二甲双胍通过抑制HK-II活性促进鼻咽癌细胞凋亡及机制研究

Aanaerobic and low glucose micro-environment surrounding the tumor contributes to tumor resistance to radio- and chemo-therapy. Hexokinase II(HK-II), the first rate-limiting enzyme of glycolysis, plays critical roles in production of ATP and macromolecules requied for tumor growth preferentially by way of glycolysis and in resisitance to therapy of tumors. Declarer previously found that under high-glucose culture conditions, high concentration of metformin inhibited the proliferation of nasopharyngeal carcinoma(NPC) cellC666-1, and induced G1 phase cell cycle arrest. While low concentration（1mM）of metformin inhibited the activity of HK-II and induced apoptosis of C666-1 cells cultured in low-glucose medium. This suggested that NPC cells were more sensitive to metformin under glucose-deficient contidition, and metformin may ameliorate resisitance to radio- and chemo-therapy of NPC. The project will be developed by cell model cultured in hypoxic and low-glucose medium to elucidate mechanism of low concentration metformin(0.01～1mM) and nude mice model that bears human radio-resistant NPC ectopic transplantaed tumor inhibiting NPC growth and proliferation by hindering glycolysis resulted from reduced transcriptional activity of HK-II mediated by mTOR/HIF-1 signaling and inducing apoptosis mediated by mitochondria.These data will add new content to the mechanism of tumor radio- and chemo-therapy resistance and provide a basis for metformin of enhancing radio-sensitivity of NPC.

肿瘤组织局部相对缺氧低糖的微环境是其对放化疗抵抗的原因之一。糖酵解首个限速酶己糖激酶（HK）-II在肿瘤组织优先利用糖酵解生成自身所需能量和生物大分子及肿瘤放化疗抵抗中起重要作用。申报者前期工作发现，高糖培养条件下，高浓度二甲双胍可抑制C666-1鼻咽癌细胞增殖，使其发生G1期阻滞；但在低糖培养条件下，低浓度二甲双胍即可抑制C666-1细胞HK-II活性，并诱导细胞凋亡。这些结果提示鼻咽癌细胞在低糖条件下对二甲双胍非常敏感，二甲双胍可能增强鼻咽癌放疗敏感性。本项目拟通过体外缺氧低糖培养的细胞实验及人放疗抵抗鼻咽癌裸鼠异位移植瘤模型，进一步阐明低浓度（0.01～1mM）二甲双胍通过抑制mTOR/HIF-1信号通路抑制HK-II转录，进而抑制糖酵解使鼻咽癌增殖受阻、并激活线粒体途径使鼻咽癌细胞凋亡的作用及分子机制，为肿瘤放化疗抵抗分子机制提供新内容，为二甲双胍用于增强鼻咽癌放疗敏感性提供依据。

我们研究发现在低糖条件下，低浓度二甲双胍（0.1-1mM）能够抑制过表达HK2的C666-1细胞的增殖，而对干扰HK2的细胞无明显抑制作用。在CNE2鼻咽癌细胞中也发现，分别将制备好的HK2过表达和干扰的慢病毒颗粒感染CNE2Z细胞，发现HK2过表达的CNE2Z的细胞周期发生了S期阻滞，而干扰HK-2后的鼻咽癌细胞细胞周期分布则无明显异常。这些结果与现有文献报道结果相反。鼻咽癌细胞亚株HONE-1经直线加速器照射11天后，最高总照射剂量为22Gy的细胞仍然存活，可是胰酶消化30min，无法将其消化下来，因此未能成功建立可供后续实验使用的放射线抵抗的HONE-1R细胞。因此，在2015年对研究内容进行了较大的调整，研究了某细胞骨架蛋白X对鼻咽癌细胞生物学功能的影响的影响。结果证明，敲低X蛋白后，能抑制鼻咽癌的增殖、并影响细胞周期分布和诱导凋亡。这将促使我们继续深入研究下去，以期发现新的鼻咽癌治疗的理论新靶点。