Significant Decrease In Bcl-2 Expression Research Articles

Potential role of Bcl2 in lipid metabolism and synaptic dysfunction of age-related hearing loss

Age-related hearing loss (ARHL) is a prevalent condition affecting millions of individuals globally. This study investigated the role of the cell survival regulator Bcl2 in ARHL through in vitro and in vivo experiments and metabolomics analysis. The results showed that the lack of Bcl2 in the auditory cortex affects lipid metabolism, resulting in reduced synaptic function and neurodegeneration. Immunohistochemical analysis demonstrated enrichment of Bcl2 in specific areas of the auditory cortex, including the secondary auditory cortex, dorsal and ventral areas, and primary somatosensory cortex. In ARHL rats, a significant decrease in Bcl2 expression was observed in these areas. RNAseq analysis showed that the downregulation of Bcl2 altered lipid metabolism pathways within the auditory pathway, which was further confirmed by metabolomics analysis. These results suggest that Bcl2 plays a crucial role in regulating lipid metabolism, synaptic function, and neurodegeneration in ARHL; thereby, it could be a potential therapeutic target. We also revealed that Bcl2 probably has a close connection with lipid peroxidation and reactive oxygen species (ROS) production occurring in cochlear hair cells and cortical neurons in ARHL. The study also identified changes in hair cells, spiral ganglion cells, and nerve fiber density as consequences of Bcl2 deficiency, which could potentially contribute to the inner ear nerve blockage and subsequent hearing loss. Therefore, targeting Bcl2 may be a promising potential therapeutic intervention for ARHL. These findings provide valuable insights into the molecular mechanisms underlying ARHL and may pave the way for novel treatment approaches for this prevalent age-related disorder.

Lemongrass Essential Oil Attenuates Perfluorooctane Sulfonate-Induced Jejunal Mucosal Injury in Rat: A Histological, Immunohistochemical, and Biochemical Study.

Perfluorooctane sulfonate (PFOS) has harmful impacts on various organs, including the intestine. Lemongrass essential oil (LGEO) has anti-inflammatory, anti-oxidant, antibacterial, and immunomodulatory effects. This study investigated the impact of PFOS on the mucosa of the jejunum of rats and evaluated LGEO's protective impact. Four groups of rats were created: control, LGEO (100 mg/kg/day), PFOS (5 mg/kg/day), and LGEO-PFOS group. The agents were given orally for 28 days. Oxidative stress parameters, pro-inflammatory cytokines, and caspase-3 were measured in jejunal homogenates. Rat jejunal sections were evaluated histologically (light and electron microscopic examination) and immunohistochemically [for tumor necrosis factor-α (TNF-α), Proliferating cell nuclear antigen (PCNA), cyclooxygenase-2 (COX2), and Bcl2]. PFOS significantly elevated oxidative stress, pro-inflammatory cytokines, caspase-3, and gene expression of nuclear factor kappa B (NF-kB) and inducible nitric oxide synthetase (iNOS). The disturbed architecture of jejunal villi and crypts was demonstrated. Immunohistochemically, a significant rise in TNF-α, PCNA, and COX2 and a significant decrease in Bcl2 expression were revealed compared to control group. Further ultrastructural alterations included dilated RER, mitochondria with destroyed cristae, vacuolated cytoplasm, and shrunken condensed nuclei of enterocytes. LGEO treatment significantly reduced these harmful effects. LGEO protected against PFOS-induced jejunal damage by reducing the oxidative, inflammatory, and apoptotic impacts.

Effects of metformin, letrozole and atorvastatin on inflammation and apoptosis in experimental peritoneal and ovarian endometriosis in the rat

Endometriosis is a common gynecological hurting disorder in which tissue is similar to the tissue that normally lines the inner layer of the uterus. It often causes fertility problems. Unfortunately, effective treatments are limited. Therefore it's important to explore an imperative and easily accessible treatment to alleviate the probable pathologies and preserve fertility in endometriosis. Consequently, we aimed to investigate the effects of metformin, letrozole, and atorvastatin on inflammation and apoptosis in experimentally induced ovarian and peritoneal endometriosis in rat models. In the present study, 35 rats were randomly divided into five groups. Group 1: sham-operated control group. Group 2: untreated endometriosis group. Group 3: given 100 mg/kg/day of oral metformin. Group 4: given 0.1 mg/kg/day of oral letrozole. Group 5: given 2.5 mg/kg/day of oral atorvastatin. At the end of the 28 days, we examined Ki67, Bax and Bcl-2 immunoexpressions in ovarian and peritoneal tissues, and IL-6, IL-8, and TNF-α levels were evaluated from the peritoneal fluid. All medical treatment groups showed a significant decrease in Ki67 expression. A significant increase in Bax expression was also observed in all samples from all medical treatment groups (other than the untreated endometriosis groups). Further, a significant decrease in Bcl-2 expression was found in all medical treatment groups. IL-6, IL-8, and TNF-α levels were significantly lower in all medical treatment groups than in the endometriosis groups. In conclusion; Metformin, letrozole, and atorvastatin showed apoptosis induction and anti-inflammatory effects on both ovarian and peritoneal endometriosis in experimental models.

PDE5 inhibitor sildenafil attenuates cardiac microRNA 214 upregulation and pro-apoptotic signaling after chronic alcohol ingestion in mice.

Abusive chronic alcohol consumption can cause metabolic and functional derangements in the heart and is a risk factor for development of non-ischemic cardiomyopathy. microRNA 214 (miR-214) is a molecular sensor of stress signals that negatively impacts cell survival. Considering cardioprotective and microRNA modulatory effects of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, we investigated the impact of chronic alcohol consumption on cardiac expression of miR-214 and its anti-apoptotic protein target, Bcl-2 and whether sildenafil attenuates such changes. Adult male FVB mice received unlimited access to either normal liquid diet (control), alcohol diet (35% daily calories intake), or alcohol + sildenafil (1mg/kg/day, p.o.) for 14weeks (n = 6-7/group). The alcohol-fed groups with or without sildenafil had increased total diet consumption and lower body weight as compared with controls. Echocardiography-assessed left ventricular function was unaltered by 14-week alcohol intake. Alcohol-fed group had 2.6-fold increase in miR-214 and significant decrease in Bcl-2 expression, along with enhanced phosphorylation of ERK1/2 and cleavage of PARP (marker of apoptotic DNA damage) in the heart. Co-ingestion with sildenafil blunted the alcohol-induced increase in miR-214, ERK1/2 phosphorylation, and maintained Bcl-2 and decreased PARP cleavage levels. In conclusion, chronic alcohol consumption triggers miR-214-mediated pro-apoptotic signaling in the heart, which was prevented by co-treatment with sildenafil. Thus, PDE5 inhibition may serve as a novel protective strategy against cardiac apoptosis due to chronic alcohol abuse.

Cystathionine β Synthase/Hydrogen Sulfide Signaling in Multiple Myeloma Regulates Cell Proliferation and Apoptosis.

Objective-To investigate cystathionine β synthase (CBS)/hydrogen sulfide (H2S) signaling in multiple myeloma (MM) patients and to identify its effect on the proliferation of U266 cells. Methods-Bone marrow samples of 19 MM patients and 23 healthy donors were collected. qRT-PCR was performed to measure the mRNA expression levels of H2S synthases, cystathionine β synthase, and cystathionine γ lyase. ELISA assays quantified the amount of H2S produced by the two enzymes CBS and CSE. CCK-8 experiment was used to investigate the influence of the CBS inhibitor amino oxyacetic acid and the CSE inhibitor propargylglycine on the proliferation of U266 cells. Flow cytometry and western blotting were performed to determine the effects of AOAA, PAG, and NaHS on cell cycle distribution as well as Caspase-3 and Bcl-2 expression. Results-Patients with MM had higher level of CBS compared with healthy donors. AOAA significantly inhibited cell proliferation in both a time and concentration dependent characteristic, whereas PAG does not. After 24 hours of treatment, AOAA significantly elevated the G0/G1 phase proportion of cells, and reduced the cell distribution in both S and G2/M phases, while NaHS accelerated cell cycle progression by reducing the relative number of cells in G0/G1 phase and increasing the proportion of cells in the G2/M phase. Moreover, AOAA abolished the impact of NaHS on cell cycle progression of U266 cells. AOAA treatment also led to a significant decrease in Bcl-2 expression and dramatic increase in Caspase-3 expression, though NaHS reversed these effects. Conclusion-CBS/H2S system might have a certain effect on the proliferation and apoptosis of MM cells.

Combination of Enasidenib and Venetoclax Shows Superior Anti-Leukemic Activity Against IDH2 Mutated AML in Patient-Derived Xenograft Models

Mutations in isocitrate dehydrogenase 2 (IDH2) promote AML pathogenesis through production of 2-hydroxyglutarate (2-HG). Enasidenib is an inhibitor of mutant IDH2 activity and induces the differentiation of IDH2-mutated leukemic blasts. In a phase I/II clinical trial, enasidenib monotherapy resulted in an overall response rate of 40% and median duration of response of 6 months in relapsed/refractory AML (Stein et. al. Blood 2017).We previously discovered that mutant IDH activity sensitizes AML cells to BCL-2 inhibition through accumulation of 2-HG (Chan et. al. Nature Medicine 2015). Pharmacologic inhibition of BCL-2 activity with venetoclax, a highly specific BH3 mimetic, preferentially targets IDH-mutated human AML cells. In a phase II clinical trial of venetoclax monotherapy, IDH-mutated relapsed/refractory AML patients had a response rate of 33% compared to 10% in IDH-wildtype patients (Konopleva et. al. Cancer Discovery 2016).Based on the above findings, we hypothesized that combination therapy with enasidenib and venetoclax may demonstrate superior anti-leukemic activity in comparison to single agents in the treatment of IDH2-mutant AML. However, given that the mechanism by which IDH mutations increase BCL-2 dependence is via 2-HG, reduction of 2-HG with enasidenib may antagonize venetoclax activity. As such, we further hypothesized that a sequential dosing schedule may be superior to concurrent dosing.To test our hypotheses, we conducted a preclinical study to evaluate the efficacy of monotherapy versus combination therapy in reducing the leukemic burden in three patient-derived xenograft (PDX) models of human IDH2-mutant AML. All three PDX models were derived from samples with co-occurring IDH2R140Q and NPM1c mutations. Engrafted animals were randomly assigned to one of six treatment arms (N=5 per arm): vehicle (arm 1), enasidenib alone (arm 2), venetoclax alone (arm 3), concurrent combination (arm 4), and sequential combinations (arms 5 and 6; see Figure 1 for details). Enasidenib and venetoclax were administered by oral gavage at a dose of 40 mg/kg twice a day and 100 mg/kg daily, respectively. Tumor burden was measured in bone marrow samples collected immediately prior to treatment and every 2 weeks during the 12-week treatment period.Concurrent combination treatment (arm 4) resulted in the greatest reduction in leukemia engraftment compared to all other treatment arms, including the sequential dosing arms, in two of the three models (#1 and #2, henceforth termed “responders”; Figure 2A). Although venetoclax monotherapy reduced engraftment in all three models, persistent disease above a threshold of 0.1% was detectable in 12 of 13 animals by flow cytometry (9 of 13 by ddPCR) after 12 weeks of treatment (Figure 2B). In contrast, disease was detectable in only 2 of 9 animals (0 of 9 by ddPCR) treated with concurrent therapy in responders. In the remaining model (#3, henceforth termed “non-responder”), combination therapy was not superior to venetoclax monotherapy but importantly, co-treatment with enasidenib did not antagonize venetoclax activity. Interestingly, enasidenib monotherapy increased expression of myeloid differentiation markers, CD15 and CD11b, only in responders, indicating that differentiation might be a precondition for responsiveness to concurrent therapy. We confirmed that the lack of response in non-responders was not due to selection of an IDH2 wildtype clone or failure to block 2-HG production by enasidenib.To gain insights into the mechanism by which enasidenib might enhance venetoclax sensitivity, we performed quantitative intracellular flow cytometry staining for the anti-apoptotic proteins BCL-2, BCL-xL and MCL-1 in leukemic cells collected after 12 weeks of treatment. Enasidenib monotherapy resulted in a significant decrease in BCL-2 expression in responders. The reduction in anti-apoptotic protein expression could potentiate mitochondrial priming and sensitization to venetoclax.In summary, our findings demonstrate that concurrent combination therapy with enasidenib and venetoclax is a promising therapeutic approach for IDH2-mutated AML. Responsiveness to combination therapy is associated with enasidenib-induced differentiation and reduction in anti-apoptotic protein expression. Our findings support ongoing and future clinical investigations in combination therapies with mutant IDH and BCL-2 inhibitors. DisclosuresCojocari:AbbVie Inc: Employment. Phillips:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. Leverson:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. MacBeth:Celgene Corporation: Employment, Equity Ownership. Nicolay:Agios: Employment. Narayanaswamy:Agios: Employment. Ronseaux:Agios: Employment. Liu:Agios: Employment, Equity Ownership. Chan:AbbVie: Research Funding; Genentech: Research Funding; Celgene: Research Funding.

Replication Study: Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia.

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fung et al., 2015), that described how we intended to replicate selected experiments from the paper "Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia" (Dawson et al., 2011). Here, we report the results of those experiments. We found treatment of MLL-fusion leukaemia cells (MV4;11 cell line) with the BET bromodomain inhibitor I-BET151 resulted in selective growth inhibition, whereas treatment of leukaemia cells harboring a different oncogenic driver (K-562 cell line) did not result in selective growth inhibition; this is similar to the findings reported in the original study (Figure 2A and Supplementary Figure 11A,B; Dawson et al., 2011). Further, I-BET151 resulted in a statistically significant decrease in BCL2 expression in MV4;11 cells, but not in K-562 cells; again this is similar to the findings reported in the original study (Figure 3D; Dawson et al., 2011). We did not find a statistically significant difference in survival when testing I-BET151 efficacy in a disseminated xenograft MLL mouse model, whereas the original study reported increased survival in I-BET151 treated mice compared to vehicle control (Figure 4B,D; Dawson et al., 2011). Differences between the original study and this replication attempt, such as different conditioning regimens and I-BET151 doses, are factors that might have influenced the outcome. We also found I-BET151 treatment resulted in a lower median disease burden compared to vehicle control in all tissues analyzed, similar to the example reported in the original study (Supplementary Figure 16A; Dawson et al., 2011). Finally, we report meta-analyses for each result.

Molecular and immunohistochemical expression of apoptotic proteins Bax, Bcl-2 and Caspase 3 in infantile hemangioma tissues as an effect of propranolol treatment

BackgroundInfantile hemangiomas (IHs) are the most common benign tumors of childhood. They are characterized by a unique clinical course with two phases, proliferation and involution, which are followed by regression. The therapy of infantile hemangiomas was revolutionized in 2008 by the introduction of propranolol, however, the mechanism of its influence on hemangiomas remains unclear. MethodsThe study included 71 patients with IHs, 27 of whom were treated with propranolol while the remaining 44 were used as a comparative group. The expression of Bcl-2, Bax and Caspase3 was determined with immunohistochemistry and mRNA of Bax, Bcl-2 and Caspase3 were assessed with the use of RT-PCR. ResultsBoth methods revealed a statistically significant decrease in Bcl-2 expression and an increase in Bax in IHs tissues after propranolol treatment. ConclusionsThe results obtained for Bax and Bcl-2 proteins may indicate a link between the effect of propranolol and apoptosis. Higher Bax and lower Bcl-2 expression in the propranolol treated group indicates a strong pro- apoptotic action countering any anti-apoptotic activity; apoptosis was indicted in IH tissue as a potential result of propranolol treatment, with potential clinical impact in other tumors.

Effect of annexin A7 suppression on the apoptosis of gastric cancer cells

Understanding the molecular mechanism of gastric cancer cell apoptosis is pivotal for the development of precise therapies targeting this disease. In the present study, we examined the effects of annexin A7 inhibition on the apoptosis of gastric cancer cells and the growth of tumour xenografts in vivo. Expression of annexin A7 in BGC823 cells was suppressed by small interference RNA, and cells apoptosis was assessed by flow cytometry. The mechanism by which annexin A7 mediates apoptosis in BGC823 cells was explored by determining the expression of key apoptosis regulators. In addition, by suppressing annexin A7 in BGC823 cells with small hairpin RNA, we studied the effects of annexin A7 inhibition on in vivo tumour growth. Our results showed that inhibiting annexin A7 expression induced more than fivefold increase in BGC823 cell apoptosis in vitro. This was in concord with a significant decrease of Bcl-2 expression and increases of Bax, Caspase-3, and Caspase-9. The activities of caspase-3 and caspase-9 were increased by 2.95 ± 0.18 and 3.70 ± 0.33 times, respectively, upon the annexin A7 downregulation in BGC823 cells. Importantly, suppressing annexin A7 showed the same apoptotic mechanism in vivo and significantly inhibited the growth of BGC823 xenografts in mice. These data suggest that annexin A7 likely protects gastric cells from apoptosis and targeting it may represent a valuable strategy in future therapeutic development.

Liver epithelial cells inhibit proliferation and invasiveness of hepatoma cells

Hepatocellular carcinoma (HCC) is a worldwide malignancy with poor prognosis. Liver progenitors or stem cells could be a potential therapy for HCC treatment since they migrate toward tumors. Rat liver epithelial (RLE) cells have both progenitor and stem cell-like properties. Therefore, our study elucidated the therapeutic effect of RLE cells in rat hepatoma cells. RLE cells were isolated from 10-day old rats and characterized for stem cell marker expression. RLE cells and rat hepatoma cells (H4-IIE-C3 cells) were co-cultured and divided into four groups with different ratios of RLE and hepatoma cells. Group A had only rat hepatoma cells as a control group. The ratios of rat hepatoma and RLE cells in group B, C and D were 5:1, 1:1 and 1:5, respectively. Effective inhibition of cell proliferation and migration was found in group D when compared to group A. There was a significant decrease in Bcl2 expression and increase in late apoptosis of rat hepatoma cells when adding more RLE cells. RLE cells reduced cell proliferation and migration of rat hepatoma cells. These results suggested that RLE cells could be used as a potential cell therapy.

Increase in Bcl2 expression of penile and prostate cells of Sprague Dawley male rats following treatment with buceng (combination of Pimpinella alpina molk with Eurycoma longifolia Jack)

Background: Treatment with buceng combination of Eurycoma longifolia Jack and Pimpinella alpine Molk has been proven to increase testosterone level, decrease apoptosis and caspase3 expression. Bcl2 is an antiapoptotic protein found in cytoplasm which inhibits cells apoptosis. This study was aimed to investigate the effect of buceng on Bcl2 expression on penile and prostate tissues of the rats. Methods: In this experimental study, 24 male Sprague Dawley rats of 90 days old, weighing ± 300 grams, were randomly assigned into four groups. Group A, normal rats. Group B, castrated rats and treated with buceng 100 mg/day, per oral (Cast-Bcg); Group C, castrated rats and treated with 2 ml of water as placebo against buceng (Cast-Plac). Group D, castrated rats, treated with mesterolone 6.75 mg/day, per oral, as exogenous testosterone (Cast-Mest). All rats were treated for 30 days. Manova test was used to analyze the different expression of Bcl2 among groups with significance level at p ≤ 0.05. Results: Castration was associated with significant decrease of Bcl2 expression in the penile and prostate tissues (53.0 and 50.9%, respectively) compared to normal rats (82.6 and 84.2%, respectively, p < 0.001). Treatment with mesterolone reversed Bcl2 expression (77.1 and 78.1%) to a near normal level. The same level of Bcl2 expression was also observed with buceng treatment (73.8 and 78.2%).Conclusion: The treatment with buceng could enhance Bcl2 expression in penile and prostate tissues, comparable to normal rats and mesterolone treated rats.

Central nervous system toxicity after acute oral formaldehyde exposure in rabbits

Formaldehyde (FA) is one of the most widely used chemical compounds in industrial field. It is described as toxic, particularly to the nervous system, the urogenital system, and the respiratory tracts. In this study, we determined the effects of acute oral exposure to FA in rabbit brain tissue. A total of 16 rabbits were selected and divided into 2 groups: formaldehyde group (group F) and control group (group C). FA was administered to group F at a rate of 40 mg/kg/day via a nasogastric tube for 5 days. Saline was similarly administered to the eight controls. All the animals were euthanized after 5 days of exposure, and brain tissue samples were collected in 10% neutral formalin and embedded in paraffin. To investigate the effects of FA on the apoptotic process, we examined active caspase-3, Bax, and Bcl-2 immunohistochemical expression and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate -biotin nick-end labeling (TUNEL) reactivity in the rabbit brains. In addition, glial fibrillary acidic protein (GFAP) was biochemically assessed in brain tissue samples for neurotoxicity. We found that FA treatment caused a significant decrease in Bcl-2 expression and an increase in active caspase-3 and Bax expressions as well as an increase in the number of TUNEL-positive apoptotic cells. The GFAP level was found to be significantly higher in group F. In conclusion, acute oral exposure to FA caused DNA damage, apoptosis, and neuronal injury in the rabbit brains.

Evaluation of apoptosis regulatory proteins in response to PUVA therapy for psoriasis

The histopathologic changes characteristic of psoriasis might be related to suppressed apoptosis. One of the actions of psoralen ultraviolet A (PUVA) in psoriasis could be exerted through induction of apoptosis of keratinocytes and lymphocytes; however, its exact molecular mechanism is still confusing. In this study, we evaluated the expression of pro-apoptotic (P53, Fas and Bax) and anti-apoptotic (Bcl-2) proteins correlating it with apoptotic index (AI) and epidermal thickness in psoriatic skin before and after PUVA therapy. Lesional and non-lesional skin biopsy specimens were obtained from 10 patients with generalized plaque psoriasis before and after 8 weeks of PUVA therapy. Histometric measurements of epidermal thickness as well as P53, Fas, Bax and Bcl-2 expressions were evaluated using immunoperoxidase technique and apoptotic cells were detected by terminal deoxynucleotide transferase (TdT) mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. After PUVA therapy, the epidermal thickness of psoriatic skin was significantly decreased (P < 0.001) and keratinocytes of psoriatic skin showed significant increased expression of P53 (P < 0.001), Fas (P < 0.001) and Bcl-2 (P < 0.001) with no significant change in Bax expression (P > 0.05). Apart from significant decrease of Bcl-2 expression (P = 0.01), no significant difference in all previous markers were encountered in lymphocytes (P53, Fas and Bax; P > 0.05) after PUVA therapy. The AI was significantly increased (P = 0.008) after PUVA therapy especially in lymphocytes (P = 0.002). The present study suggests that one of the actions of PUVA therapy in psoriasis might be exerted through induction of apoptosis especially of lymphocytes by suppression of Bcl-2 expression and of keratinocytes through P53 and Fas pathways leading to healing of psoriasis.

Polycyclic Aromatic Hydrocarbons: Role of Apoptosis in Dermatotoxic and Carcinogenic Effect in Asphalt Road Paving Workers

Asphalt contains a complex mixture of polycyclic aromatic hydrocarbons (PAHs). This work aimed firstly to assess the dermatotoxic and carcinogenic risk associated with chronic PAHs exposure, and secondly to investigate the causal relationship between PAHs exposure and cancer through studying the effect of PAHs on apoptosis. This effect was studied by examining the expression pattern of P53, Bax and Bcl-2 apoptotic proteins in skin specimens from road paving workers. The study was conducted on one hundred and fifty two male subjects classified into 122 asphalt fumes exposed workers (tested group) and 30 non exposed workers (control group); careful skin examination and skin biopsies were obtained from all participants after written consent. Biopsies were examined histopathologically and immunohistochemically. SPSS version 15.0 was used for statistical analysis. Results showed that 71 (58.19%) PAHs exposed workers had erythema, itching, excoriations, chronic dermatitis, chemical keratosis, keratoacanthoma (KA) and nine (7.38%) exposed workers had squamous cell carcinoma (SCC). Imunohistochemically, wild type P53 was significantly higher in epidermal keratinocytes of PAHs exposed normal uninvolved (non lesional) skin (t=0.51, P=0.04) and mutant type P53 was significantly higher in SCC cases (t=4.79, p=0.003) when compared with control. A significant increase in Bax expression was observed in all asphalt exposed workers when compared with the control (t=2.73, P=0.03). A significant decrease in Bcl-2 expression was noted in PAHs exposed un-involved skin (t=2.49, P=0.047) while none of the 9 tested SCC cases were positive for Bcl-2. It could be concluded that chronic exposure to PAHs fumes in asphalt workers may increase the risk for developing dermatotoxic and/ or cancer through disturbing apoptosis. Although PAHs exposure can disturb P53, Bax and Bcl-2 apoptotic protein, more extensive researches on other factors involved in controlling apoptosis as Bcl-xl, Caspase family and Fas are to be undertaken.

Plant flavone apigenin inhibits HDAC and remodels chromatin to induce growth arrest and apoptosis in human prostate cancer cells: in vitro and in vivo study.

Apigenin (4',5,7,-trihydroxyflavone), an anticancer agent, selectively toxic to cancer cells induces cell cycle arrest and apoptosis through mechanisms that have not been fully elucidated. Our studies indicate that apigenin-mediated growth inhibitory responses are due to inhibition of class I histone deacetylases (HDACs) in prostate cancer cells. Treatment of PC-3 and 22Rv1 cells with apigenin (20-40 µM) resulted in the inhibition of HDAC enzyme activity, specifically HDAC1 and HDAC3 at the protein and message level. Apigenin-mediated HDAC inhibition resulted in global histone H3 and H4 acetylation, as well as localized hyperacetylation of histone H3 on the p21/waf1 promoter. A corresponding increase was observed in p21/waf1 and bax protein and mRNA expression after apigenin exposure, consistent with the use of HDAC inhibitor, trichostatin A. The downstream events demonstrated cell cycle arrest and induction of apoptosis in both cancer cells. Studies of PC-3 xenografts in athymic nude mice further demonstrated that oral intake of apigenin at doses of 20 and 50 µg/mouse/d over an 8-wk period resulted in a marked reduction in tumor growth, HDAC activity, and HDAC1 and HDAC3 protein expression at both doses of apigenin. An increase in p21/waf1 expression was observed in apigenin-fed mice, compared to the control group. Furthermore, apigenin intake caused a significant decrease in bcl2 expression with concomitant increase in bax, shifting the bax/bcl2 ratio in favor of apoptosis. Our findings confirm for the first time that apigenin inhibits class I HDACs, particularly HDAC1 and HDAC3 and its exposure results in reversal of aberrant epigenetic events that promote malignancy. © 2011 Wiley Periodicals, Inc.

Degenerative disc disease of herniated intervertebral discs is associated with extracellular matrix remodeling, vimentin-positive cells and cell death

We studied patients with degenerative disc disease (DDD) to demonstrate that i) remodeling of the extracellular matrix (ECM) in the intervertebral disc (IVD), particularly the elastic fiber system, of subjects with herniated discs is dysregulated and that ii) it is accompanied by accelerated elastin degradation due to increased expression of matrix metalloprotease-9 (MMP-9). Moreover we wanted to obtain a deeper insight into the pathogenesis of DDD through the study of ECM calcification, DNA fragmentation using TUNEL analysis, BAX, bcl-2 and vimentin immunopositive cells. We studied herniated discs from patients of three age groups (group 1=30-40 years; group 2=40-50 years; and group 3=50-65 years) to evaluate the oxytalan fiber systemMMP-9, apoptosis and vimentin immunopositive cells. The results demonstrated the presence of oxytalan fibers in the annulus fibrosus (AF) and the nucleus pulposus (NP) of herniated discs. In the AF oxytalan fibers replaced disrupted mature elastic fibers in calcified areas, while in the NP they were mostly found in nests at the periphery of chondrocytes. MMP-9 was prevalently observed in NP nests above all in group 1 and group 3 discs while group 2 exhibited a lower MMP-9 immunostaining. Activation of the apoptotic process was demonstrated by upregulated BAX expression in group 3. BAX immunopositivity was inversely mirrored by a significant decrease in bcl-2 expression. Intermediate filament protein vimentin was strongly expressed only in group 1 samples. A large number of apoptotic TUNEL+ cells was observed in group 3 specimens. The presence of oxytalan fibers may be the result of a process of incomplete elastogenesis, or a response to mechanical stress trying to functionally replace the lack of elastic fibers. MMP-9 expression seems to relate to disc damage, while chondrocyte BAX upregulation and TUNEL+ cell staining revealed apoptosis activation regardless of patient age. Vimentin immunopositivity was clearly detected in group 1 annulus fibrosus and nucleus pulposus cells. In conclusion, as demonstrated by the vimentin-positive cells, the injured IVD has endogenous resources that can stem the DDD damage, including substitution of damaged elastic fibers by oxytalan fibers. In addition, induction of apoptosis suggests an increased cell turnover in response to repair needs.

Preoperative evaluation of P53 and bcl-2 over expression in clinical stage 1 endometrial carcinoma and their correlation with surgico-pathological data and prognosis of patients

Introduction: P53 and bcl-2 over expression was reported to affect the biology and prognosis of patients endometrial carcinoma. Methods: This prospective study included 38 patients with histologically confirmed endometrial carcinoma and staged clinically as stage I. Immunohistochemical staining of the tumor specimens obtained by dilatation and curettage (D&C) with P53 and bcl-2 monoclonal antibodies was done. The surgical, pathological and follow up data of all patients were studied. Results: There were 18 cases (47.4%) with positive P53 over expression. P53 over expression was found to be associated with increasing grade of differentiation, advanced stage, and non endometrioid type (significant), and increased depth of myometrial invasion (non-significant). It was associated with more recurrence rate (22% versus 15%) and shorter mean survival time (33.9 versus 29.2 months). Bcl-2 expression was present in24 cases (63.2%) of the studied group. There was significant decrease in bcl-2expression with poorly differentiated, advanced stage, increased depth of myometrial invasion, and non-endometrioid type. It was not significantly correlated with recurrence rate and mean survival time. Conclusion: P53 over expression in the D&C specimens was associated with adverse surgicopathological criteria, increased mortality rate, and shorter survival time in patients with endometrial carcinoma. A significant decrease in bcl-2 expression was associated with adverse surgicopathological criteria, but it was not significantly correlated with prognosis of the patients.

Immunohistochemical Study for the Expression of Bcl-2 Family Proteins in Walker 256 Carcinosarcoma Cells under the Influence of Cytostatic Drugs

Immunohistochemical study was performed to evaluate the expression of Bcl-2 family proteins (Bcl-2, Bax, and Bad) in Walker 256 carcinosarcoma cells after implantation into the thigh muscle of male Wistar rats (10(6) cells). The experiment was conducted under conditions of spontaneous tumor development and individual or combined treatment with melatonin and cyclophosphamide. The use of melatonin as monotherapy or in combination with cyclophosphamide was followed by a significant decrease in Bcl-2 expression in carcinosarcoma cells. The Bcl-2/Bax and Bcl-2/Bad ratio was significantly reduced under these conditions (particularly after combined treatment with cytostatic drugs). These changes were accompanied by a significant (by 93.61%) decrease in the volume of transplantable tumor on day 14. Daily treatment with melatonin was accompanied by significant changes in the structure of Walker 256 carcinosarcoma. It was manifested in the formation of connective tissue septa and pseudofollicles.

GLI1 Transcription Factor: A Potential Therapeutic Target in B-CLL.

B-cell Chronic Lymphocytic Leukemia (B-CLL) is characterized by accumulation of clonal lymphocytes resistant to apoptosis. Therefore, identification of molecules responsible for the increased resistance to apoptosis is warranted. The identified molecules could be further targeted to develop effective therapy. Emerging evidence on the mechanism of resistant to apoptosis in several cancers suggests that GLI1 transcription factor, target of hedgehog signaling, may have a potential role in the increased resistance to apoptosis in B-CLL. However such studies have not been reported for B-CLL. Therefore, we investigated the role of sonic hedgehog (Shh)-GLI signaling in the pathogenesis B-CLL using 38 different patient samples and different parameters. The results were analyzed based on good and poor clinical outcome B-CLL subgroups identified on the basis of cytogenetic abnormality and/or CD38 expression levels. Microarray and RT-PCR analyses demonstrated active Shh-GLI signaling molecules in B-CLL cells including GLI1. Our results demonstrate that GLI1 transcripts were significantly (p = 0.003) over-expressed in B-CLL cells of poor clinical outcome patient subgroups (17pdel, 11qdel, trisomy12; N = 20) compared to good clinical outcome (13qdel, normal karyotype; N = 18) as observed in microarray analysis and confirmation by real time PCR. In addition, similar observation was also made with poor prognosis (high CD38 expression) versus good prognosis (low CD38 expression) B-CLL subgroups. Furthermore, higher expression of GLI1 was associated with significantly (p = 0.02) shorter time to treatment compared to lower GLI1 expressing B-CLL patients as determined by Kaplan-Meier survival analysis using log rank test, suggesting the increased expression of GLI1 and its association with the disease progression in poor clinical outcome B-CLL patient sub-groups. Addition of exogenous Shh on the significantly (p<0.01) increased cell survival and up-regulation of GLI1 transcripts, and decreased cell survival and downregulation of GLI1 transcripts in the presence of cyclopamine, known inhibitor of signaling, as determined by MTT assay and RT-PCR in vitro, suggesting the role of GLI1 in the survival of B-CLL. Downregulation of GLI1 expression in B-CLL cells using antisense oligonucleotides resulted in the significantly (p<0.01) increased susceptibility of the B-CLL cells to fludarabine mediated cytotoxicity compare to fludarabine alone or antisense alone or B-CLL cells treated with irrelevant oligonucleotides as determined by MTT assay and Annexin V method for identifying apoptotic cells. There was a significant decrease in BCL2 expression in GLI1-downregulated B-CLL cells suggesting that GLI1 may regulates BCL2 and thereby regulate cell survival/apoptosis in B-CLL. Together, these results demonstrate a role for GLI1 transcription factor for the increased resistance to apoptosis and thereby disease progression in B-CLL, and hence may be a potential therapeutic target to further improve treatment for B-CLL.

Apoptotic pathway induced by noscapine in human myelogenous leukemic cells

It has been shown that noscapine, an opium-derived phthalideisoquinoline alkaloid that is currently being used as an oral antitussive drug, induces apoptosis in myeloid leukemia cells. The molecular mechanism responsible for the anticancer effects of noscapine is poorly understood. In the current study, the apoptotic effects of noscapine on two myeloid cell lines, apoptosis-proficient HL60 cells and apoptosis-resistant K562 cells, were analyzed. An increase in the activity of caspase-2, -3, -6, -8 and -9, poly(ADP ribose) polymerase cleavage, detection of phosphatidylserine on the outer layer of the cell membrane, nucleation of chromatin, and DNA fragmentation suggested the induction of apoptosis. Noscapine increased the Bax/Bcl-2 ratio with a significant decrease of Bcl-2 expression accompanied with Bcl-2 phosphorylation. Using an inhibitory approach, the activation of the caspase cascade involved in the noscapine-induced apoptosis was analyzed. We observed no inhibitory effect of the caspase-8 inhibitor on caspase-9 activity. In view of these results and taking into consideration that K562 cells are Fas-null, we suggested that caspase-8 is activated in a Fas-independent manner downstream of caspase-9. In conclusion, noscapine can induce apoptosis in both apoptosis-proficient and apoptosis-resistant leukemic cells, and it can be a novel candidate in the treatment of hematological malignancies.