Selective Affimers Recognise the BCL-2 Family Proteins BCL-xL and MCL-1 through Noncanonical Structural Motifs*.

The BCL-2 family is a challenging group of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are useful as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody-binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-xL , BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective recognition of their target BCL-2 protein. For anti-apoptotic targets BCL-xL and MCL-1, competitive inhibition of their canonical protein-protein interactions is demonstrated. Co-crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug-bound-like conformation. These proof-of-concept studies indicate that Affimers could be used as alternative templates to inspire the design of selective BCL-2 family modulators and more generally other protein-protein interaction inhibitors.

The Role of Bcl-xL Protein in Viral Infections.

Bcl-xL represents a family of proteins responsible for the regulation of the intrinsic apoptosis pathway. Due to its anti-apoptotic activity, Bcl-xL co-determines the viability of various virally infected cells. Their survival may determine the effectiveness of viral replication and spread, dynamics of systemic infection, and viral pathogenesis. In this paper, we have reviewed the role of Bcl-xL in the context of host infection by eight different RNA and DNA viruses: hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), influenza A virus (IAV), Epstein-Barr virus (EBV), human T-lymphotropic virus type-1 (HTLV-1), Maraba virus (MRBV), Schmallenberg virus (SBV) and coronavirus (CoV). We have described an influence of viral infection on the intracellular level of Bcl-xL and discussed the impact of Bcl-xL-dependent cell survival control on infection-accompanying pathogenic events such as tissue damage or oncogenesis. We have also presented anti-viral treatment strategies based on the pharmacological regulation of Bcl-xL expression or activity.

Bcl-XL: A multifunctional anti-apoptotic protein.

B-cell lymphoma-extra large (Bcl-XL) is one of the anti-apoptotic proteins of the Bcl-2 family that is localized in the mitochondria. Bcl-XL is one of the key regulators of apoptosis that can also regulate other important cellular functions. Bcl-XL is overexpressed in many cancers, and its inhibitors have shown good therapeutic effects. Bcl-XL interacts with Beclin 1, a key factor regulating autophagy. Bcl-XL is essential for the survival of neurons and plays protective roles in neuronal injuries. It can promote the growth of neurons and the correct formation of neural networks, enhance synaptic plasticity, and control neurotoxicity. Bcl-XL can also promote the transport of Ca2+ to mitochondria, increase the production of ATP, and improve metabolic efficiency. In addition, targeting Bcl-XL has shown potential value in autoimmune diseases and aging. In this review, we summarize the functions of Bcl-XL in cancer, autophagy, Ca2+ signaling, neuroprotection, neuronal growth and synaptic plasticity, energy metabolism, immunity, and senescence as revealed by investigations conducted in the past 10 years. Moreover, we list some inhibitors that have been developed based on the functions of Bcl-XL.

Differing Requirements for MALT1 Function in Peripheral B Cell Survival and Differentiation.

During a T cell-dependent immune response, formation of the germinal center (GC) is essential for the generation of high-affinity plasma cells and memory B cells. The canonical NF-κB pathway has been implicated in the initiation of GC reaction, and defects in this pathway have been linked to immune deficiencies. The paracaspase MALT1 plays an important role in regulating NF-κB activation upon triggering of Ag receptors. Although previous studies have reported that MALT1 deficiency abrogates the GC response, the relative contribution of B cells and T cells to the defective phenotype remains unclear. We used chimeric mouse models to demonstrate that MALT1 function is required in B cells for GC formation. This role is restricted to BCR signaling where MALT1 is critical for B cell proliferation and survival. Moreover, the proapoptotic signal transmitted in the absence of MALT1 is dominant to the prosurvival effects of T cell-derived stimuli. In addition to GC B cell differentiation, MALT1 is required for plasma cell differentiation, but not mitogenic responses. Lastly, we show that ectopic expression of Bcl-2 can partially rescue the GC phenotype in MALT1-deficient animals by prolonging the lifespan of BCR-activated B cells, but plasma cell differentiation and Ab production remain defective. Thus, our data uncover previously unappreciated aspects of MALT1 function in B cells and highlight its importance in humoral immunity.

IL-22 in hepatocyte's survival of Pakistani patients with end stage liver disease: an insight into IL 22 mediated hepato-regenerative pathway.

Hepatitis is the principal cause of hepatocellular carcinoma (HCC) and decompensated cirrhosis. HCC is amongst the leading causes of deaths worldwide. Current therapeutic options have proven to be unsuccessful in treating this disease due to multifactorial nature of the disease. The present study was designed to investigate the role of IL-22 mediated survival of hepatocytes during cirrhosis and HCC. Resected/explanted liver tissue samples of patients with End Stage Liver Disease were obtained from Hepato-Pancreato-Biliary Liver Transplant Unit of Sheikh Zayed Hospital, Lahore, Pakistan. Qualitative expression of IL-22, SOCS3, and IL-22 induced anti-apoptotic protein, B-cell lymphoma extra-large (Bcl-xL), were evaluated by Immunohistochemical analysis (IHC). The IHC analysis revealed significantly high expression of IL-22, SOCS3, and Bcl-xL within explanted livers of HCC patients. Overall, the expression of SOCS3 was higher than any other protein, and the expression of all proteins showed significant variation in different group of patients based on clincopathological features. The results of the current study indicated that IL-22 mediated JAK-STAT pathway i.e. liver regeneration and healing is dependent on the disease progression and type of agent responsible for causing the infection in the first place. However, quantitative analysis of these factors in future can provide further evidence of the role of this pathway in HCC for development of anti-HCC therapies.

Induction of ER Stress-Mediated Apoptosis by the Major Component 5,7,4'-Trimethoxyflavone Isolated from Kaempferia parviflora Tea Infusion.

Kaempferia parviflora (KP) is a famous medicinal plant from Thailand, and is a rich source of various kinds of methoxyflavones (MFs). Many kinds of food products such as tea, capsule, and liquor are manufactured from the rhizomes of KP. In this study, KP infusions were prepared with different brewing conditions, and the amounts of three major methoxylflavones, 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF), and 3,5,7,3',4'-pentamethoxyflavone (PMF), were analyzed. The antiproliferative activities of DMF, TMF, and PMF isolated from the brewed tea samples were evaluated. TMF was discovered to be significantly effective at inhibiting proliferation of SNU-16 human gastric cancer cells in a concentration dependent manner. TMF induced apoptosis, as evidenced by increments of sub-G1 phase, DNA fragmentation, annexin-V/PI staining, the Bax/Bcl-xL ratio, proteolytic activation of caspase-3,-7,-8, and degradation of poly (ADP-ribose) polymerase (PARP) protein. Furthermore, it was found that TMF induced apoptosis via ER stress, verified by an increase in the level of C/EBP homologous protein (CHOP), glucose regulated protein 78 (GRP78), inositol-requiring enzyme 1 α (IRE1α), activating transcription factor-4 (ATF-4), and the splice isoform of X-box-binding protein-1 (XBP-1) mRNA.

Study on the effect of integrin ανß6 on the proliferation and apoptosis of thyroid carcinoma cells.

PURPOSE: To study the effect of integrin αvß6 on the proliferation and apoptosis of thyroid carcinoma cells. METHODS: The experiment was conducted on 3 groups : the control group, the positive observation group (in which the ανß6 on the surface of the thyroid carcinoma cell line SW579 was blocked by monoclonal antibody 10D5) and the negative observation group (in which the ανß6 was dealt with the negative placebo of 10D5-the IgG2a). Cell proliferation was detected by MTT assay, apoptosis by flow cytometry and the protein levels in Caspase-3, CyclinB1 and Bcl-xl as well as the protein levels in ERK, p-ERK, JNK, p-JNK, p38 and p-p38 were detected by Western blot. RESULTS: The cell survival rates of the control group and the negative observation group were prominently higher than those of the positive observation group, following decrease in the apoptosis rates, and the differences were statistically significant (p<0.05). The protein levels in CyclinB1 and Bcl-x1 of the control group and the negative observation group were prominently higher than those of the positive observation group, whereas the levels in Caspase-3 were decreased; the differences were statistically significant (p<0.05). The protein levels in p-ERK, p-JNK and p-p38 of the control group and the negative observation group were prominently higher than those of the positive observation group, while the protein levels of ERK, JNK and p38 showed no difference. CONCLUSION: Integrin ανß6 can mediate the MAPK signal pathway of the cells and regulate the expression of CyclinB1 and the apoptosis-related proteins like Bcl-x1 and Caspase-2, thus affecting the process of the proliferation and apoptosis of thyroid carcinoma cells.

Long non-coding RNA INXS is a critical mediator of BCL-XS induced apoptosis.

BCL-X mRNA alternative splicing generates pro-apoptotic BCL-XS or anti-apoptotic BCL-XL gene products and the mechanism that regulates splice shifting is incompletely understood. We identified and characterized a long non-coding RNA (lncRNA) named INXS, transcribed from the opposite genomic strand of BCL-X, that was 5- to 9-fold less abundant in tumor cell lines from kidney, liver, breast and prostate and in kidney tumor tissues compared with non-tumors. INXS is an unspliced 1903 nt-long RNA, is transcribed by RNA polymerase II, 5'-capped, nuclear enriched and binds Sam68 splicing-modulator. Three apoptosis-inducing agents increased INXS lncRNA endogenous expression in the 786-O kidney tumor cell line, increased BCL-XS/BCL-XL mRNA ratio and activated caspases 3, 7 and 9. These effects were abrogated in the presence of INXS knockdown. Similarly, ectopic INXS overexpression caused a shift in splicing toward BCL-XS and activation of caspases, thus leading to apoptosis. BCL-XS protein accumulation was detected upon INXS overexpression. In a mouse xenograft model, intra-tumor injections of an INXS-expressing plasmid caused a marked reduction in tumor weight, and an increase in BCL-XS isoform, as determined in the excised tumors. We revealed an endogenous lncRNA that induces apoptosis, suggesting that INXS is a possible target to be explored in cancer therapies.

Odontoblastic Cell Quantification and Apoptosis within Pulp of Deciduous Teeth Versus Pulp of Permanent Teeth.

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.

Expression of apoptosis regulating proteins identifies stage II and III colon cancer patients with high risk of recurrence.

BACKGROUND AND OBJECTIVES: Deregulation of apoptosis related genes may be associated with poor outcome in cancer. Aim of the present study was to investigate the prognostic role of expression levels of apoptosis related proteins in stage II and III colon cancer. METHODS: From tumor samples of 386 stage II and III colon cancer patients, DNA was isolated and tissue microarrays were constructed. Expression of Bcl-2, Bcl-X, BAX, XIAP, Fas, FasL and c-FLIP was evaluated and PCR-based microsatellite instability analysis was performed. RESULTS: High FasL expressing tumors were associated with high disease recurrence rates in stage II colon cancer patients overall, as was low Bcl-X expression in microsatellite stable stage II patients. In stage II patients, a multivariable model based on FasL and Bcl-XL expression revealed a significant association with disease free survival (DFS). In stage III colon cancer patients, low Bcl-2, low BAX and low Fas expression levels were associated with worse outcome. In these patients a multivariable model based on angioinvasion and Bcl-2, Fas and FasL expression was significantly associated with DFS. CONCLUSIONS: Stage II patients with low Bcl-X and high FasL protein expression levels and stage III patients with low Fas, high FasL and low Bcl-2 expression could be considered as high risk for disease recurrence.

Expression of TRF2 and its prognostic relevance in advanced stage cervical cancer patients.

BACKGROUND: Telomeres are protective caps consisted of specific tandem repeats (5'-TTAGGG-3'). Shortening of telomeres at each cell division is known as "mitotic clock" of the cells, which renders telomeres as important regulators of lifespan. TRF2 is one of the critical members of shelterin complex, which is a protein complex responsible from the preservation of cap structure, and loss or mutation of TRF2 results in DNA damage, senescence or apoptosis. Since cancer is frequently associated with aberrant cell cycle progression, defective DNA repair or apoptosis pathways, TRF2 could be one likely candidate for cancer therapy. Here we investigated the prognostic role of TRF2 levels in cervical cancer patients. Fold-induction rates were evaluated with respect to median values after real-time PCR analysis. Overall survival, distant disease-free and local recurrence-free survival rates were calculated using Kaplan-Meier long rank test. RESULTS: Both five year overall- and disease-free survival rates were longer in patients with higher TRF2 expression compared to lower expression, but results were not statistically significant (69.2% vs 28.9%, respectively). Mean local recurrence-free survivals (LRF) were very close ( 58.6, CI: 44.3-72.9 vs 54.5, CI: 32.1-76.9 months) for high and low expressions, respectively. Cumulative proportion of LRF at the end of five year period was 76.9% for high and 57.1% for low TRF2 expression (P = 0.75). Statistically significant difference was found between survival ratios and Bcl-xL and p53 gene expressions, but not with TRF2. A respectable correlation between TRF2 expression and apoptosis along with distant metastasis was noted (P = 0.045 and 0.036, respectively). Additionally, high TRF2 expression levels had a positive impact in five year survival rate of stage IIIB-IVA patients (P = 0.04). CONCLUSIONS: Our results support the role of TRF2 in apoptosis and imply a positive relation with distant metastases and survival in advanced stage patients. The remarkable difference in survival periods of patients with different TRF2 expressions suggest that TRF2 may be a candidate factor to estimate survival for cervical cancer, a preliminary observation which should further be verified with a larger cohort.

Identification of differential protein binding affinities in an atropisomeric pharmaceutical compound by noncovalent mass spectrometry, equilibrium dialysis, and nuclear magnetic resonance.

Atropisomerism of pharmaceutical compounds is a challenging area for drug discovery programs (Angew. Chem., Int. Ed. 2009, 48, 6398-6401). Strategies for dealing with these compounds include raising the energy barrier to atropisomerization in order to develop the drug as a single isomer (Tetrahedron 2004, 60, 4337-4347) or reducing the barrier to rotation and developing a mixture of rapidly interconverting isomers (Chirality 1996, 8, 364-371). Commonly, however, the atropisomers will be differentiated in terms of their affinity for a given protein target, and it is therefore important to rapidly identify the most active component prior to further compound development. We present equilibrium dialysis and saturation transfer difference NMR (STD-NMR) as techniques for assessing relative affinities of an atropisomeric mixture against antiapoptotic protein targets Bcl-2 and Bcl-xL. These techniques require no prior separation of the mixture of compounds and are therefore rapid and simple approaches. We also explore the use of noncovalent mass spectrometry for determining KD values of individual atropisomers separated from the equilibrium mixture and compare the results to solution-phase measurements. Results from equilibrium dialysis, STD-NMR, and noncovalent mass spectrometry are all in excellent agreement and provide complementary information on differential binding, amplification of the strongest binders, and KD values.

Effect of methylprednisolone and edaravone administration on spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is one of the most devastating traumatic conditions that primarily affects young males with an annual incidence of 15-40 cases per million. AIM: To explore the superior neuroprotective effect of edaravone (ED) on spinal cord injury during maintenance therapy compared with methylprednisolone (MP). MATERIALS AND METHODS: Sprague-Dawley rat model of spinal cord injury was established by modified Allen's method. Total 114 rats were divided into two groups and then six subgroups individually: A1 (control group, normal saline injection within 8 h), B1 (MP group, MP injection within 8 h), C1(ED group, ED injection within 8 h), A2 (control group, normal saline injection after 8 h), B1 (MP group, MP injection after 8 h), C1 (ED group, ED injection after 8 h). Further, we investigated the changes of histopathology, caspase-3 and Bcl-xL positive cell. RESULTS: Haemorrhage, swelling, hyperaemia, gliocytes hyperplasia, inflammatory cells infiltration, vacuolar denaturation, and nucleus concentration could be observed, especially in control group. Caspase-3 positive cell was significantly decreased in MP and ED group within 8 h administration, but caspase-3 positive cell was only significantly decreased in ED group after 8 h administration. And B-cell lymphoma extra large (Bcl-xL) was significantly increased in ED group than MP group no matter within 8 h or after 8 h administration. CONCLUSIONS: More attention should be paid on the time point of MP administration, and ED administration seem to be more effective for maintenance therapy.

Exploring key parameters to detect subtle ligand-induced protein conformational changes using traveling wave ion mobility mass spectrometry.

Evidencing subtle conformational transitions in proteins occurring upon small modulator binding usually requires atomic resolution techniques (X-ray crystallography or NMR). Recently, hyphenation of ion mobility and mass spectrometry (IM-MS) has greatly enlarged the potentials for biomolecular assembly structural characterization. Using the well 3D-characterized Bcl-xL/ABT-737 protein model, we explored in the present report whether IM-MS can be used to differentiate close conformers and monitor collision cross section (CCS) differences correlating with ligand-induced conformational changes. Because comparing CCS derived from IM-MS data with 3D-computed CCS is critical for thorough data interpretation, discussing pitfalls related to protein construct similarity and missing sequence sections in PDB files was of primary importance to avoid misinterpretation. The methodic exploration of instrument parameters showed enhanced IM separation of Bcl-xL conformers by combining high wave heights and velocities with low helium and nitrogen flow rates while keeping a high He/N(2) flow rate ratio (>3). The robustness of CCS measurements was eventually improved with a modified IM calibration method providing constant CCS values regardless of instrument settings. Altogether, optimized IM-MS settings allowed a 0.4 nm(2) increase (i.e., 2%) of Bcl-xL CCS to be evidenced upon ABT-737 binding.

The downregulation of Mcl-1 via USP9X inhibition sensitizes solid tumors to Bcl-xl inhibition.

BACKGROUND: It has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. Mcl-1 is a critical survival protein in a variety of cell lineages and is critically regulated via ubiquitination. METHODS: The Mcl-1, Bcl-xL and USP9X expression patterns in human lung and colon adenocarcinomas were evaluated via immunohistochemistry. Interaction between USP9X and Mcl-1 was demonstrated by immunoprecipitation-western blotting. The protein expression profiles of Mcl-1, Bcl-xL and USP9X in multiple cancer cell lines were determined by western blotting. Annexin-V staining and cleaved PARP western blotting were used to assay for apoptosis. The cellular toxicities after various treatments were measured via the XTT assay. RESULTS: In our current analysis of colon and lung cancer samples, we demonstrate that Mcl-1 and Bcl-xL are overexpressed and also co-exist in many tumors and that the expression levels of both genes correlate with the clinical staging. The downregulation of Mcl-1 or Bcl-xL via RNAi was found to increase the sensitivity of the tumor cells to chemotherapy. Furthermore, our analyses revealed that USP9X expression correlates with that of Mcl-1 in human cancer tissue samples. We additionally found that the USP9X inhibitor WP1130 promotes Mcl-1 degradation and increases tumor cell sensitivity to chemotherapies. Moreover, the combination of WP1130 and ABT-737, a well-documented Bcl-xL inhibitor, demonstrated a chemotherapeutic synergy and promoted apoptosis in different tumor cells. CONCLUSION: Mcl-1, Bcl-xL and USP9X overexpression are tumor survival mechanisms protective against chemotherapy. USP9X inhibition increases tumor cell sensitivity to various chemotherapeutic agents including Bcl-2/Bcl-xL inhibitors.

Anticancer efficacy of simvastatin on prostate cancer cells and tumor xenografts is associated with inhibition of Akt and reduced prostate-specific antigen expression.

Prostate cancer is the second-leading cause of cancer-associated death among men in the United States. There has been renewed interest in the potential therapeutic benefits of statins for cancer. Simvastatin, a widely used generic drug for preventing cardiovascular events, is well known for its effects on cellular proliferation and inflammation, two key processes that also determine the rate of tumor growth. Although a growing body of evidence suggests that statins have the potential to reduce the risk of many cancers, there are discrepancies over the pro- and anticancer effects of statins. In the current study, we sought to investigate the effects of simvastatin on the Akt pathway in prostate cancer cells with respect to the regulation of various cell functions in vitro and tumor growth in vivo. Time- and dose-dependent effects of simvastatin on LNCaP (androgen-dependent) and PC3 (androgen-independent) cells indicate that treatment with simvastatin at concentrations as low as 25 µM was sufficient to inhibit serum-stimulated Akt activity. Akin to this, treatment with simvastatin significantly inhibited serum-induced cell migration, invasion, colony formation, and proliferation. Simvastatin-mediated effects on colony formation were rescued by adenovirus-mediated expression of constitutively active Akt (myristoylated Akt) in PC3 cell lines. A PC3 xenograft model performed in nude mice exhibited reduced tumor growth with simvastatin treatment associated with decreased Akt activity and reduced prostate-specific antigen (PSA) levels. Our findings demonstrate the therapeutic benefits of simvastatin for prostate cancer and suggest a link between simvastatin, regulation of Akt activity, and PSA expression in prostate tumors.

3,3'-Diindolylmethane inhibits prostate cancer development in the transgenic adenocarcinoma mouse prostate model.

3,3'-Diindolylmethane (DIM) is a major in vivo derivative of indole-3-carbinol, which is present in cruciferous vegetables and has been reported to possess anti-carcinogenic properties. In the present study, we examined whether DIM inhibits the development of prostate cancer using the transgenic adenocarcinoma mouse prostate (TRAMP) model. DIM feeding inhibited prostate carcinogenesis in TRAMP mice, reduced the number of cells expressing the SV40 large tumor antigen and proliferating cell nuclear antigen, and increased the number of terminal dUTP nick-end labeling-positive cells in the dorsolateral lobes of the prostate. Additionally, DIM feeding reduced the expression of cyclin A, cyclin-dependent kinase (CDK)2, CDK4, and Bcl-xL, and increased p27 and Bax expression. To assess the mechanisms by which DIM induces apoptosis, LNCaP and DU145 human prostate cancer cells were cultured with various concentrations of DIM. DIM induced a substantial reduction in the numbers of viable cells and induced apoptosis in LNCaP and DU145 cells. DIM increased the cleavage of caspase-9, -7, -3, and poly (ADP-ribose) polymerase (PARP). DIM increased mitochondrial membrane permeability and the translocation of cytochrome c and Smac/Diablo from the mitochondria. Additionally, DIM induced increases in the levels of cleaved caspase-8, truncated Bid, Fas, and Fas ligand, and the caspase-8 inhibitor Z-IETD-FMK was shown to mitigate DIM-induced apoptosis and the cleavage of caspase-3, PARP, and Bid. These results indicate that DIM inhibits prostate carcinogenesis via induction of apoptosis and inhibition of cell cycle progression. DIM induces apoptosis in prostate cancer cells via the mitochondria- and death receptor-mediated pathways.

Effect of antiviral therapy on the immunohistochemical expression of bcl-xL and bax protein in patients with HBeAg-negative chronic hepatitis B.

The effect of antiviral treatment on apoptosis in chronic hepatitis B (CHB) has not been clarified. We evaluated the hepatic immunohistochemical expression of the pro-apoptotic bax and the antiapoptotic bcl-xL protein in HBeAg-negative CHB patients before and after treatment. In our study we included 72 paired biopsies from 36 HBeAg-negative CHB patients: 29 treated (interferon-alfa: 17, adefovir: 12) and 7 untreated. Changes in expression of apoptotic proteins (D-bax, D-bcl-xL), necroinflammation and fibrosis (D-grade/D-stage) (Ishak classification) were evaluated. We found that Bax-positive compared to bax-negative biopsies had worse necroinflammation (8.2 vs. 6.7, P = 0.05) and fibrosis score (3.9 vs. 3, P = 0.036). bcl-xL-positive compared to bcl-xL-negative biopsies had lower intralobular inflammation (1.6 vs. 2.2, P = 0.03). Decreased compared to stable/increased D-bax was associated with greater improvement in necroinflammation only in treated patients (D-grade: -4.6 vs. -1.6, P = 0.05) and greater fibrosis improvement in interferon treated patients (D-stage: -0.4 vs. 0.55, P = 0.05). Increased compared to stable/decreased total apoptotic trend [D-apoptosis: (D-bax)-(D-bcl-xL)], was associated with worsening fibrosis, particularly in adefovir treated patients (D-stage: 2.3 vs. 0, P = 0.004). In the 11 patients without significant changes from 1st to 2nd biopsy, increased apoptosis was more frequent in treated than untreated cases (P = 0.046). In multivariate analysis, bax change was independently associated with change of grade (P = 0.038) and antiviral therapy (P = 0.015). In conclusions, in HBeAg-negative CHB, histological improvement after treatment is associated with decreased hepatocyte apoptosis. In patients without substantial histological changes, treatment seems to increase the apoptosis of hepatocytes, thus having a possible protective effect on hepatocarcinogenesis.

Downregulation of STAT3 signaling induces apoptosis but also promotes anti-apoptotic gene expression in human pancreatic cancer cell lines.

Signal transducer and activator of transcription 3 (STAT3) is a key regulator of cytokine signaling pathways that regulates gene expression. In pancreatic cancer, constitutive activation of STAT3 contributes to oncogenesis by preventing apoptosis through upregulation of anti-apoptotic proteins. We have examined the inhibition of STAT3 as a potential therapeutic approach in pancreatic cancer. siRNA targeting STAT3 was used to evaluate the role of STAT3 in modulating the expression of Survivin/BIRC5 and BCL-xL in the pancreatic cancer cell lines PANC-1 and BxPC-3 and induction of apoptosis. Expression of STAT3, Survivin/BIRC5, and BCL-xL on mRNA and protein level was measured by real-time RT-PCR and Western blot analysis 24, 48, and 72 h after transfection. STAT3 downregulation resulted in a decrease of cell viability in both cell lines and induced apoptosis in BxPC-3 cells. Despite significant inhibition of STAT3, the expression of the anti-apoptotic genes Survivin/BIRC5 and BCL-xL were not subsequently downregulated. Even more, the cell line BxPC-3 shows a significant increase of Survivin/BIRC5 and BCL-xL mRNA after 48-72 h as a result of STAT3 downregulation. Inactivation of STAT3 in pancreatic cancer cell lines induces apoptosis but also may promote the expression of anti-apoptotic genes.

Combining factors on protein and gene level to predict radioresponse in head and neck cancer cell lines.

BACKGROUND: Radiotherapy is the main therapy for head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems, highlighting the need for predictive markers. In this study, we evaluated selected proteins, mutations, and single nucleotide polymorphisms (SNPs) involved in apoptosis, cell proliferation, and DNA repair alone or combined as predictive markers for radioresponse in 42 HNSCC cell lines. METHODS: The expression of epidermal growth factor receptor, survivin, Bax, Bcl-2, Bcl-X(L) , cyclooxygenase-2 (COX-2), and heat shock protein 70 was analyzed by ELISA. Furthermore, mutations and SNPs in the p53 gene as well as SNPs in the MDM2, XRCC1, and XRCC3 genes were analyzed for their relation to radioresponse. To enable the evaluation of the predictive value of several factors combined, each cell line was allocated points based on the number of negative points (NNP) system, and the NNP sum was correlated with radioresponse. RESULTS: Survivin was the only factor that alone was significantly correlated with the intrinsic radiosensitivity (IR; r = 0.36, P = 0.02). The combination of survivin, Bax, Bcl-2, Bcl-X(L) , COX-2, and the p53 Arg72Pro polymorphism was found to most strongly correlate with radioresponse (r = 0.553, P < 0.001). CONCLUSION: These data indicate that the IR of 42 HNSCC cell lines can be predicted by a panel of factors on both the protein and gene levels. Moreover, among the investigated factors, survivin was the most promising biomarker of radioresponse.