Chemosensitization of HT29 and HT29-5FU Cell Lines by a Combination of a Multi-Tyrosine Kinase Inhibitor and 5FU Downregulates ABCC1 and Inhibits PIK3CA in Light of Their Importance in Saudi Colorectal Cancer

Abstract

Colorectal cancer (CRC) remains one of the main causes of death worldwide and in Saudi Arabia. The toxicity and the development of resistance against 5 fluorouracil 5FU pose increasing therapeutic difficulties, which necessitates the development of personalized drugs and drug combinations. Objectives: First, to determine the most important kinases and kinase pathways, and the amount of ABC transporters and KRAS in samples taken from Saudi CRC patients. Second, to investigate the chemosensitizing effect of LY294002 and HAA2020 and their combinations with 5FU on HT29, HT29-5FU, HCT116, and HCT116-5FU CRC cells, their effect on the three ABC transporters, cell cycle, and apoptosis, in light of the important kinase pathways resulting from the first part of this study. Methods: The PamChip® peptide micro-array profiling was used to determine the level of kinase and targets in the Saudi CRC samples. Next, RT-PCR, MTT cytotoxicity, Western blotting, perturbation of cell cycle, annexin V, and immunofluorescence assays were used to investigate the effect on CRC, MRC5, and HUVEC cells. Results: The kinase activity profiling highlighted the importance of the PI3K/AKT, MAPK, and the growth factors pathways in the Saudi CRC samples. PIK3CA was the most overexpressed, and it was associated with increased level of mutated KRAS and the three ABC transporters, especially ABCC1 in the Saudi samples. Next, combining HAA2020 with 5FU exhibited the best synergistic and resistance-reversal effect in the four CRC cells, and the highest selectivity indices compared to MRC5 and HUVEC normal cells. Additionally, HAA2020 with 5FU exerted significant inhibition of ABCC1 in the four CRC cells, and inhibition of PIK3CA/AKT/MAPK7/ERK in HT29 and HT29-5FU cells. The combination also inhibited EGFR, increased the preG1/S cell cycle phases, apoptosis, and caspase 8 in HT29 cells, while it increased the G1 phase, p21/p27, and apoptosis in HT29-5FU cells. Conclusion: We have combined the PamChip kinase profiling of Saudi CRC samples with in vitro drug combination studies in four CRC cells, highlighting the importance of targeting PIK3CA and ABCC1 for Saudi CRC patients, especially given that the overexpression of PIK3CA mutations was previously linked with the lack of activity for the anti-EGFRs as first line treatment for CRC patients. The combination of HAA2020 and 5FU has selectively sensitized the four CRC cells to 5FU and could be further studied.

Phytochemical, Cytotoxic, and Antimicrobial Evaluation of Tribulus terrestris L., Typha domingensis Pers., and Ricinus communis L.: Scientific Evidences for Folkloric Uses

“…As previously reported, the cytotoxicity of the three extracts was evaluated by MTT assay [ 39 , 40 ]. All cancer cell lines and normal fibroblast were separately cultured in 96-well plates (3 × 103/well) and incubated at 37°C overnight.…”

Growth Factors, PI3K/AKT/mTOR and MAPK Signaling Pathways in Colorectal Cancer Pathogenesis: Where Are We Now?

“…Presently, it is used in China for mCRC treatment in patients that have failed at least two prior systemic antineoplastic therapies [ 170 ]. The resistance that appears in VEGFR inhibitors seems to be attributed to receptor mutations that appear in PIK3CA/AKT, ERK, HER-2, or even EGFR [ 171 ]. The effectiveness of two monoclonal antibodies, cetuximab and panitumumab, increases in combination with fluorouracil (5-FU) plus irinotecan (FOLFIRI) and 5-FU plus oxaliplatin (FOLFOX) by acting on EGFR, leading to RAS-RAF-MEK-ERK signaling pathway inhibition in mCRC patients [ 172 ].…”

In Silico Approach Using Free Software to Optimize the Antiproliferative Activity and Predict the Potential Mechanism of Action of Pyrrolizine-Based Schiff Bases

“…Compound 16g, the most active in MTT assay ( Table 2 ), was selected for this assay. The assay was performed according to the previous report [ 36 ]. The results are represented in Figure 11 .…”

“…The effect of compound 16g on the cell cycle distribution of MCF7 was performed following the previous report [ 36 ]. Briefly, the cancer cells were cultured for 72 h with 0.00, 0.01, 0.05, and 0.10 μM final concentration of compound 16g .…”