Breast cancer (BC) has a great impact on the health of women worldwide as it affects more than 1.3 million women each year and accounts for about 14% of cancer-related deaths. Over the past two decades, significant research efforts have been initiated towards the role of melatonin and vitamin D3 in the etiology of cancer. The exact cellular and genetic mechanisms through which these compounds confer their therapeutic effect is still very ambiguous. In this study, we tried to study the therapeutic effect of vitamin D3, melatonin on MCF-7 breast cancer cell line and tried to unravel the molecular and cellular pathways that could initiate apoptosis of the cancer cells in-vitro.
The results presented demonstrate that melatonin and vitamin D3 have an optimum therapeutic dosage at 48 h of 5 nM and 0.5 nM respectively. Both MTT and LDH assay supported the therapeutic efficacy of these treatments. In most of the cytotoxic studies, MTT had been considered as the single most reliable parameter to measure cellular metabolic activity, and indicator of cellular viability, proliferation, and cytotoxicity. However, we tried to explore more at the metabolic status of these cells as a result of treatments by performing LDH assay. As, the role of the LDH enzyme in the initiation, promotion, and progression of cancer cells was well documented [14]. The cancer cells rely mostly on glycolysis with a huge intake of glucose and lactate production, the phenomena known as the Warburg effect. Lactate production in glycolysis contributes largely to malignant progressions, like replenishing NAD + for glycolysis, lowering pH for invasion/n and triggering immune escape, and above all the hypoxia conditions support angiogenesis. Lactate dehydrogenase A (LDH) converts pyruvate to lactate and activation of LDH enzyme is closely related to diverse cancers. We observed an increased amount of LDH activity in the control of MCF-7 cells after 24 h and 48 h in culture as expected and reported in other studies that cancer cells have high LDH activity [15]. However, the treatment of vitamin D3, melatonin, and in their combination resulted in significant downregulation of LDH enzyme levels indicating the therapeutic potential of these drugs.
Our data showed that melatonin and vitamin D3 were involved in the regulation of cell proliferation and apoptosis and suggested that this action could be, at least in part, ascribed to the increased expression of p53. Previous studies had shown that melatonin combined with D3 synergistically inhibits the proliferation of MCF-7 cells [16]. The human homolog of the mouse double minute gene 2 (MDM2) is known to be usually overexpressed in different types of malignancies [17]. MDM2 can ligate p53 protein by its E3 ubiquitin ligase, and the resulting ubiquitinated p53 can be transferred to the cytoplasm to be degraded by proteinases. Thus the main role of MDM2 in the progression of cancer is to silence the tumor suppresser activity of p53. The MDM2 gene is itself transcriptionally activated by p53in a regulatory loop [18]. Several therapeutic strategies have been suggested to disrupt the p53-MDM2 interactions including the use of small peptides [19] and inhibitors of ubiquitin ligase [20]. Recent studies on natural products as a safe therapeutic in the treatments of various forms of cancers have reported that melatonin and vitamin D3 could reduce MDM2 levels or to hinder the p53-MDM2 interactions [21, 22]. Enhanced production of p53 as a result of treatments is one of the goals as well as promising prognosis and indicators of the success of the treatments.
The cytotoxic effect of the melatonin in-vitro model of breast cancer in MCF-7 cells was exhibited due to the accumulation of cells in the Go/G1 phase of the cell cycle or delaying the progression to the S phase of the cell cycle [23]. This decreased cell proliferation was also accompanied by significantly increased gene expression of p53in other studies[24].
The quantitative gene expression studies using RT-PCR exhibited a significant upregulation of p53 with the vitamin D3, melatonin, and their combined treatments. Moreover, concomitant results were shown for protein expression analysis by Western blot, demonstrating that in control cells, there was no signal for p53 protein expression but cells treated with vitamin D3 showed a light band indicating the upregulation of p53, whereas melatonin treatment showed a very high signal for p53 protein expression. This effect was very significant and important for this study as it indicated that melatonin alone has a supportive upregulating effect on the P53 gene and protein expression as compared to vitamin D3 alone or in its combination. The exact molecular mechanism and the involvement of MDM2 gene expression concerning p53 expression with the treatment of vitamin D3 and melatonin warrants further investigation and opens up new avenues for future research. In this study, both of the compounds had shown to have a pro-apoptotic effect on breast cancer cells in-vitro and also indicated that both of them might have exhibited these properties through different molecular and biochemical mechanisms.