Zhang Xian-Ming, Chen Jia, Xia Yu-Gui, Xu Qiang
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210093, People's Republic of China.
Cancer Chemother Pharmacol. 2005 Mar;55(3):251-62. doi: 10.1007/s00280-004-0863-5. Epub 2004 Nov 5.
In our previous study, quercetin was found to induce apoptosis of murine melanoma B16-BL6 cells. The cellular and molecular mechanism of quercetin-induced apoptosis was investigated in the present study.
Nuclear morphology was determined by fluorescence microscopy. DNA fragmentation was analyzed by electrophoresis and quantified by the diphenylamine method. The transmembrane potential of mitochondria was measured by flow cytometry. Bcl-2, Bcl-X(L), PKC-alpha, PKC-beta, and PKC-delta were detected by Western blotting. Caspase activity was determined spectrophotometrically.
Quercetin induced the condensation of nuclei of B16-BL6 cells in a dose-dependent pattern as visualized by Hoechst 33258 and propidium iodide dying. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly enhanced apoptosis induced by quercetin, while doxorubicin, a PKC inhibitor, markedly decreased it. Both PMA and doxorubicin showed a consistent effect on the fragmentation of nuclear DNA caused by various dosages of quercetin. Quercetin dose-dependently led to loss of the mitochondrial membrane potential, which was also significantly reinforced or antagonized by PMA and doxorubicin, respectively. Moreover, PMA showed reinforcement, while doxorubicin showed significant antagonization, of the quercetin-mediated decrease in the expression of Bcl-2. Quercetin promoted caspase-3 activity in a dose-dependent manner, which was also regulated by PMA and doxorubicin with a pattern similar to that seen in their effect on apoptosis, mitochondrial membrane potential and Bcl-2 expression, but none of these were directly affected by PMA and doxorubicin. Free fatty acid and chlorpromazine, a PKC activator and inhibitor, respectively, did not interfere with these effects of quercetin. B16-BL6 cells expressed PKC-alpha, PKC-beta, and PKC-delta. Quercetin dose-dependently inhibited the expression of PKC-alpha but not that of PKC-beta and PKC-delta. Doxorubicin almost completely blocked the effect of quercetin on the expression of PKC-alpha. Quercetin was also involved in the translocation of PKC-delta from the cytosol to the nucleus. PMA enhanced the effect of quercetin on the translocation of PKC-delta.
These results indicate that quercetin induced apoptosis of murine melanoma B16-BL6 cells by injuring their mitochondria, increasing the activity of caspase-3, inhibiting the expression of Bcl-2 and PKC-alpha, and inducing the translocation of PKC-delta. Doxorubicin inhibited these effects of quercetin by blocking the decreased expression of PKC-alpha induced by quercetin while PMA increased these effects by enhancing the translocation of PKC-delta induced by quercetin.
在我们之前的研究中，发现槲皮素可诱导小鼠黑色素瘤B16 - BL6细胞凋亡。本研究对槲皮素诱导凋亡的细胞和分子机制进行了研究。
通过荧光显微镜确定核形态。通过电泳分析DNA片段化，并采用二苯胺法进行定量。通过流式细胞术测量线粒体跨膜电位。通过蛋白质免疫印迹法检测Bcl - 2、Bcl - X(L)、PKC -α、PKC -β和PKC -δ。通过分光光度法测定半胱天冬酶活性。
通过Hoechst 33258和碘化丙啶染色可见，槲皮素以剂量依赖性方式诱导B16 - BL6细胞核凝聚。蛋白激酶C（PKC）激活剂佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）显著增强槲皮素诱导的凋亡，而PKC抑制剂阿霉素则显著降低凋亡。PMA和阿霉素对不同剂量槲皮素引起的核DNA片段化均显示出一致的作用。槲皮素剂量依赖性地导致线粒体膜电位丧失，PMA和阿霉素分别对其有显著增强或拮抗作用。此外，PMA增强了槲皮素介导的Bcl - 2表达降低，而阿霉素则显著拮抗该作用。槲皮素以剂量依赖性方式促进半胱天冬酶 - 3活性，其也受PMA和阿霉素调节，调节模式与其对凋亡、线粒体膜电位和Bcl - 2表达的影响相似，但这些均不受PMA和阿霉素直接影响。游离脂肪酸和氯丙嗪分别作为PKC激活剂和抑制剂，不干扰槲皮素的这些作用。B16 - BL6细胞表达PKC -α、PKC -β和PKC -δ。槲皮素剂量依赖性地抑制PKC -α的表达，但不影响PKC -β和PKC -δ的表达。阿霉素几乎完全阻断了槲皮素对PKC -α表达的影响。槲皮素还参与PKC -δ从细胞质向细胞核的转位。PMA增强了槲皮素对PKC -δ转位的作用。
这些结果表明，槲皮素通过损伤线粒体、增加半胱天冬酶 - 3活性、抑制Bcl - 2和PKC -α表达以及诱导PKC -δ转位来诱导小鼠黑色素瘤B16 - BL6细胞凋亡。阿霉素通过阻断槲皮素诱导的PKC -α表达降低来抑制槲皮素的这些作用，而PMA通过增强槲皮素诱导的PKC -δ转位来增强这些作用。
