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Abstract
The aim of the study was to analyze the effect of hTERT gene knockdown in HNSCC cells by using novel in vitro models of head and neck cancer (HNSCC), as well as improving its personalized therapy. To obtain the most efficient knockdown siRNA, shRNA-bearing lentiviral vectors were used. The efficiency of hTERT silencing was verified with qPCR, Western blot, and immunofluorescence staining. Subsequently, the type of cell death and DNA repair mechanism induction after hTERT knockdown was assessed with the same methods, followed by flow cytometry. The effect of a combined treatment with hTERT gene knockdown on Double-Strand Breaks levels was also evaluated by flow cytometry. Results showed that the designed siRNAs and shRNAs were effective in hTERT knockdown in HNSCC cells. Depending on a cell line, hTERT knockdown led to a cell cycle arrest either in phase G1 or phase S/G2. Induction of apoptosis after hTERT downregulation with siRNA was observed. Additionally, hTERT targeting with lentiviruses, followed by cytostatics administration, led to induction of apoptosis. Interestingly, an increase in Double-Strand Breaks accompanied by activation of the main DNA repair mechanism, NER, was also observed. Altogether, we conclude that hTERT knockdown significantly contributes to the efficacy of HNSCC treatment.
Highlights
Malignant tumors of the head and neck are the sixth leading cancer worldwide, accounting for approximately 600,000 cases per year with the number of deaths reaching almost to 380,000
A significant telomere attrition was detected in the FaDu cell line after hTERT knockdown was conducted via siRNA (59%, p = 0.0134) and shRNA (79%, p = 0.0083) when compared to control samples
We found that FaDu cells with hTERT knockdown present an increased level of γH2AX when compared to control after irradiation with 1 and 2 Gy dose on day 3 (1.36 MFI, p = 0.0223 and 1.55 MFI, p = 0.0037, respectively)
Summary
Malignant tumors of the head and neck are the sixth leading cancer worldwide, accounting for approximately 600,000 cases per year with the number of deaths reaching almost to 380,000. In order to maximize radicalization of anti-tumor therapy, a combination of local treatments (surgery, radiotherapy) with chemotherapy is commonly used. Such an approach improves patients’ outcomes and increases overall survival[2]. The construction of a safe, efficient, and universal system is still a challenge[8,9,10,11] In this project we analyzed the efficiency and potency of RNA interference (siRNA and shRNA) directed against hTERT in order to eliminate cancer cells. Due to the complex nature of carcinogenesis processes and the contribution of many factors to the control of tumor growth, the possibility of using shRNA against telomerase may very well provide a novel approach when administrating cytostatics and/or radiation therapy. It might lead to a reduction in drug and radiation doses, and a decrease in harmful side effects
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