A new strategy for the therapy of pancreatic cancer by proton pump inhibitor agents

使用MTT测定和体内肿瘤模型检查巴弗洛霉素A1对体外和体内肿瘤生长的影响。本研究中使用了五种胰腺癌细胞系、七种结肠癌细胞系。MTT法测得巴弗洛霉素A1在各细胞系中的ID 50为5 ~ 40 nM。在巴弗洛霉素A1处理后24小时发现癌细胞的磷脂酰丝氨酸外化。在DNA分析中，在巴弗洛霉素A1处理后48小时检测到片段化DNA的梯状条带。形态学上，癌细胞表现出细胞质起泡和微绒毛数量减少，治疗后24小时，和凋亡的特征性形态学变化，包括核染色质凝聚和破碎，微绒毛的损失和细胞皱缩，观察治疗后48小时。Caspase 3和caspase 9蛋白在处理后48小时被激活，然而，caspase 6和caspase 7蛋白在处理期间通过Western印迹未被激活，这表明了caspase依赖性凋亡途径。这些细胞凋亡的变化没有抑制10 mM咪唑治疗。接下来，携带异种移植Capan-1细胞系肿瘤的裸鼠接受4周的巴弗洛霉素A1（1.0 mg/kg/天）。21天后，与对照组相比，该处理显著抑制肿瘤生长。25 C-灵菌红素对异种移植的Capan-1细胞系肿瘤也显示出相同的抑制作用。治疗组肿瘤细胞的组织学检查显示细胞凋亡的迹象，染色质凝聚和细胞皱缩。这些观察结果表明，巴弗洛霉素A1通过凋亡抑制人结肠癌和胰腺癌细胞的生长。

The effect of bafilomycin A1 on tumor growth in vitro and in vivo was examined using an MTT assay and an in vivo tumor model. Five pancretic cancer cell lines seven colon cancer cell lines were used in this study. The ID 5 0 of bafilomycin A1 by the MTT assay was from 5 nM to 40 nM in every cell lines. Phosphatidylserine externalization of cancer cells was found 24 hr after bafilomycin A1 treatment. In DNA analysis, a ladder of fragmented DNA was detected 48 hr after bafilomycin A1 treatment. Morphologically, cancer cells showed cytoplasmic blebbing and a decreased number of microvilli 24 hr after the treatment, and the characteristic morphological changes of apoptosis, including nuclear chromatin condensation and fragmentation, loss of microvilli and cell shrinkage, were observed 48 hr after the treatment. Caspase 3 and caspase 9 proteins were activated 48 hr after treatment, however, caspase 6 and caspase 7 proteins were not activated during the treatment by Western blotting, suggesting a mitochondria-dependent apoptotic pathway. These apoptotic changes were not inhibited by 10 mM imidazole treatment. Next, nude mice bearing a xenografted Capan-1 cell line tumor received 4 weeks of bafilomycin A1 (1.0 mg/kg/day). This treatment significantly inhibited tumor growth compared with controls after 21 days. 25C-prodigiosin also showed the same inhibitory effect on a xenografted Capan-1 cell line tumor. Histopathological examination of tumor cells in the treatment group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. These observations suggest that bafilomycin A1 inhibits the growth of human colon and pancreatic cancer cells through apoptosis.