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.

Highlights

  • Colorectal cancer (CRC) ranks third and fourth amongst the deadliest and diagnosed cancers worldwide, respectively [1]

  • One of the advantages of the anti-VEGFR drugs is that they can be prescribed for wider groups of CRC patients as they have more diverse biomarkers and predictive factors of response compared to the anti-EGFR drugs [6,7]

  • In the first part of this study, the PamChip® kinase activity profiling was used for analysis of the kinases and kinase pathways in samples taken from Saudi CRC patients, which facilitated the determination and assessment of the level of kinase activities and targets; compared to other genetic sequencing methods, which are used to identify kinases of the human genome [37]

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Summary

Introduction

Colorectal cancer (CRC) ranks third and fourth amongst the deadliest and diagnosed cancers worldwide, respectively [1]. It disturbs the metabolism of nucleosides leading to cell death, but its efficiency is marred by resistance of CRC cells to 5FU [5]. Considering specific CRC predictive biomarkers, the resistance to 5FU and many other CRC drugs can be overcomed by the use of targeted therapies including bevacizumab as the first-line anti-vascular endothelial growth factor-A (anti-VEGF-A) drug. One of the advantages of the anti-VEGFR (anti-angiogenic) drugs is that they can be prescribed for wider groups of CRC patients as they have more diverse biomarkers and predictive factors of response compared to the anti-EGFR drugs [6,7]. The overexpression of PIK3CA is one of the important predictive biomarkers for the use of anti-VEGFR drugs, but it is not the case for the anti-EGFR drugs [8,9]

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