Vikram Bhatia

VEGF-A immunohistochemical and mRNA expression in tissues and its serum levels in potentially malignant oral lesions and oral squamous cell carcinomas

The aim of the study was to investigate whether the estimation of circulating Vascular endothelial growth factor-A (VEGF-A) levels by ELISA could be used as surrogate of VEGF-A expression in tissues of pre-malignant oral lesions (PMOLs) and oral squamous cell carcinoma (OSCC) as compared to that in healthy controls. The study samples comprised of tissue and blood samples from 60 PMOLs, 60 OSCC, and 20 healthy controls. Serum VEGF-A levels were determined by an ELISA based assay (Quantikine human VEGF; R & D System, Minneapolis USA). Tissue VEGF-A expression and microvessel density (MVD) were assessed by immunohistochemistry (IHC) using antibodies against VEGF-A and CD-34 on formalin fixed paraffin embedded (FFPE) tissue sections. VEGF-A mRNA expression was analyzed by real-time PCR in snap frozen tissues. Serum VEGF-A levels and immunohistochemical VEGF-A expression were significantly high in PMOLs and OSCC in comparison with controls. VEGF mRNA gene expression showed more than 50-fold increase in PMOLs and OSCC. VEGF-A levels in serum correlated in a linear fashion with the tissue expression in oral pre-malignant and malignant lesions, suggesting that the serum levels may serve as surrogate material for tissue expression of VEGF-A.

Promoter Region Hypermethylation and mRNA Expression of MGMT and p16 Genes in Tissue and Blood Samples of Human Premalignant Oral Lesions and Oral Squamous Cell Carcinoma

Promoter methylation and relative gene expression of O6-methyguanine-DNA-methyltransferase (MGMT) and p16 genes were examined in tissue and blood samples of patients with premalignant oral lesions (PMOLs) and oral squamous cell carcinoma (OSCC). Methylation-specific PCR and reverse transcriptase PCR were performed in 146 tissue and blood samples from controls and patients with PMOLs and OSCC. In PMOL group, significant promoter methylation of MGMT and p16 genes was observed in 59% (P = 0.0010) and 57% (P = 0.0016) of tissue samples, respectively, and 39% (P = 0.0135) and 33% (P = 0.0074) of blood samples, respectively. Promoter methylation of both genes was more frequent in patients with OSCC, that is, 76% (P = 0.0001) and 82% (P = 0.0001) in tissue and 57% (P = 0.0002) and 70% (P = 0.0001) in blood, respectively. Significant downregulation of MGMT and p16 mRNA expression was observed in both tissue and blood samples from patients with PMOLs and OSCC. Hypermethylation-induced transcriptional silencing of MGMT and p16 genes in both precancer and cancer suggests important role of these changes in progression of premalignant state to malignancy. Results support use of blood as potential surrogate to tissue samples for screening or diagnosing PMOLs and early OSCC.

Endometrial Expression of Homeobox Genes and Cell Adhesion Molecules in Infertile Women With Intramural Fibroids During Window of Implantation.

This study was designed to examine the expression and cellular distribution of homeobox (HOX) genes (HOXA10 and HOXA11) and cell adhesion molecules (E-cadherin, N-cadherin, and β-catenin) during the window of implantation in infertile women with noncavity-distorting intramural (IM) fibroids (n = 18) and in fertile controls (n = 12). Quantitative real-time polymerase chain reaction and immunohistochemistry were used to evaluate the messenger RNA (mRNA) levels and protein expression, respectively. When compared to fertile controls, reduced HOXA10 and HOXA11 transcript and protein levels were observed in infertile women. However, changes only in the expression of HOXA10 mRNA (−1.72-fold; P = .03) and stromal protein (P = .001) were statistically significant. Significantly lower E-cadherin mRNA (−10.97-fold; P = .02) and protein levels were seen in infertile patients. E-cadherin immunostaining was significantly reduced both in the luminal (P = .048) and in the glandular (P = .014) epithelium of endometrium from infertile patients when compared to controls. No significant change was observed either in the mRNA levels or in the immunoexpression of N-cadherin and β-catenin. However, a trend toward lower N-cadherin expression in the luminal epithelium (P = .054) and decreased β-catenin expression in the glandular epithelium (P = .070) was observed in infertile patients. The present findings suggest that altered endometrial HOXA10 and E-cadherin mRNA and protein expression observed in infertile women with IM fibroids during the mid-secretory phase might impair endometrial receptivity leading to infertility in these patients.

Inhibition of Alpha-Glucosidase by Acacia nilotica Prevents Hyperglycemia along with Improvement of Diabetic Complications via Aldose Reductase Inhibition

Postprandial hyperglycemia is a prominent and early defect in diabetes and regulating blood glucose elevation may attenuate progression towards diabetes associated secondary complications. Here we investigated the alphaglucosidase inhibitory potential of the ethanolic extract of the stem bark of Acacia nilotica (EEAN). The EEAN showed a remarkable alpha-glucosidase inhibitory effect with IC50 value around 8 μg/ml. Kinetic studies revealed that the extract inhibited alpha-glucosidase in competitive manner and caused conformational changes in secondary structure of the enzyme protein. In vivo analysis showed that EEAN significantly suppresses the sucrose-induced postprandial glucose elevation in normal rats and exerts antihyperglycemic effect in streptozotocin (STZ)-induced diabetic rats in a dose-dependent fashion. Further, treatment of diabetic animals after 10 week of STZ-treatment with EEAN (250 mg/ kg) for 21 days, significantly reduced the elevated levels of blood glucose, %HbA1C, urea, uric acid and creatinine, and significantly increased the depressed plasma insulin level. The EEAN also showed inhibitory potential on aldose reductase activity with an IC50 of 7.5 μg/ml. The results suggest that EEAN possess antihyperglycemic activity through inhibition of alpha-glucosidase along with antidiabetogenic effect on polyol pathway through aldose reductase inhibition.

Fibroblast Growth Factor (FGF-2) and Its Receptors FGFR-2 and FGFR-3 May Be Putative Biomarkers of Malignant Transformation of Potentially Malignant Oral Lesions into Oral Squamous Cell Carcinoma.

There are several factors like angiogenesis, lymphangiogenesis, genetic alterations, mutational factors that are involved in malignant transformation of potentially malignant oral lesions (PMOLs) to oral squamous cell carcinoma (OSCC). Fibroblast growth factor-2 (FGF-2) is one of the prototypes of the large family of growth factors that bind heparin. FGF-2 induces angiogenesis and its receptors may play a role in synthesis of collagen. FGFs are involved in transmission of signals between the epithelium and connective tissue, and influence growth and differentiation of a wide variety of tissue including epithelia. The present study was undertaken to analyze expression of FGF-2 and its receptors FGFR-2 and FGFR-3 in 72 PMOLs, 108 OSCC and 52 healthy controls, and their role in risk assessment for malignant transformation of Leukoplakia (LKP) and Oral submucous fibrosis (OSMF) to OSCC. Immunohistochemistry was performed using antibodies against FGF-2, FGFR-2 and FGFR-3. IHC results were validated by Real Time PCR. Expression of FGF-2, FGFR-2 and FGFR-3 was upregulated from PMOLs to OSCC. While 90% (9/10) of PMOLs which showed malignant transformation (transformed) expressed FGF-2, only 24.19% cases (15/62) of PMOLs which were not transformed (untransformed) to OSCC expressed FGF-2. Similarly, FGFR-2 expression was seen in 16/62 (25.81%) of untransformed PMOLs and 8/10 (80%) cases of transformed PMOLs. FGFR-3 expression was observed in 23/62 (37.10%) cases of untransformed PMOLs and 6/10 (60%) cases of transformed PMOLs. A significant association of FGF-2 and FGFR-2 expression with malignant transformation from PMOLs to OSCC was observed both at phenotypic and molecular level. The results suggest that FGF-2 and FGFR-2 may be useful as biomarkers of malignant transformation in patients with OSMF and LKP.

Antihyperglycaemic and aldose reductase inhibitory potential of Acacia catechu hard wood and Tectona grandis leaves

The ethanolic extract of Acacia catechu hard wood (Ac) and the ethanolic as well as aqueous extracts of Tectona grandis leaves (Tg) showed marked antihyperglycaemic activity in both normoglycaemic and streptozotocin-induced diabetic rats at 250xa0mg/kg dose levels. The ethanolic and aqueous extracts of Ac and Tg also showed marked inhibition on AR from eye lens of normal rats with IC 50 values of 9.30 and 9.08 and 3.05, 4.51xa0μg/ml, respectively. The ethanolic as well as aqueous extracts of both Ac and Tg also showed marked inhibition on AR from eye lens of STZ-induced diabetic rats with IC50 values of 4.70 and 4.91 and 4.71 and 4.83xa0μg/ml. These results suggest that Acacia catechu hard wood and Tectona grandis leaves could be a new approach in the development of therapeutic or preventive agents for diabetic late-stage complications.

Acacia catechu hard wood: potential anti-diabetic cum anti-dyslipidemic

The ethanolic as well as aqueous extracts of the hard wood of Acacia catechu showed improvement on oral glucose tolerance post-sucrose load in normal rats and streptozotocin (STZ)-induced diabetic rats. Around 22 and 27% improvement in glucose tolerance was observed post 7 and 14 days of feeding the ethanolic extracts, respectively, on STZ-induced diabetic rats. Whereas around 17 and 26% improvement on glucose tolerance was observed post 7 and 14 days of feeding the ethanolic extract in the high fructose high fat diet (HFD) fed-low dosed STZ-treated rats. The ethanolic extract of A. catechu hard wood also showed marked anti-dyslipidemic activity on HFD fed Syrian golden hamster as evidenced by around 43 and 26% decline in serum triglycerides and total cholesterol, respectively. The ethanolic and aqueous extracts also showed marked inhibition on eye lens aldose reductase either from normal or STZ-induced diabetic rats. Further studies are warranted to isolate and identify the active ingredients from the ethanolic and aqueous extracts of A. catechu hard wood.

PI3K/Akt/mTOR signaling & its regulator tumour suppressor genes PTEN & LKB1 in human uterine leiomyomas.

Background & objectives: Despite their high occurrence and associated significant level of morbidity manifesting as spectrum of clinical symptoms, the pathogenesis of uterine leiomyomas (ULs) remains unclear. We investigated expression profile of tumour suppressor genes PTEN (phosphatase and tensin homolog deleted on chromosome ten) and LKB1 (liver kinase B1), and key signaling components of P13K (phosphatidylinositol 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) pathway in leiomyomas and adjacent normal myometrium in women of reproductive age, to explore the possibility of targeting this pathway for future therapeutic implications. Methods: Real time PCR (qPCR) was used to quantify relative gene expression levels of PTEN, Akt1, Akt2, mTOR, LKB1 and VEGFA (vascular endothelial growth factor A) in leiomyoma as compared to adjacent normal myometrium. Immunohistochemistry was subsequently performed to analyze expression of PTEN, phospho-Akt, phospho-mTOR, phospho-S6, LKB1 and VEGFA in leiomyoma and adjacent normal myometrium. Results: Significant upregulation of PTEN (2.52 fold; P=0.03) and LKB1 (3.93 fold; P=0.01), and downregulation of VEGFA (2.95 fold; P=0.01) genes were observed in leiomyoma as compared to normal myometrium. Transcript levels of Akt1, Akt2 and mTOR did not vary significantly between leiomyoma and myometrium. An increased immunoexpression of PTEN (P=0.015) and LKB1 (P<0.001) and decreased expression of VEGFA (P=0.01) was observed in leiomyoma as compared to myometrium. Immunostaining for activated (phosphorylated) Akt, mTOR and S6 was absent or low in majority of leiomyoma and myometrium. Interpretation & conclusions: Upregulation of PTEN and LKB1 in concert with negative or low levels of activated Akt, mTOR and S6 indicates that PI3K/Akt/mTOR pathway may not play a significant role in pathogenesis of leiomyoma.

Molecular and phenotypic expression of decorin as modulator of angiogenesis in human potentially malignant oral lesions and oral squamous cell carcinomas

BACKGROUNDnDecorin is an extracellular matrix, multifunctional small proteoglycan molecule in tumor stroma that has been shown to be modulator of angiogenesis. No clinical data is available so far on decorin expression and survival outcome of oral cancer.nnnAIMnThe aim of the present study was to examine molecular and phenotypic expression of two angiogenesis modulators viz. decorin and vascular endothelial growth factor-A (VEGF-A) in human potentially malignant oral lesions (PMOLs) and oral squamous cell carcinomas (OSCC) in relation to clinico-pathological variables and survival outcome.nnnMATERIALS AND METHODSnTissue biopsies were obtained from 72 PMOLs, 108 OSCC and 52 healthy controls. The PMOLs included cases of leukoplakias and oral submucous fibrosis. Immunohistochemistry was performed using antibodies against decorin, VEGF-A and CD-31. Messenger-ribonucleic acid (mRNA) expression was analyzed by using real-time polymerase chain reaction.nnnRESULTSnCytoplasmic staining of decorin was observed in the basal layer of epithelium in 53 (73.61%) cases of PMOLs and in peritumoral stroma in 55 (50.92%) cases of OSCC. None of the cases showed nuclear expression of decorin. Decorin expression both at phenotypic and molecular level was found to be down-regulated from PMOLs to OSCC. Lymph node metastasis and reduced decorin expression independently correlated with overall survival in OSCC. VEGF-A expression had no significant impact on survival outcome.nnnCONCLUSIONnMicro vessel density and VEGF-A expression were significantly associated with reduced decorin expression in tumor stroma suggesting, decorin as angiogenic modulator in OSCC. Down-regulation of decorin expression and the presence of lymph node metastasis were adverse factor independently affecting overall survival in OSCC.

Abstract A27: DNA methylation and transcriptional dysregulation of miRNA-137 and -193a in premalignant oral lesions and oral squamous cell carcinoma

Background: Oral squamous cell carcinoma (OSCC) is a multistep process arising through the progressive accumulation of multiple genetic and epigenetic events. Tobacco, bidi (tobacco flakes wrapped in a tendu leaf) smoking and alcohol consumption are established risk factors associated with OSCC and premalignant oral lesions (PMOLs) in Indian population. The microRNAs (miRNAs) are non-coding RNAs that leads to gene silencing at the post- transcriptional level by degradation or repression of target mRNA. The miRNA expression may be oncogenic or tumor suppressor and can be regulated epigenetically, either through DNA methylation or histone modification. Tumor suppressor miRNA-137 and -193a are epigenetically silenced in many cancers. In this study, we attempted to elucidate the miRNA methylation and expression in the patient of PMOLs [leukoplakia with (LKP) or without dysplasia (LKPD) and oral submucous fibrosis (OSMF)] and OSCC to determine the potential role of both miRNA as early predictive biomarkers. Both miRNAs methylation was finally correlated with the clinicopathological variables in Indian population. Material and Methods: Methylation-specific PCR for miR-137 and -193a methylation was performed in biopsy proven tissues; controls (n=34), PMOLs (n=84) and OSCC (n=84) and in corresponding blood samples. Mature miRNA expression was examined using quantitative reverse transcriptase PCR in tissue samples (10 controls, 30 PMOLs and 10 OSCC) using TaqMan chemistry based primers and probes. RNU44 was selected for normalization as an endogenous control for expression analysis. All experiments were performed in triplicate in a clinical setting of a tertiary care hospital. Results: We observed miR-137 methylation frequency of 48% in tissue and 35% in blood samples of OSCC, and 27% in tissue and 10% in blood samples of PMOL group, as compared to the control samples which showed methylation frequency of 17.6% in tissue and 2.9% in blood samples. Further, we found methylation of miR-193a in patients with OSCC, i.e., 49% in tissue and 39% in blood samples of OSCC group. In PMOL group, methylation frequency of miR-193a was observed in 27% of tissue samples and 10% of blood samples, respectively as compared to the controls which showed miR-193a methylation frequency of 14.7% in tissue and 2.9% in blood samples. The promoter methylation frequency was found higher in histologically well-differentiated OSCC as compared to moderately and poorly differentiated OSCC. Multinomial logistic regression showed tobacco and bidi smoking were significantly associated with methylation of both miRNAs in PMOLs and OSCC group. As compared to controls, significant downregulation of miRNA-137 was observed only in OSCC group (2.23 fold, p=0.049) whereas miRNA-193a was significantly downregulated in both PMOL (LKP: 3.45 fold, p=0.000; LKPD: 3.80 fold, p=0.034) and OSCC group (4.34 fold, p=0.002). Conclusion: Our results suggest that promoter DNA methylation of both miRNA-137 and -193a in tissue and corresponding blood samples of PMOLs and OSCC group may be predictive biomarkers for early detection and malignant risk in premalignant lesions. Taken together DNA methylation and miRNA downregulation, we concluded that both miRNAs are epigenetically silenced during oral carcinogenesis. Note: This abstract was not presented at the conference. Citation Format: Vikram Bhatia, Madhu Mati Goel, Annu Makker, S P Agarwal, Sandeep Kumar, Sudhir K Goel. DNA methylation and transcriptional dysregulation of miRNA-137 and -193a in premalignant oral lesions and oral squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A27.