Aberrant hydroxymethylation in promoter CpG regions of genes related to the cell cycle and apoptosis characterizes advanced chronic myeloid leukemia disease, poor imatinib respondents and poor survival.

BACKGROUND: There is strong evidence that disease progression, drug response and overall clinical outcomes of CML disease are not only decided by BCR/ABL1 oncoprotein but depend on accumulation of additional genetic and epigenetic aberrations. DNA hydroxymethylation is implicated in the development of variety of diseases. DNA hydroxymethylation in gene promoters plays important roles in disease progression, drug response and clinical outcome of various diseases. Therefore in this study, we aimed to explore the role of aberrant hydroxymethylation in promoter regions of different tumor suppressor genes in relation to CML disease progression, response to imatinib therapy and clinical outcome. METHODS: We recruited 150 CML patients at different clinical stages of the disease. Patients were followed up for 48 months and haematological/molecular responses were analysed. Haematological response was analysed by peripheral blood smear. BCR/ABL1 specific TaqMan probe based qRT-PCR was used for assessing the molecular response of CML patients on imatinib therapy. Promoter hydroxymethylation of the genes was characterized using MS-PCR. RESULTS: We observed that promoter hydroxymethylation of DAPK1, RIZ1, P16INK4A, RASSF1A and p14ARFARF genes characterize advanced CML disease and poor imatinib respondents. Although, cytokine signalling (SOCS1) gene was hypermethylated in advanced stages of CML and accumulated in patients with poor imatinib response, but the differences were not statistically significant. Moreover, we found hypermethylation of p14ARF, RASSF1 and p16INK4A genes and cytokine signalling gene (SOCS1) significantly associated with poor overall survival of CML patients on imatinib therapy. The results of this study are in agreement of the role of aberrant DNA methylation of different tumor suppressor genes as potential biomarkers of CML disease progression, poor imatinib response and overall clinical outcome. CONCLUSION: In this study, we report that promoter hydroxymethylation of DAPK1, RIZ1, P16INK4A, RASSF1A and p14ARFARF genes is a characteristic feature of CML disease progressions, defines poor imatinib respondents and poor overall survival of CML patients to imatinib therapy.

Epigenetic Silencing of DAPK1and p16 INK4a Genes by CpG Island Hypermethylation in Epithelial Ovarian Cancer Patients.

Transcriptional silencing induced by hypermethylation of CpG islands in the promoter regions of genes is believed to be an important mechanism of carcinogenesis in human cancers including epithelial ovarian cancer (EOC). Previously published data on gene methylation of EOC focused mainly on single gene or on cancer tissues. Objectives of the study were to estimate the promoter hypermethylation status of DAPK1 and p16 INK4a genes in circulating blood of EOC patients and to determine their association with clinicopathological features of EOC. This case-control study included 50 EOC patients and 20 apparently healthy and age matched female controls. Isolation of genomic DNA was carried out from peripheral venous blood. Methylation in promoter region of DAPK1 and p16 INK4a genes was determined by methylation-specific PCR. Methylation of DAPK1 was occurred in 42 out of 50 cases (84.0%) and methylation of p16 INK4a gene was occurred in 34 out of 50 cases (68.0%). Methylation of both genes was occurred in 25 cases (50.0%). Occurrence of methylation in DAPK1 and p16 INK4a genes was statistically significant (p < 0.0001) in cases compared to controls. Methylation of both genes was not statistically associated with age at diagnosis, menopausal status, histopathological types and FIGO staging of EOC. Identification of the peculiar promoter hypermethylation of DAPK1 and p16 INK4a genes might be a successful approach for ancillary diagnosis of EOC at early stage in blood sample.

ER-stress mobilization of death-associated protein kinase-1-dependent xenophagy counteracts mitochondria stress-induced epithelial barrier dysfunction.

The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation.

Effect of Subtoxic DDT Exposure on Glucose Uptake and Insulin Signaling in Rat L6 Myoblast-Derived Myotubes.

Exposure to persistent organic pollutants including dichlorodiphenyltrichloroethane (DDT) induces insulin resistance. But the mechanism is not clearly known. The present study was designed to explore the effect of subtoxic DDT exposure on (1) insulin-stimulated glucose uptake, (2) malondialdehyde (MDA) level and total antioxidant content, (3) activation of redox sensitive kinases (RSKs), and (4) insulin signaling in rat L6 myoblast-derived myotubes. Exposure to 30 mg/L and 60 mg/L of DDT for 18 hours dose dependently decreased glucose uptake and antioxidant content in myotubes and increased MDA levels. The exposures did not alter tumor necrosis factor α (TNF-α) level as determined by enzyme-linked immunosorbent assay, despite decreased messenger RNA expression following DDT exposures. Phosphorylation of c-Jun N-terminal kinases and IκBα, an inhibitory component of nuclear factor κB (NFκB), was increased, suggesting activation of RSKs. The level of tyrosine phosphorylation of insulin receptor substrate 1 and serine phosphorylation of protein kinase B (Akt) on insulin stimulation decreased in myotubes with exposure to subtoxic concentrations of DDT, but there was no change in tyrosine phosphorylation level of insulin receptors. We conclude that subtoxic DDT exposure impairs insulin signaling and thereby induces insulin resistance in muscle cells. Data show that oxidative stress-induced activation of RSKs is responsible for impairment of insulin signaling on DDT exposure.

Evaluating NISCH and CDH1 Promoter Hypermethylation in Nonsmokers, Cancer Free Smokers and Lung Cancer Patients: A Case Control Study.

Lung cancer has very high mortality due to late stage diagnosis not amenable to curative resection. Cancer specific methylation patterns of tumor suppressor genes may precede precursor lesions of lung cancer. Our aim was to evaluate the promoter hypermethylation of tumor suppressor gene NISCH and CDH1 in cfDNA from plasma of lung cancer patients and its possible correlation with smoking status and various clinicopathological parameters. Forty histopathologically confirmed lung cancer cases, thirty smoker and thirty nonsmoker controls were enrolled. Plasma cfDNA was extracted and subjected to bisulfite treatment followed by MS-PCR. Serum nischarin levels were estimated by ELISA. The frequency of promoter hypermethylation of NISCH and CDH1 was significantly higher in lung cancer patients as compared to lifelong non-smoker controls (p < 0.05). It did not vary with smoking status among cancer cases. No significant association was found with staging or histological grading. NISCH methylation was found to be significantly higher among smoker controls. Pack years and packs per day were significantly higher in the methylated group. Serum nischarin levels showed no significant association with NISCH methylation or clinicopathological variables. NISCH is highly methylated in both high risk smoker controls as well as cancerousnon-smokers and may mark the convergence of varied etiologies of lung cancer. Hence NISCH and CDH1 are highly methylated in plasma cfDNA of lung cancer patients.

Clinical Importance of Estrogen Receptor 1 (ESR1) Gene Polymorphisms and Their Expression Patterns in Coronary Artery Disease Patients: A Study from India.

The influence of Estrogen Receptor 1 (ESR1) gene -397T>C (PvuII) and -351A>G (XbaI) polymorphisms on the risk of development of coronary artery disease (CAD) in the north Indian population was analysed. We hypothesized that ESR1 gene polymorphisms may influence the susceptibility to CAD through variation in Estrogen Receptor α (ERα) expression. To assess this concept, we evaluated ERα mRNA expression in blood plasma of CAD patients. The study included hundred CAD patients who showed presence of greater than 50% luminal stenosis in at least one major coronary artery in angiography along with hundred age and sex matched healthy controls. The ESR1 polymorphisms were investigated by PCR-RFLP. Quantitative Real Time PCR was carried out for the measurement of ERα mRNA expression. The results showed that genotypic frequencies of ESR1 -397T>C and -351A>G gene polymorphisms were significantly higher in CAD patients than control subjects (p < 0.0001). A significantly increased CAD risk was also found in dominant and codominant inheritance model for both of the SNPs. In gender based analysis these findings were replicated only in male subgroup. In case of -397T>C polymorphism, the ERα mRNA expression was highest in CAD patients with wild type homozygous TT genotype (2-∆ct = 0.28). A mutant 'C' allele, dose dependent, significant decrease in trend in ERα mRNA expression was observed, with lowest expression in mutant homozygous CC genotype (2-∆ct = 0.09), and intermediate expression level in heterozygous TC genotype (2-∆ct = 0.14) subgroups of CAD patients. In conclusion, this study demonstrates a significantly heightened risk of CAD associated with the inheritance of mutant genotypes of ESR1 -397T>C and -351A>G gene polymorphisms, in the north Indian population. This is the first report of a lowered ERα mRNA expression in conjunction with the presence of mutant 'C' allele of ESR1 -397T>C polymorphism with consequent increased CAD susceptibility.

Reduced intestinal epithelial mitochondrial function enhances in vitro interleukin-8 production in response to commensal Escherichia coli.

Uncoupling of oxidative phosphorylation in epithelial mitochondria results in decreased epithelial barrier function as characterized by increased internalization of non-invasive Escherichia coli and their translocation across the epithelium. We hypothesized that the increased burden of intracellular commensal bacteria would activate the enterocyte, with the potential to promote inflammation. Treatment of human colon-derived epithelial cell lines in vitro with dinitrophenol (DNP) and commensal E. coli (strains F18, HB101) provoked increased production of interleukin (IL-8), which was not observed with conditioned medium from the bacteria, lipopolysaccharide or inert beads. The IL-8 response was inhibited by co-treatment with cytochalasin-D (blocks F-actin rearrangement), chloroquine (blocks phagosome acidification) and a MyD88 inhibitor (blocks TLR signaling), consistent with TLR-signaling mediating IL-8 synthesis subsequent to bacterial internalization. Use of the mitochondria-targeted antioxidant, mitoTEMPO, or U0126 to block ERK1/2 MAPK signalling inhibited DNP+E. coli-evoked IL-8 production. Mutations in the NOD2 (the intracellular sensor of bacteria) or ATG16L1 (autophagy protein) genes are susceptibility traits for Crohn's, and epithelia lacking either protein displayed enhanced IL-8 production in comparison to wild-type cells when exposed to DNP + E coli. Thus, metabolic stress perturbs the normal epithelial-bacterial interaction resulting in increased IL-8 production due to uptake of bacteria into the enterocyte: this potentially pro-inflammatory event is enhanced in cells lacking NOD2 or ATG16L1 that favor increased survival of bacteria within the enterocyte. We speculate that by increasing epithelial permeability and IL-8 production, reduced mitochondria function in the enteric epithelium would contribute to the initiation, pathophysiology, and reactivation of inflammatory disease in the gut.

Absence of the NOD2 protein renders epithelia more susceptible to barrier dysfunction due to mitochondrial dysfunction.

Irregular mitochondria structure and reduced ATP in some patients with IBD suggest that metabolic stress contributes to disease. Loss-of-function mutation in the nucleotide-binding oligomerization domain (NOD)-2 gene is a major susceptibility trait for IBD. Hence, we assessed if loss of NOD2 further impairs the epithelial barrier function instigated by disruption of mitochondrial ATP synthesis via the hydrogen ionophore dinitrophenol (DNP). NOD2 protein (virtually undetectable in epithelia under basal conditions) was increased in T84 (human colon cell line) cells treated with noninvasive Escherichia coli + DNP (16 h). Increased intracellular bacteria in wild-type (WT) and NOD2 knockdown (KD) cells and colonoids from NOD2-/- mice were mediated by reactive oxygen species (ROS) and the MAPK ERK1/2 pathways as determined by cotreatment with the antioxidant mitoTEMPO and the ERK inhibitor U0126: ROS was upstream of ERK1/2 activation. Despite increased E. coli in DNP-treated NOD2 KD compared with WT cells, there were no differences in the internalization of fluorescent inert beads or dead E. coli particles. This suggests that lack of killing in the NOD2 KD cells was responsible for the increased numbers of viable intracellular bacteria; a conclusion supported by evidence of reduced autophagy in NOD2 KD T84 epithelia. Thus, in a two-hit hypothesis, decreased barrier function due to dysfunctional mitochondrial is amplified by lack of NOD2 in transporting enterocytes: subsequently, greater numbers of bacteria entering the mucosa would be a significant inflammatory threat especially since individuals with NOD2 mutations have compromised macrophage and Paneth cell responses to bacteria.NEW & NOTEWORTHY Increased internalization of bacteria by epithelia with dysfunctional mitochondria (reduced ATP) is potentiated if the cells lack nucleotide-binding oligomerization domain 2 (NOD2), mutations in which are inflammatory bowel disease-susceptibility traits. Uptake of bacteria was dependent on reactive oxygen species and MAP-kinase activity, and the increased viable intracellular bacteria in NOD2-/- cells likely reflect a reduced ability to recognize and kill bacteria. Thus a significant barrier defect occurs with NOD2 deficiency in conjunction with metabolic stress that could contribute to inflammation.

PDGFRα promoter polymorphisms and expression patterns influence risk of development of imatinib-induced thrombocytopenia in chronic myeloid leukemia: A study from India.

Platelet-derived growth factor receptor has been implicated in many malignant and non-malignant diseases. Platelet-derived growth factor receptor-α is a tyrosine kinase and a side target for imatinib, a revolutionary drug for the treatment of chronic myeloid leukemia that has dramatically improved the survival of chronic myeloid leukemia patients. Given the importance of platelet-derived growth factor receptor in platelet development and its inhibition by imatinib, it was intriguing to analyze the role of platelet-derived growth factor receptor-α in relation to imatinib treatment in the development of imatinib-induced thrombocytopenia in chronic myeloid leukemia patients. We hypothesized that two known functional polymorphisms, +68GA insertion/deletion and -909C/A, in the promoter region of the platelet-derived growth factor receptor-α gene may affect the susceptibility of chronic myeloid leukemia patients receiving imatinib treatment to the development of thrombocytopenia. A case-control study was conducted among a cohort of chronic myeloid leukemia patients admitted to the Lok Nayak Hospital, New Delhi, India. A set of 100 patients of chronic myeloid leukemia in chronic phase and 100 age- and sex-matched healthy controls were studied. After initiation of imatinib treatment, the hematological response of chronic myeloid leukemia patients was monitored regularly for 2 years, in which the development of thrombocytopenia was the primary end point. Platelet-derived growth factor receptor-α promoter polymorphisms +68GA ins/del and -909C/A were studied by allele-specific polymerase chain reaction. Platelet-derived growth factor receptor-α messenger RNA expression was evaluated by quantitative real-time polymerase chain reaction. The messenger RNA expression results were expressed as 2-Δct ± standard deviation. The distribution of +68GA ins/del promoter polymorphism genotypes differed significantly between the thrombocytopenic and non-thrombocytopenic chronic myeloid leukemia patient groups (p < 0.0001). Moreover, +68GA del/del and ins/del genotypes in imatinib-treated chronic myeloid leukemia patients were associated with an increased risk of developing thrombocytopenia, with odds ratios 6.5 (95% confidence interval = 2.02-0.89, p = 0.001) and 6.0 (95% confidence interval = 2.26-15.91, p = 0.0002), respectively. Similarly, -909C/A promoter polymorphism genotype distribution also differed significantly between thrombocytopenic and non-thrombocytopenic chronic myeloid leukemia patient groups (p = 0.02), and a significantly increased risk of imatinib-induced thrombocytopenia was associated with -909C/A polymorphism mutant homozygous (AA) genotypes the odds ratio being 7.7 (95% confidence interval 1.50 to 39.91, p = 0.009). However, no significant risk of imatinib-induced thrombocytopenia was found to be associated with heterozygous genotype (-909C/A) with odds ratio 1.9 (95% confidence interval = 0.86-4.56, p = 1.14). Platelet-derived growth factor receptor-α messenger RNA expression was significantly higher in chronic myeloid leukemia patients compared to controls (p = 0.008). Moreover, patients with imatinib-induced thrombocytopenia had a significantly lower platelet-derived growth factor receptor-α messenger RNA expression, compared to patients without thrombocytopenia (p = 0.01). A differential expression of platelet-derived growth factor receptor-α messenger RNA was observed with respect to different +68 GA ins/del and -909C/A polymorphism genotypes. The +68GA deletion allele and -909A allele were significantly associated with lower expression of platelet-derived growth factor receptor-α messenger RNA. The platelet-derived growth factor receptor-α +68GA del/del, +68GA ins/del, and -909AA genotypes are associated with an increased risk of developing thrombocytopenia in imatinib-treated chronic myeloid leukemia patients. A significantly lower platelet-derived growth factor receptor-α messenger RNA expression accompanies the +68GA deletion allele in an allele dose-dependent manner. Platelet-derived growth factor receptor-α -909AA genotype is also associated with lower expression of platelet-derived growth factor receptor-α. The downregulation of platelet-derived growth factor receptor-α expression may play a causative role in imatinib-induced thrombocytopenia, a common side effect, in the subset of chronic myeloid leukemia patients with platelet-derived growth factor receptor-α +68 GA ins/del, +68 GA del/del, and -909C/A genotypes.

Butyrate enhances antibacterial effects while suppressing other features of alternative activation in IL-4-induced macrophages.

The short-chain fatty acid butyrate is produced by fermentation of dietary fiber by the intestinal microbiota; butyrate is the primary energy source of colonocytes and has immunomodulatory effects. Having shown that macrophages differentiated with IL-4 [M(IL-4)s] can suppress colitis, we hypothesized that butyrate would reinforce an M(IL-4) phenotype. Here, we show that in the presence of butyrate M(IL-4)s display reduced expression of their hallmark markers Arg1 and Ym1 and significantly suppressed LPS-induced nitric oxide, IL-12p40, and IL-10 production. Butyrate treatment likely altered the M(IL-4) phenotype via inhibition of histone deacetylation. Functionally, M(IL-4)s treated with butyrate showed increased phagocytosis and killing of bacteria, compared with M(IL-4) and this was not accompanied by enhanced proinflammatory cytokine production. Culture of regulatory T cells with M(IL-4)s and M(IL-4 + butyrate)s revealed that both macrophage subsets suppressed expression of the regulatory T-cell marker Foxp3. However, Tregs cocultured with M(IL-4 + butyrate) produced less IL-17A than Tregs cocultured with M(IL-4). These data illustrate the importance of butyrate, a microbial-derived metabolite, in the regulation of gut immunity: the demonstration that butyrate promotes phagocytosis in M(IL-4)s that can limit T-cell production of IL-17A reveals novel aspects of bacterial-host interaction in the regulation of intestinal homeostasis.

Involvement of CASP3 promoter polymorphism (-1337 C > G) in the development and progression of non-small cell lung cancer.

Downregulation of CASP3 gene expression has been observed to be associated with various malignancies, and promoter polymorphisms in the CASP3 gene may have a great impact on the CASP3 transcriptional activity. The present study aimed to analyze the possible impact of the CASP3 (-1337 C > G, rs1405937) polymorphism on the expression profile of CASP3 gene and ultimately its association in the development of non-small cell lung cancer. A case-control study of 100 non-small cell lung cancer patients and 100 cancer free healthy controls was conducted, wherein genotype and expression profile of CASP3 gene were evaluated using serum DNA and serum RNA, respectively, by primer-introduced restriction fragment analysis and real-time PCR techniques. Compared to the CASP3 CC genotype, odds ratio of 11.1 was found to be associated to the homozygous GG genotype with more than sixfold decrease of CASP3 gene expression in non-small cell lung cancer patients. Significant trend of decrease in caspase 3 expression was observed with the increase in severity of the disease. Patients with CASP3 (-1337GG) genotype had significantly shorter overall survival compared to CASP3 (-1337CC) genotype carriers. In addition, significantly poor overall survival was also reflected by patients with higher fold decrease in CASP3 gene expression. CASP3 (-1337 GG) genotype was found to be associated with significantly lower CASP3 gene expression especially among patients with advanced status of the disease, suggesting that CASP3 (-1337C > G) polymorphism may be involved in the development and progression of non-small cell lung cancer.

The conglomeration of diagnostic, prognostic and therapeutic potential of serum miR-199a and its association with clinicopathological features in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal cause of morbidity and mortality worldwide. miRNA deregulation evinces a remarkable role in ovarian cancer tumorigenesis. miRNA-199a (miR-199a) is known to be involved in cancer development and progression. Although miR-199a has been studied in various cell types, its correlation with clinicopathological features in EOC has not been documented. In this study, we identified the clinicopathological hallmarks which might be perturbed due to the downregulation of serum miR-199a in EOC. Seventy serum samples from histopathologically confirmed EOC patients and 70 controls were collected. Total RNA from serum was isolated by Trizol method, polyadenylated and reverse transcribed into cDNA. Expression level of miR-199a was detected by using miRNA qRT-PCR. Relative expression was determined with matched controls using U6 snRNA as reference. Level of miR-199a expression was compared with distinct clinicopathological features. Expression of miR-199a was found to be significantly downregulated in comparison with matched normal controls. The expression level of miR-199a was found to be significantly associated with tumor stage, lymph node metastasis, and distal metastasis. Receiver operating characteristic (ROC) curve for diagnostic potential yielded significant area under the curve (AUC) with a considerable sensitivity and specificity. ROC curves for prognosis yielded significant AUCs for histological grade, distal metastasis, lymph node status, and survival. Our findings suggest that miR-199a downregulation might be a potential indicator for disease progression promoting the aggressive tumor progression and be identified as a diagnostic marker to predict the prognosis and survival in EOC patients.

Prognostic significance of serum ERBB3 and ERBB4 mRNA in lung adenocarcinoma patients.

Serum messenger RNA (mRNA) is an emerging prognostic tool for noninvasive malignant disease prognosis, and to study serum mRNA may have importance in the prognosis and detection of disease. This study aimed to evaluate the possible prognostic role of serum ERBB3 and ERBB4 mRNA expressions in lung adenocarcinoma patients. One hundred newly diagnosed lung adenocarcinoma patients and 100 age- and sex-matched healthy controls were included. Expression was analysed by quantitative real-time PCR and overall survival was analysed by Kaplan-Meier analysis. Serum ERBB3 and ERBB4 mRNA expressions was found to be significantly associated with distant metastases and TNM stages. It was observed that patients with distant metastases had 4.8- and 3.4-fold high ERBB3 and ERBB4 expression in contrast to patients without distant metastases, respectively. It was also found that ERBB3 and ERBB4 mRNA expression was 7.7-fold and 6.7-fold high in TNM stage IV compared to TNM stage I, respectively. Significantly, 2.6-fold increased serum ERBB4 mRNA expression was found in patients with pleural effusion compared to patients without pleural effusion (p = 0.005). Lung adenocarcinoma patients with ≤8- and >8-fold increased serum ERBB3 mRNA expression had 10.0 and 5.5 months of overall median survival while serum ERBB4 mRNA with ≤10- and >10-fold increased expression showed 11.4 and 5.0 months overall median survival, respectively. ERBB3 and ERBB4 together also found to be significantly associated with poor overall median survival. Patients with ≤8 + ≤10- and >8 + >10-fold expression showed 11.3 vs 4.8 months of overall median survival, respectively. In conclusion, serum ERBB3 and ERBB4 mRNA expressions may be a prognostic marker and monitoring of serum ERBB3 and ERBB4 mRNA can be one of the predictive factors for metastases and poor overall survival of lung adenocarcinoma patients.

Serum microRNA-21 expression as a prognostic and therapeutic biomarker for breast cancer patients.

MiRNA-21 is recognized as the main active candidate and high expression in many solid tumors consequential cell proliferation, differentiation, apoptosis, and closely related to metastasis of disease. The study aimed to evaluate the serum miRNA-21 expression and therapy outcome in breast cancer patients and cell lines. Seventy-five histopathologically confirmed newly diagnosed breast cancer patients were included in the study; before and after therapy, patient's blood sample were collected and analyzed for serum microRNA-21 expression by quantitative real-time PCR. In patients, 8.9 mean fold increased microRNA-21 expression was observed compared to controls. Increased expression was found to be associated with advanced stage (11.72-fold), lymph node involvement (11.12-fold), and distant metastases (20.17-fold). After treatment significant decrease in miRNA-21 expression was observed and found to be significant (p < 0.0001). Patients treated with neoadjuvant therapy had significant impact on miRNA-21 suppression and found to be significantly associated with different clinicopathological features of patients. Increased miRNA-21 expression was also found to be significantly associated with poor survival of breast cancer patients (p = 0.002). MicroRNA-21 expression could be used as promising predictive indicators for breast cancer prognosis. MicroRNA-21 over-expression was associated with response to neoadjuvant therapy and may perhaps be considered as primary treatment choice.

Maternal and neonatal vitamin-D status in twin versus singleton pregnancies.

AIM: There is a paucity of information on vitamin D status of women with twin pregnancy and their newborns. This case-control study compared maternal and neonatal vitamin-D status in twin versus singleton pregnancies. METHODS: Subjects included 50 women with twin pregnancy delivering at >28 weeks and 50 gestational-age-matched women with singleton pregnancy delivering during the same period. Maternal and neonatal serum 25-hydroxy vitamin D [25(OH)D] was compared between the two groups using the independent Student's t-test on log values. Serum albumin-adjusted calcium, inorganic phosphate, and intact parathormone levels were also compared. RESULTS: Maternal vitamin-D deficiency (VDD; serum 25(OH)D < 30 nmol/L) was present in 90% of twin and 88% of singleton pregnancies. The prevalence of neonatal VDD was 89% in twin and 74% in singleton pregnancies (P = 0.03). Maternal serum 25(OH)D was lower in the twin group as compared to the singleton group (14.3 ± 10.47 vs 18.5 ± 12.36 nmol/L; P = 0.02). Mean serum calcium, intact parathormone, and inorganic phosphate were comparable between the women in the two groups. Maternal and neonatal 25(OH)D showed positive correlation in the two groups (P < 0.001). Mean cord blood 25(OH)D was significantly lower in the twins than in singleton newborns (14.8 ± 12.63 vs 22.6 ± 16.68 nmol/L; P = 0.002). The difference persisted even after adjustment for birthweights and maternal serum 25(OH)D. Mean serum calcium was significantly lower in the twins. CONCLUSION: Twin newborns and their mothers have higher VDD as compared to singleton newborns and their mothers in the VDD population.

Extracellular cytochrome c as a biomarker for monitoring therapeutic efficacy and prognosis of non-small cell lung cancer patients.

Non-small cell lung cancer has a devastating prognosis, and markers enabling a precise prediction of therapy response have long remained scarce. Better treatment monitoring would allow an individual's more effective patient adjusted therapy with lesser side effects and good clinical outcomes. In the present study, we monitored the serum cytochrome c levels pre- and post-chemotherapy of non-small cell lung cancer patients. Using highly sensitive enzyme-linked immunosorbent assay, we evaluated cytochrome c levels in serum of 100 non-small cell lung cancer and 100 healthy controls. We observed about threefold lower serum cytochrome c level in newly diagnosed non-small cell lung cancer patients than healthy individuals. Patients in advanced stages and grade 3 histological differentiation showed significantly low level of serum cytochrome c, and the lower levels were associated with worse survival outcome of non-small cell lung cancer patients. In addition, serum cytochrome c level was observed to be more than 13-fold higher after first cycle of conventional chemotherapy, wherein patients with higher level of serum cytochrome c before any therapy showed better response to chemotherapy in terms of significantly higher level of serum cytochrome c after first cycle of chemotherapy than patients with low level of serum cytochrome c at the time of diagnosis. Detection of serum cytochrome c levels at the time of diagnosis may be useful in suggesting disease severity and prognosis of the non-small cell lung cancer patients. Monitoring of serum cytochrome c might also serve as a sensitive apoptotic marker in vivo reflecting chemotherapy-induced cell death burden in patients with non-small cell lung cancer.

Association of p53 and mdm2 in the development and progression of non-small cell lung cancer.

MDM2 protein is an important regulator of the p53 pathway and has a large effect on the anti-tumorigenic activity of the p53. Presently, we aimed to analyze the possible association of p53 and mdm2 in the development and progression of non-small cell lung cancer. In addition, impact of an important gene promoter polymorphism of MDM2 (T309G, rs 2279744) on its gene expression and ultimately of the TP53 was investigated in non-small cell lung cancer patients. A case-control study using peripheral blood samples of 100 non-small cell lung cancer patients and 100 cancer free healthy controls was conducted. Expression profile of MDM2 and TP53 gene were evaluated by using quantitative real time polymerase chain reaction assay, and MDM2 promoter polymorphism were analyzed by amplification refractory mutation system polymerase chain reaction. Non-small cell lung cancer patients expressed more than 6-fold increased mdm2 and about 7-folds decreased in p53 expression levels compared to healthy controls. Higher fold change increase of mdm2 and/or decrease of p53 were associated with advanced status and poor clinical outcome of the patients. A significant increase in mdm2 of about 14-folds and decrease in p53 of about 16.5-fold were observed among patients with MDM2 (309GG) genotype vs just 2.2-fold increase in mdm2 and 1.9-fold decrease in p53 among patients with MDM2 (309TT) genotype. In conclusion, present study demonstrated that MDM2 (309 T > G) polymorphism may be one of the important factors for the increased expression mdm2, which was associated with down-regulation of p53 at messenger RNA (mRNA) level and ultimately may contribute in the poor clinical outcome of the non-small cell lung cancer patients, thus may prove as a promising target for the treatment of non-small cell lung cancer at molecular level.

Role of survivin re-expression in the development and progression of non-small cell lung cancer.

Survivin is highly expressed in fetal tissue and is completely absent in terminally differentiated cells, but its re-expression has been observed in most human tumors. Presently, we aimed to analyze the possible impact of the survivin gene (-31G > C, rs 9904341) promoter polymorphism on the expression profile of survivin gene and ultimately the role of survivin re-expression in the development and progression of non-small cell lung cancer. A case-control study of 100 non-small cell lung cancer patients and 100 cancer-free healthy controls was conducted. Survivin gene promoter polymorphism was analyzed by PCR-restriction fragment length polymorphisms (RFLP) technique, and the survivin expression profile was evaluated using quantitative real-time PCR assay. Compared to the survivin GG genotype, odd ratio of 3.2 (95 % CI 4.8-25.9, p = 0.004) was found to be associated to homozygous CC genotype with 15-fold increase of survivin gene expression in non-small cell lung cancer patients. Significant trend of increase in survivin expression was observed with the increase in severity of the disease. Patients with survivin (-31CC) genotype had significantly shorter overall survival compared to survivin (-31GG) genotype carriers. In addition, advanced disease status and significant poor overall survival were also reflected by patients with higher-fold increase in survivin gene expression. In conclusion, present study demonstrated that survivin (-31G > C) polymorphism may contribute to the risk of developing non-small cell lung cancer in Indian population. Survivin (-31CC) genotype was associated with significantly increased survivin gene expression and ultimately may contribute in the poor clinical outcome of non-small cell lung cancer patients, suggesting its possible significance in the development and progression of non-small cell lung cancer.

Ochronosis of the hip joint: differential diagnosis of inflammatory arthropathy with lytic lesion.

Alkaptonuria or ochronosis is a rare inborn disorder of metabolism which is characterized by deficiency of homogentisic acid oxidase. There is accumulation of homogentisic acid in the connective tissues causing brownish black pigmentation and skeletal damage. The most serious complication of this disease is crippling degenerative arthropathy which presents in late years of life. There is no definitive treatment for the condition. Symptomatic management is the main stay. Surgical management such as arthroplasty is done for severe cases of ochronosis. We report a case of ochronosis of the hip joint presenting with inflammation and lytic lesion which can create confusion and lead to error in diagnosis.