Modulation Of Bcl-2 Expression Research Articles

Neuroprotective potential of Afzelin: A novel approach for alleviating catalepsy and modulating Bcl-2 expression in Parkinson's disease therapy

The lost dopaminergic neurons in the brain prevent mobility in Parkinson's disease (PD). It is impossible to stop the disease's progress by means of symptoms management. Research focuses on oxidative stress, mitochondrial dysfunction, and neuronal degeneration. Exploration of potential neuroprotective drugs against prosurvival B-cell lymphoma 2 (Bcl-2) protein is ongoing. An investigable cause behind PD, as well as preventive measures, could be discovered considering the association between such behavioural manifestations (cataleptic behaviours) and PD. The compound Afzelin, known to guard the nervous system, was chosen for this study. The study was done on rats divided into six different groups. First, there was a control group. The other group was treated with Reserpine (RES) (1 mg/kg). The third group received RES (1 mg/kg) and levodopa (30 mg/kg). The remaining three groups were given RES (1 mg/kg) in conjunction with Afzelin at the following doses: 5 mg/kg, 10 mg/kg, and 20 mg/kg. Cataleptic behavior and mobility in rats was assessed using the rotarod, open field, and modified forced-swim tests. thiobarbituric acid reactive substances (TBARS), nitric oxide (NO), biogenic amines, and Bcl-2 level in rat tissue homogenates were considered. According to the study's findings, the rats treated through co-administration of RES and Afzelin improved significantly in their cataleptic behaviours and locomotor activity. In addition, administering Afzelin itself caused Bcl-2 expression, which could have some neuroprotection properties. This study provides meaningful information on the effectiveness of Afzelin in handling catalepsy and other degenerative neurologic disorders. As a result, other studies need to be conducted to establish the reasons behind the reactions and determine the long-term effects of Afzelin on these conditions.

Local Delivery of Azithromycin Nanoformulation Attenuated Acute Lung Injury in Mice.

Humanity has suffered from the coronavirus disease 2019 (COVID-19) pandemic over the past two years, which has left behind millions of deaths. Azithromycin (AZ), an antibiotic used for the treatment of several bacterial infections, has shown antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as against the dengue, Zika, Ebola, and influenza viruses. Additionally, AZ has shown beneficial effects in non-infective diseases such as cystic fibrosis and bronchiectasis. However, the systemic use of AZ in several diseases showed low efficacy and potential cardiac toxicity. The application of nanotechnology to formulate a lung delivery system of AZ could prove to be one of the solutions to overcome these drawbacks. Therefore, we aimed to evaluate the attenuation of acute lung injury in mice via the local delivery of an AZ nanoformulation. The hot emulsification-ultrasonication method was used to prepare nanostructured lipid carrier of AZ (AZ-NLC) pulmonary delivery systems. The developed formulation was evaluated and characterized in vitro and in vivo. The efficacy of the prepared formulation was tested in the bleomycin (BLM) -mice model for acute lung injury. AZ-NLC was given by the intratracheal (IT) route for 6 days at a dose of about one-eighth oral dose of AZ suspension. Samples of lung tissues were taken at the end of the experiment for immunological and histological assessments. AZ-NLC showed an average particle size of 453 nm, polydispersity index of 0.228 ± 0.07, zeta potential of -30 ± 0.21 mV, and a sustained release pattern after the initial 50% drug release within the first 2 h. BLM successfully induced a marked increase in pro-inflammatory markers and also induced histological changes in pulmonary tissues. All these alterations were significantly reversed by the concomitant administration of AZ-NLC (IT). Pulmonary delivery of AZ-NLC offered delivery of the drug locally to lung tissues. Its attenuation of lung tissue inflammation and histological injury induced by bleomycin was likely through the downregulation of the p53 gene and the modulation of Bcl-2 expression. This novel strategy could eventually improve the effectiveness and diminish the adverse drug reactions of AZ. Lung delivery could be a promising treatment for acute lung injury regardless of its cause. However, further work is needed to explore the stability of the formulation, its pharmacokinetics, and its safety.

BET Inhibitors in Chronic Lymphocytic Leukemia: JQ1 Synergizes with Venetoclax in Promoting Apoptosis

▪Background: The bromodomain and extra terminal BET family proteins (BRD2, BRD3, BRD4 and the testis-specific BRDT) play a critical role in mediating gene transcription and have been considered as highly promising targets in several diseases including cancer. Specifically, BET inhibitors block the transcription of key oncogenes (BCL2, C-MYC and CDK6) through the displacement of BRDs and other epigenetic modifiers from chromatin. However, while BET inhibitors were consistently described as highly effective drugs in in vitro experiments, few reports and clinical trials have already demonstrated the rapid emergence of BET inhibitors resistance.Aim: We examined the efficacy of the BET inhibitor JQ1 in CLL samples, upon short and prolonged treatments, to assess its efficacy and the potential development of resistances.Methods: MEC-1/EHEB CLL cell lines (p53 wild-type EHEB and p53-mutated MEC-1 cell lines) and primary CD19 positive lymphocytes from CLL patients were subjected to short and prolonged treatment with the BET inhibitor JQ1. Responses were measured in terms of proliferation, cell-cycle and apoptosis.Results: Treatment of CLL cell lines with the BET inhibitor JQ1 was associated with block of proliferation, cell cycle arrest and apoptosis. Molecularly, JQ1 treatment was associated to c-Myc down-modulation. Notably, treatment with JQ1 caused also loss of viability of primary patient-purified CLL cells. However, starting from 48 hours of JQ1 treatment in CLL cell lines, we observed a compensatory increase of BRDs mRNA levels, which was associated to the development of resistant clones after longer treatments (30 days). These data demonstrate that resistance to BET inhibitors can rapidly emerge and suggest that a combination therapy with other drugs should be explored. Interestingly, we observed that JQ1 was also able to modulate BCL-2 expression in CLL. We therefore assessed the effect of a combination therapy based on JQ1 in association with the BCL-2 inhibitor, Venetoclax. Intriguingly, co-treatment with JQ1 and Venetoclax significantly increased apoptosis demonstrating a synergic action.Conclusions: Our data demonstrated for the first time that: i) CLL may be effectively targeted by BET inhibitors; ii) short term treatment with BET inhibitors rapidly results in the upregulation of additional BRD family members; iii) prolonged treatment is associated with the development of resistance; IV) BET proteins modulate BCL-2 and c-MYC expression also in CLL; V) the combination therapy based of JQ1 administration in association with Venetoclax is extremely effective in CLLDiscussion: Here, we show that JQ1 treatment results in growth inhibition and apoptosis of CLL cells by down-modulating the expression of the pro-survival genes, C-MYC and BCL-2. Furthermore, our data show that treatment with JQ1 is only effective for a short time while the co-treatment with Venetoclax exerts a synergistic lethality effect in CLL. These data suggest the efficacy of a combination therapy with JQ1 and Venetoclax in refractory TP53 mutated CLL patients. DisclosuresSaglio:Roche: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

Effects of Baicalin on proliferation and apoptosis of gastric cancer cells and its relationship with c-jun amino-terminal kinase signaling pathway

Objective Effects of Baicalein on AGS cell proliferation and apoptosis in gastric cancer based on c-Jun N-terminal kinase signaling pathway. Methods Firstly, Annexin V staining was used to detect the apoptosis of AGS cells after baicalin intervention, and then cell counting kit-8 (CCK-8) was used to detect the effect of baicalin intervention on the proliferation of AGS cells after gastric cancer. Finally, Western blotting was used to detect the expression of c-jun amino-terminal kinase signaling molecules B lymphoma-2 and c-jun protein in the downstream of c-jun amino-terminal kinase signaling pathway after the intervention of baicalin. Results CCK-8 assay showed that after 48 h of baicalin intervention with 0 μmol/L (control group), 25, 50, 100 μmol/L, the proliferation activity (Mean±SD) of AGS cells in the corresponding four groups was 100.00±3.38, 79.49±4.47, 68.67±2.26, 57.02±3.28. respectively. Compared with the control group, baicalin intervention can significantly reduce the proliferation activity of AGS cells in gastric cancer with a dose-dependent effect, P<0.05. After 48 hours of intervention with baicalin at 0 μmol/L (control group), 25, 50, 100 μmol/L, Annexin V staining and flow cytometry, the apoptosis rates (Mean±SD) of AGS cells in the four groups were 1.97±0.72, 3.44±0.45, 4.11±0.35, 4.93±1.03. Compared with the control group, baicalin can significantly promote the apoptosis of AGS cells, P<0.05. Western blotting analysis showed that the expressions of B lymphocytoma-2 and c-Jun in AGS cells were decreased after baicalin treatment. Conclusion Baicalein significantly inhibited the activity of gastric cancer cells, induced apoptosis of gastric cancer cells and inhibited the growth of gastric cancer cells. And it’s significant The role of modulating bcl-2 and c-jun expression. Key words: c-Jun N-terminal kinase signaling pathway; Baicalein; Gastric cancer; Proliferation; Apoptosis

Synergistic growth inhibitory effect of deracoxib with doxorubicin against a canine mammary tumor cell line, CMT-U27.

Cyclooxygenase (COX) inhibitors have been shown to exert anti-angiogenic and anti-tumor activities on many types of malignant tumors. These anticancer properties make it worthwhile to examine the possible benefit of combining COX inhibitors with other anti-cancer agents. In the present study, we evaluated the potential of deracoxib (DER) in potentiating antitumor activity of doxorubicin (DOX) in canine mammary carcinoma cells (CMT-U27). DER (50–250 µM) enhanced the antiproliferative activity of DOX by reducing the IC50 (approximately 3- to 3.5 fold). Interaction analysis of the data showed that combinations of DOX at 0.9 µM with DER (100–250 µM) produced synergism in the CMT-U27 cell line, with a ratio index ranging from 1.98 to 2.33. In additional studies identifying the mechanism of observed synergistic effect, we found that DER strongly potentiated DOX-caused G0/G1 arrest in cell cycle progression. Also, DER (100–250 µM) augmented apoptosis induction with approximately 1.35- and 1.37- fold increases in apoptotic response caused by DOX in the cells. DER enhanced the antiproliferative effect of DOX in conjunction with induction of apoptosis by modulation of Bcl-2 expression and changes in the cell cycle of the CMT-U27 cell line. Although the exact molecular mechanism of the alterations in the cell cycle and apoptosis observed with DER and DOX combinations require further investigations, the results suggest that the synergistic effect of DOX and DER combinations in CMT therapy may be achieved at relatively lower doses of DOX with lesser side effects. Therefore, combining DER with DOX may prove beneficial in the clinical treatment of canine mammary cancer.

Transformer 2β and miR-204 regulate apoptosis through competitive binding to 3' UTR of BCL2 mRNA.

RNA-binding proteins and microRNAs are potent post-transcriptional regulators of gene expression. Human transformer 2β (Tra2β) is a serine/arginine-rich-like protein splicing factor and is now implicated to have wide-ranging roles in gene expression as an RNA-binding protein. RNA immunoprecipitation (RIP) with an anti-Tra2β antibody and microarray analysis identified a subset of Tra2β-associated mRNAs in HCT116 human colon cancer cells, many of which encoded cell death-related proteins including Bcl-2 (B-cell CLL/lymphoma 2). Tra2β knockdown in HCT116 cells decreased Bcl-2 expression and induced apoptosis. Tra2β knockdown accelerated the decay of BCL2α mRNA that encodes Bcl-2 and full-length 3' UTR, while it did not affect the stability of BCL2β mRNA having a short, alternatively spliced 3' UTR different from BCL2α 3' UTR. RIP assays with anti-Tra2β and anti-Argonaute 2 antibodies, respectively, showed that Tra2β bound to BCL2α 3' UTR, and that Tra2β knockdown facilitated association of miR-204 with BCL2α 3' UTR. The consensus sequence (GAA) for Tra2β-binding lies within the miR-204-binding site of BCL2 3' UTR. Mutation of the consensus sequence canceled the binding of Tra2β to BCL2 3' UTR without disrupting miR-204-binding to BCL2 3' UTR. Transfection of an anti-miR-204 or introduction of three-point mutations into the miR-204-binding site increased BCL2 mRNA and Bcl-2 protein levels. Inversely, transfection of precursor miR-204 reduced their levels. Experiments with Tra2β-silenced or overexpressed cells revealed that Tra2β antagonized the effects of miR-204 and upregulated Bcl-2 expression. Furthermore, TRA2β mRNA expression was significantly upregulated in 22 colon cancer tissues compared with paired normal tissues and positively correlated with BCL2 mRNA expression. Tra2β knockdown in human lung adenocarcinoma cells (A549) increased their sensitivity to anticancer drugs. Taken together, our findings suggest that Tra2β regulates apoptosis by modulating Bcl-2 expression through its competition with miR-204. This novel function may have a crucial role in tumor growth.

LRIG1 enhances chemosensitivity by modulating BCL-2 expression and receptor tyrosine kinase signaling in glioma cells.

PurposeLeucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) are an inhibitor of receptor tyrosine kinases (RTKs) that was discovered in recent years, and many studies showed that LRIG1 is a tumor suppressor gene and may be related to tumor drug resistance. In this study, we explored whether LRIG1 protein expression can improve the chemosensitivity of glioma cells and what was its mechanism.Materials and MethodsWe collected 93 cases of glioma tissues and detected the expression of LRIG1 and BCL-2. We constructed a multidrug resistance cell line U251/multidrug resistance (MDR) and examined the change of LRIG1 and BCL-2 at mRNA and protein expression levels. LRIG1 expression was upregulated in U251/MDR cells and we detected the change of multidrug resistance. Meanwhile, we changed the expression of LRIG1 and BCL-2 and explored the relationship between LRIG1 and BCL-2. Finally, we also explored the relationship between LRIG1 and RTKs.ResultsLRIG1 was negatively correlated with BCL-2 expression in glioma tissue and U251/MDR cells, and upregulation of LRIG1 can enhance chemosensitivity and inhibit BCL-2 expression. Furthermore, LRIG1 was negatively correlated with RTKs in U251/MDR cells.ConclusionThese results demonstrated that LRIG1 can improve chemosensitivity by modulating BCL-2 expression and RTK signaling in glioma cells.

Erratum to "LRIG1 Enhances Chemosensitivity by Modulating BCL-2 Expression and Receptor Tyrosine Kinase Signaling in Glioma Cells" by Guo Z, et al. (Yonsei Med J 2014;55:1196-205.)

[This corrects the article on p. 1196 in vol. 55, PMID: 25048475.].

Bovine herpesvirus type 5 infection regulates Bax/BCL-2 ratio

Bovine herpesvirus 5 (BoHV-5) is an α-herpesvirus that causes neurological disease in young cattle and is also occasionally involved in reproductive disorders. Although there have been many studies of the apoptotic pathways induced by viruses belonging to the family Herpesviridae, there is little information about the intrinsic programmed cell death pathway in host-BoHV-5 interactions. We found that BoHV-5 is able to replicate in both mesenchymal and epithelial cell lines, provoking cytopathology that is characterized by cellular swelling and cell fusion. Viral antigens were detected in infected cells by immunofluorescence assay at 48 to 96 h post-infection (p.i.). At 48 to 72 h p.i., anti-apoptotic BCL-2 antigens were found at higher levels than Bax antigens; the latter is considered a pro-apoptotic protein. Infected cells had increased BCL-2 phenotype cells from 48 to 96 h p.i., based on flow cytometric analysis. At 48 to 96 h p.i., Bax mRNA was not expressed in any of the infected cell monolayers. In contrast, BCL-2 mRNA was found at high levels at all p.i. in both types of cells. BoHV-5 replication apparently modulates BCL-2 expression and gene transcription, enhancing production of virus progeny.

HOXC6 Is Deregulated in Human Head and Neck Squamous Cell Carcinoma and Modulates Bcl-2 Expression

Homeobox C6 (HOXC6) genes belong to the homeoprotein family of transcription factors, which play an important role in morphogenesis and cellular differentiation during embryonic development. The aim of this study was to explore the role of HOXC6 in the regulation of Bcl-2 in human head and neck squamous cell carcinoma (HNSCC). The HOXC6 and Bcl-2 gene were identified as being overexpressed in HNSCC tissue and cell lines. Transfection assays demonstrated that HOXC6 increased the levels of Bcl-2 mRNA and protein. A luciferase reporter assay suggested that HOXC6 induced activity of the Bcl-2 promoter. A series of Bcl-2 promoter deletion mutants were examined and the minimal HOXC6-responsive region was identified to be in the TAAT motif (-420 bp) of the Bcl-2 promoter. Interestingly, the inhibition of HOXC6 using siRNA led to the repression of Bcl-2 expression and induced caspase-3-dependent apoptosis; overexpression of HOXC6 in HNSCC cells increased the resistance to paclitaxel-induced apoptosis. Together, our findings suggest that HOXC6 is an important mechanism of the anti-apoptotic pathway via regulation of Bcl-2 expression.

Intracellular Insulin-like Growth Factor-1 Induces Bcl-2 Expression in Airway Epithelial Cells

Bcl-2, a prosurvival protein, regulates programmed cell death during development and repair processes, and it can be oncogenic when cell proliferation is deregulated. The present study investigated what factors modulate Bcl-2 expression in airway epithelial cells and identified the pathways involved. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection showed that increased expression of IL-1β and insulin-like growth factor-1 (IGF-1) coincided with induced Bcl-2 expression compared with controls. Treatment of cultured airway epithelial cells with IL-1β and IGF-1 induced Bcl-2 expression by increasing Bcl-2 mRNA stability with no discernible changes in promoter activity. Silencing the IGF-1 expression using short hairpin RNA showed that intracellular IGF-1 (IC-IGF-1) was increasing Bcl-2 expression. Blocking epidermal growth factor receptor or IGF-1R activation also suppressed IC-IGF-1 and abolished the Bcl-2 induction. Induced expression and colocalization of IC-IGF-1 and Bcl-2 were observed in airway epithelial cells of mice exposed to LPS or cigarette smoke and of patients with cystic fibrosis and chronic bronchitis but not in the respective controls. These studies demonstrate that IC-IGF-1 induces Bcl-2 expression in epithelial cells via IGF-1R and epidermal growth factor receptor pathways, and targeting IC-IGF-1 could be beneficial to treat chronic airway diseases.

Celecoxib And Bcl-2: Emerging Possibilities For Anticancer Drug Design

Celecoxib is a multifaceted drug with promising anticancer properties. A number of studies have been conducted that implicate the compound in modulating the expression of Bcl-2 family members and mitochondria-mediated apoptosis. The growing data surrounding the role of celecoxib in the regulation of the mitochondrial death pathway provides a platform for ongoing debate. Studies that describe celecoxib's properties as a BH3 mimic or as a direct inhibitor of Bcl-2 are not available. The motivations for this review are: to provide the basis for the development of novel compounds that modulate Bcl-2 expression using celecoxib as a structural starting point and to encourage additional biological studies (such as binding and enzymatic assays) that would provide information regarding celecoxib's role as a Bcl-2 antagonist. The current review summarizes work that identifies the role of celecoxib in blocking the activity of Bcl-2.

Sperm-borne microRNA-34c is required for the first cleavage division in mouse

In mammals, the sperm deliver mRNA of unknown function into the oocytes during fertilization. The role of sperm microRNAs (miRNAs) in preimplantation development is unknown. miRNA profiling identified six miRNAs expressed in the sperm and the zygotes but not in the oocytes or preimplantation embryos. Sperm contained both the precursor and the mature form of one of these miRNAs, miR-34c. The absence of an increased level of miR-34c in zygotes derived from α-amanitin-treated oocytes and in parthenogenetic oocytes supported a sperm origin of zygotic miR-34c. Injection of miR-34c inhibitor into zygotes inhibited DNA synthesis and significantly suppressed first cleavage division. A 3' UTR luciferase assay and Western blotting demonstrated that miR-34c regulates B-cell leukemia/lymphoma 2 (Bcl-2) expression in the zygotes. Coinjection of anti-Bcl-2 antibody in zygotes partially reversed but injection of Bcl-2 protein mimicked the effect of miR-34c inhibition. Oocyte activation is essential for the miR-34c action in zygotes, as demonstrated by a decrease in 3'UTR luciferase reporter activity and Bcl-2 expression after injection of precursor miR-34c into parthenogenetic oocytes. Our findings provide evidence that sperm-borne miR-34c is important for the first cell division via modulation of Bcl-2 expression.

Effects of etomldate postconditlonlng on apoptotds In a rnt model of focal cerebral lschemia-reperhrslon In-jury

Objective To investigate the effects of etomidate postconditioning on apoptosis in a rat model of focal cerebral ischemia-reperfusion(I/R) injury.Methods Thirty-two pathogen-free male SD rats weighing 250-300 g were randomly divided into 4 groups ( n =8 each) using random number table:group sham operation ( group S); group focal cerebral I/R; group lipid emulsion (vehicle for etomidate) (group L) and group etomidate postconditioning (group Ep).Focal cerebral I/R was induced by inserting a nylon thread with rounded tip into right internal carotid artery.The thread was advanced cranially until resistance was met.Middle cerebral artery was occluded for 2 h in groups I/R,L and Ep.Normal saline,lipid emulsion and etomidate emulsion 20 mg/kg were injected peritoneally at the end of ischemia in groups I/R,L and Ep respectively.The animals were sacrificed at 24 h of reperfusion and their brains were removed for microscopic examination,assessment of apoptosis (by TUNEL) and detection of Bcl-2 and Bax expression ( by immuno-histochemistry).Apoptosis index ( AI =the number of apoptofic neurons/the total number of neurons examined × 100％ ) and Bcl-2/Bax ratio were calculated.Results I/R induced microscopic changes,significantly increased AI and Bcl-2 Bax ratio and up-regulated Bcl-2 and Bax expression in group I/R as compared with group S.Etomidate postconditioning significantly amefiorated brain damage,decreased AI,increased Bcl-2/Bax ratio,up-regulated Bcl-2 expression and down-regulated Bax expression in ischemic cerebral hemisphere in group Ep as compared with group I/R.There was no significant difference in brain damage,AI and Bcl-2 and Bax expression and Bcl-2/Bax ratio between groups I/R and L.Conclusion Etomidate postconditioning can attenuate focal cerebral ischemia-reperfusion injury in rats by inhibiting apoptosis and modulating Bcl-2 and Bax expression. Key words: Etomidate; Reperfusion injury; Brain; Apoptosis

Pretreatment with astragaloside IV protects H9c2 cells against hydrogen peroxide-induced apoptosis by scavenging of reactive oxygen species and regulation of Bcl-2 and Bax expression

Astragaloside IV (ASI), a saponin isolated from Radix Astragali, has been found to have potent cardioprotective effects. This study was designed to investigate whether ASI prevents cardiomyocytes from hydrogen peroxide (H2O2)-induced apoptosis. H9c2 cells were pretreated with different concentrations of ASI (5, 10, 20 μg/ml) for 24 h and then exposed to 100 μM H2O2 for 24 h. The cell viability were examined by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay, reactive oxygen species(ROS) generation was quantified by the 2',7'-dichlorofluorescin-diacetate (DCFH-DA) method. The activity of antioxidant enzymes, including catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were determined photometrically. Apoptotic cells were detected by Hoechst 33258 staining, annexin-V binding and by assessment of caspase-3. Protein and mRNA expression of both Bax and Bcl-2 were determined by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The cell apoptosis significantly increased after 24 h of H2O2 exposure. Pretreatment of H9c2 cells with ASI significantly increased the activities of antioxidant enzymes, scavenged ROS and reduced malondialdehyde (MDA) production. ASI increased the expression of Bcl-2, decreased the expression of Bax, and ultimately reduced H2O2-induced H9c2 cells apoptosis. In summary, these results suggest that ASI can block H2O2-induced apoptosis in H9c2 cells, and that the underlying mechanism involves in scavenging of ROS and modulating expression of Bcl-2 and Bax. Key words: Astragaloside IV, H9c2 cells, hydrogen peroxide, oxidative stress, apoptosis.

Abstract LB-46: Chemo-sensitization by modulation of Bcl-2 expression: The first example of an i-motif interactive small molecule that modulates gene expression

Abstract For the first time we provide evidence that not only illustrates the i-motif DNA secondary structure has potential to serve as a molecular target, but that within the Bcl-2 promoter this targeting can result in the modulation of gene expression and increase in chemo-sensitivity. The Bcl-2 oncoprotein is a key regulator and inhibitor of the intrinsic apoptotic pathway. Evasion of apoptosis is a well-known hallmark of cancer and is also recognized to play an important role in chemo-resistance in a variety of cancers particularly of lymphocytic origin. The significant over-expression of Bcl-2 common in low-grade lymphomas and lymphocytic leukemias has been associated with prolonged cell survival despite apoptotic stimuli and therefore is an attractive target for chemo-sensitization. Transcriptional regulation of Bcl-2 involves two promoter sites, P1 and P2, with P1 exerting the majority of control. Directly upstream of the P1 promoter is a GC-rich element capable of transitioning to DNA secondary structures. Previously we have shown the G-rich promoter element forms a mixed parallel/antiparallel G-quadruplex that has potential to adopt three different G-quadruplex structures. In recently published studies we have demonstrated that the complementary C-rich sequence has potential to form a stable i-motif structure. We now show that Bcl-2 expression can be modulated at the transcriptional level through small molecule targeting and destabilization of the i-motif. A high-throughput fluorescence resonance energy transfer (FRET) screening assay yielded a steroid derivative (compound 59) from the NCI diversity set that increased the i-motif FRET probe fluorescence by 2.5 fold indicative of substantial destabilization of the i-motif structure. The destabilization of the Bcl-2 i-motif was then confirmed with CD spectral and thermal stability analyses. As proof of principle we demonstrated that following treatment with compound 59, Bcl-2 gene expression is significantly decreased as detected by Real-Time RT-PCR in MCF-7, MCF-7 tamoxifen resistant breast cancer cell lines and the GRANTA-519 mantle cell lymphoma cell line. In addition, the GRANTA-519 cell line displays resistance to etoposide-induced apoptosis; however, in the presence of compound 59 the cells exhibited an increase in capase activity in comparison to cells that were treated with etoposide alone. These studies have been expanded to include a chemo-sensitization lymphoma cell line model in which two cell lines differ in Bcl-2 expression, low versus high, and sensitivity to etoposide-induced apoptosis, sensitive versus resistant, respectively. We anticipate from preliminary data that in response to compound 59 treatment the etoposide-resistant cell line that over-expresses Bcl-2 will have an increased apoptotic response similar to its etoposide sensitive counter-part. A Bcl-2 i-motif non-interactive compound is used as a negative control. Small molecules that target the Bcl-2 i-motif and result in the down-regulation of Bcl-2 expression have the potential to act as chemo-sensitizing agents to improve current chemotherapy in refractory patients by an alternative mechanism to ABT-737, which is in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-46.

Catalpol inhibits apoptosis in hydrogen peroxide-induced endothelium by activating the PI3K/Akt signaling pathway and modulating expression of Bcl-2 and Bax

Catalpol, an iridoid glucoside found in the root of Rehmannia glutinosa Libosch, has been demonstrated to reduce apoptosis in neuronal cell lines. Recent data suggests that catalpol also exerts anti-apoptotic effects on other cell types. The aim of the present study was to investigate whether catalpol protects against hydrogen peroxide (H 2O 2) induced apoptosis in human umbilical vein endothelial cells (HUVECs). Apoptotic cells were detected by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling, Annexin V-fluorescein isothiocyanate binding assay and by assessment of caspase-3 activity. The level of intracellular reactive oxygen species was quantified by 2′, 7′-dichlorofluorescein diacetate assay. Expression of Akt, Bad, Bcl-2 and Bax mRNA and protein was determined by real-time semiquantitative reverse transcription-polymerase chain reaction and Western blotting. Apoptosis in HUVECs was associated with increased Bax, decreased Bcl-2 activity and inactivated phosphorylation of Akt and Bad after 24 h of H 2O 2 exposure. Pre-treatment of HUVECs with catalpol significantly reduced H 2O 2-induced intracellular reactive oxygen species release. Catalpol not only increased the expression of Bcl-2, while decreasing Bax expression, but also induced Akt activation and Bad phosphorylation, and ultimately reduced H 2O 2-induced apoptosis. The protective effects of catalpol were partially inhibited by the phosphatidylinositol 3-kinase (PI3K) antagonist wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). Taken together, these results suggest that pre-treatment of HUVECs with catalpol can block H 2O 2-induced apoptosis, and that the underlying mechanism involves reactive oxygen species scavenging, activation of the PI3K/Akt–Bad signaling pathway and increased Bcl-2 and decreased Bax expression.

On the role of major vault protein in the resistance of senescent human diploid fibroblasts to apoptosis

Major vault protein (MVP), the main component of vault complex, is overexpressed in many multidrug-resistant cancer cell lines, suggesting a possible role for MVP in cell signaling and survival. In this study, we have found that MVP is markedly increased in senescent human diploid fibroblasts (HDFs) as well as in aged organs. We examined whether MVP expression might be affected by apoptotic stress in an aging-dependent manner. We treated young and senescent HDFs with apoptosis-inducing agents such as H(2)O(2), staurosporine and thapsigargin, and monitored MVP expression. We found that MVP expression is markedly reduced in young HDFs but not in senescent HDFs, in response to apoptotic stresses. Downregulation of MVP increased the sensitivity of senescent HDFs to apoptosis. Also, the level of antiapoptotic B-cell lymphoma protein-2 (Bcl-2) was significantly reduced and the accumulation of c-Jun increased in MVP knocked-down senescent HDFs. Moreover, treatment of MVP knocked-down senescent HDFs with SP600125, a specific c-Jun NH(2)-terminal kinase (JNK) inhibitor, restored the level of Bcl-2 protein. Taken together, these results suggest that MVP is important in the resistance of senescent HDFs to apoptosis by modulation of Bcl-2 expression by JNK pathway.

Docetaxel and bortezomib downregulate Bcl-2 and sensitize PC-3-Bcl-2 expressing prostate cancer cells to irradiation

Currently, docetaxel is used to treat hormone-refractory metastatic prostate cancer. Docetaxel not only inhibits microtubule formation but can also downregulate expression of Bcl-2, a known antiapoptotic oncogene. Furthermore, the 26S proteasome inhibitor bortezomib can downregulate Bcl-2 expression. Previously, we demonstrated that overexpression of Bcl-2 renders cells resistant to radiation therapy. In this study, we investigated whether treating human prostate cancer cells with docetaxel, bortezomib, or both could modulate Bcl-2 expression and whether such modulation could render Bcl-2-overexpressing cells more susceptible to radiation. PC-3-Bcl-2 and PC-3-Neo human prostate cancer cells treated with docetaxel and/or bortezomib in addition to irradiation were analyzed in vitro for proliferation, clonogenic survival, cell cycle phase distribution, and expression of Bcl-2 and Bcl-xL proteins. Docetaxel and bortezomib alone had significant cytotoxic effects. In addition, docetaxel, bortezomib, or radiation resulted in a G2M phase arrest in PC-3-Bcl-2, whereas only docetaxel or radiation did so in PC-3-Neo cells. Both cell lines were more sensitized to radiation's killing effects when treated with the combination of docetaxel and bortezomib than when treated with either agent alone. Furthermore, docetaxel and bortezomib-treated cells exhibited marked changes in the expression of Bcl-2 and Bcl-xL. This is the first study to demonstrate that docetaxel and bortezomib in combination can effectively sensitize Bcl-2-overexpressing human prostate cancer cells to radiation effects by modulating the expression of key members of the Bcl-2 family. Together, these findings warrant further evaluation of the combination of docetaxel and bortezomib in prostate cancer.

Dichloroacetate (DCA) sensitizes both wild‐type and over expressing Bcl‐2 prostate cancer cells in vitro to radiation

Bcl-2 protects cells from apoptosis and provides a survival advantage to cells over-expressing this oncogene. In addition, over expression of Bcl-2 renders cell resistant to radiation therapy. Recently, dichloroacetate (DCA) was proven to potentiate the apoptotic machinery by interacting with Bcl-2. In this study, we investigated whether treating human prostate cancer cells with DCA could modulate Bcl-2 expression and if the modulation in Bcl-2 expression could render the Bcl-2 over expressing cells more susceptible to cytotoxicity effects of radiation. PC-3-Bcl-2 and PC-3-Neo human prostate cancer cells treated with DCA in addition to irradiation were analyzed in vitro for changes in proliferation, clonogenic survival, apoptosis, cell cycle phase distribution, mitochondrial membrane potential, and expression of Bcl-2, Bcl-xL, Bax, or Bak proteins. DCA alone produced significant cytotoxic effects and was associated with G1 cell cycle arrest. Furthermore, DCA was associated with an increased rate of apoptosis. The combination of DCA with irradiation sensitized both cell lines to radiation's killing effects. Treatment of PC-3-Bcl-2 or PC-3-Neo with DCA and irradiation resulted in marked changes in various members of the Bcl-2 family. In addition, DCA therapy resulted in a significant change in mitochondria membrane potential, thus supporting the notion that DCAs effect is on the mitochondria. This is the first study to demonstrate DCA can effectively sensitize wild-type and over expressing Bcl-2 human prostate cancer cells to radiation by modulating the expression of key members of the Bcl-2 family. Together, these findings warrant further evaluation of the combination of DCA and irradiation.