Mohanty Sanghamitra, Huang Jie, Basu Alakananda
Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Clin Cancer Res. 2005 Sep 15;11(18):6730-7. doi: 10.1158/1078-0432.CCR-05-0450.
Bryostatin 1, a unique protein kinase C (PKC) activator, is already in the clinical trials. An understanding of complex regulation of PKC by bryostatin 1 is essential for effective use of bryostatin 1 in the clinic. We have previously shown that the ability of bryostatin 1 to enhance cisplatin sensitivity correlated with its ability to down-regulate PKCdelta in HeLa cells. We have investigated how bryostatin 1 influences PKCdelta regulation in cisplatin-resistant HeLa (HeLa/CP) cells, and if bryostatin 1 could be used to reverse cisplatin resistance.
Phorbol 12,13-dibutyrate (PDBu), bryostatin 1, and small interfering RNA were used to manipulate PKC level/activation status. Cell death was monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Annexin V dye-binding assay, and analysis of hypodiploid peak in a flow cytometer.
Bryostatin 1 elicited a biphasic concentration response on PKCdelta down-regulation and cisplatin-induced cell death in HeLa/CP cells; the maximum effect was achieved with 1 nmol/L bryostatin 1. Down-regulation of PKCalpha increased with increasing concentrations of bryostatin 1. PDBu induced down-regulation of PKCalpha in HeLa and HeLa/CP cells but it had little effect on PKCdelta down-regulation in HeLa/CP cells. However, both PDBu and bryostatin 1 enhanced the sensitivity of HeLa/CP cells to cisplatin. Knockdown of PKCdelta by small interfering RNA inhibited cisplatin-induced apoptosis but knockdown of PKCalpha enhanced cisplatin-induced cell death.
These results suggest that although PKCdelta acts as a proapoptotic protein, full-length PKCdelta may inhibit cisplatin-induced cell death. Thus, persistent activation/down-regulation of PKCdelta by bryostatin 1 was associated with cisplatin sensitization. Furthermore, PKCalpha acts as an antiapoptotic protein and down-regulation of PKCalpha by PDBu was associated with cellular sensitization to cisplatin.
苔藓抑素1是一种独特的蛋白激酶C（PKC）激活剂，已进入临床试验阶段。了解苔藓抑素1对PKC的复杂调节作用对于其在临床上的有效应用至关重要。我们之前已经表明，苔藓抑素1增强顺铂敏感性的能力与其下调HeLa细胞中PKCδ的能力相关。我们研究了苔藓抑素1如何影响顺铂耐药的HeLa（HeLa/CP）细胞中PKCδ的调节，以及苔藓抑素1是否可用于逆转顺铂耐药性。
使用佛波醇12,13 - 二丁酸酯（PDBu）、苔藓抑素1和小干扰RNA来调控PKC水平/激活状态。通过3 -（4,5 - 二甲基噻唑 - 2 - 基）- 2,5 - 二苯基四氮唑溴盐（MTT）法、膜联蛋白V染料结合法以及流式细胞仪分析亚二倍体峰来监测细胞死亡情况。
苔藓抑素1对HeLa/CP细胞中PKCδ的下调和顺铂诱导的细胞死亡呈现双相浓度反应；1 nmol/L的苔藓抑素1可达到最大效应水平。随着苔藓抑素1浓度的增加，PKCα的下调作用增强。PDBu可诱导HeLa和HeLa/CP细胞中PKCα的下调，但对HeLa/CP细胞中PKCδ的下调作用较小。然而，PDBu和苔藓抑素1均增强了HeLa/CP细胞对顺铂的敏感性。通过小干扰RNA敲低PKCδ可抑制顺铂诱导的细胞凋亡，但敲低PKCα则增强了顺铂诱导的细胞死亡。
这些结果表明，尽管PKCδ作为一种促凋亡蛋白，但全长PKCδ可能会抑制顺铂诱导的细胞死亡。因此，苔藓抑素1对PKCδ的持续激活/下调与顺铂增敏作用相关。此外，PKCα作为一种抗凋亡蛋白，PDBu对PKCα的下调与细胞对顺铂的增敏作用相关。
