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Purpose: Bladder cancer is the most general malignant cancer in genitourinary system, more than 90% of BCs are bladder transitional cell carcinomas (BTCC). This study aimed to investigate the clinical significance of growth arrest-specific 5 (GAS5) gene and its regulatory effects of malignant proliferation and chemotherapy resistance to doxorubicin in BTCC cells., Methods: The expression of GAS5 was detected by quantitative real-time PCR. Statistical analysis was used to determine the relationship between GAS5 expression and clinical features and the prognostic value of GAS5 for disease free survival. MTT assay was used to detect cell proliferation ability and chemosensitivity. Dual-color flow cytometric method was used to detect cell apoptosis. The expression of Bcl-2 protein was examined by western blot., Results: In this study, we found that GAS5 low-expressed in BTCC tissues and cells, and its low expression level had positive correlation with higher pathological grades of BTCC. Moreover, GAS5 was a prognostic biomarker of disease free survival for BTCC patients. GAS5 over-expression could inhibit cell proliferation of BTCC J82 and T24 cells significantly. The IC50 to doxorubicin in T24/DOX cells (resistance to doxorubicin) presented a conspicuous depression, GAS5 enhancement reduced the chemotherapy resistance to doxorubicin. GAS5 over-expression promoted apoptosis induced by doxorubicin in T24/DOX cells, and depressed the expression of anti-apoptosis protein Bcl-2. The results indicated that GAS5 regulated the chemotherapy resistance to doxorubicin via Bcl2 partly., Conclusions: In summary, lncRNA GAS5 was a prognostic biomarker of disease free survival in BTCC patients, and acted as a tumor-suppressing gene to inhibit malignant proliferation and resistance to doxorubicin in BTCC cells. LncRNA GAS5 might be a novel potential therapeutic target for BTCC.

To identify prognostic factors for patients with diffuse large B-cell lymphoma (DLBCL), specifically those classified into conflicting subgroups by Hans' and Choi's classification algorithms. We retrospectively reviewed clinical and pathological data of 154 patients diagnosed with de novo DLBCL in the First Hospital of Jilin University from January 2004 to September 2011. All cases were classified into subgroups based on Hans' and Choi's algorithms with immunohistochemical markers., Statistical Analysis Used: The correlation between various clinicopathological factors and 5-year survival rate, the correlation between those factors with the International Prognostic Index, the concordance between Hans' and Choi's approach was evaluated. The survival in different subtypes as classified by Hans' or Choi's approach was mapped., Results: The Eastern Cooperative Oncology Group (ECOG) performance score 2-5, positive Bcl-2 expression, negative CD10 expression or negative Bcl-6 expression significantly correlated with worse prognosis. The two algorithms showed good consistency (83% concordance, Kappa = 0.660, P < 0.001). By both classifications, the 5-year overall survival rate in germinal center B-cell-like subtype (GCB) lymphoma is significantly higher than that in the non-GCB subtype. There were 25 cases assigned to conflicting subtypes by the two approaches. Among these 25 cases, ECOG 2-5, positive Bcl-2 expression, negative CD10 expression, or negative Bcl-6 expression significantly correlated with worse prognosis., Conclusions: ECOG 2-5, positive Bcl-2 expression, negative CD10 expression, or negative Bcl-6 expression are independent markers for poor prognosis of DLBCL patients. There were 15% cases assigned to conflicting subgroups based on the two algorithms. For these cases, ECOG 2-5, positive Bcl-2 expression, negative CD10 expression, or negative Bcl-6 expression still significantly correlate with poor prognosis.

Purpose Double-hit lymphomas (DHLs) and double-expressor lymphomas (DELs) are subtypes of diffuse large B-cell lymphoma (DLBCL) associated with poor outcomes after standard chemoimmunotherapy. Data are limited regarding outcomes of patients with relapsed or refractory (rel/ref) DEL or DHL who undergo autologous stem-cell transplantation (ASCT). We retrospectively studied the prognostic impact of DEL and DHL status on ASCT outcomes in patients with rel/ref DLBCL. Methods Patients with chemotherapy-sensitive rel/ref DLBCL who underwent ASCT at two institutions and in whom archival tumor material was available were enrolled. Immunohistochemistry for MYC, BCL2, and BCL6 and fluorescence in situ hybridization (FISH) for MYC were performed. In cases with MYC rearrangement or copy gain, FISH for BCL2 and BCL6 was also performed. Results A total of 117 patients were included; 44% had DEL and 10% had DHL. DEL and DHL were associated with inferior progression-free survival (PFS), and DHL was associated with poorer overall survival (OS). The 4-year PFS in patients with DEL compared with those with non-DEL was 48% versus 59% ( P = .049), and the 4-year OS was 56% versus 67% ( P = .10); 4-year PFS in patients with DHL compared with those with non-DHL was 28% versus 57% ( P = .013), and 4-year OS was 25% versus 61% ( P = .002). The few patients with concurrent DEL and DHL had a poor outcome (4-year PFS, 0%). In multivariable models, DEL and DHL were independently associated with inferior PFS, whereas DHL and partial response ( v complete response) at transplant were associated with inferior OS. Conclusion DEL and DHL are both associated with inferior outcomes after ASCT in patients with rel/ref DLBCL. Although ASCT remains a potentially curative approach, these patients, particularly those with DHL, are a high-risk subset who should be targeted for investigational strategies other than standard ASCT.

Background: Oral cancer is known to be governed by the antiapototic and loss of cell adhesion properties which dictate its progression., Aim: To study the immunexpression of E-cadherin and Bcl-2 in varying TNM stages and histopathological grades of OSCC., Materials and Methods: 11 cases of well differentiated, 10 cases of moderately differentiated and 11 cases of poorly differentiated OSCC were studied immunohistochemically using archival paraffin embedded tissue specimens., Statistical Analysis: Differences between the different variables were analyzed using ANOVA test, Kruskal-Wallis test and post hoc test followed by Bonferroni test. The resulting data was analyzed using SPSS software version 19., Results: The expression of Bcl-2 and E cadherin immunopositivity was associated positively with tumor grade, high T category and Histopathological grades., Conclusions: The results of this study points to the significance of cell proliferation and invasion as a major determinant of prognosis in OSCC.

Aberrant T cell immune responses appear central to the development of systemic lupus erythematosus (SLE). We previously reported that Gαq, the alpha subunit of Gq, regulates T and B cell immune responses, promoting autoimmunity. To address whether Gαq contributes to the pathogenesis of SLE, Gαq mRNA expression was studied using real time-PCR in PBMCs and T cells from SLE patients as well as age- and sex-matched healthy controls. Our results showed that Gαq mRNA expression was decreased in PBMCs and T cells from SLE patients compared to healthy individuals. Correlation analyses showed that Gαq expression in T cells from SLE patients was associated with disease severity (as per SLE Disease Activity Index), the presence of lupus nephritis, and expression of Th1, Th2 and Th17 cytokines. In keeping with clinical results, T-helper cell subsets (Th1, Th2 and Th17) were over-represented in Gαq knockout mice. In addition, Gαq expression in SLE T cells was negatively correlated with the expression of Bcl-2, an anti-apoptotic gene, and positively correlated with the expression of Bax, a pro-apoptotic gene. These data suggest that reduced Gαq levels in T cells may promote enhanced and prolonged T cell activation, contributing to the clinical manifestations of SLE.

Renal ischemia-reperfusion injury (IRI) is a major complication in clinical practice. However, despite its frequency, effective preventive/treatment strategies for this condition are scarce. Curcumin possesses antioxidant properties and is a promising potential protective agent against renal IRI, but its poor water solubility restricts its application. In this study, we constructed curcumin-carrying distearoylphosphatidylethanolamine-polyethylene glycol nanoparticles (Cur-NPs), and their effect on HK-2 cells exposed to IRI was examined in vitro. Curcumin encapsulated in NPs demonstrated improved water solubility and slowed release. Compared with the IRI and Curcumin groups, Cur-NP groups displayed significantly improved cell viability, downregulated protein expression levels of caspase-3 and Bax, upregulated expression of Bcl-2 protein, increased antioxidant superoxide dismutase level, and reduced apoptotic rate, reactive oxygen species level, and malondialdehyde content. Results clearly showed that Cur-NPs demonstrated good water solubility and slow release, as well as exerted protective effects against oxidative stress in cultured HK-2 cells exposed to IRI.

Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. While surgery remains the mainstay of treatment in early stage CRC, chemotherapy is usually given to prolong the overall survival and improve the quality of life for metastatic colorectal cancer (mCRC). But drug resistance is one of the major hurdles of mCRC treatment, and the underlying mechanisms are still largely unknown. In this study, we show that, compared with parental cells, RhoA is up-regulated in irinotecan (CPT-11)-resistant CRC cells. Furthermore, inhibition of RhoA in drug resistant cells, at least partially, rescues the resistance against irinotecan and increases the sensitivity to other chemotherapeutic drug by inhibiting expression of MDR1, MRP1and GSTP1, promotes apoptosis by suppressing the expression of BCL-XL and Bcl-2 and increasing Bax expression, and significantly decreases side population cells. Our results suggest that, in addition to survival, proliferation, migration, adhesion, cell cycle and gene transcription, RhoA is also involved in chemoresistance by regulating the expression of membrane transporter and apoptosis protein in colorectal cancer. They raise an interesting possibility that the expression of RhoA may indicate a poor prognosis due to the high probability to therapy resistance and, on the other hand, inhibition of RhoA activity and function may overcome chemoresistance and improve the effectiveness of clinical treatment of CRC.

The YiQiFuMai powder injection (YQFM), a traditional Chinese medicine (TCM) prescription re-developed based on the well-known TCM formula Sheng-maisan, showed a wide range of pharmacological activities in cardiovascular diseases in clinics. However, its role in protection against myocardial ischemia/reperfusion (MI/R) injury has not been elucidated. The present study not only evaluated the cardioprotective effect of YQFM from MI/R injury but also investigated the potential molecular mechanisms both in vivo and in vitro. The myocardium infarct size, production of lactate dehydrogenase (LDH), creatine kinase (CK), cardiac function, TUNEL staining, and caspase-3 activity were measured. Cell viability was determined, and cell apoptosis was measured by Hoechst 33342 staining and flow cytometry. Mitochondrial membrane potential (ΔΨm) was measured, and ATP content was quantified by bioluminescent assay. Expression of apoptosis-related proteins, including Caspase-3, Bcl-2, Bax, AMPKα, and phospho-AMPKα, was analyzed by western blotting. AMPKα siRNA transfection was also applied to the mechanism elucidation. YQFM at a concentration of 1.06 g/kg significantly reduced myocardium infarct size and the production of LDH, CK in serum, improved the cardiac function, and also produced a significant decrease of apoptotic index. Further, combined treatment with compound C partly attenuated the anti-apoptotic effect of YQFM. In addition, pretreatment with YQFM ranging from 25 to 400 μg/mL markedly improved cell viability and decreased LDH release. Moreover, YQFM inhibited H9c2 apoptosis, blocked the expression of caspase-3, and modulated Bcl-2 and Bax proteins, leading to an increased mitochondrial membrane potential and cellular ATP content. Mechanistically, YQFM activated AMP-activated protein kinase (AMPK) signaling pathways whereas pretreatment with AMPK inhibitor Compound C and application of transfection with AMPKα siRNA attenuated the anti-apoptotic effect of YQFM. Our results indicated that YQFM could provide significant cardioprotection against MI/R injury, and potential mechanisms might suppress cardiomyocytes apoptosis, at least in part, through activating the AMPK signaling pathways.

Nonylphenol and short-chain nonylphenol ethoxylates such as NP 2 EO are present in aquatic environment as wastewater contaminants, and their toxic effects on aquatic species have been reported. Apoptosis has been shown to be induced by serum deprivation or copper treatment. To understand the toxicity of nonylphenol diethoxylate, we investigated the effects of NP 2 EO on apoptosis induced by serum deprivation and copper by using PC12 cell system. Nonylphenol diethoxylate itself showed no toxicity and recovered cell viability from apoptosis. In addition, nonylphenol diethoxylate decreased DNA fragmentation caused by apoptosis in PC12 cells. This phenomenon was confirmed after treating apoptotic PC12 cells with nonylphenol diethoxylate, whereas the cytochrome c release into the cytosol decreased as compared to that in apoptotic cells not treated with nonylphenol diethoxylates. Furthermore, Bax contents in apoptotic cells were reduced after exposure to nonylphenol diethoxylate. Thus, nonylphenol diethoxylate has the opposite effect on apoptosis in PC12 cells compared to nonylphenol, which enhances apoptosis induced by serum deprivation. The difference in structure of the two compounds is hypothesized to be responsible for this phenomenon. These results indicated that nonylphenol diethoxylate has capability to affect cell differentiation and development and has potentially harmful effect on organisms because of its unexpected impact on apoptosis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1389-1398, 2016., (© 2015 Wiley Periodicals, Inc.)

Tumor heterogeneity implies the possibility of significantly different expression of key pathways between primary and metastatic clones. Colon adenocarcinoma is one of the few tumors where current practice includes resection of primary and isolated organ metastases simultaneously without neoadjuvant therapy. We performed a pilot study on 28 cases of colon adenocarcinoma resected simultaneously with metastases in patients with no history of neoadjuvant therapy. We assayed matched primary and metastatic tumors from each patient with common diagnostic antibodies to Bcl-2, Cyclin D1, AMACR, and ALDH-1 by immunohistochemistry with semi-quantitative interpretation on archived formalin fixed, paraffin embedded samples. We were powered for large, consistent differences between primary and metastatic expression, and found 21 of 28 had a significant difference in expression of at least one of the four proteins, accounting for multiplicity of testing. Cyclin D1 had significantly more cases with differential metastatic:primary expression than would be expected by chance alone (p-value 0.0043), favoring higher expression in the metastatic sample. Bcl-2 and ALDH-1 had trends in this direction (p-value 0.078 each). Proportionately more cases with significant differences were identified when a liver metastasis was tested. We conclude differences in expression between metastatic and primary colon adenocarcinoma within the same patient exist, and may have therapeutic and biomarker testing consequences., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The chromosomal translocation t(14;18) in follicular lymphoma (FL) is a primary oncogenic event resulting in BCL-2 over-expression. This study investigates activity of the BH3 mimetic venetoclax (ABT-199), which targets BCL-2, and mechanisms of acquired resistance in FL.The sensitivity of FL cells to venetoclax treatment correlated with BCL-2/BIM ratio. Cells with similar expression of anti-apoptotic proteins, but with higher levels of BIM were more sensitive to the treatment. Venetoclax induced dissociation of BCL-2/ BIM complex and a decrease in mitochondrial potential. Interestingly the population of cells that survived venetoclax treatment showed increased p-ERK1/2 and p-BIM (S69), as well as a decrease in total BIM levels. Venetoclax resistant cells initially showed elevated levels of p-AKT and p-Foxo1/3a, a dissociation of BIM/BCL-2/BECLIN1 complex, and a decrease in SQSTM1/p62 level (indicating increased autophagy) together with a slight decline in BIM expression. After stable resistant cell lines were established, a significant reduction of BCL-2 levels and almost total absence of BIM was observed.The acquisition of these resistance phenotypes could be prevented via selective ERK/AKT inhibition or anti-CD20 antibody treatment, thus highlighting possible combination therapies for FL patients.

Abnormal expression of microRNAs (miRNAs) is involved in the development of and anti‑apoptotic effects in various types of human cancer. However, miRNA‑mediated regulation of oral verrucous carcinoma (OVC) remains to be elucidated. The present study aimed to investigate the expression of miR‑181b in OVC and oral squamous cell carcinoma (OSCC). The expression levels of miR‑181b were determined using reverse transcription‑quantitative polymerase chain reaction. The expression levels of B‑cell lymphoma 2 (Bcl‑2) and leucine rich repeats and immunoglobulin like domains 1 (LRIG1), were evaluated using immunohistochemical staining. The correlation between Bcl‑2 and LRIG1 expression was determined using a Pearson correlation analysis. The expression levels of miR‑181b and Bcl‑2 in OVC were significantly higher compared with normal mucosal tissue (NM); however, lower compared with the OSCC. The key target of miR‑181b was LRIG1 and it was significantly lower in OVC tissues compared with NM tissue; however this was higher when compared with OSCC tissue. The expression levels of Bcl‑2 were correlated with expression levels of LRIG1 in OVC tissues. Therefore, LRIG1 may be associated with anti‑apoptotic function in OVC tissues.

Mortality rates associated with off‑pump coronary artery bypass (CAB) are relatively high, as the majority of patients requiring CAB are at a high risk for cardiac events. The present study aimed to establish porcine models of acute myocardial ischemia, and evaluate the protective role of shunt and active perfusion. A total of 30 pigs were randomly assigned to five groups, as follows: i) Sham (control); ii) A1 (shunt; stenosis rate, 55%); iii) A2 (shunt; stenosis rate, 75%); iv) B1 (active perfusion; stenosis rate, 55%); and v) B2 (active perfusion; stenosis rate, 75%) groups. Aortic pressure (P0), left anterior descending coronary pressure (P1), and coronary effective perfusion pressure (P1/P0) were measured. The expression levels of tumor necrosis factor‑α (TNF‑α), cardiac troponin (cTnI), creatine kinase‑myocardial band (CK‑MB), interleukin (IL)‑6, IL‑10, B‑cell lymphoma 2 (Bcl‑2), and caspase‑3 were detected using enzyme‑linked immunosorbent assay or western blotting. The myocardial apoptosis rate was determined using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Ischemia models with stenosis rates of 55 and 75% were successfully constructed following suturing of the descending artery. Compared with the control, the 55 and 75% stenosis groups demonstrated significantly decreased P1/P0, increased expression levels of TNF‑α, cTnI, CK‑MB, IL‑6, IL‑10 and caspase‑3, an increased rate of myocardial apoptosis, and a decreased expression level of anti‑apoptotic protein, Bcl‑2. At 30 min following successful establishment of the model (ST segment elevation to 1 mm), group B demonstrated significantly increased P1/P0, decreased expression levels of TNF‑α, cTnI, CK‑MB, IL‑6, IL‑10 and caspase‑3, a decreased rate of myocardial apoptosis, and an increased expression level of anti-apoptotic protein, Bcl‑2. Furthermore, the current study indicated that active perfusion was more efficacious in maintaining myocardial perfusion and alleviating ischemic injury when compared with traditional shunt perfusion.

Mitochondria actively contribute to apoptotic cell death through mechanisms including the loss of integrity of the outer mitochondrial membrane, the release of intermembrane space proteins, such as cytochrome c, in the cytosol and the caspase cascade activation. This process is the result of careful cooperation not only among members of the Bcl-2 family but also dynamin-related proteins. These events are often accompanied by fission of the organelle, thus linking mitochondrial dynamics to apoptosis. Emerging evidences are suggesting a fine regulation of mitochondrial morphology by Bcl-2 family members and active participation of fission-fusion proteins in apoptosis. The debate whether in mitochondrial morphogenesis the role of Bcl-2 family members is functionally distinct from their role in apoptosis is still open and, above all, which morphological changes are associated with cell death sensitisation. This review will cover the findings on how the mitochondrial fission and fusion machinery may intersect apoptotic pathways focusing on recent advances on the key role played by Mcl-1., (© 2016 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Apoptosis suppression caused by overexpression of anti-apoptotic proteins is a central factor to the acquisition of multi-drug resistance (MDR) in breast cancer. As a highly conserved anti-apoptotic protein, Bcl-2 can initiate an anti-apoptosis response via an ERK1/2-mediated pathway. However, the details therein are still far from completely understood and a quantitative description of the associated proteins in the biological context may provide more insights into this process. Following our previous attempts in the quantitative analysis of MDR mechanisms, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics was continually employed here to describe ERK/Bcl-2-mediated anti-apoptosis. A targeted proteomics assay was developed and validated first for the simultaneous quantification of ERK1/2 and Bcl-2. In particular, ERK isoforms (i.e., ERK1 and ERK2) and their differential phosphorylated forms including isobaric ones were distinguished. Using this assay, differential protein levels and site-specific phosphorylation stoichiometry were observed in parental drug-sensitive MCF-7/WT cancer cells and drug-resistant MCF-7/ADR cancer cells and breast tissue samples from two groups of patients who were either suspected or diagnosed to have drug resistance. In addition, quantitative analysis of the time course of both ERK1/2 and Bcl-2 in doxorubicin (DOX)-treated MCF-7/WT cells confirmed these findings. Overall, we propose that targeted proteomics can be used generally to resolve more complex cellular events.

Sex steroids have been postulated to influence skin development and functions as well as its pathogenesis. MCC occurs in both sexes; however, the specific differences in pathogenesis among sexes have yet to be conclusively defined. The detailed status of sex steroid receptors (AR, PRA and PRB, and ERα, ERβ) are also unknown in MCC patients. We first immunolocalized sex steroid receptors and compared the results with immunolocalization of relevant transcription factors including SOX2, FOXA1, and Bcl-2 and Ki-67 in 18 cases of MCCs. AR, PRA, PRB, ERα, ERβ, Bcl-2, SOX2, and FOXA1 immunoreactivity was evaluated by using the modified H score method, and Ki-67 was quantified using labeling index. ERβ immunoreactivity was markedly present in all the cases of MCC examined, with relatively weak immunoreactivity of ERα, AR, PRA, and PRB. The status of ERβ immunoreactivity was also significantly correlated with Ki-67 labeling index and Bcl-2 score. These results demonstrated that ERβ could be associated with regulation of both cell proliferation and apoptosis in MCCs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To study the effect of leptin on the expression of calcium-activated neutral protease 1 (calpain-1) and B cell lymphoma-2 (Bcl-2) and apoptosis in the myocardial tissue of neonatal rats after asphyxia., Methods: A total of 48 neonatal rats were randomly and equally divided into normal control group, asphyxia group, leptin treatment groups, and calpain-1 inhibitor (CAI-1) group. The neonatal rat model of asphyxia under normal atmospheric condition was established in all groups except the control group. For the leptin treatment groups, rats received 20, 80, and 160 μg/kg leptin by intraperitoneal injection immediately after model establishment, respectively. For the CAI-1 group, rats received 10 mg/kg CAI-1 by intraperitoneal injection immediately after model establishment. For all the groups, the myocardial tissue was collected at 2 hours after model establishment. Immunohistochemistry was used to measure the expression of calpain-1 and Bcl-2. The TUNEL method was used to evaluate apoptosis of myocardial cells., Results: The expression of calpain-1 and Bcl-2 and apoptosis index (AI) were significantly higher in the asphyxia group than in the normal control group (P˂0.05). The leptin treatment groups and the CAI-1 group had significantly lower expression of calpain-1, significantly lower AI, and significantly higher expression of Bcl-2 than the asphyxia group (P˂0.05). The CAI-1 group had the largest changes in all the indices compared with the asphyxia group. However, there were no significant differences in all indices between the 160 μg/kg leptin treatment group and the CAI-1 group. After asphyxia, the expression of calpain-1 was positively correlated with AI, while the expression of Bcl-2 was negatively correlated with AI and the expression of calpain-1 (P˂0.05)., Conclusions: Leptin reduces apoptosis of myocardial cells in asphyxiated neonatal rats by the inhibition of calpain-1 activation and upregulation of Bcl-2 expression.

Purpose: Immunochemotherapy used in the treatment of non-Hodgkin diffuse large B-cell lymphoma (DLBCL) modifies the course of disease and has a positive effect on overall survival (OS). The purpose of this study was to verify the existence of the important Myd 88 mutation and other immunohistochemical factors on disease prognosis in patients with DLBCL in southeast Serbia., Methods: Immunohistochemical expression of CD10, Bcl- 2, Bcl-6, Ki-67 and MUM 1 was performed using paraffin blocks of DLBCL. Molecular-genetic study of MyD88 L265P gene polymorphism was done by isolation of genomic DNA from paraffin embedded tissue by means of polymerase chain reaction (PCR)., Results: Immunochemotherapy (rituximab+CHOP/R-CHOP) significantly improved the overall survival (OS) of patients with DLBCL compared with patients treated with CHOP alone (p<0.0001). OS in the R-CHOP group was longest in patients with International Prognostic Index (IPI) 2 score (p=0.012) and IPI 4 score (p=0.024). Patients with Bcl-2 +, and MUM 1+ benefited from R-CHOP and their expression had no effect on OS. Analysis of restriction fragment length on the genomic DNA showed a homozygous normal TT genotype., Conclusion: Addition of rituximab to CHOP standard protocol improved the OS rate in patients with DLBCL and altered the character and significance of previously recognized prognostic factors. IPI score in the immunochemotherapy era could not reveal possible predictive factors of poor prognosis which would help identify a high-risk subgroup of newly diagnosed DLBCL. In the patient population from Southeast Serbia pathological signaling pathway achieved by Myd 88 L265 mutation was not responsible for the development of DLBCL.

Objective: To explore the effects of umbilical cord blood mononuclear cells (UCBMC) transplantation on the neuronal apoptosis and the expression of Bcl-2 and Bax proteins in neonatal rats with hypoxic-ischemic brain damage (HIBD)., Methods: Seven-day-old Sprague-Dawley neonatal rats were randomly divided into normal control (N)+normal saline (NS), HIBD+NS, N+UCBMC, and HIBD+UCBMC groups. HIBD model was prepared using the classical Rice-Vannucci method. Twenty-four hours after HIBD, UCBMC were transplanted in the N+UCBMC and HIBD+UCBMC groups. Seven days after transplantation, NeuN/Cleaved-Caspase-3 double immunofluorescence staining and TUNEL methods were used to observe neural apoptosis in the cortex. The expression levels of Bax and Bcl-2 proteins were examined by Western blot analysis., Results: There were more NeuN + cleaved Caspase-3 + DAPI + and TUNEL + DAPI + cells in the HIBD+NS group compared with the N+NS and N+UCBMC groups (P<0.01). There were less NeuN + cleaved Caspase-3 + DAPI + and TUNEL + DAPI + cells in the HIBD+UCBMC group compared with the HIBD+NS group (P<0.01). The concentration of Bax protein was higher and that of Bcl-2 proteins was lower in the HIBD+NS group compared with the N+NS and N+UCBMC groups (P<0.01). The concentration of Bax protein in HIBD+UCBMC group was lower than that in the HIBD+NS group (P<0.01). The concentration of Bcl-2 protein was higher compared with the HIBD+NS, N+NS and N+UCBMC groups (P<0.05)., Conclusions: UCBMC transplantation via lateral ventricle can upregulate the expression of Bcl-2 protein and down-regulate the expression of Bax protein, thus alleviating brain neural apoptosis in neonatal rats with HIBD.

The role and clinical implication of ZRF1 in breast cancer are poorly understood. So this study is aimed to explore the role of ZRF1 in breast cancer progression. With this context, we first assessed its expression pattern in FFPE primary and metastasis breast tissue samples as well as from publicly available databases. Moreover, we also explored the survival status of patients from the publicly available database and interestingly discover that high expression of ZRF1 decreases the survival of estrogen-positive breast cancer patients more than estrogen-negative status patients. In the perspective of this, we evaluated the role ZRF1 in MCF-7 breast cancer cells and found that it's silencing by knockdown results in decreased cell proliferation as well as cell viability. Results also show that expression of ZRF1 is down regulated in the presence of estrogen-depleted conditions but independent of RAS/MEK as well as AKT axes. Moreover, the decrease in viability of MCF-7 cells was accompanied by induction of apoptosis and DNA damage, well-marked with upregulation of cleaved PARP and downregulation of BCL2 and H2AUbK119 levels. Furthermore, we also explored that knockdown of ZRF1 sensitises the effect of curcumin, observed with decrease in cell viability and dropping of IC50 value from 25 to 15 μM. This investigation thus shed a new light on the role on ZRF1 in breast cancer cells and hence can be exploited to design better therapeutic intervention.

Multiple myeloma (MM) evolves from a premalignant condition known as monoclonal gammopathy of undetermined significance (MGUS). However, the factors underlying the malignant transformation of plasmocytes in MM are not fully characterized. We report here that Eµ-directed expression of the antiapoptotic Bcl-B protein in mice drives an MM phenotype that reproduces accurately the human disease. Indeed, with age, Eµ-bcl-b transgenic mice develop the characteristic features of human MM, including bone malignant plasma cell infiltration, a monoclonal immunoglobulin peak, immunoglobulin deposit in renal tubules, and highly characteristic bone lytic lesions. In addition, the tumors are serially transplantable in irradiated wild-type mice, underlying the tumoral origin of the disease. Eµ-bcl-b plasmocytes show increased expression of a panel of genes known to be dysregulated in human MM pathogenesis. Treatment of Eµ-bcl-b mice with drugs currently used to treat patients such as melphalan and VELCADE efficiently kills malignant plasmocytes in vivo. Finally, we find that Bcl-B is overexpressed in plasmocytes from MM patients but neither in MGUS patients nor in healthy individuals, suggesting that Bcl-B may drive MM. These findings suggest that Bcl-B could be an important factor in MM disease and pinpoint Eµ-bcl-b mice as a pertinent model to validate new therapies in MM., (© 2016 Hamouda et al.)

Sebaceous neoplasms encompass a range of lesions, including benign entities such as sebaceous adenoma and sebaceoma, as well as sebaceous carcinoma. The distinction of sebaceous carcinoma from benign lesions relies on histological identification of architectural or cytological features of malignancy. In this study we have assessed the diagnostic discriminatory ability of mitotic rate and immunohistochemical markers (p53, bcl-2 and p16) in a selected group of well circumscribed sebaceous neoplasms, incorporating examples of sebaceous adenoma, sebaceoma and sebaceous carcinoma. We found that mitotic rate was significantly higher in malignant lesions as compared to benign lesions, but none of the immunohistochemical markers showed a discriminatory expression pattern. In addition, we performed a mutational analysis on the same group of lesions using next generation sequencing (NGS) technology. The most commonly mutated gene was TP53, although there was no correlation between the p53 immunohistochemical results and number or type of TP53 mutation detected. CDKN2A, EGFR, CTNNB1 and KRAS were also commonly mutated across all lesions. No particular gene, mutation profile or individual mutation could be identified which directly correlated with the consensus histological diagnosis. In conclusion, within this diagnostically challenging group of lesions, mitotic activity, but not immunohistochemical labelling for p16 or bcl-2, correlates with diagnostic category. While a number of genes potentially involved in the genesis of sebaceous neoplasia were uncovered, any molecular differences between the histological diagnostic categories remain unclear., (Copyright © 2016 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Purpose: To compare disease-free survival (DFS) after maintenance therapy with the selective protein kinase C β (PKCβ) inhibitor, enzastaurin, versus placebo in patients with diffuse large B-cell lymphoma (DLBCL) in complete remission and with a high risk of relapse after first-line therapy., Patients and Methods: This multicenter, phase III, randomized, double-blind, placebo-controlled trial enrolled patients who were at high risk of recurrence after rituximab-cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Patients (N = 758) with stage II bulky or stage III to IV DLBCL, three or more International Prognostic Index risk factors at diagnosis, and a complete response or unconfirmed complete response after 6 to 8 cycles of R-CHOP were assigned 2:1 to receive oral enzastaurin 500 mg daily or placebo for 3 years or until disease progression or unacceptable toxicity. Primary end point was DFS 3 years after the last patient entered treatment. Correlative analyses of biomarkers, including cell of origin by immunohistochemistry and PKCβ expression, with efficacy outcomes were exploratory objectives., Results: After a median follow-up of 48 months, DFS hazard ratio for enzastaurin versus placebo was 0.92 (95% CI, 0.689 to 1.216; two-sided log-rank P = .541; 4-year DFS, 70% v 71%, respectively). Independent of treatment, no significant associations were observed between PKCβ protein expression or cell of origin and DFS or overall survival., Conclusion: Enzastaurin did not significantly improve DFS in patients with high-risk DLBCL after achieving complete response to R-CHOP. Achievement of a complete response may have abrogated the prognostic significance of cell of origin by immunohistochemistry., (© 2016 by American Society of Clinical Oncology.)

Background: Nonsteroidal anti-inflammatory drugs and vitamin-D derivatives can target signaling pathways activated in basal cell carcinoma (BCC)., Objective: We investigated the efficacy of topically applied diclofenac sodium 3% gel, calcitriol 3 μg/g ointment, and a combination of both in superficial BCC (sBCC) and nodular BCC., Methods: Patients with a primary, histologically proven sBCC (n = 64) or nodular BCC (n = 64) were randomized to topical diclofenac, calcitriol, combination of both, or no topical treatment (control group). After self-application twice daily under occlusion (8 weeks), tumors were excised. Primary outcome was posttreatment expression levels of proliferation (Ki-67) and antiapoptosis (B-cell lymphoma [Bcl-2]) immunohistochemical markers. Secondary outcomes were histologic clearance, adverse events, application-site reactions, and patient compliance., Results: sBCC treated with diclofenac showed a significant decrease in Ki-67 (P < .001) and Bcl-2 (P = .001), and after combination therapy for Ki-67 (P = .012). Complete histologic tumor regression was seen in 64.3% (P = .0003) of sBCC (diclofenac) and 43.8% (P = .007) of sBCC (combination therapy) compared with 0.0% of controls. No significant changes were found in nodular BCC. Application-site reactions were mostly mild to moderate., Limitations: The sample size was small., Conclusion: Our results suggest that topical diclofenac is a promising new treatment for sBCC. Its mode of action differs from available noninvasive therapies, and thus has an additive value., (Copyright © 2016 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Benign prostatic hyperplasia (BPH) is a urologic disease that affects most of men over the age 50. But until now there is no such perfect cure without side effects. Because of diverse adverse effects, it is desirable to develop effective and long term-safety-herbal medicines to inhibit the progress of BPH. In spite of garlic's large use and a wide spectrum of studies, including anti-hyperlipidemic, cardio-protective, and anti-inflammatory activities, there was none to prove efficacy for BPH. In this study, we evaluated the efficacy of garlic to prove its suppressing effects on BPH. Garlic administration decreased relative prostate weight ratio, suppressed mRNA expression level of AR, DHT serum levels, and the growth of prostatic tissue in BPH-induced rats. Moreover, garlic administration decreased the levels of inflammatory proteins, iNOS, and COX-2 in prostatic tissue. Further investigation showed that garlic induced accumulation of death-inducing signal complex and activation of AMPK and decreased the levels of anti-apoptotic proteins, such as Bcl-2, Bcl-xL, and survivin. These results suggest that garlic may have suppressing effects on BPH and it has great potential to be developed as treatment for BPH. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Cyclophosphamide, doxorubicin, vincristine, and prednisolone plus rituximab (R-CHOP) is the standard treatment for patients with diffuse large B cell lymphoma (DLBCL). However, rituximab cannot be popularly applied in a considerable number of patients with DLBCL because of economic reasons. To develop a new regimen to improve the outcome of these patients is extremely important. In our study, sixty five patients with DLBCL were randomly assigned to thalidomide plus CHOP group (n=32) or to CHOP alone group (n=33). Objective response rates (ORR) and complete remission rates (CRR) were 96.7% and 80.6% in T-CHOP group versus 78.9 % and 57.8 % in CHOP group, respectively (P <0.05). At a median follow-up of 96 months, median PFS for T-CHOP group was still not reached yet, and in CHOP group it was 22.9 months (95% CI [0-50.4]). (P=0.163). Median overall survival (OS) for T-CHOP group was also not reached, and the estimated median OS for CHOP group was 83.5 months, the difference of OS between the two groups is not significant (p=0.263). But, in patients with Bcl-2 positive and Bcl-6 negative, the median PFS in T-CHOP group was longer than that in CHOP group (111.0 vs 8.5 months (P=0.017). In addition, thalidomide did not significantly increase the grade 3/4 toxicity of CHOP. We concluded that the addition of thalidomide to the CHOP regimen significantly improved the CRR and showed a trend of improving clinical outcome in patients with DLBCL, especially for patients with Bcl-2 positive and Bcl-6 negative B-cell phenotype, without increased toxicity., Competing Interests: The authors declare no conflicts of interest.

Dual expression of MYC and BCL2 by immunohistochemistry (IHC) is associated with poor outcome in diffuse large B-cell lymphoma (DLBCL). Dual translocation of MYC and BCL2, so-called "double-hit lymphoma," has been associated with a high risk of central nervous system (CNS) relapse; however, the impact of dual expression of MYC and BCL2 (dual expressers) on the risk of CNS relapse remains unknown. Pretreatment formalin-fixed paraffin-embedded DLBCL biopsies derived from patients subsequently treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) were assembled on tissue microarrays from 2 studies and were evaluated for expression of MYC and BCL2 by IHC. In addition, cell of origin was determined by IHC and the Lymph2Cx gene expression assay in a subset of patients. We identified 428 patients who met the inclusion criteria. By the recently described CNS risk score (CNS-International Prognostic Index [CNS-IPI]), 34% were low risk (0 to 1), 45% were intermediate risk (2 to 3), and 21% were high risk (4 or greater). With a median follow-up of 6.8 years, the risk of CNS relapse was higher in dual expressers compared with non-dual expressers (2-year risk, 9.7% vs 2.2%; P = .001). Patients with activated B-cell or non-germinal center B-cell type DLBCL also had an increased risk of CNS relapse. However, in multivariate analysis, only dual expresser status and CNS-IPI were associated with CNS relapse. Dual expresser MYC(+) BCL2(+) DLBCL defines a group at high risk of CNS relapse, independent of CNS-IPI score and cell of origin. Dual expresser status may help to identify a high-risk group who should undergo CNS-directed evaluation and consideration of prophylactic strategies., (© 2016 by The American Society of Hematology.)

Purpose: T o investigate the possible protective effect of thymoquinone (TQ) in cisplatin (CP) induced myocardial injury., Methods: A total of 28 adult male Wistar-Albino rats were randomly and equally divided into four groups as follows: Group 1 (control), Group 2 (CP at 15 mg/kg dose), Group 3 (TQ 40 mg/kg/day for two days prior to CP injection and on third day, CP at 15 mg/kg dose was intraperitoneally administered and TQ treatment continued until fifth day) and Group 4 (TQ at 40mg/kg/day dose for five days)., Results: There was a significant increment in CP group in terms of congestion, edema and pycnotic nuclei in myocardial fibers, comparing with other groups. TQ group exhibited significant increase in expression of antiapoptotic protein Bcl-2, comparing with CP group (p<0.05). In only CP administered group, expression of antiapoptotic protein Bcl-2 was lowest comparing with other groups., Conclusion: Established data indicate that cisplatin is cardiotoxic and thymoquinone may be useful in treating CP-induced cardiac injury.

Objective: To investigate Wnt11 and BCL2A1 immunohistochemical expression in complete moles and normal villi., Study Design: The expression of Wnt11 and BCL2A1 in 84 complete moles and 30 normal first-trimester villi were detected by Envision immunohistochemistry. Quantitative evaluation according to color deconvolution and immunoreactive score was performed. Data was analyzed using Kruskal-Wallis test, Pearson test, and ROC curve., Results: Of 84 complete moles, 14 developed to post-molar gestational trophoblastic neoplasia, and the others regressed spontaneously. Both proteins showed cytoplasmic pattern, whereas the DAB wt% of BCL2A1 and Wnt11 expression was highest in moles that developed to GTN, gradually reduced in spontaneously regressed moles and normal villi (all p < 0.01). We considered a 23.17% cutoff valuefor Wnt11 DAB wt% and 16.31% for BCL2A1 DAB wt% to assess molar progression to GTN. There was positive correlation between expressions of the 2 proteins (r = 0.403)., Conclusion: Our findings demonstrated immunohistochemical expression of Wnt11 and BCL2A1 in complete moles and normal villi. Both proteins may be included as part of an immunohistochemical panel to identify postmolar outcome when other trophoblastic markers yield ambiguous results.

Background/aim: Invasive lobular breast carcinoma is the second most common type of breast cancer after invasive ductal carcinoma. According to the American Cancer Society, more than 180,000 women in the United States find out they have invasive breast cancer each year. Personal history of breast cancer and certain changes in the breast are correlated with an increased breast cancer risk. The aim of this work was to analyze breastfeeding in patients with infiltrating lobular breast carcinoma, in relation with: 1) clinicopathological parameters, 2) hormonal receptors and 3) tissue-based tumor markers., Materials and Methods: The study included 80 women with ILC, 46 of which had breastfeed their children. Analyzed parameters were: age, tumor size, axillary lymph node (N), distant metastasis (M), histological grade (HG), estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), Ki-67, p53 and BCL2., Results: ILC of non-lactating women showed a larger (p = 0.009), lymph node involvement (p = 0.051) and distant metastasis (p = 0.060). They were also more proliferative tumors measured by Ki-67 (p = 0.053). Breastfeeding history did not influence the subsequent behavior of the tumor regardless of histological subtype., Conclusion: Lactation seems to influence the biological characteristics of ILC defining a subgroup with more tumor size, axillary lymph node involvement, distant metastasis and higher proliferation measured by ki-67 expression.

Aims: Hypoxia is an important factor that causes decreased local disease control as well as increased distant metastases and resistance to radiotherapy in patients with advanced nasopharyngeal carcinoma (NPC). Gambogic acid (GA), the major active ingredient of gamboge, exerts antitumor effects in vitro and in vivo. However, the molecular mechanism by which GA inhibits tumor radioresistance remains unclear. The present study aimed to investigate the radiosensitizing effects of GA on NPC and explore the underlying mechanisms., Materials and Methods: CNE-1 and CNE-2 cells exposed to hypoxia and radiation were treated with GA at different concentrations. CCK-8 assay, clonogenic assay, and flow cytometry were performed to analyze cell proliferation, colony formation, apoptosis, and cell cycle. The expression levels of hypoxia-inducible factor-1α (HIF-1α), Bcl-2, Bax, caspase-3, cyclin B1/p-cdc2 and γ-H2AX were assessed using Western blot and/or immunofluorescence analysis., Results: Results of the CCK-8 assay, clonogenic assay, and flow cytometry showed that treatment of NPC cells with growth-suppressive concentrations of GA resulted in G2/M phase arrest and apoptosis. Western blot analysis demonstrated that GA-induced cell cycle arrest and apoptosis in CNE-2 cells was associated with upregulated expression of caspase-3 and Bax and downregulated expression of Bcl-2 and cyclin B1/p-cdc2 in hypoxia. Treatment with GA markedly decreased the expression of HIF-1α under hypoxic conditions., Conclusions: The results of this study suggest that GA efficiently radiosensitizes NPC cells and the effect may be significant in hypoxic conditions.

A 55-year-old man was diagnosed with an abdominal tumor and referred to our department. Abdominal contrast-enhanced computed tomography revealed a 15-cm tumor in the retroperitoneum. The inferior vena cava was compressed ventrally, the right ureter laterally, and the right kidney toward the head. Part of the tumor was situated on the dorsal side of the abdominal aorta. A preoperative diagnosis of neurogenic tumor or a type of sarcoma was made. We performed en bloc resection of the tumor. The right kidney was also resected. We exfoliated between the tumor and inferior vena cava without temporary interruption. The final diagnosis was a solitary fibrous tumor. The patient received no adjuvant therapy. Neither recurrence nor metastasis has been detected for 8 months postoperatively.

Esophageal cancer is of high prevalence and poor prognosis. Hesperetin has been reported to exert antitumor ability by inducing apoptosis in many cancers in vitro and in vivo without obvious toxicity. However, there is no study concerning about the effect of hesperetin on esophageal cancer. In this study, we aimed to investigate whether hesperetin could induce apoptosis in esophageal cancer cells and explore its potential mechanism. We found that hesperetin induced esophageal cancer cells apoptosis in a concentration-dependent and time-dependent manner compared with the untreated cells. Hoechst 33258 staining and flow cytometry analysis showed more apoptotic cells in the hesperetin-treated group (p < 0.05, respectively). The intracellular reactive oxygen species (ROS) increased significantly, and glutathione (GSH) was depleted. The loss of △Ψ m was more tremendous in the hesperetin-treated cells. N-acetylcysteine (NAC) reduced the proapoptotic ability of hesperetin, while DL-buthionine-S, R-sulfoximine (BSO) enhanced the anticancer effect. Western blotting showed that the expression levels of cytochrome C (Cyt C) and apoptosis-inducing factor (AIF) decreased in mitochondria and increased in cytoplasm (p < 0.05). The levels of intracellular cleaved caspase-9, cleaved caspase-3, Apaf-1, Bcl-2-associated X protein (Bax), and suppressor of fused (SuFu) increased, while B cell lymphoma 2 (Bcl-2) and Survivin decreased. What is more, in xenograft tumor model, hesperetin inhibited the tumor growth significantly via induction of cell apoptosis which was detected by TUNEL assay (p < 0.05). Taken together, our study demonstrated that hesperetin could induce cell apoptosis in esophageal cancer cells via mitochondrial-mediated intrinsic pathway by accumulation of ROS.

The Aim: The creation of a mathematical model of survival in patients with colon adenocarcinoma based on multivariable analysis of the state of cancer cell nuclear apparatus., Patients and Methods: The study was performed on 141 samples of biopsy materials or material obtained during surgical treatment of the patients with colon adenocarcinoma or benign colon neoplasms with the use of histological, morphometric, densitometric, immunohistochemical and mathematical methods., Results: It has been shown that each discrete pattern of the state of adenocarcinoma cell nuclei (quantity of DNA, the number and volume of nuclear organizer regions, expression rates of Ki-67, Bcl-2 and p53) is prognostically invalid in the case of its separate use. Combination of these characteristics significantly enhances prognostic validity of the survival model. Based on equation of Cox proportional hazards, survival model of good quality for the patients with moderately and poorly differentiated adenocarcinoma and increased average DNA content in tumor cell nuclei has been created., Conclusion: The proposed survival model for colon adenocarcinoma demonstrates the quality twice superior to the model based on the use of tumor grade only (G) which in fact is presently used as a sole common independent histological criterion of prognosis.

Aims: MYC rearrangements with or without BCL2 rearrangements have been shown to be associated with poor prognosis and inferior survival in diffuse large B-cell lymphomas (DLBCL). Most of these cases are still diagnosed by fluorescent in situ hybridisation (FISH) testing, which is expensive, requires expertise and is not routinely available in all laboratories. Immunohistochemistry (IHC) is widely available and has the potential to be used as a screening test to identify cases with increased protein expression and select cases that require confirmatory testing. We correlated the expression of MYC and BCL2 by IHC with FISH studies in an attempt to define a cut-off value, which can be used by laboratories to select cases requiring confirmatory FISH testing. The prevalence of MYC-positive DLBCL and double-hit lymphoma (DHL) has also been studied., Methods: 209 cases comprising of 15 cases of Burkitt lymphoma (BL), 13 cases of intermediate BL/DLBCL and 181 cases of DLBCL were included. IHC and FISH for MYC and BCL2 were performed and the results were correlated., Results: The prevalence of MYC-positive DLBCL and MYC/BCL2DHL was 13.4% and 7.4%, respectively, in our study. Germinal-centre subtype was more common in MYC-positive DLBCL and DHL. MYC-positive DLBCL also showed higher median Ki-67 (>90%) and CD10 positivity as compared with MYC-negative cases., Conclusions: IHC can be used for screening cases, which require further confirmatory FISH testing. We recommend a cut-off value of ≥30% for MYC by IHC; however, international standardisation of these values is necessary to provide uniformity among laboratories., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Recurrences occur in 30 % of lung cancer patients after radical therapy; however, known prognostic factors are not always effective. In this study, we investigated whether the frequency of squamous non-small cell lung cancer (NSCLC) recurrence depends on the presence of reactive lesions in tumor-adjacent bronchial epithelium. Specimens of adjacent lung tissue from 104 patients with squamous NSCLC were used for the determination of basal cell hyperplasia (BCH) and squamous metaplasia (SM) and for the analysis of the expression of Ki-67, p53, Bcl-2, and CD138. We found that recurrence was observed in 36.7 % of patients with BCH combined with SM (BCH + SM+) in the same bronchus, compared with 1.8 % in patients with isolated BCH (BCH + SM-; odds ratio (OR) 31.26, 95 % confidence interval (CI) 3.77-258.60; p = 0.00002). The percentage of Ki-67-positive cells was significantly higher in BCH + SM+ than in BCH + SM- (34.9 vs. 18.3 %; effect size 2.86, 95 % CI 2.23-3.47; p = 0.003). P53 expression was also more significant in BCH + SM+ than in BCH + SM- (14.4 vs. 9.6 %; effect size 1.22, 95 % CI 0.69-1.76; p = 0.0008). In contrast, CD138 expression was lower in BCH + SM+ than in BCH + SM- (21.8 vs. 38.5 %; effect size -6.26, 95 % CI -7.31 to -5.22; p = 0.003). Based on our results, we concluded that the co-presence of reactive bronchial lesions is associated with the development of recurrent squamous NSCLC and may be a negative prognostic indicator. In addition, significant differences in Ki-67, p53, and CD138 expression exist between isolated BCH and BCH combined with SM that probably reflect part of biological differences, which could relate to the mechanism of lung cancer recurrence.

The International Prognostic Index (IPI) has been the basis for determining prognosis in patients with diffuse large B-cell lymphoma (DLBCL) for the past 20 years. The utility of the IPI must be reassessed in the era of immunochemotherapy. Seven risk factors at diagnosis were identified, and a maximum of 7 points were assigned to each patient. Four risk groups were created: low (0-1), low-intermediate (2-3), high-intermediate (4), and high (5-7). Using MYC and BCL-2 clinical data from the Drum Tower Hospital collected during the rituximab era, we performed a retrospective analysis of patients with DLBCL treated with R-CHOP and built an biological markers adjusted IPI with the goal of improving risk stratification.Clinical features from 60 adults with de novo DLBCL diagnosed from 2008-2013 were assessed for their prognostic significance. The IPI remains predictive, but it cannot identify the high-risk subgroup. Compared with the IPI, the MYC and BCL-2 adjusted-IPI (A-IPI) better discriminated patients in the high-risk subgroup (4-year overall survival [OS]: 33.3%) than did the IPI (4 year OS: 48.0%). In the era of R-CHOP treatment, MYC and BCL-2 adjusted-IPI is more powerful than the IPI for helping guide treatment planning and interpretation of clinical trials.

Objective: To provide in vitro evidence of Psorinum treatment against cancer cells in a controlled study., Methods: Effects of homeopathic Psorinum 6× on cell viability were initially determined in several cancer cell lines, including A549, HepG2 and MCF-7, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and an ethanol 6× control. The cell line that exhibited highest inhibition was selected and used in the following experiments. A range of Psorinum 6× doses was used to explore treatment effects on cell cycle arrest, cell death (apoptosis), generation of reactive oxygen species (ROS) and change in mitochondrial membrane potential (MMP) using flow cytometry and fluorescence microscopy, respectively. Expression of several signal proteins related to apoptosis and cell survival were quantified with Western blotting and confocal microscopy. Further, circular dichroism (CD) spectroscopy was used to determine possible drug-DNA interactions, as well as the induction of conformational changes., Results: Treatment of cancer cell lines with Psorinum showed greater anticancer effects in A549 cells than in others. In A549 cells Psorinum treatment inhibited cell proliferation at 24 h after treatment, and arrested cell cycle at sub-G1 stage. It also induced ROS generation, MMP depolarization, morphological changes and DNA damage, as well as externalization of phosphatidyl serine. Further, increases in p53 expression, Bax expression, cytochrome c release, along with reduction of Bcl-2 level and caspase-3 activation were observed after Psorinum 6× treatment, which eventually drove A549 cells towards the mitochondria-mediated caspase-3-dependent pathway. CD spectroscopy revealed direct interaction of Psorinum with DNA, using calf thymus-DNA as target., Conclusion: Psorinum 6× triggered apoptosis in A549 cells via both up- and down-regulations of relevant signal proteins, including p53, caspase-3, Bax and Bcl-2.

Introduction: Proliferation and apoptosis are opposing processes by which the cell numbers are kept in a delicate balance, essential for tissue homeostasis, whereas uncontrolled growth of cells is a hallmark of cancer. Papillary thyroid cancer (PTC) is the commonest type of thyroid cancer, with some PTC following an indolent course, whereas the other ones are more aggressive., Aim: To evaluate respective contribution of proliferation and apoptosis in the tumorigenesis of PTC by automated analysis., Materials and Methods: We investigated the immunolabeling of phosphorylated histone H3 (pHH3), cyclin D1, active caspase-3, and bcl-2 in thirteen cases each of metastatic PTC, follicular variant of PTC (FVPTC), papillary microcarcinoma (PMC) and well differentiated tumor of uncertain malignant potential (WDT-UMP). FVPTC cases comprised seven encapsulated and six unencapsulated cases., Results: Proliferation, as assessed by pHH3 and cyclin D1 immunolabeling, was increased in all PTC variants, including the putative precursor lesion WDT-UMP, compared to normal thyroid tissue. pHH3 was immunolabeled in more cells of metastatic PTC than of PMC and of encapsulated FVPTC. Surprisingly, metastatic PTC and unencapsulated FVPTC also demonstrated more cleaved caspase-3 immunolabeled cells than the other types. In contrast, increased expression of bcl-2 protein was seen in normal thyroid areas, encapsulated FVPTC and PMC as compared to metastatic PTC. Metastatic PTC shows higher proliferation than other types of PTC but unexpectedly also higher apoptotic levels. Similar results were also seen with unencapsulated FVPTC, thus suggesting that unencapsulated FVPTC has a potential for adverse outcome. Bcl-2 was immunolabeled in a low percentage of cells in WDT-UMP., Conclusions: The expression of the proliferative protein pHH3 together with the apoptotic marker cleaved caspase-3 may indicate an aggressive behaviour of PTC and loss of apoptosis inhibition by bcl-2 protein can further amplify the role of these proteins in tumor progression. Both cyclin D1 and bcl-2 could prove to be interesting markers of PTC precursor lesions. Automated/digital image quantification approach helps in refining the diagnostic accuracy.

Follicular lymphomas are characterized by overexpression of BCL2 which, in the large majority of cases, is due to a t(14;18) translocation which juxtaposes the BCL2 locus to the immunoglobulin heavy chain locus (IGH). Here, we report partial absence of BCL2 immunohistochemical staining in a case of FL, due to a mutation in the part of BCL2 that encodes the epitope for the most frequently used antibody against BCL2. This finding shows that mutations in BCL2 occur in an ongoing process in follicular which can give rise to unusual immunohistochemical staining patterns., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

In this study, the authors investigated the expression and significance of WTl in xenotransplanted ovarian carcinoma cell SKOV3 of nude mice treated with paclitaxel. Xenotransplanted ovarian carcinoma was established in nude mice using the SKOV3 cell line. The mice were randomized into the treatment group with paclitaxel and control group with normal sodium. The sizes of the xenotransplanted tumors were measured and the tumor specimens were confirmed by routine hemotoxylin-eosin (H&E) staining. The apoptosis index was then assayed using flow cytometry. WTl and bcl-2 expression were detected with immunohistochemistry, and WT1 mRNA expression was determined by reverse transcriptase polymerase chain reaction (RT-PCR). The authors found that the growth of the xenotransplanted tumor was inhibited by paclitaxel therapy. Compared to the control group, the apoptosis rate was significantly increased in the treatment group (p < 0.05). At the same time, the expression of WTl, bcl-2 and WTI, mRNA were significantly decreased in the paclitaxel therapy group (p < 0.05). The authors conclude that the WTl gene may play an important role during apoptosis of ovarian carcinoma and the mechanism may be closely related to bcl-2.

The classification of cutaneous follicular lymphoma (CFL) into primary cutaneous follicle center lymphoma (PCFCL) or secondary cutaneous follicular lymphoma (SCFL) is challenging. SCFL is suspected when tumor cells express BCL2 protein, reflecting a BCL2 translocation. However, BCL2 expression is difficult to assess in CFLs because of numerous BCL2+ reactive T cells. To investigate these issues and to further characterize PCFCL, we studied a series of 25 CFLs without any extracutaneous disease at diagnosis, selected on the basis of BCL2 protein expression using 2 BCL2 antibodies (clones 124 and E17) and BOB1/BCL2 double immunostaining. All cases were studied using interphase fluorescence in situ hybridization with BCL2, BCL6, IGH, IGK, IGL breakapart, IGH-BCL2 fusion, and 1p36/1q25 dual-color probes. Nineteen CFLs were BCL2 positive, and 6 were negative. After a medium follow-up of 24 (6 to 96) months, 5 cases were reclassified as SCFL and were excluded from a part of our analyses. Among BCL2+ PCFCLs, 60% (9/15) demonstrated a BCL2 break. BCL2-break-positive cases had a tendency to occur in the head and neck and showed the classical phenotype of nodal follicular lymphoma (CD10+, BCL6+, BCL2+, STMN+) compared with BCL2-break-negative PCFCLs. Del 1p36 was observed in 1 PCFCL. No significant clinical differences were observed between BCL2+ or BCL2- PCFCL. In conclusion, we show that a subset of PCFCLs harbor similar genetic alterations, as observed in nodal follicular lymphomas, including BCL2 breaks and 1p36 deletion. As BCL2 protein expression is usually associated with the presence of a BCL2 translocation, fluorescence in situ hybridization should be performed to confirm this hypothesis.

Objective: While the odontoblast ability to respond to injury in permanent teeth (PT) is well established, there is a lack of knowledge about deciduous teeth (DT). Aim of this study was to compare the odontoblasts activity within the pulp of DT versus the pulp of PT., Study Design: Dental pulp was obtained from forty-two DT and twenty-seven PT extracted from sixty-five patients (aged 6-16 years). Histomorphometry was carried out and the quantification of odontoblastic layer was assessed. Dental pulps of DT and PT were stained for anti-ssDNA, BCL-2, BCL-x, BAX, caspase3., Results: Pulps from DT were characterized by reduction of odontoblastic layer and greater occurrence of apoptotic odontoblasts. Pro-apoptotic BAX phenotype expression on odontoblasts correlated with the occurrence of numerous activated caspase3 odontoblasts in DT. The number of BAX positive cells was significantly higher compared to BCL-2 positive cells in the odontoblastic layer of the DT (p=0.03). Since BAX and BCL-2 proteins have an inverse role in the regulation of the apoptosis, this finding suggests that odontoblasts have a predominant pro-apoptotic phenotype in DT., Conclusion: According to our results, the odontoblasts of DT can be assumed to have a lower reparative activity if compared to odontoblasts of PT.

Background/aims: Amiodarone, a thyroid hormone-like molecule, can induce dyslipidemia and thyroid dysfunction. However, the effects of dronedarone on lipid metabolism and of both dronedarone and amiodarone on thyroid function and lipid metabolism remain unknown., Methods: Fifty male Sprague-Dawley rats were randomly divided into 5 groups (10 in each group): normal control (NC), amiodarone-treated (AMT), dronedarone-treated (DRT), rats treated with amiodarone combined with polyene phosphatidylcholine (AC), and rats treated with dronedarone combined with polyene phosphatidylcholine (DC). Rats were given amiodarone (120 mg/kg/d), dronedarone (120 mg/kg/d), and polyene phosphatidylcholine (200 mg/kg/d) for 13 weeks. At the end of weeks 4, 8, 12, and 13, plasma-free triiodothyronine (FT3), free thyroxine (FT4), triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) were determined. At the end of this protocol, rats were sacrificed and the thyroid glands were isolated, weighed, and examined histopathologically. The protein expression of Bcl-2 was measured by immunochemical staining. The mRNA expression of thyroglobulin (Tg), type-1 deiodinase (D1), and thyroid peroxidase (TPO) were detected by polymerase chain reaction (PCR)., Results: Compared with the NC group, FT3 and FT4 levels in the DRT and DC groups significantly increased at week 4 but declined thereafter. The AMT and AC groups had lower FT3 levels but comparable FT4 levels. The levels of TG, LDL-c, and HDL-c in the NC group were lower than those in the other groups whereas the LDL-c/HDL-c ratio was lowest in the AMT group. Bcl-2 expression significantly increased in the DRT group. The mRNA expression of Tg increased whereas the mRNA expression of D1 decreased. Dronedarone induced hyperthyroidism at the early stage and hypothyroidism at the late stage whereas amiodarone only caused hypothyroidism., Conclusion: Both dronedarone and amiodarone can induce dyslipidemia and increase the levels of TC, LDL-c, and HDL-c, and these effects may be associated with thyroid dysfunction., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Purpose: To study the antiproliferative effects of myricetin in human glioma U251 cells together with assessing its effects on cell cycle, apoptosis, apoptosis-related proteins, reactive oxygen species (ROS) generation and cell migration., Methods: Cell viability of human glioma cells after myricetin treatment was assessed by MTT assay. Phase-contrast and confocal fluorescence microscopies were used to assess the morphological changes that occured in these cells following myricetin treatment. Flow cytometry using propidium iodide (PI) and Annexin-V FITC as probes was employed to evaluate the effects on cell cycle arrest and apoptosis induction, respectively. The effect of myricetin on intracellular ROS production was measured by flow cytometry with a fluorescent probe CM-DCFH2-DA., Results: Myricetin induced a dose-dependent as well as time-dependent growth inhibitory effect in U251 human glioma cells. Myricetin treatment resulted in U251 cells detachment from adjacent cells making clusters of cells floating in the medium. Detached cells had irregular shape and incapable to maintain their membranes intact. Apoptotic cell death was induced by myricetin treatment as witnessed by fluorescence microscopy. The percentage of early and late apoptotic cells increased from 0.41% and 8.2% to 23.1% and 10.2%, 25.2% and 19.4%, to finally 36.2% and 28.4% after treatment with 15 μM, 60 μM and 120 μM of myricetin, respectively. We also observed a dose-dependent increase in Bax and Bad levels and a dose-dependent decrease in Bcl-2 and Bcl-xl expression levels following myricetin treatment. Cell cycle arrest in G2/M phase of the cell cycle was also induced by the drug treatment. A concentration-dependent ROS generation was also witnessed and a 3-fold increase of ROS production was seen after 60 μM myricetin treatment., Conclusion: Myricetin exerts anticancer effects in U251 human glioma cells by inducing mitochondrial-mediated apoptosis, G2/M phase cell cycle arrest, ROS generation and inhibition of cell migration.

Objectives: The histological differential diagnosis between epithelioid mesothelioma (EM) and reactive mesothelial hyperplasia (RMH) is not always straightforward. The aim of the present study was to search for new immunohistochemical markers to distinguish EM from RMH., Methods: We evaluated and compared the expression of apoptosis-related genes in EM and RMH by real-time RT-PCR array analysis followed by clustering of significant gene expression. Immunohistochemical staining and statistical analysis of Noxa expression in 81 cases of EM and 55 cases of RMH were performed and compared with the utility of other previously reported antibodies such as Desmin, EMA, GLUT-1, IMP-3 and CD146., Results: Noxa mRNA expression levels were found to be increased in EM when compared to RMH by RT-PCR array analysis. In the immunohistochemical analysis, Noxa showed sensitivity of 69.0%, specificity of 93.6% and positive predictive value of 93.0% as a positive marker of EM in distinguishing it from RMH, and these values were almost similar to IMP-3., Conclusion: Noxa is a marker with relatively high specificity, and can be used to distinguish EM from RMH. It would be a valuable addition to the current antibody panel used for the differential diagnosis of EM and RMH., (© 2016 S. Karger AG, Basel.)

We previously found that tyrosine kinase 2 (TYK2) signaling through its downstream effector phospho-STAT1 acts to upregulate BCL2, which in turn mediates aberrant survival of T-cell acute lymphoblastic leukemia (T-ALL) cells. Here we show that pharmacologic inhibition of heat shock protein 90 (HSP90) with a small-molecule inhibitor, NVP-AUY922 (AUY922), leads to rapid degradation of TYK2 and apoptosis in T-ALL cells. STAT1 protein levels were not affected by AUY922 treatment, but phospho-STAT1 (Tyr-701) levels rapidly became undetectable, consistent with a block in signaling downstream of TYK2. BCL2 expression was downregulated after AUY922 treatment, and although this effect was necessary for AUY922-induced apoptosis, it was not sufficient because many T-ALL cell lines were resistant to ABT-199, a specific inhibitor of BCL2. Unlike ABT-199, AUY922 also upregulated the proapoptotic proteins BIM and BAD, whose increased expression was required for AUY922-induced apoptosis. Thus, the potent cytotoxicity of AUY922 involves the synergistic combination of BCL2 downregulation coupled with upregulation of the proapoptotic proteins BIM and BAD. This two-pronged assault on the mitochondrial apoptotic machinery identifies HSP90 inhibitors as promising drugs for targeting the TYK2-mediated prosurvival signaling axis in T-ALL cells.

Breast cancer is the second leading cause of cancer-related death in women in the United States and discovery and development of safe chemopreventive drugs is urgently needed. The fruit pomegranate (Punica granatum) is gaining importance because of its various health benefits. This study was initiated to investigate chemopreventive potential of a pomegranate emulsion (PE) against 7,12-dimethylbenz(a)anthracene (DMBA) rat mammary carcinogenesis. The animals were orally administered with PE (0.2-5.0 g/kg), starting 2 wk before and 16 wk following DMBA treatment. PE exhibited a striking reduction of DMBA-induced mammary tumor incidence, total tumor burden, and reversed histopathological changes. PE dose-dependently suppressed cell proliferation and induced apoptosis in mammary tumors. Immunohistochemical studies showed that PE increased intratumor Bax, decreased Bcl2 and manifested a proapoptotic shift in Bax/Bcl2 ratio. In addition, our gene expression study showed PE-mediated upregulation of Bad, caspase-3, caspase-7, caspase-9, poly (ADP ribose) polymerase and cytochrome c in mammary tumors. Thus, PE exerts chemoprevention of mammary carcinogenesis by suppressing cell proliferation and inducing apoptosis mediated through upregulation of Bax and downregulation of Bcl2 in concert with caspase cascades. Pomegranate bioactive phytoconstituents could be developed as a chemopreventive drug to reduce the risk of breast cancer.

Objective: Prognostic parameters in determining risk groups for treatment in neuroblastoma are cellular differentiation, mitosis karyorrhexis index (MKI), N-myc amplification and age. However, additional prognosticators are still needed because patients can show unpredictable biological behavior. We aimed to study the prognostic significance of apoptotic activity in neuroblastomas., Material and Method: Thirty-five primary neuroblastoma were evaluated. The data including stage, treatment protocol, metastatic disease, survival, N-myc status, age and prognostic categorization were obtained from the clinical records. The differentiation and MKI were evaluated in hematoxylin and eosin stained slides. Apoptotic activity was assessed by both bcl-2 immunohistochemical staining and the transferase-mediated d-UTP-biotin nick end labeling (TUNEL) method. Data were correlated with established prognostic factors and clinical outcome., Results: Twenty-five (71.4%) cases were located in the adrenal. Sixteen cases showed low and 19 high MKI. Thirty-three (%94) were immunopositive for bcl-2. TUNEL staining was negative in 2 cases. Of the remaining positive 33 cases, 14 had an apoptotic index of ≤2%, 11 of 2-4% and 8 of ≥4%. Cases located in the adrenal showed higher scores of bcl-2 positivity than extra-adrenal tumors. There was no statistical significance for both bcl-2 staining and apoptotic index in correlation with cellular differentiation, MKI, N-myc amplification, age and other clinical parameters. Statistical significance was observed between bcl-2 scoring and tumor localization., Conclusion: According to our results, apoptotic activity is unlikely to be a prognostic parameter in neuroblastomas. Some studies showing significant correlations between clinical outcome and both bcl-2 immunoscoring and apoptotic index, as assessed by the TUNEL method, differences in case numbers and methodology can explain these conflicting results. Larger series and different methodologies are needed to evaluate the prognostic value of apoptotic activity in neuroblastomas.