Muscle specific kinase protects dystrophic mdx mouse muscles from eccentric contraction-induced loss of force-producing capacity.

KEY POINTS: Adeno-associated viral vector was used to elevate the expression of muscle specific kinase (MuSK) and rapsyn (a cytoplasmic MuSK effector protein) in the tibialis anterior muscle of wild-type and dystrophic (mdx) mice. In mdx mice, enhanced expression of either MuSK or rapsyn ameliorated the acute loss of muscle force associated with strain injury. Increases in sarcolemmal immunolabelling for utrophin and ß-dystroglycan suggest a mechanism for the protective effect of MuSK in mdx muscles. MuSK also caused subtle changes to the structure and function of the neuromuscular junction, suggesting novel roles for MuSK in muscle physiology and pathophysiology. ABSTRACT: Muscle specific kinase (MuSK) has a well-defined role in stabilizing the developing mammalian neuromuscular junction, but MuSK might also be protective in some neuromuscular diseases. In the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy, limb muscles are especially fragile. We injected the tibialis anterior muscle of 8-week-old mdx and wild-type (C57BL10) mice with adeno-associated viral vectors encoding either MuSK or rapsyn (a cytoplasmic MuSK effector protein) fused to green fluorescent protein (MuSK-GFP and rapsyn-GFP, respectively). Contralateral muscles injected with empty vector served as controls. One month later mice were anaesthetized with isoflurane and isometric force-producing capacity was recorded from the distal tendon. MuSK-GFP caused an unexpected decay in nerve-evoked tetanic force, both in wild-type and mdx muscles, without affecting contraction elicited by direct electrical stimulation of the muscle. Muscle fragility was probed by challenging muscles with a strain injury protocol consisting of a series of four strain-producing eccentric contractions in vivo. When applied to muscles of mdx mice, eccentric contraction produced an acute 27% reduction in directly evoked muscle force output, affirming the susceptibility of mdx muscles to strain injury. mdx muscles overexpressing MuSK-GFP or rapsyn-GFP exhibited significantly milder force deficits after the eccentric contraction challenge (15% and 14%, respectively). The protective effect of MuSK-GFP in muscles of mdx mice was associated with increased immunolabelling for utrophin and ß-dystroglycan in the sarcolemma. Elevating the expression of MuSK or rapsyn revealed several distinct synaptic and extrasynaptic effects, suggesting novel roles for MuSK signalling in muscle physiology and pathophysiology.

[Association of ambient fine particulate matters with anxiety in middle-aged and elderly people].

Objective: To investigate the association of ambient fine particulate matters (PM(2.5)) exposure with anxiety in middle-aged and elderly people in China. Methods: Using a stratified random sampling method, 5 997 middle-aged and elderly people (aged 40-89) who resided in the region for more than 2 years and had no hearing or language impairment were selected from 32 districts/counties in the key areas for air pollution prevention and control in China from October 10(th), 2017 to February 7(th), 2018. Information about demographic characteristics, socioeconomic factors and health status were collected by questionnaire survey and physical examination. The anxiety symptoms were assessed by 7-item Generalized Anxiety Disorder Scales. Three-year moving average concentrations of PM(2.5) were calculated to estimate exposure level. The multivariate logistic regression model was conducted to assess the association between PM(2.5) exposure and anxiety. The interaction of age, gender, overweight, education, smoking, drinking and chronic diseases was also analyzed by likelihood ratio test. Results: There were 2 995 (49.94%) males subjects, 4 092 (68.23%) subjects with education of secondary school or above and 2 576 (42.95%) subjects with self-reported chronic diseases among the 5 997 middle-aged and elder participants. The prevalence of anxiety was 6.64% (n=398). The mean±SD of 3-year moving average concentrations of PM(2.5), O(3) and SO(2) were (53.50±13.38), (90.58±13.26) and (40.29±12.56) µg/m(3), respectively. PM(2.5) had significantly association with anxiety, and the corresponding OR value was 1.17 (95%CI: 1.05,1.31) with a 10 µg/m(3) increment of 3-year moving average concentrations of PM(2.5). Compared with female (OR (95%CI)=1.07 (0.93,1.23)), those with no chronic diseases (OR (95%CI)=1.06 (0.93,1.20)) and those with primary school degree or below (OR (95% CI)=0.90 (0.75,1.09)), the association between PM(2.5) and anxiety was stronger among male (OR (95%CI)=1.35 (1.12,1.63)) and those with chronic diseases (OR (95%CI)=1.77 (1.31,2.38)), middle school education (OR (95%CI)=1.43 (1.22,1.67)), college education and above (OR (95%CI)=1.68 (1.05, 2.67)), all the P interaction values were <0.05. Conclusion: PM(2.5) exposure has significantly positive association with anxiety. The associations are stronger in male, people with higher educational qualifications and patients with chronic diseases.

Association of cigarette smoking with a past history and incidence of herpes zoster in the general Japanese population: the SHEZ Study.

Few studies have examined the impact of cigarette smoking on the risk for herpes zoster. The Shozu Herpes Zoster (SHEZ) Study is a community-based prospective cohort study over 3 years in Japan aiming to clarify the incidence and predictive and immunological factors for herpes zoster. We investigated the associations of smoking status with past history and incidence of herpes zoster. A total of 12 351 participants provided valid information on smoking status and past history of herpes zoster at baseline survey. Smoking status was classified into three categories (current, former, never smoker), and if currently smoking, the number of cigarettes consumed per day was recorded. The participants were under the active surveillance for first-ever incident herpes zoster for 3 years. We used a logistic regression model for the cross-sectional study on the association between smoking status and past history of herpes zoster, and a Cox proportional hazards regression model for the cohort study on the association with risk of incidence. The multivariable adjusted odd ratios (95% CI) of past history of herpes zoster for current vs. never smokers were 0·67 (0·54-0·80) for total subjects, 0·72 (0·56-0·93) for men and 0·65 (0·44-0·96) for women. The multivariable adjusted hazard ratios (95% CI) of incident herpes zoster for current vs. never smokers were 0·52 (0·33-0·81) for total subjects, 0·49 (0·29-0·83) for men and 0·52 (0·19-1·39) for women. Smoking status was inversely associated with the prevalence and incidence of herpes zoster in the general population of men and women aged ⩾50 years.

Characterization and functional analyses of a novel chicken CD8α variant X1 (CD8α1).

We provide the first description of cloning and of structural and functional analysis of a novel variant in the chicken cluster of differentiation 8 alpha (CD8a) family, termed the CD8α X1 (CD8α1) gene. Multiple alignments of CD8α1 with known CD8α and CD8ß sequences of other species revealed relatively low conservation of AA residues involved in the specific and unique structural domains among CD8α genes. For example, cysteine residues that are involved in disulfide bonding to form the V domain are conserved. In contrast, the O-linked glycosylation sites (XPXX motif) are not found in the chicken CD8α1 sequence, and the A ß strand and complementarity-determining region 1 and 2 sequences are poorly conserved between chicken CD8α1 and avian CD8α. Furthermore, the alignment showed that the transmembrane regions show relatively high sequence similarity, whereas the cytoplasmic regions show relatively low similarity, indicating poor conservation. Moreover, the motif (CXCP) that is thought to be responsible for binding the p56 lymphocyte cell kinase subunit (p56) is missing in the CD8α1 sequence. The chicken CD8α1 genomic structure is similar to that of chicken CD8α, but their protein structures differ. Phylogenetic analysis showed that chicken CD8α1 grouped with known avian CD8α sequences but was somewhat distantly related to the CD8α molecules of other species. Moreover, we analyzed the signal transduction and cytokine response to CD8α1 treatment to determine the specific biological functions of chicken CD8α1 in immune cells. The results showed that chicken CD8α1 is a key regulator of the expression of genes that are associated and cooperate with transcription factors in the major histocompatibility complex class I and II promoter regions and activates Janus kinase (JAK) 1/2, signal transducer and activator of transcription (STAT), and suppressor of cytokine signaling (SOCS) 1 signaling-related genes. Immune cells that express functional CD8α1 induce proinflammatory cytokines as well as innate immune responses. Therefore, our data indicate that CD8α1 may have immunoregulatory activity by regulating the expression of proinflammatory or anti-inflammatory cytokines via its effect on immune cells.

Association of global DNA hypomethylation with clinicopathological variables in colonic tumors of Iraqi patients.

BACKGROUND/AIM: Colorectal cancer (CRC) ranks sixth among the most common 10 cancers in Iraq. It is a foremost public health dilemma and there is improved interest in understanding the fundamental principles of its molecular biology. DNA methylation in cancer has become the issue of passionate investigation. As compared with normal cells, the malignant cells show major disruptions in their DNA methylation patterns. We aimed to assess the association of global DNA hypomethylation in colonic adenomas and carcinomas of Iraqi patients, measured by immunohistochemistry of 5-methylcytosin, with different clinicopathological variables. PATIENTS AND METHODS: Thirty tissue paraffin blocks from patients with colorectal adenomas, 30 tissue paraffin blocks from patients with colorectal adenocarcinomas, and 30 samples of apparently normal colonic tissue taken from autopsy cases as a control group were included in the present study. From each block, two sections of 5 µm thickness were taken, one section was stained with Hematoxylin and Eosin for revision of histopathological diagnosis and one section was immunohistochemically stained for 5-methylcytosine (5mC) and digitally analyzed by AperioImageScope software. RESULTS: The mean digital value of 5mC immunohistochemical expression was sequentially decreased during neoplastic progression from normal colonic tissue into adenoma and then to carcinoma. The mean digital value of 5mC expression was significantly lower in large size adenomas (≥1 cm), and those with severe dysplasia. Concerning carcinoma cases, 5mC expression was significantly lower in stage C2. CONCLUSIONS: The immunohistochemical evaluation of 5mC yields refined information on colorectal tumor biology in adenoma and carcinoma. Global DNA hypomethylation reflected by low immunohistochemical expression of 5-mC is associated with advanced colorectal adenomatous polyps suggesting that it is an early event in colorectal carcinogenesis. Also this hypomethylation can reflect bad prognosis of patients with colorectal cancer by its correlation to higher tumor stage.

Correlation of P16 (Ink4a) and CK17 to HPV (16E6+18E6) in Premalignant and Malignant Lesions of Uterine Cervix: A Clinicopathologic Study.

BACKGROUND: This research was accomplished to evaluate the IHC expression of p16 (ink4a) and CK17 in low grade cervical intraepithelial lesions (LSIL), high grade cervical intraepithelial lesions (HSIL) and invasive cervical carcinomas and to assess their correlation to HPV (16E6+18E6). METHODS: The study included (127) formalin-fixed paraffin-embedded cervical biopsies; of which 22 cases were chronic cervicitis, 24 cases were LSIL, 28 cases were HSIL and 53 cases were invasive cervical carcinomas. Sections were immunohistochemically stained for p16 (ink4a), CK17 and HPV (16E6+18E6). RESULTS: The study established a highly significant increase in IHC of expression of p16 (ink4a), CK17 and HPV (16E6+18E6) from LSIL through HSIL to invasive carcinomas (P-value˂0.001). There was non-significant association between IHC expression of all makers with age of patients; types, grade and stage of cervical carcinomas (P-value˃0.05). HPV (16E6+18E6) revealed a significantly positive correlation with p16 (ink4a) (P-value˂0.05) and a non- significant correlation with CK17 (P-value˃0.05); in LSIL, HSIL and invasive carcinoma cases. CONCLUSION: p16 (ink4a) expression directly reflects infection with high risk HPV in cervical lesions and can add a significant diagnostic accuracy in the evaluation of CIN. CK 17 is a good marker of malignant transformation, with increasing in its expression according to the severity of cervical lesions; however, it is not related to HPV infection. Both markers are not related to prognostic variables of patients with cervical carcinoma.

Vascular endothelial growth factor -634 G/C polymorphism and risk of cancer: an updated meta-analysis.

The association between vascular endothelial growth factor (VEGF) gene polymorphisms and risk of cancer has been investigated in several studies published previously; however, the individual results are inconclusive. Therefore, we performed a meta-analysis to establish evidence for an association between the VEGF -634 G/C polymorphism and risk of cancer. We searched PubMed, Medline, and Korean Studies Information Service System databases and identified 29 case-control studies, containing data of 25,324 individuals, for this meta-analysis. The odds ratio (OR) and 95% confidence interval (95%CI) were used to determine the strength of the association. Overall, no significant association was detected in the allele model (G allele vs C allele, OR = 0.98, 95%CI = 0.93-1.03), dominant model (G/G+G/C vs C/C, OR = 1.00, 95%CI = 0.90-1.11), or recessive model (G/G vs G/C+C/C, OR = 0.96, 95%CI = 0.89-1.03). The meta-analysis results suggest that the VEGF -634 G/C polymorphism may not be related to the development of cancer. However, additional studies with larger sample size are required in order to provide supporting evidence.

Variability in functional p53 reactivation by PRIMA-1(Met)/APR-246 in Ewing sarcoma.

BACKGROUND: Though p53 mutations are rare in ES, there is a strong indication that p53 mutant tumours form a particularly bad prognostic group. As such, novel treatment strategies are warranted that would specifically target and eradicate tumour cells containing mutant p53 in this subset of ES patients. METHODS: PRIMA-1(Met), also known as APR-246, is a small organic molecule that has been shown to restore tumour-suppressor function primarily to mutant p53 and also to induce cell death in various cancer types. In this study, we interrogated the ability of APR-246 to induce apoptosis and inhibit tumour growth in ES cells with different p53 mutations. RESULTS: APR-246 variably induced apoptosis, associated with Noxa, Puma or p21(WAF1) upregulation, in both mutant and wild-type p53 harbouring cells. The apoptosis-inducing capability of APR-246 was markedly reduced in ES cell lines transfected with p53 siRNA. Three ES cell lines established from the same patient at different stages of the disease and two cell lines of different patients with identical p53 mutations all exhibited different sensitivities to APR-246, indicating cellular context dependency. Comparative transcriptome analysis on the three cell lines established from the same patient identified differential expression levels of several TP53 and apoptosis-associated genes such as APOL6, PENK, PCDH7 and MST4 in the APR-246-sensitive cell line relative to the less APR-246-sensitive cell lines. CONCLUSION: This is the first study reporting the biological response of Ewing sarcoma cells to APR-246 exposure and shows gross variability in responses. Our study also proposes candidate genes whose expression might be associated with ES cells' sensitivity to APR-246. With APR-246 currently in early-phase clinical trials, our findings call for caution in considering it as a potential adjuvant to conventional ES-specific chemotherapeutics.

Antitumor activity of IL-32ß through the activation of lymphocytes, and the inactivation of NF-κB and STAT3 signals.

Cytokine and activation of lymphocytes are critical for tumor growth. We investigated whether interleukin (IL)-32ß overexpression changes other cytokine levels and activates cytotoxic lymphocyte, and thus modify tumor growth. Herein, IL-32ß inhibited B16 melanoma growth in IL-32ß-overexpressing transgenic mice (IL-32ß mice), and downregulated the expressions of anti-apoptotic proteins (bcl-2, IAP, and XIAP) and cell growth regulatory proteins (Ki-67 antigen (Ki-67) and proliferating cell nuclear antigen (PCNA)), but upregulated the expressions of pro-apoptotic proteins (bax, cleaved caspase-3, and cleaved caspase-9). IL-32ß also inhibited colon and prostate tumor growth in athymic nude mice inoculated with IL-32ß-transfected SW620 colon or PC3 prostate cancer cells. The forced expression of IL-32ß also inhibited cell growth in cultured colon and prostate cancer cells, and these inhibitory effects were abolished by IL-32 small interfering RNA (siRNA). IL-10 levels were elevated, but IL-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α) levels were reduced in the tumor tissues and spleens of IL-32ß mice, and athymic nude mice. The number of cytotoxic T (CD8(+)) and natural killer (NK) cells in tumor tissues, spleen, and blood was significantly elevated in IL-32ß mice and athymic nude mice inoculated with IL-32ß-transfected cancer cells. Constituted activated NF-κB and STAT3 levels were reduced in the tumor tissues of IL-32ß mice and athymic nude mice, as well as in IL-32ß-transfected cultured cancer cells. These findings suggest that IL-32ß inhibits tumor growth by increasing cytotoxic lymphocyte numbers, and by inactivating the NF-κB and STAT3 pathways through changing of cytokine levels in tumor tissues.

Immunohistochemical expression of matrix metalloproteinase-7 in human colorectal adenomas using specified automated cellular image analysis system: a clinicopathological study.

BACKGROUND/AIM: To evaluate the immunohistochemical expression of matrix metalloproteinase-7 (MMP-7) in colorectal adenomas, and to correlate this expression with different clinicopathological parameters. PATIENTS AND METHODS: The study was retrospectively designed. Thirty three paraffin blocks from patients with colorectal adenoma and 20 samples of non-tumerous colonic tissue taken as control group were included in the study. MMP-7 expression was assessed by immunohistochemistry method. The scoring of immunohistochemical staining was conducted utilizing a specified automated cellular image analysis system (Digimizer). RESULTS: The frequency of positive immunohistochemical expression of MMP-7 was significantly higher in adenoma than control group (45.45% versus 10%) (P value < 0.001). Strong MMP-7 staining was mainly seen in adenoma cases (30.30%) in comparison with control (0%) the difference is significant ( P < 0.001). The three digital parameters of MMP-7 immunohistochemical expression (Area (A), Number of objects (N), and intensity (I)) were significantly higher in adenoma than control. Mean (A and I) of MMP-7 showed a significant correlation with large sized adenoma (≥ 1cm) ( P < 0.05), also a significant positive correlation of the three digital parameters (A, N, and I) of MMP-7 expression with villous configuration and severe dysplasia in colorectal adenoma had been identified ( P < 0.05). CONCLUSION: MMP-7 plays an important role in the growth and malignant conversion of colorectal adenomas as it is more likely to be expressed in advanced colorectal adenomatous polyps with large size, severe dysplasia and villous histology. The use of automated cellular image analysis system (Digmizer) to quantify immunohistochemical staining yields more consistent assay results, converts semi-quantitative assay to a truly quantitative assay, and improves assay objectivity and reproducibility.

4-O-methylhonokiol, a PPARγ agonist, inhibits prostate tumour growth: p21-mediated suppression of NF-κB activity.

BACKGROUND AND PURPOSE: The effects of 4-O-methylhonokiol (MH), a constituent of Magnolia officinalis, were investigated on human prostate cancer cells and its mechanism of action elucidated. EXPERIMENTAL APPROACH: The anti-cancer effects of MH were examined in prostate cancer and normal cells. The effects were validated in vivo using a mouse xenograft model. KEY RESULTS: MH increased the expression of PPARγ in prostate PC-3 and LNCap cells. The pull-down assay and molecular docking study indicated that MH directly binds to PPARγ. MH also increased transcriptional activity of PPARγ but decreased NF-κB activity. MH inhibited the growth of human prostate cancer cells, an effect attenuated by the PPARγ antagonist GW9662. MH induced apoptotic cell death and this was related to G(0) -G(1) phase cell cycle arrest. MH increased the expression of the cell cycle regulator p21, and apoptotic proteins, whereas it decreased phosphorylation of Rb and anti-apoptotic proteins. Transfection of PC3 cells with p21 siRNA or a p21 mutant plasmid on the cyclin D1/ cycline-dependent kinase 4 binding site abolished the effects of MH on cell growth, cell viability and related protein expression. In the animal studies, MH inhibited tumour growth, NF-κB activity and expression of anti-apoptotic proteins, whereas it increased the transcriptional activity and expression of PPARγ, and the expression of apoptotic proteins and p21 in tumour tissues. CONCLUSIONS AND IMPLICATION: MH inhibits growth of human prostate cancer cells through activation of PPARγ, suppression of NF-κB and arrest of the cell cycle. Thus, MH might be a useful tool for treatment of prostate cancer.

Immunohistochemical expression of PCNA and CD34 in colorectal adenomas and carcinomas using specified automated cellular image analysis system: a clinicopathologic study.

BACKGROUND/AIM: To evaluate the immunohistochemical expression of proliferating cell nuclear antigen (PCNA) and CD34 in colorectal adenomas and carcinomas, and to correlate this expression with different clinicopathologic parameters. MATERIALS AND METHODS: The study was retrospectively designed. A total of 86 tissue samples, including 33 paraffin blocks from patients with colorectal adenomas, 33 paraffin blocks from patients with colorectal adenocarcinomas, and a control group of 20 samples of nontumerous colonic tissue, were included in the study. From each block, 3 sections of 5 µm thickness were taken, 1 section was stained with hematoxylin and eosin (H and E) and the other 2 sections were stained immunohistochemically for PCNA and CD34. Scoring of the immunohistochemical staining was performed using a specified automated cellular image analysis system (Digimizer). RESULTS: PCNA expression was significantly increased in a sequence of normal mucosa-adenoma-carcinoma. It was significantly higher in adenomas ≥ 1 cm and those with severe dysplasia, and it showed a significant positive correlation with grade and lymph node involvement in colorectal carcinoma. CD34 showed significantly higher expression in carcinoma than adenoma and in adenoma than in the control group. CD34 expression showed a significant correlation with adenomas carrying severe dysplasia and large-sized adenomas (≥1 cm). It was significantly correlated with tumor grade, lymphovascular invasion, and lymph node involvement in colorectal carcinoma. CONCLUSION: PCNA plays an important role in colorectal neoplastic progression and can be utilized as ancillary marker for the risk of malignant transformation in colorectal adenomas as it correlates with high grade dysplasia and size. Intratumoral quantification of the mean (A and N) of CD34 in colorectal carcinoma reflects the grade of tumors and can predict lymph node involvement and lymphovascular invasion, to make a useful additional prognostic factor.

A selective inhibitor of the immunoproteasome subunit LMP2 induces apoptosis in PC-3 cells and suppresses tumour growth in nude mice.

BACKGROUND: Although the proteasome is a validated anticancer target, the clinical application of its inhibitors has been limited because of inherent systemic toxicity. To broaden clinical utility of proteasome inhibitors as anticancer agents, it is critical to develop strategies to selectively target proteasomes in cancer cells. The immunoproteasome is an alternative form of the constitutive proteasome that is expressed at high levels in cancer tissues, but not in most normal cells in the body. METHODS: To validate the immunoproteasome as a chemotherapeutic target, an immunoproteasome catalytic subunit LMP2-targeting inhibitor and siRNA were used. The sensitivity of PC-3 prostate cancer cells to these reagents was investigated using viability assays. Further, a xenograft model of prostate cancer was studied to test the in vivo effects of LMP2 inhibition. RESULTS: A small molecule inhibitor of the immunoproteasome subunit LMP2, UK-101, induced apoptosis of PC-3 cells and resulted in significant inhibition (~50-60%) of tumour growth in vivo. Interestingly, UK-101 did not block degradation of IκBα in PC-3 cells treated with TNF-α, suggesting that its mode of action may be different from that of general proteasome inhibitors, such as bortezomib, which block IκBα degradation. CONCLUSION: These results strongly suggest that the immunoproteasome has important roles in cancer cell growth and thus provide a rationale for targeting the immunoproteasome in the treatment of prostate cancer.

Effects of nicotine on apoptosis in human gingival fibroblasts.

AIM: Cigarette smoke is a complex mixture of more than 4700 chemical compounds including free radicals and oxidants and it is a world widely known problem to health. Nicotine is the major compound of tobacco and known as the cause of gingivitis and periodontitis. It induces intracellular oxidative stress recognized as the important agent in the damage of biological molecules. The aim of this study is to clarify the cytotoxic pathway of nicotine in human gingival fibroblasts (HGFs). METHODS: Human gingival fibroblasts stimulated by nicotine were used as an in vitro model. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell viability and reactive oxygen species (ROS) generation was assessed with 2,7-dichlorofluoroscein diacetate (DCF-DA). Morphological change was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay, stained with 4,6-diamidino-2-phenylindole (DAPI). To delineate the roles of extracellular signal-regulated kinase (ERK), P38 and c-Jun N-terminal kinase (JNK), Western blot and caspase-3 (CASP3) activity assay were performed. RESULTS: Exposure of the human gingival fibroblasts to nicotine reduced cell viability by time and dose dependent and increased the generation of ROS. It also showed morphological evidence of increased apoptosis, resulted in transient activation of JNK and ERK concomitant with activation of P38, and stimulated apoptosis as evidenced by CASP3 activation and Poly ADP ribose polymerase (PARP) cleavage. CONCLUSION: These results suggest that nicotine induces apoptosis through the ROS generation and CASP3 dependent pathways in HGFs.

IL-32γ inhibits cancer cell growth through inactivation of NF-κB and STAT3 signals.

Several studies have shown physiological functions of interleukin (IL)-32, a novel cytokine. However, the role of IL-32 in cancer development has not been reported. In this study, we showed that IL-32γ inhibited tumor growth in IL-32γ-overexpressing transgenic mice inoculated with melanoma as well as colon tumor growth in xenograft nude mice inoculated with IL-32γ-transfected colon cancer cells (SW620). The inhibitory effect of IL-32γ on tumor growth was associated with the inhibition of constitutive activated nuclear transcription factor-κB (NF-κB) and of signal transducer and activator of transcription 3 (STAT3). The expression of antiapoptotic, cell proliferation and tumor-promoting genes (bcl-2, X-chromosome inhibitor of apoptosis protein (IAP), cellular IAP and cellular FADD-like IL-1ß-converting enzyme-inhibitory protein, cyclin D), cyclin-dependent kinase 4, cycolooxygenase-2 and inducible nitric oxide synthase was decreased, whereas the expression of apoptotic target genes (caspase-3 and -9, bax) increased. In tumor, spleen and blood, the number of cytotoxic CD8(+) T cells and CD57(+) natural killer cells and the levels of IL-10 increased, but that of tumor necrosis factor-α (TNF-α), IL-1ß and IL-6 decreased. We also found that forced overexpression of IL-32γ inhibited colon cancer cell (SW620 and HCT116) growth accompanied with the inhibition of activated NF-κB and STAT3 in vitro. In addition, when IL-32γ was knocked down by small interfering RNA (siRNA) or neutralized with an anti-IL-32γ antibody, IL-32γ-induced colon cancer cell growth inhibition, the IL-32γ-induced decrease of TNF-α, IL-1 and IL-6 production, and the increase of IL-10 production were abolished. However, siRNA of NF-κB and STAT3 augmented IL-32γ-induced colon cancer cell growth inhibition. These findings indicate significant pathophysiological roles of IL-32γ in cancer development.

Hsa-mir-145 is the top EWS-FLI1-repressed microRNA involved in a positive feedback loop in Ewing's sarcoma.

EWS-FLI1 is a chromosome translocation-derived chimeric transcription factor that has a central and rate-limiting role in the pathogenesis of Ewing's sarcoma. Although the EWS-FLI1 transcriptomic signature has been extensively characterized on the mRNA level, information on its impact on non-coding RNA expression is lacking. We have performed a genome-wide analysis of microRNAs affected by RNAi-mediated silencing of EWS-FLI1 in Ewing's sarcoma cell lines, and differentially expressed between primary Ewing's sarcoma and mesenchymal progenitor cells. Here, we report on the identification of hsa-mir-145 as the top EWS-FLI1-repressed microRNA. Upon knockdown of EWS-FLI1, hsa-mir-145 expression dramatically increases in all Ewing's sarcoma cell lines tested. Vice versa, ectopic expression of the microRNA in Ewing's sarcoma cell lines strongly reduced EWS-FLI1 protein, whereas transfection of an anti-mir to hsa-mir-145 increased the EWS-FLI1 levels. Reporter gene assays revealed that this modulation of EWS-FLI1 protein was mediated by the microRNA targeting the FLI1 3'-untranslated region. Mutual regulations of EWS-FLI1 and hsa-mir-145 were mirrored by an inverse correlation between their expression levels in four of the Ewing's sarcoma cell lines tested. Consistent with the role of EWS-FLI1 in Ewing's sarcoma growth regulation, forced hsa-mir-145 expression halted Ewing's sarcoma cell line growth. These results identify feedback regulation between EWS-FLI1 and hsa-mir-145 as an important component of the EWS-FLI1-mediated Ewing's sarcomagenesis that may open a new avenue to future microRNA-mediated therapy of this devastating malignant disease.

Outcomes of endoscopic sinus surgery in an elderly population: comparison with those in an adult population.

OBJECTIVE: The aim of this study was to compare the surgical outcomes of endoscopic sinus surgery in two patient groups: adults younger than 65 years of age and adults aged 65 years or older. DESIGN: A retrospective study. SETTING: This study was conducted at an academic tertiary referral centre. PARTICIPANTS: One hundred and eighty consecutive older patients (> or =65 years), and 180 adult patients (>15, <65 years) with chronic rhinosinusitis who underwent endoscopic sinus surgery under local anaesthesia between 1997 and 2007 were enrolled in this study. MAIN OUTCOME MEASURES: Recurrence and complication rates were analyzed. Data were analyzed using the Mann-Whitney test, Fisher's Exact test, the Pearson Chi-Square test, and univariate and multivariate logistic regression. P < 0.05 was considered statistically significant. RESULTS: When looked at without control for other factors the surgical complication rates were higher in the older group patients (31%) compared with the younger patients (11%). However, multivariate logistic regression analysis showed that preoperative duration of symptoms, length of follow-up, and diabetes mellitus were positively correlated with complications. In this analysis, the older patients, once controlled for these factors, were less likely to have complications. Regarding recurrence, in multivariate analysis there was no difference between the older and the younger group with preoperative duration and length of postoperative follow-up having a small effect. CONCLUSIONS: Comorbidity especially diabetes mellitus and duration of the condition should be the factors considered preoperatively to predict the likely hood of complications rather than whether the patient is younger or older that 65 years of age.

A small nuclear RNA, hdm365, is the major processing product of the human mdm2 gene.

mdm2 encodes for an E3 ubiquitin ligase targeting constitutively expressed p53 for proteasomal degradation. Several protein isoforms have been described for human MDM2 (HDM2), some of which may correspond to splicing variants detectable by RT-PCR in many tumors. Upon cellular stress, p53 becomes resistant to MDM2 and, in a feedback loop, up-regulates mdm2 transcription. The physiological relevance of stress-induced mdm2 gene activity is not well understood. We describe a small nuclear RNA of 365 bases comprised of the first five hdm2 exons and lacking polyadenylation. hdm365 precedes full-length hdm2 RNA expression after induction by p53 and accumulates to significant levels in the nucleus, detectable at the site of hdm2 transcription and processing only. Considering a 10-fold lower stability and high steady-state levels of the novel RNA species, hdm365 appears to be the major processing product of hdm2 transcripts. hdm365 induction was observed after ectopic expression of p53 and after DNA damaging treatment of tumor cell lines, primary fibroblasts and lymphocytes, and was not related to apoptosis. Corresponding truncated transcripts were observed in hdm2 amplified cells. High stress-inducible expression levels, absence of a corresponding protein, and nuclear localisation of hdm365 suggest a novel RNA-based function for hdm2.