Anti-lung Cancer Activity of Synthesized Substituted 1,4-Benzothiazines: An Insight from Molecular Docking and Experimental Studies.

BACKGROUND: Thiazine, a 6-membered distinctive heterocyclic motif with sulfur and nitrogen atoms, is one of the heterocyclic compounds that functions as a core scaffold in a number of medicinally significant molecules. Small thiazine-based compounds may operate simultaneously on numerous therapeutic targets and by employing a variety of methods to halt the development, proliferation, and vasculature of cancer cells. We have, herein, reported a series of substituted 1,4 benzothiazines as potential anticancer agents for the treatment of lung cancer. METHODS: In order to synthesize 2,3-disubstituted-1,4 benzothiazines in good yield, a facile green approach for the oxidative cycloaddition of 2-amino benzenethiol and 1,3-dicarbonyls employing a catalytic amount of ceric ammonium nitrate has been devised. All the molecules have been characterized by spectral analysis and tested for anticancer activity against the A-549 lung cancer cell line using various functional assays. Further in silico screening of compound 3c against six crucial inflammatory molecular targets, such as Il1-α (PDB ID: 5UC6), Il1- ß (PDB ID: 6Y8I), Il6 (PDB ID: 1P9M), vimentin (PDB ID: 3TRT), COX-2 (PDB ID: 5KIR), Il8 (PDB ID: 5D14), and TNF-α (PDB ID: 2AZ5), was done using AutoDock tool. RESULTS: Among the synthesized compounds, propyl 3-methyl-3,4-dihydro-2H-benzo[b][1,4]thiazine-2- carboxylate (3c) was found to be most active based on cell viability assays using A-549 lung cancer cell line and was found to effectively downregulate various pro-inflammatory genes, like Il1-α, Il1-ß, Il6, vimentin, COX-2, Il8, and TNF-α in vitro. The ability of the molecule to effectively suppress the proliferation and migration of lung cancer cells in vitro has been further demonstrated by the colony formation unit assay and wound healing assay. Molecular docking analysis showed the maximal binding affinity (- 7.54 kcal/mol) to be exhibited by compound 3c against IL8. CONCLUSION: A green unconventional route for the synthesis of 2,3-disubstituted-1,4 benzothiazines has been developed. All the molecules were screened for their activity against lung cancer and the data suggested that the presence of an additional unbranched alkyl group attached to the thiazine ring increased their activity. Also, in vitro and in silico modeling confirmed the anti-cancer efficiency of compound 3c, encouraging the exploration of such small molecules against cancer.

Investigating dysregulation of TGF-ß1/SMAD3 signaling in Atopic Dermatitis: A Molecular and Immunohistochemical Analysis".

Atopic Dermatitis (AD) is a persistent and recurring inflammatory condition affecting the skin. An expanding corpus of evidence indicates the potential participation of TGF-ß1 in the modulation of inflammation and tissue remodeling in AD. The primary objective of this study was to examine the aberrant modulation of TGF-ß1/SMAD3 signaling through a comprehensive analysis of their molecular and protein expression profiles. The study encompassed an aggregate of 37 participants, which included 25 AD patients and 12 controls. The assessment of mRNA and protein levels of TGF-ß1 and SMAD3 was conducted utilizing quantitative real-time PCR and immunohistochemistry, whereas serum IgE and vitamin D levels were estimated by ELISA and chemiluminescence, respectively. Quantitative analysis demonstrated a 2.5-fold upregulation of TGF-ß1 mRNA expression in the lesional AD skin (p<0.0001). Immunohistochemistry also exhibited a comparable augmented pattern, characterized by moderate to strong staining intensities. In addition, TGF-ß1 mRNA showed an association with vitamin D deficiency in serum (p<0.02), and its protein expression was linked with the disease severity (p<0.01) Furthermore, a significant decrease in the expression of the SMAD3 gene was observed in the affected skin (p = 0.0004). This finding was further confirmed by evaluating the protein expression and phosphorylation of SMAD3, both of which exhibited a decrease. These findings suggest that there is a dysregulation in the TGF-ß1/SMAD3 signaling pathway in AD. Furthermore, the observed augmentation in mRNA and protein expression of TGF-ß1, along with its correlation with the disease severity, holds considerable clinical significance and emphasizes its potential role in AD pathogenesis.

Analysis of intronic SNP (rs4147358) and expression of SMAD3 gene in Atopic Dermatitis: A case-control study.

BACKGROUND: Atopic Dermatitis (AD) is a multifactorial cutaneous disorder associated with chronic inflammation of the skin. Growing evidence points to TGF-ß/SMAD signaling as a key player in mediating inflammation and the subsequent tissue remodeling, often resulting in fibrosis. This study investigates the role of a core transcription factor involved in TGF-ß signaling i.e., SMAD3 genetic variants (rs4147358) in AD predisposition and its association with SMAD3 mRNA expression, serum IgE levels, and sensitization to various allergens in AD patients. METHODS: A total of 246 subjects including 134 AD cases and 112 matched healthy controls were genotyped for SMAD3 intronic SNP by PCR-RFLP. mRNA expression of SMAD3 was determined by quantitative Real-Time PCR (qRT-PCR), Vitamin-D levels by chemiluminescence, and total serum IgE levels by ELISA. In-vivo allergy testing was performed for the evaluation of allergic reactions to house dust mites (HDM) and food allergens. RESULTS: A significantly higher frequency of mutant genotype AA (cases: 19.4% vs controls: 8.9%) (OR = 2.8, CI = 1.2 - 6.7, p = 0.01) was observed in AD cases. The mutant allele 'A' also showed a 1.9-fold higher risk for AD compared to the wild allele 'C' indicating that the carriers of the A allele have a higher risk for AD predisposition (OR-1.9, CI = 1.3-2.8, p < 0.001). In addition, quantitative analysis of SMAD3 mRNA in peripheral blood showed 2.8-fold increased expression in AD cases as compared to healthy controls. Stratification analysis revealed the association of the mutant AA genotype with deficient serum Vitamin D levels (p = 0.02) and SMAD3 mRNA overexpression with HDM sensitization (p = 0.03). Furthermore, no significant association of genotypes with SMAD3 mRNA expression was observed. CONCLUSION: Our study indicates that SMAD3 intronic SNP bears a significant risk of AD development. Moreover, overexpression of SMAD3 mRNA and its association with HDM sensitization highlights the possible role of this gene in AD pathogenesis.

Unveiling the TGF- ß1 paradox: Significant implication of TGF- ß1 promoter variants and its mRNA and protein expression in atopic dermatitis.

BACKGROUND: Atopic Dermatitis (AD) is a chronic inflammatory skin disorder with evidence of lichenification in later stages. There is mounting evidence supporting the role of TGF- ß1 in mediating inflammation as well as subsequent tissue remodeling, often resulting in fibrosis. Given the role of genetic variants in the differential expression of TGF-ß1 in various diseases, this study seeks to ascertain the role of TGF-ß1 promoter variants (rs1800469 and rs1800468) in AD susceptibility, as well as their association with TGF- ß1 mRNA expression, TGF- ß1 serum levels and skin prick test positivity in Atopic Dermatitis patients. METHODS: An aggregate of 246 subjects including 134 AD cases and 112 matched healthy controls were genotyped for TGF-ß1 promoter polymorphisms by PCR-RFLP. TGF- ß1 mRNA was quantified by quantitative Real-Time PCR (qRT-PCR), Vitamin-D levels by chemiluminescence, and serum TGF- ß1, and total IgE levels were determined by ELISA. In-vivo allergy testing was performed for the evaluation of allergic reactions to house dust mites and food allergens. RESULTS: A higher frequency of TT genotypes of rs1800469 (OR = 7.7, p = 0.0001) and GA+AA genotypes of rs1800468 (OR-4.4, p < 0.0001) were observed in AD cases than those in controls. Haplotype analysis demonstrated that TG haplotype carriers had an increased risk of AD (p = 0.013). Quantitative analysis revealed a significant upregulation of both mRNA (p = 0.0002) and serum levels (p < 0.0001) of TGF- ß1 with a substantial positive correlation between them (Correlation coefficient=0.504; p = 0.01). Moreover, serum TGF-ß1 levels were associated with quality of life (p = 0.03), the severity of the disease (p = 0.03), and House dust mite allergy (p = 0.01) whereas TGF-ß1 mRNA levels positively correlated with disease severity(p = 0.02). Stratification analysis revealed that the TT genotype of rs1800469 was associated with higher IgE levels (p = 0.01) and eosinophil percentage(p = 0.007) whereas the AA genotype of rs1800468 correlated with elevated serum IgE levels (p = 0.01). Besides, no significant association of genotypes with mRNA and serum expression of TGF-ß1 was observed. CONCLUSION: Our study indicates that TGF-ß1 promoter SNPs bear a significant risk of AD development. Moreover, upregulation of TGF-ß1 mRNA and serum levels and their association with disease severity, quality of life, and HDM allergy suggests its role as a diagnostic/prognostic biomarker that could help in the development of new therapeutic and prevention strategies.

Gene variants and mRNA expression analysis of SOCS3 and its association with serum IL-4 levels in atopic diseases.

BACKGROUND: The suppressors of cytokine signaling (SOCS) are a class of negative regulators for several aspects of cytokine signaling that have been attributed to the pathophysiology of inflammatory disorders. Given the role of the SOCS3 gene in regulating Th2 cell proliferation, our study aimed to analyze two SOCS3 SNPs viz. rs8074003 and rs7222391, and their potential influence on IL-4 levels and SOCS3 mRNA expression besides analyzing the interaction of the SOCS3 genotypes with the various clinicopathological parameters. METHODS: A total of 314 subjects including 154 atopic cases and 160 healthy controls were genotyped for SOCS3 polymorphisms by PCR-RFLP. SOCS3 mRNA was quantified by Real-Time PCR. The serum IL-4 and total IgE levels were determined by ELISA and Vitamin-D levels were quantified by chemiluminescence. RESULTS: The CC genotype of rs8074003 was more frequent in atopic cases and posed a 3- fold risk of atopy (p = 0.001) whereas CG and GG genotypes were widespread in the controls (p = 0.1). For the other SNP rs7222391, there was no difference in genotypic and allelic distribution. The SOCS3 mRNA expression and serum IL-4 levels were substantially increased in the atopic cases with a significant positive correlation between them (p < 0.05). CONCLUSION: SOCS3 SNP rs8074003 poses a convincing risk of atopic disease development. The SOCS3 expression and IL-4 levels were up-regulated in total atopy and in its different presentations. It seems plausible to target SOCS3 and IL-4 as a potential target for the diagnosis of atopy and for the development of reliable personalized therapeutic strategies to control atopic conditions.

Association of epidermal differentiation complex (EDC) genetic variants with House Dust Mite sensitization in Atopic Dermatitis Patients.

The etiopathogenesis of AD is multifactorial and defects of the skin barrier, which physiologically constitutes the natural protection, are associated with the disease phenotype. The identification of the genetic and environmental factors paving the way for impaired barrier function is therefore important in developing new therapeutic and prevention strategies. MATERIAL AND METHODS: Confirmed 100 cases were tested against 106 controls for filaggrin mutation and LELP-1 polymorphism by PCR-RFLP and chain termination sequencing. Total IgE and Vitamin D were estimated by ELISA. House dust mite sensitization was assessed by an in-vivo skin prick test. RESULTS: FLG deletion (2282del4) was present in 4% of the patients and all these were heterozygous carriers, whereas FLG null mutation (R501X) was not present in any of the cases. In the control group, both the mutations were not found. CT genotype and T allele of LELP-1 (rs7534334) were significantly associated with elevated IgE levels, early-onset, HDM sensitization, and disease severity (P < 0.05). However, the genotypic and allelic distribution of LELP-1 among the cases and controls was found to be insignificant. CONCLUSION: The low frequency of 2282del4 deletion and the absence of R501X mutation suggest that filaggrin deficiency does not confer a major risk for AD in the Indian population. However, significant association of LELP-1 (rs7534334) variant allele with clinical variables may serve as a novel biomarker for the severity of Atopic Dermatitis as well as an indicator for the allergen-specific immunotherapy and hence bears important clinical implications and needs to study on larger sample size and diverse populations.

Oral contraceptive use increases risk of inflammatory and coagulatory disorders in women with Polycystic Ovarian Syndrome: An observational study.

Polycystic ovarian syndrome (PCOS) is a multispectral disorder requiring lifelong management. Its pathophysiology is still being explored which makes its treatment options restrained. Present study explores impact of oral contraceptive mode of treatment on metabolic, hormonal, inflammation and coagulation profile of PCOS women. 50 subjects diagnosed with Rotterdam criteria receiving no drug treatment served as controls whereas 50 subjects receiving only OCPs (Ethinyl estradiol 0.03 mg, Levonorgestrel 0.15 mg) as a mode of treatment at least for six-months served as cases. Ferriman-Gallwey score and hormonal profile improved on OCP treatment. However, parameters like weight, Body mass index, waist-hip ratio, Oral glucose tolerance test, lipid profile, insulin, HOMA-IR, adiponectin, interleukin1ß, visfatin, resistin, tissue factor, PT and APTT showed considerable derangements in OCP group. All above parameters are associated with the risk of diabetes mellitus, dyslipidemia, coronary vascular disease, cancers, hypercoagulable state, venous thromboembolism and thrombotic events. Long-term use of OCPs needs to be considered carefully for PCOS patients who are already burdened with associated risk factors. This study was conducted in a region where women do not have much access to high-end screening and diagnostic facilities that further exacerbates their clinical outcomes. Large scale, long-term studies need to be designed to further evaluate safety use of OCPs in PCOS women.

Clinical profile in arrhythmogenic cardiomyopathy and a recessive plakophilin-2 gene mutation.

OBJECTIVE: Arrhythmogenic cardiomyopathy (ACM) is not an uncommon cause of cardiac morbidity in Kashmir valley. This study was designed to document various clinical features and to sequence exons 11 and 12 of plakophilin 2 (PKP2) gene in these patients. METHODS: ACM patients who attended cardiology outpatient department of our institute from January 2014 to April 2015 were included in the study. Their records were reviewed. Controls were randomly selected, who had no history or family history of cardiac illness and had a normal cardiac examination. A blood sample was also taken from both the groups for sequencing of exon 11 and 12 of PKP2 gene. ACM patients were followed up until July 2016. RESULTS: Eleven ACM patients and seven controls were included in the study. Most common mode of presentation was ventricular tachycardia (VT). Two patients had left ventricular (LV) systolic dysfunction. One patient had a splice site mutation in exon 12 of PKP2 gene and one patient died during follow-up. One of the controls had an intronic variation that has no pathogenic significance vis-à-vis ACM. CONCLUSION: Our study describes various clinical parameters in ACM patients and a recessive plakophilin 2 mutation after a limited PKP2 gene sequencing.

SNP and Haplotype Analysis of Vascular Endothelial Growth Factor (VEGF) Gene in Lung Cancer Patients of Kashmir

Vascular endothelial growth factor (VEGF) is a major mediator of angiogenesis involving tumor growth and metastasis. In this large case-control study, we investigated whether functional polymorphisms (+405C>G, +936C>T) in the VEGF gene are associated with the risk of lung cancer. The study investigates the association between variants of VEGF gene and lung cancer. We performed single nucleotide polymorphism (SNP), haplotype and linkage disequilibrium studies on 100 patients and 128 healthy controls with 2 SNPs in the VEGF gene. The results were analyzed using logistic regression models, adjusted for age and sex. No Significant association was detected between individual SNPs and lung cancer using all the models of inheritance (codominant, dominant, recessive, over dominant and additive) for finding an association between genotypes and the cancer risk. The P values obtained for two markers were nonsignificant (P>0.05). Haplotype analysis produced additional support for the non-association of individual haplotypes/ all haplotypes with the cancer risk (Global association P=0.56). Our findings suggest the non-involvement of genetic variants (+405C>G, +936C>T) of the VEGF gene in the etiology of lung cancer.

Association of IL1 beta gene polymorphism and allograft functions in renal transplant recipients :a case control study from Kashmir Valley.

BACKGROUND: Cytokines have been found to be the important mediators during renal graft outcome. Therefore, we designed this study to investigate the role of recipients' IL-1 ß promoter (-511) and IL-1 ß exon-5 (+3954) polymorphisms with the risk of graft outcome. METHODOLOGY: We enrolled one hundred recipients of living-related renal transplants together with the age and sex matched controls from the healthy population not having any renal abnormality for this study. Genotype frequencies of the IL-1 ß promoter (-511) and IL-1 ß exon-5 (+3954) were analyzed using PCR-RFLP technique. RESULTS: Our results revealed significant differences in the healthy control group and patient group in IL 1ß +3954 (p < 0.001). The frequency of variant type TT genotype was higher in RE group as compared to SGF and showed 4 fold risk of rejection (OR = 4.54, p < 0.069) although p value was not significant. The frequency of wild type CC genotype and CT was not significant (p value 0.89 and 0.74 respectively). CONCLUSION: Our findings suggest that there is a prevalence of mutated allele of IL-1 gene cluster in our population, which may be responsible for renal dysfunction.

Single nucleotide polymorphisms, haplotype association and tumour expression of the vascular endothelial growth factor (VEGF) gene with lung carcinoma.

VEGF contains several polymorphic sites known to influence its expression. We examined the possible association between+405(-634)C>G,+936C>T,-2578C>A and lung cancer in 199 Kashmiri patients and 401 healthy controls. VEGF+405CG,+936CT+TT and-2578CA genotypes were significantly associated with lung cancer risk compared to VEGF+405CC,+936CC and-2578AA+CC genotypes [OR=0.07 (0.04-0.13), P<0.0001, OR=0.36 (0.25-0.52), P<0.0001 and 0.08 (0.05-0.13), P<0.0001]. Haplotype analysis revealed that CGA and TGA haplotypes of VEGF gene conveys the risk for lung cancer [OR=0.18 (0.10-0.33), P<0.0001 and 0.07 (0.03-0.13), P<0.0001]. VEGF expression revealed non-significant association with the genotypes of the three SNPs. In conclusion, the SNPs examined appear to influence lung cancer susceptibility while as genotypes of the SNPs don't appear to have significant association with VEGF mRNA expression in lung tumours.

Chronic urticaria merits serum vitamin D evaluation and supplementation; a randomized case control study.

BACKGROUND: Several studies suggest that Vitamin D (Vit-D3) supplementation reduces Chronic Urticaria (CU) symptoms. OBJECTIVES: Evaluation of serum 25-hydroxyvitamin-D (25 (OH)2D) level and assessment of therapeutic effect of VitD3in CU patients. METHODS: 192 subjects were stratified according to the baseline 25(OH)2D levels and subsequently randomized into three subgroups to receive Vit-D3 alone (VD) or antihistamine and systemic corticosteroid (H+S) or VitD3 with antihistamine and systemic corticosteroid (VD+H+S) for 6 weeks between July 2012 to Oct 2014. 130 healthy controls (HC) were followed without any intervention. The patients were evaluated for reduction in urticarial symptoms using visual analogue scale (VAS) and 5-D itch score. RESULTS: Low serum levels of 25 (OH)2D was observed in 91% of CU patients and 64% of the healthy controls (P < 0.0001). VAS and 5-D Score in subgroups VD, H + S and VD + H + S decreased significantly from 6 · 7 ± 0 · 043, 6 · 6 ± 0 · 42 and 6 · 68 ± 0 · 40 at baseline to 5 · 2 ± 0 · 70 (P = 0 · 0088), 3 · 3 ± 0 · 50 (P < 0 · 0001) and1 · 86 ± 0 · 39 (P < 0 · 0001) after treatment and from 14 · 5 ± 0 · 72, 13 · 9 ± 0 · 77 and 13 · 9 ± 0 · 221 to 12 · 06 ± 1 · 10 (P = 0 · 0072), 8 · 1 ± 1 · 13 (P < 0 · 0001) and 5 · 01 ± 0 · 94 (P < 0 · 0001) respectively. CONCLUSIONS: CU patients have low serum 25(OH)2D levels and Vit-D3 supplementation in combination with antihistamine and systemic corticosteroid show elevated response in resolving the symptoms of CU. This study also warrants that each subject with CU should be screened for serum 25 (OH)2D levels before starting a treatment.

Significant impact of IL-6 -174G/C but inverse relation with -634 C/G polymorphism in patients with non-small cell lung cancer in Kashmiri population.

To study the possible role of proinflammatory interleukin 6 -174 G>C (rs 1800795) and -634 C>G (rs 1800796) polymorphism in the pathogenesis of non-small cell lung cancer (NSCLC). A total of 190 NSCLC patients and 200 healthy controls were evaluated for polymorphic analysis of -174 G/C and -634 C/G by PCR-RFLP followed by DNA sequencing. A significant association was observed in the genotypic and allelic distribution of IL-6 -174 G/C in the NSCLC group as compared to control group [OR = 2.7 (1.77-4.11), p < 0.0001]. Smokers with the -174C allele were found to be significantly associated with NSCLC (p = 0.01), while 634C/G SNP showed an inverse relation [OR-0.4, p < 0.0001]. The present investigation revealed a significant association of the IL6 -174 G/C gene promoter polymorphism with NSCLC, and thus, the IL-6 -174G/C genotype can be considered as one of the biological markers in the etiology of NSCLC.

A disintegrin and metalloprotease 33 polymorphism association with COPD in long-term tobacco smokers of the ethnic Kashmiri population of India.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by an interaction of various environmental influences especially cigarette smoking and genetic determinants. The prevalence of this disease is ever increasing and characterization of the genetic determinants of the disease has been undertaken globally. The 'A disintegrin and metalloprotease 33' (ADAM 33) gene is one candidate gene that has been studied. OBJECTIVE: Our objective was to investigate whether single nucleotide polymorphisms in ADAM33 gene are associated with COPD in long-term tobacco smokers in the ethnic Kashmiri population of northern India. MATERIALS AND METHODS: This was a randomized case-control study, which included 78 stable COPD (GOLD stage11-IV) patients, who were compared with 77 age- and sex-matched long-term tobacco smokers (>20 pack years) without any evidence of COPD. Polymorphic analysis for three single nucleotide polymorphisms (SNPs), (T1, T2, and Q1) of the ADAM33 gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by sequencing. The data were analyzed by descriptive statistics and comparative evaluation was done by parametric/non-parametric tests. RESULTS: The analysis of the T1, T2, and Q1 SNPs, revealed that the frequencies of the T2GG, T1GG, and the Q1AG genotypes were significantly higher in patients with COPD in comparison with the controls (P < 0.001). Similarly, the T1G and T2G allele frequency was higher in the patients than in the controls (p = 0.177 and 0.43, respectively). CONCLUSION: Three SNPs of the ADAM33 gene were significantly associated with COPD in the Kashmiri population of India. This study establishes the possible role of ADAM33 SNPS in the causation of COPD. Further studies across different geographical areas in the country will unravel the contribution of this gene in the causation of COPD in India.

Stroke-induced immune depression-a randomized case control study in Kashmiri population of North India.

BACKGROUND: Stroke leads to transient immunedepression, which leads to increased incidence of poststroke infections. Because infection is one of the most common causes of increased mortality in patients with stroke, this study was undertaken to document immunedepression after stroke in our population. METHODS: A case-controlled study wherein 39 patients with acute ischemic stroke in the age group of 18 and 60 years without any evidence of previous immunedepression were included. Interleukin 6 (IL-6) and interleukin 10 (IL-10) levels were checked in plasma in both the groups on day 3 and day 45. Also Cortisol and epinephrine levels were checked in the urine samples collected on day 3 and day 8. RESULTS: No significant difference was seen between the IL-6 and the IL-10 levels in samples collected on day 3 between the controls and cases, whereas Cortisol and norepinephrine were significantly raised in samples collected on day 3 in cases who developed infection as compared with controls. CONCLUSIONS: The higher levels of urinary cortisol and norepinephrine were observed in patients with stroke who developed infections, which indirectly reflected increased amount of stroke related stress. Furthermore, the levels of plasma IL-6 and IL-10 were also elevated in the same group of patients, which means transformation of immunecompetence to immunedepression, which is responsible for higher mortality. Subsequently on recovery from infection the plasma levels of interleukins and urinary cortisol and norepinephrine did not show any difference, which indirectly means recovery of the immune system on recovery from acute stage of stroke. Mortality in the patients with infection was increased than controls.

COX-2 overexpression and -8473 T/C polymorphism in 3' UTR in non-small cell lung cancer.

A new class of compounds targeting cyclooxygenase 2 (COX-2) together with other different clinically used therapeutic strategies has recently shown a promise for the chemoprevention of several solid tumors including lung cancer. The aim was to study the possible role of COX-2 -8473 T/C NP and its expression in the pathogenesis of non-small cell lung cancer. One hundred ninety non-small cell lung cancer (NSCLC) patients and 200 healthy age-, sex-, and smoking-matched controls were used for polymorphic analysis, and 48 histopathologically confirmed NSCLC patients were analyzed for COX-2 messenger RNA (mRNA) and protein expression. Our results showed that the frequencies of variant genotypes 8473 CT/CC were significantly less common in the cases (30.0%) than in the controls (36%), suggesting that the 8473 C variant allele is related with lower susceptibility in NSCLC (OR = 0.79, 95% CI 0.54-1.4). However, the frequency of COX-2 -8473 TC and CC genotypes were significantly associated with age in NSCLC (P = 0.02). Quantitative real-time expression analysis showed a significant increase in the COX-2 mRNA in tumor tissues as compared to their adjacent normal tissues [delta cycle threshold (ΔCT) = 9.25 ± 4.67 vs 5.63 ± 3.85, P = 0.0001]. Multivariate logistic regression analyses revealed that the COX-2 expression was associated significantly with age (P = 0.044). Also, an increasing trend was observed in stages I and II and in female patients compared to stages III and IV and male patients, respectively, but no statistical significance was observed. However, COX-2 mRNA expression shown no association with the -8473 C variant allele. Our findings indicate that the COX-2 T8473C polymorphism may contribute to NSCLC cancer susceptibility in the Kashmiri population, while our expression analysis revealed a significant increase of COX-2 in tumor tissues as compared to their adjacent normal tissues, suggesting that it could become an important therapeutic marker in NSCLC in the future.

Association of interleukin 1 beta (IL-1ß) polymorphism with mRNA expression and risk of non small cell lung cancer.

INTRODUCTION: Interleukin 1 beta (IL- 1ß), a key proinflammatory cytokine encoded by the interleukin 1 beta gene, has been associated with chronic inflammation and plays an important role in lung inflammatory diseases including lung cancer. Elevated levels of Interleukin 1proteins, in particular interleukin 1 beta greatly enhance the intensity of the inflammatory response. AIM: To study the role of interleukin 1 beta-31C > T and -511 T > C polymorphism in the pathogenesis of non small cell lung cancer (NSCLC). MATERIALS AND METHODS: One hundred and ninety non small cell lung cancer patients and 200 healthy age, sex, smoking and dwelling matched controls were used for polymorphic analysis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by sequencing. Normal tissues of 48 histopathologically confirmed non small cell lung cancer patients were taken for mRNA expression analysis. Quantitation of interleukin 1 beta was carried out by quantitative real time PCR. RESULT: The T/T genotype of interleukin 1 beta-31 gene was significantly associated with increased risk of NSCLC [(P = 0.001, OR - 2.8 (95%CI 1.52-5.26)]. The interleukin 1 beta - 511 T > C does not show any difference between the NSCLC and control group (P = 0.3, OR - 0.72 (95%CI 0.41-1.28). Quantitative analysis of mRNA showed significant association with interleukin 1 beta T allele as compared to the interleukin 1 beta-31C allele (P = 0.006). CONCLUSION: We conclude that lung cancer risk genotype interleukin 1 beta-31TT results in increased expression of interleukin 1 beta mRNA in lung cancer patients. Our data suggest that this genotype (IL1ß -31TT) in the interleukin 1 beta regulatory region provide a microenvironment with elevated inflammatory stimuli and thus increasing the risk for lung cancer.

Role of vitamin D receptor (VDR) polymorphisms in susceptibility to multiple myeloma in ethnic Kashmiri population.

BACKGROUND: Vitamin D regulates many biological processes including bone metabolism, innate immune response, and cell proliferation and differentiation by binding to its receptor VDR. Vitamin D receptor (VDR) gene polymorphisms have been associated with many cancers like breast, colorectal, prostate, and skin. The main aim of this study was to determine whether VDR polymorphisms (ApaI, BsmI and FokI) are associated with increased risk of multiple myeloma. METHODS: We designed a case control study where 75 multiple myeloma cases were studied for VDR polymorphisms (ApaI, BsmI and FokI) against 150 controls taken from general population. The polymorphisms of VDR gene were investigated using PCR-RFLP method. RESULTS: We did not find any significant association between ApaI and BsmI polymorphisms and multiple myeloma risk (P>0.05), but FokI polymorphism was significantly associated with increased risk for multiple myeloma. We also found a significant association between the ff variant genotype with creatinine levels, albumin levels, and Durie-Salmon stage III. CONCLUSION: Our findings suggest that the FokI polymorphism is involved in the increased susceptibility to development and progression in multiple myeloma in the ethnic Kashmiri population. Furthermore these results suggest that ff genotype is associated with higher risk for developing multiple myeloma.

WITHDRAWN: Single nucleotide polymorphisms, haplotype association and tumour expression of the vascular endothelial growth factor (VEGF) gene with lung carcinoma.

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Vascular endothelial growth factor A gene (VEGFA) polymorphisms and expression of VEGFA gene in lung cancer patients of Kashmir Valley (India).

The vascular endothelial growth factor (VEGF) is a major mediator of angiogenesis involving tumor growth and metastasis. Polymorphisms in the VEGF gene may regulate VEGF production. In this case-control study, we investigated whether functional polymorphisms (+405 C > G and +936 C > T) in the VEGF gene are associated with the risk of lung cancer. Genomic DNA was isolated from the blood of 100 lung cancer patients and 150 healthy controls, and total RNA was isolated from 48 tumor tissues and adjacent normal lung tissues. Two DNA polymorphisms (+405 C > G and +936 C > T) in the 3'-untranslated regions (3'-UTR) and 5'-untranslated regions (5'-UTR) of vascular endothelial growth factor A (VEGFA) were studied using PCR-RFLP method, and mRNA expression of VEGFA was studied by quantitative real-time PCR. Polymorphisms in the 5'-UTR (+405 C > G) and 3'-UTR (+936 C > T) did not show significant difference between lung cancer cases and control samples (P = 0.11 and P = 0.09, respectively). VEGF +405 CG and GG are significantly more in age group >50 years old, in all grades, and in early pathological stages (P = 0.04, P = 0.03, and P = 0.006, respectively). Also, increased expression of VEGFA mRNA was noted in tumorous compared to non-tumorous tissue (P < 0.0001). Overexpression of the gene was considered at ΔC (T) > 6.0. Within the group of patients with conventional tumor, those with histology other than squamous cell carcinoma (SCC) had a higher level of VEGFA mRNA expression than SCC patients (P = 0.04). Overexpression of VEGFA mRNA was noted in lung cancer and more so in lung cancer with adenocarcinoma and large cell carcinoma histology and in pathological stages III and IV. VEGFA +405 C > G SNP showed an association with age, pathological grade, and stage.