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Background: Translationally controlled tumour protein (TCTP) is a multifunctional protein elevated in multiple cancers. However, studies on its role in oral carcinogenesis and prognosis are rare. We recently reported the role of its interacting partner, MCL1, in oral cancer progression and outcome. Hence, the present study aimed to assess TCTP expression in oral tumorigenesis and its association with patient outcomes alone and in combination with MCL1. Methods: TCTP expression was assessed by immunohistochemistry and immunoblotting in oral tissues and cells, respectively. Cell viability post siRNA/dihydroartemisinin treatment was analysed by tetrazolium salt assay. Cell survival, invasion and tumorigenic potential post TCTP knockdown were assessed by clonogenic, Matrigel and soft‐agar assays, respectively. The association of TCTP with patient outcome was analysed by Kaplan–Meier and Cox regression. Results: TCTP was significantly overexpressed in oral premalignant lesions (p < 0.0001), oral tumours (p < 0.0001) and oral dysplastic and cancer cells versus normal oral mucosa and also in recurrent (p < 0.05) versus non‐recurrent oral tumours. Further, elevated TCTP was significantly (p < 0.05) associated with poor recurrence free survival (RFS) and poor overall survival (OS; hazard ratio = 2.29; p < 0.05). Intriguingly, the high co‐expression of TCTP and MCL1 further reduced the RFS (p < 0.05) and OS (p < 0.05; hazard‐ratio = 3.49; p < 0.05). Additionally, TCTP knockdown decreased survival (p < 0.05), invasion (p < 0.01) and in vitro tumorigenic potential (p < 0.0001). Dihydroartemisinin treatment reduced TCTP levels and viability of oral cancer cells. Conclusion: Our studies demonstrate an oncogenic role of TCTP in oral cancer progression and poor outcome. Thus, TCTP may be a potential prognostic marker and therapeutic target in oral cancers. [ABSTRACT FROM AUTHOR]

Simple Summary: Our electron microscopic (EM) findings indicate that hydrodynamic based plasmid transfer to the hepatocytes possibly occurs via active or passive endocytosis with the plasmid entrapment in membrane bound multiple vesicle and vacuoles. Our ultramorphological study on the vacuole containing mouse hepatocytes did not show disrupted plasma membrane or other signs of cellular damage, thus keeping out the mechanism like membrane poration, but cannot be entirely excluded at the micro level. Independent of the plasmid itself or its enclosed components, we have also found the vesicle formations is a non-specific process. Therefore, it remains to be clarified as how the active or passive mechanisms are involved, leading to the vesicle generation and DNA-entry to the nucleus of the hepatocytes. Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of live mice or rats. The DNA constructs are composed of coupled plasmids, while one contains the gene of interest that stably integrate into the hepatocyte genome with help of the other consisting sleeping beauty transposase system. The rapid injection of a large volume of DNA-solution through the tail vein induces an acute cardiac congestion that refluxed into the liver, mainly in acinus zone 3, also found through our EM study. Although, HT mediated hydrodynamic force can permeabilizes the fenestrated sinusoidal endothelium of liver, but the mechanism of plasmid incorporation into the hepatocytes remains unclear. Therefore, in the present study, we have hydrodynamically injected 2 mL volume of empty plasmid (transposon vector) or saline solution (control) into the tail vein of anesthetized C57BL/6J/129Sv mice. Liver tissue was resected at different time points from two animal group conditions, i.e., one time point per animal (1, 5, 10–20, 60 min or 24 and 48 hrs after HT) or multiple time points per animal (0, 1, 2, 5, 10, 20 min) and quickly fixed with buffered 4% osmium tetroxide. The tissues fed with only saline solution was also resected and fixed in the similar way. EM evaluation from the liver ultrathin sections reveals that swiftly after 1 min, the hepatocytes near to the central venule in the acinus zone 3 shows cytoplasmic membrane-bound vesicles. Such vesicles increased in both numbers and size to vacuoles and precisely often found in the proximity to the nucleus. Further, EM affirm these vacuoles are also optically empty and do not contain any electron dense material. Although, some of the other hepatocytes reveals sign of cell damage including swollen mitochondria, dilated endoplasmic reticulum, Golgi apparatus and disrupted plasma membrane, but most of the hepatocytes appeared normal. The ultrastructural findings in the mice injected with empty vector or saline injected control mice were similar. Therefore, we have interpreted the vacuole formation as nonspecific endocytosis without specific interactions at the plasma membrane. [ABSTRACT FROM AUTHOR]

Radiotherapy is an important treatment modality for oral cancer. However, development of radioresistance is a major hurdle in the efficacy of radiotherapy in oral cancer patients. Identifying predictors of radioresistance is a challenging task and has met with little success. The aim of the present study was to explore the differential spectral profiles of the established radioresistant sublines and parental oral cancer cell lines by Raman spectroscopy. We have established radioresistant sublines namely, 50Gy-UPCI:SCC029B and 70Gy-UPCI:SCC029B from its parental UPCI:SCC029B cell line, by using clinically admissible 2Gy fractionated ionizing radiation (FIR). The developed radioresistant character was validated by clonogenic cell survival assay and known radioresistance-related protein markers like Mcl-1, Bcl-2, Cox-2 and Survivin. Altered cellular morphology with significant increase (p<0.001) in the number of filopodia in radioresistant cells with respect to parental cells was observed. The Raman spectra of parental UPCI:SCC029B, 50Gy-UPCI:SCC029B and 70Gy-UPCI:SCC029B cells were acquired and spectral features indicate possible differences in biomolecules like proteins, lipids and nucleic acids. Principal component analysis (PCA) provided three clusters corresponding to radioresistant 50Gy, 70Gy-UPCI:SCC029B sublines and parental UPCI:SCC029B cell line with minor overlap, which suggest altered molecular profile acquired by the radioresistant cells due to multiple doses of irradiation. The findings of this study support the potential of Raman spectroscopy in prediction of radioresistance and possibly contribute to better prognosis of oral cancer. [ABSTRACT FROM AUTHOR]

Objective: In the rat, the pancreatic islet transplantation model is an established method to induce hepatocellular carcinomas (HCC), due to insulin-mediated metabolic and molecular alterations like increased glycolysis and de novo lipogenesis and the oncogenic AKT/mTOR pathway including upregulation of the transcription factor Carbohydrate-response element-binding protein (ChREBP). ChREBP could therefore represent an essential oncogenic co-factor during hormonally induced hepatocarcinogenesis. Methods: Pancreatic islet transplantation was implemented in diabetic C57Bl/6J (wild type, WT) and ChREBP-knockout (KO) mice for 6 and 12 months. Liver tissue was examined using histology, immunohistochemistry, electron microscopy and Western blot analysis. Finally, we performed NGS-based transcriptome analysis between WT and KO liver tumor tissues. Results: Three hepatocellular carcinomas were detectable after 6 and 12 months in diabetic transplanted WT mice, but only one in a KO mouse after 12 months. Pre-neoplastic clear cell foci (CCF) were also present in liver acini downstream of the islets in WT and KO mice. In KO tumors, glycolysis, de novo lipogenesis and AKT/mTOR signalling were strongly downregulated compared to WT lesions. Extrafocal liver tissue of diabetic, transplanted KO mice revealed less glycogen storage and proliferative activity than WT mice. From transcriptome analysis, we identified a set of transcripts pertaining to metabolic, oncogenic and immunogenic pathways that are differentially expressed between tumors of WT and KO mice. Of 315 metabolism-associated genes, we observed 199 genes that displayed upregulation in the tumor of WT mice, whereas 116 transcripts showed their downregulated expression in KO mice tumor. Conclusions: The pancreatic islet transplantation model is a suitable method to study hormonally induced hepatocarcinogenesis also in mice, allowing combination with gene knockout models. Our data indicate that deletion of ChREBP delays insulin-induced hepatocarcinogenesis, suggesting a combined oncogenic and lipogenic function of ChREBP along AKT/mTOR-mediated proliferation of hepatocytes and induction of hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

Radiotherapy is an integral part of oral cancer treatment, either alone or in combination with surgery. But, during radiotherapy, oral tumours of a subset of patients develop radioresistance creating a major obstruction towards its efficacy. The aim of this study was to establish radioresistant cell lines from different oral subsites using clinically admissible low dose radiation and profile them by proteomic and transcriptomic approaches to identify proteins/genes associated with radioresistance in oral cancer. We have established three radioresistant oral cancer cell lines; 70Gy-AW13516, 70Gy-AW8507 (Tongue-SCC) and 70Gy-SCC029B (Buccal-SCC). Radiation was given in 2Gy dose by 60Co-? linear accelerator (Bhabhatron-2) up to total dose of 70Gy to each and radioresistant character was assessed by clonogenic assay. Proteomic profiling of parental/resistant cells was done by 2D-gel electrophoresis. Gels were analysed by PDQuest software and differential spot identities revealed by MALDITOF/ TOF. To explore changes at transcript levels; cDNA microarray was performed by Affymetirx Gene Chip array and analysed by Gene Spring GX-12.5. MS identified 106 differential proteins among three parental/radioresistant cell lines with significant MS/MS score. A panel of 8 common proteins across three sets; GRP78, STIP1, PGP, PKM2, GRP94, PDIA3, HSP70-1A/B and HSPA8 were selected and validated by Real time-PCR. Further, statistically significant genes (>3 fold) from radioresistant cells of different sub sites (Tongue & Buccal) were mapped by David & Panther functional tools. Pathway analysis revealed genes related to altered cell survival & apoptosis pathways like; Apoptosis signalling, PI3K, P38 and Wnt signalling. Proteomic and transcriptomic profiling of established oral radioresistant cells may provide new insights into the mechanisms underlying clinical radioresistance & help in identifying radioresistance related biomarkers. [ABSTRACT FROM AUTHOR]