Bassant M. Barakat

Thymoquinone up-regulates PTEN expression and induces apoptosis in doxorubicin-resistant human breast cancer cells.

The use of innocuous naturally occurring compounds to overcome drug resistance and cancer recalcitrance is now in the forefront of cancer research. Thymoquinone (TQ) is a bioactive constituent of the volatile oil derived from seeds of Nigella sativa Linn. TQ has shown promising anti-carcinogenic and anti-tumor activities through different mechanisms. However, the effect of TQ on cell signaling and survival pathways in resistant cancer cells has not been fully delineated. Here, we report that TQ greatly inhibits doxorubicin-resistant human breast cancer MCF-7/DOX cell proliferation. TQ treatment increased cellular levels of PTEN proteins, resulting in a substantial decrease of phosphorylated Akt, a known regulator of cell survival. The PTEN expression was accompanied with elevation of PTEN mRNA. TQ arrested MCF-7/DOX cells at G2/M phase and increased cellular levels of p53 and p21 proteins. Flow cytometric analysis and agarose gel electrophoresis revealed a significant increase in Sub-G1 cell population and appearance of DNA ladders following TQ treatment, indicating cellular apoptosis. TQ-induced apoptosis was associated with disrupted mitochondrial membrane potential and activation of caspases and PARP cleavage in MCF-7/DOX cells. Moreover, TQ treatment increased Bax/Bcl2 ratio via up-regulating Bax and down-regulating Bcl2 proteins. More importantly, PTEN silencing by target specific siRNA enabled the suppression of TQ-induced apoptosis resulting in increased cell survival. Our results reveal that up-regulation of the key upstream signaling factor, PTEN, in MCF-7/DOX cells inhibited Akt phosphorylation, which ultimately causes increase in their regulatory p53 levels affecting the induction of G2/M cell cycle arrest and apoptosis. Overall results provide mechanistic insights for understanding the molecular basis and utility of the anti-tumor activity of TQ.

Tangeretin sensitizes cisplatin-resistant human ovarian cancer cells through downregulation of phosphoinositide 3-kinase/akt signaling pathway.

Combination of innocuous dietary components with anticancer drugs is an emerging new strategy for cancer chemotherapy to increase antitumor responses. Tangeretin is a citrus flavonoid known to inhibit cancer cell proliferation. Here, we show an enhanced response of A2780/CP70 and 2008/C13 cisplatin-resistant human ovarian cancer cells to various combination treatments of cisplatin and tangeretin. Pretreatment of cells with tangeretin before cisplatin treatment synergistically inhibited cancer cell proliferation. This combination was effective in activating apoptosis via caspase cascade as well as arresting cell cycle at G(2)-M phase. Moreover, phospho-Akt and its downstream substrates, e.g., NF-kappaB, phospho-GSK-3beta, and phospho-BAD, were downregulated upon tangeretin-cisplatin treatment. The tangeretin-cisplatin-induced apoptosis in A2780/CP70 cells was increased by phosphoinositide-3 kinase (PI3K) inhibition and siRNA-mediated Akt silencing, but reduced by overexpression of constitutively activated Akt and GSK-3beta inhibition. The overall results indicated that tangeretin exposure preconditions cisplatin-resistant human ovarian cancer cells for a conventional response to low-dose cisplatin-induced cell death occurring through downregulation of PI3K/Akt signaling pathway. Thus, effectiveness of tangeretin combinations, as a promising modality in the treatment of resistant cancers, warrants systematic clinical studies.

Naringenin Protects HaCaT Human Keratinocytes Against UVB-induced Apoptosis and Enhances the Removal of Cyclobutane Pyrimidine Dimers from the Genome†

Many naturally occurring agents are believed to protect against UV‐induced skin damage. In this study, we have investigated the effects of naringenin (NG), a naturally occurring citrus flavonone, on the removal of UVB‐induced cyclobutane pyrimidine dimers (CPD) from the genome and apoptosis in immortalized p53‐mutant human keratinocyte HaCaT cells. The colony‐forming assay shows that treatment with NG significantly increases long‐term cell survival after UVB irradiation. NG treatment also protects the cells from UVB‐induced apoptosis, as indicated by the absence of the 180 base pair DNA ladders and the appearance of sub‐G1 peak using agarose gel electrophoresis and flow cytometric analysis, respectively. The UVB‐induced poly (ADP‐ribose) polymerase‐1 (PARP‐1) cleavage, caspase activation and Bax/Bcl2 ratio were modulated following NG treatment, indicating an antiapoptotic effect of NG in UVB‐damaged cells that occurs at least in part via caspase cascade pathway. Moreover, treatment of UVB‐irradiated HaCaT cells with NG enhances the removal of CPD from the genome, as observed by both direct quantitation of CPD in genomic DNA and immuno‐localization of the damage within the nuclei. The study provides a molecular basis for the action of NG as a promising natural flavonoid in preventing skin aging and carcinogenesis.

The p38 Mitogen-activated Protein Kinase Augments Nucleotide Excision Repair by Mediating DDB2 Degradation and Chromatin Relaxation

The p38 MAPK is a family of serine/threonine protein kinases that play important roles in cellular responses to external stress signals, e.g. UV irradiation. To assess the role of p38 MAPK pathway in nucleotide excision repair (NER), the most versatile DNA repair pathway, we determined the efficiency of NER in cells treated with p38 MAPK inhibitor SB203580 and found that p38 MAPK is required for the prompt repair of UV-induced DNA damage CPD. We further investigated the possible mechanism through which p38 MAPK regulates NER and found that p38 MAPK mediates UV-induced histone H3 acetylation and chromatin relaxation. Moreover, p38 MAPK also regulates UV-induced DDB2 ubiquitylation and degradation via phosphorylation of the target protein. Finally, our results showed that p38 MAPK is required for the recruitment of NER factors XPC and TFIIH to UV-induced DNA damage sites. We conclude that p38 MAPK regulates chromatin remodeling as well as DDB2 degradation for facilitating NER factor assembly.

Overexpression of DDB2 enhances the sensitivity of human ovarian cancer cells to cisplatin by augmenting cellular apoptosis.

Cisplatin is one of the most widely used anticancer agents, displaying activity against a wide variety of tumors. However, development of drug resistance presents a challenging barrier to successful cancer treatment by cisplatin. To understand the mechanism of cisplatin resistance, we investigated the role of damaged DNA binding protein complex subunit 2 (DDB2) in cisplatin‐induced cytotoxicity and apoptosis. We show that DDB2 is not required for the repair of cisplatin‐induced DNA damage, but can be induced by cisplatin treatment. DDB2‐deficient noncancer cells exhibit enhanced resistance to cell growth inhibition and apoptosis induced by cisplatin than cells with fully restored DDB2 function. Moreover, DDB2 expression in cisplatin‐resistant ovarian cancer cell line CP70 and MCP2 was lower than their cisplatin‐sensitive parental A2780 cells. Overexpression of DDB2 sensitized CP70 cells to cisplatin‐induced cytotoxicity and apoptosis via activation of the caspase pathway and downregulation of antiapoptotic Bcl‐2 protein. Further analysis indicates that the overexpression of DDB2 in CP70 cells downregulates Bcl‐2 expression through decreasing Bcl‐2 mRNA level. These results suggest that ovarian cancer cells containing high level of DDB2 become susceptible to cisplatin by undergoing enhanced apoptosis.

Protective Effect of Boswellic Acids against Doxorubicin-Induced Hepatotoxicity: Impact on Nrf2/HO-1 Defense Pathway

The current study aimed to investigate the potential protective role of boswellic acids (BAs) against doxorubicin- (DOX-) induced hepatotoxicity. Also, the possible mechanisms underlying this protection; antioxidant, as well as the modulatory effect on the Nrf2 transcription factor/hem oxygenase-1 (Nrf2/HO-1) pathway in liver tissues, was investigated. Animals were allocated to five groups: group 1: the saline control, group 2: the DOX group, animals received DOX (6 mg/kg, i.p.) weekly for a period of three weeks, and groups 3–5: animals received DOX (6 mg/kg, i.p.) weekly and received protective doses of BAs (125, 250, and 500 mg/kg/day). Treatment with BAs significantly improved the altered liver enzyme activities and oxidative stress markers. This was coupled with significant improvement in liver histopathological features. BAs increased the Nrf2 and HO-1 expression, which provided protection against DOX-induced oxidative insult. The present results demonstrated that BAs appear to scavenge ROS and inhibit lipid peroxidation and DNA damage of DOX-induced hepatotoxicity. The antioxidant efficacy of BAs might arise from its modulation of the Nrf2/HO-1 pathway and thereby protected liver from DOX-induced oxidative injury.

An optimized dose of raspberry ketones controls hyperlipidemia and insulin resistance in male obese rats: Effect on adipose tissue expression of adipocytokines and Aquaporin 7

ABSTRACT Obesity constitutes a major worldwide problem in which hyperlipidemia and insulin resistance represents adverse metabolic consequences of it. The present study was conducted to elucidate the role of raspberry ketones (RKs) in controlling body weight gain, hyperlipidemia and insulin resistance in male obese rats through affecting the expression of various adipocytokines. As Aquaporin‐7 is co‐related with the expression of various adipocytokines and has recently emerged as a modulator of adipocyte metabolism, the present study evaluated the effect of RKs on adipose tissue expression of aquaporin‐7(AQP7) in high‐fat (HF) diet‐fed rats. Groups of male rats were assigned to normal, HF diet‐fed control rats and RKs‐treated (250 and 500 mg/kg) groups. RKs administration effectively abrogated hyperlipidemia and oxidative burden and enhanced insulin sensitivity. In addition, treatment with RKs ameliorated adipose tissue and liver indices and the reduced adipocyte diameters. Moreover, administration of the low dose of RKs ameliorated the expression of apelin and its receptor, and visfatin with upregulating adiponectin expression compared to HF diet control rats. However, both doses effectively downregulated leptin expression. It was obvious that both RKs doses revealed effectiveness in upregulating the AQP7 expression. The present data suggest the promising therapeutic role of RKs in HF diet‐induced obesity that is likely attributable, at least in part, to upregulation of AQP7 expression.