P-glycoprotein (Pgp), encoded by the ABCB1 gene, is a well-characterized efflux transporter responsible for multidrug resistance (MDR) in cancer cells. This study investigates the functional and molecular role of Pgp in mediating resistance to chemotherapeutic agents in human colorectal adenocarcinoma HCT116 cells. Using two independently derived resistant sublines—HCT116tax (paclitaxel-resistant) and HCT116nut (Nutlin-3a-resistant)—we systematically evaluated the contribution of Pgp to drug efflux, cross-resistance, and survival under therapeutic stress.
Functional assays using Hoechst 33342 accumulation revealed that both resistant lines exhibited significantly reduced intracellular dye retention compared to parental HCT116wt cells. This reduction was most pronounced in HCT116tax, suggesting enhanced efflux activity. Inhibition experiments with tariquidar—a specific Pgp blocker—resulted in a 1.9-fold increase in Hoechst fluorescence in HCT116tax cells, confirming Pgp-mediated efflux. Sodium orthovanadate, a broad-spectrum inhibitor of ATP-dependent transporters, also increased dye retention, particularly in HCT116tax (2.3-fold), indicating involvement of additional ABC transporters beyond Pgp.
Gene expression analysis via real-time RT-PCR showed marked upregulation of ABCB1 mRNA in both resistant lines: 10-fold in HCT116tax and 19-fold in HCT116nut. Western blotting corroborated these findings, demonstrating elevated Pgp protein levels in resistant cells. The correlation between increased Pgp expression and enhanced efflux confirms its central role in MDR. Moreover, pharmacological inhibition of Pgp with tariquidar restored sensitivity to paclitaxel and Nutlin-3a in resistant cells, as evidenced by decreased GI50 values and increased apoptosis rates.
Interestingly, while HCT116nut cells were highly resistant to Nutlin-3a (RI = 4.1), they also showed moderate resistance to paclitaxel (RI = 1.6). Conversely, HCT116tax cells displayed strong resistance to paclitaxel (RI = 3.2) and notable cross-resistance to Nutlin-3a (RI = 1.9). These data suggest that Pgp activation confers non-selective protection against diverse drugs, regardless of mechanism of action. The ability of Pgp to export structurally unrelated compounds—including cytostatics, targeted agents, and fluorescent dyes—underscores its role as a universal defense system.
Apoptosis analysis using Muse Annexin V & Dead Cell Kit revealed that resistant cells had significantly lower levels of early and late apoptosis after treatment with either agent.182498-32-4 SMILES This was consistent with reduced drug accumulation due to active efflux.1643489-24-0 SMILES Cell cycle analysis further confirmed delayed G2/M arrest in paclitaxel-treated resistant cells, reinforcing the notion that diminished intracellular drug concentration impairs mitotic disruption.PMID:30725832
These results demonstrate that P-glycoprotein is a dominant mediator of multidrug resistance in colorectal cancer cells. Its overexpression not only reduces the efficacy of primary therapies but also undermines subsequent treatments through cross-resistance. Targeting Pgp with inhibitors such as tariquidar may reverse resistance and enhance therapeutic outcomes. The established resistant cell models provide essential tools for evaluating next-generation MDR modulators and designing rational combination regimens aimed at overcoming efflux-mediated resistance in colon cancer.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com