Anticlastogenic and anti-apoptotic potential of <i>Eulophia gracilis</i> extract in arsenic-induced oxidative liver damage in rats

Sodium arsenite exposure at concentration >5 μM may induce embryotoxic and teratogenic effects in animal models. Long-term health effects of sodium arsenite from contaminated drinking water may result in different forms of cancer and neurological abnormalities. As cancer development processes seem to be originated in stem cells, we have chosen to examine the effects of sodium arsenite on signaling pathways and the corresponding transcription factors that regulate cell viability and self-renewal in mouse embryonic stem cells (ESC) and mouse neural stem/precursor cells. We demonstrated that the crucial signaling pathway, which was substantially suppressed by sodium arsenite exposure (4 μM) in ESC, was the PI3K-AKT pathway linked with numerous downstream targets that control cell survival and apoptosis. Furthermore, the whole core transcription factor circuitry that control self-renewal of mouse ESC (Stat3-P-Tyr705, Oct4, Sox2 and Nanog) was strongly down-regulated by sodium arsenite (4 μM) exposure. This was followed by G2/M arrest and induction of the mitochondrial apoptotic pathway that might be suppressed by caspase-9 and caspase-3 inhibitors. In contrast to mouse ESC with very low endogenous IL6, mouse neural stem/precursor cells (C17.2 clone immortalized by v-myc) with high endogenous production of IL6 exhibited a strong resistance to cytotoxic effects of sodium arsenite that could be decreased by inhibitory anti-IL6 antibody or Stat3 inhibition. In summary, our data demonstrated suppression of self-renewal and induction of apoptosis in mouse ESC by sodium arsenite exposure, which was further accelerated due to simultaneous inhibition of the protective PI3K-AKT and Stat3-dependent pathways.

AIM: To investigate the prevalence of the expression of Bcl-2 and Bax proteins and its correlation with mammographical imaging signs in patients with breast cancer. METHODS: Fifty-four breast cancers and twenty-six benign diseases were proved by pathologic methods and all cases underwent mammography. Immunohistochemical technique was performed to measure the expression of Bcl-2 and Bax proteins in these tissues. The correlation of imaging signs with the expression of Bcl-2 and Bax proteins in breast cancer and benign lesions was analyzed. RESULTS: The expressions of Bcl-2 and Bax proteins in the breast cancer were higher than those in benign breast diseases: 41/54 (76%) vs. 8/26 (31%) and 32/54 (59%) vs. 5/26 (19%), P = 0.0001 and 0.0008, respectively. The expressions of Bcl-2 and Bax proteins in breast tumor with spiculate sign were higher than those in breast tumors without spiculate sign: 26/30 (87%) vs. 15/24 (63%) and 22/30 (73%) vs. 10/24 (42%), P = 0.039 and 0.0186, respectively. The expression of Bcl-2 protein in breast tissues with clustered calcifications was higher than that without calcifications: 21/23 (91%) vs. 18/31 (58%), P = 0.007. CONCLUSION: Breast cancer has a higher rate of expression of Bcl-2 and Bax than benign breast diseases. Spiculate sign and clustered calcifications of breast cancer were closely related to the expressions of Bcl-2 and/or Bax proteins and these signs may reflect the biological behavior of breast tumor cells.

Transgenic mice expressing high levels of the BclxL or Bcl2 proteins in the male germinal cells show a highly abnormal adult spermatogenesis accompanied by sterility. This appears to result from the prevention of an early and massive wave of apoptosis in the testis, which occurs among germinal cells during the first round of spermatogenesis. In contrast, sporadic apoptosis among spermatogonia, which occurs in normal adult testis, is not prevented in adult transgenic mice. The physiological early apoptotic wave in the testis is coincident, in timing and localization, with a temporary high expression of the apoptosis-promoting protein Bax, which disappears at sexual maturity. The critical role played by the intracellular balance, probably hormonally controlled, of the BclxL and Bax proteins (Bcl2 is apparently not expressed in normal mouse testis) in this early apoptotic wave is shown by the occurrence of a comparable testicular syndrome in mice defective in the bax gene. The apoptotic wave appears necessary for normal mature spermatogenesis to develop, probably because it maintains a critical cell number ratio between some germinal cell stages and Sertoli cells, whose normal functions and differentiation involve an elaborate network of communication.

Effect of Gonadotropin-Releasing Hormone Analogs on Bcl-2 and Bax expression in endometrial cultures from patients with endometriosis

Bcl-2 expression by eosinophils in a patient with hypereosinophilia

Objective To investigate the expression of survivin and bcl-2 in human squamous cell carcinoma (SCC) lesions and cell line SCL-1. Methods Tissue samples from 60 patients with SCC and 10 normal human controls were immunohistochemically stained to detect the expressions of survivin and bcl-2.Western blot was used to measure the expressions of bcl-2 and survivin proteins in HaCaT human keratinocytes and SCL-1 human squamous cell carcinoma cells. Results In normal control tissues, there was no expressions of survivin or bcl-2, while in SCC, the expression rates of bcl-2 and survivin were 70% and 60%, respectively,and there was no statistical correlation between the expressions of bcl-2 and survivin (P >0.05). Neither the expression of survivin nor that of bcl-2 was correlated to patients' age, gender or lesional site (all P >0.05). A statistical correlation was observed between the pathological stage in patients and expression of bcl-2 as well as between lymph node metastasis and expression of survivin (both P < 0.05). Western blot analysis revealed a significant increase in the expression of survivin and bcl-2 in SCL-1 cells compared with HaCaT cells. Con-clusion In SCC, survivin and bcl-2 seem to play their roles via different anti-apoptotic pathways. Key words: Neoplasms; squamous; cell; Survivins; Genes; bcl-2; Cells; cultured

We explored the role of the BCL-2 proto-oncogene in the life cycle of trophoblast cells by examining: 1) the patterns of BCL-2 expression in normal placenta at various gestational ages and in specimens of hydatidiform moles and choriocarcinomas, and 2) the effects of cyclic adenosine monophosphate (cAMP) treatment of JEG-3 choriocarcinoma cells, which induces differentiated functions, on BCL-2. BCL-2 protein was localized by indirect immunofluorescence and immunoperoxidase staining of tissue sections and cells using monoclonal and polyclonal antibodies. Western and Northern blotting were used to assess BCL-2 and p53 protein and mRNA levels, respectively. JEG-3 cells were transfected with a BCL-2 expression plasmid to establish that BCL-2 protein could be expressed at high levels in this cell type. BCL-2 immunostaining was most prominent in the syncytiotrophoblast of normal placenta. It was found in syncytiotrophoblast of complete and partial hydatidiform moles, whereas cytotrophoblast staining was weak. BCL-2 immunostaining was also barely detectable in choriocarcinoma cells (JEG-3 cells) and a primary choriocarcinoma. However, BCL-2 protein could be transiently overexpressed in JEG-3 cells by transfection with an expression plasmid. Western blot analysis revealed low levels of BCL-2 in JEG-3 cells and a rise in BCL-2 protein in placental extracts from 10 weeks' gestation to term. In contrast, p53 protein was abundant in JEG-3 cells and normal placenta at 10 weeks' gestation, but low at term, BCL-2 transcripts were substantially more abundant in term placenta than in JEG-3 cells. Treatment of JEG-3 cells with 8-Br-cAMP, which induces genes characteristic of the syncytiotrophoblast, raised BCL-2 protein approximately twofold, whereas p53 mRNA declined. We conclude that: 1) There is a differentiation-dependent pattern of BCL-2 expression in the placenta, with the protein being most abundant in terminally differentiated trophoblast cells; 2) there appears to be an inverse relation between BCL-2 and p53 expression in trophoblast; and 3) cAMP regulates BCL-2 protein in trophoblast cells. We speculate that the expression of BCL-2 in terminally differentiated trophoblast cells, and hence resistance to apoptotic cell death, may be one mechanism by which trophoblast mass is preserved during pregnancy. Conversely, the relatively low expression of BCL-2 in choriocarcinoma cells may render them more susceptible to apoptosis.

Photodynamic therapy (PDT) in early squamous cell carcinoma of the bronchus has been shown to result in complete response (CR) and cure. However, local recurrence after PDT develops frequently even after complete remission. Because the effect of PDT had been reported to depend on apoptosis, and apoptosis is inhibited by bcl-2 protein, the relationship between the expression of bcl-2 protein and local recurrence after PDT was examined immunohistochemically. From 1983 to 1997, 50 patients with 59 early squamous cell carcinoma of the bronchus received PDT, and a CR was obtained in 43 lesions (72.8%). As there was no recurrence among tumours that were disease-free for more than 2 years, in this study the tumours were defined as cured when recurrence did not occur 2 years subsequent to the receiving of PDT. Of these CR lesions, 31 carcinomas (53.4%) resulted in a cure. Bcl-2 immunoreactivity was detected in 23 tumours (46.9%) and p53 immunoreactivity was detected in 22 tumours (44.9%). When all tumours were divided into either a large tumour with a longitudinal tumour length of 10 mm or more, or a small tumour with a length of less than 10 mm, the large tumour expressed more bcl-2 protein than the small tumour (P = 0.0155). The degree of bcl-2 expression was significantly related with tumour size (P = 0.0155). The expression of bcl-2 and p53 protein was not associated with the cure rate due to PDT. Tumour length and T status in TNM staging were significantly related to the cure by univariate analysis. T status was the only predictor of the cure according to mutivariate analysis. Of 42 CR lesions, the expression of neither bcl-2 nor p53 protein was associated with local recurrence; only T status was significantly associated (P = 0.008). The relationship between the expression of oncoprotein and local recurrence after PDT was not documented in this study. The success of PDT may depend on the exact assessment of tumour size under optimized PDT illumination. © 2000 Cancer Research Campaign

Objective To investigate the expressions of B7-H1 and Bcl-2 proteins in epithelial ovarian cancer, and to explore the association of the expressions of B7-H1 and Bcl-2 with clinicopathological features. Methods The expressions of B7-H1 and Bcl-2 proteins were detected by immunohistochemistry in 80 cases of epithelial ovarian cancer tissues and 20 normal ovary tissues. The association of the expressions of B7-H1 and Bcl-2 with the clinicopathological features was analyzed. Results The positive expression rates of B7-H1 and Bcl-2 in epithelial ovarian cancer tissues were 77.5% (62/80) and 58.8% (47/80), both higher than 15.0% (3/20) and 10.0% (2/20) in normal ovary tissues with significant difference (χ2=27.473, P<0.05; χ2=15.216, P<0.05). Both of Bcl-2 and B7-H1 expressions in epithelial ovarian cancer tissues were negatively correlated with the differentiation degree of epithelial ovarian cancer (χ2=9.367, P<0.01; χ2=11.702, P<0.01). The Bcl-2 expression in epithelial ovarian cancer tissues was positively correlated with the FIGO stage (χ2=7.766, P<0.01). The expression of B7-H1 was positively correlated with the expression of Bcl-2 in epithelial ovarian cancer (r=0.400, P<0.01). Conclusion The expressions of B7-H1 and Bcl-2 are up-regulated in epithelial ovarian cancer and they correlate to each other positively. The expressions of B7-H1 and Bcl-2 are correlated with the invasion and metastasis of epithelial ovarian cancer. The detection of B7-H1 combined with Bcl-2 may have an important clinical significance in the diagnosis and treatment for patients with epithelial ovarian cancer. Key words: Ovarian neoplasms; Gene; B7-H1; Bcl-2; Immunohistochemistry

Bcl-2 and p53 Protein Expression, Apoptosis, and p53 Mutation in Human Epithelial Ovarian Cancers

Endemic and sporadic Burkitt's lymphoma differ in site and age of presentation, and Epstein-Barr virus (EBV) association. This study aimed to describe the clinical presentation, EBV association and p53 expression of Burkitt's lymphoma in 59 Malaysian patients. Expression of Bcl-2, retinoblastoma and Ki67 proteins was also investigated. EBV was detected by EBER in situ hybridization. Expression of p53, Bcl-2, pRb and Ki67 proteins was detected by immunohistochemical staining. Polymerase chain reaction was employed for EBV subtyping and detection of translocation t (14; 18). The male to female ratio was 3.9: 1. The most common age group was children younger than 15 years (72.9%), with a mean of 15.8y. The disease is more common in ethnic Chinese. EBV association is 33.3% and all were infected with type-A virus. Expressions of p53, Bcl-2, pRb and Ki67 proteins were detected in 42/49 (85.7%), 9/54 (16.7%), 47/51 (92.2%) and 35/39 (89.7%) of the biopsies, respectively. Translocation t (14; 18) was not detected in cases expressing the Bcl-2 protein. Clinical presentation and EBV association of Burkitt's lymphoma in Malaysian patients corresponds with that of sporadic Burkitt's lymphoma. This study also reveals that a substantial proportion of cases express high levels of p53 and retinoblastoma proteins, in contrast to the Bcl-2 protein.

Objective To evaluate the role of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway in mild head hypothermia-induced reduction of global cerebral ischemia-reperfusion (I/R) injury in rats. Methods Sixty male Sprague-Dawley rats, weighing 250-280 g, were randomly divided into 5 groups (n=12 each) using a random number table: sham operation group (group S), group I/R, mild hypothermia + I/R group (group H), mild hypothermia + I/R + solvent control group (group DM), and mild hypothermia + I/R + PI3K inhibitor LY294002 group (group LY). Global cerebral I/R was induced by modified four-vessel occlusion method described by Pulsinelli. In H group, when the hippocampal temperature was decreased to 33℃ using nasopharyngeal cooling, the bilateral common carotid arteries were occluded for 15 min followed by reperfusion, and hippocampal hypothermia was maintained at 32.5-33.5 ℃ for 1 h. In DM and LY groups, DMSO and LY294002 5 μl were injected into the left ventricle, respectively, and 20 min later the other procedures were similar to those previously described in group H. At 8 h of reperfusion, 6 rats were sacrificed, and hippocampal specimens were obtained to detect the expression of pFoxO3a, Bcl-2 and Bax (by immuno-histochemistry). The expression of phosphor-Akt (p-Akt) was determined by Western blot. Results Compared with group S, the expression of p-Akt, pFoxO3a and Bcl-2 was significantly up-regulated in I/R, H and DM groups, the expression of Bcl-2 was down-regulated, and the ratio of Bcl-2/Bax was increased in H and DM groups, and the ratio of Bcl-2/Bax was decreased in I/R and LY groups. Compared with group I/R, the expression of p-Akt, pFoxO3a and Bcl-2 was significantly up-regulated, the expression of Bcl-2 was down-regulated, and the ratio of Bcl-2/Bax was increased in H and DM groups. Compared with H and DM groups, the expression of p-Akt, pFoxO3a and Bcl-2 was down-regulated, the expression of Bax was up-regulated, and the ratio of Bcl-2/Bax was decreased in group LY. Conclusion PI3K/Akt signaling pathway is involved in reduction of global cerebral I/R injury by mild head hypothermia in rats. Key words: Phosphoinositide-3-kinase; Protein-serine-threonine kinases; Hypothermia, induced; Reperfusion injury; Brain

Bcl-6 and Bcl-2 protein expression in diffuse large B-cell lymphoma and follicular lymphoma: Correlation with 3q27 and 18q21 chromosomal abnormalities

Chrysin (CHR) is a food-based bioactive ingredient whereas, sodium arsenite (SA) is one of the major contaminant in drinking water. When ingested, SA contributes to tissue damage due to bioactivation by S-adenosyl methionine (SAM)-dependent methyltransferase. Hence, the needs to nullify this effect by investigating the potentials of CHR on SA-induced genotoxicity in rats. The experiment was divided into two successive stages (ameliorative and preventive, curative studies) for 1 week. Rats were divided into four groups: distilled water, 10mg/kg SA, 10mg/kg CHR and co-administration. In stage 2, the experimental groups were given either CHR or SA for 1 week, and treated in reversed order for additional week. Lipid peroxidation, protein carbonyl and DNA fragmentation in liver, blood brain and bone marrow cells micronucleus were assayed for using standard protocols. Molecular docking of SAM-dependent methyltransferase in the presence of CHR was conducted. CHR significantly ( p < 0.05) decreased the level of lipid peroxidation, protein carbonyls and DNA fragmentation in blood, liver and brain tissues as against group treated with SA. It also significantly ( p<0.05) reduced the level of micronuclei generated in bone marrow cells. The effects of CHR were shown to be ameliorative, preventive and curative in nature. Furthermore, CHR was able to dock (with binding energy of -24.81 kcal/mol and predicted inhibition kinetic constant (Ki) of 0.959 µM) into the active site of SAM-dependent methyltransferase with strong hydrogen bond and hydrophobic interactions. The study might have unravelled the potentials of CHR against SA-induced chromosomal and DNA damage, which might be due to inhibition of SAM-dependent methyltransferase.

Concurrent BCL2 and MYC Protein Expression by Immunohistochemistry Determines Clinical Outcome In DLBCL Patients Treated with R-CHOP