Mechanisms of dihydrochalcone-induced human cancer cell death

Abstract

Dihydrochalcone derivatives (DHCs), a sub-class of chalcone, possess several biological activities such as anti-microorganism, antioxidant, anti-inflammation, and anti-cancer activities. There are many DHCs that have been reported for their induction of cancer cell death via various pathways. DHCs in this study consist of 4′,6′-dihydroxy-2′,4-dimethoxy-5’-(2″-hydroxybenzyl)dihydrochalcone (DHC-1), a natural DHC, was extracted from leaves and twigs of Cyathostemma argenteum and 2′,6′-dihydroxy-4,4′-dimethoxydihydrochalcone (synthetic DHC, called calomelanone or DHC-2). The aims of this study were to determine the cytotoxic effects of DHCs on various cancer cell types including human breast cancer cells (MDA-MB-231 and MCF-7 cells) and human hepatocellular carcinoma HepG2 cells and to investigate the modes of cell death and mechanisms involved. The cytotoxic effect of DHCs was determined by MTT assay and it was found that both DHCs, DHC-1 and DHC-2, reduced cell viability of MDA-MB-231 (aggressive) and MCF-7 (non-aggressive) cells, whereas they were less toxic to normal cells, viz., peripheral blood mononuclear cells (PBMCs) and murine fibroblasts NIH3T3 cells. In addition, both DHCs also induced both breast cancer cells early apoptosis, which was examined by using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining and employed flow cytometry technique. Due to DHC-1 was cytotoxic towards breast cancer cells more than DHC-2, thus DHC-1 was selected for further studies. PI staining was utilized to characterize mode of cell death and cell morphology. Condensed nuclei and apoptotic bodies were found after DHC-1 treatment in both breast cancer cells. DHC-1 enhanced the cell cycle checkpoint genes, atm and atr, leading to the upregulation of BH3-only pro-apoptotic proteins including Bim, Bad, Bid and tBid. These proteins triggered mitochondrial transmembrane potential (ΔΨm) loss and activated caspase-9, an initiator caspase in intrinsic apoptosis pathway. DHC-1 also induced caspase-8 activity, which is also an initiator caspase of extrinsic apoptosis pathway. Moreover, DHC-1 induced ER stress by up-regulating the calcium levels in both cytosol and mitochondria. In addition, DHC-1 promoted apoptosis by EGFR/MAPK suppression. DHC-1 combined with imatinib, sorafenib and smac mimetic (BV6) showed the synergistic effects in MDA-MB-231 cells and additive effects in MCF-7 cells. In conclusion, DHC-1 induced breast cancer MDA-MB-231 and MCF-7 cells apoptosis via intrinsic, extrinsic, and ER stress pathways. Furthermore, inhibition of EGFR/MAPK signaling pathway was found in MCF-7 cells. Therefore, DHC-1 might be applied for alternative treatment for breast cancer patients. The cytotoxic effect of DHC-2 was investigated in hepatocellular carcinoma HepG2 cells. The HepG2 cell viability was inhibited after DHC-2 treatment at 48 and 72 h but not at 24 h. The levels of hydrogen peroxide (H2O2) or peroxide radicals (O2•−2) and superoxide anion radicals (O2•−) were increased in DHC-2-treated HepG2 cells resulting in cell stress and damage. DHC-2 stimulated apoptosis under oxidative stress condition by inducing the expression of Bax and Bak (pro-apoptotic proteins) whereas Bcl-xL (anti-apoptotic protein) was suppressed. The reduction of ΔΨm and increased caspase-9, -8 and -3 activities were found in DHC-2-treated HepG2 cells. Autophagic vacuoles, a character of autophagic cells, was found after DHC-2 treatment at 24 h. DHC-2 promoted autophagy mechanism by reduced expression of Akt, an autophagy regulator protein, and increasing the expression of Atg5, autophagic protein markers. DHC-2 induced autophagy to protect HepG2 cells from cell death. Nevertheless, co-treatment of DHC-2 with an autophagy inhibitor, 3-methyladenine (3-MA), could inhibit HepG2 cell viability. In conclusion, DHC-2-induced intrinsic and extrinsic pathways via ROS generation or oxidative stress. DHC-2 also induced autophagy mechanism for HepG2 cell survival. Combination of DHC-2 with 3-MA might be applied as anti-cancer agents for HCC patients. In summary, both DHC derivatives possessed the anticancer effects either on human breast or liver cancer via intrinsic and extrinsic pathway of apoptotic cell death. However, in hepatocellular carcinoma HepG2 cells in the short incubation time, the cells underwent autophagy which promoted cell survival.