Regulation Of Anti-apoptotic Genes Research Articles

Apoptotic and antioxidant activities of extracts of Spondias monbin stem bark in dimethylhydrazine (DMH)-induced colorectal carcinogenesis in Wistar rats

BackgroundColorectal cancer is the third most prevalent malignant neoplasm in the world and remains a leading cause of death, particularly in western nations. This study aimed to evaluate the apoptotic and antioxidant activities of Spondias mombin stem bark extracts in dimethylhydrazine (DMH) induced colon cancer in Wistar rats as well as to investigate the binding affinities of epicatechin, kaempferol and Lunamarin against p53 and caspase 3 proteins. MethodsA total of thirty-five (35) male Wistar rats were used for this experiment. They were grouped into five groups: Group 1 (normal control), Group 2 (DMH only- negative control), Group 3 (DMH + Xeloda - Standard Drug), Group 4 (DMH + 500 mg/kg bwt crude hydroethanol extracts), Group 5 (DMH + 500 mg bwt crude ethanol extracts. ResultsThe results showed high expression of proapoptotic genes which are Caspase-3 genes, B cell lymphoma 2 genes, and p53 genes and there was down regulation of antiapoptotic genes - Bcl like protein 4 genes. Also, the extracts of S. mombin stem bark attenuate the decreased antioxidant status (CAT, SOD and GSH) of DMN-induced colorectal cancer in rats and a significant decrease in the malondialdehyde levels. Molecular docking of the compounds (epicatechin, kaempferol and Lunamarin) against caspase 3 proteins possesses strong binding affinities with binding energies of -7.1, -7.0 and -7.5 kcal/mol respectively. However, kaempferol had similar binding energy(-7.0 kcal/mol) when docked against p53 gene. ConclusionTherefore, from the study, it was concluded that Spondias mombin stem bark has apoptotic and antioxidant properties.

Control of ER-positive breast cancer by ERα expression inhibition, apoptosis induction, cell cycle arrest using semisynthetic isoeugenol derivatives

New semi-synthetic effective and safe anticancer agents isoeugenol derivatives were synthesized, characterized, and screened for their cytotoxic activity against MCF-7. Moreover, their selective cytotoxicity was assessed against MCF-10A. Three derivatives, 2, 8 and 10 were significantly more active than the reference drug 5-FU with IC50 values of 6.59, 8.07 and 9.63 and 30.93 μM, respectively. Also interestingly, these derivatives demonstrated some degree of selectivity to cancer cells over normal cells. Furthermore, derivative 2 was subjected to other in vitro experiments against MCF-7 where it inhibited colony formation by 87.5% and lowered ERα concentration to 395.7 pg/mL compared to 1129 pg/mL in untreated control cells. In continuation of the investigation, the apoptotic activity of compound 2, was assessed where it significantly enhanced total apoptotic cell death by 9.16-fold (18.70% compared to 1.64% for the untreated MCF-7 control cells) and arrested the cell cycle at the G2/M phase. Furthermore, the molecular mechanism of apoptotic activity was investigated at both the gene (RT-PCR) and protein (western plotting) levels where upregulation of pro-apoptotic and down regulation of anti-apoptotic genes was detected. Additionally, compound 2 treatment enhanced the antioxidant (GSH, CAT, SOD) activities. Finally, in vivo experiments verified the effective anticancer activity of compound 2 through inhibition of tumor proliferation by 47.6% compared to 22.9% for 5-FU and amelioration of the hematological, biochemical, and histopathological examinations near normal. In effect, compound 2 can be viewed as a promising semi-synthetic derivative of isoeugenol with some degree of selectivity for management of breast cancer through apoptotic induction and ERα downregulation.

Use of Secondary Metabolites Derived from Aspergillus Species as Anticancer Agents and Related Histological and Genetic Alterations: In Vitro Study

The present work aimed to use natural derived fungal secondary metabolites as anticancer agents concerning cytotoxicity, apoptotic, genetic and histopathological profile. It was noticed that Asp. Terrius (Asp. T), Flavous (Asp. Fl) and Fumegatgus (Asp. Fu) induced variable toxic potential that was cell type, secondary metabolite type and concentration dependent. CaCo-2 cells showed less sensitivity than HuH-7 and in turn the IC50 was variable. Also, the apoptotic potential of Asp. species derived fungal secondary metabolites was proven via detection of up regulated pro apoptotic genes and down regulation of anti-apoptotic genes. The expression rate was cell type dependent. Concurrently apoptotic profile was accompanied with cellular DNA accumulation at the G2/M phase, an insignificant accumulation during the G0/G1 phase but there was not during the Pre-G1 and S phases. Also, there were a characteristic apoptotic features of treated cells presented as abnormal intra-nuclear eosinophyllic structures, necrotic cells with mixed euchromatin and heterochromatin, ruptured cell membranes, intranuclear eosinophyllic structures, apoptotic cells with irregular cellular and nuclear membranes, peripheral chromatin condensation and necrotic swollen cells with mixed euchromatin and heterochromatin. It can be concluded that A. secondary metabolites are promising agents can be used as a supplementary agents to current cancer drugs regimen applied.

In Vitro Evaluation of Anticancer Potential of Echispyramidum Venom (Viperidae) and Related Genetic and Apoptotic Profile Alterations

Background: cancer is basically a disease of uncontrolled cell division. Its development and progression are usually linked to a series of changes in the activity of cell cycle regulators. Objective: the present study aimed to investigate the anticancer potential of Echispyramidum venom against HepG2 and HCT 116 cancer cell lines in comparison with the Cisplatin as positive control. Patients and Methods: cell viability performed using MTT assay was cell type and concentration dependent was inversely proportional with the tow substances (Echispyramidum) venom as well as Cisplatin had IC50 of 11.8 and 112.7 µg/mL and 13.4 and 71.2 µg/mL developed in HCT116 and HepG2 cell lines respectively. Results: both of Echispyramidum venom and Cisplatin showed DNA accumulation at the Pre-G1 and G2/ M Phases of cell cycle. Apoptotic profile of treated cells showed up regulation of apoptotic genes (P53, Bax and Casp-3) and down regulation of anti-apoptotic gene (Bcl-2) in a significant way compared with untreated cell control. Also, apoptotic profile was assured via cellular changes detected microscopically in H&E stained cells. Microscopic examination showed apoptotic and slightly necrotic features in case of venoms while Cisplatin induced a number of apoptotic bodies and necrotic cell colonies. Conclusion: it can be concluded that Echispyramidum venom has a clear anticancer potential to human liver and colorectal cancer cells that was proven through the cell cycle arrest profile, induced apoptosis detected via detection of the levels of expression of pro and anti-apoptotic genes, as well as histological alteration detected.

In Vitro Evaluation of Anticancer Potential of Echispyramidum Venom (Viperidae) and Related Genetic and Apoptotic Profile Alterations

Background: cancer is basically a disease of uncontrolled cell division. Its development and progression are usually linked to a series of changes in the activity of cell cycle regulators. Objective: the present study aimed to investigate the anticancer potential of Echispyramidum venom against HepG2 and HCT 116 cancer cell lines in comparison with the Cisplatin as positive control. Patients and Methods: cell viability performed using MTT assay was cell type and concentration dependent was inversely proportional with the tow substances (Echispyramidum) venom as well as Cisplatin had IC50 of 11.8 and 112.7 µg/mL and 13.4 and 71.2 µg/mL developed in HCT116 and HepG2 cell lines respectively. Results: both of Echispyramidum venom and Cisplatin showed DNA accumulation at the Pre-G1 and G2/ M Phases of cell cycle. Apoptotic profile of treated cells showed up regulation of apoptotic genes (P53, Bax and Casp-3) and down regulation of anti-apoptotic gene (Bcl-2) in a significant way compared with untreated cell control. Also, apoptotic profile was assured via cellular changes detected microscopically in H&E stained cells. Microscopic examination showed apoptotic and slightly necrotic features in case of venoms while Cisplatin induced a number of apoptotic bodies and necrotic cell colonies. Conclusion: it can be concluded that Echispyramidum venom has a clear anticancer potential to human liver and colorectal cancer cells that was proven through the cell cycle arrest profile, induced apoptosis detected via detection of the levels of expression of pro and anti-apoptotic genes, as well as histological alteration detected.

Cabazitaxel antiproliferative mechanism of action in U87MG human glioblastoma cells: a promising cell-cycle phase-specific radiosensitizer

Introduction: One mechanism of cell cycle manipulation and mitotic catastrophe is arrest at G2/M phase of cell cycle. Cabazitaxel, a mitotic inhibitor agent, is a second-generation semisynthetic taxane. An expected anti-neoplastic effect of Cabazitaxel is cell cycle perturbation and alteration of microtubule dynamics. In contrast to other taxane compounds, Cabazitaxel is a poor substrate for P-gp efflux transporters, hence, penetrates easily across the blood brain barrier (BBB). In the present study, we investigate its distinct antiproliferative mechanism of action and radiosensitizing potential against U87MG glioblastoma cells. Materials and Methods: In order to evaluate the effect of Cabazitaxel, cells were treated with different concentrations of the drug at different time points and then cytotoxicity and cell cycle were assessed using MTT and flow cytometry assays, respectively. Comparison of induced apoptosis and necrosis in population of cells was depicted by Annexin/PI assay. In order to investigate the expression of genes involved in apoptosis real-time polymerase chain reaction (PCR) method was used. Results: A consistent G2/M arrest was exerted by Cabazitaxel. The drug resulted in a concentration- and time-dependent toxicity and a shap decline in cell viabiliity. EC50 and IC50 in cells treated with Cabazitaxel for 24 hours exactly approved each other. The cytotoxicity response of U87MG cells increased following Cabazitaxel-IR administration. On the other hand, IC50 of Cabazitaxel-IR in comparison with 24 or 48 hours treated cells showed a significant decrease. Concurrently, these observations were further supported by up regulation of pro- apoptotic genes and down regulation of anti-apoptotic genes. Conclusion: To our Knowledge, no straightforward explanation thus far has addressed the effect of Cabazitaxel applied before radiation on U87MG cells. This report provides the first preclinical evidence supporting that Cabazitaxel can render cells more susceptible to the cytotoxicity of radiation and potentiates the effect of RT. In the near future, Cabazitaxel could potentially be administered in combination modalities as a potential therapeutic compound and also promising cell cycle-specific radiosensitizer to stall tumor growth.

MiR-509-3p promotes cisplatin-induced apoptosis in ovarian cancer cells through the regulation of anti-apoptotic genes.

Previous observations have implicated miR-509-3p's ability in regulating cisplatin-triggered apoptosis in ovarian cancer. However, the underlying mechanisms were not fully understood. The roles of miR-509-3p in cellular apoptosis were assessed through MTT and DAPI assays. The confirmation of the regulation of BCL2 family members by miR-509-3p was investigated by luciferase reporter assay, western blot, quantitative real-time PCR and rescue experiments. MiR-509-3p can decrease the IC50 values of cisplatin and promote apoptosis in ovarian cancer cells. Furthermore, on a panel of anti-apoptotic proteins, we identified that miR-509-3p could regulate BCL2, BCL2L2 and MCL1 via their 3'UTRs. Our study demonstrates that miR-509-3p could sensitize ovarian cancer cells to cisplatin treatment by targeting multiple anti-apoptosis genes including BCL2.

The molecular mechanism of the anticancer effect of Artonin E in MDA-MB 231 triple negative breast cancer cells.

Nature has provided us with a wide spectrum of disease healing phytochemicals like Artonin E, obtained from the root bark of Artocarpus elasticus. This molecule had been predicted to be drug-like, possessing unique medicinal properties. Despite strides made in chemotherapy, prognosis of the heterogenous aggressive triple negative breast cancer is still poor. This study was conducted to investigate the mechanism of inhibition of Artonin E, a prenylated flavonoid on MDA-MB 231 triple negative breast cancer cell, with a view of mitigating the hallmarks displayed by these tumors. The anti-proliferative effect, mode of cell death and the mechanism of apoptosis induction were investigated. Artonin E, was seen to effectively relinquish MDA-MB 231 breast cancer cells of their apoptosis evading capacity, causing a half-maximal growth inhibition at low concentrations (14.3, 13.9 and 9.8 μM) after the tested time points (24, 48 and 72 hours), respectively. The mode of cell death was observed to be apoptosis with defined characteristics. Artonin E was seen to induce the activation of both extrinsic and intrinsic caspases initiators of apoptosis. It also enhanced the release of total reactive oxygen species which polarized the mitochondrial membrane, compounding the release of cytochrome c. Gene expression studies revealed the upregulation of TNF-related apoptosis inducing ligand and proapoptotic genes with down regulation of anti-apoptotic genes and proteins. A G2/M cell cycle arrest was also observed and was attributed to the observed upregulation of p21 independent of the p53 status. Interestingly, livin, a new member of the inhibitors of apoptosis was confirmed to be significantly repressed. In all, Artonin E showed the potential as a promising candidate to combat the aggressive triple negative breast cancer.

Targeting ceramide metabolic pathway induces apoptosis in human breast cancer cell lines

The sphingolipid ceramide is a pro apoptotic molecule of ceramide metabolic pathway and is hydrolyzed to proliferative metabolite, sphingosine 1 phosphate by the action of acid ceramidase. Being upregulated in the tumors of breast, acid ceramidase acts as a potential target for breast cancer therapy. We aimed at targeting this enzyme with a small molecule acid ceramidase inhibitor, Ceranib 2 in human breast cancer cell lines MCF 7 and MDA MB 231. Ceranib 2 effectively inhibited the growth of both the cell lines in dose and time dependant manner. Morphological apoptotic hallmarks such as chromatin condensation, fragmented chromatin were observed in AO/EtBr staining. Moreover, ladder pattern of fragmented DNA observed in DNA gel electrophoresis proved the apoptotic activity of Ceranib 2 in breast cancer cell lines. The apoptotic events were associated with significant increase in the expression of pro-apoptotic genes (Bad, Bax and Bid) and down regulation of anti-apoptotic gene (Bcl 2). Interestingly, increase in sub G1 population of cell cycle phase analysis and elevated Annexin V positive cells after Ceranib 2 treatment substantiated its apoptotic activity in MCF 7 and MDA MB 231 cell lines. Thus, we report Ceranib 2 as a potent therapeutic agent against both ER+ and ER− breast cancer cell lines.

Overexpression of Telomerase Protects Human and Murine Lung Epithelial Cells from Fas- and Bleomycin-Induced Apoptosis via FLIP Upregulation.

High doses of bleomycin administered to patients with lymphomas and other tumors lead to significant lung toxicity in general, and to apoptosis of epithelial cells, in particular. Apoptosis of alveolar epithelium is an important step in the pathogenesis of bleomycin-induced pulmonary fibrosis. The Fas-FasL pathway is one of the main apoptotic pathways involved. Telomerase is a ribonucleoprotein RNA-dependent DNA polymerase complex consisting of an RNA template and a catalytic protein, telomerase reverse transcriptase (TERT). Telomerase also possess extra-telomeric roles, including modulation of transcription of anti-apoptotic genes, differentiation signals, and more. We hypothesized that telomerase overexpression affects Fas-induced epithelial cell apoptosis by an extra-telomeric role such as regulation of anti-apoptotic genes, specifically FLICE-like inhibitory protein (FLIP). Telomerase in mouse (MLE) and human (A549) lung epithelial cell lines was upregulated by transient transfection using cDNA hTERT expression vector. Telomerase activity was detected using a real-time PCR-based system. Bleomycin, and bleomycin-induced Fas-mediated apoptosis following treatment with anti-Fas activating mAb or control IgG, were assessed by Annexin V staining, FACS analysis, and confocal microscopy; caspase cleavage by Western blot; FLIP or Fas molecule detection by Western blot and flow cytometry. hTERT transfection of lung epithelial cells resulted in a 100% increase in their telomerase activity. Fas-induced lung epithelial cell apoptosis was significantly reduced in hTERT-transfected cells compared to controls in all experiments. Lung epithelial cells with increased telomerase activity had higher levels of FLIP expression but membrane Fas expression was unchanged. Upregulation of hTERT+ in human lung epithelial cells and subsequent downregulation of FLIP by shFLIP-RNA annulled hTERT-mediated resistance to apoptosis. Telomerase-mediated FLIP overexpression may be a novel mechanism to confer protection from apoptosis in bleomycin-exposed human lung epithelial cells.

Fatty acid esters of phloridzin induce apoptosis of human liver cancer cells through altered gene expression.

Phloridzin (phlorizin or phloretin 2′-O-glucoside) is known for blocking intestinal glucose absorption. We have investigated the anticarcinogenic effect of phloridzin and its novel derivatives using human cancer cell lines. We have synthesised novel acylated derivatives of phloridzin with six different long chain fatty acids by regioselective enzymatic acylation using Candida Antarctica lipase B. The antiproliferative effects of the new compounds were investigated in comparison with the parent compounds, phloridzin, aglycone phloretin, the six free fatty acids and chemotherapeutic drugs (sorafenib, doxorubicin and daunorubicin) using human hepatocellular carcinoma HepG2 cells, human breast adenocarcinoma MDA-MB-231 cells and acute monocytic leukemia THP-1 cells along with normal human and rat hepatocytes. The fatty acid esters of phloridzin inhibited significantly the growth of the two carcinoma and leukemia cells while similar treatment doses were not toxic to normal human or rat hepatocytes. The antiproliferative potency of fatty esters of phloridzin was comparable to the potency of the chemotherapeutic drugs. The fatty acid esters of phloridzin inhibited DNA topoisomerases IIα activity that might induce G0/G1 phase arrest, induced apoptosis via activation of caspase-3, and decreased ATP level and mitochondrial membrane potential in HepG2 cells. Based on the high selectivity on cancer cells, decosahexaenoic acid (DHA) ester of phloridzin was selected for gene expression analysis using RT2PCR human cancer drug target array. Antiproliferative effect of DHA ester of phloridzin could be related to the down regulation of anti-apoptotic gene (BCL2), growth factor receptors (EBFR family, IGF1R/IGF2, PDGFR) and its downstream signalling partners (PI3k/AKT/mTOR, Ras/Raf/MAPK), cell cycle machinery (CDKs, TERT, TOP2A, TOP2B) as well as epigenetics regulators (HDACs). These results suggest that fatty esters of phloridzin have potential chemotherapeutic effects mediated through the attenuated expression of several key proteins involved in cell cycle regulation, DNA topoisomerases IIα activity and epigenetic mechanisms followed by cell cycle arrest and apoptosis.

Up-regulation of anti-apoptotic genes confers resistance to the novel anti-leukaemic compound PEP005 in primary AML cells.

We showed previously that PEP005 induced apoptosis in leukaemic cell lines and blasts from patients with acute myeloid leukaemia (AML). Here we assess the anti-leukeamic effects of PEP005 in vivo and determine the mechanism of resistance of PEP005 non-responsive cells. We used 2 human xenograft mouse models of AML to assess the anti-leukaemic effects of PEP005 in vivo. Expression microarray analysis of primary AML blasts following treatment with PEP005 was used to determine patterns of gene expression that conferred resistance. PEP005 significantly reduced tumour burden in two human leukaemia mouse xenograft models. We also assessed responsiveness of 33 AML samples to PEP005, with 78% of the samples entering apoptosis at 100nM. Resistance to PEP005 was not restricted to a particular AML subtype. Expression microarray analysis of resistant samples following treatment with PEP005 revealed a significant up regulation of the anti-apoptotic genes Bcl-2A1, Mcl-1, and PHLDA1 which was verified using RT-PCR. We conclude that PEP005 shows broad efficacy against AML subtypes and that up regulation of anti-apoptotic genes underlies resistance to this agent and could be used to screen for patients unlikely to benefit from a therapeutic regime involving PEP005.

Cell Type-Dependent Function of Atf5: Where We Go from Here

Copyright: © 2012 Lengel KL, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Uncontrollable cell proliferation and suppressed apoptosis are hallmarks of tumorigenic transformation [1-3]. Deregulation of genes controlling cell proliferation and survival plays an important role in the process. For instance, apoptotic genes are frequently down-regulated while anti-apoptotic genes are highly expressed in a number of cancer cell types; artificial down regulation of anti-apoptotic genes or up regulation of apoptotic genes are often sufficient to eradicate those cancer cells. The activating transcription factor 5 (ATF5 or ATFx) is a member of the ATF/CREB transcription factor family. Although ATF5 is known to regulate cell cycle progression [4-7], cell survival [5-13], autophagy [14], cell fate determination [15-17] and cellular stress response [13-19], and it is likely involved in the development of schizophrenia [20-22] and chronic lymphocytic leukemia [23], only a few of its targets have been reported and the mechanism of ATF5 function remains largely unknown and occasionally controversial.

Cerastes cerastes and Vipera lebetina Snake Venoms Apoptotic – Stimulating Activity to Human Breast Cancer Cells and Related Gene Modulation

Apoptosis occurs normally during development and aging as a homeostatic mechanism to maintain cell populations. Dysregulation of apoptosis can disrupt the equilibrium between cell growth and cell death leading to the development of cancer. Thus, the investigation of new biological apoptotic activators could play an important role in cancer therapy. In the present study, Cerastes cerastes and Vipera lebetina snake venoms were evaluated for their ability to activate apoptosis in cancer cells where test venoms exhibited a concentration and time dependent cytotoxic effect on breast cancer (MCF-7) cells. Typical apoptotic morphological features were demonstrated in venom treated cells detected via transmission electron microscope. In addition, flow cytometric analysis showed an increase in the percentage of apoptotic cells post 24 h treatment relative to venom concentrations. At the molecular level, test venoms induced apoptosis were mediated by up regulation of pro-apoptotic genes (p53 & Bax) and down regulation of anti-apoptotic gene (Bcl-2) in MCF-7 cells, indicating that these venoms could serve as apoptotic stimulators, presenting a novel and potential therapeutic strategy for cancer treatment.