Thymoquinone Inhibits Bone Metastasis of Breast Cancer Cells Through Abrogation of the CXCR4 Signaling Axis.

Overexpression of chemokine receptor type 4 (CXCR4) has been found to be associated with increased cell proliferation, metastasis and also act as an indicator of poor prognosis in patients with breast cancer. Therefore, new agents that can abrogate CXCR4 expression have potential against breast cancer metastasis. In this study, we examined the potential effect of thymoquinone (TQ), derived from the seeds of Nigella sativa, on the expression and regulation of CXCR4 in breast cancer cells. TQ was found to inhibit the expression of CXCR4 in MDA-MB-231 triple negative breast cancer (TNBC) cells in a dose- and time-dependent manner. It was noted that suppression of CXCR4 by TQ was possibly transcriptionally regulated, as treatment with this drug caused down-regulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and suppression of NF-κB binding to the CXCR4 promoter. Pretreatment with a proteasome inhibitor and/or lysosomal stabilization did not affect TQ induced suppression of CXCR4. Down-regulation of CXCR4 was further correlated with the inhibition of CXCL12-mediated migration and invasion of MDA-MB-231 cells. Interestingly, it was observed that the deletion of p65 could reverse the observed anti-invasive/anti-migratory effects of TQ in breast cancer cells. TQ also dose-dependently inhibited MDA-MB-231 tumor growth and tumor vascularity in a chick chorioallantoic membrane assay model. We also observed TQ (2 and 4 mg/kg) treatment significantly suppressed multiple lung, brain, and bone metastases in a dose-dependent manner in a metastasis breast cancer mouse model. Interestingly, H&E and immunohistochemical analysis of bone isolated from TQ treated mice indicated a reduction in number of osteolytic lesions and the expression of metastatic biomarkers. In conclusion, the results indicate that TQ primarily exerts its anti-metastatic effects by down-regulation of NF-κB regulated CXCR4 expression and thus has potential for the treatment of breast cancer.

Inhibiting Glycine Decarboxylase Suppresses Pyruvate-to-Lactate Metabolism in Lung Cancer Cells.

Glycine decarboxylase (GLDC) gene is frequently upregulated in various types of cancer including lung, prostate and brain. It catabolizes glycine to yield 5,10-methylenetetrahydrofolate, an important substrate in one-carbon metabolism for nucleotide synthesis. In this study, we used exon splicing modulating steric hindrance antisense oligonucleotide (shAON) to suppress GLDC expression and investigated its effect on pyruvate metabolism via hyperpolarized carbon-13 magnetic resonance spectroscopy (MRS). The MRS technique allows us to study in vivo metabolic flux in tumor tissues with/without GLDC-shAON intervention. Here, we show that GLDC-shAON treatment is able to suppress lung cancer cell growth and tumorigenesis, both in vitro and in vivo. The carbon-13 MRS results indicated that the conversion of pyruvate into lactate in GLDC-shAON-treated tumor tissues was significantly reduced, when compared with the control groups. This observation corroborated with the reduced activity of lactate dehydrogenase and pyruvate dehydrogenase in GLDC-shAON-treated lung cancer cells and tumor tissues. Glycolysis stress test showed that extracellular acidification rate was significantly suppressed after GLDC-shAON treatment. Besides lung cancer, the antitumor effect of GLDC-shAON was also observed in brain, liver, cervical, and prostate cancer cell lines. Furthermore, it enhanced the treatment efficacy of cisplatin in lung cancer cells. Taken together, our findings illustrate that pyruvate metabolism decreases upon GLDC inhibition, thereby starving cancer cells from critical metabolic fuels.

Downregulating serine hydroxymethyltransferase 2 (SHMT2) suppresses tumorigenesis in human hepatocellular carcinoma.

Serine-glycine biosynthetic pathway diverts the glycolytic intermediate 3-phosphoglycerate to synthesize serine and glycine, of which the latter was found to correlate with cancer cell proliferation. Increased de novo biosynthesis of glycine by serine hydroxymethyltransferase 2 (SHMT2) is the central mechanism to fuel one-carbon pools supporting tumorigenesis. However, the therapeutic potential in targeting SHMT2 in hepatocellular carcinoma (HCC) is unknown. In this study we showed that SHMT2 inhibition significantly suppressed liver tumorigenesis. In vitro, SHMT2-knockdown was found to reduce cell growth and tumorigenicity in Huh-7 and HepG2 liver cancer cells. Moreover SHMT2-knockdown Huh-7 cells failed to form tumor xenograft after subcutaneous inoculation into nude mice. Similarly, inducible SHMT2 inhibition, via doxycycline-added drinking water, was found to reduce tumor incidence and tumor growth in a human tumor xenograft mouse model. SHMT2-knockdown increased the susceptibility of Huh-7 cells to doxorubicin suggesting its potential in combination chemotherapy. Through isotopomer tracing of [2-13C] glycine metabolism, we demonstrated that SHMT2 activity is associated with cancer phenotype. However, overexpression of SHMT2 was insufficient to transform immortalized hepatic cells to malignancy, suggesting that SHMT2 is one of the building blocks in liver cancer metabolism but does not initiate malignant transformation. Moreover, our results suggest that glycine, but not 5,10-methylenetetrahydrofolate, from the SHMT2-mediated enzymatic reaction is instrumental in tumorigenesis. Indeed, we found that SHMT2-knockdown cells exhibited increased glycine uptake. Taken together, our data suggest that SHMT2 may be a potential target in the treatment of human HCC.

The anti-cancer activities of Vernonia amygdalina extract in human breast cancer cell lines are mediated through caspase-dependent and p53-independent pathways.

Breast cancer is currently the leading cause of cancer-related deaths among women globally. Notably, medicinal plant extracts may be a potential source for treatments of breast cancer. Vernonia amygdalina (VA) is a woody shrub reported to have not only diverse therapeutic effects but also anti-cancer properties. However, current research about the mechanisms of the anti-cancer potential of VA has been limited. This study aimed to investigate the mechanisms of action of VA that underlie its anti-cancer effects in human breast cancer cell lines (MCF-7 and MDA-MB-231 cells). Results from MTT assay revealed that VA inhibits the proliferation of MCF-7 and MDA-MB-231, in a time- and dose-dependent manner. The underlying mechanism of this growth inhibition involved the stimulation of cell-type specific G1/S phase cell cycle arrest in only MCF-7 cells, and not in MDA-MB-231 cells. While the growth arrest was associated with increased levels of p53 and p21, and a concomitant decrease in the levels of cyclin D1 and cyclin E, it was shown that VA causes cell cycle arrest through a p53-independent pathway as tested by the wild type p53 inhibitor, pifithrin-α. Furthermore, this study revealed that VA induces apoptosis in the two cell lines, as indicated by the increase in Annexin V-positive cells and sub-G1 population, and that this VA-induced apoptosis occurred through both extrinsic and intrinsic apoptotic pathways. The apoptosis in MCF-7 cells was also likely to be caspase-dependent and not p53 transcriptional-dependent. Given that approximately 70% of diagnosed breast cancers express ER-α, a crucial finding was that VA inhibits the expression of ER-α and its downstream player, Akt, highlighting the potential clinical significance of VA. Moreover, VA exhibits synergism when combined with doxorubicin, suggesting that it can complement current chemotherapy. Overall, this study demonstrates the potential applications of VA as an anti-cancer drug for breast cancer treatment.

Thymoquinone inhibits tumor growth and induces apoptosis in a breast cancer xenograft mouse model: the role of p38 MAPK and ROS.

Due to narrow therapeutic window of cancer therapeutic agents and the development of resistance against these agents, there is a need to discover novel agents to treat breast cancer. The antitumor activities of thymoquinone (TQ), a compound isolated from Nigella sativa oil, were investigated in breast carcinoma in vitro and in vivo. Cell responses after TQ treatment were assessed by using different assays including MTT assay, annexin V-propidium iodide staining, Mitosox staining and Western blot. The antitumor effect was studied by breast tumor xenograft mouse model, and the tumor tissues were examined by histology and immunohistochemistry. The level of anti-oxidant enzymes/molecules in mouse liver tissues was measured by commercial kits. Here, we show that TQ induced p38 phosphorylation and ROS production in breast cancer cells. These inductions were found to be responsible for TQ's anti-proliferative and pro-apoptotic effects. Moreover, TQ-induced ROS production regulated p38 phosphorylation but not vice versa. TQ treatment was found to suppress the tumor growth and this effect was further enhanced by combination with doxorubicin. TQ also inhibited the protein expression of anti-apoptotic genes, such as XIAP, survivin, Bcl-xL and Bcl-2, in breast cancer cells and breast tumor xenograft. Reduced Ki67 and increased TUNEL staining were observed in TQ-treated tumors. TQ was also found to increase the level of catalase, superoxide dismutase and glutathione in mouse liver tissues. Overall, our results demonstrated that the anti-proliferative and pro-apoptotic effects of TQ in breast cancer are mediated through p38 phosphorylation via ROS generation.

Thymoquinone: potential cure for inflammatory disorders and cancer.

Thymoquinone is an active ingredient isolated from Nigella sativa and has been investigated for its anti-oxidant, anti-inflammatory and anticancer activities in both in vitro and in vivo models since its first extraction in 1960s. Its anti-oxidant/anti-inflammatory effect has been reported in various disease models, including encephalomyelitis, diabetes, asthma and carcinogenesis. Moreover, thymoquinone could act as a free radical and superoxide radical scavenger, as well as preserving the activity of various anti-oxidant enzymes such as catalase, glutathione peroxidase and glutathione-S-transferase. The anticancer effect(s) of thymoquinone are mediated through different modes of action, including anti-proliferation, apoptosis induction, cell cycle arrest, ROS generation and anti-metastasis/anti-angiogenesis. In addition, this quinone was found to exhibit anticancer activity through the modulation of multiple molecular targets, including p53, p73, PTEN, STAT3, PPAR-γ, activation of caspases and generation of ROS. The anti-tumor effects of thymoquinone have also been investigated in tumor xenograft mice models for colon, prostate, pancreatic and lung cancer. The combination of thymoquinone and conventional chemotherapeutic drugs could produce greater therapeutic effect as well as reduce the toxicity of the latter. In this review, we summarize the anti-oxidant/anti-inflammatory and anticancer effects of thymoquinone with a focus on its molecular targets, and its possible role in the treatment of inflammatory diseases and cancer.

Anticancer activity of thymoquinone in breast cancer cells: possible involvement of PPAR-γ pathway.

Thymoquinone (TQ), an active ingredient of Nigella sativa, has been reported to exhibit anti-oxidant, anti-inflammatory and anti-tumor activities through mechanism(s) that is not fully understood. In this study, we report the anticancer effects of TQ on breast cancer cells, and its potential effect on the PPAR-γ activation pathway. We found that TQ exerted strong anti-proliferative effect in breast cancer cells and, when combined with doxorubicin and 5-fluorouracil, increased cytotoxicity. TQ was found to increase sub-G1 accumulation and annexin-V positive staining, indicating apoptotic induction. In addition, TQ activated caspases 8, 9 and 7 in a dose-dependent manner. Migration and invasive properties of MDA-MB-231 cells were also reduced in the presence of TQ. Interestingly, we report for the first time that TQ was able to increase PPAR-γ activity and down-regulate the expression of the genes for Bcl-2, Bcl-xL and survivin in breast cancer cells. More importantly, the increase in PPAR-γ activity was prevented in the presence of PPAR-γ specific inhibitor and PPAR-γ dominant negative plasmid, suggesting that TQ may act as a ligand of PPAR-γ. Also, we observed using molecular docking analysis that TQ indeed formed interactions with 7 polar residues and 6 non-polar residues within the ligand-binding pocket of PPAR-γ that are reported to be critical for its activity. Taken together, our novel observations suggest that TQ may have potential implication in breast cancer prevention and treatment, and show for the first time that the anti-tumor effect of TQ may also be mediated through modulation of the PPAR-γ activation pathway.