Anti-drug resistance, anti-inflammation, and anti-proliferation activities mediated by melatonin in doxorubicin-resistant hepatocellular carcinoma: in vitro investigations

Abstract

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 IC50 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.

Disulfidptosis and ferroptosis related genes predict prognosis and personalize treatment for hepatocellular carcinoma

“…The overexpression of ABCC1 actively removes drug conjugates from cellular compartments, fostering the progression of chemotherapy resistance in HCC, leading to poor prognosis in patients ( 24 - 26 ). The downregulation or inhibition of ABCC1 renders resistant HCC cells more susceptible to the cytotoxic effects of doxorubicin and other chemotherapeutic agents, highlighting its potential as both a prognostic indicator and a therapeutic target ( 27 , 28 ). SLC7A11 serves as a crucial transporter involved in the synthesis of glutathione, thereby playing a pivotal role in maintaining redox homeostasis to counteract ferroptosis.…”

Anti-proliferative effect of melatonin in human hepatoma HepG2 cells occurs mainly through cell cycle arrest and inflammation inhibition

“…Although inflammation is important for cancer proliferation and metastasis 33 , multiple studies confirmed the anti-inflammatory role of melatonin treatment in various in vitro cancer models treated with 1 mM melatonin for 24 h via down-regulation of individual inflammatory mediators, such as NF-kB 34 , IL-1α, IL-1β, TNF-α, COX-2, IL-8 and MCP-1 9 , the present study is the first one which indicates the anti-inflammatory effect of 1 mM melatonin through targeting forty inflammatory mediated factors by using Human inflammation antibody array membrane. This inhibition could be mediated through the increased expression level of the P53 gene, as p53 has been reported to inhibit inflammatory responses, and functional loss of p53 causes excessive inflammatory reactions 15,33 .…”

“…Briefly, RT-PCR program steps was set up as shown in Table 1, for a sample with total volume of 15 µl containing 7.5 µl 2× sensiFAST SYBR No-ROX mix, 0.3 µl Ribosafe RNase Inhibitor, 0.15 µl Reverse transcriptase, 3.85 µl H 2 O, 2 µl RNA sample, and 0.6 µl of each specific primer sequence as indicated in Table 2. All RT-PCRs were performed in duplicate, and the gene copy numbers were normalized to 100,000 copies of the housekeeping beta-actin gene 15 .…”

“…Human inflammation antibody array membrane (40 targets) for quantification detection of inflammatory mediators in a response to 1 mM melatonin treatment. According to Hamed et al 15 , after HepG2 cells were seeded onto 6-well plates (300,000 cells/well), cells were treated with 1 mM mela- www.nature.com/scientificreports/ tonin for 24 h, and then lysed with RIPA lysis buffer supplemented with protease inhibitor cocktail (Invitrogen, USA) for total protein extraction. Total protein concentration was estimated using the BCA protein assay kit (Cat# 23225; Thermo scientific, USA) by following the manufacturer's instructions.…”