Abstract
BackgroundColorectal carcinoma is one of the most deadly cancers that requests effective and safe chemotherapy. Evaluation of natural product-based anticancer drugs as adjuvant treatment with fewer side effects is largely unexplored research fields. Herbal melanin (HM) is an extract of the seed coats of Nigella sativa that modulates an inflammatory response through toll-like receptor 4 (TLR4). This TLR4 receptor is also involved in the modulation of apoptosis. We therefore explored the anticancer potential of HM and specifically its effect on the molecular mechanisms underlying adenocarcinoma and metastatic colorectal cancer (mCRC) cell death in vitro.MethodsCell viability was evaluated using the MTT assay. Cellular reactive oxygen species (ROS), glutathione levels, and apoptotic status were assessed using fluorometric and colorimetric detection methods. HM-induced apoptotic and other signaling pathways were investigated using Western blot technology and mitochondrial transition pore assay kit. TLR4 receptor downregulation and blockade were performed using siRNA technology and neutralizing antibody, respectively.ResultsOur results showed that HM inhibited the proliferation of the colorectal adenocarcinoma HT29 and mCRC SW620 cell lines. Furthermore, HM enhanced ROS production and decreased glutathione levels. HM-induced apoptosis was associated with mitochondrial outer membrane permeability and cytochrome c release, inhibition of the Bcl2 family proteins, and activation of caspase-3/-7. In addition, HM modulated MAPK pathways by activating the JNK pathway and by inhibiting ERK phosphorylation. TLR4 receptor downregulation enhanced HM-induced apoptosis while TLR4 receptor blockade partially alleviated HM-inhibited ERK phosphorylation.ConclusionAltogether, these findings indicate that HM exerts pro-apoptotic effects and inhibits MAPK pathway through TLR4 in mCRC and colorectal adenocarcinoma cells, suggesting HM as a promising natural-based drug for the treatment of colorectal cancer.
Highlights
Colorectal carcinoma is one of the most deadly cancers that requests effective and safe chemotherapy
Herbal melanin (HM) inhibits colorectal adenocarcinoma and metastatic colorectal cancer (mCRC) cell proliferation Colorectal adenocarcinoma HT29 and mCRC SW620 cells were treated with various concentrations (5–200 μg/ ml) of HM for 24 h and a dose-dependent inhibition of the cell viability was assessed in each cell line, in comparison with high cell viability of untreated cells (Fig. 1a, b)
Using xCELLigence RTDP in order to measure the cell proliferation in real-time based on the cell number, an anti-proliferative effect of HM on colorectal adenocarcinoma HT29 cells treated with HM at 50 and 100 μg/
Summary
Colorectal carcinoma is one of the most deadly cancers that requests effective and safe chemotherapy. Herbal melanin (HM) is an extract of the seed coats of Nigella sativa that modulates an inflam‐ matory response through toll-like receptor 4 (TLR4). This TLR4 receptor is involved in the modulation of apoptosis. As chemotherapy induces toxic effects and targeted therapy is very expensive, it is necessary to develop novel therapeutic drugs that might eliminate advanced colorectal adenocarcinoma and mCRC cells [8, 9]. In vitro studies on different CRC cell lines demonstrated the anti-proliferative effects of phytochemicals, including green tea extracts [16], soybeans [17], garlic [18] or Chinese gold thread [19]. Cell growth inhibition and apoptosis induction may result from increased reactive oxygen species (ROS), activation of adenosine monophosphate (AMP)-activated protein kinase with VEGF reduction, inhibition of insulin-like growth factor-I receptor signaling, or NF-κB pathway inactivation [16,17,18,19]
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