Schwartz G K, Haimovitz-Friedman A, Dhupar S K, Ehleiter D, Maslak P, Lai L, Loganzo F, Kelsen D P, Fuks Z, Albino A P
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
J Natl Cancer Inst. 1995 Sep 20;87(18):1394-9. doi: 10.1093/jnci/87.18.1394.
Protein kinase C (PKC) is a family of enzymes that function in processes relevant to carcinogenesis, tumor cell metastasis, and apoptosis. Safingol, an optical isomer (the L-threo enantiomer) of dihydrosphingosine, is a specific inhibitor of PKC and might represent a novel agent for anticancer therapy. Preclinical animal studies show that safingol alone has a minimal effect on tumor cell growth, but combining this compound with conventional chemotherapy agents dramatically potentiates their antitumor effects. It has been suggested that many chemotherapeutic agents exert their antitumor effects by inducing apoptosis.
We wanted to determine the extent to which safingol, alone or in combination with a standard chemotherapeutic agent (mitomycin C [MMC]), would promote apoptosis in gastric cancer cells in vitro. Furthermore, we investigated whether the induction of apoptosis in the treated cells was affected by their p53 tumor suppressor status or their drug-resistance status.
SK-GT-5 (p53-deficient and MMC-resistant) and MKN-74 (p53 wild-type and MMC-sensitive) gastric cancer cells were exposed to either no drug, safingol (50 microM) alone, MMC (5 micrograms/mL) alone, or a combination of safingol (50 microM) and MMC (5 micrograms/mL). In some experiments, cells were exposed simultaneously to safingol and the PKC activator, 3-phorbol 12-myristate 13-acetate (PMA), prior to treatment with MMC. Apoptosis was measured by two methods: 1) quantitative fluorescence microscopy of nuclear chromatin condensation in cells stained with the dye, bisbenzamide trihydrochloride (Hoechst-33258), and 2) terminal deoxynucleotidyl transferase (TdT) labeling of the 3'-OH ends of DNA fragments produced in apoptotic cells.
As determined by quantitative fluorescence microscopy, exposure of SK-GT-5 cells to safingol alone induced apoptosis in 2% +/- 1% (mean +/- SD) of the cells, and MMC alone increased that level to 18% +/- 1%. However, the combination of safingol and MMC induced apoptosis in 39% +/- 1% of the cells (P < .001, for the drug combination versus MMC alone). With MKN-74 cells, safingol alone induced apoptosis in 8% +/- 3% of the cells, whereas MMC alone induced apoptosis in 40% +/- 4% of treated cells and the combination of safingol and MMC induced apoptosis in 83% +/- 4% of the cells. Similar results were obtained with the TdT assay. Simultaneous exposure of cells to safingol and PMA abrogated the safingol-mediated enhancement of MMC-induced apoptosis.
The PKC inhibitor safingol enhances the cytotoxic effect of the chemotherapeutic agent MMC in gastric cancer cells by promoting drug-induced apoptosis. The induction of apoptosis occurs regardless of the p53 status or the drug-resistance status of the cells.
蛋白激酶C（PKC）是一类在与致癌作用、肿瘤细胞转移和凋亡相关的过程中发挥作用的酶家族。沙芬戈是二氢神经鞘氨醇的一种旋光异构体（L-苏式对映体），是PKC的特异性抑制剂，可能是一种新型抗癌治疗药物。临床前动物研究表明，单独使用沙芬戈对肿瘤细胞生长的影响极小，但将该化合物与传统化疗药物联合使用可显著增强其抗肿瘤作用。有人提出，许多化疗药物通过诱导凋亡发挥其抗肿瘤作用。
我们想确定沙芬戈单独或与标准化疗药物（丝裂霉素C [MMC]）联合使用时，在体外促进胃癌细胞凋亡的程度。此外，我们研究了处理后细胞中凋亡的诱导是否受其p53肿瘤抑制状态或耐药状态的影响。
将SK-GT-5（p53缺陷且对MMC耐药）和MKN-74（p53野生型且对MMC敏感）胃癌细胞分别不做处理、单独暴露于沙芬戈（50微摩尔）、单独暴露于MMC（5微克/毫升）或沙芬戈（50微摩尔）与MMC（5微克/毫升）联合使用。在一些实验中，在用MMC处理之前，细胞先同时暴露于沙芬戈和PKC激活剂12-O-十四酰佛波醇-13-乙酸酯（PMA）。通过两种方法测量凋亡：1）用双苯甲酰胺三盐酸盐（Hoechst-33258）染色后对细胞中核染色质凝聚进行定量荧光显微镜观察，以及2）对凋亡细胞中产生的DNA片段的3'-OH末端进行末端脱氧核苷酸转移酶（TdT）标记。
通过定量荧光显微镜观察确定，单独将SK-GT-5细胞暴露于沙芬戈可诱导2%±1%（平均值±标准差）的细胞凋亡，单独使用MMC可将该水平提高到18%±1%。然而，沙芬戈与MMC联合使用可诱导39%±1%的细胞凋亡（药物联合组与单独使用MMC组相比，P <.001）。对于MKN-74细胞，单独使用沙芬戈可诱导8%±3%的细胞凋亡，而单独使用MMC可诱导40%±4%的处理细胞凋亡，沙芬戈与MMC联合使用可诱导83%±4%的细胞凋亡。TdT检测也得到了类似结果。细胞同时暴露于沙芬戈和PMA可消除沙芬戈介导的MMC诱导凋亡的增强作用。
PKC抑制剂沙芬戈通过促进药物诱导的凋亡增强化疗药物MMC对胃癌细胞的细胞毒性作用。无论细胞的p53状态或耐药状态如何，均可诱导凋亡。
