PI3K/Akt信号通路在PPAR-γ激动剂诱导的HepG2细胞周期阻滞及凋亡中的作用研究
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摘要
目的:肝细胞癌(Hepatocellular Carcinoma,HCC)是世界上第五大常见恶性肿瘤,高居全世界癌症死因的第三位。由于高发病率和庞大的人口基数,肝癌在我国情况尤其严峻,我国是全球肝癌发病率最高的国家,肝癌为我国第二位恶性肿瘤致死病因。由于该病起病隐匿、进展迅速、早期就诊率低,60%-70%的患者在确诊时已处于肿瘤晚期,丧失了手术治疗的机会,只能进行姑息性的对症支持治疗。肝癌对放疗和化疗的敏感性低,易产生多药耐药,且毒副作用较大,迫切需要新的治疗方法及药物提高肝癌特别是晚期肝癌患者的生存期及生存质量。因此探讨肝癌的发病机制,寻找新的治疗靶点及有效的治疗药物,有十分重要的意义。
过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor-γ,PPAR-γ)是核激素受体超家族中的成员,PPAR-γ与人类恶性肿瘤的关系及其配体的治疗作用正受到广泛的研究。最近研究发现PPAR-γ激动剂噻唑啉二酮类药物(Thiazolidinediones,TZDs)具有抑制肿瘤细胞生长,诱导肿瘤细胞凋亡,抑制肿瘤血管生成,降低肿瘤侵袭力等作用,此类作用与PPAR-γ受体激活后通过多种途径调控癌基因和抑癌基因有密切关系,但具体分子机制并不清楚。
研究认为PPAR-γ激动剂的抑癌作用可能通过上调PTEN实现。PTEN基因是人们发现的第一个具有磷脂酶活性的新型抑癌基因,可负性调节PI3K/Akt信号转导通路,在抑制细胞生长、分化,诱导细胞凋亡等方面发挥重要的作用。近期研究发现人类肿瘤细胞内Akt的高水平活化可导致Skp2的积聚,并将Skp2定义为Akt下游的靶蛋白。而Skp2介导的泛素-蛋白酶体降解途径在P27~(kip1)降解中起关键作用,Skp2的积聚将导致P27~(kip1)降解加速。P27~(kip1)蛋白是细胞周期抑制性蛋白,可负性调控细胞周期进程,诱导肿瘤细胞凋亡。由此我们推测,肿瘤细胞中可能存在PTEN-PI3K/Akt-Skp2-P27~(kip1)信号转导通路,PI3K/Akt通路在其中发挥重要作用,PPAR-γ激动剂的抑癌作用有可能通过此通路实现。
本实验检测了PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)蛋白在人肝癌组织中的表达情况,分析它们与肝癌临床病理特征的关系及相互关系,结合随访资料探讨其表达的意义及预后价值。选取肝癌细胞系HepG2,观察PI3K/Akt信号转导通路阻断剂Wortmannin、PPAR-γ激动剂罗格列酮(Rosiglitazone,ROZ)及抑制剂GW9662对HepG2细胞的增殖、凋亡及细胞周期的影响,并检测PTEN/PI3K/Akt通路及P27~(kip1)、Skp2的变化,探讨PI3K/Akt信号通路在PPAR-γ激动剂诱导的肝癌细胞周期阻滞及凋亡中的作用,以期揭示PPAR-γ激动剂诱导肝癌细胞凋亡、调控细胞周期的可能分子机制。通过构建肝癌裸鼠移植瘤模型,观察罗格列酮对荷瘤裸鼠的抑瘤作用及其对PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)蛋白表达的影响,研究罗格列酮的体内抑瘤作用及可能机制,为PPAR-γ激动剂应用于肿瘤的临床治疗提供理论基础及实验依据。
方法:
1肝癌组织中PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)蛋白的表达及其意义
收集河北医科大学第四医院肝胆外科手术切除并经病理证实的、随访资料完整的原发性肝癌标本78例,全部标本组织学类型均为肝细胞肝癌。所有患者术前均未经过任何抗肿瘤治疗。标本切除离体立即固定于10%中性甲醛溶液,石蜡包埋。由两位有经验病理医师进行病理组织学诊断。免疫组织化学方法联合检测78例肝癌组织及21例正常肝组织中PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)蛋白的表达,分析其与肝癌临床病理特征的关系及相互关系,并结合随访资料探讨它们在肝癌中表达的意义及预后价值。本研究随访时间1.0~61.3个月,中位随访时间为26.1个月。
2研究PI3K的特异性抑制剂Wortmannin对人肝癌细胞株HepG2细胞周期和凋亡的影响,检测PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1) mRNA及蛋白的变化,探讨其可能的作用机制
以不含胎牛血清的RPMI-1640培养基常规培养人肝癌细胞株HepG2。采用MTT法检测不同浓度的Wortmannin(0、10、50、100、200nmol/L)在3h、10h、24h三个时间点对HepG2细胞的增殖抑制作用;流式细胞术(FCM)测定细胞周期变化及细胞凋亡率;提取不同药物浓度及作用时间的细胞总RNA,RT-PCR检测PPAR-γ、PTEN、Skp2及P27~(kip1) mRNA的表达变化。提取不同药物浓度及作用时间的细胞总蛋白,Western blot检测PPAR-γ, PTEN, Akt, pAkt, Skp2和P27~(kip1)蛋白的表达变化。3研究PPAR-γ激动剂罗格列酮和抑制剂GW9662对人肝癌细胞株HepG2细胞周期和凋亡的影响,检测PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)mRNA及蛋白的变化,探讨其可能的作用机制
常规培养人肝癌细胞株HepG2。采用MTT法检测不同浓度的罗格列酮(0、1、10、50、100μmol/L)在24h、48h、72h三个时间点对HepG2细胞的增殖抑制作用;FCM测定细胞周期变化及细胞凋亡率;提取不同药物浓度及作用时间的细胞总RNA,进行RT-PCR检测并分析PPAR-γ、PTEN、Skp2及P27~(kip1) mRNA的表达变化。提取不同药物浓度及作用时间的细胞总蛋白,进行Western blot检测并分析PPAR-γ, PTEN, Akt, pAkt, Skp2和P27~(kip1)蛋白的表达变化。联合加入GW9662及罗格列酮(取0、5μmol/LGW9662+50μmol/LROZ、1μmol/LGW9662+50μmol/LROZ、50μmol/LROZ共4组)作用48h后,重复上述实验。
4罗格列酮对人肝癌细胞裸鼠移植瘤生长的影响
取4×10~6个肝癌细胞株HepG2细胞悬液接种于BALB/c-nu/nu裸小鼠的左侧肩胛部皮下构建肝癌细胞裸鼠移植瘤。10只裸鼠随机分为实验组和对照组(每组5只)。实验组给予30mg/kg罗格列酮(生理盐水溶解)每天一次灌胃,对照组给予相应体积生理盐水每天一次灌胃。给药5周后处死,取瘤块称重。实验过程中每隔3天测量肿瘤的最长径(a)和最短径(b),计算裸鼠移植瘤体积:V=a*b2/2,绘制肿瘤生长曲线,计算重量抑瘤率和体积抑瘤率。HE染色光镜下观察移植瘤形态学变化;FCM测定细胞周期变化及细胞凋亡率;Western blot检测并分析PPAR-γ, PTEN, Akt, pAkt, Skp2和P27~(kip1)蛋白的表达变化。
结果:
1肝癌组织中PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)蛋白的表达及其意义
免疫组织化学检测显示在肝癌组织中,PPAR-γ、Akt、pAkt及Skp2蛋白的表达率分别为51.3%、66.7%、43.6%及47.4%,显著高于正常肝组织中的表达(P<0.05),PTEN及P27~(kip1)蛋白表达率分别为42.3%及34.6%,表达水平明显下调(P<0.05)。PPAR-γ蛋白在肿瘤直径>5cm组(66.7%)、门静脉癌栓组(88.9%)的阳性表达率显著升高(P<0.05);PPAR-γ蛋白的阳性表达率与TNM分期有关,分期越晚阳性率越高(P<0.01)。PPAR-γ蛋白表达与患者预后无关(χ2=1.843,P=0.175)。PTEN蛋白在肿瘤直径>5cm组(19.0%)、门静脉癌栓组(0%)、侵及周围脏器或淋巴结转移组(0%)的表达率显著降低(P<0.05);PTEN蛋白的表达率与TNM分期有关,分期越晚表达率越低(P<0.01)。PTEN蛋白高表达组生存期明显长于低表达组(χ2=13.764,P=0.000)。Akt及pAkt蛋白在肿瘤直径>5cm组、侵及周围脏器或淋巴结转移组的表达率显著升高(P<0.05);Akt及pAkt蛋白的表达率与TNM分期有关,分期越晚表达率越高(P<0.01)。Akt及pAkt蛋白低表达组生存期明显长于高表达组(P=0.000)。Skp2蛋白在肿瘤直径>5cm组(66.7%)、门静脉癌栓组(100%)的表达率显著升高(P<0.01);Skp2蛋白的表达率与TNM分期有关,分期越晚表达率越高(P<0.01)。Skp2蛋白低表达组生存期明显长于高表达组(χ2=25.470,P=0.000)。P27~(kip1)蛋白在肿瘤直径>5cm组(19.0%)、单发肿瘤组(28.8%)的阳性表达率显著降低(P<0.05);P27~(kip1)蛋白的阳性表达率与TNM分期有关,分期越晚阳性表达率越低(P<0.01)。P27~(kip1)蛋白表达与患者预后无关(χ2=2.830,P=0.093)。
统计学相关分析显示:在肝癌组织中,PPAR-γ蛋白与PTEN、Skp2、pAkt及P27~(kip1)蛋白表达无相关性(P>0.05);PTEN与Akt蛋白的表达呈显著负相关(r=-0.385,P=0.000),与pAkt蛋白的表达呈显著负相关(r=-0.334,P=0.003),与Skp2蛋白的表达呈显著负相关(r=-0.294,P=0.009),与P27~(kip1)蛋白的表达呈显著正相关(r=0.413,P=0.000);Skp2与pAkt蛋白的表达呈显著正相关(r=0. 356,P=0.001),与P27~(kip1)蛋白的表达呈显著负相关(r=-0.313,P=0.005)。
2研究Wortmannin对人肝癌细胞株HepG2的影响,探讨其可能的作用机制
MTT结果显示,Wortmannin可显著抑制HepG2细胞的增殖,且呈时间和剂量依赖关系,最大抑制率可达(74.93±3.25)%;FCM检测结果显示,Wortmannin可诱导HepG2细胞的凋亡,使HepG2细胞的G0/G1期比例显著升高(P<0.05),S期比例显著降低(P<0.05),呈时间和剂量依赖关系;随着药物浓度的增加和作用时间的延长,RT-PCR检测发现,Skp2 mRNA表达量逐渐减少(P<0.05),而PPAR-γ、PTEN及P27~(kip1)mRNA表达量无明显变化;Western blot检测发现,P27~(kip1)蛋白的表达量逐渐增加,pAkt和Skp2蛋白的表达量逐渐减少(P<0.05),PPAR-γ、PTEN和Akt蛋白的表达量无明显变化。
3研究罗格列酮和GW9662对人肝癌细胞株HepG2细胞的影响,探讨其可能的作用机制MTT结果显示,罗格列酮可显著抑制HepG2细胞的增殖,且呈时间
和剂量依赖关系;FCM检测结果显示,罗格列酮可诱导HepG2细胞的凋亡,使HepG2细胞的G0/G1期比例显著升高(P<0.05),S期比例显著降低(P<0.05),呈时间和剂量依赖关系;随着药物浓度的增加和作用时间的延长,RT-PCR检测发现,PPAR-γ及PTEN mRNA表达量逐渐增加,Skp2 mRNA表达量逐渐减少(P<0.05),而P27~(kip1)mRNA表达量无明显变化;Western blot检测发现,PPAR-γ、PTEN和P27~(kip1)蛋白的表达量逐渐增加,pAkt和Skp2蛋白的表达量逐渐减少(P<0.05),Akt蛋白的表达量无明显变化。联合GW9662作用于HepG2细胞后,罗格列酮抑制细胞增殖、诱导凋亡及细胞周期阻滞的作用明显减弱,其上调PTEN和P27~(kip1),下调pAkt和Skp2的作用同时减弱。
4罗格列酮对人肝癌细胞裸鼠移植瘤生长的影响
成功构建人肝癌裸鼠移植瘤模型,成瘤率达100%。实验结束时,实验组移植瘤的体积及重量均明显小于对照组(P<0.05),体积抑瘤率为52.13%,重量抑瘤率为65.63%;光镜下可见实验组移植瘤内大片细胞坏死,细胞排列松散,结缔组织增生明显,伴纤维化改变,局部可见淋巴细胞浸润;与对照组相比,实验组移植瘤细胞凋亡率明显升高(19.42±2.70)%,G0/G1期细胞比例显著增高,S期细胞比例明显降低(P<0.05);实验组pAkt及Skp2蛋白表达量明显低于对照组,P27~(kip1)蛋白的表达量则明显升高(P<0.05),PPAR-γ及PTEN蛋白的表达量均高于对照组,但无统计学意义(P>0.05)。
结论:
1首次联合检测PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)蛋白在肝癌中的表达,证实PPAR-γ、Akt、pAkt及Skp2蛋白在肝癌组织中的表达水平明显升高,PTEN及P27~(kip1)蛋白的表达水平明显降低,此变化与肝癌的发生、发展及侵袭、转移等恶性生物学行为密切相关。PTEN、Akt、pAkt和Skp2可作为提示患者预后的指标。
2 Wortmannin可抑制HepG2细胞的增殖,诱导凋亡并引发G0/G1期阻滞。阻断PI3K/Akt信号通路可下调Skp2、上调P27~(kip1)蛋白的表达,PI3K/Akt信号通路可能通过Skp2对P27~(kip1)蛋白的降解进行调控,可能存在PI3K/Akt-Skp2-P27~(kip1)信号通路。
3罗格列酮可抑制HepG2细胞的增殖,诱导细胞凋亡并引发G0/G1期阻滞。罗格列酮可上调PTEN的表达,抑制PI3K/Akt信号通路,下调Skp2并促进P27~(kip1)的表达。提示罗格列酮的抑瘤作用可能通过PTEN-PI3K/Akt-Skp2-P27~(kip1)信号转导通路实现。联合PPAR-γ抑制剂GW9662后,罗格列酮的作用明显减弱,证实罗格列酮是通过激活PPAR-γ发挥作用的。
4罗格列酮能显著抑制人肝癌裸鼠移植瘤的生长,且无明显的毒副作用,其体内抑瘤作用及机制与体外相似。PPAR-γ激动剂有可能为肿瘤临床治疗提供新的策略。
Purpose: Hepatocellular carcinoma (HCC) is a significant public health problem and the fifth common cancer and the third leading cause of cancer related death in world. Due to the high morbidity rate and a huge population base, incidence rate of HCC has been becoming the most in China, where HCC is the second leading cause of cancer death now. Due to the onset occult disease, rapid progress, and ineffective treatment, 60%-70% of patients are usually diagnosed with advanced cancer and not available for the surgical treatment when they go to hospital. They have to suffer from a palliative symptomatic treatment finally. Since HCC has the lower radiotherapy and chemotherapy sensitivity and results in the higher multi-drug resistant and toxic side effects, it is very important to develop new treatments strategy and medicines for the patients with HCC to achieve a good survival rates and quality of life. Therefore, understanding the mechanism of pathogenesis of HCC might contribute the achievements of new therapeutic targets and effective drugs. As we knew, the imbalance between cell proliferation and death is considered to be an early and important event in carcinogenic process, so it is desirable to develop new strategy to induce apoptosis, proliferation inhibition, and cell cycle arrest in tumor cells.
Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a member of the nuclear receptor superfamily. Recent studies have shown that PPAR-γis associated with differentiation and apoptosis in various human cancers. PPAR-γis capable of being activated by Thiazolidinediones (TZDs) and the activated PPAR-γhas been reported to inhibit cancer cell growth and lead to G1 cell cycle arrest. In lung, prostate, colon, thyroid, gastric and pancreatic cancers, PPAR-γactivation inhibited cell growth in a dose-dependent manner. However, the mechanism by which PPAR-γaffects cell proliferation or differentiation remains unclear.
Some studies showed that PPAR-γagonists may play anticancer role by up-regulating PTEN. PTEN gene is found to the first tumor suppressor genes with a new type of phospholipase activity, which serves as a negative regulator of PI3K/Akt signaling pathway in inhibiting cell proliferation and differentiation and promoting apoptosis. Recent studies found that down-regulation of PTEN showed PTEN/Akt-dependent regulation of Skp2 and P27~(kip1). Skp2 is the main determinant in the PI3K/Akt-dependent regulation of P27~(kip1) and plays a key role in the degradation of P27~(kip1). P27~(kip1) belongs to cell cycle inhibitory proteins, which negatively regulates cell cycle progression and induce apoptosis of tumor cells through cyclin-CDKI complex. Taken together, it is possible that PPAR-γactivation regulating PTEN/PI3K/Akt/Skp2/P27~(kip1) signaling pathway plays an important role in the anti-tumor effects, such as inducing cell cycle arrest and apoptosis.
In this study, we investigated the PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1) protein expression in human liver tumor tissue, analyzed the relationship between their expression and clinical pathological characteristics, and explored the prognostic significance of those in HCC. In order to test the hypothesis that the molecular mechanism of anticancer role of PPAR-γagonist is due to induce HepG2 cell cycle arrest and apoptosis through regulating PTEN/PI3K/Akt signaling pathway, in vitro and in vivo studies were processed. In vitro studies have been carried out to observe the effects of PPAR-γagonist Rosiglitazone(ROZ) and antagonist GW9662, as well as PI3K/Akt signaling pathway inhibitor Wortmannin on proliferation, apoptosis and cell cycle of HepG2 cell line and detect the activation of PTEN/PI3K/Akt signaling pathway and P27~(kip1) and Skp2 expression. In vivo growth of implanted HepG2 cells in nude mice was monitored after oral treatment with ROZ to further confirm the antitumor effect the PPAR-γagonist. This study might provide insight the possible mechanism of PPAR-γagonists in the clinical treatment of HCC.
Method:
1 To explore the significance of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1) expression on the carcinogenesis and prognosis of HCC
Samples were obtained from 78 HCC patients who had undergone operations at the Fourth Hospital of Hebei Medical University, between 2004 and 2007. The patients were diagnosed histologically and follow-up information intact. The length of the patients’follow-up time ranged from 1 to 61.3 months, and median survival at last follow-up was 31.2 months. None of the patients had received any chemotherapy before surgery. Specimens removed from the body were immediately fixed in 10% neutral formaldehyde solution, embedded in paraffin. Immunohistochemestry was used to assess the expression of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1) in 78 cases of HCC and 21 cases of normal liver tissue. The relationship between the expression of key proteins related to PTEN/PI3K/Akt/Skp2/P27~(kip1) signaling pathway and the clinicpathological parameters in HCC was also analyzed. The prognostic significance combined with follow-up information was statistically explored with Kaplan-Meier.
2 Effects of PI3K inhibitor Wortmannin on cell cycle arrest and apoptosis in hepatocellular carcinoma cell line.
Hepatocellular carcinoma cell line HepG2 cultured in non-FBS RPMI-1640 medium were incubated with different concentrations of Wortmannin (0, 10, 50, 100 and 200nmol/L) for 3h, 10h and 24h. MTT was explored to evaluate cell proliferation. Alterations of cell cycle and apoptosis were detected by FCM. Expression of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1) was detected by Western blot and RT-PCR.
3 Effects of ROZ on cell cycle arrest and apoptosis in HepG2 and the activation of PPAR-γ-PTEN-PI3K/Akt-Skp2-P27~(kip1) signaling pathway.
HepG2 cultured in 10%FBS RPMI-1640 medium were incubated with different concentrations of ROZ (0, 1, 10, 50 and 100μmol/L) for 24h, 48h and 72h. Alterations of cell cycle and apoptosis were detected by FCM. Expression of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1) was detected by Western blot and RT-PCR.
HepG2 were incubated with both GW9662 and ROZ (0, 5μmol/L GW9662 and 50μmol/L ROZ, 1μmol/L GW9662 and 50μmol/L ROZ, 50μmol/L ROZ) for 48h. Then the same experiments were repeated as above.
4 Effect of PPAR-γagonist ROZ on tumor growth in vivo A HepG2 cell suspension (4×106 cells suspended in 0.2ml of media) was injected subcutaneously into the left scapular of each BALB/c-nu/nu nude mouse. Animals were randomly divided into two groups (5 animals per group): one group received ROZ (30mg/kg/day) and the other received vehicle only. All study medications were given by oral gavages once a day starting from day 5 after injection to the end of week 5. Tumor size was measured once 3 days with callipers in two dimensions: length (a) and width (b). Tumor volume was calculated using the formula (V=a*b2/2). At the end of week 5, all mice were sacrificed by cervical dislocation. Tumors were dissected from the body and the tumor growth curve and inhibition rate were also calculated. All samples for Western blot were frozen immediately in liquid nitrogen and stored at -80℃for analysis. Paraffin embedded tissues were processed and the morphological alterations of tumors were examined by light microscope following HE staining. The cell cycle and apoptosis of tumor single cell suspension were measured by FCM analysis Expression of PPAR-γ, PTEN, Skp2 and P27~(kip1) in tumors was detected by Western blot.
Results:
1 Immunohistochemical analysis
1.1 PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1) expression in HCC
The positive rates of PPAR-γ、Akt、pAkt and Skp2 expression in HCC were significantly higher than those in normal liver tissue groups (P<0.05), while PTEN and P27~(kip1) expression in HCC were significantly lower than those in normal liver tissue groups (P<0.05). Positive PPAR-γexpression was correlated with tumor size, cancer-embolus in portal vein and TNM stage. The positive rates of PPAR-γexpression in the cases with cancer-embolus in portal vein, larger tumor size (>5cm) and advanced TNM stages were much higher than those of the control (P<0.05). Low PTEN expression was correlated with tumor size, cancer-embolus in portal vein, invasion or metastasis and TNM stage. The rates of low PTEN expression in the cases with cancer-embolus in portal vein, larger tumor size (>5cm), invasion or metastasis and advanced TNM stages were much higher than those of the control (P<0.05). High Akt/pAkt expression was respectively correlated with tumor size, invasion or metastasis and TNM stage. The rates of high Akt/pAkt expression in the cases with invasion or metastasis, larger tumor size (>5cm) and advanced TNM stages were much higher than those of the control respectively (P<0.05). High Skp2 expression was correlated with tumor size, cancer-embolus in portal vein and TNM stage. The rates of high Skp2 expression in the cases with cancer-embolus in portal vein, larger tumor size (>5cm) and advanced TNM stages were much higher than those of the control (P<0.05). Positive P27~(kip1) expression was correlated with tumor size, tumor amount and TNM stage. The positive rates of P27~(kip1) expression in the cases with single tumor, larger tumor size (>5cm) and advanced TNM stages were much lower than those of the control (P<0.05).
1.2 Survival analysis.
Patients with low PTEN expression, high Akt expression, high pAkt expression and high Skp2 expression had a significantly worse patients’survival time than those with converse expression respectively (P<0.05, log-rank test).
No significant difference was observed in patients’survival time between the two groups distinguished with PPAR-γ/P27~(kip1) expression respectively (P>0.05, log-rank test).
1.3 The relationship between expression of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1)
According to the analysis of Spearman rank correlation, PTEN expression in HCC was negatively correlated with the expression of Akt, pAkt and Skp2 respectively, while positively correlated with the expression of P27~(kip1).
Skp2 expression in HCC was positively correlated with the expression of pAkt, while negatively correlated with the expression of P27~(kip1). No significant correlation was observed in HCC between PPAR-γand PTEN、Skp2、pAkt and P27~(kip1) expression.
2 The effects of Wortmannin on HepG2 and possible mechanism.
MTT results showed that Wortmannin significantly inhibited the proliferation of HepG2 cells, with a time- and dose-dependent manner, the maximum inhibitory rate was (74.93±3.25)%; FCM results showed that Wortmannin induced the apoptosis of HepG2 cells; The ratio of cells in G0/G1 phase was significantly increased (P<0.05), while that in S phase was significantly decreased (P<0.05), a significant dose- and time-depended response correlation could be also found. It was showed that wortmannin could induce an arrest of cell cycle in G0/G1 phase. Moreover, with the increasing concentration of wortmannin and prolonging of treatment time, Skp2 expression at both protein and mRNA level was shown decreasing (P<0.05), while there were no difference detected in the PPAR-γ, PTEN and Akt expression by Western blot and RT-PCR. However, western blot analysis indicated that treatment with wortmannin resulted in an increased expression of P27~(kip1) at protein level, but a decreased expression of pAkt and Skp2.
3 The effects of ROZ and GW9662 on HepG2 and possible mechanism
MTT results showed that ROZ can significantly inhibit the proliferation of HepG2 cells, with a time- and dose-dependent; FCM results showed that ROZ induced the apoptosis of HepG2 cells; The ratio of cells in G0/G1 phase was significantly increased (P<0.05), while that in S phase was significantly decreased (P<0.05), a significant dose- and time-depended response correlation could be found. It was showed that ROZ could induce an arrest of cell cycle in G0/G1 phase. We also found PPAR-γand PTEN mRNA expression were increased gradually and Skp2 mRNA expression was decreased in HepG2 cells upon different concentrations and different time of ROZ treatment (P<0.05). Though P27~(kip1) mRNA expression was no significant difference upon ROZ treatment, but the expression of P27~(kip1) protein was significantly increased; Moreover, Western blot analysis indicated that ROZ increased PPAR-γand PTEN expression and decreased pAkt and Skp2 protein expression (P<0.05). The ROZ-induced anti-cancer effect was significantly reduced when the cells were treated with both ROZ and PPAR-γantagonist GW9662.
4 Effects of ROZ on tumor growth in vivo
Subcutaneous tumor growth was first palpable 5 days after injection of HepG2. At day 36, there was a significant difference in tumor volume between ROZ treated mice and control mice (0.82±0.12cm3 v 1.39±0.22cm3; p<0.05). Hence the difference between the two groups was more pronounced at week 6 (1.24±0.26cm3 v 2.59±0.79cm3; p<0.05). The volume inhibition rate was 52.13% and the weight inhibition rate was 65.63% when study finished. None of the study animals died during the study. The histopathologic alterations in ROZ treated group mice showed lots of necrotic tumor cells, cells arranging loosely, fibrous hyperplasia, and lymphocyte infiltration in local tumor tissues; Compared with the control, apoptosis rate in ROZ treated group was significantly higher (19.42±2.70)%, and the ratio of cells in G0/G1 phase was significantly increased, while that in S phase cells was decreased significantly (P<0.05); pAkt and Skp2 protein expression in the ROZ treated group was significantly lower than that in control group, but P27~(kip1) protein expression was significantly increased (P<0.05). PPAR-γand PTEN protein expression were higher in the ROZ treated group, but this change did not reach statistical significance.
Conclusion:
1 PPAR-γ, Akt, pAkt and Skp2 expression were increased in hepatocellular carcinoma while PTEN and P27~(kip1) expression were decreased. Higher expression of PPAR-γ, Akt, pAkt, and Skp2, and lower expression of PTEN and P27~(kip1) play an important role in the development, invasion and metastasis of HCC. PTEN, Akt, pAkt, and Skp2 might serve as the key prognostic factors in HCC.
2 Wortmannin could inhibit HepG2 cells proliferation; induce apoptosis and G0/G1 phase arrest. Blocking PI3K/Akt signaling in HepG2 cells resulted in decreased Skp2 protein expression and increased P27~(kip1), which suggesting that Skp2 might be involved in the PI3K/Akt-dependent regulation of P27~(kip1).
3 ROZ could inhibit HepG2 cells proliferation; induce apoptosis and G0/G1 phase arrest. ROZ could up-regulate the expression of PTEN, subsequent induce the inhibition of the PI3K/Akt signaling, decrease Skp2 and increase P27~(kip1) expression. The ROZ-induced anti-cancer effect was significantly inhibited by PPAR-γantagonist GW9662. It is suggested that the anti-cancer effects of ROZ in HepG2 cells might be mediated by PPAR-γ-PTEN-PI3K/Akt-Skp2- P27~(kip1) signaling pathway.
4 ROZ could significantly inhibit the growth of human hepatocellular carcinoma xenografts in nude mice without obvious side effects. As showing the same effects and mechanisms as in vitro, in vivo studies supported the possibility that PPAR-γagonists might become a new clinical treatment for the cancer strategy.
过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor-γ,PPAR-γ)是核激素受体超家族中的成员,PPAR-γ与人类恶性肿瘤的关系及其配体的治疗作用正受到广泛的研究。最近研究发现PPAR-γ激动剂噻唑啉二酮类药物(Thiazolidinediones,TZDs)具有抑制肿瘤细胞生长,诱导肿瘤细胞凋亡,抑制肿瘤血管生成,降低肿瘤侵袭力等作用,此类作用与PPAR-γ受体激活后通过多种途径调控癌基因和抑癌基因有密切关系,但具体分子机制并不清楚。
研究认为PPAR-γ激动剂的抑癌作用可能通过上调PTEN实现。PTEN基因是人们发现的第一个具有磷脂酶活性的新型抑癌基因,可负性调节PI3K/Akt信号转导通路,在抑制细胞生长、分化,诱导细胞凋亡等方面发挥重要的作用。近期研究发现人类肿瘤细胞内Akt的高水平活化可导致Skp2的积聚,并将Skp2定义为Akt下游的靶蛋白。而Skp2介导的泛素-蛋白酶体降解途径在P27~(kip1)降解中起关键作用,Skp2的积聚将导致P27~(kip1)降解加速。P27~(kip1)蛋白是细胞周期抑制性蛋白,可负性调控细胞周期进程,诱导肿瘤细胞凋亡。由此我们推测,肿瘤细胞中可能存在PTEN-PI3K/Akt-Skp2-P27~(kip1)信号转导通路,PI3K/Akt通路在其中发挥重要作用,PPAR-γ激动剂的抑癌作用有可能通过此通路实现。
本实验检测了PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)蛋白在人肝癌组织中的表达情况,分析它们与肝癌临床病理特征的关系及相互关系,结合随访资料探讨其表达的意义及预后价值。选取肝癌细胞系HepG2,观察PI3K/Akt信号转导通路阻断剂Wortmannin、PPAR-γ激动剂罗格列酮(Rosiglitazone,ROZ)及抑制剂GW9662对HepG2细胞的增殖、凋亡及细胞周期的影响,并检测PTEN/PI3K/Akt通路及P27~(kip1)、Skp2的变化,探讨PI3K/Akt信号通路在PPAR-γ激动剂诱导的肝癌细胞周期阻滞及凋亡中的作用,以期揭示PPAR-γ激动剂诱导肝癌细胞凋亡、调控细胞周期的可能分子机制。通过构建肝癌裸鼠移植瘤模型,观察罗格列酮对荷瘤裸鼠的抑瘤作用及其对PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)蛋白表达的影响,研究罗格列酮的体内抑瘤作用及可能机制,为PPAR-γ激动剂应用于肿瘤的临床治疗提供理论基础及实验依据。
方法:
1肝癌组织中PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)蛋白的表达及其意义
收集河北医科大学第四医院肝胆外科手术切除并经病理证实的、随访资料完整的原发性肝癌标本78例,全部标本组织学类型均为肝细胞肝癌。所有患者术前均未经过任何抗肿瘤治疗。标本切除离体立即固定于10%中性甲醛溶液,石蜡包埋。由两位有经验病理医师进行病理组织学诊断。免疫组织化学方法联合检测78例肝癌组织及21例正常肝组织中PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)蛋白的表达,分析其与肝癌临床病理特征的关系及相互关系,并结合随访资料探讨它们在肝癌中表达的意义及预后价值。本研究随访时间1.0~61.3个月,中位随访时间为26.1个月。
2研究PI3K的特异性抑制剂Wortmannin对人肝癌细胞株HepG2细胞周期和凋亡的影响,检测PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1) mRNA及蛋白的变化,探讨其可能的作用机制
以不含胎牛血清的RPMI-1640培养基常规培养人肝癌细胞株HepG2。采用MTT法检测不同浓度的Wortmannin(0、10、50、100、200nmol/L)在3h、10h、24h三个时间点对HepG2细胞的增殖抑制作用;流式细胞术(FCM)测定细胞周期变化及细胞凋亡率;提取不同药物浓度及作用时间的细胞总RNA,RT-PCR检测PPAR-γ、PTEN、Skp2及P27~(kip1) mRNA的表达变化。提取不同药物浓度及作用时间的细胞总蛋白,Western blot检测PPAR-γ, PTEN, Akt, pAkt, Skp2和P27~(kip1)蛋白的表达变化。3研究PPAR-γ激动剂罗格列酮和抑制剂GW9662对人肝癌细胞株HepG2细胞周期和凋亡的影响,检测PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)mRNA及蛋白的变化,探讨其可能的作用机制
常规培养人肝癌细胞株HepG2。采用MTT法检测不同浓度的罗格列酮(0、1、10、50、100μmol/L)在24h、48h、72h三个时间点对HepG2细胞的增殖抑制作用;FCM测定细胞周期变化及细胞凋亡率;提取不同药物浓度及作用时间的细胞总RNA,进行RT-PCR检测并分析PPAR-γ、PTEN、Skp2及P27~(kip1) mRNA的表达变化。提取不同药物浓度及作用时间的细胞总蛋白,进行Western blot检测并分析PPAR-γ, PTEN, Akt, pAkt, Skp2和P27~(kip1)蛋白的表达变化。联合加入GW9662及罗格列酮(取0、5μmol/LGW9662+50μmol/LROZ、1μmol/LGW9662+50μmol/LROZ、50μmol/LROZ共4组)作用48h后,重复上述实验。
4罗格列酮对人肝癌细胞裸鼠移植瘤生长的影响
取4×10~6个肝癌细胞株HepG2细胞悬液接种于BALB/c-nu/nu裸小鼠的左侧肩胛部皮下构建肝癌细胞裸鼠移植瘤。10只裸鼠随机分为实验组和对照组(每组5只)。实验组给予30mg/kg罗格列酮(生理盐水溶解)每天一次灌胃,对照组给予相应体积生理盐水每天一次灌胃。给药5周后处死,取瘤块称重。实验过程中每隔3天测量肿瘤的最长径(a)和最短径(b),计算裸鼠移植瘤体积:V=a*b2/2,绘制肿瘤生长曲线,计算重量抑瘤率和体积抑瘤率。HE染色光镜下观察移植瘤形态学变化;FCM测定细胞周期变化及细胞凋亡率;Western blot检测并分析PPAR-γ, PTEN, Akt, pAkt, Skp2和P27~(kip1)蛋白的表达变化。
结果:
1肝癌组织中PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)蛋白的表达及其意义
免疫组织化学检测显示在肝癌组织中,PPAR-γ、Akt、pAkt及Skp2蛋白的表达率分别为51.3%、66.7%、43.6%及47.4%,显著高于正常肝组织中的表达(P<0.05),PTEN及P27~(kip1)蛋白表达率分别为42.3%及34.6%,表达水平明显下调(P<0.05)。PPAR-γ蛋白在肿瘤直径>5cm组(66.7%)、门静脉癌栓组(88.9%)的阳性表达率显著升高(P<0.05);PPAR-γ蛋白的阳性表达率与TNM分期有关,分期越晚阳性率越高(P<0.01)。PPAR-γ蛋白表达与患者预后无关(χ2=1.843,P=0.175)。PTEN蛋白在肿瘤直径>5cm组(19.0%)、门静脉癌栓组(0%)、侵及周围脏器或淋巴结转移组(0%)的表达率显著降低(P<0.05);PTEN蛋白的表达率与TNM分期有关,分期越晚表达率越低(P<0.01)。PTEN蛋白高表达组生存期明显长于低表达组(χ2=13.764,P=0.000)。Akt及pAkt蛋白在肿瘤直径>5cm组、侵及周围脏器或淋巴结转移组的表达率显著升高(P<0.05);Akt及pAkt蛋白的表达率与TNM分期有关,分期越晚表达率越高(P<0.01)。Akt及pAkt蛋白低表达组生存期明显长于高表达组(P=0.000)。Skp2蛋白在肿瘤直径>5cm组(66.7%)、门静脉癌栓组(100%)的表达率显著升高(P<0.01);Skp2蛋白的表达率与TNM分期有关,分期越晚表达率越高(P<0.01)。Skp2蛋白低表达组生存期明显长于高表达组(χ2=25.470,P=0.000)。P27~(kip1)蛋白在肿瘤直径>5cm组(19.0%)、单发肿瘤组(28.8%)的阳性表达率显著降低(P<0.05);P27~(kip1)蛋白的阳性表达率与TNM分期有关,分期越晚阳性表达率越低(P<0.01)。P27~(kip1)蛋白表达与患者预后无关(χ2=2.830,P=0.093)。
统计学相关分析显示:在肝癌组织中,PPAR-γ蛋白与PTEN、Skp2、pAkt及P27~(kip1)蛋白表达无相关性(P>0.05);PTEN与Akt蛋白的表达呈显著负相关(r=-0.385,P=0.000),与pAkt蛋白的表达呈显著负相关(r=-0.334,P=0.003),与Skp2蛋白的表达呈显著负相关(r=-0.294,P=0.009),与P27~(kip1)蛋白的表达呈显著正相关(r=0.413,P=0.000);Skp2与pAkt蛋白的表达呈显著正相关(r=0. 356,P=0.001),与P27~(kip1)蛋白的表达呈显著负相关(r=-0.313,P=0.005)。
2研究Wortmannin对人肝癌细胞株HepG2的影响,探讨其可能的作用机制
MTT结果显示,Wortmannin可显著抑制HepG2细胞的增殖,且呈时间和剂量依赖关系,最大抑制率可达(74.93±3.25)%;FCM检测结果显示,Wortmannin可诱导HepG2细胞的凋亡,使HepG2细胞的G0/G1期比例显著升高(P<0.05),S期比例显著降低(P<0.05),呈时间和剂量依赖关系;随着药物浓度的增加和作用时间的延长,RT-PCR检测发现,Skp2 mRNA表达量逐渐减少(P<0.05),而PPAR-γ、PTEN及P27~(kip1)mRNA表达量无明显变化;Western blot检测发现,P27~(kip1)蛋白的表达量逐渐增加,pAkt和Skp2蛋白的表达量逐渐减少(P<0.05),PPAR-γ、PTEN和Akt蛋白的表达量无明显变化。
3研究罗格列酮和GW9662对人肝癌细胞株HepG2细胞的影响,探讨其可能的作用机制MTT结果显示,罗格列酮可显著抑制HepG2细胞的增殖,且呈时间
和剂量依赖关系;FCM检测结果显示,罗格列酮可诱导HepG2细胞的凋亡,使HepG2细胞的G0/G1期比例显著升高(P<0.05),S期比例显著降低(P<0.05),呈时间和剂量依赖关系;随着药物浓度的增加和作用时间的延长,RT-PCR检测发现,PPAR-γ及PTEN mRNA表达量逐渐增加,Skp2 mRNA表达量逐渐减少(P<0.05),而P27~(kip1)mRNA表达量无明显变化;Western blot检测发现,PPAR-γ、PTEN和P27~(kip1)蛋白的表达量逐渐增加,pAkt和Skp2蛋白的表达量逐渐减少(P<0.05),Akt蛋白的表达量无明显变化。联合GW9662作用于HepG2细胞后,罗格列酮抑制细胞增殖、诱导凋亡及细胞周期阻滞的作用明显减弱,其上调PTEN和P27~(kip1),下调pAkt和Skp2的作用同时减弱。
4罗格列酮对人肝癌细胞裸鼠移植瘤生长的影响
成功构建人肝癌裸鼠移植瘤模型,成瘤率达100%。实验结束时,实验组移植瘤的体积及重量均明显小于对照组(P<0.05),体积抑瘤率为52.13%,重量抑瘤率为65.63%;光镜下可见实验组移植瘤内大片细胞坏死,细胞排列松散,结缔组织增生明显,伴纤维化改变,局部可见淋巴细胞浸润;与对照组相比,实验组移植瘤细胞凋亡率明显升高(19.42±2.70)%,G0/G1期细胞比例显著增高,S期细胞比例明显降低(P<0.05);实验组pAkt及Skp2蛋白表达量明显低于对照组,P27~(kip1)蛋白的表达量则明显升高(P<0.05),PPAR-γ及PTEN蛋白的表达量均高于对照组,但无统计学意义(P>0.05)。
结论:
1首次联合检测PPAR-γ、PTEN、Akt、pAkt、Skp2及P27~(kip1)蛋白在肝癌中的表达,证实PPAR-γ、Akt、pAkt及Skp2蛋白在肝癌组织中的表达水平明显升高,PTEN及P27~(kip1)蛋白的表达水平明显降低,此变化与肝癌的发生、发展及侵袭、转移等恶性生物学行为密切相关。PTEN、Akt、pAkt和Skp2可作为提示患者预后的指标。
2 Wortmannin可抑制HepG2细胞的增殖,诱导凋亡并引发G0/G1期阻滞。阻断PI3K/Akt信号通路可下调Skp2、上调P27~(kip1)蛋白的表达,PI3K/Akt信号通路可能通过Skp2对P27~(kip1)蛋白的降解进行调控,可能存在PI3K/Akt-Skp2-P27~(kip1)信号通路。
3罗格列酮可抑制HepG2细胞的增殖,诱导细胞凋亡并引发G0/G1期阻滞。罗格列酮可上调PTEN的表达,抑制PI3K/Akt信号通路,下调Skp2并促进P27~(kip1)的表达。提示罗格列酮的抑瘤作用可能通过PTEN-PI3K/Akt-Skp2-P27~(kip1)信号转导通路实现。联合PPAR-γ抑制剂GW9662后,罗格列酮的作用明显减弱,证实罗格列酮是通过激活PPAR-γ发挥作用的。
4罗格列酮能显著抑制人肝癌裸鼠移植瘤的生长,且无明显的毒副作用,其体内抑瘤作用及机制与体外相似。PPAR-γ激动剂有可能为肿瘤临床治疗提供新的策略。
Purpose: Hepatocellular carcinoma (HCC) is a significant public health problem and the fifth common cancer and the third leading cause of cancer related death in world. Due to the high morbidity rate and a huge population base, incidence rate of HCC has been becoming the most in China, where HCC is the second leading cause of cancer death now. Due to the onset occult disease, rapid progress, and ineffective treatment, 60%-70% of patients are usually diagnosed with advanced cancer and not available for the surgical treatment when they go to hospital. They have to suffer from a palliative symptomatic treatment finally. Since HCC has the lower radiotherapy and chemotherapy sensitivity and results in the higher multi-drug resistant and toxic side effects, it is very important to develop new treatments strategy and medicines for the patients with HCC to achieve a good survival rates and quality of life. Therefore, understanding the mechanism of pathogenesis of HCC might contribute the achievements of new therapeutic targets and effective drugs. As we knew, the imbalance between cell proliferation and death is considered to be an early and important event in carcinogenic process, so it is desirable to develop new strategy to induce apoptosis, proliferation inhibition, and cell cycle arrest in tumor cells.
Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a member of the nuclear receptor superfamily. Recent studies have shown that PPAR-γis associated with differentiation and apoptosis in various human cancers. PPAR-γis capable of being activated by Thiazolidinediones (TZDs) and the activated PPAR-γhas been reported to inhibit cancer cell growth and lead to G1 cell cycle arrest. In lung, prostate, colon, thyroid, gastric and pancreatic cancers, PPAR-γactivation inhibited cell growth in a dose-dependent manner. However, the mechanism by which PPAR-γaffects cell proliferation or differentiation remains unclear.
Some studies showed that PPAR-γagonists may play anticancer role by up-regulating PTEN. PTEN gene is found to the first tumor suppressor genes with a new type of phospholipase activity, which serves as a negative regulator of PI3K/Akt signaling pathway in inhibiting cell proliferation and differentiation and promoting apoptosis. Recent studies found that down-regulation of PTEN showed PTEN/Akt-dependent regulation of Skp2 and P27~(kip1). Skp2 is the main determinant in the PI3K/Akt-dependent regulation of P27~(kip1) and plays a key role in the degradation of P27~(kip1). P27~(kip1) belongs to cell cycle inhibitory proteins, which negatively regulates cell cycle progression and induce apoptosis of tumor cells through cyclin-CDKI complex. Taken together, it is possible that PPAR-γactivation regulating PTEN/PI3K/Akt/Skp2/P27~(kip1) signaling pathway plays an important role in the anti-tumor effects, such as inducing cell cycle arrest and apoptosis.
In this study, we investigated the PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1) protein expression in human liver tumor tissue, analyzed the relationship between their expression and clinical pathological characteristics, and explored the prognostic significance of those in HCC. In order to test the hypothesis that the molecular mechanism of anticancer role of PPAR-γagonist is due to induce HepG2 cell cycle arrest and apoptosis through regulating PTEN/PI3K/Akt signaling pathway, in vitro and in vivo studies were processed. In vitro studies have been carried out to observe the effects of PPAR-γagonist Rosiglitazone(ROZ) and antagonist GW9662, as well as PI3K/Akt signaling pathway inhibitor Wortmannin on proliferation, apoptosis and cell cycle of HepG2 cell line and detect the activation of PTEN/PI3K/Akt signaling pathway and P27~(kip1) and Skp2 expression. In vivo growth of implanted HepG2 cells in nude mice was monitored after oral treatment with ROZ to further confirm the antitumor effect the PPAR-γagonist. This study might provide insight the possible mechanism of PPAR-γagonists in the clinical treatment of HCC.
Method:
1 To explore the significance of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1) expression on the carcinogenesis and prognosis of HCC
Samples were obtained from 78 HCC patients who had undergone operations at the Fourth Hospital of Hebei Medical University, between 2004 and 2007. The patients were diagnosed histologically and follow-up information intact. The length of the patients’follow-up time ranged from 1 to 61.3 months, and median survival at last follow-up was 31.2 months. None of the patients had received any chemotherapy before surgery. Specimens removed from the body were immediately fixed in 10% neutral formaldehyde solution, embedded in paraffin. Immunohistochemestry was used to assess the expression of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1) in 78 cases of HCC and 21 cases of normal liver tissue. The relationship between the expression of key proteins related to PTEN/PI3K/Akt/Skp2/P27~(kip1) signaling pathway and the clinicpathological parameters in HCC was also analyzed. The prognostic significance combined with follow-up information was statistically explored with Kaplan-Meier.
2 Effects of PI3K inhibitor Wortmannin on cell cycle arrest and apoptosis in hepatocellular carcinoma cell line.
Hepatocellular carcinoma cell line HepG2 cultured in non-FBS RPMI-1640 medium were incubated with different concentrations of Wortmannin (0, 10, 50, 100 and 200nmol/L) for 3h, 10h and 24h. MTT was explored to evaluate cell proliferation. Alterations of cell cycle and apoptosis were detected by FCM. Expression of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1) was detected by Western blot and RT-PCR.
3 Effects of ROZ on cell cycle arrest and apoptosis in HepG2 and the activation of PPAR-γ-PTEN-PI3K/Akt-Skp2-P27~(kip1) signaling pathway.
HepG2 cultured in 10%FBS RPMI-1640 medium were incubated with different concentrations of ROZ (0, 1, 10, 50 and 100μmol/L) for 24h, 48h and 72h. Alterations of cell cycle and apoptosis were detected by FCM. Expression of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1) was detected by Western blot and RT-PCR.
HepG2 were incubated with both GW9662 and ROZ (0, 5μmol/L GW9662 and 50μmol/L ROZ, 1μmol/L GW9662 and 50μmol/L ROZ, 50μmol/L ROZ) for 48h. Then the same experiments were repeated as above.
4 Effect of PPAR-γagonist ROZ on tumor growth in vivo A HepG2 cell suspension (4×106 cells suspended in 0.2ml of media) was injected subcutaneously into the left scapular of each BALB/c-nu/nu nude mouse. Animals were randomly divided into two groups (5 animals per group): one group received ROZ (30mg/kg/day) and the other received vehicle only. All study medications were given by oral gavages once a day starting from day 5 after injection to the end of week 5. Tumor size was measured once 3 days with callipers in two dimensions: length (a) and width (b). Tumor volume was calculated using the formula (V=a*b2/2). At the end of week 5, all mice were sacrificed by cervical dislocation. Tumors were dissected from the body and the tumor growth curve and inhibition rate were also calculated. All samples for Western blot were frozen immediately in liquid nitrogen and stored at -80℃for analysis. Paraffin embedded tissues were processed and the morphological alterations of tumors were examined by light microscope following HE staining. The cell cycle and apoptosis of tumor single cell suspension were measured by FCM analysis Expression of PPAR-γ, PTEN, Skp2 and P27~(kip1) in tumors was detected by Western blot.
Results:
1 Immunohistochemical analysis
1.1 PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1) expression in HCC
The positive rates of PPAR-γ、Akt、pAkt and Skp2 expression in HCC were significantly higher than those in normal liver tissue groups (P<0.05), while PTEN and P27~(kip1) expression in HCC were significantly lower than those in normal liver tissue groups (P<0.05). Positive PPAR-γexpression was correlated with tumor size, cancer-embolus in portal vein and TNM stage. The positive rates of PPAR-γexpression in the cases with cancer-embolus in portal vein, larger tumor size (>5cm) and advanced TNM stages were much higher than those of the control (P<0.05). Low PTEN expression was correlated with tumor size, cancer-embolus in portal vein, invasion or metastasis and TNM stage. The rates of low PTEN expression in the cases with cancer-embolus in portal vein, larger tumor size (>5cm), invasion or metastasis and advanced TNM stages were much higher than those of the control (P<0.05). High Akt/pAkt expression was respectively correlated with tumor size, invasion or metastasis and TNM stage. The rates of high Akt/pAkt expression in the cases with invasion or metastasis, larger tumor size (>5cm) and advanced TNM stages were much higher than those of the control respectively (P<0.05). High Skp2 expression was correlated with tumor size, cancer-embolus in portal vein and TNM stage. The rates of high Skp2 expression in the cases with cancer-embolus in portal vein, larger tumor size (>5cm) and advanced TNM stages were much higher than those of the control (P<0.05). Positive P27~(kip1) expression was correlated with tumor size, tumor amount and TNM stage. The positive rates of P27~(kip1) expression in the cases with single tumor, larger tumor size (>5cm) and advanced TNM stages were much lower than those of the control (P<0.05).
1.2 Survival analysis.
Patients with low PTEN expression, high Akt expression, high pAkt expression and high Skp2 expression had a significantly worse patients’survival time than those with converse expression respectively (P<0.05, log-rank test).
No significant difference was observed in patients’survival time between the two groups distinguished with PPAR-γ/P27~(kip1) expression respectively (P>0.05, log-rank test).
1.3 The relationship between expression of PPAR-γ, PTEN, Akt, pAkt, Skp2 and P27~(kip1)
According to the analysis of Spearman rank correlation, PTEN expression in HCC was negatively correlated with the expression of Akt, pAkt and Skp2 respectively, while positively correlated with the expression of P27~(kip1).
Skp2 expression in HCC was positively correlated with the expression of pAkt, while negatively correlated with the expression of P27~(kip1). No significant correlation was observed in HCC between PPAR-γand PTEN、Skp2、pAkt and P27~(kip1) expression.
2 The effects of Wortmannin on HepG2 and possible mechanism.
MTT results showed that Wortmannin significantly inhibited the proliferation of HepG2 cells, with a time- and dose-dependent manner, the maximum inhibitory rate was (74.93±3.25)%; FCM results showed that Wortmannin induced the apoptosis of HepG2 cells; The ratio of cells in G0/G1 phase was significantly increased (P<0.05), while that in S phase was significantly decreased (P<0.05), a significant dose- and time-depended response correlation could be also found. It was showed that wortmannin could induce an arrest of cell cycle in G0/G1 phase. Moreover, with the increasing concentration of wortmannin and prolonging of treatment time, Skp2 expression at both protein and mRNA level was shown decreasing (P<0.05), while there were no difference detected in the PPAR-γ, PTEN and Akt expression by Western blot and RT-PCR. However, western blot analysis indicated that treatment with wortmannin resulted in an increased expression of P27~(kip1) at protein level, but a decreased expression of pAkt and Skp2.
3 The effects of ROZ and GW9662 on HepG2 and possible mechanism
MTT results showed that ROZ can significantly inhibit the proliferation of HepG2 cells, with a time- and dose-dependent; FCM results showed that ROZ induced the apoptosis of HepG2 cells; The ratio of cells in G0/G1 phase was significantly increased (P<0.05), while that in S phase was significantly decreased (P<0.05), a significant dose- and time-depended response correlation could be found. It was showed that ROZ could induce an arrest of cell cycle in G0/G1 phase. We also found PPAR-γand PTEN mRNA expression were increased gradually and Skp2 mRNA expression was decreased in HepG2 cells upon different concentrations and different time of ROZ treatment (P<0.05). Though P27~(kip1) mRNA expression was no significant difference upon ROZ treatment, but the expression of P27~(kip1) protein was significantly increased; Moreover, Western blot analysis indicated that ROZ increased PPAR-γand PTEN expression and decreased pAkt and Skp2 protein expression (P<0.05). The ROZ-induced anti-cancer effect was significantly reduced when the cells were treated with both ROZ and PPAR-γantagonist GW9662.
4 Effects of ROZ on tumor growth in vivo
Subcutaneous tumor growth was first palpable 5 days after injection of HepG2. At day 36, there was a significant difference in tumor volume between ROZ treated mice and control mice (0.82±0.12cm3 v 1.39±0.22cm3; p<0.05). Hence the difference between the two groups was more pronounced at week 6 (1.24±0.26cm3 v 2.59±0.79cm3; p<0.05). The volume inhibition rate was 52.13% and the weight inhibition rate was 65.63% when study finished. None of the study animals died during the study. The histopathologic alterations in ROZ treated group mice showed lots of necrotic tumor cells, cells arranging loosely, fibrous hyperplasia, and lymphocyte infiltration in local tumor tissues; Compared with the control, apoptosis rate in ROZ treated group was significantly higher (19.42±2.70)%, and the ratio of cells in G0/G1 phase was significantly increased, while that in S phase cells was decreased significantly (P<0.05); pAkt and Skp2 protein expression in the ROZ treated group was significantly lower than that in control group, but P27~(kip1) protein expression was significantly increased (P<0.05). PPAR-γand PTEN protein expression were higher in the ROZ treated group, but this change did not reach statistical significance.
Conclusion:
1 PPAR-γ, Akt, pAkt and Skp2 expression were increased in hepatocellular carcinoma while PTEN and P27~(kip1) expression were decreased. Higher expression of PPAR-γ, Akt, pAkt, and Skp2, and lower expression of PTEN and P27~(kip1) play an important role in the development, invasion and metastasis of HCC. PTEN, Akt, pAkt, and Skp2 might serve as the key prognostic factors in HCC.
2 Wortmannin could inhibit HepG2 cells proliferation; induce apoptosis and G0/G1 phase arrest. Blocking PI3K/Akt signaling in HepG2 cells resulted in decreased Skp2 protein expression and increased P27~(kip1), which suggesting that Skp2 might be involved in the PI3K/Akt-dependent regulation of P27~(kip1).
3 ROZ could inhibit HepG2 cells proliferation; induce apoptosis and G0/G1 phase arrest. ROZ could up-regulate the expression of PTEN, subsequent induce the inhibition of the PI3K/Akt signaling, decrease Skp2 and increase P27~(kip1) expression. The ROZ-induced anti-cancer effect was significantly inhibited by PPAR-γantagonist GW9662. It is suggested that the anti-cancer effects of ROZ in HepG2 cells might be mediated by PPAR-γ-PTEN-PI3K/Akt-Skp2- P27~(kip1) signaling pathway.
4 ROZ could significantly inhibit the growth of human hepatocellular carcinoma xenografts in nude mice without obvious side effects. As showing the same effects and mechanisms as in vitro, in vivo studies supported the possibility that PPAR-γagonists might become a new clinical treatment for the cancer strategy.
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1 Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics. 2002. CA Cancer J Clin. 2005, 55:74-108
2 Roy HK, Olusola BF, Clemens DL, et al. Akt Proto-oncogene overexpression is an early event during sporadic colon carcinogenesis. Carcinogenesis, 2002, 23:201-205
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