Nabendu Murmu

CUGBP2 plays a critical role in apoptosis of breast cancer cells in response to genotoxic injury.

Abstract: Posttranscriptional control of gene expression plays a key role in regulating gene expression in cells undergoing apoptosis. Cyclooxygenase‐2 (COX‐2) is a crucial enzyme in the conversion of arachidonic acid to prostaglandin E2 (PGE2) and is significantly upregulated in many types of adenocarcinomas. COX‐2 overexpression leads to increased PGE2 production, resulting in increased cellular proliferation. PGE2 enhances the resistance of cells to ionizing radiation. Accordingly, understanding mechanisms regulating COX‐2 expression may lead to important therapeutic advances. Besides transcriptional control, COX‐2 expression is significantly regulated by mRNA stability and translation. We have previously demonstrated that RNA binding protein CUGBP2 binds AU‐rich sequences to regulate COX‐2 mRNA translation. In the current study, we have determined that expression of both COX‐2 mRNA and CUGBP2 mRNA are induced in MCF‐7 cells, a breast cancer cell line, following exposure to 12 Gy γ‐irradiation. However, only CUGBP2 protein is induced, but COX‐2 protein levels were not altered. Silencer RNA (siRNA)‐mediated inhibition of CUGBP2 reversed the block in COX‐2 protein expression. Furthermore, MCF‐7 cells underwent apoptosis in response to radiation injury, which was also reversed by CUGBP2 siRNAs. These data suggest that CUGBP2 is a critical regulator of the apoptotic response to genotoxic injury in breast cancer cells.

Activation of Apoptosis by 1-Hydroxy-5,7-Dimethoxy-2-Naphthalene-Carboxaldehyde, a Novel Compound from Aegle marmelos

We have identified a natural compound that activates apoptosis of epithelial cancer cells through activation of tumor necrosis factor-alpha (TNF-alpha), TNF receptor (TNFR)-associated death domain (TRADD), and caspases. The molecule 1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxaldehyde (HDNC, marmelin) was isolated and characterized from ethyl acetate fraction of extracts of Aegle marmelos. HDNC treatment inhibited the growth of HCT-116 colon cancer tumor xenografts in vivo. Immunostaining for CD31 showed that there was a significant reduction in microvessels in the HDNC-treated animals, coupled with decreased cyclooxygenase-2, interleukin-8, and vascular endothelial growth factor mRNA. Using hexoseaminidase assay, we determined that HDNC inhibits proliferation of HCT-116 colon and HEp-2 alveolar epithelial carcinoma cells. Furthermore, the cancer cells showed increased levels of activated caspase-3 and induced G(1) cell cycle arrest, which was suppressed by caspase-3 inhibitors. HDNC induced TNF-alpha, TNFR1, and TRADD mRNA and protein expression. Moreover, caspase-8 and Bid activation, and cytochrome c release, were observed, suggesting the existence of a cross-talk between death receptor and the mitochondrial pathways. HDNC inhibited AKT and extracellular signal-regulated kinase phosphorylation both in cells in culture and in tumor xenografts. In addition, electrophoretic mobility shift assay and luciferase reporter assays showed that HDNC significantly suppressed TNF-alpha-mediated activation and translocation of nuclear factor-kappaB (NF-kappaB). This was further confirmed by Western blot analysis of nuclear extracts wherein levels of RelA, the p65 component of NF-kappaB, were significantly less in cells treated with HDNC. Together, the data suggest that the novel compound HDNC (marmelin) is a potent anticancer agent that induces apoptosis during G(1) phase of the cell cycle and could be a potential chemotherapeutic candidate.

Expression of metallothionein-1 (MT-1) mRNA in the rat testes and liver after cadmium injection.

Metallothioneins (MTs) belong to the family of stress proteins that are present in the majority of living organisms. The MTs play an important task in detoxifying heavy metals. The mammalian scrotal testis is known to be susceptible to cadmium (Cd) exposure. The present work focuses on the MT-1 isoform and aims to ascertain and confirm previous findings to answer whether rodent testes indeed contain MT-1 mRNA, whether its level is increased with Cd injection in liver and testes, and lastly what is the relative difference in the expression of MT-1 mRNA in liver and testes both with and without Cd injection. Adult male Wistar rats weighing 270–290 g received a subcutaneous injection of 4.0 μmol Cd/kg and were sacrificed by cervical dislocation 6 h later. RNA was isolated from testes as well as the liver. There were 2 replicates per treatment for RNA analyses. MT-1 mRNA levels were determined by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis and then assessed by densitometry scanning. The results of RT-PCR clearly demonstrated that the rodent testes express MT-1 mRNA. The densitometry data shows that the expression of MT-1 mRNA increases with Cd treatment in testes. The relative level of MT1-mRNA is greater in the control-liver than in the control-testes. However, upon Cd injection, the level of testes MT-1 mRNA increases 2.16 fold. These results suggest that the testes respond to Cd for at least 6 h post injection through a transcriptional mechanism.

CDKN2A-p53 mediated antitumor effect of Lupeol in head and neck cancer

PurposeThe tumor suppressor protein p53 is known to control cell cycle arrest and apoptosis. Lupeol is a phytochemical that has been found to induce apoptosis in different cancer types through the extrinsic pathway. As yet, however, its role in the induction of cell cycle arrest and apoptosis through the intrinsic pathway in head and neck cancer has not been investigated. Here, we aimed at understanding the mechanism underlying the antitumor effect of Lupeol in head and neck cancer.MethodsThe antitumor effect of Lupeol on oral and laryngeal carcinomas was assessed using two in vitro 2D cell line models (HEp-2, UPCI:SCC-131) and, subsequently, an ex vivo 3D tumor explant culture platform that maintains key features of the native tumor microenvironment. The mechanism underlying Lupeol-mediated antitumor responses was delineated using MTT, colony formation, flow cytometry, immunofluorescence, Western blotting and immunohistochemistry assays.ResultsWe found that Lupeol induced an enhanced expression of p53 in both cell line models tested and, subsequently, cell cycle arrest at the G1 phase. In addition we found that, following Lupeol treatment, p53 induced Bax expression and activated the intrinsic apoptotic pathway (as measured by Caspase-3 cleavage). Interestingly, Lupeol was also found to trigger G1 cell cycle arrest through up-regulation of the expression of CDKN2A, but not p21, resulting in inhibition of CyclinD1. In an ex vivo platform Lupeol was found to impart a potent antitumor response as defined by inhibition of Ki67 expression, decreased cell viability and concomitant activation (cleavage) of Caspase-3. Finally, we found that Lupeol can re-sensitize primary head and neck squamous cell carcinoma (HNSCC) tumor samples that had clinically progressed under a Cisplatin treatment regimen.ConclusionTogether, our data indicate that Lupeol may orchestrate a bifurcated regulation of neoplastic growth and apoptosis in head and neck cancers and may serve as a promising agent for the management of tumors that have progressed on a platinum-based treatment regimen.

Resveratrol Alleviates Cadmium-Induced Damage and Overexpression of Epidermal Growth Factor Receptor and its Downstream Signaling Proteins in the Reproductive System of Male Swiss Albino Mice

Nowadays, exposure to heavy metals and their detrimental effects in humans are grave health concerns. In this study, we investigated the protective effect of resveratrol (RES) against CdCl2 (cadmium chloride)-induced impairment of spermatogenesis, histopathological alterations, and the up-regulation of epidermal growth factor receptor (EGFR) signaling cascade in Swiss albino mice. Two different doses of CdCl2 were injected intraperitoneally into two groups of mice, and in the third group RES was administered orally before injecting CdCl2 (3 times/wk) for 14 days. Sperm motility, count, vitality, and morphology were analyzed. Hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and western blot analyses were performed on testis tissue. In CdCl2-administered animals, significant perturbations of spermatogenesis and histoarchitecture of seminiferous tubules were observed. p-EGFR, p-AKT, AKT1/2/3, NF-κβ (p50), and COX-2 of the EGFR cascade were up-regulated. Although there was significant negative correlation between percentage of motile cells and protein expression, we found positive correlation between morphologically abnormal cells and overexpression of proteins in CdCl2-only treated groups. Marked improvement of sperm parameters and histopathological damages as well as down-regulation of the EGFR signaling cascade were observed in the RES-pretreated mice. Hence, the present study elucidates that RES protects against CdCl2-induced perturbation of spermatogenesis and overexpression of EGFR and its downstream signaling proteins.

cAMP regulated EPAC1 supports microvascular density, angiogenic and metastatic properties in a model of triple negative breast cancer

Breast cancer is a leading cause of cancer-related mortality in women. Triple-negative breast cancer (TNBC; HER2-, ER-/PR-) is an aggressive subtype prone to drug resistance and metastasis, which is characterized by high intratumor microvascular density (iMVD) resulting from angiogenesis. However, the mechanisms contributing to the aggressive phenotypes of TNBC remain elusive. We recently reported that down-regulation of exchange factor directly activated by cyclic AMP (cAMP), also known as EPAC1, leads to a reduction in metastatic properties including proliferation and cell migration in TNBC cell lines. Here, we report that EPAC1 supports TNBC-induced angiogenesis, tumor cell migration and invasiveness as well as pro-metastatic phenotypes in endothelial cells induced through the tumor secretome. Using an approach that integrates proteomics with bioinformatics and gene ontologies, we elucidate that EPAC1 supports a tumor-secreted network of angiogenic, cell adhesion and cell migratory pathways. Using confocal microscopy, we show that signaling molecules involved in focal adhesion, including Paxillin and MENA, are down-regulated in the absence of EPAC1, and electric cell substrate impedance sensing technique confirmed a role for EPAC1 on TNBC-induced endothelial cell permeability. Finally, to provide a translational bridge, we studied iMVD and therapy response using a primary human tumor explant assay, CANscriptTM, which suggests a link between therapy-modulated neovascularization and drug sensitivity. These data provide mechanistic insight into the role of EPAC1 in regulating the tumor microenvironment, iMVD and cancer cell-induced angiogenesis, a dynamic mechanism under drug pressure that may associate to treatment failure.

Reversing effect of Lupeol on vasculogenic mimicry in murine melanoma progression

Vasculogenic mimicry, an endothelia-independent tumor microcirculation has been found in various cancers and is thought to be achieved by cancer stem like cells. Dacarbazine resistance is one of the most common features of melanoma and recent studies suggest that the mode of resistance is closely related to the formation of vasculogenic mimicry. In our work, we examined the anticancer effect of Lupeol, a novel phytochemical with Dacarbazine in vivo and in vitro. Results demonstrated adequate cytotoxicity followed by down regulation of CD 133 expression in Lupeol treated B16-F10 cell line. In solid tumor model the drug also inhibited vasculogenic mimicry along with angiogenesis by altering both the cancer stem cell as well as the endothelial progenitor cell population. Lupeol hindered the maturation of bone marrow derived endothelial progenitors and thus, retarded the formation of rudimentary tumor microvessels. Notably, Dacarbazine treatment demonstrated unresponsiveness to B16-F10 cells in both in vivo and in vitro model via upregulation of CD 133 expression and increased formation of vasculogenic mimicry tubes. Together, these data indicate that Lupeol alone can become a proficient agent in treating melanoma, inhibiting vasculogenic mimicry and might play a significant role in subduing Dacarbazine induced drug resistance.

Supra-physiological concentration of glyoxylate inhibits proliferation of human colon cancer cells through oxidative stress

Aims: The cytotoxic response of an intermediate metabolite glyoxylate (Glx) on colon carcinoma has been evaluated in vitro. Main methods: The anti‐proliferative effect of Glx was assessed on HT‐29 and HCT‐116 cells by performing MTT assay as well as beta‐hexosaminidase assay. Evaluation of apoptotic event of Glx treated cells was measured by flow cytometry using annexin‐V/PI staining. The mitochondrial membrane potential and level of ROS were estimated using DiOC6(3)/CCCP and DCFH‐DA method, respectively. The assessment of catalase, LDH and IDH were performed. Key findings: The results of MTT assay indicated that treatment with Glx significantly inhibited the proliferation of HT‐29 and HCT‐116 cells. Beta‐hexosaminidase assay also confirmed the inhibition of cellular viability. The dose‐dependent Glx treatment indicated lowering the colony forming ability of HT‐29 and HCT‐116 cells. Flow cytometric data demonstrated the significant increment of late apoptotic event after Glx treatment. In addition, substantial LDH activity was noticed in both the colon cancer cells whereas the IDH activity was unaltered after extra‐cellular addition of Glx. Further, dissipation of mitochondrial membrane potential and subsequently elevated ROS generation was also detected in the Glx treated colon cancer cells. However, gradual elevation of catalase activities indicated that Glx treatment on colon cancer cells exhibit oxidative stress. Significance: This study depicts that supra‐physiological concentration of Glx inhibits the proliferation of colon cancer cells due to oxidative stress.

Ionizing Radiation Increases the Circulatory Endothelial Progenitor Cell Population in Glottic Cancer Patients

Background: Cancer of the throat or larynx is one of the predominant cancer types in India and Intensity Mediated Radiation Therapy (IMRT) is the most crucial treatment regimen against the disease. We aimed to examine the effect of Circulating Endothelial Progenitor (CEP) cells in Laryngeal cancer patients undergoing radiation therapy. Patients: Five Laryngeal cancer patients were selected for the work. All patients were suffering from Squamous Cell Carcinoma (SCC) of the glottis and admitted to the institute with CT1 and CT2 stage of tumor with no lymph node metastasis (CN0). After thorough check up the patients were subjected to conventional mononodal radiotherapy (IMRT), fractionated doses to the highest dose (1.8-2 Gy per fraction). Peripheral Blood Mononuclear Cells (PBMC) was isolated from all 5 patients and flow cytometric analysis (using anti CD 44 and CD309 monoclonal antibody) was performed before and after completion of the entire course. Results: Results showed upregulation of the CEPs in all cases after exposure to radiotherapy. Additional immunofluorescence staining confirmed the rapid increase of these cells indicating a strong correlation between the exposure to ionizing radiation and stem cell expression in patients suffering from Glottic cancer. Conclusion: Increase in the CEPs suggests that the tissue damage due to radiation exposure activate the stem cell niche and allures CEPs in the peripheral blood for angiogenesis. Further prospective assessments are warranted.

Cannabis Smoke Causes Up-Regulation of Akt and Bax Protein in Subfertile Patients Sperm Cells

Background: Emerging worldwide evidences in support of adverse effects of cannabis smoke indicate its significant role in declining male fertility. The aim of the present study was to compare the percentage of damaged sperm cells and the expression profiles of cell survival protein p-Akt and pro-apoptotic protein Bax in non-smoker, tobacco smoke addicted and cannabis smoke addicted subfertile subjects. Method: Semen samples were collected from 80 male subjects of reproductive age group in Southern Assam of North-East India. 46 (57.5%), 25 (31.25 %) and 9 (11.325%) of these subjects were found to be cigarette smokers, cannabis smokers and non-smokers respectively. ROS levels in semen samples were measured by chemiluminescence assay. Sperm DNA integrity were assessed by acridine orange test, toluidine blue staining and TUNEL assay. Expression profiles of p-Akt and Bax were observed by flow cytometry and western blot analysis. Results: Among three groups, the cannabis smoke addicted subjects showed the highest level of seminal ROS production along with the highest percentage of sperm DNA damage, chromatin abnormalities and apoptotic cells. High expression of Bax and low expression of p-Akt was observed in non-smoker and tobacco smoke addicted subjects. Conversely, cannabis smoke addicted group showed the highest expression of both p-Akt and Bax proteins. Conclusion: The present study indicates cannabis smoke addiction to be more detrimental for male reproductive health compared to the tobacco smoke. The over-expression of both Akt and Bax proteins among cannabis smokers suggest that the up-regulation of pro-survival protein Akt, during sperm meiotic division could have triggered the oxidative apoptosis of sperm cells via the up-regulation of pro-apoptotic protein Bax.