapy is necessary.The primary aim of this study is to explore the mechanisms for HCC to escape immune surveillance after radiation and biotherapy,leading to HCC pathogenesis and development.
Many studies demonstrate that the presence of proinflammatory cytokines and persistent chronic inflammation in the tumor microenvironment lead to or promote cancer pathogenesis and development.Recently,studies have showed that Toll-like receptor 4 (TLR4)signaling in tumor cells can mediate tumor cell immune escape and tumor progression,companied by immune suppressive cytokine production and apoptosis resistance.
High-mobility group box-1(HMGB1)was originally identified as a DNA-binding protein that functions as a structural co-factor critical for proper transcriptional regulation in somatic cells.During the immunologic challenge,inflammatory cells and immune cells are activated,resulting in HMGB1 being packed into lysosomes and liberation into the extracellular environment;HMGB1 also can be "passively released" into the extracellular milieu by necrotic and damaged somatic cells.Once in the extracellular milieu,HMGB1 can act as an proinflammatory cytokine to activate endothelial cells, promoting angiogenesis and extravascular emigration of inflammatory cells and stem cells,thereby initiating inflammation by activation key signaling pathways involved in the regulation of cell differentiation,growth,motility and death.So,HMGB1 has also been implicated in disease states,including Alzheimer's,sepsis,ischemia-reperfusion, autoimmune diseases.Recent studies indicate that HMGB1 is associated with the tumor proliferation and metastasis,and HMGB1 has been defined as an endogenous ligand of TLR4.So,we investigated whether HMGB1 released by dying HCC cells is a paracrine factor for HCC development after radiotherapy,chemotherapy or biotherapy.
In our study,we found that supernatants from dying HCC cell line SMMC-7721 induced by radiation(30 Gy-co-ray)or TRAIL treatment could significantly induce production of immunpsuppressive cytokine VEGF and chemokines MIP-3α,IP-10 and MCP-1 by SMMC-7721 cells.To determine whether HMGB1 released from dying SMMC-7721 cells could contribute to the effects,silencing of HMGB1 in SMMC-7721 cells was performed.HMGB1 knock-down dying cells have a greatly reduced ability to promote MIP-3αprodcution,which proves that the release of HMGB1 can signal the demise of a cell to its neighbors.To further confirm the role of HMGB1 in dying cell supernatant for the induction of MIP-3α,stimulation of SMMC-7721 cells with recombinant HMGB1 also could dramatically induce the MIP-3αexpression,indicating HMGB1 in the supematant responsible for the induction of immunosuppressive cytokines and chamokines.To clarify whether HMGB1 was released by apoptotic or dying SMMC-7721 cells,Annexin V/PI apoptosis assay of SMMC-7721 ceils with radiation and TRAIL treatment showed that the dying cells rather than apoptotic cells could release HMGB1 responsible for the tumor immune escape,because the supernatant from dying cells could promote MIP-3αexpression in SMMC-7721 but apoptotic cells could not. These data show that HMGB1 in the supernatant from dying HCC cells after radiotherapy or TRAIL treatment is responsible for the induction of the immunosuppressive factors by HCC cells in paracrine manner.
Since HMGB1 could mediate the growth and spread of gliomas,so we are wondering whether HMGB1 in the supernatant from dying HCC cells could promote HCC proliferation.We found HMGB1 promoted the growth of SMMC-7721 cells as detected by MTT assay.The evaluation of cell proliferation by BrdU incorporation also confirmed the promotion of the growth of SMMC-7721 cells by HMGB1 stimulation.Then we investigated whether HMGB1 could promote the cell cycles and thus increasing the cell proliferation.Cell cycle analysis by PI staining revealed that recombinant HMGB1 could promote transition of G1 to S phase entry,which might be responsible for the HCC growth.These evidences suggested that the dying cell with radiation or biotherapy contribute to the tumor cell growth by releasing high levels of HMGB1 which can promote cell cycle.
Since cyclin A,D E are required for progression through the G1 and S entry,we then examined by Western blot whether cyclin A,D and E were affected by HMGB1 stimulation.We found that cyclin D was dramatically increased in SMMC-7721 cells after HMGB1 stimulation,without alternation of cyclin A and cyclin E.In addition,as p53,p21 and p27 all negatively regulate cell cycle progression by inhibition of the cyclin E/cyclin-dependent kinase activity,we speculated that these negative regulators might play a role in the process of cell cycle entry promoted by HMGB1.Western blot showed that HMGB1 led to significant down-regulation of p27,but not p21 and p53. Because only phosphorylated p27 on T187 can be recognized by ubiquitin protein ligase for consequent ubiquition and degradation,we therefore tested whether p27 phosphorylation was affected by HMGB1 stimulation.Western blot showed that phosphorylation on p27(T187)increased in SMMC-7721 cells in response to HMGB1. Together,the data suggest that decrease of p27 and increase of cyclinD might be responsible for the promotion of cells cycle and consequent HCC cell proliferation and growth by HGMB1 released from dying HCC cells.
For the effectiveness of chemotherapy or radiotherapy,one of major obstacles is the resistance of cancer cells to anti-tumor drugs.We speculated that HMGB1 might be involved in the increased anti-apoptosis effect of HCC cells after chemotherapyor radiotherapy.SMMC-7721 cells were pretreated by HMGB1,and then induced by TRAIL.Annexin V/PI analysis revealed that HMGB1 reduced apoptosis induction of HCC cells by TRAIL.To further elucidate the mechanism for the induction of apoptosis resistance by HMGB1,pro-or anti-apoptotic proteins were analyzed by Western blot.We found that upon stimulation of HMGB1,anti-apoptotic protein expression of Bcl-xL,but not Bcl-2,Bax or Bak,was significantly upregulated in SMMC-7721 cells.These data suggest that upregulation of Bcl-xL expression by HMGB1 might be the mechanism to avoid the TRAIL-induced apoptosis and contribute to HCC cell survival.
As observed above,recombinant HMGB1 and the HMGB1-containing supernatant from dying HCC cells promote the production of immunosuppressive cytokine VEGF and chemokines MIP-3α,IP-10 and MCP-1 by SMMC-7721 HCC cells.In addition,HMGB1 is identified as one of the endogenous ligands of TLR4.To further confirm that TLR4/MyD88 signaling pathway mediates HMGB1-induced immune escape,we established siRNA TLR4 and siRNA MyD88 of SMMC-7721 cells.RT-PCR showed that, in response to HMGB1 or supernatant from dying HCC cells,TLR4 or MyD88-silenced SMMC-7721 cells have a greatly reduced ability to express MIP-3αmRNA.Together, these results suggest that the increase of MIP-3αexpression induced by recombinant HMGB1 and supernatant from dying cells is mediated through TLR4/MyD88 pathway. In addition,similarly to LPS stimulation,HMGB1 could activate p38 MAPK,ERK1/2, JNK,and NF-κB pathways in SMMC-7721 HCC cells.Therefore,we investigated whether MAPK or NF-κB signal pathways were involved in the cell cycle progression and apoptotic resistance in human HCC cells in response to HMGB1.Western blot assay showed that HMGB1-induced phosphorylation of p27 and subsequent degradation,and cyclin D expression were significantly abrogated by the specific inhibitor of MEK1/2 (PD98059)and ERK1/2(U0126).However,the NF-κB inhibitor PDTC decreased the HMGB1-upregulated Bcl-xL,So,MEK1/2 and ERK1/2 kinase activation is essential for HMGB1-promoted cell cycle progress,and NF-κB activation is required for apoptotic resistance of HCC cells.These data demonstrate that HMGB1/TLR4-mediated MEK1/2/ ERK1/2 and NF-κB activation is responsible for the promotion of HCC cell production of immunosuppressive factors and induction of apoptosis resistance.
In conclusion,our findings provide the direct evidence that,during chemotherapy or radiotherapy,HMGB1 released by dying HCC cells could induce production of the immunosuppressive cytokine VEGF and chemokines MIP-3α,IP-10 and MCP-1 expression in HCC cells.HMGB1 promotes the cell cycle transition of G1-S phase by enhancing cyclinD expression,and phosphorylation of p27-T187 and subsequent proteasomal degradation in HCC cells.Furthermore,up-regulation of anti-apoptotic protein Bcl-xL contributes to the recombinant HMGB1-triggered HCC cell resistance of TRAIL-induced apoptosis,indicating that HMGB1 is closely related to tumor immune escape and development.HMGB1 fails to induce MIP-3αexpression in HCC cells with either silence of HMGB1 or TLR4/MyD88,further confirming the HMGB1/TLR4/MyD88-dependent pathway mediates induction of immune escape and progression of HCC after chemotherapy or radiotherapy.Therefore,our findings provide the direct evidence that,during radiotherapy or chemotherapy,the necrotic cells-induced immune escape and apoptosis resistance promotes HCC progression by HMGBI/TLR4/MyD88 dependant pathways,which imply a potential application of HMGB1 neutralization or interfering in combination with chemotherapy in the treatment of hepatocellular carcinoma.
研究结果发现,人HT29和鼠CT26结肠癌细胞组成性地表达较高水平的TLR4,提示TLR4信号可能促进结肠癌细胞免疫逃逸。首先我们用RT-PCR方法检测TLR4信号对免疫抑制因子表达谱的影响,结果显示,LPS刺激后,LPS上调CT26细胞中IL-6和COX-2的表达,随后证明LPS刺激能够促进IL-6和PGE2的合成,这些因子不仅抑制免疫细胞功能而且能够促进肿瘤转移、浸润和存活。然后,我们研究了Rapamycin这种抗炎药物是否能够抑制TLR4介导结肠癌细胞IL-6和PGE2的生成。RT-PCR和ELISA检测都证实,在Rapamycin存在情况下,TLR4-介导的IL-6和COX-2表达以及IL-6和PGE2合成均显著降低,提示Rapamycin能够显著抑制TLR4介导免疫抑制介质的生成。
由于IL-6和PGE2是非常重要的调节多种细胞增殖、凋亡、迁移和浸润的分子,因此,我们进一步研究是否LPS能够通过IL-6和PGE2自分泌促进细胞转移和浸润,以及是否Rapamycin抑制了LPS诱导IL-6和PGE2的产生,从而降低CT26细胞的活力。为了证实这一推断,我们在下层小室分别加入重组鼠IL-6或PGE2,发现外源性的IL-6和PGE2能够逆转Rapamycin对CT26细胞迁移和浸润的抑制效应,而且,同时加入IL-6和PGE2时,几乎全部恢复Rapamycin的上述抑制效应。结果表明,Rapamycin能够抑制LPS诱导结肠癌细胞的迁移和浸润,因而,在临床治疗时,Rapamycin可能通过抑制结肠癌细胞分泌IL-6和PGE2而发挥抑制结肠癌转移的抗肿瘤效应。
我们先前的实验发现,TLR4能够诱导人肺癌细胞的凋亡抵抗,那么Rapamycin是否能够逆转LPS诱导结肠癌的凋亡抵抗效应呢?Annixin V/PI分析表明,LPS能够降低OXL或DXR所诱导人HT29和鼠CT26结肠癌细胞的凋亡效应,进一步研究发现,Rapamycin能够逆转TLR4所介导的这一凋亡抵抗,表明Rapamycin能够增强肿瘤细胞对抗肿瘤药物的敏感性,表现出抗肿瘤效应。
Rapamycin抑制TLR4信号促进CT26结肠癌细胞IL-6、PGE2分泌并降低LPS诱导的凋亡抵抗。那么,其可能的分子信号机制如何呢?首先我们利用FACS对TLR4受体膜表面的表达进行分析,发现Rapamycin能够抑制TLR4在细胞膜上的表达,但对TLR4的转录水平没有影响,提示Rapamycin可能通过转录后调节如抑制TLR4的糖基化或向胞膜的转运过程而降低膜表面的表达,Rapamycin介导TLR4的下调可能削弱了LPS所诱导肿瘤的免疫逃逸。随后我们对TLR4介导的MAPKs、Akt和NF-κB信号途径进行了研究。结果发现,在CT26细胞中,Rapamycin对LPS诱导的MAPKs激酶p38、ERK1/2、JNK1/2的活化没有影响,但显著抑制了Akt的磷酸化及NF-κB信号途径。提示Rapamycin可能通过抑制这两条信号通路从而抑制IL-6和PGE2的合成及凋亡抵抗逆转。为了证实这一点,Akt和NF-κB信号分子特异性的抑制剂LY209400及PDTC处理阻断信号后,ELISA检测免疫抑制性因子的生成,结果显示与Rapamycin相似,LY209400及PDTC均能有效抑制IL-6和PGE2的分泌;同样地,LY209400及PDTC均能有效逆转LPS对肿瘤药物OXL和DXR的抵抗作用。可推断Rapamycin作用于Akt和NF-κB信号分子抑制TLR4介导的CT26分泌IL-6和PGE2及OXL和DXR诱导的凋亡抵抗。
以上研究结果显示,Rapamycin作用于Akt和NF-κB信号分子打破肿瘤细胞的免疫逃逸,Rapamycin是PI3K/Akt通路有效的抑制剂,但对于NF-κB抑制机制还不清楚。众所周知,I-κB的磷酸化及降解促进NF-κB的核转位。I-κB上游的激酶为IKKα/β,IKKα/β是Akt的调节靶分子,这些通路参与对外界的炎症应答。我们发现LY209400(Akt的抑制剂)与Rapamycin一样均能够抑制IKKα/β和NF-κB的活化,说明Rapamycin通过抑制LPS诱导的Akt/IKKα/β/NF-κB的级联信号抑制结肠癌细胞免疫逃逸和转移。
综上所述,Rapamycin能够降低结肠癌TLR4膜表面的表达和抑制Akt/IKKα/β/NF-κB级联通路,从而抑制了TLR4介导的结肠癌细胞自分泌IL-6和PGE2及其所诱导的肿瘤迁移和浸润,并逆转了TLR4所介导结肠癌细胞凋亡抵抗。本实验结果从新的角度解释了Rapamycin通过抑制参与免疫逃逸的因子的形成及增强抗肿瘤药物敏感性从而打破免疫逃逸以发挥抗肿瘤效应,进一步提示了Rapamycin可用于结肠癌的治疗。
目前,肿瘤治疗主要依赖于手术、放疗和化疗,然而,临床观察发现,在某些情况下,放化疗可以促进肝癌发展,然而其内在机制依然不明。因此,为了提高治疗效果,探究在治疗过程中HCC的生物学功能及肿瘤恶化机制尤为重要。本文所研究旨在探讨在放疗或生物治疗过程中,肝癌的免疫逃逸和发展机制。
诸多研究表明,肿瘤微环境中,持续的炎症刺激和慢性感染导致或促进肿瘤发生、发展。近年来发现,表达于肿瘤中的TLR4受体通过诱导肿瘤细胞释放免疫抑制性的因子和凋亡抵抗从而介导免疫逃逸。
高频率重组蛋白-1(High-mobility group box-1,HMGB1)最初被鉴定为DNA体细胞转录调控协同因子。免疫刺激时,炎症细胞和免疫细胞被活化导致HMGB1被包装到溶酶体然后释放到胞外环境;坏死或受损的体细胞以被动的方式释放HMGB1。一旦释放到胞外,HMGB1发挥致炎因子作用活化内皮细胞,促进血管形成,炎症细胞和干细胞向血管外的迁移,通过活化一系列关键信号途径从而启动炎症反应,调节细胞的活化、生长及死亡。因此,HMGB1涉及到多种疾病,包括帕会森、脓血症、缺血再灌注及自身免疫疾病。HMGB1到胞外环境且与肿瘤增殖和转移有关。最近研究表明,HMGB1与肿瘤增殖和转移有关,而且,HMGB1是TLR4的内源性配体,因此,我们研究了在放疗和生物治疗过程中由坏死细胞释放的HMGB1是否能够作为一个旁分泌因子促进肝癌的发展。
我们研究发现,在放疗和TRAIL治疗过程中,源于死亡的肝癌细胞株SMMC-7721上清能够诱导SMMC-7721细胞表达免疫抑制性细胞因子VEGF和趋化因子MIP-3α、IP-10和MCP-1。为了确定是否死亡细胞释放的HMGB1诱导了这种炎症效应,我们干扰了SMMC-7221细胞HMGB1,结果发现,HMGB1干扰的坏死细胞的上清对MIP-3α的诱导能力显著减弱,证实了死亡细胞释放HMGB1并将信号传递于其周边细胞。重组的HMGB1也能促进高水平MIP-3α的表达,进一步表明了上清中的HMGB1促进了免疫抑制性细胞因子和趋化因子的表达。为了明确HMGB1是由凋亡细胞还是坏死细胞释放HMGB1,利用放射和TRAIL处理SMMC-7721,Annixin V/PI凋亡检测表明,是死亡细胞而非凋亡细胞释放的HMGB1促进肿瘤细胞的免疫逃逸,因为源于死亡细胞的上清能够促进SMMC-7721表达,但凋亡细胞上清不能诱导其表达。这些研究结果表明,在放疗和TRAIL治疗过程中,死亡肝癌细胞上清中所含的HMGB1促进了免疫抑制过程。
由于HMGB1能够介导神经节瘤的生长和扩散,所以我们研究了HMGB1和死亡细胞上清是否能够促进肝癌细胞增殖。MTT检测发现,HMGB1能够促进SMMC-7721增殖,BrdU参入法检测细胞增殖进一步证实死亡细胞上清和HMGB1能够促进肝癌细胞生长。然后,我们研究了是否HMGB1通过加速细胞周期进程从而促进肝癌细胞增殖。细胞周期分析表明,重组HMGB1加速G1向S期的转化,从而促进肝癌细胞的生长。这些实验结果提示,由于放疗和化疗诱导死亡细胞可能通过释放高水平HMGB1加速细胞周期进程从而促进肿瘤的生长和恶化。
由于细胞周期素A,D和E是G1向S相转化所必需分子,然后利用蛋白印记方法检测是否HMGB1影响细胞周期素A,D,E的表达,我们发现,HMGB1刺激SMMC-7721后,细胞周期素cyclinD的表达显著增加,但对细胞周期素A和E的表达没有影响。另外,因为p53、p21和p27都是负调节细胞周期进程的分子,因此我们推测可能这些分子在这一过程中发挥重要作用。Western blot显示HMGB1显著下调p27的表达,但不影响p21和p53的表达。因为只有p27在T187位点的磷酸化能被泛素化并被蛋白酶体识别,因此我们检测了是否HMGB1影响p27的磷酸化。Western blot表明,HMGB1刺激后,SMMC-7721细胞中p27—T187磷酸化显著增加。综合以上结果,提示下调p27及增强cyclinD可能是HMGB1加速肝癌细胞周期进程从而促进肝癌细胞增殖和生长的机制之一。
在治疗肿瘤的过程中,最主要的瓶颈之一是对抗肿瘤药物的耐受从而不能杀伤肿瘤。我们推测HMGB1可能参与肿瘤的抗凋亡效应。SMMC-7721细胞用HMGB1预处理,然后TRAIL再行行刺激,Annixin V/PI分析发现,HMGB1降低了TRAIL所诱导SKMC-7721的凋亡。为了进一步阐明HMGB1导致凋亡抵抗效应的机制,用蛋白印迹检测了促调亡和抗凋亡蛋白,我们发现,HMGB1刺激SMMC-7721后,抗凋亡蛋白Bcl-xL显著上调,但Bcl-2、Bax或Bak没有变化。这些结果提示,HMGB1诱导Bcl-xL表达很可能是其促进肝癌细胞逃避TRAIL诱导的凋亡从而促进肝癌细胞存活的机制之一。
如前面所述,重组HMGB1和源于死亡细胞的上清能够促进肝癌SMMC-7721细胞免疫抑制细胞因子VEGF和趋化因子MIP-3α,IP-10和MCP-1的表达。另外,HMGB1是TLR4内源性配体,因而,为了进一步证实HMGB1是否通过TLR4/MyD88信号途径而介导免疫逃逸的诱导,我们构建了针对于SMMC-7721细胞TLR4和MyD88的干扰载体。RT-PCR检测表明,HMGB1或死亡细胞上清处理TLR4或MyD88缺陷的SMMC-7721细胞后,对MIP-3αmRNA诱导表达能力显著降低,说明重组的HMGB1和死亡细胞的上清介导MIP-3α表达依赖于TLR4/MyD88信号途径。
另外,与LPS刺激相似,HMGB1能够活化SMMC-7721细胞p38MAPK、ERK1/2、JNK和NF-κB信号通路。因此,我们研究了HMGB1是否通过MAPK或NF-κB参与细胞周期和凋亡抵抗的调节。Western blot检测表明MEK1/2(PD98059)和ERK1/2(U0126)抑制剂能够显著抑制HMGB1诱导的SMMC-7721细胞p27磷酸化及其降解以及周期素D1的表达。而NF-κB抑制剂PDTC下调HMGB1所诱导的抗凋亡蛋白Bcl-xL的表达。因此,HMGB1/TLR4诱导的肝癌细胞周期转化是由MEK1/2/ERK1/2信号通路介导,而N-κB是促进HMGB1诱导肝癌细胞凋亡抵抗的关键信号分子。因此,HMGB1/TLR4信号介导肝癌细胞凋亡抵抗和加速细胞周期转化过程,从而促进肝癌免疫逃逸和进一步发展。
综上所述,由30 Gy-co-ray放疗或TRAIL处理所导致死亡的肝癌细胞的分泌上清能够反馈性地诱导肝癌细胞表达免疫抑制细胞因子VEGF和趋化因子MIP-3α、IP-10和MCP-1,死亡的人肝细胞癌细胞释放的HMGB1介导了此种效应。HMGB1通过增强cyclinD表达及p27-T187磷酸化和降解而促进了肝癌细胞G1—S期的进程。另外,HMGB1可通过上调抗凋亡蛋白Bcl-xL而增强肝癌细胞对TRAIL诱导凋亡的抵抗,提示,放化疗导致死亡的肝癌细胞释放的HMGB1参与了肿瘤的逃避免疫和凋亡抵抗。干扰HMGB1或TLR4/MyD88之后,死亡的肝癌细胞以及重组HMGB1均不能有效诱导肝癌细胞表达MIP-3α,进一步证实了HMGB1/TLR4/MyD88途径依赖的放化疗促进肝癌的免疫逃逸作用。因此,我们研究结果提供的实验结果直接证明了在放化疗过程中坏死的肝癌细胞能够通过释放HMGB1并通过TLR4/MyD88依赖途径介导了肝癌免疫逃逸和凋亡抵抗,提示通过中和或干扰肝癌细胞中HMGB1并与化疗联合应用可能有助于肝癌的治疗。
Toll-like receptors(TLRs)are expressed in many kinds of immune cells,including antigen-presenting cells dendritic cells(DC),macrophages and B cells.Also,recent studies show that TLRs can be expressed by tumor cells,and TLR signaling contributes to tumor immune esacpe and promotes tumor metastasis.However,the mechanisms by which TLR-triggered tumor immune esacpe and tumor progression remain to be fully understood.Inspired by the data that the chronic inflammation may induce tumorigenesis of colon cancer,in this study,we investigated the TLR expression in colon cancer cells and then what's the function of TLR signaling in the immune esacape and apoptosis resistance of colon cancer cells.Our findings demonstrate that both human HT29 and mouse CT26 colon cancer cells express high level of TLR4,suggesting TLR4 signaling might contribute to immune escape.Firstly, we screened production of the immune suppressive cytokines by TLR4-ligated colon cancer cells through RT-PCR and ELISA.The data showed that the expression of IL-6, COX-2 and production of IL-6,PGE2 were significantly upregulated by CT26 colon cancer cells stimulated with LPS.It's well known that IL-6 and PGE2 can suppress the function of immune cells and promote tumor cell migration,invasion and survival. Then,we investigated whether Rapamycin,anti-inflammatory regent,affected TLR4-mediated IL-6 and PGE2 production by colon cancer cells.The data of RT-PCR, real-time PCR and ELSIA demonstrated that Rapamycin inhibited IL-6 and COX-2 expression,and IL-6 and PGE2 production by CT26 cells stimulated with LPS.These data indicated that Rapamycin could effectively suppress TLR4-mediated production of immunosuppressive mediators by colon cancer cells.
As IL-6 and PGE2 are both the important mediators that regulate cell proliferation, apoptosis,migration and invasion of several cell types,so we wondered whether LPS could promote migration and invasion of CT26 cells,and whether inhibition of LPS-induced IL-6 and PGE2 production by Rapamycin was responsible for the deceased mobility of CT26 cells.We added recombinant mIL-6 or PGE2 in the lower chamber and observed both exogenous IL-6 and PGE2 could reverse the inhibitory effect of Rapamycin on CT26 cell migration and invasion,and simultaneous addition of IL-6 and PGE2 almost totally reversed the inhibitory effect of Rapamycin.These results indicated that LPS-induced IL-6 and PGE2 can promote invasion of colon cancer cells,and Rapamycin may inhibit TLR4-promoted invasion of colon cancer cells by suppressing cancer cell autocrine production of IL-6 and PGE2.
Our previous study demonstrated that TLR4 could induce apoptosis resistance of lung cancer cells.We wondered whether Rapamycin could reduce TLR-induced apoptosis resistance of cancer cells.Annixin V/PI analysis demonstrated LPS stimulation could significantly reduce the apoptosis of both human HT29 and murine CT26 colon cancer cells induced by OXL or DXR.In addition,Rapamycin could increase apoptosis of LPS-induced CT26 cancer cells to OXL or DXR treatment,indicating that Rapamycin could reduce TLR4-evoked apoptotic resistance in colon cancer cells, and enhance the sensitivity of cancer cells to anti-tumor drugs,and thus exhibiting anti-tumor effect.
Then we explored the mechanism for Rapamycin to inhibite TLR4-triggered IL-6 and PGE2 production and apoptosis resistance of CT26 cells.Firstly,Western blot and FACS analysis demonstrated that Rapamycin could inhibit significantly surface expression of TLR4 in CT26 cells stimulated by LPS without alternation TLR4 mRNA expression,suggesting that TLR4 distribution might be modulated via post-transcriptional regulation such as TLR4 membrane transport or its glycosylation modification.These data suggested that Rapamycin-mediated downregulation of TLR4 expression may contribute to the suppressive effect of Rapamycin on tumor immune escape.Then we investigated which TLR4 signaling pathway involved in the inhibitory effect of Rapamycin on immune escape.Western blot analysis showed that LPS-mediated phosphorylation of p38,JNK,ERK1/2 MAP kinase remained unchanged in the presence of Rapamycin,but the activation of Akt and NF-κB were significantly disrupted by Rapamycin,indicating that Akt and NF-κB inactivation were involved in the process.To confirm the inhibitory effect of Rapamycin,CT26 cells were incubated with the PI3K/Akt(LY294002)and NF-κB specific inhibitor (PDTC)in presence LPS.Similar to Rapamycin,both LY294002 and PDTC significantly inhibited LPS-induced IL-6 and PGE2 secretion,and reverse the apoptosis resistance to OXL and DXR treatment for colon cancer cells.These results demonstrated that inactivation of Akt and NF-κB pathways is involved in the suppressive effect of Rapamycin on the LPS-increased IL-6,PGE2 secretion and apoptosis resistance.
As above demonstrated,Rapamycin inhibited Akt and NF-κB activation triggered by LPS.Rapamycin is the potent inhibitor of PI3K/Akt pathway.Up to now,how Rapamycin inhibits LPS-induced NF-κB activity remains to be fully understood. NF-κB activation is regulated by I-κBαphosphorylation and subsequent I-κBαdegradation,allowing NF-κB to translocate into the nucleus.I-κBαphosphorylation is regulated by I-κB kinase(IKKα/β).It is generally accepted that activation of IKKα/βis the regulatory target for Akt,which involved in cellular signaling in response to pro-inflammatory stimuli.We found that Rapamycin suppresses the LPS-induced Akt/IKKα/β/NF-κB cascade pathway,thus blocking TLR-triggered the immune escape and apoptosis resistance of tumor cells.
In summary,Rapamycin could suppress the TLR4-induced IL-6,PGE2 secretion and inhibit autocrine IL-6,PGE2-mediated migration and invasion,and reverse TLR4-induced apoptosis resistance of colon cancer cells through downregulation of surface TLR4 expreesion of colon cancer cells and inhibition of Akt/IKKα/β/NF-κB pathways.Our results have provided the new insight into the mechansim for the antitumor effect of Rapamycin,and indicate the potential application of Rapamycin in colon cancer treatment.
Surgery,radiotherapy and chemotherapy are all conventional approaches for cancer treatments.However,clinical observations suggest that sometimes radiotherapy and chemotherapy may promote progression of hepatocellular carcinoma(HCC).However, up to now,the underlying mechanisms remain unknown.Therefore,to improve treatment outcomes of HCC,a better understanding of mechanisms that underlie the behavior of HCC and contribute to HCC progression induced by radiotherapy and chemotherapy is necessary.The primary aim of this study is to explore the mechanisms for HCC to escape immune surveillance after radiation and biotherapy,leading to HCC pathogenesis and development.
Many studies demonstrate that the presence of proinflammatory cytokines and persistent chronic inflammation in the tumor microenvironment lead to or promote cancer pathogenesis and development.Recently,studies have showed that Toll-like receptor 4 (TLR4)signaling in tumor cells can mediate tumor cell immune escape and tumor progression,companied by immune suppressive cytokine production and apoptosis resistance.
High-mobility group box-1(HMGB1)was originally identified as a DNA-binding protein that functions as a structural co-factor critical for proper transcriptional regulation in somatic cells.During the immunologic challenge,inflammatory cells and immune cells are activated,resulting in HMGB1 being packed into lysosomes and liberation into the extracellular environment;HMGB1 also can be "passively released" into the extracellular milieu by necrotic and damaged somatic cells.Once in the extracellular milieu,HMGB1 can act as an proinflammatory cytokine to activate endothelial cells, promoting angiogenesis and extravascular emigration of inflammatory cells and stem cells,thereby initiating inflammation by activation key signaling pathways involved in the regulation of cell differentiation,growth,motility and death.So,HMGB1 has also been implicated in disease states,including Alzheimer's,sepsis,ischemia-reperfusion, autoimmune diseases.Recent studies indicate that HMGB1 is associated with the tumor proliferation and metastasis,and HMGB1 has been defined as an endogenous ligand of TLR4.So,we investigated whether HMGB1 released by dying HCC cells is a paracrine factor for HCC development after radiotherapy,chemotherapy or biotherapy.
In our study,we found that supernatants from dying HCC cell line SMMC-7721 induced by radiation(30 Gy-co-ray)or TRAIL treatment could significantly induce production of immunpsuppressive cytokine VEGF and chemokines MIP-3α,IP-10 and MCP-1 by SMMC-7721 cells.To determine whether HMGB1 released from dying SMMC-7721 cells could contribute to the effects,silencing of HMGB1 in SMMC-7721 cells was performed.HMGB1 knock-down dying cells have a greatly reduced ability to promote MIP-3αprodcution,which proves that the release of HMGB1 can signal the demise of a cell to its neighbors.To further confirm the role of HMGB1 in dying cell supernatant for the induction of MIP-3α,stimulation of SMMC-7721 cells with recombinant HMGB1 also could dramatically induce the MIP-3αexpression,indicating HMGB1 in the supematant responsible for the induction of immunosuppressive cytokines and chamokines.To clarify whether HMGB1 was released by apoptotic or dying SMMC-7721 cells,Annexin V/PI apoptosis assay of SMMC-7721 ceils with radiation and TRAIL treatment showed that the dying cells rather than apoptotic cells could release HMGB1 responsible for the tumor immune escape,because the supernatant from dying cells could promote MIP-3αexpression in SMMC-7721 but apoptotic cells could not. These data show that HMGB1 in the supernatant from dying HCC cells after radiotherapy or TRAIL treatment is responsible for the induction of the immunosuppressive factors by HCC cells in paracrine manner.
Since HMGB1 could mediate the growth and spread of gliomas,so we are wondering whether HMGB1 in the supernatant from dying HCC cells could promote HCC proliferation.We found HMGB1 promoted the growth of SMMC-7721 cells as detected by MTT assay.The evaluation of cell proliferation by BrdU incorporation also confirmed the promotion of the growth of SMMC-7721 cells by HMGB1 stimulation.Then we investigated whether HMGB1 could promote the cell cycles and thus increasing the cell proliferation.Cell cycle analysis by PI staining revealed that recombinant HMGB1 could promote transition of G1 to S phase entry,which might be responsible for the HCC growth.These evidences suggested that the dying cell with radiation or biotherapy contribute to the tumor cell growth by releasing high levels of HMGB1 which can promote cell cycle.
Since cyclin A,D E are required for progression through the G1 and S entry,we then examined by Western blot whether cyclin A,D and E were affected by HMGB1 stimulation.We found that cyclin D was dramatically increased in SMMC-7721 cells after HMGB1 stimulation,without alternation of cyclin A and cyclin E.In addition,as p53,p21 and p27 all negatively regulate cell cycle progression by inhibition of the cyclin E/cyclin-dependent kinase activity,we speculated that these negative regulators might play a role in the process of cell cycle entry promoted by HMGB1.Western blot showed that HMGB1 led to significant down-regulation of p27,but not p21 and p53. Because only phosphorylated p27 on T187 can be recognized by ubiquitin protein ligase for consequent ubiquition and degradation,we therefore tested whether p27 phosphorylation was affected by HMGB1 stimulation.Western blot showed that phosphorylation on p27(T187)increased in SMMC-7721 cells in response to HMGB1. Together,the data suggest that decrease of p27 and increase of cyclinD might be responsible for the promotion of cells cycle and consequent HCC cell proliferation and growth by HGMB1 released from dying HCC cells.
For the effectiveness of chemotherapy or radiotherapy,one of major obstacles is the resistance of cancer cells to anti-tumor drugs.We speculated that HMGB1 might be involved in the increased anti-apoptosis effect of HCC cells after chemotherapyor radiotherapy.SMMC-7721 cells were pretreated by HMGB1,and then induced by TRAIL.Annexin V/PI analysis revealed that HMGB1 reduced apoptosis induction of HCC cells by TRAIL.To further elucidate the mechanism for the induction of apoptosis resistance by HMGB1,pro-or anti-apoptotic proteins were analyzed by Western blot.We found that upon stimulation of HMGB1,anti-apoptotic protein expression of Bcl-xL,but not Bcl-2,Bax or Bak,was significantly upregulated in SMMC-7721 cells.These data suggest that upregulation of Bcl-xL expression by HMGB1 might be the mechanism to avoid the TRAIL-induced apoptosis and contribute to HCC cell survival.
As observed above,recombinant HMGB1 and the HMGB1-containing supernatant from dying HCC cells promote the production of immunosuppressive cytokine VEGF and chemokines MIP-3α,IP-10 and MCP-1 by SMMC-7721 HCC cells.In addition,HMGB1 is identified as one of the endogenous ligands of TLR4.To further confirm that TLR4/MyD88 signaling pathway mediates HMGB1-induced immune escape,we established siRNA TLR4 and siRNA MyD88 of SMMC-7721 cells.RT-PCR showed that, in response to HMGB1 or supernatant from dying HCC cells,TLR4 or MyD88-silenced SMMC-7721 cells have a greatly reduced ability to express MIP-3αmRNA.Together, these results suggest that the increase of MIP-3αexpression induced by recombinant HMGB1 and supernatant from dying cells is mediated through TLR4/MyD88 pathway. In addition,similarly to LPS stimulation,HMGB1 could activate p38 MAPK,ERK1/2, JNK,and NF-κB pathways in SMMC-7721 HCC cells.Therefore,we investigated whether MAPK or NF-κB signal pathways were involved in the cell cycle progression and apoptotic resistance in human HCC cells in response to HMGB1.Western blot assay showed that HMGB1-induced phosphorylation of p27 and subsequent degradation,and cyclin D expression were significantly abrogated by the specific inhibitor of MEK1/2 (PD98059)and ERK1/2(U0126).However,the NF-κB inhibitor PDTC decreased the HMGB1-upregulated Bcl-xL,So,MEK1/2 and ERK1/2 kinase activation is essential for HMGB1-promoted cell cycle progress,and NF-κB activation is required for apoptotic resistance of HCC cells.These data demonstrate that HMGB1/TLR4-mediated MEK1/2/ ERK1/2 and NF-κB activation is responsible for the promotion of HCC cell production of immunosuppressive factors and induction of apoptosis resistance.
In conclusion,our findings provide the direct evidence that,during chemotherapy or radiotherapy,HMGB1 released by dying HCC cells could induce production of the immunosuppressive cytokine VEGF and chemokines MIP-3α,IP-10 and MCP-1 expression in HCC cells.HMGB1 promotes the cell cycle transition of G1-S phase by enhancing cyclinD expression,and phosphorylation of p27-T187 and subsequent proteasomal degradation in HCC cells.Furthermore,up-regulation of anti-apoptotic protein Bcl-xL contributes to the recombinant HMGB1-triggered HCC cell resistance of TRAIL-induced apoptosis,indicating that HMGB1 is closely related to tumor immune escape and development.HMGB1 fails to induce MIP-3αexpression in HCC cells with either silence of HMGB1 or TLR4/MyD88,further confirming the HMGB1/TLR4/MyD88-dependent pathway mediates induction of immune escape and progression of HCC after chemotherapy or radiotherapy.Therefore,our findings provide the direct evidence that,during radiotherapy or chemotherapy,the necrotic cells-induced immune escape and apoptosis resistance promotes HCC progression by HMGBI/TLR4/MyD88 dependant pathways,which imply a potential application of HMGB1 neutralization or interfering in combination with chemotherapy in the treatment of hepatocellular carcinoma.
引文
1.Sprent J,Kishimoto H.The thymus and negative selection.Immunol Rev.2002;185:126-135.
2.Van Parijs L,Abbas AK.Homeostasis and self-tolerance in the immune system:turning lymphocytes off.Science.1998;280:243-248.
3.Coussens LM,Werb Z.Inflammation and cancer.Nature.2002,420:860-867
4.Balkwill F,Mantovani A.Inflammation and cancer:back to Virchow? Lancet.2001;357:539-545.
5.Itzkowitz SH,Yio X.Inflammation and cancer IV.Colorectal cancer in inflammatory bowel disease:the role of inflammation.Am J Physiol Gastrointest Liver Physiol.2004;287:7-17.
6.Seril DN,Lia J,Yang GY,Yang CS.Oxidative stress and ulcerative colitisassociated carcinogenesis:studies in humans and animal models.Carcinogenesis.2003;24:353-362.
7.Eaden J,Abrams K,Ekbom A,Jackson E,Mayberry J.Colorectal cancer prevention in ulcerative colitis:a case control study.Aliment Pharmacol Ther.2000;14:145-153.
8.Block TM,Mehta AS,Fimmel CJ,Jordan R.Molecular viral oncology of hepatocellular carcinoma.Oncogene.2003;22:5093-5107.
9.Okada F.Inflammation and free radicals in tumor development and progression.Redox Rep.2002;7:357-368.
10.Aleman M,Schierloh P,de la Barrera SS,Musella RM,Saab MA,Baldini M,Abbate E,Sasiain MC.Mycobacterium tuberculosis triggers apoptosis in peripheral neutrophils involving toll-like receptor 2 and p38 mitogen protein kinase in tuberculosis patients.Infect Immun.2004;72:5150-5158.
11.Rad R,Brenner L,Krug A,Voland P,Mages J,Lang R,Schwendy S,Reindl W,Dossumbekova A,Ballhorn W,Wagner H,Schmid RM,Bauer S,Prinz C.Toll-like receptor-dependent activation of antigen-presenting cells affects adaptive immunity to Helicobacter pylori.Gastroenterology.2007;133:150-163.
12.Archer KA,Roy CR.MyD88-dependent responses involving toll-like receptor 2are important for protection and clearance of Legionella pneumophila in a mouse model of Legionnaires' disease.Infect Immun.2006;74:3325-3333.
13.O'Connell RM;Vaidya SA;Perry AK;Saha SK;Dempsey PW;Cheng G,Immune activation of type Ⅰ IFNs by Listeria monocytogenes occurs independently of TLR4,TLR2,and receptor interacting protein 2 but involves TNFR-associated NF kappa B kinase-binding kinase 1.J Immunol.2005;174:1602-1607.
14.Taro Kawail.Innate immune recognition of viral infection.Nat Immunol.2006;7:131-137.
15.Cianchi F,Cortesini C,Fantappiè O,et al.Cyclooxygenase-2 activation mediates the proangiogenic effect of nitric oxide in colorectal cancer.Clin Cancer Res.2004;10:2694-704.
16.Wang,J.H.,Manning,B.J.,Wu,Q.D.,Blankson,S.,Bouchier-Hayes,D.,Redmond,H.P.,.Endotoxin/lipopolysaccharide activates NF-κB and enhances tumor cell adhesion and invasion through α2β4 integrin-dependent mechanism.J Immunol.2003;170:795-804.
17.Cook DN,Pisetsky DS,Schwartz DA.Toll-like receptors in the pathogenesis of human disease.Nat Immunol.2004;5:975-979.
18.Teachey,D.T.,Obzut,D.A.,Axsom,K.,Choi,J.K.,Goldsmith,K.C.,Hall,J.,Hulitt,J.,Manno,C.S.,Maris,J.M.,Rhodin,N.,Sullivan,K.E.,Brown,V.I.,Grupp,S.A.Rapamycin improves lymphoproliferative disease in murine autoimmune lymphoproliferative syndrome(ALPS).Blood.2006;108:1965-1971.
19.Hackstein,H.,Taner,T.,Zahorchak,A.F.,Morelli,A.E.,Logar,A.J.,Gessner,A.Thomson,A.W.Rapamycin inhibits IL-4-induced dendritic cell maturation in vitro and dendritic cell mobilization and function in vivo.Blood.2003;101:4457-4463.
20.Hartford,C.M.,Ratain,M.J.Rapamycin:something old,something new,sometimes borrowed and now renewed.Clin.Pharmacol Ther.2007;82:381-388.
21.Buck E,Eyzaguirre A,Brown E,Petti F,McCormack S,Haley JD,Iwata KK,Gibson NW,Griffin G..Rapamycin synergizes with the epidermal growth factor receptor inhibitor erlotinib in non-small-cell lung,pancreatic,colon,and breast tumors.Mol Cancer Ther.2006;5:2676-2684.
22.Zhang,J.F.,Liu,J.J.,Lu,M.Q.,Cai,C.J.,Yang,Y.,Li,H.,Xu,C.,Chen,G.H..Rapamycin inhibits cell growth by induction of apoptosis on hepatocellular carcinoma cells in vitro.Transpl.Immunol.2007;17:162-168.
23.Abraham,R.T.,Gibbons,J.J.The mammalian target of rapamycin signaling pathway:twists and turns in the road to cancer therapy.Clin.Cancer Res.2007;13:3109-3114.
24.Cianchi F,Cortesini C,Fantappiè O,Messerini L,Sardi I,Lasagna N,Perna F,Fabbroni V,Di Felice A,Perigli G,Mazzanti R,Masini E..Cyclooxygenase-2activation mediates the proangiogenic effect of nitric oxide in colorectal cancer.Clin Cancer Res.2004;10:2694-2704.
25.Adachi Y,Aoki C,Yoshio-Hoshino N,Takayama K,Curiel DT,Nishimoto N.Interleukin-6 induces both cell growth and VEGF production in malignant mesotheliomas.Int.J.Cancer.2006;119:1303-1311.
26.Mahic M,Yaqub S,Johansson CC,Taskén K,Aandahl EM.FOXP3+CD4+CD25+adaptive regulatory T cells express cyclooxygenase-2 and suppress effector T cells by a prostaglandin E2-dependent mechanism.J Immunol.2006;177:246-254.
27.Clinchy B,Fransson A,Druvefors B,Hellsten A,Hakansson A,Gustafsson B,Sjodahl R,Hakansson L.Preoperative interleukin-6 production by mononuclear blood cells predicts survival aider radical surgery for colorectal carcinoma.Cancer.2007;109:1742-1749.
28.Boffa,D.J.,Luan,F.,Thomas,D.,Yang,H.,Sharma,V.K.,Lagman,M.,Suthanthiran,M.Rapamycin inhibits the growth and metastatic progression of non-small cell lung cancer.Clin Cancer Res.2004;10:293-300.
29.Wang LH,Chan JL,Li W.Rapamycin together with herceptin significantly increased anti-tumor efficacy compared to either alone in ErbB2 over expressing breast cancer cells.Int J Cancer.2007;121:157-164.
30.He,W.,Liu,Q.,Wang,L.,Chen,W.,Li,N.,Cao,X.,TLR4 signaling promotes immune escape of human lung cancer cells by inducing immunosuppressive cytokines and apoptosis resistance.Mol.Immunol.2007;44:2850-2859.
31.An,H.,Yu,Y.,Zhang,M.,Xu,H.,Qi,R.,Cao,X..Involvement of ERK,p38 and NF-kappaB signal transduction in regulation of TLR2,TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells.Immunology.2002;106:38-45.
32.Dos Santos S,Delattre AI,De Longueville F,Bult H,Raes M.Gene expression profiling of LPS-stimulated murine macrophages and role of the NF-kappaB and PI3K/mTOR signaling pathways.Ann N Y Acad Sci.2007;1096:70-77.
33.Gobert AP,Vareille M,Glasser AL,Hindré T,de Sablet T,Martin C.Shiga toxin produced by enterohemorrhagic Escherichia coli inhibits PI3K/NF-kappaB signaling pathway in globotriaosylceramide-3-negative human intestinal epithelial cells.J Immunol.2007;178:8168-8174.
34.Lee SJ,Lim KT.UDN glycoprotein regulates activities of manganese-superoxide dismutase,activator protein-1,and nuclear factor-kappaB stimulated by reactive oxygen radicals in lipopolysaccharide-stimulated HCT-116 cells.Cancer Lett.2007;254:274-287.
35.Molteni M,Marabella D,Orlandi C,Rossetti C.Melanoma cell lines are responsive in vitro to lipopolysaccharide and express TLR-4.Cancer Lett.2006;235:75-83.
36.Sinha P,Clements VK,Fulton AM,Ostrand-Rosenberg S.Prostaglandin E2promotes tumor progression by inducing myeloid-derived suppressor cells.Cancer Res.2007;67:4507-4513.
37.Srivastava A,Henneke P,Visintin A,Morse SC,Martin V,Watkins C,Paton JC,Wessels MR,Golenbock DT,Malley R.The apoptotic response to pneumolysin is Toll-like receptor 4 dependent and protects against pneumococcal disease.Infect Immun.2005;73:6479-6487.
38.Castellone MD,Teramoto H,Gutkind JS.Cyclooxygenase-2 and colorectal cancer chemoprevention:the beta-catenin connection.Cancer Res.2006;66:11085-11088.
39.Myung,S.J.,Rerkom R.M.,Yanm M.,Platzer,P.,Guda,K.,Dotson,A.,Lawrence,E.,Dannenberg,A.J.,Lovgren,A.K.,Luo,G.,Pretlow,T.P.,Newman,R.A.,Willis,J.,Dawson,D.,Markowitz,S.D.15-Hydroxyprostaglandin dehydrogenase is an in vivo suppressor of colon tumorigenesis.Proc.Natl.Acad.Sci.2006;103:12098-12102.
40.Aggarwal BB,Sethi G,Ahn KS,Sandur SK,Pandey MK,Kunnumakkara AB,Sung B,Ichikawa H.Targeting signal-transducer-and-activator-of-transcription-3for prevention and therapy of cancer:modern target but ancient solution.Ann N Y Acad Sci.2006;1091:151-169.
56.Yuan F,Gu L,Guo S,Wang C,Li GM.Evidence for involvement of HMGB1protein in human DNA mismatch repair.J Biol Chem.2004;279:20935-20940.
41.Takada Y,Ichikawa H,Badmaev V,Aggarwal BB.Acetyl-11-keto-beta-boswellic acid potentiates apoptosis,inhibits invasion,and abolishes osteoclastogenesis by suppressing NF-kappa B and NF-kappa B-regulated gene expression.J Immunol.2006;176:3127-3140.
42.Kim SW,Oleksyn DW,Rossi RM,Jordan CT,Sanz I,Chen L,Zhao J.Protein kinase C-associated kinase is required for NF-{kappa}B signaling and survival in diffuse large B-cell lymphoma cells.Blood.2008;111:1644-1653.
43.Giri S,Rattan R,Singh AK,Singh I.The 15-deoxy-delta12,14-prostaglandin J2inhibits the inflammatory response in primary rat astrocytes via down-regulating multiple steps in phosphatidylinositol 3-kinase-Akt-NF-kappaB-p300 pathway independent of peroxisome proliferator-activated receptor gamma.J Immunol.2004;173:5196-5208.
44.Chung YL,Jian JJ,Cheng SH,Tsai SY,Chuang VP,Soong T,Lin YM,Horng CF.Sublethal irradiation induces vascular endothelial growth factor and promotes growth of hepatoma cells:implications for radiotherapy of hepatocellular carcinoma.Clin Cancer Res.2006;12:2706-2715.
45.Volp K,BrezniceanuML,Bosser S,et al.Increased expression of high mobility group box 1(HMGB1)is associated with an elevated level of the antiapoptotic c-IAP2 protein in human colon carcinomas.Gut.2006;55:234-242.
46.Melvin VS,Harrell C,Adelman JS,Kraus WL,Churchill M,Edwards DP.The role of the C-terminal extension(CTE)of the estrogen receptor alpha and beta DNA binding domain in DNA binding and interaction with HMGB.J Biol Chem.2004;279:14763-14771.
47.Chung YL,Jian JJ,Cheng SH,Tsai SY,Chuang VP,Soong T,Lin YM,Horng CF. Sublethal irradiation induces vascular endothelial growth factor and promotes growth of hepatoma cells:implications for radiotherapy of hepatocellular carcinoma.Clin Cancer Res.2006;12:2706-2715.
48.Scaffidi P,MisteliT,BianchiME.Release of chromatin protein HMGB1 by necrotic cells triggers inflammation.Nature.2002;418:191-195.
49.Yuan F,Gu L,Guo S,Wang C,Li GM.Evidence for involvement of HMGB1protein in human DNA mismatch repair.J Biol Chem.2004;279:20935-20940.
50.Tsung A,Sahai R,Tanaka H,Nakao A,Fink MP,Lotze MT,Yang H,Li J,Tracey KJ,Geller DA,Billiar TR.The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion.J Exp Med.2005;201:1135-1143.
51.Ellerman JE,Brown CK,de Vera M,Zeh HJ,Billiar T,Rubartelli A,Lotze MT.Masquerader:high mobility group box-1 and cancer.Clin Cancer Res.2007;13:2836-2348.
52.Ito N,DeMarco RA,Mailliard RB,Han J,Rabinowich H,Kalinski P,Stolz DB,Zeh HJ 3rd,Lotze MT.Cytolytic cells induce HMGB1 release from melanoma cell lines.J Leukoc Biol.2007;8:75-83.
53.Dong Xda E,Ito N,Lotze MT,Demarco RA,Popovic P,Shand SH,Watkins S,Winikoff S,Brown CK,Bartlett DL,Zeh HJ 3rd.High mobility group box I (HMGB1)release from tumor cells after treatment:implications for development of targeted chemoimmunotherapy.J Immunother(1997).2007;30:596-606.
54.Revesz L.Effect of tumour cells killed by X-rays upon the growth of admixed viable cells.Nature.1956;178:1391-1392.
55.André F,Delaloge S,Tursz T,Kroemer G,Zitvogel L.Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy.Nature Medicine.2007;13:1050-1059.
56.Golob M,Buettner R,Bosserhoff AK.Characterization of a transcription factor binding site,specifically activating MIA transcription in melanoma.J Invest Dermatol.2000;115:42-47.
57.Jacob K,Wach F,HolzapfelU,et al.In vitro modulation of human melanoma cell invasion and proliferation by all-trans-retinoic acid.Melanoma Res.1998;8:211-219.
58.Takada M,Hirata K,Ajiki T,Suzuki Y,Kuroda Y.Expression of receptor for advanced glycation end products(RAGE)and MMP-9 in human pancreatic cancer cells.Hepatogastroenterology.2004;51:928-930.
59.Chen K,Huang J,Gong W,Iribarren P,Dunlop NM,Wang JM.Toll-like receptors in inflammation,infection and cancer.Int Immunopharmacol.2007;7:1271-1285.
60.Edgar,B.A.Orr-Weaver TL.Endoreplication cell cycles:more for less.Cell.2001;105(3):297-306.
61.Pagano,M.Cell cycle regulation by the ubiquitin pathway.FASEB J.1997;11:1067-1075.
62.Apetoh L,Ghiringhelli F,Tesniere A,Obeid M,Ortiz C,Criollo A,Mignot G,Maiuri MC,Ullrich E,Saulnier P,Yang H,Amigorena S,Ryffel B,Barrat FJ,Saftig P,Levi F,Lidereau R,Nogues C,Mira JP,Chompret A,Joulin V,Clavel-Chapelon F,Bourhis J,André F,Delaloge S,Tursz T,Kroemer G,Zitvogel L. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med. 2007; 13:1050-1059.
63. Campoli, M., Ferrone, S., Zea, A.H., Rodriguez, P.C., Ochoa, A.C., 2005. Mechanisms of tumor evasion. Cancer Treat. Res. 123: 61-88.
64. Harmey, J.H., Bucana, C.D., Lu, W., Byrne, A.M., McDonnell, S., Lynch, C., Bouchier-Hayes, D., Dong, Z. Lipopolysaccharide-induced metastatic growth is associated with increased angiogenesis, vascular permeability and tumor cell invasion. Int. J. Cancer. 2002;101: 415-422.
65. Bonnotte, M. Crittenden, N. Larmonier, M. Gough, R.G. Vile, MIP-3alpha transfection into a rodent tumor cell line increases intratumoral dendritic cell infiltration but enhances (facilitates) tumor growth and decreases immunogenicity. J. Immunol. 2004; 173:4929-4935.
66. Conti, B.J. Rollins, CCL2 (monocyte chemoattractant protein-1) and cancer Semin. Cancer Biol. 2004; 14:149-54.
67. Locati M, Deuschle U, Massardi ML, Martinez FO, Sironi M, Sozzani S, Bartfai T, Mantovani A. Analysis of the gene expression profile activated by the CC chemokine ligand 5/RANTES and by lipopolysaccharide in human monocytes. J. Immunol. 2002;168:3557-3562.
68. Talmadge JE, Donkor M, Scholar E. inflammatory cell infiltration of tumors: Jekyll or Hyde. Cancer Metastasis Rev. 2007;26:373-400.
69. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, Lee JH, Ciarallo S, Catzavelos C, Beniston R, Franssen E, Slingerland JM.PKB/Akt phosphorylates p27,impairs nuclear import of p27 and opposes p27-mediated G1arrest.Nat Med.2002;8:1153-1160.
1.Hashimoto,C.Hudson,K.L.Anderson,K.V.The Toll gene of Drosophila,required for dorsal-ventral embryonic polarity appears to encode a transmembrane protein.Cell.1988;52:269-279.
2.Medzhitov R,Preston-Hurlburt P,Janeway Jr CA.A human homologue of the Drosophila TbU proteins signals activation of adaptive immunity.Nature.1997;388:394-397.
3.Dunne A,O Neill L A.The interleukin 一 1 receptor/Toll like receptor supeffamily:signal transduction during inflammation and host defense,sci sTKE.2003;171:3-7
4.Takeda K,Kaisho T,Akira S.Toll-like receptors.Annu Rev Immunol.2003;21:335-337.
5.Cook DN,Pisetsky DS,Schwartz DA.Toll-like receptors in the pathogenesis of human disease.Nat Immunol.2004;5:975-995.
6.O'Neill LA.How Toll-like receptors signal:what we know and what we don't know.Curr Opin Immunol.2006;25:687-693.
7.Zheng S L,Augustsson-Baiter K,Chang B.Sequence variants of Toll like receptor 4 are associated with prostate cancer risk:results from the cancer prostate in Sweden study.Cancer Res.2004;64:2918-2922.
8.Song C,Chen LZ,Zhang RH,Yu XJ,Zeng YX.Functional variant in the 3'-untranslated region of Toll-like receptor4 is associated with nasopharyngeal carcinoma risk.Cancer Biol Ther.2006;5:1285-1291.
9.Zhou XX,Jia WH,Shen GP,Qin HD,Yu XJ,Chen LZ,Feng QS,Shugart YY,Zeng YX.Sequence variants in toll-like receptor 10 are associated with nasopharyngeal carcinoma risk.Cancer Epidemiol Biomarkers Prev.2006;15:862-866.
10.Ohara T,Morishita T,Suzuki H,Hibi T.Heterozygous Thr135 Ala polymorphism at leucine-rich repeat(LRR)in genomic DNA of toll-like receptor 4 in patients with poorly-differentiated gastric adenocarcinomas.Int J Mol Med.2006;18:59-63.
11.Hold GL,Rabkin CS,Chow WH,Smith MG,Gammon MD,Risch HA,Vaughan TL,McColl KE,Lissowska J,Zatonski W,Schoenberg JB,Blot WJ,Mowat NA,Fraumeni JF Jr,El-Omar EM.A functional polymorphism of toll-like receptor 4gene increases risk of gastric carcinoma and it sprecursors.Gastroenterology.2007;132:905-912.
12.Nieters A,Beckmann L,Deeg E,Becker N.Gene polymorphisms in Toll-like receptors,interleukin-10,and intedeukin-10 receptor alpha and lymphoma risk.Genes Immun.2006;7:615-624.
13.Marshall BJ,Warren JR,Goodwin CS.Duodenal ulcer relapse after eradication of Campylobacter pylori.Lancet.1989;1:836-837.
14.Marshall BJ,Windsor HM.The relation of Helicobacter pylori to gastric adenocarcinoma and lymphoma:pathophysiology,epidemiology,screening,clinical presentation,treatment,and prevention.Med Clin North Am.2005;89:313-344.
15.Lax AJ.Opinion:Bacterial toxins and cancer-a case to answer? Nat Rev Microbiol.2005;3:343-349.
16.Pagano JS,Blaser M,Buendia MA,Damania B,Khalili K,Raab-Traub N,Roizman B.Infectious agents and cancer:criteria for a causal relation.Semin.Cancer Biol.2004;14:453-471.
17.Finch,C.E.,Crimmins,E.M.Inflammatory exposure and historical changes in human life-spans.Science.2004;305:1736-1739.
18.Zhang GM,Xiong H,Feng ZH.Listeria monocytogenes promotes tumor growth via tumor cell toll-like receptor 2 signaling.Cancer Res.2007;67:4346-4352.
19.Huang B,Zhao J,Li H,He KL,Chen Y,Chen SH,Mayer L,Unkeless JC,Xiong H.Toll-like receptors on tumor cells facilitate evasion of immunesurveillance.Cancer Res.2005;65:5009-5014.
20.Huang B,Zhao J,Unkeless JC,Feng ZH,Xiong H.TLR signaling by tumor and immune cells:a double-edged sword Oncogene.2008;27:218-224.
21 Droemann D,Albrecht D,Gerdes J,Ulmer AJ,Branscheid D,Vollmer E,Dalhoff K,Zabel P,Goldmann T.Human lung cancer cells express functionally active Toll-like receptor 9.Respir Res.2005;6:1-7.
22.SchmausserB,AndrulisM,EndrichS,Muller-Hermelink HK,EckM.Toll-like receptors TLR4,TLR5 and TLR9 on gastric carcinoma cells:an implication for interaction with Helicobacterpylori.Int J Med Microbiol.2005;295:179-185.
23.Szczepanski M,Stelmachowska M,Stryczynski L,Golusinski W,SamaraH,Mozer-Lisewskaletal.Assessment of expression of toll-like receptors 2,3 and 4 in laryngeal carcinoma.Eur Arch Otorhinolaryngol.2007;264:525-530.
24.Lee JW,Choi JJ,Seo ES,Kim MJ,Kim WY,Choi CH,Kim TJ,Kim BG,Song SY,Bae DS.Increased toll-like receptor 9 expression in cervical neoplasia.Mol Carcinog 46:941-947.Mol Carcinog.2007;46:941-947.
25.Hassan F,Islam S,Tumurkhuu G,Naiki Y,Koide N,Mori I,Yoshida T,Yokochi T.Intracellular expression of toll-like receptor 4 in neuroblastoma cells and their unresponsiveness to lipopolysaccharide.BMC Cancer.2006;6:281-287.
26.Ilvesaro JM,Merrell MA,Swain TM,Davidson J,Zayzafoon M,Harris KW,Selander KS.Toll like receptor-9 agonists stimulate prostate cancer invasion in vitro.Prostate.2007;67:774-781.
27.Merrell MA,Ilvesaro JM,Lehtonen N,Sorsa T,Gehrs B,Rosenthal E,Chen D,Shackley B,Harris KW,Selander KS.Toll-like receptor 9 agonists promote cellular invasion by increasing matrixmetalloproteinase activity.Mol Cancer Res.2006;4:437-447.
28.Bohnhorst J,Rasmussen T,Moen SH,Flφttum M,Knudsen L,Bφrset M,Espevik T,Sundan A.Toll-like receptors mediate proliferation and survival of multiple myeloma cells.Leukemia.2006;20:1138-1144.
29.Jego G,Bataille R,Geffroy-Luseau A,Descamps G,PellatDeceunynck C. Pathogen-associated molecular patterns are growth and survival factors for human myeloma cells through Toll-like receptors.Leukemia.2006;20:1130-1137.
30.Bauer AK,Dixon D,DeGraff LM,Cho HY,Walker CR,Malkinson AM,Kleeberger SR.Toll-like receptor.4 in butylated hydroxytoluene-induced mouse pulmonary,inflammation and tumorigenesis.J Natl Cancer Inst.2005;97:1778-1781.
31.He W,Liu Q,Wang L,Chen W,Li N,Cao X.TLR4 signaling promotes immune escape of human lung cancer cells by inducingimmunosuppressive cytokines and apoptosis resistance..Mol Immunol.2007;44:2850-2859.
32.Wang L,Liu Q,Sun Q,Zhang C,Chen T,Cao X.TLR4 signaling in cancer cells promotes chemoattraction of immature dendritic cells via autocrine CCL20.Biochem Biophys Res Commun.2008;366:852-856.
33.Kelly MG,Alvero AB,Chen R,Silasi DA,Abrahams VM,Chan S,Visintin I,Rutherford T,Mor G.TLR-4 signaling promotes tumor growth and paclitaxel chemoresistance in ovarian cancer.Cancer Res.2006;66:3859-3868.
34.Yuan ZQ,Feldman RI,Sussman GE,Coppola D,Nicosia V,Cheng JQ.AKT2inhibition of cisplatin-induced JNK/p38 and Bax activation by phosphorylation of ASK1:implication of AKT2 in chemoresistance.J Biol Chem.2003;278:23432-23440.
35.Dan HC,Jiang K,Coppola D,Hamilton A,Nicosia SV,Sebti SM,Cheng JQ.Phosphatidylinositol-3-OH kinase/AKT and survivin pathways as critical targets for geranylgeranyltransferase Ⅰ inhibitor-induced apoptosis.Oncogene.2004a; 23:706-715.
36.Jego G,Bataille R,Geffroy-Luseau A,Descamps G,PellatDeceunynck C.Pathogen-associated molecular patterns are growth and survival factors for human myeloma cells through Toll-like receptors.Leukemia.2006;20:1130-1137.
37.Merrell MA,Ilvesaro JM,Lehtonen N,Sorsa T,Gehrs B,Rosenthal E,Chen D,Shackley B,Harris KW,Selander KS.Toll-like receptor 9 agonists promote cellular invasion by increasing matrixmetalloproteinase activity.Mol Cancer Res.2006;4:437-447.
38.Harmey JH,Bucana CD,Lu W,Byrne AM,McDonnell S,Lynch C,Bouchier-Hayes D,Dong Z.Lipopolysaccharide-induced metastatic growth is associated with increased angiogenesis,vascular permeability and tumor cell invasion.Int J Cancer.2002;101:415-422.
39.Wang JH,Manning BJ,Wu QD,Blankson S,Bouchier-Hayes D,Redmond HP.Endotoxin/lipopolysaccharide activates NF-kappaB and enhances tumor cell adhesion and invasion through a beta 1 integrin-dependent mechanism.J Immunol.2003;170:795-804.
40.Zheng S L.,Augustsson-Baiter K,Chang B.Sequence variants of Toll like receptor 4 are associated with prostate cancer risk:results from the cancer prostate in Sweden study.Cancer Res.2004;64:2918-2922.
41.Tsan MF.Toll- like receptors,inflammation and cancer.Semin Cancer Biol.2006;16:32-37.
42.Johnson GB,Brunn GJ,Platt JL.Cutting edge:an endogenous pathway to systemic inflammatory response syndrome(SIRS)-like reactions through Toll-like receptor 4.J Immunol.2004;172:20-24.
43.Brunn GJ,Bungum MK,Johnson GB,Platt JL.Conditional signaling by Toll-like receptor 4.FASEB J.2005;19:872-874.
44.Bianchi ME.Significant(re)location:how to use chromatin and/or abundant proteins as messages of life and death.Trends Cell Biol.2004;14:287-293.
45.Bonaldi T,Talamo F,Scaffidi P,Ferrera D,Porto A,Bachi A,Rubartelli A,Agresti A,Bianchi ME.Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion.EMBO J.2003;22:5551-5560.
46.Zeh HJ Ⅲ,Lotze MT.Addicted to death:invasive cancer and the immune response to unscheduled cell death.J Immunother.2005;28:1-9.
47.Rauvala H,Huttunen HJ,Fages C,Kaksonen M,Kinnunen T,Imai S,Raulo E,Kilpelainen I.Heparinbinding proteins HB-GAM(pleiotrophin)and amphoterin in the regulation of cellmotility.Matrix Biol.2000;19:377-387.
48.Tsung A,Sahai R,Tanaka H,Nakao A,Fink MP,Lotze MT,Yang H,Li J,Tracey KJ,Geller DA,Billiar TR.The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion.J Exp Med.2005;201:1135-1143.
49.Rouhiainen A,Kuja-Panula J,Wilkman E,Pakkanen J,Stenfors J,Tuominen RK,Lepantalo M,Carpén O,Parkkinen J,Rauvala H..Regulation of monocyte migration by amphoterin(HMGB1).Blood.2004;104:1174-1182.
50. Messmer D, Yang H, Telusma G, Knoll F, Li J, Messmer B, Tracey KJ, Chiorazzi N. High mobility group box proteinl: an endogenous signal for dendritic cell maturation and Th1 polarization. J Immunol. 2004; 173:307-313.
51. Yu W, KJ, Ossowski L. Reduction in surface urokinase receptor forces malignant cells into a protracted state of dormancy. J Cell Biol. 1997;3:767-777.
52. Taguchi A, Blood DC, del Toro G, Canet A, Lee DC, Qu W, Tanji N, Lu Y, Lalla E, Fu C, Hofmann MA, Kislinger T, Ingram M, Lu A, Tanaka H, Hori O, Ogawa S, Stern DM, Schmidt AM. Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases. Nature. 2000;405:354-360.
53. Poser I, Golob M, Buettner R, Bosserhoff AK. Upregulation of HMG1 leads to melanoma inhibitory activity expression in malignant melanoma cells and contributes to their malignancy phenotype. Mol Cell Biol. 2003;23:2991-2998.
54. Volp K, BrezniceanuML, Bosser S, et al. Increased expression of high mobility group box 1 (HMGB1) is associated with an elevated level of the antiapoptotic C-IAP2 protein in human colon carcinomas. Gut. 2006;55:234-242.
55. Melvin VS, Harrell C, Adelman JS, Kraus WL, Churchill M, Edwards DP. The role of the C-terminal extension (CTE) of the estrogen receptor alpha and beta DNA binding domain in DNA binding and interaction with HMGB. J Biol Chem. 2004;279:14763-14771.
56. Chung YL, Jian JJ, Cheng SH, Tsai SY, Chuang VP, Soong T, Lin YM, Horng CF. Sublethal irradiation induces vascular endothelial growth factor and promotes growth of hepatoma cells: implications for radiotherapy of hepatocellular carcinoma. Clin Cancer Res. 2006; 12:2706-2715.
57. Chung YL, Jian JJ, Cheng SH, Tsai SY, Chuang VP, Soong T, Lin YM, Horng CF. Sublethal irradiation induces vascular endothelial growth factor and promotes growth of hepatoma cells: implications for radiotherapy of hepatocellular carcinoma. Clin Cancer Res. 2006; 12:2706-2715.
58. Ellerman JE, Brown CK, de Vera M, Zeh HJ, Billiar T, Rubartelli A, Lotze MT.Masquerader: high mobility group box-1 and cancer. Clin Cancer Res. 2007;13:2836-2348.
59. Ito N, DeMarco RA, Mailliard RB, Han J, Rabinowich H, Kalinski P, Stolz DB, Zeh HJ 3rd, Lotze MT.Cytolytic cells induce HMGBl release from melanoma cell lines. J Leukoc Biol. 2007;8:75-83.
60. Dong Xda E, Ito N, Lotze MT, Demarco RA, Popovic P, Shand SH, Watkins S, Winikoff S, Brown CK, Bartlett DL, Zeh HJ 3rd. High mobility group box I (HMGB1) release from tumor cells after treatment: implications for development of targeted chemoimmunotherapy. J Immunother (1997). 2007;30:596-606.
61. Revesz L. Effect of tumour cells killed by X-rays upon the growth of admixed viable cells. Nature. 1956;178:1391-1392.
62. Andre F, Delaloge S, Tursz T, Kroemer G, Zitvogel L. Toll-like receptor 4—dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nature Medicine. 2007; 13:1050-1059.
2.Van Parijs L,Abbas AK.Homeostasis and self-tolerance in the immune system:turning lymphocytes off.Science.1998;280:243-248.
3.Coussens LM,Werb Z.Inflammation and cancer.Nature.2002,420:860-867
4.Balkwill F,Mantovani A.Inflammation and cancer:back to Virchow? Lancet.2001;357:539-545.
5.Itzkowitz SH,Yio X.Inflammation and cancer IV.Colorectal cancer in inflammatory bowel disease:the role of inflammation.Am J Physiol Gastrointest Liver Physiol.2004;287:7-17.
6.Seril DN,Lia J,Yang GY,Yang CS.Oxidative stress and ulcerative colitisassociated carcinogenesis:studies in humans and animal models.Carcinogenesis.2003;24:353-362.
7.Eaden J,Abrams K,Ekbom A,Jackson E,Mayberry J.Colorectal cancer prevention in ulcerative colitis:a case control study.Aliment Pharmacol Ther.2000;14:145-153.
8.Block TM,Mehta AS,Fimmel CJ,Jordan R.Molecular viral oncology of hepatocellular carcinoma.Oncogene.2003;22:5093-5107.
9.Okada F.Inflammation and free radicals in tumor development and progression.Redox Rep.2002;7:357-368.
10.Aleman M,Schierloh P,de la Barrera SS,Musella RM,Saab MA,Baldini M,Abbate E,Sasiain MC.Mycobacterium tuberculosis triggers apoptosis in peripheral neutrophils involving toll-like receptor 2 and p38 mitogen protein kinase in tuberculosis patients.Infect Immun.2004;72:5150-5158.
11.Rad R,Brenner L,Krug A,Voland P,Mages J,Lang R,Schwendy S,Reindl W,Dossumbekova A,Ballhorn W,Wagner H,Schmid RM,Bauer S,Prinz C.Toll-like receptor-dependent activation of antigen-presenting cells affects adaptive immunity to Helicobacter pylori.Gastroenterology.2007;133:150-163.
12.Archer KA,Roy CR.MyD88-dependent responses involving toll-like receptor 2are important for protection and clearance of Legionella pneumophila in a mouse model of Legionnaires' disease.Infect Immun.2006;74:3325-3333.
13.O'Connell RM;Vaidya SA;Perry AK;Saha SK;Dempsey PW;Cheng G,Immune activation of type Ⅰ IFNs by Listeria monocytogenes occurs independently of TLR4,TLR2,and receptor interacting protein 2 but involves TNFR-associated NF kappa B kinase-binding kinase 1.J Immunol.2005;174:1602-1607.
14.Taro Kawail.Innate immune recognition of viral infection.Nat Immunol.2006;7:131-137.
15.Cianchi F,Cortesini C,Fantappiè O,et al.Cyclooxygenase-2 activation mediates the proangiogenic effect of nitric oxide in colorectal cancer.Clin Cancer Res.2004;10:2694-704.
16.Wang,J.H.,Manning,B.J.,Wu,Q.D.,Blankson,S.,Bouchier-Hayes,D.,Redmond,H.P.,.Endotoxin/lipopolysaccharide activates NF-κB and enhances tumor cell adhesion and invasion through α2β4 integrin-dependent mechanism.J Immunol.2003;170:795-804.
17.Cook DN,Pisetsky DS,Schwartz DA.Toll-like receptors in the pathogenesis of human disease.Nat Immunol.2004;5:975-979.
18.Teachey,D.T.,Obzut,D.A.,Axsom,K.,Choi,J.K.,Goldsmith,K.C.,Hall,J.,Hulitt,J.,Manno,C.S.,Maris,J.M.,Rhodin,N.,Sullivan,K.E.,Brown,V.I.,Grupp,S.A.Rapamycin improves lymphoproliferative disease in murine autoimmune lymphoproliferative syndrome(ALPS).Blood.2006;108:1965-1971.
19.Hackstein,H.,Taner,T.,Zahorchak,A.F.,Morelli,A.E.,Logar,A.J.,Gessner,A.Thomson,A.W.Rapamycin inhibits IL-4-induced dendritic cell maturation in vitro and dendritic cell mobilization and function in vivo.Blood.2003;101:4457-4463.
20.Hartford,C.M.,Ratain,M.J.Rapamycin:something old,something new,sometimes borrowed and now renewed.Clin.Pharmacol Ther.2007;82:381-388.
21.Buck E,Eyzaguirre A,Brown E,Petti F,McCormack S,Haley JD,Iwata KK,Gibson NW,Griffin G..Rapamycin synergizes with the epidermal growth factor receptor inhibitor erlotinib in non-small-cell lung,pancreatic,colon,and breast tumors.Mol Cancer Ther.2006;5:2676-2684.
22.Zhang,J.F.,Liu,J.J.,Lu,M.Q.,Cai,C.J.,Yang,Y.,Li,H.,Xu,C.,Chen,G.H..Rapamycin inhibits cell growth by induction of apoptosis on hepatocellular carcinoma cells in vitro.Transpl.Immunol.2007;17:162-168.
23.Abraham,R.T.,Gibbons,J.J.The mammalian target of rapamycin signaling pathway:twists and turns in the road to cancer therapy.Clin.Cancer Res.2007;13:3109-3114.
24.Cianchi F,Cortesini C,Fantappiè O,Messerini L,Sardi I,Lasagna N,Perna F,Fabbroni V,Di Felice A,Perigli G,Mazzanti R,Masini E..Cyclooxygenase-2activation mediates the proangiogenic effect of nitric oxide in colorectal cancer.Clin Cancer Res.2004;10:2694-2704.
25.Adachi Y,Aoki C,Yoshio-Hoshino N,Takayama K,Curiel DT,Nishimoto N.Interleukin-6 induces both cell growth and VEGF production in malignant mesotheliomas.Int.J.Cancer.2006;119:1303-1311.
26.Mahic M,Yaqub S,Johansson CC,Taskén K,Aandahl EM.FOXP3+CD4+CD25+adaptive regulatory T cells express cyclooxygenase-2 and suppress effector T cells by a prostaglandin E2-dependent mechanism.J Immunol.2006;177:246-254.
27.Clinchy B,Fransson A,Druvefors B,Hellsten A,Hakansson A,Gustafsson B,Sjodahl R,Hakansson L.Preoperative interleukin-6 production by mononuclear blood cells predicts survival aider radical surgery for colorectal carcinoma.Cancer.2007;109:1742-1749.
28.Boffa,D.J.,Luan,F.,Thomas,D.,Yang,H.,Sharma,V.K.,Lagman,M.,Suthanthiran,M.Rapamycin inhibits the growth and metastatic progression of non-small cell lung cancer.Clin Cancer Res.2004;10:293-300.
29.Wang LH,Chan JL,Li W.Rapamycin together with herceptin significantly increased anti-tumor efficacy compared to either alone in ErbB2 over expressing breast cancer cells.Int J Cancer.2007;121:157-164.
30.He,W.,Liu,Q.,Wang,L.,Chen,W.,Li,N.,Cao,X.,TLR4 signaling promotes immune escape of human lung cancer cells by inducing immunosuppressive cytokines and apoptosis resistance.Mol.Immunol.2007;44:2850-2859.
31.An,H.,Yu,Y.,Zhang,M.,Xu,H.,Qi,R.,Cao,X..Involvement of ERK,p38 and NF-kappaB signal transduction in regulation of TLR2,TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells.Immunology.2002;106:38-45.
32.Dos Santos S,Delattre AI,De Longueville F,Bult H,Raes M.Gene expression profiling of LPS-stimulated murine macrophages and role of the NF-kappaB and PI3K/mTOR signaling pathways.Ann N Y Acad Sci.2007;1096:70-77.
33.Gobert AP,Vareille M,Glasser AL,Hindré T,de Sablet T,Martin C.Shiga toxin produced by enterohemorrhagic Escherichia coli inhibits PI3K/NF-kappaB signaling pathway in globotriaosylceramide-3-negative human intestinal epithelial cells.J Immunol.2007;178:8168-8174.
34.Lee SJ,Lim KT.UDN glycoprotein regulates activities of manganese-superoxide dismutase,activator protein-1,and nuclear factor-kappaB stimulated by reactive oxygen radicals in lipopolysaccharide-stimulated HCT-116 cells.Cancer Lett.2007;254:274-287.
35.Molteni M,Marabella D,Orlandi C,Rossetti C.Melanoma cell lines are responsive in vitro to lipopolysaccharide and express TLR-4.Cancer Lett.2006;235:75-83.
36.Sinha P,Clements VK,Fulton AM,Ostrand-Rosenberg S.Prostaglandin E2promotes tumor progression by inducing myeloid-derived suppressor cells.Cancer Res.2007;67:4507-4513.
37.Srivastava A,Henneke P,Visintin A,Morse SC,Martin V,Watkins C,Paton JC,Wessels MR,Golenbock DT,Malley R.The apoptotic response to pneumolysin is Toll-like receptor 4 dependent and protects against pneumococcal disease.Infect Immun.2005;73:6479-6487.
38.Castellone MD,Teramoto H,Gutkind JS.Cyclooxygenase-2 and colorectal cancer chemoprevention:the beta-catenin connection.Cancer Res.2006;66:11085-11088.
39.Myung,S.J.,Rerkom R.M.,Yanm M.,Platzer,P.,Guda,K.,Dotson,A.,Lawrence,E.,Dannenberg,A.J.,Lovgren,A.K.,Luo,G.,Pretlow,T.P.,Newman,R.A.,Willis,J.,Dawson,D.,Markowitz,S.D.15-Hydroxyprostaglandin dehydrogenase is an in vivo suppressor of colon tumorigenesis.Proc.Natl.Acad.Sci.2006;103:12098-12102.
40.Aggarwal BB,Sethi G,Ahn KS,Sandur SK,Pandey MK,Kunnumakkara AB,Sung B,Ichikawa H.Targeting signal-transducer-and-activator-of-transcription-3for prevention and therapy of cancer:modern target but ancient solution.Ann N Y Acad Sci.2006;1091:151-169.
56.Yuan F,Gu L,Guo S,Wang C,Li GM.Evidence for involvement of HMGB1protein in human DNA mismatch repair.J Biol Chem.2004;279:20935-20940.
41.Takada Y,Ichikawa H,Badmaev V,Aggarwal BB.Acetyl-11-keto-beta-boswellic acid potentiates apoptosis,inhibits invasion,and abolishes osteoclastogenesis by suppressing NF-kappa B and NF-kappa B-regulated gene expression.J Immunol.2006;176:3127-3140.
42.Kim SW,Oleksyn DW,Rossi RM,Jordan CT,Sanz I,Chen L,Zhao J.Protein kinase C-associated kinase is required for NF-{kappa}B signaling and survival in diffuse large B-cell lymphoma cells.Blood.2008;111:1644-1653.
43.Giri S,Rattan R,Singh AK,Singh I.The 15-deoxy-delta12,14-prostaglandin J2inhibits the inflammatory response in primary rat astrocytes via down-regulating multiple steps in phosphatidylinositol 3-kinase-Akt-NF-kappaB-p300 pathway independent of peroxisome proliferator-activated receptor gamma.J Immunol.2004;173:5196-5208.
44.Chung YL,Jian JJ,Cheng SH,Tsai SY,Chuang VP,Soong T,Lin YM,Horng CF.Sublethal irradiation induces vascular endothelial growth factor and promotes growth of hepatoma cells:implications for radiotherapy of hepatocellular carcinoma.Clin Cancer Res.2006;12:2706-2715.
45.Volp K,BrezniceanuML,Bosser S,et al.Increased expression of high mobility group box 1(HMGB1)is associated with an elevated level of the antiapoptotic c-IAP2 protein in human colon carcinomas.Gut.2006;55:234-242.
46.Melvin VS,Harrell C,Adelman JS,Kraus WL,Churchill M,Edwards DP.The role of the C-terminal extension(CTE)of the estrogen receptor alpha and beta DNA binding domain in DNA binding and interaction with HMGB.J Biol Chem.2004;279:14763-14771.
47.Chung YL,Jian JJ,Cheng SH,Tsai SY,Chuang VP,Soong T,Lin YM,Horng CF. Sublethal irradiation induces vascular endothelial growth factor and promotes growth of hepatoma cells:implications for radiotherapy of hepatocellular carcinoma.Clin Cancer Res.2006;12:2706-2715.
48.Scaffidi P,MisteliT,BianchiME.Release of chromatin protein HMGB1 by necrotic cells triggers inflammation.Nature.2002;418:191-195.
49.Yuan F,Gu L,Guo S,Wang C,Li GM.Evidence for involvement of HMGB1protein in human DNA mismatch repair.J Biol Chem.2004;279:20935-20940.
50.Tsung A,Sahai R,Tanaka H,Nakao A,Fink MP,Lotze MT,Yang H,Li J,Tracey KJ,Geller DA,Billiar TR.The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion.J Exp Med.2005;201:1135-1143.
51.Ellerman JE,Brown CK,de Vera M,Zeh HJ,Billiar T,Rubartelli A,Lotze MT.Masquerader:high mobility group box-1 and cancer.Clin Cancer Res.2007;13:2836-2348.
52.Ito N,DeMarco RA,Mailliard RB,Han J,Rabinowich H,Kalinski P,Stolz DB,Zeh HJ 3rd,Lotze MT.Cytolytic cells induce HMGB1 release from melanoma cell lines.J Leukoc Biol.2007;8:75-83.
53.Dong Xda E,Ito N,Lotze MT,Demarco RA,Popovic P,Shand SH,Watkins S,Winikoff S,Brown CK,Bartlett DL,Zeh HJ 3rd.High mobility group box I (HMGB1)release from tumor cells after treatment:implications for development of targeted chemoimmunotherapy.J Immunother(1997).2007;30:596-606.
54.Revesz L.Effect of tumour cells killed by X-rays upon the growth of admixed viable cells.Nature.1956;178:1391-1392.
55.André F,Delaloge S,Tursz T,Kroemer G,Zitvogel L.Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy.Nature Medicine.2007;13:1050-1059.
56.Golob M,Buettner R,Bosserhoff AK.Characterization of a transcription factor binding site,specifically activating MIA transcription in melanoma.J Invest Dermatol.2000;115:42-47.
57.Jacob K,Wach F,HolzapfelU,et al.In vitro modulation of human melanoma cell invasion and proliferation by all-trans-retinoic acid.Melanoma Res.1998;8:211-219.
58.Takada M,Hirata K,Ajiki T,Suzuki Y,Kuroda Y.Expression of receptor for advanced glycation end products(RAGE)and MMP-9 in human pancreatic cancer cells.Hepatogastroenterology.2004;51:928-930.
59.Chen K,Huang J,Gong W,Iribarren P,Dunlop NM,Wang JM.Toll-like receptors in inflammation,infection and cancer.Int Immunopharmacol.2007;7:1271-1285.
60.Edgar,B.A.Orr-Weaver TL.Endoreplication cell cycles:more for less.Cell.2001;105(3):297-306.
61.Pagano,M.Cell cycle regulation by the ubiquitin pathway.FASEB J.1997;11:1067-1075.
62.Apetoh L,Ghiringhelli F,Tesniere A,Obeid M,Ortiz C,Criollo A,Mignot G,Maiuri MC,Ullrich E,Saulnier P,Yang H,Amigorena S,Ryffel B,Barrat FJ,Saftig P,Levi F,Lidereau R,Nogues C,Mira JP,Chompret A,Joulin V,Clavel-Chapelon F,Bourhis J,André F,Delaloge S,Tursz T,Kroemer G,Zitvogel L. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med. 2007; 13:1050-1059.
63. Campoli, M., Ferrone, S., Zea, A.H., Rodriguez, P.C., Ochoa, A.C., 2005. Mechanisms of tumor evasion. Cancer Treat. Res. 123: 61-88.
64. Harmey, J.H., Bucana, C.D., Lu, W., Byrne, A.M., McDonnell, S., Lynch, C., Bouchier-Hayes, D., Dong, Z. Lipopolysaccharide-induced metastatic growth is associated with increased angiogenesis, vascular permeability and tumor cell invasion. Int. J. Cancer. 2002;101: 415-422.
65. Bonnotte, M. Crittenden, N. Larmonier, M. Gough, R.G. Vile, MIP-3alpha transfection into a rodent tumor cell line increases intratumoral dendritic cell infiltration but enhances (facilitates) tumor growth and decreases immunogenicity. J. Immunol. 2004; 173:4929-4935.
66. Conti, B.J. Rollins, CCL2 (monocyte chemoattractant protein-1) and cancer Semin. Cancer Biol. 2004; 14:149-54.
67. Locati M, Deuschle U, Massardi ML, Martinez FO, Sironi M, Sozzani S, Bartfai T, Mantovani A. Analysis of the gene expression profile activated by the CC chemokine ligand 5/RANTES and by lipopolysaccharide in human monocytes. J. Immunol. 2002;168:3557-3562.
68. Talmadge JE, Donkor M, Scholar E. inflammatory cell infiltration of tumors: Jekyll or Hyde. Cancer Metastasis Rev. 2007;26:373-400.
69. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, Lee JH, Ciarallo S, Catzavelos C, Beniston R, Franssen E, Slingerland JM.PKB/Akt phosphorylates p27,impairs nuclear import of p27 and opposes p27-mediated G1arrest.Nat Med.2002;8:1153-1160.
1.Hashimoto,C.Hudson,K.L.Anderson,K.V.The Toll gene of Drosophila,required for dorsal-ventral embryonic polarity appears to encode a transmembrane protein.Cell.1988;52:269-279.
2.Medzhitov R,Preston-Hurlburt P,Janeway Jr CA.A human homologue of the Drosophila TbU proteins signals activation of adaptive immunity.Nature.1997;388:394-397.
3.Dunne A,O Neill L A.The interleukin 一 1 receptor/Toll like receptor supeffamily:signal transduction during inflammation and host defense,sci sTKE.2003;171:3-7
4.Takeda K,Kaisho T,Akira S.Toll-like receptors.Annu Rev Immunol.2003;21:335-337.
5.Cook DN,Pisetsky DS,Schwartz DA.Toll-like receptors in the pathogenesis of human disease.Nat Immunol.2004;5:975-995.
6.O'Neill LA.How Toll-like receptors signal:what we know and what we don't know.Curr Opin Immunol.2006;25:687-693.
7.Zheng S L,Augustsson-Baiter K,Chang B.Sequence variants of Toll like receptor 4 are associated with prostate cancer risk:results from the cancer prostate in Sweden study.Cancer Res.2004;64:2918-2922.
8.Song C,Chen LZ,Zhang RH,Yu XJ,Zeng YX.Functional variant in the 3'-untranslated region of Toll-like receptor4 is associated with nasopharyngeal carcinoma risk.Cancer Biol Ther.2006;5:1285-1291.
9.Zhou XX,Jia WH,Shen GP,Qin HD,Yu XJ,Chen LZ,Feng QS,Shugart YY,Zeng YX.Sequence variants in toll-like receptor 10 are associated with nasopharyngeal carcinoma risk.Cancer Epidemiol Biomarkers Prev.2006;15:862-866.
10.Ohara T,Morishita T,Suzuki H,Hibi T.Heterozygous Thr135 Ala polymorphism at leucine-rich repeat(LRR)in genomic DNA of toll-like receptor 4 in patients with poorly-differentiated gastric adenocarcinomas.Int J Mol Med.2006;18:59-63.
11.Hold GL,Rabkin CS,Chow WH,Smith MG,Gammon MD,Risch HA,Vaughan TL,McColl KE,Lissowska J,Zatonski W,Schoenberg JB,Blot WJ,Mowat NA,Fraumeni JF Jr,El-Omar EM.A functional polymorphism of toll-like receptor 4gene increases risk of gastric carcinoma and it sprecursors.Gastroenterology.2007;132:905-912.
12.Nieters A,Beckmann L,Deeg E,Becker N.Gene polymorphisms in Toll-like receptors,interleukin-10,and intedeukin-10 receptor alpha and lymphoma risk.Genes Immun.2006;7:615-624.
13.Marshall BJ,Warren JR,Goodwin CS.Duodenal ulcer relapse after eradication of Campylobacter pylori.Lancet.1989;1:836-837.
14.Marshall BJ,Windsor HM.The relation of Helicobacter pylori to gastric adenocarcinoma and lymphoma:pathophysiology,epidemiology,screening,clinical presentation,treatment,and prevention.Med Clin North Am.2005;89:313-344.
15.Lax AJ.Opinion:Bacterial toxins and cancer-a case to answer? Nat Rev Microbiol.2005;3:343-349.
16.Pagano JS,Blaser M,Buendia MA,Damania B,Khalili K,Raab-Traub N,Roizman B.Infectious agents and cancer:criteria for a causal relation.Semin.Cancer Biol.2004;14:453-471.
17.Finch,C.E.,Crimmins,E.M.Inflammatory exposure and historical changes in human life-spans.Science.2004;305:1736-1739.
18.Zhang GM,Xiong H,Feng ZH.Listeria monocytogenes promotes tumor growth via tumor cell toll-like receptor 2 signaling.Cancer Res.2007;67:4346-4352.
19.Huang B,Zhao J,Li H,He KL,Chen Y,Chen SH,Mayer L,Unkeless JC,Xiong H.Toll-like receptors on tumor cells facilitate evasion of immunesurveillance.Cancer Res.2005;65:5009-5014.
20.Huang B,Zhao J,Unkeless JC,Feng ZH,Xiong H.TLR signaling by tumor and immune cells:a double-edged sword Oncogene.2008;27:218-224.
21 Droemann D,Albrecht D,Gerdes J,Ulmer AJ,Branscheid D,Vollmer E,Dalhoff K,Zabel P,Goldmann T.Human lung cancer cells express functionally active Toll-like receptor 9.Respir Res.2005;6:1-7.
22.SchmausserB,AndrulisM,EndrichS,Muller-Hermelink HK,EckM.Toll-like receptors TLR4,TLR5 and TLR9 on gastric carcinoma cells:an implication for interaction with Helicobacterpylori.Int J Med Microbiol.2005;295:179-185.
23.Szczepanski M,Stelmachowska M,Stryczynski L,Golusinski W,SamaraH,Mozer-Lisewskaletal.Assessment of expression of toll-like receptors 2,3 and 4 in laryngeal carcinoma.Eur Arch Otorhinolaryngol.2007;264:525-530.
24.Lee JW,Choi JJ,Seo ES,Kim MJ,Kim WY,Choi CH,Kim TJ,Kim BG,Song SY,Bae DS.Increased toll-like receptor 9 expression in cervical neoplasia.Mol Carcinog 46:941-947.Mol Carcinog.2007;46:941-947.
25.Hassan F,Islam S,Tumurkhuu G,Naiki Y,Koide N,Mori I,Yoshida T,Yokochi T.Intracellular expression of toll-like receptor 4 in neuroblastoma cells and their unresponsiveness to lipopolysaccharide.BMC Cancer.2006;6:281-287.
26.Ilvesaro JM,Merrell MA,Swain TM,Davidson J,Zayzafoon M,Harris KW,Selander KS.Toll like receptor-9 agonists stimulate prostate cancer invasion in vitro.Prostate.2007;67:774-781.
27.Merrell MA,Ilvesaro JM,Lehtonen N,Sorsa T,Gehrs B,Rosenthal E,Chen D,Shackley B,Harris KW,Selander KS.Toll-like receptor 9 agonists promote cellular invasion by increasing matrixmetalloproteinase activity.Mol Cancer Res.2006;4:437-447.
28.Bohnhorst J,Rasmussen T,Moen SH,Flφttum M,Knudsen L,Bφrset M,Espevik T,Sundan A.Toll-like receptors mediate proliferation and survival of multiple myeloma cells.Leukemia.2006;20:1138-1144.
29.Jego G,Bataille R,Geffroy-Luseau A,Descamps G,PellatDeceunynck C. Pathogen-associated molecular patterns are growth and survival factors for human myeloma cells through Toll-like receptors.Leukemia.2006;20:1130-1137.
30.Bauer AK,Dixon D,DeGraff LM,Cho HY,Walker CR,Malkinson AM,Kleeberger SR.Toll-like receptor.4 in butylated hydroxytoluene-induced mouse pulmonary,inflammation and tumorigenesis.J Natl Cancer Inst.2005;97:1778-1781.
31.He W,Liu Q,Wang L,Chen W,Li N,Cao X.TLR4 signaling promotes immune escape of human lung cancer cells by inducingimmunosuppressive cytokines and apoptosis resistance..Mol Immunol.2007;44:2850-2859.
32.Wang L,Liu Q,Sun Q,Zhang C,Chen T,Cao X.TLR4 signaling in cancer cells promotes chemoattraction of immature dendritic cells via autocrine CCL20.Biochem Biophys Res Commun.2008;366:852-856.
33.Kelly MG,Alvero AB,Chen R,Silasi DA,Abrahams VM,Chan S,Visintin I,Rutherford T,Mor G.TLR-4 signaling promotes tumor growth and paclitaxel chemoresistance in ovarian cancer.Cancer Res.2006;66:3859-3868.
34.Yuan ZQ,Feldman RI,Sussman GE,Coppola D,Nicosia V,Cheng JQ.AKT2inhibition of cisplatin-induced JNK/p38 and Bax activation by phosphorylation of ASK1:implication of AKT2 in chemoresistance.J Biol Chem.2003;278:23432-23440.
35.Dan HC,Jiang K,Coppola D,Hamilton A,Nicosia SV,Sebti SM,Cheng JQ.Phosphatidylinositol-3-OH kinase/AKT and survivin pathways as critical targets for geranylgeranyltransferase Ⅰ inhibitor-induced apoptosis.Oncogene.2004a; 23:706-715.
36.Jego G,Bataille R,Geffroy-Luseau A,Descamps G,PellatDeceunynck C.Pathogen-associated molecular patterns are growth and survival factors for human myeloma cells through Toll-like receptors.Leukemia.2006;20:1130-1137.
37.Merrell MA,Ilvesaro JM,Lehtonen N,Sorsa T,Gehrs B,Rosenthal E,Chen D,Shackley B,Harris KW,Selander KS.Toll-like receptor 9 agonists promote cellular invasion by increasing matrixmetalloproteinase activity.Mol Cancer Res.2006;4:437-447.
38.Harmey JH,Bucana CD,Lu W,Byrne AM,McDonnell S,Lynch C,Bouchier-Hayes D,Dong Z.Lipopolysaccharide-induced metastatic growth is associated with increased angiogenesis,vascular permeability and tumor cell invasion.Int J Cancer.2002;101:415-422.
39.Wang JH,Manning BJ,Wu QD,Blankson S,Bouchier-Hayes D,Redmond HP.Endotoxin/lipopolysaccharide activates NF-kappaB and enhances tumor cell adhesion and invasion through a beta 1 integrin-dependent mechanism.J Immunol.2003;170:795-804.
40.Zheng S L.,Augustsson-Baiter K,Chang B.Sequence variants of Toll like receptor 4 are associated with prostate cancer risk:results from the cancer prostate in Sweden study.Cancer Res.2004;64:2918-2922.
41.Tsan MF.Toll- like receptors,inflammation and cancer.Semin Cancer Biol.2006;16:32-37.
42.Johnson GB,Brunn GJ,Platt JL.Cutting edge:an endogenous pathway to systemic inflammatory response syndrome(SIRS)-like reactions through Toll-like receptor 4.J Immunol.2004;172:20-24.
43.Brunn GJ,Bungum MK,Johnson GB,Platt JL.Conditional signaling by Toll-like receptor 4.FASEB J.2005;19:872-874.
44.Bianchi ME.Significant(re)location:how to use chromatin and/or abundant proteins as messages of life and death.Trends Cell Biol.2004;14:287-293.
45.Bonaldi T,Talamo F,Scaffidi P,Ferrera D,Porto A,Bachi A,Rubartelli A,Agresti A,Bianchi ME.Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion.EMBO J.2003;22:5551-5560.
46.Zeh HJ Ⅲ,Lotze MT.Addicted to death:invasive cancer and the immune response to unscheduled cell death.J Immunother.2005;28:1-9.
47.Rauvala H,Huttunen HJ,Fages C,Kaksonen M,Kinnunen T,Imai S,Raulo E,Kilpelainen I.Heparinbinding proteins HB-GAM(pleiotrophin)and amphoterin in the regulation of cellmotility.Matrix Biol.2000;19:377-387.
48.Tsung A,Sahai R,Tanaka H,Nakao A,Fink MP,Lotze MT,Yang H,Li J,Tracey KJ,Geller DA,Billiar TR.The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion.J Exp Med.2005;201:1135-1143.
49.Rouhiainen A,Kuja-Panula J,Wilkman E,Pakkanen J,Stenfors J,Tuominen RK,Lepantalo M,Carpén O,Parkkinen J,Rauvala H..Regulation of monocyte migration by amphoterin(HMGB1).Blood.2004;104:1174-1182.
50. Messmer D, Yang H, Telusma G, Knoll F, Li J, Messmer B, Tracey KJ, Chiorazzi N. High mobility group box proteinl: an endogenous signal for dendritic cell maturation and Th1 polarization. J Immunol. 2004; 173:307-313.
51. Yu W, KJ, Ossowski L. Reduction in surface urokinase receptor forces malignant cells into a protracted state of dormancy. J Cell Biol. 1997;3:767-777.
52. Taguchi A, Blood DC, del Toro G, Canet A, Lee DC, Qu W, Tanji N, Lu Y, Lalla E, Fu C, Hofmann MA, Kislinger T, Ingram M, Lu A, Tanaka H, Hori O, Ogawa S, Stern DM, Schmidt AM. Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases. Nature. 2000;405:354-360.
53. Poser I, Golob M, Buettner R, Bosserhoff AK. Upregulation of HMG1 leads to melanoma inhibitory activity expression in malignant melanoma cells and contributes to their malignancy phenotype. Mol Cell Biol. 2003;23:2991-2998.
54. Volp K, BrezniceanuML, Bosser S, et al. Increased expression of high mobility group box 1 (HMGB1) is associated with an elevated level of the antiapoptotic C-IAP2 protein in human colon carcinomas. Gut. 2006;55:234-242.
55. Melvin VS, Harrell C, Adelman JS, Kraus WL, Churchill M, Edwards DP. The role of the C-terminal extension (CTE) of the estrogen receptor alpha and beta DNA binding domain in DNA binding and interaction with HMGB. J Biol Chem. 2004;279:14763-14771.
56. Chung YL, Jian JJ, Cheng SH, Tsai SY, Chuang VP, Soong T, Lin YM, Horng CF. Sublethal irradiation induces vascular endothelial growth factor and promotes growth of hepatoma cells: implications for radiotherapy of hepatocellular carcinoma. Clin Cancer Res. 2006; 12:2706-2715.
57. Chung YL, Jian JJ, Cheng SH, Tsai SY, Chuang VP, Soong T, Lin YM, Horng CF. Sublethal irradiation induces vascular endothelial growth factor and promotes growth of hepatoma cells: implications for radiotherapy of hepatocellular carcinoma. Clin Cancer Res. 2006; 12:2706-2715.
58. Ellerman JE, Brown CK, de Vera M, Zeh HJ, Billiar T, Rubartelli A, Lotze MT.Masquerader: high mobility group box-1 and cancer. Clin Cancer Res. 2007;13:2836-2348.
59. Ito N, DeMarco RA, Mailliard RB, Han J, Rabinowich H, Kalinski P, Stolz DB, Zeh HJ 3rd, Lotze MT.Cytolytic cells induce HMGBl release from melanoma cell lines. J Leukoc Biol. 2007;8:75-83.
60. Dong Xda E, Ito N, Lotze MT, Demarco RA, Popovic P, Shand SH, Watkins S, Winikoff S, Brown CK, Bartlett DL, Zeh HJ 3rd. High mobility group box I (HMGB1) release from tumor cells after treatment: implications for development of targeted chemoimmunotherapy. J Immunother (1997). 2007;30:596-606.
61. Revesz L. Effect of tumour cells killed by X-rays upon the growth of admixed viable cells. Nature. 1956;178:1391-1392.
62. Andre F, Delaloge S, Tursz T, Kroemer G, Zitvogel L. Toll-like receptor 4—dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nature Medicine. 2007; 13:1050-1059.