Effect of Calomelanone, a Dihydrochalcone Analogue, on Human Cancer Apoptosis/Regulated Cell Death in an In Vitro Model

Abstract

Calomelanone, 2 ′ ,6 ′ -dihydroxy-4,4 ′ -dimethoxydihydrochalcone, possesses anticancer activities. This study was conducted to investigate the cytotoxic effect of calomelanone, a dihydrochalcone analogue, on human cancer cells and its associated mechanisms. The cytotoxic effect of calomelanone was measured by MTT assay. Annexin V-FITC/propidium iodide and DiOC6 staining that employed flow cytometry were used to determine the mode of cell death and reduction of mitochondrial transmembrane potential (MTP), respectively. Caspase activities were measured using specific substrates and colorimetric analysis. The expression levels of Bcl-2 family proteins were determined by immunoblotting. Reactive oxygen species were also measured using 2 ′ ,7 ′ -dihydrodichlorofluorescein diacetate and dihydroethidium (fluorescence dyes). Calomelanone was found to be toxic towards various human cancer cells, including acute promyelocytic HL-60 and monocytic leukemic U937 cells, in a dose-dependent manner at 24 h and human hepatocellular HepG2 cells at 48 h. However, the proliferation of HepG2 cells increased at 24 h. Calomelanone was found to induce apoptosis in HL-60 and U937 at 24 h and HepG2 apoptosis at 48 h via the intrinsic pathway by inducing MTP disruption. This compound also induced caspase-3, caspase-8, and caspase-9 activities. Calomelanone upregulated proapoptotic Bax and Bak and downregulated antiapoptotic Bcl-xL proteins in HepG2 cells. Moreover, signaling was also associated with oxidative stress in HepG2 cells. Calomelanone induced autophagy at 24 h of treatment, which was evidenced by staining with monodansylcadaverine (MDC) to represent autophagic flux. This was associated with a decrease of Akt (survival pathway) and an upregulation of Atg5 (the marker of autophagy). Thus, calomelanone induced apoptosis/regulated cell death in HL-60, U937, and HepG2 cells. However, it also induced autophagy in HepG2 depending on duration, dose, and type of cells. Thus, calomelanone could be used as a potential anticancer agent for cancer treatment. Nevertheless, acute and chronic toxicity should be further investigated in animals before conducting investigations in human patients.

Molecular Mechanisms of Antiproliferative Effects of Natural Chalcones

“…Moreover, the process of autophagy has also been initiated in calomelanone-treated cells. These effects were associated with decreased expression of Akt [ 205 ]. Recently, Song and co-workers [ 206 ] studied antiproliferative effect of ISL in hepatocellular carcinoma.…”

Programmed Cell Death Alterations Mediated by Synthetic Indole Chalcone Resulted in Cell Cycle Arrest, DNA Damage, Apoptosis and Signaling Pathway Modulations in Breast Cancer Model

“…Among others, the anticancer effect of both natural and synthetic chalcones has been intensively studied in the last decade [ 13 , 14 , 15 , 16 ]. Although the mechanism of the antiproliferative action of chalcones has not yet been fully elucidated, numerous studies documented pleiotropic mode of action [ 17 , 18 , 19 , 20 ] including cell cycle arrest, DNA damage, induction of reactive oxygen species production, modulation of various signaling pathways, etc. [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 ].…”

LCT-3d Induces Oxidative Stress-Mediated Apoptosis by Upregulating Death Receptor 5 in Gastric Cancer Cells

“…To further confirm whether LCT-3d treatment activated mitochondrial pathway, we detected mitochondria-related proteins in gastric cancer cells. Bcl-2 family proteins are the major regulators and effectors of the mitochondrial apoptotic pathway, which include anti-apoptotic proteins such as Bcl-2 and pro-apoptotic proteins such as Bax (26)(27)(28)(29). The oligomerization of Bax and Bak proteins promote the increase of the mitochondrial membrane permeability, which leading to the release of cytochrome c into the cytosol, subsequently cell apoptosis occurred through Caspase cascade.…”