FBW7基因通过GSDME介导的焦亡增强紫杉醇对胰腺癌的抗肿瘤作用研究

The research on FBW7 gene enhances antitumor effect of paclitaxel on pancreatic cancer through GSDME-mediated pyroptosis

背景与目的:胰腺癌是恶性程度极高的肿瘤,多数患者在就诊时已处于晚期,全身化疗是重要的治疗手段,但是化疗耐药给临床治疗带来很多困扰。紫杉醇作为常用的化疗药物可以诱导肿瘤细胞发生凋亡,含F-框WD重复域蛋白7(F-box and WD repeat domain containing 7,FBW7)基因是一种抑癌基因,该基因的功能丧失会导致肿瘤发生、发展。研究表明,FBW7基因具有促进肿瘤细胞凋亡和抑制肿瘤增殖的作用。此外,该基因还被证实可以促进凋亡和通过铁死亡方式增加化疗药物的效果。焦亡是一种由焦孔素(gasdermin,GSDM)蛋白介导的细胞程序性死亡模式,这种细胞死亡往往还伴有炎症反应。本研究旨在探讨FBW7基因是否可以通过焦亡促进紫杉醇发挥抗肿瘤作用。方法:采用慢病毒转染构建FBW7过表达的PANC-1细胞株,采用免疫组织化学法检测临床标本中FBW7和GSDME基因表达的相关性,采用实时荧光定量聚合酶链式反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)和蛋白质印迹法(Western blot)检测mRNA表达和蛋白质水平。光镜下观察细胞经紫杉醇处理后的形态变化,采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)检测紫杉醇对细胞活力的影响,采用流式细胞术、乳酸脱氢酶(lactate dehydrogenase,LDH)释放实验检测紫杉醇对肿瘤细胞的影响。Western blot检测经紫杉醇处理后肿瘤细胞中caspase-3和GSDME的活化情况。结果:RTFQPCR和Western blot检测结果表明,FBW7基因过表达可增加GSDME的表达。免疫组织化学检测结果表明,在体内FBW7和GSDME基因表达呈正相关。流式细胞术和LDH释放实验检测结果显示,FBW7过表达的胰腺癌细胞系PANC-1系经过紫杉醇药物刺激后,细胞死亡的水平较空载体(empty vector,EV)组显著增加。光镜下可观察到具有焦亡表现的FBW7过表达的PANC-1细胞株显著多于EV组细胞。CCK-8实验结果显示,FBW7过表达的细胞株在紫杉醇处理后细胞活性显著低于EV组细胞。Western blot检测结果显示,紫杉醇处理胰腺癌细胞系PANC-1后,FBW7过表达细胞株的活化caspase-3(activecaspase-3)和GSDME-NT蛋白水平增加,证明其有更加明显的活化。FBW7基因可以通过caspase-3/GSDME信号转导通路诱导的细胞焦亡增加PANC-1细胞对紫杉醇的敏感性。结论:FBW7可以通过上调GSDME的表达,增加胰腺癌细胞对紫杉醇诱导的细胞焦亡从而增加其对紫杉醇的敏感性,这种作用可能是通过caspase-3/GSDME信号转导通路实现的。

Background and purpose:Pancreatic cancer is a highly malignant disease.Most patients are in advanced stage upon diagnosis.Systemic chemotherapy is an important treatment method,but the chemotherapy drug resistance to tumors brings many problems to clinical treatment.As a commonly used chemotherapy drug,paclitaxel can induce apoptosis in tumor cells.The FBW7 gene is a tumor suppressor gene,and the loss of its function can lead to tumor occurrence and progression.Research has shown that it has the effect of promoting tumor cell apoptosis and inhibiting tumor proliferation.In addition,this gene has been proven to promote apoptosis and ferroptosis,which increase the effect of chemotherapy drugs.Pyroptosis is a programmed cell death mode mediated by gasdermin(GSDM)protein,and this cell death is often accompanied by inflammatory reactions.This study aimed to investigate whether FBW7 gene can promote the anti-tumor effect of paclitaxel by increasing pyroptosis.Methods:A PANC-1 cell line overexpressing FBW7 was constructed using lentivirus transfection.The correlation between FBW7 and GSDME gene expressions was detected by immunohistochemistry in clinical samples,and the expression levels of mRNA and protein were detected by real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)and Western blot.We observed the morphological changes of cells treated with paclitaxel under light microscopy.Cell counting kit-8(CCK-8)was used to detect the effect of paclitaxel on cell viability,and flow cytometry and lactate dehydrogenase(LDH)release assay were performed to detect the effect of paclitaxel on cell death.Western blot was used to detect caspase-3 and GSDME activation after paclitaxel treatment.Results:RTFQ-PCR and Western blot experiments showed that overexpression of FBW7 gene increased the expression of GSDME.Immunohistochemical staining of pathological sections of clinical patients also showed that the expressions of FBW7 and GSDME genes was positively correlated in vivo.Flow cytometry and LDH release experiments showed that the level of cell death in pancreatic cancer cell line PANC-1 overexpressing FBW7 was significantly increased compared with its empty vector(EV)cells after being treated with paclitaxel.Under light microscopy,it was observed that the number of cells with pyroptosis was significantly higher in PANC-1 cell lines overexpressing FBW7 than in EV cells.The CCK-8 experiment results showed that the cell viability was significantly lower in FBW7 overexpressed cell lines than in EV cells after paclitaxel treatment.Western blot experiment results showed that after pancreatic cancer cell line PANC-1 was treated with paclitaxel,the protein expressions of active-caspase-3 and GSDME-NT in FBW7 overexpression cell lines increased,which proved that they had more obvious activation,indicating that the FBW7 gene can increase the sensitivity of PANC-1 cells to paclitaxel through caspase-3/GSDME signaling pathway induced cell pyroptosis.Conclusion:FBW7 can increase the sensitivity of pancreatic cancer cells to paclitaxel by increasing the expression of GSDME,which is realized through caspase-3/GSDME pathway.