Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al-Qura University, Makkah 21955, Saudi Arabia.
Medicinal and Aromatic Plants Research Institute, National Center for Research, Khartoum 2424, Sudan.
Molecules. 2021 Jan 11;26(2):334. doi: 10.3390/molecules26020334.
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.
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 HAA 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.
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.
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 HAA 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, HAA 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 preG/S cell cycle phases, apoptosis, and caspase 8 in HT29 cells, while it increased the G phase, p21/p27, and apoptosis in HT29-5FU cells.
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 HAA and 5FU has selectively sensitized the four CRC cells to 5FU and could be further studied.
首先，确定从沙特 CRC 患者样本中提取的最重要的激酶和激酶途径，以及 ABC 转运蛋白和 KRAS 的数量。其次，根据本研究第一部分得出的重要激酶途径，研究 LY294002 和 HAA 的化学增敏作用及其与 5FU 在 HT29、HT29-5FU、HCT116 和 HCT116-5FU CRC 细胞中的组合对三种 ABC 转运蛋白、细胞周期和凋亡的影响。
使用 PamChip 肽微阵列分析确定沙特 CRC 样本中的激酶和靶标水平。接下来，使用 RT-PCR、MTT 细胞毒性、Western blot、细胞周期扰动、膜联蛋白 V 和免疫荧光分析研究对 CRC、MRC5 和 HUVEC 细胞的影响。
激酶活性分析突出了 PI3K/AKT、MAPK 和生长因子途径在沙特 CRC 样本中的重要性。PIK3CA 的过度表达最为明显，与沙特样本中突变型 KRAS 和三种 ABC 转运蛋白（尤其是 ABCC1）水平的增加有关。接下来，HAA 与 5FU 联合在四种 CRC 细胞中表现出最佳的协同和耐药逆转作用，与 MRC5 和 HUVEC 正常细胞相比，具有最高的选择性指数。此外，HAA 与 5FU 在四种 CRC 细胞中显著抑制 ABCC1，并抑制 HT29 和 HT29-5FU 细胞中的 PIK3CA/AKT/MAPK7/ERK。该组合还抑制 EGFR，增加 HT29 细胞的 preG/S 细胞周期阶段、细胞凋亡和半胱天冬酶 8，同时增加 HT29-5FU 细胞的 G 期、p21/p27 和细胞凋亡。
我们将沙特 CRC 样本的 PamChip 激酶分析与四种 CRC 细胞中的体外药物组合研究相结合，突出了针对 PIK3CA 和 ABCC1 治疗沙特 CRC 患者的重要性，特别是考虑到 PIK3CA 突变的过表达与抗 EGFRs 缺乏活性有关，抗 EGFRs 作为 CRC 患者的一线治疗。HAA 与 5FU 的联合选择性地使四种 CRC 细胞对 5FU 敏感，可进一步研究。
