Metformin and thymoquinone co‐treatment enhance 5‐fluorouracil cytotoxicity by suppressing the PI3K/mTOR/HIF1α pathway and increasing oxidative stress in colon cancer cells

Abstract

This study investigated the chemotherapeutic effects of 5‐fluorouracil (5‐FU), metformin (Met), and/or thymoquinone (TQ) single/dual/triple therapies in the HT29, SW480 and SW620 colon cancer (CRC) cell lines. Cell cycle/apoptosis were measured by flow cytometry. The gene and protein expression of apoptosis (PCNA/survivin/BAX/Cytochrome‐C/Caspase‐3) and cell cycle (CCND1/CCND3/p21/p27) molecules, the PI3K/mTOR/HIF1α oncogenic pathway, and glycolysis regulatory enzymes were measured by quantitative‐PCR and Western blot. Markers of oxidative stress were also measured by colorimetric assays. Although all treatments induced anti‐cancer effects related to cell cycle arrest and apoptosis, the triple therapy showed the highest pro‐apoptotic actions that coincided with the lowest expression of CCND1/CCND3/PCNA/survivin and the maximal increases in p21/p27/BAX/Cytochrome‐C/Caspase‐3 in all cell lines. The triple therapy also revealed the best suppression of the PI3K/mTOR/HIF1α pathway by increasing its endogenous inhibitors (PTEN/AMPKα) in all cell lines. Moreover, the lowest expression of lactate dehydrogenase and pyruvate dehydrogenase kinase‐1 with the highest expression of pyruvate dehydrogenase were seen with the triple therapy, which also showed the highest increases in oxidative stress markers (ROS/RNS/MDA/protein carbonyl groups) alongside the lowest antioxidant levels (GSH/CAT) in all cell lines. In conclusion, this is the first study to reveal enhanced anti‐cancer effects for metformin/thymoquinone in CRC that were superior to all monotherapies and the other dual therapies. However, the triple therapy approach showed the best tumoricidal actions related to cell cycle arrest and apoptosis in all cell lines, possibly by enhancing oxidative glycolysis and augmenting oxidative stress through stronger modulation of the PI3K/mTOR/HIF1α oncogenic network.

Synthesis and Characterization of Thymoquinone‐Oxime (TQ‐Ox) from Thymoquinone and Evaluation of its Cytotoxic, Genotoxic, and Apoptotic Potential in Lung Cancer Cells (A549) in vitro

“…The results of TQÀ Ox's elemental analysis supported the synthetic constructions. The proposed structures were further validated by the 1H and 13 C-NMR data as well as the elemental analysis findings of TQ-Ox. [17] In order to estimate the pharmacokinetic properties of thymol, TQ, and TQÀ Ox, the SwissADME online database was used (http://www.swissadme.ch/).…”

“…At 13.39 ppm, a proton from the hydroxyl group was also found. 9 carbon peaks were seen in the 13 C-NMR spectra of TQÀ Ox, including 3 methyl carbon peaks in the aliphatic area between 27 and 17 ppm and 6 aromatic carbon peaks between 186 and 118 ppm. The results of TQÀ Ox's elemental analysis supported the synthetic constructions.…”

“…[17] Figure 1 shows the TQÀ Ox NMR image and synthesis process. In order to record the 1 H-NMR and 13 C-NMR spectra, a three-channel NMR spectrometer was set up. The elemental analysis was done by the TUBITAK, Marmara Research Center's Instrumental Analysis Laboratory.…”

“…To purify the raw material, hexane washing was employed. Following thorough purification, TQÀ Ox was characterized using 1 H-NMR, 13 C-NMR, and elemental analysis.…”

“…This is supported by experimental studies emphasizing the robust molecular interactions of oximes. [11] The literature contains many studies examining TQ's potential as an anticancer agent in vitro and in vivo; [12][13][14][15][16] however, there is only one study on ovarian cancer related to TQ-oxime (TQÀ Ox). [17] Notably, our study marks the first exploration of TQÀ Ox's effects on a lung cancer cell line.…”