以第二型鈉-葡萄糖轉運蛋白抑制劑增強紫杉醇對卵巢癌細胞引起之細胞凋亡

Abstract

癌細胞攝取較多葡萄糖，儘管在有氧的環境下主要依賴來自糖解作用的能量而非氧化磷酸化，此現象稱為Warburg effect。因此在抗癌藥的開發中葡萄糖的轉運蛋白可能是具潛力的標的物。 我們發現傳統的化療藥微管抑制劑紫杉醇和鈉依賴型葡萄糖共同運輸蛋白抑制劑canagliflozin可以協同抑制ES-2卵巢癌細胞和HepG2肝癌細胞的生長。Canagliflozin顯著提升紫杉醇引起的凋亡作用，並產生較多的cleaved PARP, caspase-7和caspase-9，同時也減少Bcl-2和增加Bim的量。此外canagliflozin可顯著提升低濃度紫杉醇誘導產生的非整倍體細胞。在細胞週期蛋白的表現中，經24小時處理，cyclin B1, PLK1和phospho-histone H3在10 nM TX/30 M Cana合併處理後蛋白量降低，而這些蛋白除了PLK1外，其他都在48 h後提升。協同作用也可在紫杉醇和其他葡萄糖轉運蛋白抑制劑觀察到，包含第一型葡萄糖轉運蛋白抑制劑WZB117和另一種鈉依賴型葡萄糖共同運輸蛋白抑制劑dapagliflozin，而這些抑制劑皆可抑制第一型葡萄糖轉運蛋白的活性。此外，canagliflozin增加被紫杉醇引起的異常分裂細胞和具小核的細胞。DNA損壞程度也在合併處理後顯著提升，這可能是引起細胞凋亡的原因也可能是結果。Canagliflozin減少高濃度紫杉醇引起的cyclin B1表現和BUBR1磷酸化，這可能和spindle assembly checkpoint的去活化有關。另外，合併處理降低p70S6K和4EBP1的磷酸化以及c-myc的量。最後，canagliflozin也可和其他微管抑制劑vincristine和vinblastine在ES-2細胞中產生協同作用。總結，非整倍體細胞的產生、葡萄糖轉運蛋白抑制和DNA損傷可能是canagliflozin能加強紫杉醇導致細胞凋亡的原因，而我們這個發現有可能可以應用到臨床的癌症治療上。

Cancer cells consume more glucose and rely on energy produced from glycolysis instead of the oxidative phosphorylation pathway even in the aerobic circumstance, and this phenomenon is called the Warburg effect. Hence, the glucose transporters responsible for importing glucose to cells would be potential targets for anticancer drug development. Here we demonstrated that the traditional anti-tubulin chemotherapeautic drug, paclitaxel (TX), and the sodium dependent glucose cotransporter 2 (SGLT2) inhibitor, canagliflozin (Cana), can synergistically suppress the growth of an ovarian cancer cell line, ES-2, and a hepatocellular carcinoma cell line, HepG2. Canagliflozin augemented paclitaxel-induced apoptosis and increased the levels of cleaved PARP, caspase-7 and caspase-9. Because of increased cleaved caspase-9, we explored the intrinsic pathway and revealed downregulation of Bcl-2 and upregulation of Bim. Low concentration of paclitaxel elevated the population of aneuploid cells, and the combination treatment further enhanced this effect in a time dependent manner. In addition, cyclin B1, PLK1, and phospho-histone H3 (p-histone H3), were downregulated in the combination group (10 nM TX/30 M Cana) after 24 h treatment, and levels of cyclin B1 and p-histone H3 but not PLK1 were upregulated after 48 h treatment. The synergistic effect could be observed in the combination of paclitaxel and another SGLT2 inhibitor, dapagliflozin, and a GLUT1 inhibitor, WZB117, and these glucose inhibitors including canagliflozin could inhibit GLUT1 activity. Furthermore, canagliflozin increased paclitaxel-induced abnormal mitotic cells and accumulation of cells with fragmented nuclei. DNA damage was also observed which may lead to apoptosis or could be a consequence of DNA fragmentation following apoptosis. Canagliflozin could decrease the level of cyclin B1 and compromise the activity of spindle assembly checkpoint, which then reduced mitotic cells and p-BUBR1 induced by 1 M paclitaxel. The combination treatment also downregulated p-p70S6K, p-4EBP1 and c-myc which regulate cell growth and protein synthesis. Finally, the growth inhibitory effect of vinblastine and vincristine could also be enhanced by canagliflozin in ES-2 cells. Taken together, the induction of aneuploid cells, glucose inhibition and induction of DNA damage may eventually lead to apoptosis in ES-2 cells. These molecular changes may explain why canagliflozin could enhance paclitaxel-induced cytotoxicity and our findings may have clinical implications.