Triptolide诱导c-FLIP选择性剪切在调控TRAIL耐药胰腺癌细胞凋亡中的机制研究

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States with a five year survival of less than 5%. The poor outcome of this disease is due to lack of effective chemotherapeutic options. TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, selectively kills a wide range of cancer cells, while leaving the normal cells unaffected. However, many cancers, including pancreatic cancer, are resistant to TRAIL therapy. TRAIL initiates the extrinsic apoptotic pathway by activation of pro-caspase-8. An important modulator of caspase-8, c-FLIP, plays a key role in resistance to death receptor-mediated apoptosis in many cancer cells. Triptolide, a diterpene triepoxide extracted from the Chinese herb Tripterygium wilfordii decreases viability of pancreatic cancer cells in vitro and reduce growth and metastases of tumors in vivo. Previous data from our group has shown that a combination of low doses of TRAIL and triptolide induces significant pancreatic cancer cell death compared with either treatment alone. We therefore evaluated the effect of triptolide on c-FLIP and found that triptolide regulates c-FLIP splicing in pancreatic cancer cells. This down-regulation of c-FLIP alone allowed TRAIL to induce caspase-8 activation. However, the underlying mechanism of c-FLIP splicing by triptolide is still unclear. Our previous NSFC grant revealed that non-receptor tyrosine kinase Fyn regulates RNA-binding protein hnRNP A2/B1 expression, which further participates in Bcl-X alternative splicing. Others have shown that hnRNP A2/B1 also regulates c-FLIP splicing and triptolide down-regulates tyrosine kinase JAK and Bcr/Abl activity. Since tyrosine kinases have similar structural domains, we speculate that triptolide also regulates Fyn activity and in turn, regulates c-FLIP splicing. On the basis of our previous work, we hypothesized that triptolide modulates c-FLIP alternative splicing by down-regulation of non-receptor tyrosine kinase Fyn and RNA binding protein hnRNP A2/B1. The down-regulation of c-FLIP splicing sensitizes TRAIL resistance pancreatic cancer cells apoptosis by activating caspase-8. Since TRAIL is already in use against several cancers, understanding the mechanism by which triptolide sensitizes pancreatic cancer cells to TRAIL may result in a novel therapeutic strategy against pancreatic cancer.

胰腺癌恶性程度高，对化疗药物耐药是预后差的重要原因。胰腺癌的Fas相关死亡域蛋白样白介素-1β转换酶抑制蛋白c-FLIP过表达抑制死亡受体通路关键环节Caspase-8激活，导致对TRAIL耐药。我们前期研究发现，Triptolide对c-FLIP的剪切调控诱导Caspase-8活化，致敏TRAIL耐药胰腺癌细胞凋亡，但机制仍不清楚。酪氨酸激酶Fyn通过调控hnRNP A2/B1，参与下游基因剪切调控。本研究基于前期工作发现，提出Triptolide通过抑制Fyn活性，下调hnRNP A2/B1，参与c-FLIP选择性剪切，激活Caspase-8，最终诱导胰腺癌细胞凋亡的假说。本研究拟从组织细胞学水平，探索Triptolide诱导c-FLIP选择性剪切的信号通路，并通过动物实验在体证实。本研究有望阐明Triptolide突破胰腺癌细胞TRAIL耐受屏障的机制，为胰腺癌的治疗提供新思路。

背景：雷公藤甲素（TPL）能增强胰腺癌细胞中肿瘤坏死因子相关凋亡诱导配体（TRAIL）的敏感性，目前对其作用机制的研究仅限于TPL能否影响TRAIL的相关下游信号通路。这还远远不足以让我们完全理解TPL在胰腺癌中致敏TRAIL的机制。在本研究中，我们旨在寻找TPL调节的TRAIL上游信号通路，以进一步探索TPL致敏TRAIL的调节机制。方法：采用基因芯片分析和贴壁细胞计数。采用Western blot实验、细胞增殖实验、裸鼠成瘤实验、流式、透射电镜等方法，分析了Pumilio-RNA结合家族成员1（Pum1）在TPL致敏TRAIL过程中的作用。结果：TPL和TPL+TRAIL均能降低PUM1的表达，但TRAIL不能降低PUM1的表达。PUM1沉默可以致敏低浓度TRAIL抑制胰腺癌细胞增殖并促进其凋亡。PUM1的过表达逆转了TPL致敏TRAIL抑制胰腺癌细胞增殖并促进其凋亡的作用。此外，我们发现TPL和PUM1沉默致敏低浓度TRAIL，从而诱导胰腺癌细胞自噬激活。此外，我们发现PUM1的过表达逆转了TPL致敏TRAIL，对胰腺癌细胞自噬激活的影响。最后，我们的研究结果表明，PUM1通过自噬调控胰腺癌细胞的增殖和凋亡。结论：体内、体外实验均证明，抑制PUM1可以致命TRAIL，提示PUM1可能是提高肿瘤细胞对TRAIL敏感性的新靶点。此外，我们的研究结果表明，TPL通过下调胰腺癌细胞中PUM1的表达来增强TRAIL敏感性，从而激活自噬。这一新的发现可能有助于探索TRAIL在肿瘤细胞敏感性上治疗的新方案。