p53-dependent Fas expression is critical for Ginsenoside Rh2 triggered caspase-8 activation in HeLa cells

We have recently reported that Ginsenoside Rh2 (G-Rh2) induces the activation of two initiator caspases, caspase-8 and caspase-9 in human cancer cells. However, the molecular mechanism of its death-inducing function remains unclear. Here we show that G-Rh2 stimulated the activation of both caspase-8 and caspase-9 simultaneously in HeLa cells. Under G-Rh2 treatment, membrane death receptors Fas and TNFR1 are remarkably upregulated. However, the induced expression of Fas but not TNFR1 was contributed to the apoptosis process. Moreover, significant increases in Fas expression and caspase-8 activity temporally coincided with an increase in p53 expression in p53-non-mutated HeLa and SK-HEP-1 cells upon G-Rh2 treatment. In contrast, Fas expression and caspase-8 activity remained constant with G-Rh2 treatment in p53-mutated SW480 and PC-3 cells. In addition, siRNA-mediated knockdown of p53 diminished G-Rh2-induced Fas expression and caspase-8 activation. These results indicated that G-Rh2-triggered extrinsic apoptosis relies on p53-mediated Fas over-expression. In the intrinsic apoptotic pathway, G-Rh2 induced strong and immediate translocation of cytosolic BAK and BAX to the mitochondria, mitochondrial cytochrome c release, and subsequent caspase-9 activation both in HeLa and in SW480 cells. p53-mediated Fas expression and subsequent downstream caspase-8 activation as well as p53-independent caspase-9 activation all contribute to the activation of the downstream effector caspase-3/-7, leading to tumor cell death. Taken together, we suggest that G-Rh2 induces cancer cell apoptosis in a multi-path manner and is therefore a promising candidate for anti-tumor drug development.

Effect of valproic acid sodium on proliferation and apoptosis of acute T-lymphoblastic leukemia Jurkat cells / 中国实验血液学杂志

This study was aimed to investigate the effects of valproic acid sodium (VPA) on the proliferation and apoptosis of acute T-lymphoblastic leukemia Jurkat cells. Jurkat cells were treated with different concentration of VPA. Proliferation-inhibition curve was assayed and plotted by CCK-8 method and the cell apoptosis was detected by flow cytometry with Annexin V/PI double staining. The expression level of anti-apoptotic gene BCL-2 and pro-apoptosis gene Bak1 were detected by semi-quantitative RT-PCR. The results showed that the VPA inhibited the proliferation of Jurkat cells in concentration-dependent manner. As compared with the control group, the apoptosis of cells increased along with adding concentration of VPA; VPA could decrease the expression of BCL-2 gene, but did not show obvious effect on the expression of Bak1. It is concluded that the VPA can inhibit proliferation of Jurkat cells which possibly associates with the decrease of BCL-2 expression.

Harringtonine induces apoptosis in NB4 cells through down-regulation of Mcl-1 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the growth inhibition effect, cytotoxicity and apoptotic induction of harringtonine (HT) in human acute promyelocytic leukemia (APL) NB4 cells,and the related mechanism.</p><p><b>METHODS</b>NB4 cells were treated with HT. Total cell numbers were counted by hemocytometer, and cell viabilities were determined by trypan blue exclusion. Apoptotic cells were determined by fluorescence microscopy and FACS after staining with AO and EB or PI, respectively. The cleavage of PARP and the activation of Bax and the expression of anti-apoptotic proteins were determined by Western Blot. siRNA was used to silence the expression of target genes. Primary cells were isolated following Ficoll-Hypaque density gradient centrifugation method.</p><p><b>RESULTS</b>HT inhibited cell growth and induced apoptosis of NB4 cells in a dose- and time-dependent manner. Apoptosis induced by HT was correlated with the down-regulation of Mcl-1 and the cleavage of PARP, while HT did not affect the protein level of Bax and Bak or change the protein level of Bcl-2. The silence of Bcl-XL sensitized HT-induced apoptosis in NB4 cells.Apoptosis induced by HT in primarily cultured APL cells was also correlated with the down-regulation of Mcl-1.</p><p><b>CONCLUSION</b>HT inhibits cell growth and induces apoptosis in NB4 cells and primarily cultured APL cells, which may be associated with down-regulation of Mcl-1.</p>

Expression of Bim, Bax and Bak in the process of gingipain-induced osteoblast apoptosis / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To establish osteoblast apoptosis model induced by gingipains, and to examine the expression of pro-apoptotic protein Bcl-2 interacting mediator (Bim), Bcl-2 associated X protein (Bax) and Bcl-2 antagonist/killer (Bak).</p><p><b>METHODS</b>Gingipain and gingipain acticity were extracted and measured. Mouse osteoblast cell line MC3T3-E1 cells were cultured in the presence of 0.453, 0.906, 1.812 U/L gingipains for 0, 16, 24 and 48 h. Apoptosis was examined by 4',6-diamidino-2-phenylindole (DAPI) staining or annexin V/propidine iodide (PI) staining.Protein expression of Bim, Bax and Bak was determined by Western blotting after osteoblasts were cultured with 1.812 U/L gingipain for 0, 4, 8, 16, 24 and 48 h. Osteoblasts were cultured with 1.812 U/L gingipain which had been inhibited with N-alpha-tosyl L-lysyl-chlorom ethylketone (TLCK). Western blotting was used to detect Bim expression and DAPI staining to measure apoptosis.</p><p><b>RESULTS</b>Arginine-specific proteinases (Rgp) activity was (18.11 ± 2.11) U/L and specific proteinases (Kgp) was (1.02 ± 0.25) U/L. Percentage of osteoblast apoptosis induced by 1.812 U/L gingipain rose to (6.31 ± 0.37)% after 16 h, and reached (11.20 ± 0.35)% at 24 h and (10.80 ± 0.46)% after 48 h with DAPI staining. Annexin V/PI staining supported the result from DAPI staining.Bim protein level increased during osteoblast apoptosis, the relative fold rose to (0.31 ± 0.03) after 4 h (about 2 fold compared to control), peaking at 24 h (0.57 ± 0.05, 3-4 fold compared to control). Proteinase inhibitor TLCK effectively blocked the activity of gingipain and inhibited up-regulation of Bim induced by gingipains from (0.58 ± 0.04) to (0.14 ± 0.03). The percentage of osteoblast apoptosis decreased from (11.20 ± 0.35)% to (4.31 ± 0.38)% in the presence of TLCK. Expression of Bax remained unchanged when cells were cultured with or without gingipains. Bak was under the detectable level in MC3T3-E1.</p><p><b>CONCLUSIONS</b>1.812 U/L gingipains induced osteoblast apoptosis. Protein expression of Bim was up-regulated during cell apoptosis and was down-regulated when gingipain inhibited with TLCK, suggesting that Bim was involved in osteoblast apoptosis induced by gingipain. Inhibition of Bim protein expression protected osteoblast from apoptosis.</p>

Protective Effects of Oleic Acid Against Palmitic Acid-Induced Apoptosis in Pancreatic AR42J Cells and Its Mechanisms

Palmitic acid (PAM), one of the most common saturated fatty acid (SFA) in animals and plants, has been shown to induce apoptosis in exocrine pancreatic AR42J cells. In this study, we investigated cellular mechanisms underlying protective effects of oleic acid (OLA) against the lipotoxic actions of PAM in AR42J cells. Exposure of cells to long-chain SFA induced apoptotic cell death determined by MTT cell viability assay and Hoechst staining. Co-treatment of OLA with PAM markedly protected cells against PAM-induced apoptosis. OLA significantly attenuated the PAM-induced increase in the levels of pro-apoptotic Bak protein, cleaved forms of apoptotic proteins (caspase-3, PARP). On the contrary, OLA restored the decreased levels of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, and Mcl-1) in PAM-treated cells. OLA also induced up-regulation of the mRNA expression of Dgat2 and Cpt1 genes which are involved in triacylglycerol (TAG) synthesis and mitochondrial beta-oxidation, respectively. Intracellular TAG accumulation was increased by OLA supplementation in accordance with enhanced expression of Dgat2 gene. These results indicate that restoration of anti-apoptotic/pro-apoptotic protein balance from apoptosis toward cell survival is involved in the cytoprotective effects of OLA against PAM-induced apoptosis in pancreatic AR42J cells. In addition, OLA-induced increase in TAG accumulation and up-regulation of Dgat2 and Cpt1 gene expressions may be possibly associated in part with the ability of OLA to protect cells from deleterious actions of PAM.

miR-125b promotes proliferation of human acute myeloid leukemia cells by targeting Bak1 / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate miR- 125b regulation mechanism by identifying miR-125b target genes and its function in acute myeloid leukemia (AML).</p><p><b>METHODS</b>The bioinformatics software and database were applied to predict and analyze target genes of miR-125b. The vector contained the target gene 3'-UTR portion cloned into a luciferase reporter construct. A luciferase reporter assay was performed following co-transfection of small molecular miR-125b mimics and target gene wild-type or mutant plasmid into HEK-293T cells. Further in leukemia cell lines NB4 and HL-60, the protein level of target gene was measured by Western blot after overexpression miR-125b. Finally, the viabilities of NB4 and HL-60 cells were measured by CCK-8 assay at 24 h, 48 h, 72 h, 96 h after electroporation.</p><p><b>RESULTS</b>Bcl-2-antagonist/killer 1 (Bak1), a pro-apoptotic gene, was a target gene of miR-125b by software predicts. Reporter vector containing the 3'-UTR Bak1 wild and mutation sites were co-transfected with small molecule analogues of miR-125b in HEK-293T cells. Dual luciferase reporter gene assay system showed that miR-125b significantly suppresses the reporter gene activity containing Bak1 3'-UTR by about 53.8% (P<0.05), but it didn't suppresses the reporter gene activity containing 3'-UTR Bak1 mutation. Western blot showed that miR-125b mimics significantly down-regulated the expression of Bak1 in human leukemia cell lines NB4 and HL-60. Meanwhile, the growth rate of cells treated with miR-125b obviously increased compared with that in control by CCK-8 test (P<0.05).</p><p><b>CONCLUSION</b>Our findings strongly indicated that BAK1 was a downstream target gene of miR-125b, and miR-125b promoted proliferation in human AML cells at least partially by targeting Bak1, so we speculated that miR-125b as an oncogene could be a potential therapeutic target for treating AML.</p>

Expression of MCL-1 and BAK proteins in nutritional anemia and its clinical significance / 中国实验血液学杂志

This study was aimed to investigate the relation of MCL-1 and BAK proteins with incidence and development of nutritional anemia (NA) and their clinical significance. The MCL-1 and BAK protein levels in serum of 66 patients with NA were determined by using ELISA. Eighteen healthy people were randomly selected as normal controls. The results indicated that: (1) as compared with normal control group, the expression level of MCL-1 protein in 3 NA groups (iron-deficiency anemia, macrocytic anemia, mixed anemia) significantly decreased (P < 0.001), while the expression level of BAK protein obviously increased (P < 0.001), but the expression level of MCL-1 and BAK proteins among 3 NA groups showed no obvious differences; (2) the MCL-1 protein expression level increased and BAK protein expression level decreased in 3 NA groups after treatment (P < 0.05). (3) there was negative correlation of expression levels of MCL-1 protein with BAK protein in NA group (r = -0.858 P < 0.05). It is concluded that the MCL-1 and BAK proteins may play an important role in the incidence and development of NA, and can be used as the assist index for defining diagnosis and evaluate prognosis of NA.

Human Bop is a novel BH3-only member of the Bcl-2 protein family

One group of Bcl-2 protein family, which shares only the BH3 domain (BH3-only), is critically involved in the regulation of programmed cell death. Herein we demonstrated a novel human BH3-only protein (designated as Bop) which could induce apoptosis in a BH3 domain-dependent manner. Further analysis indicated that Bop mainly localized to mitochondria and used its BH3 domain to contact the loop regions of voltage dependent anion channel 1 (VDAC1) in the outer mitochondrial membrane. In addition, purified Bop protein induced the loss of mitochondrial transmembrane potential (Δψm) and the release of cytochrome c. Furthermore, Bop used its BH3 domain to contact pro-survival Bcl-2 family members (Bcl-2, Bcl-X(L), Mcl-1, A1 and Bcl-w), which could inhibit Bop-induced apoptosis. Bop would be constrained by pro-survival Bcl-2 proteins in resting cells, because Bop became released from phosphorylated Bcl-2 induced by microtubule-interfering agent like vincristine (VCR). Indeed, knockdown experiments indicated that Bop was partially required for VCR induced cell death. Finally, Bop might need to function through Bak and Bax, likely by releasing Bak from Bcl-X(L) sequestration. In conclusion, Bop may be a novel BH3-only factor that can engage with the regulatory network of Bcl-2 family members to process intrinsic apoptotic signaling.

Low apoptotic index & bak expression in EBV-associated childhood classical Hodgkin lymphoma.

Background & objectives: Association of Epstein-Barr virus (EBV) with Hodgkin lymphoma (HL) is particularly high in low-income countries, and resistance to apoptosis might play a role in pathogenesis and survival. Data from previous studies are not consistent, and none is available in children. Thus this study was undertaken on Indian children with classical Hodgkin lymphoma to assess the significance of bcl-2, bak and p53 expression, and apoptotic index in relation with EBV status and treatment outcome with chemotherapy alone. Methods: Children (age<15 yr) with classical HL (n=143) were included in the study. Bcl-2, bak, p53, Ki67 and latent membrane protein-1 (LMP1) were detected by immunohistochemistry in pre-treatment lymph node biopsies. Apoptotic index was assessed by TdT-dUTP nick-end labelling (TUNEL). Results: Bcl-2, bak, p53 were expressed above positivity threshold in 83.3, 94.0 and 7.1 per cent of the cases respectively. More than 10 per cent of apoptotic tumour cells were seen in 60.4 per cent of the cases. 131 (91.6%) cases were EBV associated. EBV-positive cases had a significantly lower mean bak expression (p=0.001) and a lower apoptotic index, without higher proliferation index. Advanced stage showed a borderline association with bcl-2 expression in >25 per cent of tumour cells and p53 negative tumours. In univariate analysis, p53 positive cases, which were significantly associated with B symptoms, had a poorer overall survival (P=0.03) while low proliferation index was associated with poorer failurefree survival. Neither EBV status nor any of the apoptotic parameters studied showed independent association with survival. Interpretation & conclusion: EBV detection in children with classical Hodgkin lymphoma was associated with significant lower bak expression and with lower spontaneous apoptosis of H-RS cells suggesting that EBV-LMP1 might downregulate bak pro-apoptotic protein. this needs to be substantiated further.

Inhibitory effect of RNA interference targeting BaxBak on apoptosis of human granulosa cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of small interfering RNA (siRNA) targeting Bax-Bak on the apoptosis of human granulosa cells.</p><p><b>METHODS</b>Human granulosa cells were transfected with Bax-siRNA and Bak-siRNA either alone or in comibnation, and the cell morphological changes were obsered and the cell apoptosis was detected with flow cytometry. Western blotting was performed to examine the changes in Bax and Bak expressions in the transfected cells.</p><p><b>RESULTS</b>Western blotting demonstrated significantly weakened expressions of Bax and Bak in the transfected cells. The cell morphology of the cells tranfected with Bak siRNA and with both Bak and Bax siRNA remained normal; the cells with exclusive Bax siRNA transfection presented with basically normal cell morphology, but black spots were noted in the cytoplasm. In the positive and negative control groups, the cells became rounded and shrank with expanded intercellular spaces and numerous black spots in the cytoplasm. Flow cytometry showed apoptotic indexes of 3.44% and 3.97% in cells transfected with Bak siRNA and Bax-Bak siRNA, respectively, significantly lower than that in the negative group. Bax siRNA transfection resulted in an apoptotic index of 19.98%, similar to that in the negative group.</p><p><b>CONCLUSION</b>Interference of the expression of Bak gene inhibits the apoptosis of human granulosa cells, and the inhibitory effect can be enhanced by simultaneous Bax interference, which, when used alone, does not obviosuly inhibit the apoptosis of human granulosa cells.</p>

The Bax BH3 peptide H2-H3 promotes apoptosis by inhibiting Bcl-2's pore-forming and anti-Bax activities in the membrane / 生物医学工程学杂志

Pore-formation and protein-protein interactions are considered to play critical roles in the regulation of apoptosis by Bcl-2 family proteins. During the initiation of apoptosis, the anti-apoptotic Bcl-2 and the pro-apoptotic Bax form different pores to regulate the permeability of mitochondrial outer membrane, playing their opposite functions. Overexpression of Bcl-2 has been found in various cancer cells, therefore it is gaining widespread interest to discover small molecules to compromise Bcl-2 function for anti-cancer treatment. Since Bax binds to Bcl-2's hydrophobic groove via its BH3 domain (composed of helices 2 and 3), by which their functions are inhibited each other, the H2-H3 peptide that contains the functional BH3 domain of Bax has been considered as a potential Bcl-2 antagonist. We recently reported that Bax peptide H2-H3 promotes cell death by inducing Bax-mediated cytochrome c release and by antagonizing Bcl-2's inhibitory effect on Bax. However, the mechanism of how H2-H3 inhibits the anti-apoptotic activity of Bcl-2 remains poorly understood. To address this question, we reconstituted the Bcl-2 or Bax pore-forming process in vitro. We found that H2-H3 inhibited Bcl-2's pore formation and neutralized Bcl-2's inhibitory effect on Bax pore formation in the membrane, whereas the mutant H2-H3 peptide that does not induce apoptosis in cells was shown to have no effect on Bcl-2's activities. Thus, inhibiting Bcl-2's pore-forming and anti-Bax activities in the membrane is strongly correlated with H2-H3's pro-apoptosis function in cells.

Stimulatory heterotrimeric G protein augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 human lung cancer cells

Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the alpha subunit of Gs (GalphasQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GalphasQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked GalphasQL-stimulated Bak reporter luciferase activity. Expression of GalphasQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Galphas activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Galphas augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.

effects of resveratrol and tannic acid on apoptosis in colon adenocarcinoma cell line

To investigate the effects of resveratrol and tannic acid on apoptosis, and Bcl-2 homologous antagonist/killer [Bak] and fas associated death domain [FADD] proteins in the CaCo-2 cell line. In the present study, resveratrol and tannic acid were administrated in the CaCo-2 cell line at doses of 25, 50, and 100 uM. The CaCo-2 cells were grown and cultured in the Medical Biology Department, Eskisehir Osmangazi University, Eskisehir, Turkey in 2007. The effects of these agents on apoptotic index were determined by Apop Taq peroxidase kit and their effects on the ratios of Bak and FADD proteins by the immunohistochemical staining method at 24, 48, and 72 hours. Stained and non-stained cells in 30 separate areas of the 3 separate chamber slides, prepared for each group, were counted. The percentage of apoptosis, and Bak and FADD proteins was calculated with the control. Mean +/- standard error values were calculated for the 3 experiments. Apoptotic index, Bak protein percentage ratio, and FADD protein percentage ratio values in all groups that received tannic acid and resveratrol increased when compared within the groups. This increase was found to be time and dose independent in all parameters. Cells undergo apoptosis in 2 pathways [mitochondrial and death receptor] in resveratrol and tannic acid induced CaCo-2 cells

Selection of bak siRNA sequences and its influence on Al-induced apoptosis of SH-SY5Y cell line / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To find the optimal design of small interfering RNA compounds, transfection concentration and transfection time to reduce the Al-induced apoptosis in SH-SY5Y cells.</p><p><b>METHODS</b>Three siRNA sequences on bak gene were designed and transfected into SH-SY5Y cells, which were treated at various concentrations of aluminum. Cell viability was detected by CCK-8 kit on different siRNA sequences, various transfection concentrations, and diverse transfection courses. Transfection efficiency was determined by fluorescent staining of CY3, and interference efficiency was measured by QRT-PCR. Besides, immunohistochemical staining was used to express Bak protein content. Finally, apoptotic rate and necrotic rate in Al treated SH-SY5Y cells transfecting by the selected bak siRNA 1 were detected.</p><p><b>RESULTS</b>Based on the viability of siRNA sequences, siRNA 1 was selected as the optimal siRNA sequences. The optimal transfection concentration was 10 nmol/L, and the optimal time course was 24 h after transfection. The transfection efficiency was above 90% and the interference efficiency with bak gene was 57.76%. Furthermore, there was significant transfection effect on Bak protein. The apoptotic rate in Al treated SH-SY5Y cells were significantly decreased by bak siRNA 1 transfection.</p><p><b>CONCLUSION</b>Apoptosis is one of the major cell death pathways in SH-SY5Y cells induced by aluminum. When chemically synthesized siRNA is inducted to neural cells, it can significantly reduce bak gene level, decrease Bak protein expression and apoptotic rate, which may serve as the basis for preventing neural cells apoptosis and inhibiting the development of neurodegenerative diseases.</p>

Apoptosis suppressing protein Bcl-2 expression in rheumatoid synovium

It has been suggested that defective apoptosis plays a role in the development of autoimmune. The aim of this study is to investigate the apoptosis suppressing protein Bcl-2 in the rheumatoid synovum. Twenty synovium biopsy specimens were studied histopathologically and immuno-histochemically. Positive immunoreactivity was observed in 16 cases [80%] the infiltrated lymphocytes and in 11 cases [55%] in the lymphoid follicles, while, in 8 cases [40%] deep synovial fibroblasts showed marked Bcl-2 positively. The superficial synovial cell lining was only positive in 5 cases [25%].The apoptotic process is suppressed in rheumatoid arthritis as triggered by Bcl-2 oncoprotein. The defective control of apoptosis. [programmed cell death] as well excessive proliferation may he of central importance in the pathogenesis of rheumatoid arthritis

Tacrolimus (FK506) Induced Apoptotic Signal Transduction Pathway

PURPOSE: This study examined the effects of Tacrolimus (FK506) on the expression of the apoptotic signal transduction proteins of Jurkat human T-lymphocytes. METHODS: The cell viability was examined by a MTT assay, DAPI stain, enzyme activity of caspase family proteins, and western blotting for Bcl-2, Bak, Fas, and Fas-L. The cells were cultured in the presence or absence of FK506. FK506 induced cell death was confirmed to be apoptosis by the observation of nuclear fragmentation. RESULTS: The viability of Jurkat cells was decreased by the addition of FK506 in a dose- and time- dependent manner. The FK506 induced activation of caspase-3 protease was observed. FK506 didn't increase the catalytic activity of caspase -6, -8, and -9 proteases of Jurkat cells in a time-dependent manner. The viability was improved when a caspase-3 inhibitor was added. However, the caspase-9 inhibitor did not affect the viability. Bak protein expression was increased, and the Bcl-2 protein was decreased for some time. The expression of Fas and Fas-L were unaffected by FK506. CONCLUSION: FK506 induces dose- and time-dependent apoptotic cell death, and enhances the apoptosis of Jurkat cell by increasing the expression of Bak and caspase-3.

Effect of Rapamycin on the Cell Cycle Arrest of T Lymphocytes

PURPOSE: Rapamycin (RPM) and its analogues are known for their potent immunosuppressant and anti-proliferative properties, which stem from their ability to modulate the signal transduction pathways involved in cell cycle progression from the G1 to S phase. Thus, RPM has been shown to inhibit the proliferation of a number of non-immune cell types, including hepatocytes, vascular smooth cells and fibroblasts. In addition to its effects on proliferation, RPM may also play a role in the regulation of apoptosis under certain circumstances. METHODS: The effects of RPM on the activation, proliferation and expression of cytotoxic effector molecules were examined on Molt-4 human T-lymphocyte by determining its effects on apoptosis, cell viability, reactive oxygen species (ROS) production and mitochondrial dysfunction. Cells were cultured in the presence or absence of RPM, and then analyzed by Flow cytometry after staining with PI (propidium iodide). RESULTS: The viability of Molt-4 T cells dose- and time-dependently decreased on the addition of RPM. CONCLUSION: RPM induced cytotoxicity was characterized by G2/M phase cell cycle arrest. In addition, a pharmacological scavenging study of ROS, including H2O2, revealed the cytotoxicity was mainly induced by the generation of ROS, which might modulate the expression of Bak protein and mitochondrial dysfunction.

Expression of cyclin-D1 and apoptosis regulating protein BCL-2 in infiltrating ductal breast carcinoma [NOS]

Breast carcinoma is the most frequently diagnosed carcinoma affecting females contributing for about 30% of all female cancers in Egypt Estrogen and progesterone receptors [ER and PR] have been used as tools to identify patients eligible for adjuvant treatments, yet they present some limits, as the existence of dysfunctional receptors made some ER positive tumours non-responsive to antiestrogen therapy. In breast cancer, Bcl-2 expression has been reported to be associated with better outcomes in patients treated with either hormone- or chemo-therapy. Also, the expression of the protein cyclin D1 is detected in up to 50% of primary breast cancers. Cyclin D1 over expression has been strongly associated with well-differentiated, estrogen receptor-positive ductal tumors The present work aimed at determining the expression of cyclin D1 and bcl-2 proteins in some cases of infiltrating breast ductal carcinoma not otherwise specified [NOS,] and to assess any possible relationship between each marker and estrogen receptor status of the tumour tissue. Thirty cases of infiltrating ductal carcinoma [IDC], not otherwise specified [NOS], 17 cases of which were associated with axillary lymph node metastases, in addition to 5 cases of florid epithelial hyperplasia were included in the study. For all studied cases routine histopathological examination and immunohistochemical staining by ER, bcl-2 and cyclin D1 antibodies were done. ER status showed positive ER immunoreactivity in 21 cases [70%] out of 30 [2 cases/2 were grade I, 14 cases /18 were grade II and 5 cases/10 were grade III]. Positive Bcl-2 immunoreactivity was observed in 23 cases of the 30 IDC cases [76.66%]: 2 cases/2 were of grade I, 16 cases/18 of grade II and 5 cases/10 were of grade III. o A statistically significant relation was found between bcl-2 immunoreactivity and ER expression in the malignant cases [p<0.0001]. Cyclin D1 expression was detected in 20 cases of IDC [66.66%], staining was nuclear and diffuse: 2 cases out of 2 of WD carcinoma and 4 cases of MD carcinoma out of 18 showed strong staining [score 1], 10 cases of MD carcinoma out of 18 showed moderate staining [score 2], and 4 cases out of 10 of PD carcinoma showed weak staining [score 3].All the cyclin D1 positive 20 cases were also ER positive [100%]. A statistically significant relation was found between cyclin D1 immunoreactivity and ER expression [p<0.0004]. Bcl-2 was strongly associated with ER positivity in the majority of breast carcinomas but over expression of cyclin D1 protein correlated with poor prognosis and it was demonstrated that its over expression distinguished malignant breast carcinomas from premalignant breast lesions as the less differentiated high grade tumors exhibited a more intense nuclear stain and the non-neoplastic epithelial components were not stained

Mechanism of apoptosis induced by specific COX-2 inhibitor SC236 in gastric cancer cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the underlying mechanism of apoptosis-inducing effect of a specific COX-2 inhibitor SC236 in gastric cancer cells.</p><p><b>METHODS</b>Western blot analysis was used to measure apoptosis-related proteins, cytochrome c, and caspase-3. The catalytic activity of the caspases was measured using a colorimetric assay.</p><p><b>RESULTS</b>Treatment of AGS gastric cancer cells with SC236 caused a significant elevation of the pro-apoptotic protein Bak, release of cytochrome c to the cytosol, and activation of caspase-3. A specific caspase-3 inhibitor, z-DEVD-fmk, blocked SC236-induced apoptosis.</p><p><b>CONCLUSION</b>SC236 inhibits cell growth and induces apoptosis in gastric cancer cells at least partly through the up-regulation of Bak, stimulation of cytochrome c release, and activation of caspase-3.</p>

The Mechanism of Intracellular Signal Pathway that Baicalin Hydrate Elevate Chemotherapeutic Response of Cervical Carcinoma / 대한산부인과학회잡지

Baicalin is flavonoid and major component of PC-SPES. Flavonoids including baicalin have been reported to not only function as anti-oxidant but also cause cytotoxic effect. Baicalin hydrate has been reported to induce cell death, however the mechanism by which baicalin hydrate induces the apoptosis of cancer cells is still unclear. To evaluate the mechanistic insights of apoptosis by baicalin hydrate, we tested the activities of apoptosis signaling pathway in HeLa cells. The viability of HeLa and HeLa s3 cells was markedly decreased by baicalin hydrate in a dose- and time- dependent method. Baicalin hydrate induced the apoptotic death of HeLa cells, which was characterized by the chromatin condensation of the nuclei and phosphorylation of histone H2AX. Baicalin hydrate increased the sub-G1 DNA content of HeLa cell lines. Baicalin hydrate digested Bid protein, increased Bak protein level and also, induced mitochondrial dysfunction disrupted as shown as the mitochondrial membrane potential. It activated caspase-3, thereby resulted in cleavage of poly (ADP) ribose polymerase (PARP).