Evaluation of infliximab-induced genotoxicity and possible action on BCL-2 and P53 genes.

Although the last pandemic created an urgency for development of vaccines, there was a continuous and concerted effort to search for therapeutic medications among existing drugs with different indications. One of the medications of interest that underwent this change was infliximab (IFM). This drug is used as an anti-inflammatory, predominantly in patients with Crohn 's disease, colitis ulcerative, and rheumatoid arthritis. In addition to these patients, individuals infected with Coronavirus Disease (COVID-19) were administered this chimeric monoclonal antibody (IMF) to act as an immunomodulator for patients in the absence of comprehensive research. Consequently, the present study aimed to examine the genotoxic effects attributed to IFM treatment employing different assays in vivo using mouse Mus musculus. Therefore, IFM was found to induce genotoxic effects as evidenced by the comet assay but did not demonstrate genotoxic potential utilizing mouse bone marrow MN test. The results of evaluating the expression of the P53 and BCL-2 genes using RT-qPCR showed stimulation of expression of these genes at 24 hr followed by a decline at 48 hr. Although the comet assay provided positive results, it is noteworthy that based upon negative findings in the micronucleus test, the data did not demonstrate significant changes in the genetic material that might affect the therapeutic use of IFM. The stimulation of expression of P53 and BCL-2 genes at 24 hr followed by a decline at 48 hr suggest a transient, if any, effect on genetic material. However, there is still a need for more research to more comprehensively understand the genotoxic profile of this medication.

Empagliflozin reduces the adverse effects of diabetes mellitus on testicular tissue in type 2 diabetic Rats: A stereological and biochemical study.

Empagliflozin as an antioxidant decreases blood glucose and insulin resistance in type 2 diabetes mellitus. Base on the empagliflozin antioxidant properties we decided to investigate the its effects on the testis histological changes through stereological techniques and biochemical evaluations in T2 diabetes mellitus rats. Rats were divided into: control, diabetes mellitus (DM, streptozotocin + nicotinamide) and diabetes mellitus + empagliflozin (DM + EMPA, 10 mg/kg/day) groups. 56 days after inducing diabetes mellitus testis histological changes and serum biochemical factors along with the level of Bax, Bcl2 and Nrf2 genes expression in the testicular tissue were assessed. A significant decrease in the mean total volume of testis and its components, the level of Bcl2 and Nrf2 gene expression (p < 0.001) along with a significant increase in the level of IL-6, TNF-α, MDA, Bax gene expression were observed in the DM group compared to the control group (p < 0.001). In the DM + EMPA group, the mean total volume of testis and its components, the level of Bcl2 gene expression (p< 0.01) and Nrf2 (p < 0.001) significantly increased whereas the mean level of IL-6 (p < 0.01), TNF-α (p < 0.001), MDA (p < 0.001), Bax (p < 0.001) gene expression significantly decreased compared to the DM group. Our results showed that empagliflozin, by improving the antioxidant defense system, can reduce testicular inflammation and apoptosis and partly prevent the adverse effects of diabetes mellitus on testicular tissue.

Apoptotic effects of human amniotic fluid mesenchymal stem cells conditioned medium on human MCF-7 breast cancer cell line.

Introduction: Breast cancer, as the most common malignancy among women, is shown to have a high mortality rate and resistance to chemotherapy. Research has shown the possible inhibitory role of Mesenchymal stem cells in curing cancer. Thus, the present work used human amniotic fluid mesenchymal stem cell-conditioned medium (hAFMSCs-CM) as an apoptotic reagent on the human MCF-7 breast cancer cell line. Methods: Conditioned medium (CM) was prepared from hAFMSCs. After treating MCF-7 cells with CM, a number of analytical procedures (MTT, real-time PCR, western blot, and flow cytometry) were recruited to evaluate the cell viability, Bax and Bcl-2 gene expression, P53 protein expression, and apoptosis, respectively. Human fibroblast cells (Hu02) were used as the negative control. In addition, an integrated approach to meta-analysis was performed. Results: The MCF-7 cells' viability was decreased significantly after 24 hours (P < 0.0001) and 72 hours (P < 0.05) of treatment. Compared with the control cells, Bax gene's mRNA expression increased and Bcl-2's mRNA expression decreased considerably after treating for 24 hours with 80% hAFMSCs-CM (P = 0.0012, P < 0.0001, respectively); an increasing pattern in P53 protein expression could also be observed. The flow cytometry analysis indicated apoptosis. Results from literature mining and the integrated meta-analysis showed that hAFMSCs-CM is able to activate a molecular network where Bcl2 downregulation stands in harmony with the upregulation of P53, EIF5A, DDB2, and Bax, leading to the activation of apoptosis. Conclusion: Our finding demonstrated that hAFMSCs-CM presents apoptotic effect on MCF-7 cells; therefore, the application of hAFMSCs-CM, as a therapeutic reagent, can suppress breast cancer cells' viabilities and induce apoptosis.

Methotrexate loaded in alginate beads for controlled drug release against breast cancer.

Methotrexate (MTX), as a folate antagonist is used for breast cancer chemotherapy, but its application due to the adverse side effects was limited. In this study, MTX were encapsulated in magnetic alginate beads coated with glutaraldehyde to control its release in order to reduce the side effects and improve its stability. The complex was characterized by physicochemical studies. The encapsulation efficiency was 75 % and the complex showed acceptable controlled release behavior. The cell cytotoxicity assessed using methylthiazol tetrazolium (MTT) method showed that magnetic alginate beads-MTX, in lower dosage has higher anticancer effect compared to the free MTX. The real-time polymerase chain reaction (PCR) was used to evaluate apoptotic factors Bcl2 associated X gene (Bax), B-cell lymphoma 2 (Bcl-2), and neuroinflammatory marker tumor necrosis factor-alpha (TNF-α) genes expression level on the treated cells. The findings demonstrated the significant increase of expression of Bax and a significant decrease in the expressions of Bcl-2 and TNF-α in Michigan cancer foundation-7 (MCF-7) cells. These results indicated that the developed drug can overcome the side effects of MTX and offer a controlled drug release for a sustained period with the long-term treatment of breast cancer.

Assessment of gentamicin and cisplatin-induced kidney damage mediated via necrotic and apoptosis genes in albino rats.

BACKGROUND: Gentamicin (GM) is a low-cost, low-resistance antibiotic commonly used to treat gram-negative bacterial diseases. Cisplatin (Csp) is a platinum-derived anti-neoplastic agent. This experiment aimed to identify the early signs of gentamicin and cisplatin-induced nephrotoxicity in rats. Thirty Wistar rats were divided into three groups of 10: a control group, which received no treatment; a gentamicin group administered by a dose of (100 mg/kg, IP) for 7 consecutive days, and a cisplatin group was administered intraperitoneal in a dose of (1.5 mg/kg body weight) repeated twice a week for 3 weeks. RESULTS: Both experimental groups exhibited increased levels of creatinine, urea, and uric acid, with the cisplatin-treated group showing higher levels than the gentamicin group. Experimental groups also exhibited significantly increased Malondialdehyde (MDA), reduced glutathione (GSH), and glutathione peroxidase (GSH-Px) with more pronounced effects in the cisplatin-treated group. Further, both experimental groups exhibited significant up-regulation of Tumor Necrosis Factor α (TNF-α), caspase-3, and Bax and down regulation of Bcl-2. CONCLUSION: These findings confirm the use of necrotic, apoptotic genes as early biomarkers in the detection of tubular kidney damage. Further, cisplatin was shown to have a greater nephrotoxic effect than gentamicin; therefore, its use should be constrained accordingly when co-administered with gentamicin.

Pharmacological Effects of Guava (Psidium guajava L.) Seed Polysaccharides: GSF3 Inhibits PC-3 Prostate Cancer Cell Growth through Immunotherapy In Vitro.

The inhibitory effects of purified fractions isolated from guava seed polysaccharides (GSPS) including guava seed polysaccharide fraction 1 (GSF1), GSF2, and GSF3 on prostate cancer cells remain unclear. To clarify the anti-prostate cancer potential, GSPS, GSF1, GSF2, and GSF3 were isolated using Sepharose 6B gel filtration chromatography to assay their inhibitory effects on prostate PC-3 cell growth with direct action or indirect immunotherapy using either splenocyte conditioned media (SCM) or macrophage conditioned media (MCM). Correlations between cytokine profiles in the conditioned media and pro-apoptotic gene expression levels in the corresponding treated PC-3 cells were analyzed. Results showed that GSPS, GSF1, GSF2, and GSF3, particularly GSF3, through either direct action or indirect treatments using SCM or MCM, significantly (p < 0.05) inhibited PC-3 cell growth. GSF3 direct treatments increased pro-apoptotic Bax/anti-apoptotic Bcl-2 mRNA expression ratios in corresponding treated PC-3 cells. Either SCM or MCM cultured with GSF3 increased Fas mRNA expression levels in corresponding treated PC-3 cells. Both Th2-polarized and anti-inflammatory cytokine IL-10 either secreted in SCM or MCM were positively correlated with Fas mRNA expression levels in corresponding treated PC-3 cells. Our results suggest that GSF3 is a potent biological response modifier to decrease PC-3 cell growth through inducing apoptosis.

Aronia melanocarpa Ameliorates Adrenal Cytoarchitecture Against the Hexavalent Chromium-Induced Injury.

Hexavalent chromium is a toxin that penetrates the cell, triggering reactive oxygen species (ROS) production. Aronia melanocarpa, due to its proanthocyanidins, anthocyanins, and phenolic acid contents, is a valuable antioxidant. The aim was to observe the influence of hexavalent chromium Cr(VI) on the adrenal gland, and if this impact can be recovered by the administration of A. melanocarpa. Accordingly, 36 rats were divided into six groups: control; Aronia; Cr receiving Cr(VI) in distilled water for 3 months; CrA receiving a mix of Cr(VI) and A. melanocarpa at 2.5% aqueous extract for 3 months; Cr2 receiving, for 3 months, Cr(VI) in distilled water, and next, for 1 month, only distilled water; and respectively, CrA2 receiving, for 3 months, Cr(VI) in distilled water, followed by 1 month of Aronia at 2.5% extract administration. The adrenal gland samples were examined toward histological and molecular assessment, and results were statistically analyzed (ANOVA). Hexavalent chromium induced changes in the adrenal cortex expressed by focal or diffuse hypertrophies, cytoplasmic vacuolization (due to lipidic accumulation), and cells' shape and size alteration, including necrosis. These structural alterations were carried by Bax and Bcl2 gene expression: the Bax gene expression levels, increased significantly (p < 0.001) in all experimental groups, except the Aronia group, compared with control. In the Cr2, CrA, and CrA2 groups, notable reduction of Bax gene expression (p < 0.001) was reported compared with the Cr group. Regarding the Bcl2 gene expression (p < 0.001), a significant increase was observed in the experimental groups, compared with the control. The Bcl2 expression level had a similar pattern to Bax gene, consequently trying to compensate its overexpression. Aronia administered concomitantly, or after Cr(VI), diminished structural changes and expression of the studied genes, thus reducing the Bax/Bcl2 ratio and suggesting that the active ingredients from Aronia are capable of blocking apoptotic cascade induced by the pathway of Bax and Bcl2 proteins.

Associação do polimorfismo genético no gene BCL2 no Acidente Vascular Cerebral Hemorrágico/Aneurisma / Association of genetic polymorphism in the BCL2 gene in/with Hemorrhagic Stroke/Aneurysm / Asociación de polimorfismo genético en el gen BCL2 en/con el accidente cerebrovascular hemorrágico/aneurisma

Objetivo: Verificar a distribuição do polimorfismo do gene BCL2 (rs1801018), em sua região codante, em pacientes portadores de Acidente Vascular Cerebral Hemorrágico (AVCh)/Aneurisma. Além de associar o presente polimorfismo as manifestações clinicas da doença. Método: O estudo foi conduzido com 158 participantes de pesquisa. Os grupos foram pareados quanto ao sexo e idade. A genotipagem foi conduzida pela técnica PCR-RFLP. Após o cálculo das frequências alélicas e genotípicas de cada grupo, foram utilizados testes estatísticos apropriados para cada tipo de comparação. O nível de significância adotado foi de 5%. Resultados: Os dados indicaram que a frequência dos genótipos apresentou uma diferença estatisticamente significante entre o grupo caso e controle, encontrando-se o genótipo ala43ala na maioria dos participantes de ambos os grupos. Conclusão: A presença do alelo mutante (trh) foi vista como um fator protetor para AVCh/aneurisma. Porém, estudos em outras populações devem ser realizados para se obter uma melhor compreensão sobre a doença AVCh/aneurisma.

Objective: Verify the distribution of the BCL2 gene polymorphism (rs1801018), located in its coding region, in patients with Hemorrhagic Stroke (HS)/Aneurysm (A). Furthermore, to associate this polymorphism with the HS/A clinical manifestations. Method: The study was conducted with 158 research participants and the groups matched by sex and age. Genotyping was done by the PCR-RFLP technique. After calculating the allele and genotype frequencies of each group, appropriate statistical tests were performed for each comparison type with the adopted significance level of 5%. Results: The data indicated that the frequency of the genotypes showed a statistically significant difference between the case and control group, and the ala43ala genotype was found in most participants in both groups. Conclusion: The presence of the mutant allele (trh) was observed as a protective factor for HS/A. However, studies in other populations should be performed to obtain a better understanding of this disease.

Objetivo: Objetivo: Verificar la distribución del polimorfismo del gen BCL2 (rs1801018), en ubicado su región de codificación, en pacientes con accidente cerebrovascular hemorrágico (ACVh)/aneurisma. Asimismo, asociar el presente polimorfismo con las manifestaciones clínicas de la enfermedad. Método: El estudio se realizó con 158 participantes, y los grupos fueron agrupados por sexo y edad. La determinación del genotipo se realizó mediante la técnica PCR-RFLP. Después de calcular las frecuencias de alelos y genotipos de cada grupo, se realizaron pruebas estadísticas apropiadas para cada tipo de comparación con el nivel de significación adoptado de 5%. Resultados: Los datos indicaron que la frecuencia de los genotipos mostró una diferencia estadísticamente significativa entre el grupo caso y el control, con el genotipo ala43ala encontrado en la mayoría de los participantes de ambos grupos. Conclusión: La presencia del alelo mutante (trh) fue visto como un factor protector para el ACVh/aneurisma. Sin embargo, se deben realizar más estudios en otras poblaciones para obtener una mejor comprensión de la enfermedad de ACVh/aneurisma.

Amelioration of fenitrothion induced oxidative DNA damage and inactivation of caspase-3 in the brain and spleen tissues of male rats by N-acetylcysteine.

N-acetylcysteine (NAC) has largely been used as an effective chemo- protective agent owing to their beneficial effect in restoring several physiological parameters and relieving oxidative stress. Interestingly, it has been suggested that NAC mechanisms of action extend beyond being a precursor to the antioxidant glutathione and that they may involve several neurotropic and inflammatory pathways. Exposure to fenitrothion, an organophosphorus insecticide, promotes oxidative stress and induces several deleterious changes in the immune response and various tissues including cerebrum and spleen. The main objective of our study was to investigate ameliorative efficacy of N-acetylcysteine for immunological and neurological alterations and oxidative DNA damage induced by fenitrothion toxicity in cerebrum and spleen tissues of male rats. Our results revealed that oral exposure to fenitrothion for 30 days caused a reduction in the erythrocyte count in addition to leukocytosis, lymphocytosis, and neutrophilia. Also, this route of administration increased the serum levels of LDH, TNF-α, and IL-2 with reduction in serum immunoglobulins (IgG & IgM) concentrations. Furthermore, a significant downregulation in the antioxidant markers (GSH & SOD) with an elevation of free radical (MDA) levels were noticed. Regarding the brain, fenitrothion administration inhibited AchE activity and increased brain GABA, serotonin and dopamine levels. Moreover, it induced an elevation in oxidative DNA damage indicated by 8-hydroxy 2-deoxyguanosine (8OH2dG) and mRNA expression of pro-apoptotic genes, including Bax, and p53, but Bcl-2 expression was reduced. N-acetylcysteine co-treatment restored the normal physiological tone in most of these parameters. Immunostaining for GFAP and Caspase-3 markers in the brain and spleen tissues were increased respectively. In conclusion, N-acetylcysteine supplementation has an ameliorative effect against immunotoxic, neurotoxic and oxidative DNA damage induced by fenitrothion exposure.

The effects of coadministration of tilorone dihydrochloride and culture supernatants from Lactobacillus reuteri on the mouse hepatoma cell line.

CONTEXT: Tilorone dihydrochloride is a therapeutic agent with a different mechanism in cancer. The species of Lactobacillus have an important role in cytotoxic effect. AIMS: Because of unknown effects of tilorone and culture supernatants from Lactobacillus reuteri on hepatoma, the aim of this study is to evaluate apoptotic, cytotoxic, and therapeutic effects of tilorone on mouse hepatoma cell line with and without culture supernatants from L. reuteri. MATERIALS AND METHODS: To do so, after cell line culture, cells were divided into different groups such as negative control, treatment with four doses of tilorone, positive control of supernatant (single dose), and combination therapy groups of different doses of tilorone with supernatant (constant doses), for 48 h. All groups were studied with pathologic tests, biochemical study, tetrazolium dye (3-(4, 5- dimethylthiazol -2-yl)-2, 5-diphenyltetrazolium bromide [MTT]) assay, and absolute real-time-polymerase chain reaction (RT-PCR) were done to assess Bax and Bcl-2 genes expression, as molecular studies. RESULTS: MTT assay results revealed that the tilorone tissue culture IC50 (TCIC50) on the Hepa1-6 cell line was 50 µg/ml. RT-PCR analysis showed that tilorone dihydrochloride induced upregulation and downregulation in expression of Bax and Bcl-2, respectively. Simultaneous, antioxidant effect has also seen in a way that prevented necrosis, in biochemical analysis. These results were dose dependent and statistically significant compared to the control group. CONCLUSIONS: Based on these results, it appeared that this agent could be a good candidate for further evaluation as effective chemotherapy acting through the induction of apoptosis in hepatoma. The cell death caused through bacterial supernatant was rather necrosis than apoptosis.

MYC translocation partner gene determines survival of patients with large B-cell lymphoma with MYC- or double-hit MYC/BCL2 translocations.

In large B-cell lymphoma (LBCL) MYC- and MYC/BCL2 double-hit (DH) translocations have been associated with inferior survival. We hypothesised that the negative prognostic impact of MYC translocation was determined by an immunoglobulin MYC translocation partner gene (IG-MYC), as opposed to a non-immunoglobulin partner gene (nonIG-MYC). In a prospective, unselected cohort of 237 LBCL patients MYC and BCL2 translocations were identified by fluorescent in situ hybridisation (FISH) with split probes. MYC translocation partner gene was identified by IGH/MYC fusion probes and/or kappa/lambda split probes. Clinical data were collected from patient files. MYC translocation was identified in 28/225 patients. IG-MYC translocation partner gene was identified in 12/24 patients. DH translocation was identified in 23/228 patients. IG-MYC translocation partner gene was identified in 9/19 DH patients. Neither MYC-nor DH translocation showed correlation with survival. However, MYC translocation with IG-MYC translocation partner gene was associated with worse OS compared with both MYC translocation with nonIG-MYC translocation partner gene (P = 0.02) as well as absence of MYC translocation (P = 0.03). In patients with DH a similar, however, stronger correlation was seen (P = 0.003 and P = 0.0004 respectively). MYC - or DH translocation with nonIG-MYC translocation partner gene was not associated with worse overall survival (P = 0.2 and P = 0.3 respectively). Most patients received Rituximab (86%) and CHOP/CHOP-like chemotherapy regimes (81%). We suggest that prognostic stratification of LBCL patients by MYC and/or DH translocations should include identification of MYC translocation partner gene because approximately half of the cases harbour nonIG-MYC translocation partner genes with no or minor influence on survival.

Berberine, an isoquinoline alkaloid, inhibits streptozotocin-induced apoptosis in mouse pancreatic islets through down-regulating Bax/Bcl-2 gene expression ratio.

Diabetes may cause apoptosis in pancreatic islets. Berberine is an isoquinoline alkaloid used for its pharmacological functions including anti-inflammation. However, the berberine effect on pancreatic islets is still not clear. This study is aimed at clarifying the protective mechanism in berberine against islet cell apoptosis. This study established in vitro experimental models using streptozotocin (STZ)-treated primary pancreatic islet cells from ICR mice to unravel the protective mechanism of berberine on islets. The Bax/Bcl-2 (pro-/anti-apoptotic) genes expression in the islets was determined using real-time quantitative polymerase chain reaction assay. The results showed that berberine administration at one time or before STZ-stimulation significantly (P<0.05) down-regulated the Bax/Bcl-2 genes expression ratio, compared to those in STZ-treatment alone group. Our results suggest that berberine's anti-apoptotic effect on pancreatic primary islets is through down-regulating the Bax/Bcl-2 genes expression ratio in both concurrent and preventive manners.

Salvia miltiorrhiza bge. f. alba up-regulates Bcl-2 and inhibits apoptosis of human umbilical vein endothelial cells / 第二军医大学学报

Objective To study the influence of Salvia miltiorrhiza bge. f. alba(SMA) on apoptosis and Bcl-2 expression in human umbilical vein endothelial cell (HUVEC) in vitro. Methods HUVECs were isolated using perfusion and enzyme digestion methods, and the obtained cells were identified by morphological observation and VIII = Ag immunoreactivity examination. The cells in the exponential phase of growth were treated with H2O2 and different concentrations of SM A (high dose, 0.10 g/ml, low dose, 0.01 g/ml). The cell apoptosis was determined by flow cytometric analysis and Bcl-2 expression was examined by immunofluorescence mehtod. Results SMA significantly decreased the H2O2-induced apoptosis of HUVECs (P<0.01 in high dose group and P<0.05 in low dose group), and significantly increased the expression of Bcl-2 in high dose group(P<0.01). Conclusion SMA can inhibit H2O2-induced apoptosis of HUVEC, which might be associated with the increase of Bcl-2 expression. Salvia miltiorrhiza bge. f. alba; umbilical veins; endothelial cell; apoptosis; bcl-2 genes.

RTKN2 Induces NF-KappaB Dependent Resistance to Intrinsic Apoptosis in HEK Cells and Regulates BCL-2 Genes in Human CD4(+) Lymphocytes.

The gene for Rhotekin 2 (RTKN2) was originally identified in a promyelocytic cell line resistant to oxysterol-induced apoptosis. It is differentially expressed in freshly isolated CD4(+) T-cells compared with other hematopoietic cells and is down-regulated following activation of the T-cell receptor. However, very little is known about the function of RTKN2 other than its homology to Rho-GTPase effector, rhotekin, and the possibility that they may have similar roles. Here we show that stable expression of RTKN2 in HEK cells enhanced survival in response to intrinsic apoptotic agents; 25-hydroxy cholesterol and camptothecin, but not the extrinsic agent, TNFα. Inhibitors of NF-KappaB, but not MAPK, reversed the resistance and mitochondrial pro-apoptotic genes, Bax and Bim, were down regulated. In these cells, there was no evidence of RTKN2 binding to the GTPases, RhoA or Rac2. Consistent with the role of RTKN2 in HEK over-expressing cells, suppression of RTKN2 in primary human CD4(+) T-cells reduced viability and increased sensitivity to 25-OHC. The expression of the pro-apoptotic genes, Bax and Bim were increased while BCL-2 was decreased. In both cell models RTKN2 played a role in the process of intrinsic apoptosis and this was dependent on either NF-KappaB signaling or expression of downstream BCL-2 genes. As RTKN2 is a highly expressed in CD4(+) T-cells it may play a role as a key signaling switch for regulation of genes involved in T-cell survival.

Effect of Human Bcl-2 Gene Expression on the Peripheral Atherosclerotic Lesions of Apolipoprotein E-Deficient Mouse

BACKGROUND AND OBJECTIVES: Bcl-2 protein is related to the inhibition of apoptosis via the mitochondrial pathway and Bcl-2's anti-oxidant effect. During the development of atherosclerosis, apoptosis is known to play an important role in the pathophysiologic behavior of atherosclerotic vascular disease in the medium-sized arteries. Apoptosis may be a compensatory reaction to regulate the cellular density of various tissues during the cellular proliferation process such as happens with tissue injury and during the development of atherosclerosis. The consequences of apoptosis in atherosclerosis may be related to the formation of an acellular lipid core, plaque instability and the loss of vascular wall integrity and remodeling. We sought to determine the effect of Bcl-2 gene expression on the development of primary atherosclerosis in apolipoprotein E deficient mouse, which is one of the typical animal models that are used for the development of peripheral atherosclerosis. MATERIALS AND METHODS: Bcl-2 transgenic mice were cross hybridized with apolipoprotein E deficient mice. Systemic analysis of the distribution and severity of their atherosclerotic lesions was done by dissecting microscopy, and the histological characteristics of the lesions were evaluated in normal chow-fed, 9-month-old apolipoprotein-E deficient/Bcl-2 transgenic mice (n=6) and apolipoprotein-E deficient mice (n=6). RESULTS: The distribution and severity of atherosclerotic lesions at the peripheral arteries were less in the apolipoprotein-E deficient/Bcl-2 transgenic mice. Acellular lipid core formation, destruction of the smooth muscle cell layers in the media and infiltration of inflammatory cells in the adventitia were much less in the apolipoprotein-E deficient/Bcl-2 transgenic mice. The lipid profile was similar in both groups. CONCLUSION: The effect of Bcl-2 gene expression on the peripheral atherosclerosis was related with the inhibition or the delay of atherosclerotic lesion progression, such as the reduction of amount of the acellular lipid core, maintenance of vascular smooth muscle cell integrity and the reduction of adventitial inflammation, and this was achieved regardless of serum cholesterol level.

Induction of Apoptosis by a Novel Bcl-2 Protein, Bak-like without a BH3 Domain

OBJECTIVE: In a variety of physiologic settings, cells are eliminated by apoptosis, a genetically encoded process of cellular suicide. Bak, a member of the Bcl-2 protein family, accelerates apoptosis by an unknown mechanism. Here, we describe the identification and characterization of a complementary DNA that encodes a previously unknown Bcl-2 homologue designated Bak-like. METHODS: We identified a splicing variant of Bak with a lacked BH3 domain from human full-length cDNA bank. The expression of Bak-like was examined by northern blot analysis and polymerase chain reaction. To investigate whether Bak-like might arise from alternative splicing of mRNA of Bak, Southern blot analysis was executed. Apoptosis in transfected HeLa cells was analyzed by direct counting of viable cells. We examined the location of Bak-like in individual living cells by using EGFP fusion constructs and confocal microscope. RESULTS: Bak-like cDNA coded a protein consisting of 101 amono acid, and conserved BH1 and BH2 domains like Bak but not BH3 domain. Bak-like mRNA was about 2.4kb similar to bak. Bak-like was assumed to be an alternative splicing variant of Bak and to concern with promotion of apoptosis. GFP-bak-like markedly changed its intracellular distribution, relocating within cells during apoptosis from a diffuse to a punctate pattern. CONCLUSION: Our results define a novel splicing form of the bak gene and demonstrate that this variant without a conserved BH3 domain appears to contain the BH1 and BH2 domains and the transmembrane sequence for apoptosis induction by channel-forming Bcl-2 proteins. Like Bak, Bak-like gene product primarily enhanced apoptotic cell death following an appropriate stimulus.

Expression of Bcl-2 and Bax in cis-Diamminedichloroplatinum (II)-Resistant Bladder Cancer Cell Lines / 대한비뇨기과학회지

PURPOSE: Development of drug resistance has been the major obstacle in cis-Diamminedichloroplatinum (II) (cisplatin)-based combination chemotherapy in the treatment of advanced bladder cancer for which a variety of mechanisms has been suggested. We investigated to determine the changes of expression of apoptotic regulator proteins Bcl-2 and Bax in cisplatin-resistant bladder cancer cell lines and the reversibility of chemoresistance with antisense oligonucleotide against Bcl-2. MATERIALS AND METHODS: In T24, J82, 253J, 253J-BV and HT-1376 bladder cancer cell lines, we established cisplatin-resistance using stepwise exposure to cisplatin. The changes of Bcl-2 and Bax proteins in the resistant cell lines were determined by Western blot. Then, after administration of antisense oligonucleotide targeting the Bcl-2 coding sequence to the T24, T24-R1, and T24-R2 cell lines with lipofectamine, changes of Bcl-2 expression were determined along with cisplatin cytotoxicity before and after transfection. RESULTS: We confirmed the acquisition of cisplatin resistance in all 5 cell lines as the percent increase of IC50 in each cell lines were 210%, 175%, 181%, 280% and 153%, respectively. The expression of Bcl-2 protein increased in all 5 cisplatin-resistant cell lines, while the expressions of Bax decreased in 4 of 5 cisplatin-resistant cell lines. Treatment with antisense oligonucleotide significantly enhanced the cytotoxicity of cisplatin in T24, T24-R1 and T24-R2 cell lines. CONCLUSIONS: These results suggest that the up-regulation of Bcl-2 expression as well as down-regulation of Bax expression may be one of the mechanisms of cisplatin resistance in bladder cancer cells, and antisense Bcl-2 oligonucleotide may be helpful in chemotherapy of bladder cancer by reversing cisplatin resistance.

Mechanism of Azaline B-induced Apoptosis of Ventral Prostate in Rat / 대한비뇨기과학회지

PURPOSE: Androgen deprivation triggers a sequence of events that activates apoptotic cell death of the androgen-dependent epithelial cells within the rat ventral prostate, ultimately resulting in the involution of the gland. To investigate the mechanism of azaline B-dependent apoptosis in the rat ventral prostate, the regulation of apoptosis-related genes were examined. MATERIALS AND METHODS: Azaline B was subcutaneously injected in Sprague-Dawley rat. Fas receptor(Fas), Fas ligand(FasL), bcl-2 mRNA, and protein levels were detected by RT-PCR and Western blot. Azaline B-dependent apoptosis was determined by TUNEL and DNA fragmentation assay. Transacting factor of FasL promoter was identified by DNA footprinting and DNA mobility shift assay. RESULTS: The prostate regressed after azaline B treatment in rat, and the involuted ventral prostate regenerated after testosterone pretreatment. Apoptosis of the ventral prostate was detected by TUNEL assay and apoptotic DNA fragmentation assay after azaline B treatment. The levels of Fas and FasL mRNA and protein increased after azaline B treatment. In DNase I footprinting assay with FasL promoter using nuclear extract prepared from control prostate, at least two sites were protected: SP-1 binding site at -283bp and prostate-unidentified factor(P-UF) binding site at -247bp. SP-1 binding activity vanished in the nuclear extract prepared from azaline B-treated rats. In the DNA mobility shift assay, SP-1 binding activity decreased after azaline B treatment. Bcl-2 mRNA and protein were downregulated after azaline B treatment. CONCLUSIONS: These results suggest that Fas/FasL system and Bcl-2 are important to azaline B-dependent apoptosis in rat ventral prostate and that SP-1 is related to azaline B-dependent regulation of the FasL gene.