Modulation of the Main Resistance-Associated ABC Transporter's Expression by Plant Flavonol Isorhamnetin

Abstract

Background/Objectives: Multidrug resistance is one the leading problems in cancer treatment, where the overexpression of P-gp and other drug efflux pumps is regarded as the primary cause. With the intention to develop transporter inhibitors, natural products such as phenolics have shown great potential and diverse attention recently. Among these, isorhamnetin (ISO), an O-methylated flavonol, is predominantly found in the fruits and leaves of various plants. Thus, this study aimed to investigate the effects of ISO on the mRNA expression of membrane transporters P-gp, BCRP, MRP 1, 2, and 5, the protein expression of P-gp, as well as the GSTP1 and GSH content in DLD1 and HCT-116 colon cancer cells. Methods: The cytotoxic effect of isorhamnetin is assessed using an MTT test, while qPCR and immunocytochemistry methods were used to determine gene and protein expression levels. The concentration of reduced glutathione was determined using the colorimetric method. Results: Based on the results, ISO can modulate the expression of transporters responsible for the resistance development (all transporters on the transcriptional level were downregulated in DLD1 cells, while only MRP1 on HCT-116 cells, and reduced P-gp protein expression on both investigated cell lines). Increased glutathione content in treated cells and GSTP1 expression suggest metabolizing the ISO and potential ejection with GSH-dependent pumps. Conclusions: Thus, in future experiments, ISO as a natural medicinal compound could be used as a chemosensitizer to prevent or overcome membrane transporter-mediated drug resistance.

Keywords: BCRP; MDR; MRPs; P-gp; cancer cell resistance; isorhamnetin.

Figure 1

Effect of ISO on the viability of DLD-1 and HCT-116 cells. The results are presented as the mean ± standard error of three independent experiments. * Statistically significant difference (p < 0.05) compared to control values.

Figure 2

Expression of the mRNA of genes for membrane transporters in DLD1 and HCT-116 cells, under the influence of ISO (IC₂₅ value) compared to the control (value 1), 24 h after treatment. * Statistically significant difference (p < 0.05) concerning control values.

Figure 3

mRNA expression of the genes for GSTP1 in DLD1 and HCT-116 cells under the influence of ISO (IC₂₅ value) compared to the control (value 1), 24 h after treatment. * Statistically significant difference (p < 0.05) concerning control values.

Figure 4

GSH concentration in DLD1 and HCT-116 cells under the influence of ISO (IC₂₅), 24 h after applied treatment. The results are presented in nmol/mL and calculated per number of viable cells. * Statistically significant difference (p < 0.05) concerning control values.

Figure 5

The protein level of P-gp in DLD1 and HCT-116 cells. The results are presented as the mean ± standard error of three independent experiments. * Statistically significant difference (p < 0.05) compared to control.

Figure 6

Potential mechanism of the combined therapy of ISO and the appropriate chemotherapeutic agent in a malignant cell; red arrow transporter expression inhibition pathway, green arrow transporter expression induction pathway, and blue arrow chemotherapeutics pathway.

Figure 7

The potential of ISO as an antagonist in combined therapy with chemotherapeutics; red arrow chemotherapeutic pathway, blue arrow isorhamnetin pathway.