二氯乙酸盐与二甲双胍相互增敏抗宫颈癌的作用及分子机制研究

It has been paid close attention to the antitumor efficacy of dichloroacetate (DCA) and metformin (Met) via regulating metabolism. However, it is unclear about the effects of the combined use of both DCA and Met on cervical cancer. Our preliminary exploration showed that 1) DCA and Met sensitized each other in killing cervical cancer cells, 2) Met dramatically suppressed DCA-mediated Mcl-1 upregulation, and 3) DCA significantly attenuated Met-induced lactate accumulation and repressed the phosphorylation of PDHE1α (pyruvate dehydrogenase E1 alpha). According to the above innovative findings, together with previous reports, we hypothesize that Met-mediated sensitization to DCA may be mainly through regulating "AMPK (adenosine monophosphate activated protein kinase) → mTOR (mammalian target of rapamycin) → Mcl-1 (myeloid cell leukemia 1)" pathway, and DCA-induced sensitization to Met may be mainly via regulating "PDK (pyruvate dehydrogenase kinase) → PDH (pyruvate dehydrogenase) → lactate" pathway. In the present project, so many technologies (such as gene silence and overexpression, flow cytometry, glucose uptake, lactate assay, etc.) will be used to verify the above hypothesis. Subsequently, cervical cancer cells and tumor-bearing mice will be used to evaluate the significance of regulating "AMPK→mTOR→Mcl-1"and "PDK→PDH→lactate" pathways for DCA and Met sensitizing each other in anti-cervical cancer, which hopes to provide scientific evidences for developing novel methods for the treatment of cervical cancer via regulating metabolism.

二氯乙酸盐（DCA）与二甲双胍（Met）通过调节代谢而抗肿瘤的作用倍受关注，但二者联合用于宫颈癌的研究未见报道。我们前期探索发现：①DCA与Met可相互增敏对方杀伤宫颈癌细胞的能力；②Met可抑制DCA诱导的Mcl-1上调；③DCA可抑制Met诱导的乳酸堆积，同时能降低PDHE1α的磷酸化水平。基于这些新发现并结合有关文献，提出如下假设：Met增敏DCA可能主要是通过调节"AMPK→mTOR→Mcl-1"通路而实现的，而DCA增敏Met的主要机制可能是调控了"PDK→PDH→乳酸"通路。本项目将借助基因沉默与过表达、流式细胞术、葡萄糖摄取、乳酸监测等一系列技术，首先证实上述假设的成立，进而在宫颈癌细胞模型和荷瘤鼠模型上探讨调控"AMPK→mTOR→Mcl-1和PDK→PDH→乳酸"这两条通路在DCA与Met相互增敏抗宫颈癌中的重要性，旨在为探索通过调节代谢而治疗宫颈癌的新措施提供科学依据。

二氯乙酸盐（dichloroacetate, DCA）与二甲双胍（metformin, Met）通过调节代谢而抗肿瘤的作用倍受关注，但DCA介导的保护性自噬和Met诱导的乳酸堆积等缺点分别削弱了二者各自杀伤肿瘤细胞的能力。因此，若能设法克服二者各自的不足，无疑有助于提高他们抗肿瘤的效应。在本项目中，我们发现：①DCA与Met可协同抑制宫颈癌细胞和卵巢癌细胞的生长、促进癌细胞凋亡。相关机制研究显示，Met可通过抑制DCA诱导的抗凋亡蛋白Mcl-1和保护性自噬而增敏DCA，而DCA则可通过抑制Met诱导的过量葡萄糖消耗和乳酸堆积而增敏Met。裸鼠荷瘤实验证实，DCA与Met可在体内协同抑制移植瘤生长，并在移植瘤组织中下调Mcl-1和磷酸化丙酮酸脱氢酶E1α的水平，促进移植瘤细胞凋亡。以上发现为探索以DCA与Met相互增敏为基础的抗肿瘤新策略提供了科学依据；②在宫颈癌细胞中，环氧合酶-2（cyclooxygenase-2, COX2）的抑制剂塞来昔布可提高癌细胞对DCA的敏感性。相关机制研究发现，DCA可通过降低RNA结合蛋白QKI（quaking）的水平而抑制COX2 mRNA的降解，从而使COX2蛋白的水平升高；过表达QKI可显著减少DCA诱导的COX2，用塞来昔布抑制COX2可增敏DCA杀伤宫颈癌细胞的效应。裸鼠荷瘤实验显示，塞来昔布可在体内增敏DCA抑制移植瘤生长，并在移植瘤组织中上调QKI、下调COX2，从而促进移植瘤细胞凋亡。以上结果表明，上调COX2是宫颈癌细胞抵抗DCA的一个新机制，提示联用COX2抑制剂和DCA可能有益于宫颈癌的治疗。.此外，我们还发现：盐酸吡柔比星（pirarubicin HCl, THP）可在宫颈癌细胞中通过下调miR34c-5p而升高自噬调节关键蛋白ATG4B（autophagy related 4B）的水平，从而促进癌细胞发生保护性自噬，导致癌细胞对THP的敏感性降低。裸鼠荷瘤实验显示，THP可在体内抑制移植瘤生长，并在移植瘤组织中下调miR34c-5p的水平、升高ATG4B的水平。这些结果表明，在THP处理的宫颈癌细胞中存在“miR34c-5p→ATG4B→自噬”信号通路，该通路至少部分解释了宫颈癌患者对THP不敏感的原因，提示该通路可能是增敏THP抗宫颈癌的新靶点。. 就以上有关研究结果已发表科研论著3篇、会议论文4篇。