Herbal Melanin Inhibits Real-Time Cell Proliferation, Downregulates Anti-Apoptotic Proteins and Upregulates Pro-Apoptotic p53 Expression in MDA-MB-231 and HCT-116 Cancer Cell Lines.

Background and Objectives: Cancer is the second-most-important deadly disease in the world, leading to severe socioeconomic consequences and posing a public threat. Consequently, breast and colorectal cancers are significant cancer types that affect women and men more commonly, respectively. Treatment failure or recurrent diseases frequently occur due to resistance, in addition to the side effects of the currently available anticancer agents. Therefore, in this study, herbal melanin anticancer activity was investigated against human breast adenocarcinoma (MDA-MB-231) and human colorectal (HCT 116) cell proliferation and the expression of downregulated anti-apoptotic proteins and upregulated pro-apoptotic p53. Materials and Methods: MDA-MB-231 and HCT 116 cells were monitored for their real-time proliferation properties using Xcelligence. Herbal melanin of various concentrations significantly inhibited MDA-MB-231 and HCT 116 cell proliferation. Then, the expression of proapoptotic and anti-apoptotic proteins such as p53, Bcl-2 and Bcl-xl was studied using Western blotting. Results: The Bcl-2 and Bcl-xl expressions were downregulated, while the p53 expression was upregulated after treatment with herbal melanin. Similarly, the expression of apoptotic proteins such as Bcl-2, Bcl-xl, XIAP, Survivin, Bid, Bax, p53, Cytochrome C, PARP genes and mRNA was studied after herbal melanin treatment using real-time PCR, which revealed the downregulation of Bcl-2, Bcl-xl, XIAP and Survivin and the upregulation of Bid, Bax, p53, Cytochrome C and PARP apoptotic protein expression. Also, caspase 3 and 9 expressions were monitored after the treatment with herbal melanin, which revealed the upregulation of both the MDA-MB-231 and HCT 116 cell types. Conclusions: Overall, herbal melanin can be used as an alternative anticancer agent against the MDA-MB-231 and HCT 116 cell types.

Herbal melanin modulates PGE2 and IL-6 gastroprotective markers through COX-2 and TLR4 signaling in the gastric cancer cell line AGS.

We reported a gastric anti-ulcerogenic effect of the Nigella sativa (L.)-derived herbal melanin (HM) using rat models. However, the molecular mechanisms underlying this HM gastroprotective effect remain unknown. Cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E2 (PGE2) and toll-like receptor 4 (TLR4)-mediated interleukin-6 (IL-6) production and secretion play major roles in gastric mucosal protection. In the current study, the human gastric carcinoma epithelial cell line AGS was used as a model to investigate the effect of HM on TLR4, COX-2, glycoprotein mucin 4 protein and gene expression using immuno-cyto-fluorescence staining, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gastroprotective markers PGE2 and IL-6 production and secretion were also assessed using an enzyme-linked immunosorbent assay (ELISA). Bacterial lipopolysaccharides (LPS), well-known inducers of TLR4, COX-2, PGE2 and IL-6 expression, were used as a positive control. We showed that HM upregulated its main receptor TLR4 gene and protein expression in AGS cells. HM increased, in a dose- and time-dependent manner, the secretion of PGE2 and the expression of COX-2 mRNA and protein, which was detected in the nucleus, cytoplasm and predominantly at the intercellular junctions of the AGS cells. In addition, HM enhanced IL-6 production and secretion, and upregulated the mucin 4 gene expression, the hallmarks of gastroprotection. To check whether HM-induced PGE2 and IL-6 through TLR4 signaling and COX-2 generated, AGS cells were pre-treated with a TLR4 signaling inhibitor TAK242 and the COX-2 inhibitor NS-398. A loss of the stimulatory effects of HM on COX-2, PGE2 and IL-6 production and secretion was observed in TAK242 and NS-398-pre-treated AGS cells, confirming the role of TLR4 signaling and COX-2 generated in the HM gastroprotective effects. In conclusion, our results showed that HM enhances TLR4/COX-2-mediated secretion of gastroprotective markers PGE2 and IL-6, and upregulates mucin 4 gene expression in the human gastric epithelial cell line AGS, which may contribute to the promising beneficial gastroprotective effect of HM for human gastric prevention and treatment.

Herbal melanin induces interleukin-1ß secretion and production by human THP-1 monocytes via Toll-like receptor 2 and p38 MAPK activation.

Herbal melanin (HM), extracted from Nigella sativa, is known for its immunogenic properties through the modulation of cytokine production via Toll-like receptor (TLR)4. TLRs play a crucial role in the host defense through the regulation of innate and adaptive immune responses. However, the potential effect of HM on the production of interleukin-1ß (IL-1ß), the main immunoregulatory cytokine secreted by activated monocytes, has not been reported. The present study aimed to investigate the effects of HM on IL-1ß secretion and production, detected by enzyme-linked immunosorbent assay, western blotting and mRNA expression monitored by reverse transcription-PCR, in human monocytes and a monocytic cell line, THP-1. Signaling pathways involved in the HM-induced IL-1ß production was investigated in the THP-1 cells. It was shown that HM upregulated the IL-1ß mRNA in the THP-1 cells and induced the secretion of IL-1ß in the monocytes and THP-1 cells, in a dose-dependent manner, compared to the untreated cells. HM increased the protein expression of IL-1ß, TLR2, the main receptor for IL-1ß production, and activated p38 mitogen-activated protein kinase (MAPK), a key mediator for stress-induced IL-1ß gene expression. The blockade of the p38 MAPK pathway, with the pharmacological inhibitor SB202190, and TLR2 receptor with a neutralization antibody, resulted in the decrease of HM-induced IL-1ß production in THP-1 cells. The TLR4 receptor blockade also decreased HM-induced IL-1ß production, but to a lesser extent than TLR2 blockade. In conclusion, the present study demonstrated that HM stimulates IL-1ß production in monocytes and THP-1 cells, in a TLR2/p38 MAPK pathway-dependent manner, suggesting promising immunoregulatory potentials of HM against inflammatory-associated diseases.

Distinct anti-proliferative effects of herbal melanin on human acute monocytic leukemia THP-1 cells and embryonic kidney HEK293 cells.

BACKGROUND: Herbal melanin (HM) is a dark pigment extracted from the seed coat of Nigella sativa L. and known to exert biological effects via toll-like receptor 4 (TLR4). Recently, TLR4 was described as involved in natural programmed cell death (apoptosis). Tumor and embryonic cells are used as in vitro cellular models for drug and anti-cancer agent screening. To date, no cytotoxic studies have been reported of HM in TLR4-positive acute monocytic leukemia THP-1 cells compared to TLR4-negative human embryonic kidney HEK293 cells. METHODS: We studied the anti-proliferative effects of several HM concentrations on THP-1 and HEK293 cells by evaluating cell viability using the CellTiter-Glo® luminescent assay, assessing the TLR4 expression level, determining the apoptotic status, and analyzing the cell cycle distribution using flow cytometry. Apoptotic pathways were investigated using mitochondrial transition pore opening, caspase activity assays and immunoblot technology. RESULTS: Low HM concentrations did not affect THP-1 cell viability, but high HM concentrations (62.5-500 µg/mL) did decrease THP-1 cell viability and induced G0/G1 phase cell cycle arrest. Only at the highest concentration (500 µg/mL), HM slightly increased the TLR4 expression on the THP-1 cell surface, concomitantly upregulated TLR4 whole protein and gene expression, and induced apoptosis in THP-1 cells via activation of the extrinsic and intrinsic pathways. No change of apoptotic status was noticed in TLR4-negative HEK293 cells, although HM decreased HEK293 cell viability and induced cell growth arrest in the G2 phase. CONCLUSION: HM exerts distinct anti-proliferative effects on human acute monocytic leukemia and embryonic kidney cells mainly through cell cycle interference in a TLR4-independent manner and through apoptosis induction in a TLR4-dependent manner, as observed in only the THP-1 cells.

Herbal melanin inhibits colorectal cancer cell proliferation by altering redox balance, inducing apoptosis, and modulating MAPK signaling.

BACKGROUND: Colorectal 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. METHODS: Cell 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. RESULTS: Our 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. CONCLUSION: Altogether, 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.

Renal injury, nephrolithiasis and Nigella sativa: A mini review.

OBJECTIVE: The incidence and prevalence of kidney stone is increasing worldwide. After the first recurrence the risk of subsequent relapses is higher and the time period between relapses is shortened. Urinary stones can be severely painful and make a huge economic burden. The stone disease may increase the vulnerability of patients to other diseases such as renal failure. Medicinal herbs are rich sources of antioxidants which are increasingly consumed globally for their safety, efficacy and low price. Nigella sativa is a spice plant that is widely used for prevention and treatment of many ailments in Muslim countries and worldwide. This review aims at investigation of the effects of Nigella sativa on renal injury and stone formation. MATERIALS AND METHODS: The scientific resources including PubMed, Scopus, and Google scholar were searched using key words such as: nephrolithiasis, urolithiasis, kidney/renal stone, renal injury, renal failure, urinary retention and black seed, black cumin, Nigella sativa and thymoquinone. RESULTS: N. sativa and its main component, thymoquinone showed positive effects in prevention or curing kidney stones and renal failure through various mechanism such as antioxidative, anti-inflammatory, anti-eicosanoid and immunomodulatory effects. The putative candidate in many cases has been claimed to be thymoquinone but it seems that at least in part, particularly in kidney stones, the herbal melanin plays a role which requires further investigation to prove. CONCLUSION: N. sativa and its components are beneficial in prevention and curing of renal diseases including nephrolithiasis and renal damages.