Dudeja Vikas, Mujumdar Nameeta, Phillips Phoebe, Chugh Rohit, Borja-Cacho Daniel, Dawra Rajinder K, Vickers Selwyn M, Saluja Ashok K
Department of Surgery, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Gastroenterology. 2009 May;136(5):1772-82. doi: 10.1053/j.gastro.2009.01.070. Epub 2009 Feb 6.
BACKGROUND & AIMS: Heat shock proteins (HSPs) are highly conserved and serve a multitude of functions that mediate cell survival. HSP70, the only inducible form of the 70-kilodalton subfamily of HSPs, is overexpressed in pancreatic cancer cells and has been shown to inhibit caspase-dependent apoptosis. We aimed to elucidate the mechanism by which HSP70 inhibits apoptosis in cancer cells.
HSP70 expression was down-regulated in cultured pancreatic cancer cells by exposure to quercetin, triptolide, or short interfering RNAs. Intracellular Ca2+, cytosolic cathepsin B activity, caspase-3 activity, cell viability, and lysosome integrity were measured using colorimetric assays. Immunofluorescence assays were used to localize cathepsin B and Lamp2. BAPTA-AM was used to chelate intracellular Ca2+.
Inhibition of HSP70 increased intracellular Ca2+ levels in pancreatic and colon cancer cell lines and led to loss of lysosome integrity in pancreatic cancer cells. The release of intracellular Ca2+ and lysosomal enzymes activated caspase-dependent apoptosis independently and simultaneously.
HSP70 inhibits apoptosis in cancer cells by 2 mechanisms: attenuation of cytosolic calcium and stabilization of lysosomes. HSP70-mediated cell survival might occur in other types of cancer cells.
热休克蛋白（HSPs）高度保守，具有多种介导细胞存活的功能。HSP70是HSPs 70千道尔顿亚家族中唯一的可诱导形式，在胰腺癌细胞中过表达，且已显示其可抑制半胱天冬酶依赖性凋亡。我们旨在阐明HSP70抑制癌细胞凋亡的机制。
通过用槲皮素、雷公藤内酯醇或小干扰RNA处理培养的胰腺癌细胞，下调HSP70表达。使用比色法测定细胞内钙离子、胞质组织蛋白酶B活性、半胱天冬酶-3活性、细胞活力和溶酶体完整性。免疫荧光测定法用于定位组织蛋白酶B和Lamp2。使用BAPTA-AM螯合细胞内钙离子。
抑制HSP70可增加胰腺和结肠癌细胞系中的细胞内钙离子水平，并导致胰腺癌细胞溶酶体完整性丧失。细胞内钙离子和溶酶体酶的释放独立且同时激活半胱天冬酶依赖性凋亡。
HSP70通过两种机制抑制癌细胞凋亡：降低胞质钙水平和稳定溶酶体。HSP70介导的细胞存活可能也发生在其他类型的癌细胞中。
