Mechanistic investigation of synergistic interaction of tocopherol succinate with a quinoline-based inhibitor of mammalian target of rapamycin

Abstract

Objectives Cancer monotherapy is associated with various limitations; therefore, combination chemotherapy is widely explored for optimum drug efficacy. In this study, 4-(N-Phenyl-N′-substituted benzenesulfonyl)-6-(4-hydroxyphenyl) quinoline-based mammalian target of rapamycin (mTOR) inhibitor (IIIM-4Q) was investigated in combination with tocopherol succinate (TOS), and the mechanism of cytotoxicity was elucidated. Methods The cytotoxic potential of IIIM-4Q and TOS was evaluated in five cell lines. Further, to understand the mechanism of cytotoxicity of IIIM-4Q, TOS and their combination, various studies including morphological analysis using scanning electron microscopy and 6-diamidino-2-phenylindole (DAPI) staining, estimation of reactive oxygen species (ROS) level, measurement of mitochondrial membrane potential (MMP), in-vitro cell migration assay, Western blotting and staining with acridine orange (AO) for autophagy detection were performed. Key findings Investigated combination was synergistic in nature and exhibited greater oxidative stress and mitochondrial dysfunction in pancreatic cancer cells. The migration potential of MIA PaCa-2 cells was significantly mitigated under the influence of this combination, and morphological changes such as chromatin condensation and nuclear blebbing were observed. Also, poly (adenosine diphosphate-ribose) polymerase cleavage and caspase-3 activation were observed in IIIM-4Q and TOS combination-treated cells. Conclusions The investigated combination synergistically inhibited proliferation of MIA PaCa-2 cells through simultaneous induction of autophagy followed by apoptosis, and this combination demonstrated potential for further translational studies.

Antifibrotic Agent Mediated Tumor Microenvironment Modulation and Improved Nanomedicine Delivery in Solid Tumor

“…The cells were again washed and stained using 1 μg/mL DAPI dye for 15 min. After rinsing the cells with PBS, media was added to each well and the cells were visualized under an inverted fluorescence microscope at 60X (FLoid Cell Imaging Station, Life Technologies) . The % apoptotic cells were calculated using the following formula: % Apoptotic 0.25em cells = no . of 0.25em apoptotic 0.25em cells total 0.25em no . of 0.25em cells × 100 …”

“…ROS generation in MIA PaCa-2 cells with or without treatment was estimated using DCFH-DA dye . Concisely, cells were seeded (1 × 10 5 cells/well) and treated with GEM GEM-HSA NPs, LST, and LST-HSA NPs up to 48 h at concentrations mentioned in Section .…”

“…The developed colonies were then washed with PBS and fixed using 4% paraformaldehyde. The cells were washed again and incubated with 0.5% crystal violet dye for 20–30 min . The excessive dye was washed out and images were captured.…”

“…The images captured were labeled as 0 h images. Later, the cells were treated with drugs and nanoparticles for 48 h in low serum (1% FCS) containing media for suppressing cell proliferation under serum starvation conditions, and images were acquired again under a bright field microscope following PBS washing . The images captured were labeled as 48 h images. % Wound closure = 100 − true[ wound 0.25em area 0.25em after 0.25em 48 0.25em normalh 0.25em treatment wound 0.25em area 0.25em at 0.25em 0 0.25em normalh × 100 true] …”

“…The cytotoxic potential of native drugs and nanoparticles was determined using SRB (Sulforhodamine B) assay. 25 MIA PaCa-2 cells were grown in DMEM media in a T-75 cell culture flask and were seeded in 96-well plates at a density of 7000 cells/well. The cells were allowed to adhere for the next 24 h. The cells were then treated with 50, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, and 0.005 μg/mL concentrations of GEM as well as GEM-HSA NPs (concentration equivalent to the GEM) and 500, 200, 100, 50, 30, 10, and 1 μg/mL concentrations of LST as well as LST-HSA NPs (concentration equivalent to the LST).…”