Role of non-coding RNAs in head and neck squamous cell carcinoma: A narrative review.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with high recurrence, metastasis, and poor treatment outcome. Recent studies have reported that non-coding RNA (ncRNA) might play critical role in regulating different types of cancer. MicroRNAs (miRs) are short ncRNAs (20-25 nucleotides) responsible for post-transcriptional regulation of gene expression and may have a role in oncogenesis by acting as oncomiRs or tumor suppressor miRs. Long non-coding RNAs (lncRNAs) are heterogenous group of ncRNAs more than 200 nucleotides long, can act in cis and/or in trans, and have been also implicated in carcinogenesis. These molecules have been suggested to be promising candidates as diagnostic and prognostic biomarkers and for development of novel therapeutic approaches. In this review, we have summarized recent findings on role of these ncRNAs in HPV-negative (HPV-ve) and HPV-positive (HPV+ve) HNSCC. The available literature supports differential expression of both microRNAs and long non-coding RNAs, which include oncogenic ncRNAs (miR-21, miR-31, miR-155, miR-211, HOTAIR, and MALAT1) and tumor suppressor ncRNAs (let7d, miR-17, miR-375, miR-139, and MEG3) in HPV+ve HNSCC tumors as compared to HPV-ve tumors and they have distinct role in the pathophysiology of these two types of HNSCCs.

Detection of active human papilloma virus-16 in head and neck cancers of Asian North Indian patients.

BACKGROUND: Head and neck cancers (HNC) are one of the most common cancers in India. Human papillomavirus (HPV) has been identified as an emerging risk factor for HNC. METHODS: The present study was carried out to determine the active form of HPV-16 using a combination of PCR, viral load determination, HPV-16 E7 mRNA expression, p16, p53, and pRB immuno-histochemistry (IHC). RESULTS: A total of 226 HNC patients were enrolled in the present study. Sixty-seven (29.7%) of HNC cases were found to be HPV DNA positive. Thirty-two (14%) cases were HPV-16 DNA positive and 20 (9%) cases expressed HPV-16 E7 mRNA. HPV-16 mRNA/p16 positive cases had significantly increased viral load and integrated HPV-16 DNA. In summary, of total HNC patients, 6% cases were positive for both HPV-16 DNA and p16, and 5% were positive for both E7 mRNA and p16 IHC. We observed similar HPV-16 DNA/E7mRNA prevalence in oropharynx and oral cavity sites, however, oropharynx SCC had significantly higher viral load. CONCLUSION: Our results show low prevalence of active HPV-16 in North Indian HNC patients. HPV-16 E7 mRNA expression correlated with p16 nuclear positivity and increased viral load. Therefore, E7 mRNA expression may be used as a good surrogate indicator for active form of HPV-16 infection.

Lipid profile and fat patterning in children at a mean of 8.8 years after Kawasaki disease: a study from Northern India.

OBJECTIVES: Kawasaki disease (KD) is an acute vasculitis that can result in coronary artery abnormalities (CAA). Higher risk of atherosclerosis has also been documented in those who do not develop CAA. We report herein the lipid profile and fat patterning in children with KD in a cohort from Northern India at a mean follow-up of 8.8 years after the acute stage. There is a paucity of literature on this aspect of KD. METHODS: Twenty children, who had developed KD at least 5 years previously were enrolled along with age- and sex-matched controls. Cases and controls underwent anthropometric assessment using standardised techniques and instruments. Lipids were assayed only in the cases. RESULTS: There was no significant difference in weight, height, mid-upper arm circumference, waist circumference, hip circumference and waist-to-hip ratio between cases and controls. Skinfold thickness (ST) at triceps, subscapular, midaxillary and suprailiac regions was similar in cases and controls. Biceps and medial calf ST was, however, significantly higher among girls with KD in 10-14.9 years age group. On comparison with cut-offs enumerated by the National Cholesterol Education Program (NCEP), 2 children with KD had borderline while 1 had undesirable levels of total cholesterol. Undesirable triglyceride levels were seen in 12 children. Ten children had HDL levels <35 mg/dl while 1 had borderline LDL levels. CONCLUSIONS: Lipid abnormalities at a mean of 8.8 years after KD suggest that these patients may be prone to premature atherosclerosis. There were no significant differences in the anthropometric parameters and most of the ST.

Role of autophagy in head and neck cancer and therapeutic resistance.

Head and neck squamous cell carcinomas (HNSCC) are one of the most common cancers worldwide, accounting for almost 50% of all malignancies in developing nations. Autophagy is a catabolic process involving turnover of long-lived proteins and organelles and is an important mechanism for cell survival under stress conditions. Autophagy has been shown to play a pivotal role in etio-pathogenesis of several cancers. Autophagy and apoptosis may be triggered by common upstream signals, and sometimes this results in combined autophagy and apoptosis, or defective apoptosis rendering immortalized epithelial cells highly tumorigenic. Autophagy has been found to buffer metabolic stress and may help in cell survival; however, inhibiting autophagy under conditions of nutrient limitation can restore cell death to apoptosis-refractory tumors. Therefore, autophagy acts as a double-edged sword in cancer therapeutics. Role of autophagy in pathophysiology and as a potential cancer therapeutics is a subject of intensive research. This review will focus on the role of autophagy and how it contributes to the pathogenesis and overcoming therapeutic resistance in HNSCC.

Hypermethylation of carcinogen metabolism genes, CYP1A1, CYP2A13 and GSTM1 genes in head and neck cancer.

OBJECTIVES: To investigate the role of aberrant hypermethylation of carcinogen metabolism pathway genes, CYP1A1, CYP2A13 and GSTM1 in head and neck cancer independently as well as its relation to tobacco and alcohol consumption and CYP1A1 and CYP2A13 polymorphisms in Indian population. METHODS: Seventy-three histologically confirmed head and neck cancer patients undergoing treatment in Postgraduate Institute of Medical Education and Research, Chandigarh, India were recruited. Non-cancerous tissues were obtained from 19 trauma subjects undergoing maxillofacial surgery. Methylation-specific PCR was performed to determine the methylation status of selected genes. RESULTS: The aberrant hypermethylation of CYP1A1, CYP2A13 and GSTM1 genes was found in cancer tissues with frequency of about 39.7%, 27.4%, and 58.1%, respectively, and in normal healthy tissues with a frequency of about 10.5%, 15.8%, and 20.0%, respectively. Hypermethylation of CYP1A1 (P 0.027) and GSTM1 (P 0.010) showed significant association with head and neck cancer. We also observed significant interaction between smoking and methylation status of CYP1A1 (P 0.029) and CYP2A13 (P -0.034) in head and neck cancer. No association was observed between methylation status and alcohol consumption, clinical features and genetic polymorphisms of CYP1A1 and CYP2A13. CONCLUSIONS: Hypermethylation of carcinogen metabolism pathway genes independently and in interaction with smoking is associated with increased risk of head and neck cancer.

Polymorphisms in CYP2A13 and UGT1A7 genes and head and neck cancer susceptibility in North Indians.

OBJECTIVES: To examine role of genetic variants of CYP2A13 and UGT1A7 genes, involved in activation and detoxification of tobacco carcinogens, with risk of head and neck cancer as well as to assess the potential modifying role of gene-gene and gene-environment interactions. METHODS: 203 head and neck cancer patients and 201 healthy controls were genotyped for functional polymorphisms of CYP2A13 and UGT1A7 genes using polymerase chain reaction-restriction fragment length polymorphism, denaturing high-performance liquid chromatography and sequencing. RESULTS: We identified two novel polymorphisms T478C and T494C in CYP2A13 gene which were associated with significantly reduced risk of cancer (OR 0.37; 95% CI 0.19-0.71; P < 0.05). A CYP2A13 haplotype carrying variant alleles of T478C/T494C was found to be associated with reduced risk of head and neck cancer (OR 0.42; 95% CI 0.22-0.78; P = 0. 005). Mutant 'T' allele of CYP2A13 C578T polymorphism was found to be present in cancer patients only. A sevenfold increased risk of cancer was observed in smokers with UGT1A7 low activity genotypes (OR 7.01; 95% CI 1.02-48.37; P < 0.05). UGT1A7 haplotype carrying C allele (T622C) showed 10-fold increased risk of cancer (OR 10.12; 95% CI 1.29-79.4; P < 0.05). CONCLUSION: Interplay between genetic variants of CYP2A13 and UGT1A7 genes and smoking may modulate susceptibility to head and neck cancer.

High frequency of heterozygosity in GJB2 mutations among patients with non-syndromic hearing loss.

OBJECTIVE: To determine the prevalence of GJB2 mutations among subjects with congenital, non-syndromic, sensorineural hearing loss, within a north Indian population. MATERIALS AND METHODS: This was a case-control study in which the frequencies of the three most prevalent GJB2 mutations (35delG, W24X and 167delT) were studied. Polymerase chain reaction restriction fragment length polymorphism assays were performed to detect these mutations. The entire coding region of the GJB2 gene was sequenced in all patients, and also in any of their family members who showed GJB2 mutations. RESULTS: The 35delG mutation was found to be the most prevalent mutation (21 per cent), followed by the W24X mutation (7 per cent). This is the first report of the 35delG mutation in an Indian population. One patient was a compound heterozygote for 35delG/W24X. The 167delT mutation was not observed in any patient. CONCLUSIONS: These findings challenge the classical view that the W24X variant of the GJB2 gene represents a single 'founder' mutation.

Effect of ACE inhibitors and beta-blockers on homocysteine levels in essential hypertension.

Recent studies have shown that antihypertensive drugs like diuretics increase plasma homocysteine (Hcy) levels. However, the effect of other antihypertensive drugs on plasma Hcy levels has not been tested extensively. The aim of present study was to investigate the effect of antihypertensive therapy (AHT) on Hcy levels in essential hypertensive subjects. A case-control study of 273 patients with essential hypertension (EH) and 103 normotensive controls was undertaken. Plasma Hcy levels were measured before and after 6 weeks of AHT. The genotyping of MTHFR C677T polymorphism was performed by polymerase chain reaction-restriction fragment length polymorphism. Angiotensin-converting enzyme (ACE) inhibitors and beta-blockers significantly decreased and hydrochlorothiazides significantly increased the plasma Hcy levels in hypertensive patients (P<0.05). No significant association between MTHFR C677T genotypes and changes in Hcy levels in response to antihypertensive was observed in EH patients. The decrease in Hcy induced by beta-blockers and ACE inhibitors observed in our study may be due to the improvement of endothelial function along with improved renal function. Thus, our results suggest that ACE inhibitors and beta-blockers may provide additional beneficial therapeutic effects to the EH patients by decreasing Hcy levels.

Oxidative status in stable renal transplantation.

Increased oxidative stress and hyperhomocysteinemia are frequently observed in patients with end-stage renal disease. The effects of kidney transplantation on oxidative state are incompletely understood. With an aim to evaluate the prevalence and severity of oxidative stress in living donor renal transplant recipients, we conducted a cross-sectional study. Thirty-five renal transplant recipients (mean age 34 years; body mass index 21.93 +/- 1.92) with normal renal function (mean serum creatinine 1.41 +/- 0.33 mg%) were enrolled in the study. All patients were on cyclosporine-based immunosuppression. We assessed serum nitric oxide (NO) levels, plasma total homocysteine levels (tHCy), and malonaldehyde (MDA) levels. We evaluated the antioxidant power ferric reducing ability of plasma (FRAP) assay. The mean duration to the first sampling was 9.23 months after transplantation. Fourteen age- and sex-matched normotensive people were used as controls. The mean tHCy was significantly higher among patients (15.29 +/- 0.66 mmol/L compared with controls (9.58 +/- 2.90 mmol/L; P < .05). The MDA levels in patients (6.405 +/- 2.05 nmol/mL) were comparable to controls (6.093 +/- 1.93 nmol/mL; P = .099). The status of antioxidative power as measured by FRAP showed a trend to higher antioxidative status (697.57 +/- 103.07 mmol/L) in patients compared with controls (518 +/- 120.99 mmol/L; P = NS). The mean NO levels in patients (545.01 +/- 281.49 mmol/mL) were significantly higher than controls (183.49 +/- 64.53 nmol/mL; P < .05). Stable renal transplant recipients display a pattern of increased oxidant stress that may be counterbalanced by an enhanced antioxidant mechanisms.

Hyperhomocysteinemia as a risk factor for ischemic stroke: an Indian scenario.

BACKGROUND: Hyperhomocysteinemia has been proposed as an important risk factor for ischemic stroke worldwide, but data available from the Indian subcontinent is scarce. AIM: To study homocysteine levels in patients with ischemic stroke and compare it with age- and sex-matched controls. SETTINGS AND DESIGN: Case-control prospective study. MATERIALS AND METHODS: Fifty-seven patients with ischemic stroke and 30 controls were recruited for the study. They were subdivided into two subgroups (< 40 years and> 40 years of age) and plasma fasting total homocysteine (tHcy) levels were measured. STATISTICAL ANALYSIS USED: Student's 't' test and chi-square test. RESULTS: The tHcy were significantly high in patients with stroke, compared to controls (9.91 +/- 2.25 vs 8.00 +/- 2.74 micromol/l; P vs 8.45 +/- 2.72 micromol/l; P = 0.01) and female patients compared to controls (9.08 +/- 1.81 vs 6.79 +/- 2.60 micromol/l; P = 0.04). The tHcy levels were significantly high in patients with hypertension compared to normotensive patients (10.96 vs 9.49 micromol/l; P = 0.01) and smokers compared to nonsmokers (11.17 vs 9.33 micromol/l; P = 0.01). CONCLUSIONS: Hyperhomo-cysteinemia emerged as an important independent risk factor for ischemic stroke. A strong positive correlation was also observed between hypertension, smoking, and high-tHcy levels in the present study.

Biochemical and molecular markers in renal cell carcinoma: an update and future prospects.

Cancer is a big problem in the developed world as well as in developing countries. Renal cell carcinoma (RCC) accounts for approximately 3% of adult malignancies and 90-95% of neoplasms arising from the kidney. RCC is more common in men than in women (2:1), and it most often occurs in patients between the ages of 50-70 years. In all cancers the cancerous cells release particular kind of proteins (called tumour markers) and blood tests are used to detect the presence of these markers. These tumour markers nowadays are an area of interest for oncologists who search for a possible solution in the detection and treatment of RCC. Different kinds of biochemical and molecular markers such as ferritin, MN/CA9, apoptotic index, p53, IL-2, gamma-enolase, CD44, CD95, chromosome instability and loss of heterozygosity have been tested in RCC, but so far no marker fulfils one or the other criteria to be considered as an ideal marker for RCC. This review gives basic and updated information about the different kinds of biomarkers studied in RCC and about the role implementation of genomics and proteomics in RCC.

Enalapril acts through release of nitric oxide in patients with essential hypertension.

BACKGROUND: Endothelial dysfunction has been proposed as an etiological agent in the pathogenesis of essential hypertension. Amongst the various antihypertensive drugs, angiotensin converting enzyme inhibitors (ACEI) have been implicated in modifying the vascular endothelium by the release of mediators that include bradykinin, nitric oxide, prostaglandins and thromboxane A2. MATERIALS AND METHODS: To study the mechanism of action of enalapril, an ACEI, serum reactive nitrite intermediates (RNI) and citrulline, by products of nitric oxide metabolism were measured before and after treatment with enalapril in 25 consecutive patients of essential hypertension. RESULTS: Following treatment serum RNI intermediate increased from a pretreatment value of 164.5 +/- 20.2 nmol/mL to a post treatment value of 266.9 +/- 47.3 nmol/mL (p < 0.05), however there was no significant change in the levels of citrulline (p > 0.1). There was no significant correlation between the severity of hypertension and serum RNI. Serum RNI levels were lower in the postmenopausal women but did not reach statistical significance. CONCLUSIONS: It is postulated that enalapril exhibits its antihypertensive property through release of nitric oxide.

Calcium supplementation in pre-eclampsia.

OBJECTIVE: The objective of this prospective, open, randomised trial was to determine the role of calcium supplementation in preventing pre-eclampsia. METHODS: One hundred uncomplicated normotensive primigravidae were enrolled in the study before 20 weeks of pregnancy. Fifty each were randomised to receive either two gram elemental calcium daily from 20 weeks of gestation to delivery (study group) or no calcium supplementation (control group). Prior to 20 weeks of gestation each underwent a complete clinical and laboratory evaluation. Serum and urine calcium was measured first at 20 weeks of gestation and then at 24-28 weeks and at 32-36 weeks of pregnancy. RESULTS: Patient characteristics at the start of therapy were similar in the two groups. Blood pressure profile was similar throughout pregnancy in the groups. The incidence of pre-eclampsia was similar (18% in the study group and 16% in the control group), but severe pre-eclampsia was significantly less in the study group. There was no significant difference between the two groups with regards to intra- and postpartum characteristics, perinatal outcome and maternal or fetal side effects. Serum and urinary calcium levels did not differ between the two groups. CONCLUSION: While calcium supplementation did not lower the incidence of pre-eclampsia it did reduce its severity.

Serum and urine nitrite and citrulline levels among patients with systemic lupus erythematosus: a possible addition to activity parameters?

Nitric oxide (NO) plays a significant role in the inflammatory process and has been implicated in several autoimmune disorders. This study was carried out prospectively to estimate the levels of nitrite and citrulline in the serum and urine, as surrogate markers of NO production, among patients with systemic lupus erythematosus (SLE). Forty-seven patients and 44 age- and sex-matched, healthy volunteers were studied. Nitrite and citrulline were measured in serum and urine by spectrophotometry.Median serum nitrite and citrulline levels and urine citrulline levels were higher among patients as compared with controls (p < 0.05). Patients with skin involvement stood out and had higher median serum and urine citrulline levels (p < 0.05). Disease activity correlated with steroid dosage, serum nitrite levels, and serum and urine citrulline levels (p < 0.05). Steroid dosage correlated with serum citrulline level (p < 0.05). Serum and urine citrulline levels correlated with each other (p < 0.01). In the subset of 13 individuals with renal involvement, serum and urine citrulline levels correlated with each other (p < 0.01) as did urine nitrite and citrulline levels (p < 0.05).NO production is increased among patients with SLE, and this increase correlates with disease activity and dosage of steroids used. The addition of a urine test to measure NO production as a marker of disease activity using simple spectrophotometry can be a valuable adjunct to other tests, can obviate the need for drawing a blood sample for this purpose, and can be repeated as often as necessary.

Elevated nitric oxide production in patients with primary Sjögren's syndrome.

Nitric oxide (NO) production is elevated in patients with inflammatory disorders. We have previously shown increased NO production in patients with rheumatoid arthritis and systemic lupus erythematosus. In this study we used nitrite and citrulline levels as surrogate markers of NO production in patients with primary Sjögren's syndrome (SS) and measured their levels by spectrophotometry. Fifteen patients and 15 age- and sex-matched controls were studied. Mean nitrite levels in patients were 582.3+/-208.3 nmol/ml, but those in controls were significantly lower, at 203.2-106.9 nmol/ml (p<0.001). Citrulline levels were 2820.4+/-933.9 nmol/ml in patients and were significantly higher than 217.4+/-144.8 nmol/ml, the levels in controls (p<0.0001). Mean levels of both nitrite and citrulline were significantly higher in patients with arthritis than in those who had no joint manifestations (p<0.05). There was no correlation between NO production and other variables, such as age, disease duration, drug therapy and antinuclear antibodies or rheumatoid factor positivity. Increased NO production may be partly a reflection of the presence of arthritis in five patients. It is concluded that there is increased NO production in patients with primary SS, especially if they have associated arthritis.

Pentoxiphylline reduces nitric oxide production among patients with HIV infection.

Tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) levels are elevated among patients with human immunodeficiency virus (HIV) infection. TNF-alpha is known to lower NO production. In this study we used a TNF-alpha inhibitor, pentoxiphylline, to treat patients with HIV infection who were free of opportunistic infections and see if NO production was altered with this drug. NO production was determined by spectrophotometric analysis using nitrite and citrulline as surrogate markers and TNF-alpha levels were determined by ELISA before and after 4 weeks of the treatment. Nineteen patients (ten males, mean age 36.6+/-5.2 years) and 16 age and sex matched healthy controls were studied. Mean CD4 counts of patients were 206.5 mm(3). Nitrite level among patients at recruitment was 99.7+/-26.5 nmol/ml (range 50-167 nmol/ml) and was significantly higher than 46.4+/-16.2 nmol/ml; the value of healthy controls (P<0.05). Patient levels declined significantly to 44. 2+/-19.7 nmol/ml (range 10-106.6 nmol/ml) following 4 weeks of therapy (P<0.01). Citrulline level at recruitment was 810.8+/-425.8 nmol/ml (range 366.6-1888.7 nmol/ml), which was significantly higher than 488.6+/-224.5 nmol/ml, the level of controls (P<0.01). There was a statistically significant decrease in these levels among patients to 533.6+/-299.5 nmol/ml (range 250-163.4 nmol/ml) after 4 weeks of therapy (P<0.01). TNF-alpha levels showed a significant decline in the OD values from 0.34+/-0.22 at the start of therapy to 0.24+/-0.18 (P<0.05). We conclude that the use of pentoxiphylline is associated with decrease in TNF-alpha levels and NO production.

Effect of oral magnesium supplementation on blood pressure, platelet aggregation and calcium handling in deoxycorticosterone acetate induced hypertension in rats.

OBJECTIVE: To study the effect of oral magnesium supplementation on blood pressure, platelet aggregation and platelet calcium handling in deoxycorticosterone acetate (DOCA)-induced hypertension in rats. DESIGN AND METHODS: Rats were divided into four groups of 20 each. Drug treatments were given for a 6-week period. Control rats were vehicle treated. In the second group, DOCA, 15 mg/kg, was injected subcutaneously twice weekly with 1% NaCl used instead of drinking water. The third group was given magnesium oxide (MgO), 1 g/kg daily, orally by gavage. The fourth group was given MgO along with DOCA and 1% NaCl. Blood pressure and heart rate were measured weekly. Platelet aggregation, intracellular calcium, calcium uptake and calcium efflux studies were performed at the end of sixth week. Serum magnesium concentration, plasma levels of reactive nitrogen intermediates (RNI) and citrulline were also measured RESULTS: There was a significant rise in blood pressure in the DOCA-treated rats. Magnesium prevented the gradual rise in blood pressure when given along with DOCA, but had no effect in normotensive rats. Heart rate did not show any significant change. Platelet aggregation was significantly reduced in all the treatment groups compared to the control group. DOCA treatment produced a significant increase in the intracellular calcium concentration as well as the calcium uptake compared to the control group. Magnesium supplementation inhibited the increased intracellular calcium concentration and calcium uptake in DOCA-treated rats. RNI and citrulline levels were elevated in all the treatment groups. Serum magnesium levels were significantly higher in the magnesium-treated and DOCA plus magnesium-treated rats. CONCLUSIONS: Magnesium supplementation prevents blood pressure elevation in DOCA hypertensive rats. These effects are associated with inhibition of platelet calcium uptake and decreased intracellular free calcium concentration.

Intestinal mucins: the binding sites for Salmonella typhimurium.

Mucus-bacterial interactions in the gastrointestinal tract and their impact on subsequent enteric infections are poorly delineated. In the present study, we have examined the binding of Salmonella typhimurium to rat intestinal mucus and characterized a mucus protein (Mucus-Rs) which specifically binds to S. typhimurium. Both virulent (1402/84), and avirulent (SF 1835) S. typhimurium were observed to bind to crude mucus, however, the virulent strain showed 6 fold more binding as compared to avirulent strain. Fractionation of crude mucus on sepharose CL-6B resolved it into three major peaks. Maximal bacterial binding was observed with a high mol. wt. glycoprotein corresponding to neutral mucin. SDS-PAGE of purified protein (termed Mucus-Rs) under non reducing conditions showed it to be a homogenous glycoprotein (mol. wt. 250 kDa), while under reducing conditions, three bands corresponding to mol. wt. of 118,75 and 60 kDa were observed. Pretreatment of Mucus-Rs with pronase, trypsin and sodium metaperiodate markedly inhibited bacterial binding. GLC analysis of Mucus-Rs showed it to contain Mannose, Glucose, Galactose, Glucosamine, Galactosamine and Sialic acid as main sugars. Competitive binding in the presence of various sugars and lectins indicated the involvement of mannose in the mucus-bacterial interactions. The Mucus-Rs binding was highly specific for S. typhimurium; no significant binding was seen with E. coli and V. cholerae. Thus, we conclude that S. typhimurium specifically binds to a 250 kDa neutral mucin of intestinal tract. This binding appears to occur via specific adhesin-receptor interactions involving bacterial pili and mannose of neutral mucin.