自噬在涎腺腺样囊性癌化疗中的作用及其相关蛋白与临床预后的相关性
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摘要
研究目的
1.分析应用自噬抑制剂对顺铂(cis-Dichlorodiamineplatinum,DDP)诱导涎腺腺样囊性癌细胞死亡和细胞凋亡的影响。
2.分析应用转染技术沉默自噬基因抑制自噬对涎腺腺样囊性癌细胞死亡和细胞凋亡的影响。
3.分析Bcl-2在DDP诱导涎腺腺样囊性癌细胞自噬和细胞凋亡中的变化。
4.分析P53-AMPK-mTOR相关蛋白是否参与了DDP诱导的自噬反应。
5.分析自噬抑制剂在DDP治疗ACC-M移植瘤中的作用。
研究方法
1.MTT实验及流式细胞仪分别检测应用自噬抑制剂或沉默自噬基因抑制自噬前后DDP诱导涎腺腺样囊性癌细胞死亡和细胞凋亡的变化。Western blot检测凋亡蛋白actived-caspase-3。
2.电镜及荧光显微镜检测DDP诱导涎腺腺样囊性癌细胞自噬的变化。Western blot检测自噬标志蛋白LC3。
3.Western blot检测Bcl-2及P53-AMPK-mTOR相关蛋白。
4.构建ACC-M移植瘤,检测应用自噬抑制剂前后肿瘤体积及重量的变化,免疫组化检测实体瘤中自噬标志蛋白LC3的变化。
研究结果
1.3-MA增加DDP诱导涎腺腺样囊性癌细胞死亡,同时促进癌细胞凋亡。
MTT结果显示DDP处理ACC-M细胞的24小时IC50为11.34±0.92ugμ/mL因此,在随后的实验中使用,采用浓度为10μg/mL DDP处理ACC-M24小时。与对照组相比,DDP疗组的ACC-M细胞活力下降了约61.02%；3-甲基腺嘌呤(3-methyladenine,3-MA)和DDP联合治疗组的细胞死亡率增加了约78.73%。3-MA组的细胞死亡率较对照组无明显差别。
用流式细胞仪AnnexinV-FITC和碘化丙啶(PI)染色检测细胞凋亡；用Western blot检测actived-caspase-3.与DDP组相比,DDP和3-MA联合处理组的ACC-M细胞凋亡率显著增加,从19.08%增加到约29.72%。通过Western blot检测下游凋亡通路的capase-3的活性形式cis-Dichlorodiamineplatinum actived-caspase-3.与DDP组相比,3-MA和DDP联合治疗组中的actived-caspase-3增加了约36.36%。
3-MA是一个众所周知的自噬抑制剂,它可抑制type I PI3K活性,而type IP13K是白噬体形成过程中必不可少的因子；DDP可诱导细胞自噬,这些在我们的实验中被再次证实。我们在电子显微镜下发现DDP诱导ACC-M细胞产生的自噬体。为了证实3-MA可抑制顺铂诱导ACC-M细胞自噬,我们用GFP-LC3质粒转染细胞并检测GFP分布。LC3是一种微管相关蛋白,是自噬体的重要组成部分。GFP-LC3质粒转染细胞己成为一个检测自噬体有效的手段。在对照组中,观察到ACC-M细胞胞浆中散在点状荧光,而DDP处理的细胞表现出点状荧光增加,且表达点状荧光的细胞数明显增加。点状荧光增加表明自噬细胞的存在。研究发现DDP和3-MA联合治疗组与顺铂组相比,表达点状荧光细胞减少了约64.29%。
LC3-Ⅰ转换成LC3-Ⅱ的程度与细胞自噬的水平正相关,Western blot检测LC3-II, LC3-II带密度代表自噬水平。顺铂治疗诱导ACC-M细胞LC3-Ⅱ表达,但3-MA使其表达减少约52.83%。除了LC3用作自噬的检测外,p62/SQSTM1蛋白作为LC3和泛素化底物之间的连接,也被用于自噬的检测。Western blot检测发现DDP和3-MA联合治疗组与顺铂治疗组相比,p62的蛋白水平增加了约36.51%。
总体而言,顺铂(DDP)治疗诱导了ACC-M细胞自噬,但3-MA抑制了细胞自噬和增强了DDP诱导ACC-M的细胞死亡,增加了caspase途径的细胞凋亡。
2.Beclin-1siRNA促进涎腺腺样囊性癌的细胞凋亡,增加癌细胞死亡。
Beclin-1是白噬过程中的一个关键的起始蛋白质。为了更直接的观察抑制自噬的对涎腺腺样囊性癌细胞的影响,我们使用Beclin-1siRNA降低内源性beclin-1的表达。转染后24小时,Western blot检测到干扰后ACC-M细胞中内源性Beclin-1与对照组相比,减少了约56.60%。
此外,采用流式细胞仪AnnexinV-PE和7AAD的染色检测细胞凋亡率和Western blot检测actived-caspase-3。与DDP组相比,DDP和Beclin-1siRNA联合组ACC-M细胞的凋亡率显著增加：从18.78%增加到30.41%。此外,与DDP组相比,DDP和Beclin-1siRNA联合组actived-caspase-3增加了35.19%。
在我们的实验中证实了DDP诱导细胞自噬和Beclin-1siRNA抑制自噬。我们观察到,与DDP组相比,DDP与Beclin-1siRNA联合组中ACC-M自噬细胞减少了约80.32%。DDP组ACC-M细胞中LC3-Ⅱ明显增加,但与DDP组相比,DDP和Beclin-1siRNA联合组LC3-Ⅱ减少了约61.11%。相应地,与DDP治疗相比,DDP和Beclin-1siRNA联合组的p62蛋白表达增加了约100%。
总体而言,顺铂(DDP)诱导了ACC-M细胞自噬,Beclinl siRNA抑制细胞自噬和增强了DDP诱导ACC-M细胞死亡,提高了caspase途径细胞凋亡。
3.Bcl-2蛋白可能是细胞凋亡和自噬之间的联系蛋白；P53-AMPK-mTOR信号通路可能参与了DDP诱导的自噬反应。
以上结果发现DDP诱导的细胞凋亡和自噬之间可能有串扰,因此我们检测了Bcl-2的表达。通过Western blot分析,我们发现DDP可提高ACC-M细胞中Bcl-2的表达,而Beclin-1siRNA可使增加的Bcl-2减少。因此,在我们的实验中Bcl-2蛋白可能是自噬和凋亡途径之间的联系蛋白。
此外,我们探索了DDP诱导自噬的基本机制。通过Western blot法,我们发现在ACC-M细胞中DDP处理组肿瘤抑制基因P53和AMP激活的蛋白激酶(AMPK)磷酸化增加,哺乳动物雷帕霉素靶蛋白(mTOR)磷酸化显著下降。
4.DDP和3-MA联合对ACC-M移植瘤的抗癌作用。
为了进一步验证自噬在体内涎腺腺样囊性癌细胞的作用,我们构建了ACC-M裸鼠移植瘤。结果显示各分组之间荷瘤鼠体重无显著差异。与顺铂组及空白对照组相比,顺铂和3-MA联合组的肿瘤生长被显著抑制。与空白对照相比,3-MA组的肿瘤生长没有明显变化。30天时,与DDP组相比较,顺铂和3-MA联合组的小鼠肿瘤体积和肿瘤重量分别显著减少了53.64%和59.75%。免疫组织化学分析结果示DDP组移植瘤组织中LC3的表达明显高于其他三组。这些结果表明DDP和3-MA联合能够抑制涎腺腺样囊性癌细胞自噬及其移植瘤的生长。
结论
1.3-MA增加DDP诱导涎腺腺样囊性癌细胞死亡,同时促进癌细胞凋亡。
2.Beclin-1siRNA促进涎腺腺样囊性癌的细胞凋亡,增加癌细胞死亡。
3.Bcl-2蛋白可能是细胞凋亡和自噬之间的联系蛋白；P53-AMPK-mTOR信号可能参与了DDP诱导的自噬反应。
4.DDP和3-MA联合对ACC-M移植瘤的抗癌作用。
自噬是细胞内源性途径,它在细胞应激和营养缺乏状态下可维持能量平衡和高分子合成,促进细胞存活。内质网应激是在细胞应激和营养缺乏状态下生理功能的中断导致未折叠蛋白的积累和诱导未折叠蛋白应答的过程。内质网应激和自噬均与人类癌症有关。我们研究了自噬相关蛋白(LC3和Beclin1)和内质网应激相关蛋白(GRP78)在头颈部涎腺腺样囊性癌组织中的表达。79例头颈部腺样囊性癌组织(?)组织标本制成组织微阵列芯片用于免疫组化。LC3的表达与淋巴结转移(P=0.016)及TNM分期(P=.021)显着相关。Beclin1的表达组织增长模式(P=0.002),组织学分级(P<.001)及survivial (P<.001)显着相关。GRP78的表达组织增长模式(P=0.019),组织学分级(P=0.019)及survivial (P=0.001)显着相关。LC3的表达与Beclin1的表达(P<0.001)呈正相关；LC3和Beclin1的表达与GRP78的表达呈正相关(P=0.035)(P=0.008)。我们研究了在腺样囊性癌中LC3, Beclin1和GRP78的表达及其与临床病理因素和总生存期的关系。这些结果表明,LC3, Beclin1和GRP78可能在腺样囊性癌肿瘤发生发展中扮演一个重要的角色,且Beclin1和GRP78能成为判断涎腺腺样囊性癌患者预后标志。
Objective
1. To analyze the effect of autophagy inhibitors during DDP-induced cell death and apoptosis of salivary adenoid cystic carcinoma.
2. To analyze the effect of silence autophagy gene during DDP-induced cell death and apoptosis of salivary adenoid cystic carcinoma.
3. To detect the change of bcl-2during DDP-induced cell autophagy and apoptosis of salivary adenoid cystic carcinoma.
4. To analyze whether the P53-AMPK-mTOR related protein is involved in DDP-induced autophagy reaction or not.
5. To analyze anticancer effect of DDP and autophagy inhibitor combination against ACC-M xenografts.
Methods
1. MTT and flow cytometry were used to detect DDP-induced death and apoptosis of adenoid cystic carcinoma after using autophagy autophagy inhibitors or silence autophagy gene. Western blot was used to analyze apoptosis protein changes.
2. Electron microscopy and fluorescence microscopy to detect DDP-induced autophagy changes in adenoid cystic carcinoma.
3. Western blot was used to detect Bcl-2and P53-AMPK-mTOR protein.
4. To build ACC-M xenografts and then detect the volume and weight changes, immunohistochemical analysis was used to detected tumor autophagy change.
Results
1. DDP-induced salivary adenoid cystic carcinoma cell death by enhancing apoptosis was augmented by3-MA treatment. The IC50of ACC-M cell was11.34±0.92μg/mL for24h DDP treatment, which was examined via MTT assay. Therefore, we used10μg/mL DDP in ACC-M for24h in the subsequent experiment. By DDP treatment, viability of ACC-M decreased to 61.02%and using both3-MA and DDP increased cell death to78.73%. There was no significantly different cell death by treatment of3-MA alone in ACC-M cell.
In addition, apoptosis was examined by FCM and immunoblotting of actived-caspase-3. We used AnnexinV-FITC and propidium iodide (PI) staining assay to observe the apoptotic cell death. Compared with the DDP group, the apoptosis rate of the ACC-M cells that were treated with DDP and3-MA significantly increased from19.08%to29.72%. Furthermore, actived-caspase-3was examined by immunoblotting, which is active form of capase-3in downstream apoptosis pathway. DDP with addition of3-MA made actived-caspase-3increased36.36%compared with DDP group.
DDP induces autophagy and3-MA is a well known autophagy inhibitor that inhibits the activity of type I PI3K (a kinase that is essential for vesicle nucleation, the first phase of autophagosome formation), which were confirmed in our experiment. Electron microscopic analysis of DDP-induced ACC-M cells indicated autophagosome. To determine whether3-MA inhibition combined with DDP induces autophagy in ACC-M cells, we transfected the cells with GFP-LC3plasmid and detected the distribution of GFP. LC3is a microtubule-associated protein that is a critical component of the autophagosome. The use of the GFP-tagged LC3plasmid has become an effective marker for the autophagosome. Diffuse cytoplasmic localization of GFP-LC3was observed in untreated ACC-M cells, whereas DDP-treated cells showed increased punctate fluorescence. Increases in punctate fluorescence indicated the presence of autophagic cells. We observed combination (DDP plus3-MA group) reduced about64.29%compared with DDP group.
The change of LC3protein was also observed by immunoblotting. Since the extent of conversion of LC3-Ⅰ to LC3-Ⅱ is correlated to the level of autophagy, LC3-Ⅰ and LC3-Ⅱ were detected by western blot, and the autophagic level was demonstrated LC3-Ⅱ band density. LC3-Ⅱ was induced by DDP treatment in ACC-M cells, but3-MA reduced them about52.83%. In addition to LC3, p62/SQSTM1as a marker is also used by immunoblotting. The p62protein serves as a link between LC3and ubiquitinated substrates. P62becomes incorporated into the completed autophagosome and is degraded in autolysosomes. Correspondingly, the level of p62protein increased about36.51%with DDP added3-MA, compared with DDP treatment.
Overall, DDP treatment induced autophagy in ACC-M cells, but addition of3-MA inhibited autophagy and enhanced DDP-induced ACC-M cell death by enhancing the caspase-mediated apoptosis pathway.
2. DDP-induced cell death by increasing apoptosis in salivary adenoid cystic carcinoma cell was enhanced by beclin-1siRNA.
Beclin-1is a critical initial protein in the process of autophagy. To observe the influence of autophagy more directly, we used beclin-1siRNA to decrease endogenous beclin-1expression. By immunoblotting, endogenous beclin-1was reduced by56.60%in ACC-M cells, compared with the control, at24h after transfection with beclin-1siRNA or the universal control siRNA.
In addition, apoptosis was examined by FCM and immunoblotting of actived-caspase-3. We used AnnexinV-PE and7AAD staining to observe the apoptotic cell death. Compared with the DDP group, the apoptosis rate of the ACC-M cells that were treated with DDP and beclin-1siRNA significantly increased from18.78%to30.41%. Furthermore, DDP with addition of beclin-1siRNA made actived-caspase-3increased35.19%compared with DDP group.
DDP induces autophagy and beclin-1siRNA inhibits autophagy, which were confirmed in our experiment. We observed the autophagic cells of combination (DDP plus beclin-1siRNA group) reduced about80.32%compared with DDP group. Similarly, LC3-Ⅱ was induced by DDP treatment in ACC-M cells, but beclin-1siRNA reduced them about61.11%. Correspondingly, the level of p62protein increased about100%with DDP added beclin-1siRNA, compared with DDP treatment.
Overall, DDP treatment induced autophagy in ACC-M cells, but addition of beclin1siRNA inhibited autophagy and enhanced DDP-induced ACC-M cell death by enhancing the caspase-mediated apoptosis pathway.
3. Bcl-2may be a link between apoptosis and autophagy and P53-AMPK-mTOR may participate in DDP-induced autophagy response.
Since it is strongly suggested that there is crosstalk between DDP-induced apoptosis and autophagy, we checked expression of the bcl-2which is reportedly implicated in both apoptosis and autophagy. By immunoblot analysis, we found that DDP increased the expression of in ACC-M cells. Use of beclin-1siRNA combination reduced the increase of bcl-2. Therefore, bcl-2may be a link between autophagy and the apoptosis pathways in our experiment.
In addition, it is necessary to examine the underlying mechanism of DDP-induced autophagy. By immunoblotting, we found that with DDP treatment, expression and phosphorylation of tumour suppressor p53and AMP-activated protein kinase (AMPK) increased significantly and the mammalian target of rapamycin (mTOR), a gatekeeper of autophagy decreased also decreased in ACC-M cells.
4. Anticancer effect of DDP and3-MA combination against ACC-M xenografts
To further verify the effects of autophagy in vivo, ACC-M xenografts in nude mice were established. There were no differences in body weight between groups. Tumor growth was significantly inhibited by DDP combined with3-MA compared with the the control, DDP treatment. There is no significant influence on tumor growth treated with3-MA. On day30, compared with the DDP group, tumor volume and tumor weight of combination group mice were significantly reduced by53.64%and59.75%. The result of immunohistochemical analysis showed that expression of LC3significantly induced in combination therapy compared with DDP group. These results indicated that DDP,3-MA combination could inhibit autophagy and the growth of xenografts.
Conclusion
1. DDP-induced salivary adenoid cystic carcinoma cell death by enhancing apoptosis was augmented by3-MA treatment.
2. DDP-induced cell death by increasing apoptosis in salivary adenoid cystic carcinoma cell was enhanced by beclin-1siRNA.
3. Bcl-2may be a link between apoptosis and autophagy and P53-AMPK-mTOR may participate in DDP-induced autophagy response.
4. Anticancer effect of DDP and3-MA combination against ACC-M xenografts.
Autophagy is the endogenous cellular pathway which facilitates cellular survival via maintaining energy homeostasis and macromolecular synthesis during cellular stress and nutrient deprivation. Endoplasmic reticulum(ER) stress is the process that disruption of these physiological functions leads to accumulation of unfolded proteins and induces the unfolded protein response (UPR). ER stress and autophagy are involved in human cancer. We investigated the expression of autophagic proteins (LC3and beclin1) and ER stress-related protein (GRP78) in head and neck adenoid cystic carcinoma tissue. Tissue samples from79cases of head and neck adenoid cystic carcinoma tissue were utilized for immunohistochemistry. LC3expression was significantly correlated with lymph node involvement (P=.016) and TNM (P=.021). Beclin1expression was significantly correlated with histological growth pattern (P=.002), histological grade (P<.001) and longer survivial (P<.001). GRP78expression was significantly correlated with histological growth pattern (P=.019), histological grade (P=.019) and longer survivial (P=.001). LC3expression was positively correlated with beclin1expression (P<.001); LC3and beclin1expression was positively correlated with GRP78expression respectively (P=.035)(P=.008). Our study showed that the expression of LC3, beclin1and GRP78in adenoid cystic carcinoma and its relation with clinicopathologic factors and overall survival. These results suggest that LC3, beclin1and GRP78may play an important role in tumorigenesis of adenoid cystic carcinoma and beclin1and GRP78may serve as new prognostic indicators for outcome of the patients with adenoid cystic carcinoma.
1.分析应用自噬抑制剂对顺铂(cis-Dichlorodiamineplatinum,DDP)诱导涎腺腺样囊性癌细胞死亡和细胞凋亡的影响。
2.分析应用转染技术沉默自噬基因抑制自噬对涎腺腺样囊性癌细胞死亡和细胞凋亡的影响。
3.分析Bcl-2在DDP诱导涎腺腺样囊性癌细胞自噬和细胞凋亡中的变化。
4.分析P53-AMPK-mTOR相关蛋白是否参与了DDP诱导的自噬反应。
5.分析自噬抑制剂在DDP治疗ACC-M移植瘤中的作用。
研究方法
1.MTT实验及流式细胞仪分别检测应用自噬抑制剂或沉默自噬基因抑制自噬前后DDP诱导涎腺腺样囊性癌细胞死亡和细胞凋亡的变化。Western blot检测凋亡蛋白actived-caspase-3。
2.电镜及荧光显微镜检测DDP诱导涎腺腺样囊性癌细胞自噬的变化。Western blot检测自噬标志蛋白LC3。
3.Western blot检测Bcl-2及P53-AMPK-mTOR相关蛋白。
4.构建ACC-M移植瘤,检测应用自噬抑制剂前后肿瘤体积及重量的变化,免疫组化检测实体瘤中自噬标志蛋白LC3的变化。
研究结果
1.3-MA增加DDP诱导涎腺腺样囊性癌细胞死亡,同时促进癌细胞凋亡。
MTT结果显示DDP处理ACC-M细胞的24小时IC50为11.34±0.92ugμ/mL因此,在随后的实验中使用,采用浓度为10μg/mL DDP处理ACC-M24小时。与对照组相比,DDP疗组的ACC-M细胞活力下降了约61.02%；3-甲基腺嘌呤(3-methyladenine,3-MA)和DDP联合治疗组的细胞死亡率增加了约78.73%。3-MA组的细胞死亡率较对照组无明显差别。
用流式细胞仪AnnexinV-FITC和碘化丙啶(PI)染色检测细胞凋亡；用Western blot检测actived-caspase-3.与DDP组相比,DDP和3-MA联合处理组的ACC-M细胞凋亡率显著增加,从19.08%增加到约29.72%。通过Western blot检测下游凋亡通路的capase-3的活性形式cis-Dichlorodiamineplatinum actived-caspase-3.与DDP组相比,3-MA和DDP联合治疗组中的actived-caspase-3增加了约36.36%。
3-MA是一个众所周知的自噬抑制剂,它可抑制type I PI3K活性,而type IP13K是白噬体形成过程中必不可少的因子；DDP可诱导细胞自噬,这些在我们的实验中被再次证实。我们在电子显微镜下发现DDP诱导ACC-M细胞产生的自噬体。为了证实3-MA可抑制顺铂诱导ACC-M细胞自噬,我们用GFP-LC3质粒转染细胞并检测GFP分布。LC3是一种微管相关蛋白,是自噬体的重要组成部分。GFP-LC3质粒转染细胞己成为一个检测自噬体有效的手段。在对照组中,观察到ACC-M细胞胞浆中散在点状荧光,而DDP处理的细胞表现出点状荧光增加,且表达点状荧光的细胞数明显增加。点状荧光增加表明自噬细胞的存在。研究发现DDP和3-MA联合治疗组与顺铂组相比,表达点状荧光细胞减少了约64.29%。
LC3-Ⅰ转换成LC3-Ⅱ的程度与细胞自噬的水平正相关,Western blot检测LC3-II, LC3-II带密度代表自噬水平。顺铂治疗诱导ACC-M细胞LC3-Ⅱ表达,但3-MA使其表达减少约52.83%。除了LC3用作自噬的检测外,p62/SQSTM1蛋白作为LC3和泛素化底物之间的连接,也被用于自噬的检测。Western blot检测发现DDP和3-MA联合治疗组与顺铂治疗组相比,p62的蛋白水平增加了约36.51%。
总体而言,顺铂(DDP)治疗诱导了ACC-M细胞自噬,但3-MA抑制了细胞自噬和增强了DDP诱导ACC-M的细胞死亡,增加了caspase途径的细胞凋亡。
2.Beclin-1siRNA促进涎腺腺样囊性癌的细胞凋亡,增加癌细胞死亡。
Beclin-1是白噬过程中的一个关键的起始蛋白质。为了更直接的观察抑制自噬的对涎腺腺样囊性癌细胞的影响,我们使用Beclin-1siRNA降低内源性beclin-1的表达。转染后24小时,Western blot检测到干扰后ACC-M细胞中内源性Beclin-1与对照组相比,减少了约56.60%。
此外,采用流式细胞仪AnnexinV-PE和7AAD的染色检测细胞凋亡率和Western blot检测actived-caspase-3。与DDP组相比,DDP和Beclin-1siRNA联合组ACC-M细胞的凋亡率显著增加：从18.78%增加到30.41%。此外,与DDP组相比,DDP和Beclin-1siRNA联合组actived-caspase-3增加了35.19%。
在我们的实验中证实了DDP诱导细胞自噬和Beclin-1siRNA抑制自噬。我们观察到,与DDP组相比,DDP与Beclin-1siRNA联合组中ACC-M自噬细胞减少了约80.32%。DDP组ACC-M细胞中LC3-Ⅱ明显增加,但与DDP组相比,DDP和Beclin-1siRNA联合组LC3-Ⅱ减少了约61.11%。相应地,与DDP治疗相比,DDP和Beclin-1siRNA联合组的p62蛋白表达增加了约100%。
总体而言,顺铂(DDP)诱导了ACC-M细胞自噬,Beclinl siRNA抑制细胞自噬和增强了DDP诱导ACC-M细胞死亡,提高了caspase途径细胞凋亡。
3.Bcl-2蛋白可能是细胞凋亡和自噬之间的联系蛋白；P53-AMPK-mTOR信号通路可能参与了DDP诱导的自噬反应。
以上结果发现DDP诱导的细胞凋亡和自噬之间可能有串扰,因此我们检测了Bcl-2的表达。通过Western blot分析,我们发现DDP可提高ACC-M细胞中Bcl-2的表达,而Beclin-1siRNA可使增加的Bcl-2减少。因此,在我们的实验中Bcl-2蛋白可能是自噬和凋亡途径之间的联系蛋白。
此外,我们探索了DDP诱导自噬的基本机制。通过Western blot法,我们发现在ACC-M细胞中DDP处理组肿瘤抑制基因P53和AMP激活的蛋白激酶(AMPK)磷酸化增加,哺乳动物雷帕霉素靶蛋白(mTOR)磷酸化显著下降。
4.DDP和3-MA联合对ACC-M移植瘤的抗癌作用。
为了进一步验证自噬在体内涎腺腺样囊性癌细胞的作用,我们构建了ACC-M裸鼠移植瘤。结果显示各分组之间荷瘤鼠体重无显著差异。与顺铂组及空白对照组相比,顺铂和3-MA联合组的肿瘤生长被显著抑制。与空白对照相比,3-MA组的肿瘤生长没有明显变化。30天时,与DDP组相比较,顺铂和3-MA联合组的小鼠肿瘤体积和肿瘤重量分别显著减少了53.64%和59.75%。免疫组织化学分析结果示DDP组移植瘤组织中LC3的表达明显高于其他三组。这些结果表明DDP和3-MA联合能够抑制涎腺腺样囊性癌细胞自噬及其移植瘤的生长。
结论
1.3-MA增加DDP诱导涎腺腺样囊性癌细胞死亡,同时促进癌细胞凋亡。
2.Beclin-1siRNA促进涎腺腺样囊性癌的细胞凋亡,增加癌细胞死亡。
3.Bcl-2蛋白可能是细胞凋亡和自噬之间的联系蛋白；P53-AMPK-mTOR信号可能参与了DDP诱导的自噬反应。
4.DDP和3-MA联合对ACC-M移植瘤的抗癌作用。
自噬是细胞内源性途径,它在细胞应激和营养缺乏状态下可维持能量平衡和高分子合成,促进细胞存活。内质网应激是在细胞应激和营养缺乏状态下生理功能的中断导致未折叠蛋白的积累和诱导未折叠蛋白应答的过程。内质网应激和自噬均与人类癌症有关。我们研究了自噬相关蛋白(LC3和Beclin1)和内质网应激相关蛋白(GRP78)在头颈部涎腺腺样囊性癌组织中的表达。79例头颈部腺样囊性癌组织(?)组织标本制成组织微阵列芯片用于免疫组化。LC3的表达与淋巴结转移(P=0.016)及TNM分期(P=.021)显着相关。Beclin1的表达组织增长模式(P=0.002),组织学分级(P<.001)及survivial (P<.001)显着相关。GRP78的表达组织增长模式(P=0.019),组织学分级(P=0.019)及survivial (P=0.001)显着相关。LC3的表达与Beclin1的表达(P<0.001)呈正相关；LC3和Beclin1的表达与GRP78的表达呈正相关(P=0.035)(P=0.008)。我们研究了在腺样囊性癌中LC3, Beclin1和GRP78的表达及其与临床病理因素和总生存期的关系。这些结果表明,LC3, Beclin1和GRP78可能在腺样囊性癌肿瘤发生发展中扮演一个重要的角色,且Beclin1和GRP78能成为判断涎腺腺样囊性癌患者预后标志。
Objective
1. To analyze the effect of autophagy inhibitors during DDP-induced cell death and apoptosis of salivary adenoid cystic carcinoma.
2. To analyze the effect of silence autophagy gene during DDP-induced cell death and apoptosis of salivary adenoid cystic carcinoma.
3. To detect the change of bcl-2during DDP-induced cell autophagy and apoptosis of salivary adenoid cystic carcinoma.
4. To analyze whether the P53-AMPK-mTOR related protein is involved in DDP-induced autophagy reaction or not.
5. To analyze anticancer effect of DDP and autophagy inhibitor combination against ACC-M xenografts.
Methods
1. MTT and flow cytometry were used to detect DDP-induced death and apoptosis of adenoid cystic carcinoma after using autophagy autophagy inhibitors or silence autophagy gene. Western blot was used to analyze apoptosis protein changes.
2. Electron microscopy and fluorescence microscopy to detect DDP-induced autophagy changes in adenoid cystic carcinoma.
3. Western blot was used to detect Bcl-2and P53-AMPK-mTOR protein.
4. To build ACC-M xenografts and then detect the volume and weight changes, immunohistochemical analysis was used to detected tumor autophagy change.
Results
1. DDP-induced salivary adenoid cystic carcinoma cell death by enhancing apoptosis was augmented by3-MA treatment. The IC50of ACC-M cell was11.34±0.92μg/mL for24h DDP treatment, which was examined via MTT assay. Therefore, we used10μg/mL DDP in ACC-M for24h in the subsequent experiment. By DDP treatment, viability of ACC-M decreased to 61.02%and using both3-MA and DDP increased cell death to78.73%. There was no significantly different cell death by treatment of3-MA alone in ACC-M cell.
In addition, apoptosis was examined by FCM and immunoblotting of actived-caspase-3. We used AnnexinV-FITC and propidium iodide (PI) staining assay to observe the apoptotic cell death. Compared with the DDP group, the apoptosis rate of the ACC-M cells that were treated with DDP and3-MA significantly increased from19.08%to29.72%. Furthermore, actived-caspase-3was examined by immunoblotting, which is active form of capase-3in downstream apoptosis pathway. DDP with addition of3-MA made actived-caspase-3increased36.36%compared with DDP group.
DDP induces autophagy and3-MA is a well known autophagy inhibitor that inhibits the activity of type I PI3K (a kinase that is essential for vesicle nucleation, the first phase of autophagosome formation), which were confirmed in our experiment. Electron microscopic analysis of DDP-induced ACC-M cells indicated autophagosome. To determine whether3-MA inhibition combined with DDP induces autophagy in ACC-M cells, we transfected the cells with GFP-LC3plasmid and detected the distribution of GFP. LC3is a microtubule-associated protein that is a critical component of the autophagosome. The use of the GFP-tagged LC3plasmid has become an effective marker for the autophagosome. Diffuse cytoplasmic localization of GFP-LC3was observed in untreated ACC-M cells, whereas DDP-treated cells showed increased punctate fluorescence. Increases in punctate fluorescence indicated the presence of autophagic cells. We observed combination (DDP plus3-MA group) reduced about64.29%compared with DDP group.
The change of LC3protein was also observed by immunoblotting. Since the extent of conversion of LC3-Ⅰ to LC3-Ⅱ is correlated to the level of autophagy, LC3-Ⅰ and LC3-Ⅱ were detected by western blot, and the autophagic level was demonstrated LC3-Ⅱ band density. LC3-Ⅱ was induced by DDP treatment in ACC-M cells, but3-MA reduced them about52.83%. In addition to LC3, p62/SQSTM1as a marker is also used by immunoblotting. The p62protein serves as a link between LC3and ubiquitinated substrates. P62becomes incorporated into the completed autophagosome and is degraded in autolysosomes. Correspondingly, the level of p62protein increased about36.51%with DDP added3-MA, compared with DDP treatment.
Overall, DDP treatment induced autophagy in ACC-M cells, but addition of3-MA inhibited autophagy and enhanced DDP-induced ACC-M cell death by enhancing the caspase-mediated apoptosis pathway.
2. DDP-induced cell death by increasing apoptosis in salivary adenoid cystic carcinoma cell was enhanced by beclin-1siRNA.
Beclin-1is a critical initial protein in the process of autophagy. To observe the influence of autophagy more directly, we used beclin-1siRNA to decrease endogenous beclin-1expression. By immunoblotting, endogenous beclin-1was reduced by56.60%in ACC-M cells, compared with the control, at24h after transfection with beclin-1siRNA or the universal control siRNA.
In addition, apoptosis was examined by FCM and immunoblotting of actived-caspase-3. We used AnnexinV-PE and7AAD staining to observe the apoptotic cell death. Compared with the DDP group, the apoptosis rate of the ACC-M cells that were treated with DDP and beclin-1siRNA significantly increased from18.78%to30.41%. Furthermore, DDP with addition of beclin-1siRNA made actived-caspase-3increased35.19%compared with DDP group.
DDP induces autophagy and beclin-1siRNA inhibits autophagy, which were confirmed in our experiment. We observed the autophagic cells of combination (DDP plus beclin-1siRNA group) reduced about80.32%compared with DDP group. Similarly, LC3-Ⅱ was induced by DDP treatment in ACC-M cells, but beclin-1siRNA reduced them about61.11%. Correspondingly, the level of p62protein increased about100%with DDP added beclin-1siRNA, compared with DDP treatment.
Overall, DDP treatment induced autophagy in ACC-M cells, but addition of beclin1siRNA inhibited autophagy and enhanced DDP-induced ACC-M cell death by enhancing the caspase-mediated apoptosis pathway.
3. Bcl-2may be a link between apoptosis and autophagy and P53-AMPK-mTOR may participate in DDP-induced autophagy response.
Since it is strongly suggested that there is crosstalk between DDP-induced apoptosis and autophagy, we checked expression of the bcl-2which is reportedly implicated in both apoptosis and autophagy. By immunoblot analysis, we found that DDP increased the expression of in ACC-M cells. Use of beclin-1siRNA combination reduced the increase of bcl-2. Therefore, bcl-2may be a link between autophagy and the apoptosis pathways in our experiment.
In addition, it is necessary to examine the underlying mechanism of DDP-induced autophagy. By immunoblotting, we found that with DDP treatment, expression and phosphorylation of tumour suppressor p53and AMP-activated protein kinase (AMPK) increased significantly and the mammalian target of rapamycin (mTOR), a gatekeeper of autophagy decreased also decreased in ACC-M cells.
4. Anticancer effect of DDP and3-MA combination against ACC-M xenografts
To further verify the effects of autophagy in vivo, ACC-M xenografts in nude mice were established. There were no differences in body weight between groups. Tumor growth was significantly inhibited by DDP combined with3-MA compared with the the control, DDP treatment. There is no significant influence on tumor growth treated with3-MA. On day30, compared with the DDP group, tumor volume and tumor weight of combination group mice were significantly reduced by53.64%and59.75%. The result of immunohistochemical analysis showed that expression of LC3significantly induced in combination therapy compared with DDP group. These results indicated that DDP,3-MA combination could inhibit autophagy and the growth of xenografts.
Conclusion
1. DDP-induced salivary adenoid cystic carcinoma cell death by enhancing apoptosis was augmented by3-MA treatment.
2. DDP-induced cell death by increasing apoptosis in salivary adenoid cystic carcinoma cell was enhanced by beclin-1siRNA.
3. Bcl-2may be a link between apoptosis and autophagy and P53-AMPK-mTOR may participate in DDP-induced autophagy response.
4. Anticancer effect of DDP and3-MA combination against ACC-M xenografts.
Autophagy is the endogenous cellular pathway which facilitates cellular survival via maintaining energy homeostasis and macromolecular synthesis during cellular stress and nutrient deprivation. Endoplasmic reticulum(ER) stress is the process that disruption of these physiological functions leads to accumulation of unfolded proteins and induces the unfolded protein response (UPR). ER stress and autophagy are involved in human cancer. We investigated the expression of autophagic proteins (LC3and beclin1) and ER stress-related protein (GRP78) in head and neck adenoid cystic carcinoma tissue. Tissue samples from79cases of head and neck adenoid cystic carcinoma tissue were utilized for immunohistochemistry. LC3expression was significantly correlated with lymph node involvement (P=.016) and TNM (P=.021). Beclin1expression was significantly correlated with histological growth pattern (P=.002), histological grade (P<.001) and longer survivial (P<.001). GRP78expression was significantly correlated with histological growth pattern (P=.019), histological grade (P=.019) and longer survivial (P=.001). LC3expression was positively correlated with beclin1expression (P<.001); LC3and beclin1expression was positively correlated with GRP78expression respectively (P=.035)(P=.008). Our study showed that the expression of LC3, beclin1and GRP78in adenoid cystic carcinoma and its relation with clinicopathologic factors and overall survival. These results suggest that LC3, beclin1and GRP78may play an important role in tumorigenesis of adenoid cystic carcinoma and beclin1and GRP78may serve as new prognostic indicators for outcome of the patients with adenoid cystic carcinoma.
引文
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