Alterations In Caspase Research Articles

Effect of Silibinin on the Expression of Mir-20b, Bcl2L11, and Erbb2 in Breast Cancer Cell Lines.

This study aimed to evaluate the comparative effect of silibinin (SB) on the expression of MiR‑20b and BCL2L11 in T47D and MCF-7 cell lines. Molecular simulation studies were carried out to analyze Erbb2, as a potential target of SB, to direct the breast cancer cells toward apoptosis. At first, cell viability, apoptosis, and cell cycle arrest-inducing capacity of SB were examined using MTT and flow cytometry analysis, respectively. Real-time PCR (RT-PCR) was employed to assess the effect of SB on BCL2L11, Phosphatase and tensin homolog (PTEN), and Caspase 9 mRNarrest-indu. Moreover, alterations in Caspase 9 protein expression were determined using Western blot analysis. Finally, AutoDockVina software was used to dock the SB/ MiR‑20b and SB/ erb-b2 receptor tyrosine kinase 2 (Erbb2) interaction. The obtained data revealed the potent cytotoxicity of SB in both T47D and MCF-7 cells through apoptosis induction and cell cycle arrest. SB-treated cells also showed downregulation of MiR‑20b and high expression of BCL2L11, PTEN, and Caspase 9 mRNA compared to non-treated cancer cells. Computational docking showed a strong interaction between SB/ MiR‑20b and SB/Erbb2. It can be concluded that SB had a strong anti-tumorigenic activity through BCL2L11upregulation and MiR‑20b down expression, maybe through targeting the PTEN and interacting with Erbb2, which resulted in apoptotic induction and cell cycle arrest.

Role of Mcl-1 in regulation of cell death in human induced pluripotent stem cell-derived cardiomyocytes in vitro

Targeting the anti-apoptotic protein Mcl-1 holds a promise to improve therapy of multiple types of Mcl-1 dependent cancers but raises concerns of on-target cardiotoxicity due to the presence and reported role of Mcl-1 in heart. Herein, we investigated the importance of Mcl-1 in the survival and contractile function of human pluripotent stem cell-derived cardiomyocytes in culture. Effective knockdown of Mcl-1 with siRNAs reproducibly resulted in early (measured at Day 3) marginal alterations in caspase 3/7 activity, LDH leakage, ATP content and cellular impedance. After 14 days of Mcl-1 knockdown, loss of mitochondrial membrane potential, deteriorating effects on mitochondrial ultrastructure, and alterations in beat rate and amplitude were revealed. Inhibition of Bcl-xL by siRNA-mediated knockdown or selective inhibitors did not cause any overt cellular responses except for a minimal increase in caspase 3/7 activity; however, loss of Mcl-1 concomitant with down-regulated Bcl-xL activated apoptosis and caused extensive cell death as reflected by an 80% loss in cell index, activation of caspase-3 with associated PARP cleavage, and a decrease in beat amplitude and mitochondrial membrane potential after 3 days of Mcl-1/Bcl-xL knockdown., Together, these findings suggest that Mcl-1 and Bcl-xL provide duplicate safeguard measures in maintaining structural and functional integrity of cardiomyocytes. Hence, BH3-mimetic drugs targeting Mcl-1 may be well tolerated in the presence of intact Bcl-xL.

In vitro changes in mitochondrial potential, aggresome formation and caspase activity by a novel 17‐β‐estradiol analogue in breast adenocarcinoma cells

2-Methoxyestradiol, a natural metabolite of estradiol, exerts antiproliferative and antitumour properties in vitro and in vivo. Because of its low oral bioavailability, several promising analogues of 2-methoxyestradiol have been developed. In this study, the in vitro influence of the compound, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (C19), a non-commercially available 17-β-estradiol analogue, was tested on the breast adenocarcinoma MCF-7 cell line. The in vitro influence of 24 h exposure to 0.18 μM of C19 on MCF-7 cells was evaluated on cell morphology, cell cycle progression and possible induction of apoptosis and autophagy. Polarization-optical transmitted light differential interference contrast and fluorescence microscopy revealed the presence of cells blocked in metaphase, occurrence of apoptotic bodies and compromised cell density in C19-treated cells. Hallmarks of autophagy, namely an increase in the number of acidic vacuoles and lysosomes, were also observed in C19-treated samples. An increase in the number of cells present in the sub-G1 fraction, as well as a reduction in mitochondrial membrane potential was observed. No significant alterations in caspase 8 activity were observed. A twofold increase in aggresome formation was observed in C19-treated cells. C19 induced both apoptosis and autophagy in MCF-7 cells.

Methylmercury inhibits electron transport chain activity and induces cytochrome c release in cerebellum mitochondria

The involvement of oxidative stress has been suggested as a mechanism for toxicity caused by methylmercury (MeHg). One of the major critical sites for oxidative stress is the mitochondria. In this research, to clarify the target site in mitochondria affected by MeHg, the individual activities of the mitochondrial electron transport chain (ETC) (I∼IV) were examined in the liver, cerebrum and cerebellum of MeHg-intoxicated rats. In addition, to elucidate the mechanism underlying MeHg toxicity, cytochrome c release, caspase 3 activity and histological study were examined in the cerebrum and cerebellum. The cerebellum was found to be an exclusive tissue in which significant MeHg-induced alterations were observed. The complex II activity in the cerebellum mitochondria significantly decreased after MeHg exposure. Cytochrome c release from mitochondria increased only in the cerebellum by MeHg exposure. However, no significant alterations in caspase 3 activity or histological structure were found in brain tissues. These results suggest that MeHg acts on the constituents of complex II in the cerebellum, and induces mitochondrial dysfunction, leading to a release of cytochrome c from mitochondria. These events were considered to occur at the early stage of MeHg intoxication.

Alterations of caspase 9 mRNA gene expression in survivors and nonsurvivors of severe sepsis

Sepsis-induced lymphocyte apoptosis plays a fundamental role in the pathophysiology of sepsis. Recent animal models of sepsis have identified anomalies in the extrinsic apoptotic pathway, a key pathological occurrence in sepsis [1]. Specifically apoptosis markers such as caspases 1, 3, 8, 9 and FADD have been shown to be significant in animal models of infection [2]. We investigated mRNA transcription of these markers in a human model of severe sepsis. We hypothesized that ICU mortality from severe sepsis is associated with distinctive gene expression of extrinsic apoptosis markers.

Involvement of P53 and Bax/Bad triggering apoptosis in thioacetamide-induced hepatic epithelial cells.

Thioacetamide (TAA) has been used in studying liver fibrosis and cirrhosis, however, the mechanisms of TAA-induced apoptosis in liver are still unclear. The hepatic epithelial cell line clone 9 was cultured and treated with TAA to investigate the causes of cell death. The cell viability of TAA-induced clone 9 cells was determined using MTT assay. Total cellular GSH in TAA-induced clone 9 cells was measured using a slight modification of the Tietze assay. The activity of caspase 3 in TAA-induced clone 9 cells was monitored by the cleavage of DEVD-p-nitroanaline. TUNEL assay and flow cytometry were applied for the determination of DNA fragmentation and the proportion of apoptosis in TAA-induced clone 9 cells, respectively. The alterations of caspase 3, Bad, Bax and Phospho-P53 contents in TAA-induced clone 9 cells were measured by Western blot. The experimental data indicated that TAA caused rat hepatic epithelial cell line clone 9 cell death in a dose-and time-dependent manner; 60% of the cells died (MTT assay) within 24 h after 100 mg/L TAA was applied. Apoptotic cell percentage (TUNEL assay) and caspase 3 activities were highest after 100 mg/L TAA was added for 8 h. The release of GSH and the elevation in caspase content after TAA treatment resulted in clone 9 cell apoptosis via oxidative stress and a caspase-dependent mechanism. The phospho-p53, Bax and Bad protein expressions in clone 9 cells were increased after TAA treatment. These results reveal that TAA activates p53, increases caspase 3, Bax and Bad protein contents, perhaps causing the release of cytochrome c from mitochondria and the disintegration of membranes, leading to apoptosis of cells.

CD44 promotes resistance to apoptosis in murine colonic epithelium

Dysregulated expression of CD44 isoforms occurs consistently in colon carcinogenesis, and this change occurs also in most other types of cancer. One of the basic features of malignant transformation is the acquisition of resistance to apoptosis. We previously found that the colonic epithelium of mice, deficient in CD44 is predisposed to apoptosis. In this study, we asked whether the expression of CD44 alters the response of the colon to an apoptotic stimulus, and what are the mechanisms involved. For this, we assessed the susceptibility of the murine colon to apoptosis by total body irradiation to induce apoptosis. Apoptotic and concomitant changes relevant to the mechanisms of apoptosis were monitored by molecular markers of apoptosis. We found enhanced susceptibility to apoptosis in CD44 deficient colonic epithelium based on an increase in the number of apoptotic bodies, and activation of caspase 3. This was not associated with alterations in proliferations as shown by comparable Ki-67 expression and BrdU labeling. Furthermore, upregulated active caspase 3 in CD44 deficient colon was accompanied by concomitant molecular alterations in caspase 9 and not caspase 8, and this indicated the involvement of the mitochondrial pathway in apoptosis execution. Overall, this is the first report demonstrating CD44 mediated resistance to apoptosis in the colonic epithelium in vivo. This implicates CD44 in promoting cell transformation into a malignant phenotype, in conjunction with other anti-apoptotic factors.

CD44 promotes resistance to apoptosis in human colon cancer cells

Overexpression of CD44, especially its variant isoforms, occurs consistently in colon cancer, as compared to autologous normal colon, and this change occurs also in most other types of cancer. One of the basic features of malignant transf ormation is the acquisition of resistance to apoptosis. In this study, we asked whether the expression of CD44 and some of its variant isoforms commonly found in colon cancer participate in resistance to apoptosis and what are the mechanisms involved. A human colon cancer cell line, SW620, which does not express CD44 was stably transfected with standard, v3-10, and v8-10 containing isoforms of CD44. Mock-transfected and CD44-transfected cells were exposed to etoposide to induce apoptosis. Apoptotic and concomitant changes relevant to the mechanisms of apoptosis were monitored by flow cytometry, DNA fragmentation, and immunoblot analyses. It was observed that resistance to apoptosis induced by etoposide is promoted by CD44 expression in SW620, and this resistance is better sustained by the full variant isoform, v3-10. Concomitant alterations in caspase 9, caspase 3, Bcl-xl, and Bak indicated that the resistance to apoptosis in this model involved the mitochondrial pathway. The differential response of CD44 transfectants was associated with a downregulation of pRb and phosphorylated AKT. The results of this study are consistent with the conclusion that expression of variant CD44 isoforms which is characteristic of colon cancer, and most other types of cancer, confers a selective advantage to resist apoptosis, thereby promoting cell transformation into a malignant phenotype, in conjunction with other anti-apoptotic factors.