Co-expression of galectin-3 and vimentin in triple negative breast cancer cells promotes tumor progression, metastasis and survival.

BACKGROUND: Lack of druggable targets and complex expression heterogeneity of known targets is common among TNBC subtypes. An enhanced expression of galectin-3 in TNBCs has already been documented. We have observed a tumor progression-dependent galectin-3 expression in TNBCs compared to adjacent epithelium and non TNBCs. OBJECTIVE: To unravel the association of galectin- 3 in tumor progression, aggressiveness and drug resistance in TNBC patients. METHODS: Galectin-3 expression in 489 breast cancer tissues was correlated with clinicopathological features and the results were validated in cell lines and mouse model by silencing galectin-3 using shRNA and the proteins were profiled by western blot and qRT-PCR. Protein interaction was analyzed by GFP Trap and Mass spectrometry. RESULTS: Galectin-3 expression correlated with tumor stage in TNBC and a lower galectin-3 expression was associated with poor patient survival. The positive correlation between galectin-3, vimentin and CD44 expression, pinpoints galectin-3 contribution to epithelial to mesenchymal transition, drug resistance and stemness. Vimentin was found as an interacting partner of galectin-3. Duplexing of galecin-3 and vimentin in patient samples revealed the presence of tumor cells co-expressing both galectin-3 and vimentin. In vitro studies also showed its role in tumor cell survival and metastatic potential, elementary for tumor progression. In vivo studies further confirmed its metastatic potential. CONCLUSIONS: Tumor progression dependent expression pattern of galectin 3 was found to indicate prognosis. Co-expression of galectin-3 and vimentin in tumor cells promotes tumor dissemination, survival and its metastatic capability in TNBCs.

Genomic perspective of triazole resistance in clinical and environmental Aspergillus fumigatus isolates without cyp51A mutations.

Aspergillus fumigatus is the most common etiologic agent of primarily all clinical manifestations of aspergillosis. A steady increase in the number of azole resistant A. fumigatus (ARAF) isolates from environment and clinical samples leading to therapeutic failures in clinical settings have alarmed the mycologists and clinicians worldwide. Although mutations in azole target cyp51A gene have been implicated in conferring azole resistance in A. fumigatus, recent studies have demonstrated occurrence of azole resistant strains without cyp51A mutations. In this study, next generation sequencing techniques and the expression profiling of transporter genes with single nucleotide polymorphisms (SNPs) in clinical and environmental ARAF isolates with (G54E) and without known cyp51A mutations was undertaken to understand the genetic background and role of transporters in azole resistance. The raw reads of four ARAF strains when mapped to Af293 reference genome (>100X depth) covered at least 93.1% of the reference genome. Among all four strains, a total of 212,711 SNPs was identified with 37,829 were common in at least two isolates. The expression analysis suggested the overexpression of MFS transporter, namely, mfsC in all ARAF isolates. None of the resistant strain showed significant upregulation of cyp51A and cyp51B gene. On the other hand, abcD was upregulated (5-fold) in the isolates with cyp 51A mutation (G54E). The whole genome sequence analysis showed the presence of two previously described amino acid substitutions S269F and F390Y in HMG1 gene in a clinical panazole resistant strain without cyp51A mutations. These mutations have been previously associated with azole resistance in A. fumigatus strains without cyp51A mutations. Further, several punctual mutations and a large-segment deletion among different strains were observed suggesting the involvement of resistance mechanisms other than cyp51A.

The role of inflammation in cervical cancer.

Knowledge regarding cervical cancer and human papillomavirus is expanding rapidly. Inflammation subsequent to viral infection is a driving force that accelerates cancer development. The infiltrated immune cells and their secretory cytokines along with chemokines and growth factors greatly contribute the malignant traits of cervical cancer. A better understanding of the mechanisms related to inflammation and cancer progression in terms of pathogen survival, cancer development, progression, and metastasis will lead to innovative approach for treating cancer.

Efficacy of a herbal mouthwash for management of periodontitis and radiation-induced mucositis - A consolidated report of two randomized controlled clinical trials.

BACKGROUND: Oral diseases like periodontitis and mucositis often require home care using topical agents in the form of mouthwashes. Many herbal mouthwashes are found to be beneficial; however lack proper scientific evidence to support their use. OBJECTIVES: Study 1 evaluated clinical efficacy of herbal mouthwash in the management of chronic periodontitis in comparison with chlorhexidine mouthwash. Study 2 aimed at assessment of herbal mouthwash in patients of radiation-induced mucosititis. METHODS: The novel herbal mouthwash used in the present study wa prepared from extracts of five plants namely Emblica Officinalis, Terminalia chebula, Terminalia bellerica, Glycyrrhiza glabra, and Azadirachta indica. 50 periodontitis patients were randomly allocated to two groups. As per allocation, they were instructed to use either herbal mouthwash or chlorhexidine mouthwash twice daily for two weeks after nonsurgical periodontal therapy. Similarly, patients with radiation-induced mucositis were randomly given herbal mouthwash and soda saline mouthwash. Intergroup and intragroup comparisons of continuous variables were conducted using paired and unpaired t-tests. Categorical variables were compared using the chi-square test. RESULTS: Significant reductions in gingival bleeding, plaque accumulation, and pocket depth were noticed in periodontitis patients in both groups. Patients reported acceptable taste, freshness, and satisfaction after the use of herbal mouthwash. The herbal mouthwash group noticed a significant reduction in the severity of radiation-induced mucositis and analgesic requirements. The intensity of pain, dryness of mouth, oral hygiene, and need for the use of antibiotic and antifungal during radiotherapy was not significant among the groups. CONCLUSION: The results of this preliminary clinical trial support the use of the tested herbal formulation mouthwash as an adjunct in the treatment of periodontitis as well as radiation-induced mucositis. CLINICAL TRIAL REGISTRATION NUMBER: For Study 1: CTRI/2019/10/021574, Study 2: CTRI/2020/04/024851.

Evaluation of immune response to single dose of quadrivalent HPV vaccine at 10-year post-vaccination.

BACKGROUND: The recent World Health Organization recommendation supporting single-dose of HPV vaccine will significantly reduce programmatic cost, mitigate the supply shortage, and simplify logistics, thus allowing more low- and middle-income countries to introduce the vaccine. From a programmatic perspective the durability of protection offered by a single-dose will be a key consideration. The primary objectives of the present study were to determine whether recipients of a single-dose of quadrivalent HPV vaccine had sustained immune response against targeted HPV types (HPV 6,11,16,18) at 10 years post-vaccination and whether this response was superior to the natural antibody titres observed in unvaccinated women. METHODS: Participants received at age 10-18 years either one, two or three doses of the quadrivalent HPV vaccine. Serology samples were obtained at different timepoints up to 10 years after vaccination from a convenience sample of vaccinated participants and from age-matched unvaccinated women at one timepoint. The evolution of the binding and neutralizing antibody response was presented by dose received. 10-year durability of immune responses induced by a single-dose was compared to that after three doses of the vaccine and in unvaccinated married women. RESULTS: The dynamics of antibody response among the single-dose recipients observed over 120 months show stabilized levels 18 months after vaccination for all four HPV types. Although the HPV type-specific (binding or neutralizing) antibody titres after a single-dose were significantly inferior to those after three doses of the vaccine (lower bounds of GMT ratios < 0.5), they were all significantly higher than those observed in unvaccinated women following natural infections (GMT ratios: 2.05 to 4.04-fold higher). The results correlate well with the high vaccine efficacy of single-dose against persistent HPV 16/18 infections reported by us earlier at 10-years post-vaccination. CONCLUSION: Our study demonstrates the high and durable immune response in single-dose recipients of HPV vaccine at 10-years post vaccination.

An ex vivo evaluation of the efficacy of andrographolide in modulating differential expression of transcription factors and target genes in periodontal cells and its potential role in treating periodontal diseases.

ETHANOPHARMACOLOGICAL RELEVANCE: Andrographolide is a herbal extract traditionally used in South Asian countries for treating inflammatory diseases. AIM OF THE STUDY: To evaluate the efficacy of andrographolide in management of periodontal disease which is a highly prevalent oral disease. MATERIALS AND METHODS: Periodontal ligament fibroblasts (PDLF) were cultured from healthy and diseased periodontium using explant culture methods. The safe dose of AG was determined using MTT assay. LPS (lipopolysaccharide) of the most important periodontopathogen, P gingivalis was used to activate NF-κB and STAT3 in PDLF. The efficacy of AG in inhibiting NF-κB and STAT3 was analyzed using immunofluorescence. Down regulation of expression of target genes of these transcription factors related to inflammation and bone resorption were analyzed using real time PCR. RESULTS: AG up to the concentration of 25µM was found to be safe as determined by MTT assay. Statistically significant activation of NF-κB and STAT3 in cultured PDLF was observed in diseased group compared to healthy controls before and after LPS challenge. 5µM AG pretreatment significantly inhibited activation of NF-κB and STAT3 and down regulated expression of inflammatory and bone resorptive genes in cultured PDLF. CONCLUSIONS: The findings of the present study propose the adjunctive use of a novel herbal drug andrographolide as a promising host modulation agent for periodontal therapy by inhibiting NF-κB and STAT3 activation and inhibition of inflammation and bone resorption related genes.

Single nucleotide polymorphisms of DNA repair genes XRCC1 and XPD and its molecular mapping in Indian oral cancer.

Tobacco users with diminished ability to repair somatic mutations may be more susceptible to tobacco attributable cancers. The distribution of single nucleotide polymorphisms (SNPs) in DNA repair genes XRCC1 and XPD in 110 oral carcinoma cases, 84 leukoplakia and 110 controls belonging to the Travancore South Indian population were examined. SNPs investigated included Arg194Trp, Arg280His, and Arg399Gln of the XRCC1 gene and Lys751Gln of the XPD gene. In addition, one of the variants positions, A399G, was mapped onto the BRCT I domain model built by comparative modeling (threading). Presence of the polymorphic variant of XRCC1 codon 194 and 399 and XPD was associated with increased risk of oral cancer compared to the wild genotype. Smokers and betel quid chewers with the variant allele of XRCC1 399 codon and XPD also exhibited increased risk of oral cancer. The A399G variant position mapped onto the surface of the BRCT I domain provides a possible rationale for altered XRCC1 function. These results suggest that polymorphisms in functionally important repair genes, specifically, those that map onto the protein surface may alter protein function without significantly affecting its structure.

Interleukin 2 production in lymphocyte cultures: a rapid test for cancer-associated immunodeficiency in malignant cervical neoplasia.

Peripheral blood lymphocytes of patients with malignant cervical neoplasia were studied for their capacity to produce interleukin-2 (IL-2) upon in vitro mitogen stimulation. Patients with early disease (Stage I/IIA) showed levels comparable to normal controls but those with advanced disease showed reduced levels, the reduction increasing with tumour load. These findings excellently correlated with our previous observations on a depressed cellular immunity in these patients. On follow up of the patients it was noticed that those who developed recurrent disease showed sustained lower levels of IL-2 production compared to those who remained disease free. These results stress the importance of a deranged immune system in these patients and also shows the clinical and prognostic value of such an assay.

Immune reactive proteins as prognostic and clinical markers in malignant cervical neoplasia.

The significance of immune reactive proteins in malignant cervical neoplasia, with particular reference to its prognostic and clinical relevance, was studied in 229 patients. All stages of the disease showed elevated levels of circulating immune complexes (CIC). Patients with stages I/IIA showed elevated levels of IgG, whereas those with stages III and IV showed depressed IgM levels. In the case of IgA, the situation was reversed: patients with advanced disease showed increased levels. Radiotherapy resulted in highly elevated levels of CIC, IgG, and IgA. However, in patients who remained disease-free after radiation treatment, levels of these proteins came down, often falling below pretreatment levels. On the other hand, patients developing recurrent disease showed sustained, high levels of CIC and IgA, with IgG as an isolated case in stage I/IIA. The present study therefore shows that although these increases were non-tumour-specific, patients with persistent elevation of CIC and IgA levels were at much greater risk of developing recurrent disease, thereby proving the merit of these substances as markers in prognosticating the course of disease in malignant cervical neoplasia.

Radiation associated eosinophilia and monocytosis in carcinoma of the uterine cervix: a simple reliable clinical and prognostic indicator.

Clinical and prognostic significance of radiation associated eosinophilia (RAE) and radiation associated monocytosis (RAM) was evaluated in 176 patients with squamous cell carcinoma of the uterine cervix followed up after radiotherapy. Significant RAE was noticed in patients with Stages I/IIA and IIB who remained disease-free after treatment. On the contrary, patients belonging to these two stages but who developed recurrent disease showed absence of RAE. These patients as well as those belonging to Stage III showed considerably higher levels of RAM as compared to those patients remaining disease-free. These observations showed the usefulness of these two simple inexpensively carried out parameters in prognosticating disease course in malignant cervical neoplasia.

Oncogenic microRNAs as biomarkers of oral tumorigenesis and minimal residual disease.

OBJECTIVES: Classical diagnostic methods are not sensitive enough in detecting oral lesions that may progress to cancer and in assessing minimal residual disease (MRD) in oral surgical margins. Altered expression of microRNAs (miRNAs) contributes to human cancer, including oral cancer. Although there are many studies on microRNAs in oral cancer, there is no reported study comparing the expression of microRNAs during oral tumor progression and in oral surgical margins. MATERIALS AND METHODS: This study analyzed the expression of 72 miRNAs that were reported (till June 2011) to be differentially expressed in oral cancer, during phases of oral cancer progression and in oral surgical margins. RESULTS: Of the 72 miRNAs analyzed, four (hsa-miR-125a, hsa-miR-184, hsa-miR16 and hsa-miR-96) showed a common pattern of expression in both sets of tissues. We further analyzed the downstream target genes of hsa-miR-16 BCL2 and CCND1. The in silico network analysis of these four microRNAs and their target genes revealed presence of genes involved in tumor progression and transcription factors. CONCLUSIONS: The findings suggest that the combinatorial regulation by these miRNAs and their target transcription factors might play a substantial role in oral tumorigenesis. Here we report for the first time that a decreased expression of hsa-miR-125a, hsa-miR-184 and hsa-miR-16 and an increased expression of hsa-miR-96 could be useful in predicting oral tumorigenesis and importantly in the detection of MRD and decision-making process for postoperative treatment modalities.

Identification of heat shock protein 90 inhibitors to sensitize drug resistant side population tumor cells using a cell based assay platform.

Current cancer therapeutics are identified based on initial tumor regression screens that mostly kill differentiated tumor cells, sparing the rare cancer stem cells (CSCs). Being rare and difficult to characterize, it remains a challenge to identify compounds active against them. Side population (SP) cells identified in multiple cancer cell line panels expressing mitochondrial Cytochrome C-EGFP were evaluated for identifying possible drug candidates utilizing high-throughput imaging. We identified heat shock protein 90 inhibitors as potential agents to sensitize SP cells to anticancer drugs. Hsp90 inhibitors induced down regulation of Akt leading to proteasomal degradation of survivin and consequent mitochondrial apoptosis. A successful screening platform for identifying compounds targeting drug resistant side population cells was developed.

Meso-tetrakis(p-sulfonatophenyl)N-confused porphyrin tetrasodium salt: a potential sensitizer for photodynamic therapy.

A water-soluble derivative of N-confused porphyrin (NCP) was synthesized, and the photodynamic therapeutic (PDT) application was investigated by photophysical and in vitro studies. High singlet oxygen quantum yield in water at longer wavelength and promising IC(50) values in a panel of cancer cell lines ensure the potential candidacy of the sensitizer as a PDT drug. Reactive oxygen species (ROS) generation on PDT in MDA-MB 231 cells and the apoptotic pathway of cell death was illustrated using different techniques.

Coexpression of Notch1 and NF-kappaB signaling pathway components in human cervical cancer progression.

OBJECTIVES: Features of deregulated Notch1 signaling and NF-kappaB activation have independently been reported in cervical cancers. Here, we have extended these observations and examined both these pathways simultaneously in human cervical cancer tissue. Further, we have investigated the potential cross-talk between these pathways in a human cervical cancer derived cell line CaSki, which mirrors features of Notch activation as in the majority of human cervical cancers. METHODS: Cervical tissue samples were analyzed for the expression of Notch1, Jagged 1, Hes1, pAKT, NF-kappaB p50, NF-kappaB p65, IkappaB-alpha, Bcl-2, CyclinD1, Cdk9, c-Fos, and p53 by immunohistochemistry. A total of 352 samples were analyzed which included 69 normal cervical tissue, 132 preinvasive lesions and 151 squamous cell carcinomas of the uterine cervix. Dual immunofluorescent analysis was performed to evaluate the coexpression of Notch1 and NF-kappaB. Transcriptional reporter assays and xenografts were undertaken with CaSki cells. RESULTS: Features of Notch1 activation as measured by intracellular Notch1, high levels of Jagged1, Hes1 and Cdk9 were paralleled by nuclear translocation of both NF-kappaB p50 and p65 with target gene expression (IkappaB-alpha, Bcl-2, and CyclinD1) in human cervical cancer sections. Reporter assays in CaSki cells are consistent with Notch being an upstream regulator of NF-kappaB. Further, the xenografts recreate key aspects of human cancer tissue. CONCLUSIONS: Results from this study suggest that there is a co-activation of Notch1 and NF-kappaB signaling pathways at the cellular level in the majority of human cervical cancers, with Notch as an upstream regulator.

Genetic polymorphism of CYP1A1, CYP2D6, GSTM1 and GSTT1 and susceptibility to acute lymphoblastic leukaemia in Indian children.

BACKGROUND: Biotransformation plays a crucial role in carcinogen activity and many genetic polymorphisms in xenobiotic metabolising enzymes have been associated with an increased risk of cancer. Such polymorphisms can lead to considerable variation in the activities of these enzymes, which are crucial in carcinogen and drug metabolism. These variations could play a role in the risk of developing paediatric acute lymphoblastic leukaemia (ALL) by their varying action on environmental carcinogens. PROCEDURE: The present study looked for two polymorphisms (m1 and m2) in the CYP1A1, CYP2D6*4 genes and deletions of the glutathione S-transferases (GSTM1 and GSTT1) in 118 paediatric ALL patients and 118 age matched control children. The polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to study gene polymorphisms. RESULTS: In children with ALL, CYP1A1 m1 polymorphism was evident in 42.4% of subjects and CYP1A1 m2 in 37.3%. These were significantly different from the results obtained for control children (20.3% for CYP1A1 m1 and 19.5% for m2). Subjects with CYP1A1 m1 homozygous variant had a sixfold risk and CYP1A1 m2 a fourfold risk. In contrast, CYP2D6*4 was more prevalent in the controls than in the cases. Subjects with GSTM1 deletions had increased risk of ALL (OR = 2.1, P = 0.009). The odds ratios for both CYP1A1 m1 and m2 homozygous polymorphisms being associated with childhood ALL was 5.67 (95% CI = 2.11-15.27). The odds ratios for both GSTM1 and GSTT1 deletions being associated with ALL was 2.78 (95% CI = 0.67-11.56). CONCLUSIONS: These results suggest that genetic polymorphisms of xenobiotic metabolising enzymes appear to influence susceptibility to childhood ALL.

Development of an immunological staging system to prognosticate disease course in malignant cervical neoplasia.

A multiparameter analysis of immune function was done on patients with squamous cell carcinoma of the uterine cervix to look for any variable(s) that could be correlated with the clinical stage of the disease. Five immunological variables, viz., CD4+ lymphocytes, CD4/CD8 ratio, natural killer cells, concanavalin A-induced suppressor index, and circulating immune complexes, were found to consistently vary with tumor load. When these variables were subjected to a multiple regression and multivariate analysis, an equation for a diagnostic index curve was derived. Application of this equation led to an immunological staging system which could be used as an excellent prognostic indicator. The immunological staging system showed that patients classified into a particular clinical (FIGO) stage behaved in a heterogeneous way immunologically and that patients developing recurrent disease could easily be identified from those remaining disease free, even before treatment. Subsequent follow-up of these patients further confirmed this observation, with the recurrent disease group easily identifiable. These results point out the immense potential of such a staging system and the importance of immunological evaluation in the preliminary management of patients with malignant cervical neoplasia.

Interleukin 2 and alpha interferon induced in vitro modulation of spontaneous cell mediated cytotoxicity in patients with cancer of the uterine cervix undergoing radiotherapy.

In vitro modulation of spontaneous cell mediated cytotoxicity by interferon and interleukin 2 was carried out using peripheral blood lymphocytes from patients with cancer of the uterine cervix before and at different intervals after commencement of radiation treatment. A total of 150 patients with various stages of the disease were included and cytotoxicity was measured using the single cell cytotoxic assay. These results indicate a beneficial effect in vitro of interleukin 2 and interferon in augmenting spontaneous cell mediated cytotoxicity, a possibly vital antitumour immune mechanism in patients with relatively early cervix cancer. Natural killer cell, lymphokine activated killer cell and interferon activated killer cell activity was depressed immediately following radiotherapy. The activity of these cell types later on increased above pretreatment levels in patients with stages I, IIA and IIB. A similar rebound above pretreatment levels was not observed in patients with stages III and IV.