Changes In Bcl-2 Protein Levels Research Articles

Exploring the interplay between miRNAs, apoptosis and viral load, in Dengue virus infection

Dengue virus (DENV) is a major global health concern, causing millions of infections annually. Understanding the cellular response to DENV infection is crucial for developing effective therapies. This study provides an in-depth analysis of the cellular response to Dengue virus (DENV) infection, with a specific focus on the interplay between microRNAs (miRNAs), apoptosis, and viral load across different DENV serotypes. Utilizing a variety of cell lines infected with four DENV serotypes, the research methodically quantifies viral load, and the expression levels of miRNA-15, miRNA-16, and BCL2 protein, alongside measuring apoptosis markers. Methodologically, the study employs quantitative PCR for viral load and miRNA expression analysis, and Western blot for apoptosis and BCL2 detection, with a statistical framework that includes ANOVA and correlation analysis to discern significant differences and relationships. The findings reveal that despite similar viral loads across DENV serotypes, DENV-2 exhibits a marginally higher load. A notable upregulation of miRNA-15 and miRNA-16 correlates positively with increased viral load, suggesting their potential role in modulating viral replication. Concurrently, a marked activation of caspases 3 and 7, along with changes in BCL2 protein levels, underscores the role of apoptosis in the cellular response to DENV infection. Conclusively, the study enhances the understanding of miRNA involvement in DENV pathogenesis, highlighting miRNA-15 and miRNA-16 as potential regulatory agents in viral replication and apoptosis. These findings pave the way for further exploration into miRNA-based therapeutic strategies against DENV infection.

Abstract 903: Comparison of the citrus flavonoid naringenin and metformin for effects on breast cancer in obese ovariectomized mice

Abstract Metformin has been shown to decrease breast cancer incidence in diabetic women, suggesting that improvement of metabolic status may be a useful approach to decrease breast cancer risk. As the putative cellular target of metformin, AMP-activated protein kinase (AMPK) not only regulates energy metabolism in liver, muscle, and adipose tissue but also inhibits protein synthesis and induces cell cycle arrest and apoptosis. Naringenin is a flavonoid abundant in citrus fruits that may have beneficial effects on metabolic health and tumorigenesis. Our in vitro data showed that naringenin inhibited proliferation of E0771 murine breast cancer cells, increased AMPK activity, and reduced expression of cell cycle regulatory proteins Cyclin D1 and anti-apoptotic Bcl-2 protein. In this study, we evaluated whether dietary naringenin acts like metformin to suppress mammary tumor growth in a mouse model of postmenopausal obesity. Ovariectomized female C57BL/6J mice were fed a high-fat diet (60% energy as fat) for 3 weeks to induce obesity. Then mice were randomized to experimental diets of HF (continued the high-fat diet), LN (high fat + 1% wt/wt naringenin in diet), HN (high fat + 3% wt/wt naringenin in diet), and Met (high fat + 180 mg/kg metformin in water) for 5 weeks. After 2 weeks of treatment, 5 × 10(5) E0771 cells were injected into one thoracic mammary fat pad. Mice were maintained on study diets for another 3 weeks. For all mice tumor weight correlated with weight gain in the first 5 weeks prior to cell inoculation (r = 0.51, p < 0.01). There was no difference in fasting glucose levels among the groups on week 8. No difference was observed between the HF and LN groups regarding final body weight, food intake, tissue weights, and tumor mass. HN significantly decreased final body weight, food intake, and adipose depot mass. mRNA expression of two inflammatory cytokines, interleukin 6 and monocyte chemoattractant protein-1 were decreased in perigonadal fat of the HN group. Although tumor growth (cm2) decreased over time in HN mice, there was no significant difference in tumor mass between HF and HN groups at necropsy (p = 0.4). HN did not affect AMPK activity in tumor tissue, but decreased Bcl-2 protein expression. Additionally, dietary naringenin resulted in a dose ­dependent accumulation in plasma, tumor, and mammary fat. In comparison to naringenin enriched diets, metformin significantly reduced tumor growth and final tumor weight, without affecting body weight, food intake, and tissue weights. Metformin decreased protein expression of Cyclin D1 in mammary tumors. However, we did not observe AMPK activation and changes in Bcl-2 protein levels in tumors. Taken together, these data suggest that metformin may inhibit mammary tumor growth without affecting metabolic status as assessed by body weight, adiposity, and fasting glucose. There may be different mechanisms of action for naringenin and metformin to alter mammary tumorigenesis. Citation Format: Jia-Yu Ke, Yung-Hsuan Hsiao, Shana R. Straka, Lisa D. Yee, Martha A. Belury. Comparison of the citrus flavonoid naringenin and metformin for effects on breast cancer in obese ovariectomized mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 903. doi:10.1158/1538-7445.AM2015-903

Polycystin-1 Protein Level Determines Activity of the Gα12/JNK Apoptosis Pathway

Mutations in PKD1 are the most common cause of autosomal dominant polycystic kidney disease (ADPKD). The protein product of PKD1 (polycystin-1 (PC1)) is a large transmembrane protein with a short intracellular C terminus that interacts with numerous signaling molecules, including Galpha(12). Cyst formation in ADPKD results from numerous cellular defects, including abnormal cilia, changes in polarity, and dysregulated apoptosis and proliferation. Recently, we reported increased apoptosis in Madin-Darby canine kidney (MDCK) cells through Galpha(12) stimulation of JNK and degradation of the anti-apoptotic protein Bcl-2 (Yanamadala, V., Negoro, H., Gunaratnam, L., Kong, T., and Denker, B. M. (2007) J. Biol. Chem. 282, 24352-24363). Herein, we confirm this pathway in Galpha(12)-silenced MDCK cells and utilize MDCK cell lines harboring either overexpressed or silenced PC1 to demonstrate that PC1 expression levels determine activity of the JNK/Bcl-2 apoptosis pathway. PC1-overexpressing MDCK cells were resistant to thrombin/Galpha(12)-stimulated apoptosis, JNK activation, and Bcl-2 degradation. In contrast, PC1-silenced MDCK cells displayed enhanced thrombin-induced apoptosis, JNK activity, and Bcl-2 degradation. In pulldown experiments, PC1 bound to Galpha(12), but not the related Galpha(13) subunit, and thrombin-stimulated MDCK cells led to increased interaction of Galpha(12) with the PC1 C terminus. In transient transfection assays, a PC1 C-terminal mutant lacking the G protein-binding domain was uncoupled from PC1-inhibited apoptosis. PC1 expression levels may be increased or decreased in ADPKD, and these findings suggest a mechanism in which levels of PC1 expression modulate Galpha(12)/JNK-stimulated apoptosis. Taken together, these findings are consistent with a set point model in which PC1 expression levels regulate specific G protein signaling pathways important to cyst development.

Inhibin a increases apoptosis in early ovarian antral follicles of diethylstilbestrol-treated rats.

The purpose of this study was to evaluate the role of inhibin A in follicular development and apoptosis-related mechanisms in preantral and early antral follicles from prepubertal diethylstilbestrol (DES)-treated rats. Granulosa cells isolated from the ovaries of 23- to 25-day-old rats were cultured in serum-free medium containing FSH (20 ng/ml), transforming growth factor beta (5 ng/ml), and estradiol (50 ng/ml) in the presence or absence of different concentrations of recombinant human inhibin A. (3)H-Thymidine incorporation was decreased in the presence of Inh, but no significant changes were observed in progesterone and estradiol levels in culture medium. An increase in low molecular weight DNA fragmentation indicative of apoptosis and an increase in the levels of Bax protein with no changes in Bcl-2 protein levels were evident in early antral follicles incubated for 24 h with Inh. For each animal, Inh (0.5 micro g/ovary) was injected intrabursally in one ovary, and the contralateral ovary served as a control. Ovarian histology revealed an inhibitory effect of Inh treatment on the follicular development induced by DES. At 24 h after Inh injection, the number of preantral follicles was increased compared with controls, whereas the number of early antral follicles was decreased. In addition, in vivo Inh treatment caused an increase in the percentage of apoptotic cells in preantral and early antral follicles. These results suggest that inhibin produced by the dominant follicle may act as a paracrine factor inhibiting the growth of neighboring follicles, thus participating in the mechanism of follicular selection.

G(1) phase-dependent expression of bcl-2 mRNA and protein correlates with chemoresistance of human cancer cells.

Recent experiments suggest an interconnection between cell proliferation and programmed cell death (apoptosis), although the detailed molecular mechanisms remain unclear. We have hypothesized that expression of some apoptosis regulators is cell cycle-dependent, which in turn influences tumor cell chemosensitivity in a cell cycle-dependent fashion. To test these hypotheses, we synchronized human leukemia Jurkat T, Neo (using aphidicolin), breast cancer MCF-7, normal fibroblast, and simian virus 40-transformed cells (by aphidicolin or serum starvation), and measured levels of several Bcl-2 family proteins. The highest expression of Bcl-2 protein was found in the G(1) phase of all the five cell lines tested. In contrast, levels of Bax protein remained relatively unchanged in four of the cell lines, and levels of Bcl-X(L), Bcl-X(S), and Bak proteins showed little or no cell cycle-dependent changes in Jurkat T cells. Similar to the changes in Bcl-2 protein levels, its mRNA expression was also G(1) phase-specific, whereas the level of a Bcl-2 cleavage activity remained constitutive. When treated with an anticancer drug (etoposide or cisplatin) or the kinase inhibitor staurosporin, the cells containing a high G(1) population and a high Bcl-2 protein level were much more resistant to the induced apoptosis than the cells containing a high S phase population and a low Bcl-2 protein level. Constitutive overexpression of Bcl-2 protein in Jurkat T cells completely blocked the S phase-associated sensitivity to these apoptosis stimuli. The cell cycle-dependent Bcl-2 protein expression seems to contribute to the regulation of chemosensitivity and apoptotic commitment of human tumor cells.

Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression.

Previously we reported that proteasome inhibitors were able to overcome Bcl-2-mediated protection from apoptosis. Here we show that inhibition of the proteasome activity in Bcl-2-overexpressing cells accumulates the proapoptotic Bax protein to mitochondria/cytoplasm, where it interacts to Bcl-2 protein. This event was followed by release of mitochondrial cytochrome c into the cytosol and activation of caspase-mediated apoptosis. In contrast, proteasome inhibition did not induce any apparent changes in Bcl-2 protein levels. In addition, treatment with a proteasome inhibitor increased levels of ubiquitinated forms of Bax protein, without any effects on Bax mRNA expression. We also established a cell-free Bax degradation assay in which an in vitro-translated, (35)S-labeled Bax protein can be degraded by a tumor cell protein extract, inhibitable by addition of a proteasome inhibitor or depletion of the proteasome or ATP. The Bax degradation activity can be reconstituted in the proteasome-depleted supernatant by addition of a purified 20S proteasome or proteasome-enriched fraction. Finally, by using tissue samples of human prostate adenocarcinoma, we demonstrated that increased levels of Bax degradation correlated well with decreased levels of Bax protein and increased Gleason scores of prostate cancer. Our studies strongly suggest that ubiquitin/proteasome-mediated Bax degradation is a novel survival mechanism in human cancer cells and that selective targeting of this pathway should provide a unique approach for treatment of human cancers, especially those overexpressing Bcl-2.

Nitrogen mustard up-regulates Bcl-2 and GSH and increases NTP and PCr in HT-29 colon cancer cells.

We hypothesized that unexplained increases in nucleoside triphosphates (NTP) observed by 31P magnetic resonance spectroscopy (MRS) after treatment of tumours by DNA-damaging agents were related to chemotherapy-induced up-regulation of the bcl-2 gene and DNA damage prevention and repair processes. To test this hypothesis, we treated HT-29 cells with 10(-4) M nitrogen mustard (HN2) and performed sequential perchloric acid extractions in replicate over 0-18 h. By reference to an internal standard (methylene diphosphonic acid), absolute changes in 31P-detectable high-energy phosphates in these extracts were determined and correlated with changes in bcl-2 protein levels, cell viability, cell cycle, apoptosis and total cellular glutathione (GSH) (an important defence against DNA damage from alkylating agents). After HN2 administration, bcl-2 protein levels in the HT-29 cell line rose at 2 h. Cell viability declined to 25% within 18 h, but apoptosis measured using fluorescence techniques remained in the 1-4% range. Increased cell division was noted at 4 h. Two high-energy interconvertible phosphates, NTP (P < or = 0.006) and phosphocreatine (PCr) (P < or = 0.0002), increased at 2 h concurrently with increased levels of bcl-2 protein and glutathione. This study demonstrates that bcl-2 and glutathione are up-regulated by HN2 and links this to a previously unexplained 31P MRS phenomenon: increased NTP after chemotherapy.