Autophagy inhibition contributes to the synergistic interaction between EGCG and doxorubicin to kill the hepatoma Hep3B cells.
(-)-Epigallocatechin-3-O-gallate(EGCG), the highest catechins from green tea, has promisingly been found to sensitize the efficacy of several chemotherapy agents like doxorubicin (DOX) in hepatocellular carcinoma (HCC) treatment. However, the detailed mechanisms by which EGCG augments the chemotherapeutic efficacy remain unclear. Herein, this study was designed to determine the synergistic impacts of EGCG and DOX on hepatoma cells and particularly to reveal whether the autophagic flux is involved in this combination strategy for the HCC. Electron microscopy and fluorescent microscopy confirmed that DOX significantly increased autophagic vesicles in hepatoma Hep3B cells. Western blot and trypan blue assay showed that the increasing autophagy flux by DOX impaired about 45% of DOX-induced cell death in these cells. Conversely, both qRT-PCR and western blotting showed that EGCG played dose-dependently inhibitory role in autophagy signaling, and that markedly promoted cellular growth inhibition. Amazingly, the combined treatment caused a synergistic effect with 40 to 60% increment on cell death and about 45% augmentation on apoptosis versus monotherapy pattern. The DOX-induced autophagy was abolished by this combination therapy. Rapamycin, an autophagic agonist, substantially impaired the anticancer effect of either DOX or combination with EGCG treatment. On the other hand, using small interference RNA targeting chloroquine autophagy-related gene Atg5 and beclin1 to inhibit autophagy signal, hepatoma cell death was dramatically enhanced. Furthermore, in the established subcutaneous Hep3B cells xenograft tumor model, about 25% reduction in tumor growth as well as 50% increment of apoptotic cells were found in combination therapy compared with DOX alone. In addition, immunohistochemistry analysis indicated that the suppressed tendency of autophagic hallmark microtubule-associated protein light chain 3 (LC3) expressions was consistent with thus combined usage in vitro. Taken together, the current study suggested that EGCG emerges as a chemotherapeutic augmenter and synergistically enhances DOX anticancer effects involving autophagy inhibition in HCC.
New Drug Research & Development Center, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, P. R. China ; Department of Gastroenterology, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, P. R. China ; Pharmacy School of Guangxi Medical University, Nanning, Guangxi, P. R. China.
Li
L
Chen
Department of Gastroenterology, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, P. R. China.
Hui-Lan
HL
Ye
Department of Gastroenterology, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, P. R. China.
Guo
G
Zhang
Department of Gastroenterology, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, P. R. China.
Wen-Min
WM
Yao
Department of Gastroenterology, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, P. R. China.
Xing-Zhou
XZ
Chen
Department of Gastroenterology, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, P. R. China.
Fa-Can
FC
Zhang
Pharmacy School of Guangxi Medical University, Nanning, Guangxi, P. R. China.
Gang
G
Liang
D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000473:pathology* / Q000648:ultrastructure; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000008:administration & dosage / Q000494:pharmacology*; D004357:Drug Synergism; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000473:pathology* / Q000648:ultrastructure; D051379:Mice; D008819:Mice, Nude; D015854:Up-Regulation / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
D004317:Doxorubicin; D002392:Catechin; C045651:epigallocatechin gallate