Apoptosis of murine melanoma B16-BL6 cells induced by quercetin targeting mitochondria, inhibiting expression of PKC-alpha and translocating PKC-delta
Authors: Jia Chen
Xian-Ming Zhang
Yu-Gui Xia
Qiang Xu
Journal: Cancer Chemotherapy and Pharmacology (2005)
Discipline: CLINICAL MEDICINE
Keywords: quercetin; apoptosis; B16-BL6 cells; mitochondria; caspase-3
Affiliations: State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210093, People's Republic of China.

Abstract:  Purpose: 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. Methods: 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. Results: 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. Conclusions: 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. 

Retracted: No

PubPeer comments:
 Figure 9 and Figure 10. * Unexpected similarities between lanes. Boxes of the same color highlight lanes that look similar. Part of a set of 29 papers reported to the journal in the summer of 2015 with a friendly reminder in April 2017, but no answer. With the exception of one paper, the journal did not correct or retract a single paper from this set. ![file] , 
 Correction for several type III duplications, April 2021, found here: https: link.springer.com article 10.1007 s00280-021-04287-1  *In the original publication of the article, there were several inadvertent errors in the Fig. 9a, b and Fig. 10c, d. The corrected Figs. 9 and 10 are given below.* ,