Role and regulation of GLUT1/3 during oral cancer progression and therapy resistance.

OBJECTIVE: This study aimed to determine whether glucose transporter-1/3 (GLUT1/3) increased expression could contribute to oral tumor severity. Furthermore, this study detected whether GLUT1/3 mRNA/protein was associated with oncogenic transcription factors (HIF1α, AP1 and NFκB) and whether by blocking GLUT1 along with cisplatin could sensitize drug-resistant OSCC cells. DESIGN: We used 120 post-operated human tissue samples, including 35 primary tumors (PT), 43 invasive tumors (N1-3), 17 recurrent chemoradiation-resistant tumors (RCRT), and 25 PT-adjacent normal tissues (AN). The cisplatin-resistant (CisR-SCC4/9) cells were generated using a drug escalation strategy from parental SCC4/9 cells. The BAY-876 treatment blocked GLUT1 in OSCC cells. Western Blot, Immunohistochemistry, and reverse transcription polymerase chain reaction (RT-PCR) were used to detect various proteins and mRNA. Cell survival was determined by MTT assay. RESULTS: GLUT1/3 expression was observed more in PT over AN tissue (PT > AN), N1-3 > PT, and .RCRT > PT. GLUT1 expression was maximum in the RCRT group and CisR-SCC4/9 cells over their parental counterpart, linked with tumor size (p=0.0037) and loco-regional invasiveness (p=0.0422). GLUT1/3 mRNA/protein was correlated (positively) with oncogenic transcription factors (TFs) like HIF1α, AP1 and NFκB. We found the degree of positive correlation of these TFs with GLUT1/3 was in the order c-Jun > HIF1α > Fra-2 > NFκB > c-Fos. Treatment of BAY-876 and cisplatin-induced cell death in both CisR-SCC4/9 cells, possibly by triggering apoptosis and autophagy. CONCLUSION: Collectively, our results demonstrated increased GLUT1/3 overexpression linked with oral tumor severity like invasion and therapy resistance, and it was powered mainly by c-Jun (AP1). Blocking GLUT1 receptors and cisplatin application can sensitize CisR-OSCC cells.

Evidence-based effectiveness of herbal treatment modality for recurrent aphthous ulcers - A systematic review and meta-analysis.

Recurrent Apthous Ulcers (RAU) has affected mankind through time immemorial. It is the most commonly prevalent oral mucosal lesion manifesting as painful ulcers involving non - keratinised oral mucosa. This review was done to assess herbal intervention in RAU patients for outcomes of ulcer size and pain intensity. Literature search of published articles in Medline, Scopus, Ovid and Journal of Web upto August 2020 were reviewed for the pre-described outcomes. Revman 5.4 software was used for study analysis. Total 9 articles were finally chosen for qualitative analysis. Meta analytic comparison demonstrated the ulcer reduction (CI = -2.22 to - 0.09; p <0.001) and pain intensity (CI = -4.60 to - 0.08; p <0.001) was reduced in the herbal group as compared to the controls. A definite evidence of herbal intervention was noted in alleviating RAU signs and symptoms.

First Report of Stem Rot Caused by Athelia rolfsii on Curry Leaf Tree (Murraya koenigii) in Tripura, India.

Murraya koenigii is an important medicinal plant of India and commonly known as curry leaf tree grown in tropical and subtropical regions. The leaves of curry tree are used as a herb due to the presence of following important active constituent bismahanine, murrayanine, murrayafoline-A, bi-koeniquinone-A, murrayazolidine etc. (Jain et al. 2017). During mid-July 2019, stem rot disease symptoms were observed on curry leaf trees at the College of Agriculture, Lembucherra, Tripura (India). The disease symptoms consisted of rotting, wilting and blighting with disease incidence ranging from 8 to 10%. Initially, infected plants gradually withered and white mycelia mats appeared on the surface of the lower stem at the soil line. Infected stem samples were collected and surface was sterilized with 0.25% sodium hypochlorite for 1 min, washed thrice with sterilized distilled water and placed in Petri plates containing 2% water agar. After three days of incubation at 26°C, hyphae produced from plant bits were transferred into Petri plates containing potato dextrose agar. Ten isolates were collected from the diseases samples. Pure cultures were obtained as abundant, aerial and white mycelia with round to irregular sclerotia of 0.8 to 1.5 mm in diam. The sclerotia were initially white in color but later turned into brown color. The pathogen was identified as Athelia rolfsii based on morphology (Aycock 1966). To confirm the identification, the genomic DNA was extracted from a mycelia mat of the isolates using ZR fungal/Bacterial DNA miniprep kit (Irvine, CA) and the internal transcribed spacer (ITS) region of rDNA was amplified using the universal primers, ITS1 and ITS4 (White et al. 1990). A 550 bp PCR product was sequenced and showed 99% similarity with Athelia rolfsii isolate (GenBank accession MH854711).The generated sequence was submitted to GenBank (Accession MT535585). After identification of the pathogen a pot experiment was conducted to confirm the pathogenicity. Earthen pots (29 cm. diam.) were filled with sterilized soil and kept in a green house. Ten curry leaf plants (50 days old) were grown from seed in the separate pot were inoculated with 15-day old mycelia mats prepared in potato dextrose broth. The stem of each curry plant were artificially injured with the help of sterile blade and wrapped with moistened sterilized cotton containing the mycelial mat. Five curry leaf plants artificially injured and inoculated with sterilized distilled water were used as control. The Earthen pots with plants were individually covered with plastic bags and kept in the green house at 26°C for approximately 15 days. The inoculated plants started showing symptoms of stem rot six days after inoculation and started drying onward. The symptoms of stem rot on the inoculated plants were similar to those observed in the field. The fungus was re-isolated from the inoculated plants and A. rolfsii identification was confirmed based on morphology. No symptoms were observed on the control plants. The obtained culture was deposited in the Indian Type Culture Collection, Division of Plant Pathology, ICAR - Indian Agricultural Research Institute, New Delhi, India (ITC-8666). To the best of our knowledge this is the first report of stem rot disease of curry leaf plant caused by A. rolfsii in India and worldwide. Due to medicinal, flavour and aroma properties, it is regularly used in India. Curry leaf plant is regularly used as a medical herb in India and therefore this disease poses a significant risk to production.

Chronic Exposure to Chewing Tobacco Induces Metabolic Reprogramming and Cancer Stem Cell-Like Properties in Esophageal Epithelial Cells.

Tobacco in its smoke and smokeless form are major risk factors for esophageal squamous cell carcinoma (ESCC). However, molecular alterations associated with smokeless tobacco exposure are poorly understood. In the Indian subcontinent, tobacco is predominantly consumed in chewing form. An understanding of molecular alterations associated with chewing tobacco exposure is vital for identifying molecular markers and potential targets. We developed an in vitro cellular model by exposing non-transformed esophageal epithelial cells to chewing tobacco over an eight-month period. Chronic exposure to chewing tobacco led to increase in cell proliferation, invasive ability and anchorage independent growth, indicating cell transformation. Molecular alterations associated with chewing tobacco exposure were characterized by carrying out exome sequencing and quantitative proteomic profiling of parental cells and chewing tobacco exposed cells. Quantitative proteomic analysis revealed increased expression of cancer stem cell markers in tobacco treated cells. In addition, tobacco exposed cells showed the Oxidative Phosphorylation (OXPHOS) phenotype with decreased expression of enzymes associated with glycolytic pathway and increased expression of a large number of mitochondrial proteins involved in electron transport chain as well as enzymes of the tricarboxylic acid (TCA) cycle. Electron micrographs revealed increase in number and size of mitochondria. Based on these observations, we propose that chronic exposure of esophageal epithelial cells to tobacco leads to cancer stem cell-like phenotype. These cells show the characteristic OXPHOS phenotype, which can be potentially targeted as a therapeutic strategy.

Intracellular zinc flux causes reactive oxygen species mediated mitochondrial dysfunction leading to cell death in Leishmania donovani.

Leishmaniasis caused by Leishmania parasite is a global threat to public health and one of the most neglected tropical diseases. Therefore, the discovery of novel drug targets and effective drug is a major challenge and an important goal. Leishmania is an obligate intracellular parasite that alternates between sand fly and human host. To survive and establish infections, Leishmania parasites scavenge and internalize nutrients from the host. Nevertheless, host cells presents mechanism like nutrient restriction to inhibit microbial growth and control infection. Zinc is crucial for cellular growth and disruption in its homeostasis hinders growth and survival in many cells. However, little is known about the role of zinc in Leishmania growth and survival. In this study, the effect of zinc on the growth and survival of L.donovani was analyzed by both Zinc-depletion and Zinc-supplementation using Zinc-specific chelator N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) and Zinc Sulfate (ZnSO4). Treatment of parasites with TPEN rather than ZnSO4 had significantly affected the growth in a dose- and time-dependent manner. The pre-treatment of promastigotes with TPEN resulted into reduced host-parasite interaction as indicated by decreased association index. Zn depletion resulted into flux in intracellular labile Zn pool and increased in ROS generation correlated with decreased intracellular total thiol and retention of plasma membrane integrity without phosphatidylserine exposure in TPEN treated promastigotes. We also observed that TPEN-induced Zn depletion resulted into collapse of mitochondrial membrane potential which is associated with increase in cytosolic calcium and cytochrome-c. DNA fragmentation analysis showed increased DNA fragments in Zn-depleted cells. In summary, intracellular Zn depletion in the L. donovani promastigotes led to ROS-mediated caspase-independent mitochondrial dysfunction resulting into apoptosis-like cell death. Therefore, cellular zinc homeostasis in Leishmania can be explored for new drug targets and chemotherapeutics to control Leishmanial growth and disease progression.