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.
Highlights
Among the primary forms of liver cancer, hepatocellular carcinoma (HCC) is the most commonly diagnosed primary malignant tumor of the liver with high rates of incidence around the world [1]
Since several natural compounds or derivatives are known to be toxic to cancer cells [28], the cytotoxic effect of calomelanone was determined on various cancer cells
The results indicated that calomelanone was able to inhibit HL-60 and U937 cell viability in a dose-dependent manner after incubation for 24 h, but not HepG2 cells at 24 h (Figure 2)
Summary
Among the primary forms of liver cancer, hepatocellular carcinoma (HCC) is the most commonly diagnosed primary malignant tumor of the liver with high rates of incidence around the world [1]. The risk factors for primary liver cancer include hepatitis B virus and hepatitis C viral infection, alcohol consumption, tobacco, oral contraceptives, and aflatoxin [2]. Leukemia is a cancer of the hematopoietic system resulting in abnormal proliferation of white or red blood cells [3]. Leukemia is frequently found in childhood and is known to be caused by genetic, radioactive, infectious, and environmental factors [4]. Cancer can be treated with surgery, radiation, chemotherapy, and immune therapy. For the treatment of leukemia, patients need to receive chemotherapy, radiation, or bone marrow transplants. The goal of these treatments is to induce cancer cell death through regulated cell death
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