Abstract B44: Radiation therapy induced a supra-additive cytotoxic effect in cervical cancer cell line when combined with mTOR inhibitors
Authors: Assad Daniele Xavier , Borges Gabriel Alvares , Avelino Samuel , Guerra Eliete Neves da Silva 
Published: May 2019

Abstract
The PI3K/AKT/mTOR signaling pathway is frequently activated in HPV-positive squamous cell carcinomas, such as cervical (CC) and head and neck tumors. This study aimed to investigate the biologic effects of mTOR inhibitors associated with radiation therapy (RT) in a cervical cancer cell line (HeLa). A human keratinocytes cell line (HaCaT) was used as control. Temsirolimus, everolimus, resveratrol, curcumin, and epigallocatechin gallate (EGCG) were the mTOR inhibitors used as treatment. MTT cell viability assay was performed to obtain dose-response curves. A nonlinear regression was applied to dose-response curves, in order to calculate the 50% cell cytotoxicity rate (CC50) for each treatment. To evaluate radiosensitization, cells were pretreated with mTOR inhibitors at CC50 followed by RT at 2Gy dose. Mann-Whitney test was used to compare the irradiated and nonirradiated groups. Cell death profile after treatment with temsirolimus, resveratrol, and curcumin was also assessed with flow cytometry. Kruskal-Wallis test with Dunn's post-test was applied on flow cytometry results to compare the control to the treated groups within each cell death profile. Everolimus, temsirolimus, EGCG, resveratrol, and curcumin were cytotoxic to the cervical cancer cell line. The lowest CC50 values after both 24 and 48 hours of treatment were seen in the groups of everolimus with HeLa (28.31 µM and 22 µM, respectively) and temsirolimus with HaCaT (26.54 µM and 12.85 µM). Resveratrol resulted in the highest CC50 values for both cell lines (325.8 µM and 144.6 µM for 24 and 48 hours in HeLa and 355.3 µM and 260.3 µM for 24 and 48 hours in HaCaT). An additive effect was observed when RT was associated with mTOR inhibitors. Cell viability at CC50 for the drug alone and combined to RT were: 64.44% and 33.88% for EGCG; 63.28% and 33.41% for resveratrol; 52.62% and 36.55% for curcumin; 54.06% and 35.04% for temsirolimus; 50.06% and 37.37% for everolimus. These results showed that radiation induced a statistically significant (p &lt; 0.01) supra-additive cytotoxic effect in the cervical cancer cell line when combined with mTOR inhibitors. A Tumor Selectivity Index (TSI) was calculated for each mTOR inhibitor. After a 24-hour treatment, only EGCG and resveratrol were more cytotoxic to HeLa than to HaCaT, with TSIs of 1.41 and 1.09, respectively. After 48 hours of treatment, resveratrol, curcumin, and everolimus were also more cytotoxic to HeLa when compared to HaCaT, with TSIs of 1.8, 3.25, and 3.77, respectively. The TSI results showed that resveratrol was selective for the cancer cell line in both 24 and 48 hours, even though its CC50 was very high. After 24 hours, temsirolimus induced late apoptosis or necrosis in HeLa cells. Based on these data, new studies with mTOR inhibitors as treatment options for cervical cancer are recommended, mainly combined to radiotherapy. Diet-derived mTOR inhibitors may be powerful strategies for the treatment of cervical cancer, particularly if its selectivity for cancer cell lines is confirmed in further studies. Considering the role of HPV oncoproteins and the activation of the PI3K/AKT/mTOR pathway in the pathogenesis of cervical cancer, mTOR inhibitors could be an important therapy in the drug armamentarium for cervical cancer.
Citation Format: Daniele Xavier Assad, Gabriel Alvares Borges, Samuel Avelino, Eliete Neves da Silva Guerra. Radiation therapy induced a supra-additive cytotoxic effect in cervical cancer cell line when combined with mTOR inhibitors [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B44.