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Hepatocellular carcinoma (HCC) is the major life-threatening primary liver malignancy in both sexes all over the world. Unfortunately, the majority of patients are diagnosed at later stages because HCC does not elicit obvious symptoms during its early incidence. Consequently, most individuals escape the first-line HCC treatments and are treated with chemotherapy. Regrettably, the therapeutic outcomes for those patients are usually poor because of the development of multidrug resistance phenomena. Furthermore, most anti-HCC therapies cause severe undesired side effects that notably interfere with the life quality of such patients. Accordingly, there is an important need to search for an alternative therapeutic drug or adjuvant which is more efficient with safe or even minimal side effects for HCC treatment. Melatonin was recently reported to exert intrinsic antitumor activity in different cancers. However, the regulatory pathways underlying the antitumor activity of melatonin are poorly understood in resistant liver cells. Furthermore, a limited number of studies have addressed the therapeutic role of melatonin in HCC cells resistant to doxorubicin chemotherapy. In this study, we investigated the antitumor effects of melatonin in doxorubicin-resistant HepG2 cells and explored the regulatory pivotal targets underlying these effects. To achieve our aim, an MTT assay was used to calculate the 50% inhibitory concentration of melatonin and evaluate its antiproliferative effect on resistant cells. Additionally, qRT-PCR was used to quantify genes having a role in drug resistance phenotype (ABCB1, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, and ABCG2); apoptosis (caspases-3, and -7, Bcl2, Bax, and p53); anti-oxidation (NRF2); expression of melatonin receptors (MT1, MT2, and MT3); besides, programmed death receptor PD-1 gene. The active form of the caspase-3 enzyme was estimated by ELISA. A human inflammatory antibody membrane array was employed to quantify forty inflammatory factors expressed in treated cells. We observed that melatonin inhibited the proliferation of doxorubicin-resistant HepG2 cells in a dose-dependent manner after 24-h incubation time with a calculated IC 50 greater than 10 mM (13.4 mM), the expression levels of genes involved in drug resistance response (ABCB1, ABCC1, ABCC5, and ABCG2) were downregulated. Also, the expression of caspase-3, Caspase-7, NRF2, and p53 genes were expressed at higher levels as compared to control (DMSO-treated cells). An active form of caspase-3 was confirmed by ELISA. Moreover, the anti-inflammatory effect of melatonin was detected through the calculated fold change to control which was reduced for various mediators that have a role in the inflammation pathway. The current findings introduce melatonin as a promising anti-cancer treatment for human-resistant HCC which could be used in combination with current chemotherapeutic regimens to improve the outcome and reduce the developed multidrug resistance., (© 2023. The Author(s).)

Hepatocellular carcinoma (HCC) is the major lethal primary liver malignant worldwide. Although, melatonin has various antitumor bioactivities; there is a requirement for more investigations to elucidate the not discussed effects, and the controversial responses of the treatment with melatonin on targets mediated in HCC. To achieve the aim of the present study, HCC-HepG2 cells were treated with different concentrations of melatonin at various time intervals. The selected minimal proliferation inhibition doses of melatonin were then incubated with cells to examine the arresting effect of melatonin on dividing cells using flow cytometry. Furthermore, the molecular patterns of genes that contributed to apoptosis, drug resistance development, antioxidation, and melatonin crossing were quantified by qRT-PCR. Additionally, the Human inflammation antibody array membrane (40 targets) was used to check the anti-inflammatory effect of melatonin. Our results validated that, melatonin shows anti-proliferative action through preserving cells in G0/G1 phase (P < 0.001) that is associated with a highly significant increase in the expression level of the P53 gene (P < 0.01). On contrary, as a novelty, our data recorded decreases in expression levels of genes involved in the pro-apoptotic pathway; with a significant increase (P < 0.05) in the expression level of an anti-apoptotic gene, Bcl2. Interestingly, we detected observed increases in the expression levels of genes responsible for conferring drug resistance including ABCB1, ABCC1, and ABCC5. Our study proved the anti-inflammatory activity of 1 mM melatonin in HCC-HepG2 cells. Accordingly, we can conclude that melatonin facilitates the anti-proliferation of cells at doses of 1 mM, and 2.5 mM after 24 h. This action is initiated through cell cycle arrest at G0/G1 phase via increasing the expression of P53, but independently on apoptosis. Collectively, melatonin is an effective anti-inflammatory and anti-proliferative promising therapy for the treatment of HCC. However, its consumption should be cautious to avoid the development of drug resistance and provide a better treatment strategy., (© 2023. The Author(s).)