Cell Death Mechanisms of the Promising Anticancer Compound Gallotannin

The polyphenolic hydrolyzable tannin, gallotannin (GT), also known as tannic acid, possesses interesting anticarcinogenic properties. An evidence from experimental studies suggests that GT is effective against multiple cancer types. Gallotannin has been shown to induce programmed cell death in a wide variety of cancers including colon, breast, prostate, and liver, among others. Apoptosis, cellular senescence, autophagy, and necrosis are the main mechanisms by which GT can suppress cancer progression. In addition, GT is a potent inhibitor of many proliferation pathways. Herein, this chapter provides a summary of our current knowledge about GT’s programmed cell death mechanisms against cancer

Radiosensitization by 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide under oxia and hypoxia in human colon cancer cells

BACKGROUND: The sensitizing effects of 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide (DCQ) and ionizing radiation (IR) were determined in four colon cancer cells and in FHs74Int normal intestinal cells. METHODS: Cell cycle modulation, TUNEL assay, clonogenic survival and DNA damage were examined under oxia or hypoxia. Effects on apoptotic molecules and on p-Akt and Cox-2 protein expression were investigated. RESULTS: The four cell lines responded differently to DCQ+IR; HT-29 cells were most resistant. Combination treatment caused significant increases in preG(1 )(apoptosis) in HCT-116, while G(2)/M arrest occurred in DLD-1. DCQ potentiated IR effects more so under hypoxia than oxia. Pre-exposure of DLD-1 to hypoxia induced 30% apoptosis, and G(2)/M arrest in oxia. The survival rate was 50% lower in DCQ+IR than DCQ alone and this rate further decreased under hypoxia. FHs74Int normal intestinal cells were more resistant to DCQ+IR than cancer cells.Greater ssDNA damage occurred in DLD-1 exposed to DCQ+IR under hypoxia than oxia. In oxia, p-Akt protein expression increased upon IR exposure and drug pre-treatment inhibited this increase. In contrast, in hypoxia, exposure to IR reduced p-Akt protein and DCQ restored its expression to the untreated control. Apoptosis induced in hypoxic DLD-1 cells was independent of p53-p21 modulation but was associated with an increase in Bax/Bcl-2 ratio and the inhibition of the Cox-2 protein. CONCLUSION: DCQ is a hypoxic cell radiosensitizer in DLD-1 human colon cancer cells

Epigenetic Regulation of p21cip1/waf1 in Human Cancer

p21cip1/waf1 is a central regulator of cell cycle control and survival. While mutations are rare, it is commonly dysregulated in several human cancers due to epigenetic mechanisms influencing its transcriptional control. These mechanisms include promoter hypermethylation as well as additional pathways such as histone acetylation or methylation. The epigenetic regulators include writers, such as DNA methyltransferases (DNMTs); histone acetyltransferases (HATs) and histone lysine methyltransferases; erasers, such as histone deacetylases (HDACs); histone lysine demethylases [e.g., the Lysine Demethylase (KDM) family]; DNA hydroxylases; readers, such as the methyl-CpG-binding proteins (MBPs); and bromodomain-containing proteins, including the bromo- and extraterminal domain (BET) family. We further discuss the roles that long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play in the epigenetic control of p21cip1/waf1 expression and its function in human cancers

The quinoxaline di-N-oxide DCQ blocks breast cancer metastasis in vitro and in vivo by targeting the hypoxia inducible factor-1 pathway

BACKGROUND: Although tumor hypoxia poses challenges against conventional cancer treatments, it provides a therapeutic target for hypoxia-activated drugs. Here, we studied the effect of the hypoxia-activated synthetic quinoxaline di-N-oxide DCQ against breast cancer metastasis and identified the underlying mechanisms. METHODS: The human breast cancer cell lines MCF-7 (p53 wildtype) and MDA-MB-231 (p53 mutant) were treated with DCQ under normoxia or hypoxia. Drug toxicity on non-cancerous MCF-10A breast cells was also determined. In vitro cellular responses were investigated by flow cytometry, transfection, western blotting, ELISA and migration assays. The anti-metastatic effect of DCQ was validated in the MDA-MB-231 xenograft mouse model. RESULTS: DCQ selectively induced apoptosis in both human breast cancer cells preferentially under hypoxia without affecting the viability of non-cancerous MCF-10A. Cancer cell death was associated with an increase in reactive oxygen species (ROS) independently of p53 and was inhibited by antioxidants. DCQ-induced ROS was associated with DNA damage, the downregulation of hypoxia inducible factor-1 alpha (HIF-1α), and inhibition of vascular endothelial growth factor (VEGF) secretion. In MCF-7, HIF-1α inhibition was partially via p53-activation and was accompanied by a decrease in p-mTOR protein, suggesting interference with HIF-1α translation. In MDA-MB-231, DCQ reduced HIF-1α through proteasomal-dependent degradation mechanisms. HIF-1α inhibition by DCQ blocked VEGF secretion and invasion in MCF-7 and led to the inhibition of TWIST in MDA-MB-231. Consistently, DCQ exhibited robust antitumor activity in MDA-MB-231 breast cancer mouse xenografts, enhanced animal survival, and reduced metastatic dissemination to lungs and liver. CONCLUSION: DCQ is the first hypoxia-activated drug showing anti-metastatic effects against breast cancer, suggesting its potential use for breast cancer therapy

antiproliferative activities of artemisia herba alba ethanolic extract in human colon cancer cell line hct116

Artemisia herba-alba (AHE) is a plant commonly used in traditional medicine for the treatment of various ailments. Here, we investigated the antioxidant and antitumor activity of the aqueous and ethanol extracts of AHE in human colon cancer HCT116 cells. The antioxidant activity was measured by DCFH assay, while antitumor effects were assessed by cell viability assays, cell cycle progression by flow cytometry, and DNA fragmentation analysis in addition to investigating the expression of key cell cycle and apoptotic proteins. While the aqueous extract had no antineoplastic effects, the ethanol extract significantly decreased HCT116 viability (IC50 of 51mg/mL at 24 h) and inhibited the production of reactive oxygen species (ROS). Treatment of HCT116 cells with the ethanol extract also caused dramatic increase in the PreG1 population with concomitant decrease in cycling cells, provoked DNA fragmentation, significant increase in the expression levels of p53 and Bax proteins and activated pro-apoptotic caspase-3. The results obtained suggest that the ethanol extract of AHE could be used as an easily accessible source of natural antioxidants and as potential phytochemicals against colon cancer.</p

Copper chelation selectively kills colon cancer cells through redox cycling and generation of reactive oxygen species

Background: Metals including iron, copper and zinc are essential for physiological processes yet can be toxic at high concentrations. However the role of these metals in the progression of cancer is not well defined. Here we study the anti-tumor activity of the metal chelator, TPEN, and define its mechanism of action.Methods: Multiple approaches were employed, including cell viability, cell cycle analysis, multiple measurements of apoptosis, and mitochondrial function. In addition we measured cellular metal contents and employed EPR to record redox cycling of TPEN-metal complexes. Mouse xenografts were also performed to test the efficacy of TPEN in vivo.Results: We show that metal chelation using TPEN (5μM) selectively induces cell death in HCT116 colon cancer cells without affecting the viability of non-cancerous colon or intestinal cells. Cell death was associated with increased levels of reactive oxygen species (ROS) and was inhibited by antioxidants and by prior chelation of copper. Interestingly, HCT116 cells accumulate copper to 7-folds higher levels than normal colon cells, and the TPEN-copper complex engages in redox cycling to generate hydroxyl radicals. Consistently, TPEN exhibits robust anti-tumor activity in vivo in colon cancer mouse xenografts.Conclusion: Our data show that TPEN induces cell death by chelating copper to produce TPEN-copper complexes that engage in redox cycling to selectively eliminate colon cancer cells. © 2014 Fatfat et al.; licensee BioMed Central Ltd

Thymoquinone-induced conformational changes of PAK1 interrupt prosurvival MEK-ERK signaling in colorectal cancer

Background Thymoquinone (TQ) was shown to reduce tumor growth in several cancer models both in vitro and in vivo. So far only a few targets of TQ, including protein kinases have been identified. Considering that kinases are promising candidates for targeted anticancer therapy, we studied the complex kinase network regulated by TQ. Methods Novel kinase targets influenced by TQ were revealed by in silico analysis of peptide array data obtained from TQ-treated HCT116wt cells. Western blotting and kinase activity assays were used to determine changes in kinase expression patterns in colorectal cancer cells (HCT116wt, DLD-1, HT29). To study the viability/apoptotic effects of combining the PAK1 inhibitor IPA-3 and TQ, crystal violet assay and AnnexinV/PI staining were employed. Interactions between PAK1 and ERK1/2 were investigated by co-immunoprecipitation and modeled by docking studies. Transfection with different PAK1 mutants unraveled the role of TQ-induced changes in PAK1 phosphorylation and TQ´s effects on PAK1 scaffold function. Results Of the 104 proteins identified, 50 were upregulated ≥2 fold by TQ and included molecules in the AKT-MEK-ERK1/2 pathway. Oncogenic PAK1 emerged as an interesting TQ target. Time-dependent changes in two PAK1 phosphorylation sites generated a specific kinase profile with early increase in pPAKThr212 followed by late increase in pPAKThr423. TQ induced an increase of pERK1/2 and triggered the early formation of an ERK1/2-PAK1 complex. Modeling confirmed that TQ binds in the vicinity of Thr212 accompanied by conformational changes in ERK2-PAK1 binding. Transfecting the cells with the non-phosphorylatable mutant T212A revealed an increase of pPAKThr423 and enhanced apoptosis. Likewise, an increase in apoptosis was observed in cells transfected with both the kinase-dead K299R mutant and PAK1 siRNA. Using structural modeling we suggest that TQ interferes also with the kinase domain consequently disturbing its interaction with pPAKThr423, finally inhibiting MEK-ERK1/2 signaling and disrupting its prosurvival function. pERK1/2 loss was also validated in vivo. Conclusions Our study shows for the first time that the small molecule TQ directly binds to PAK1 changing its conformation and scaffold function. Because TQ affects the central RAF/MEK/ERK1/2 pathway, the combination of TQ with targeted therapies is worth considering for future anticancer treatments

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 antioxidant 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 antiproliferative and pro-apoptotic effects of TQ in breast cancer are mediated through p38 phosphorylation via ROS generation