El-Shaimaa A. Arafa

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.

Diosmin Protects against Ethanol-Induced Gastric Injury in Rats: Novel Anti-Ulcer Actions

Alcohol consumption has been commonly associated with gastric mucosal lesions including gastric ulcer. Diosmin (DIO) is a natural citrus flavone with remarkable antioxidant and anti-inflammatory features that underlay its protection against cardiac, hepatic and renal injuries. However, its impact on gastric ulcer has not yet been elucidated. Thus, the current study aimed to investigate the potential protective effects of DIO against ethanol-induced gastric injury in rats. Pretreatment with DIO (100 mg/kg p.o.) attenuated the severity of ethanol gastric mucosal damage as evidenced by lowering of ulcer index (UI) scores, area of gastric lesions, histopathologic aberrations and leukocyte invasion. These actions were analogous to those exerted by the reference antiulcer sucralfate. DIO suppressed gastric inflammation by curbing of myeloperoxidase (MPO) and tumor necrosis factor-α (TNF-α) levels along with nuclear factor kappa B (NF-κB) p65 expression. It also augmented the anti-inflammatory interleukin-10 (IL-10) levels. Meanwhile, DIO halted gastric oxidative stress via inhibition of lipid peroxides with concomitant enhancement of glutathione (GSH), glutathione peroxidase (GPx) and the total antioxidant capacity (TAC). With respect to gastric mucosal apoptosis, DIO suppressed caspase-3 activity and cytochrome C (Cyt C) with enhancement of the anti-apoptotic B cell lymphoma-2 (Bcl-2) in favor of cell survival. These favorable actions were associated with upregulation of the gastric cytoprotective prostaglandin E2 (PGE2) and nitric oxide (NO). Together, these findings accentuate the gastroprotective actions of DIO in ethanol gastric injury which were mediated via concerted multi-pronged actions, including suppression of gastric inflammation, oxidative stress and apoptosis besides boosting of the antioxidant and the cytoprotective defenses.

Camel’s milk ameliorates TNBS-induced colitis in rats via downregulation of inflammatory cytokines and oxidative stress

Current treatment strategies for inflammatory bowel diseases (IBD) are associated with several adverse effects, and thus, the search for effective agents with minimal side effects merits attention. Camels milk (CM) is endowed with antioxidant/anti-inflammatory features and has been reported to protect against diabetes and hepatic injury, however, its effects on IBD have not been previously explored. In the current study, we aimed to investigate the potential alleviating effects of CM against TNBS-induced colitis in rats. CM (10 ml/kg b.i.d. by oral gavage) effectively suppressed the severity of colon injury as evidenced by amelioration of macroscopic damage, colon weight/length ratio, histopathological alterations, leukocyte influx and myeloperoxidase activity. Administration of CM mitigated the colonic levels of TNF-α and IL-10 cytokines. The attenuation of CM to colon injury was also associated with suppression of oxidative stress via reduction of lipid peroxides and nitric oxide along with boosting the antioxidant defenses through restoration of colon glutathione and total anti-oxidant capacity. In addition, caspases-3 activity, an apoptotic marker, was inhibited. Together, our study highlights evidences for the promising alleviating effects of CM in colitis. Thus, CM may be an interesting complementary approach for the management of IBD.

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.

OSU-A9 inhibits angiogenesis in human umbilical vein endothelial cells via disrupting Akt–NF-κB and MAPK signaling pathways

Since the introduction of angiogenesis as a useful target for cancer therapy, few agents have been approved for clinical use due to the rapid development of resistance. This problem can be minimized by simultaneous targeting of multiple angiogenesis signaling pathways, a potential strategy in cancer management known as polypharmacology. The current study aimed at exploring the anti-angiogenic activity of OSU-A9, an indole-3-carbinol-derived pleotropic agent that targets mainly Akt-nuclear factor-kappa B (NF-κB) signaling which regulates many key players of angiogenesis such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Human umbilical vein endothelial cells (HUVECs) were used to study the in vitro anti-angiogenic effect of OSU-A9 on several key steps of angiogenesis. Results showed that OSU-A9 effectively inhibited cell proliferation and induced apoptosis and cell cycle arrest in HUVECs. Besides, OSU-A9 inhibited angiogenesis as evidenced by abrogation of migration/invasion and Matrigel tube formation in HUVECs and attenuation of the in vivo neovascularization in the chicken chorioallantoic membrane assay. Mechanistically, Western blot, RT-PCR and ELISA analyses showed the ability of OSU-A9 to inhibit MMP-2 production and VEGF expression induced by hypoxia or phorbol-12-myristyl-13-acetate. Furthermore, dual inhibition of Akt-NF-κB and mitogen-activated protein kinase (MAPK) signaling, the key regulators of angiogenesis, was observed. Together, the current study highlights evidences for the promising anti-angiogenic activity of OSU-A9, at least in part through the inhibition of Akt-NF-κB and MAPK signaling and their consequent inhibition of VEGF and MMP-2. These findings support OSU-A9s clinical promise as a component of anticancer therapy.

Sensitization of Hepatocellular Carcinoma Cells to Apo2L/TRAIL by a Novel Akt/NF-κB Signalling Inhibitor

Hepatocellular carcinoma (HCC) cells are intrinsically resistant to tumour necrosis factor‐related apoptosis ligand (Apo2L/TRAIL), in part, due to the compensatory activation of nuclear factor‐kappaB (NF‐κB). To broaden the clinical utilization of Apo2L/TRAIL in HCC, OSU‐A9, a potent indole‐3‐carbinol‐derived Akt/NF‐κB signalling inhibitor was used to overcome the intrinsic resistance. The antitumour effects of OSU‐A9, Apo2L/TRAIL and the therapeutic combination were assessed by MTT assay, caspase activation and PARP cleavage, and the synergistic interactions were determined by Calcusyn analysis. NF‐κB reporter gene and RT‐PCR were tested for the activation of NF‐κB and the expression of death receptors (DR)4 and 5. OSU‐A9 could sensitize HCC cells to Apo2L/TRAIL with high potency through down‐regulation of Akt/NF‐κB signalling. OSU‐A9 dose‐dependently reduced Akt phosphorylation and the expression and nuclear localization of RelA/p65, accompanied by parallel decreases in the expression of NF‐κB target products, including Bcl‐xL, Mcl‐1, cIAP1, cIAP2 and survivin. Moreover, OSU‐A9 increased DR5 expression through a reactive oxygen species (ROS)‐dependent mechanism. Concertedly, these mechanisms underlie the synergistic interaction between OSU‐A9 and Apo2L/TRAIL in mediating apoptotic death in HCC cells. The ability of OSU‐A9 to accentuate Apo2L/TRAIL‐induced apoptosis by inactivating Akt/NF‐κB signalling might foster a promising therapeutic strategy for HCC.

Tangeretin Alleviates Cisplatin-Induced Acute Hepatic Injury in Rats: Targeting MAPKs and Apoptosis

Despite its broad applications, cisplatin affords considerable nephro- and hepatotoxicity through triggering inflammatory and oxidative stress cascades. The aim of the current investigation was to study the possible protective effects of tangeretin on cisplatin-induced hepatotoxicity. The impact of tangeretin on cisplatin-evoked hepatic dysfunction and histopathologic changes along with oxidative stress, inflammatory and apoptotic biomarkers were investigated compared to silymarin. Tangeretin pre-treatment significantly improved liver function tests (ALT and AST), inhibited cisplatin-induced lipid profile aberrations (total cholesterol and triglycerides) and diminished histopathologic structural damage in liver tissues. Tangeretin also attenuated cisplatin-induced hepatic inflammatory events as indicated by suppression of tumor necrosis factor-α (TNF-α) and enhancement of interleukin-10 (IL-10). Meanwhile, it lowered malondialdehyde (MDA), nitric oxide (NO) and nuclear factor erythroid 2-related factor 2 (NRF-2) levels with restoration of glutathione (GSH), and glutathione peroxidase (GPx). Regarding mitogen-activated protein kinase (MAPK) pathway, tangeretin attenuated cisplatin-induced increase in phospho-p38, phospho-c-Jun N-terminal kinase (p-JNK) and phospho-extracellular signal-regulated kinase (p-ERK1/2) in liver tissues. In addition, tangeretin downregulated Bax expression with augmentation of Bcl-2 promoting liver cell survival. Our results highlight the protective effects of tangeretin against cisplatin-induced acute hepatic injury via the concerted modulation of inflammation, oxidative stress, MAPKs and apoptotic pathways.