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.
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.
In India, oral cancer is the most common cancer in males and ranks third among females, attributed predominantly to the use of smokeless tobacco. Radiotherapy is an integral part of oral cancer treatment either alone or in combination with surgery and chemotherapy. However the development of radioresistance creates a hurdle in the efficacy of radiotherapy. Therefore, exploring the differential molecular profile of established radioresistant versus parental oral cancer cells may help in predicting the clinical effectiveness of radiotherapy. The present study aims to profile the radioresistant cell lines established by low dose radiation from their parental oral cancer cell lines, to identify the molecules and the possible mechanisms associated with radioresistance in oral cancer. We have established three radioresistant oral cancer cell lines i.e. 70Gy-AW13516, 70Gy-AW8507 and 70Gy-SCC029B derived from their parental AW13516, AW8507 and SCC029B cell lines respectively using in-vitro fractionated ionizing radiation (FIR). A clinically admissible 2Gy radiation dose was given using a 60Co-γ linear accelerator (Bhabhatron-2, ACTREC) upto 70Gy totally for each of the cell lines. The radioresistant character was determined by clonogenic cell survival assay and proteomic profiling of parental versus radioresistant cells was done by two-dimensional gel electrophoresis. Gels were analysed by PD-Quest software (Bio-Rad) and differential spot identities were revealed by MALDI-TOF/TOF (Bruker Ultra flex-II). To explore changes at transcript level; cDNA microarray was performed by Affymetirx Gene Chip array and analysed by Gene Spring GX-12.5 software. Mass spectrometry (MS) identified a total of 106 differentially expressed proteins among the three replicate sets of each parental and radioresistant 2-D gels with significant MS/MS score. Some of the differentially expressed proteins like Prohibitin, Moesin, PCNA, Keratin-8, Vimentin, HSP-70 and 14-3-3 sigma were validated by western blotting. We further, transiently knocked down Moesin expression by siRNA strategies and demonstrated a significant decrease in the radioresistant character of 70Gy-AW13516 cells as compared to control siRNA treated cells, indicating a role for Moesin in acquired radioresistance of oral cancer cells. Also differentially expressed genes (>3 fold) of radioresistant cells, were mapped by David (NIH) & Panther (USC) functional tools. The pathway analysis revealed genes related to cell survival (PI3K, P38), Wnt and Apoptosis signalling. The proteomic and transcriptomic profiling of radioresistant oral cancer cell lines have revealed several radioresistance associated molecules that may be utilised in predicting cellular response to radiotherapy and the pathways identified may provide new insights in understanding the mechanism underlying clinical radioresistance in oral cancers. Citation Format: Tanuja R. Teni, Mohd Yasser, Sagar Pawar. Identification of potential molecular targets related to radioresistance in human oral cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3320. doi:10.1158/1538-7445.AM2015-3320
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