Zuo Jin Wan Reverses the Resistance of Colorectal Cancer to Oxaliplatin by Regulating the MALAT1/miR-200s/JNK Signaling Pathway

Abstract

Background: Oxaliplatin (L-OHP) is a common chemotherapy drug used in the treatment of colorectal cancer (CRC). Our previous work showed that Zuo Jin Wan (ZJW), a traditional Chinese medicine prescription, could improve sensitivity to L-OHP in the treatment of CRC, but the detailed mechanism is not clear. In previous mechanistic studies, we found that the miR-200s expression in CRC is associated with L-OHP sensitivity through regulation of MDR1/p-gp and the downstream c-JunN-terminal kinase (JNK) signaling pathway. Moreover, lncRNA-MALAT1 offers great potential in the regulation of drug resistance by interacting with miR-200s. Therefore, in this work, we explored whether ZJW could reverse L-OHP resistance in CRC by regulating MALAT1, miR-200s, and the downstream signaling pathway.

Methods: Cell Counting Kit-8 and flow cytometry were used to detect the effects of ZJW combined with L-OHP on chemotherapy tolerance and cell apoptosis of HCT116/L-OHP cells. Western blotting and quantitative real-time PCR (qRT-PCR) were used to detect the activation of the JNK signaling pathway and the protein and mRNA expression levels of the drug resistance-related MDR1/ABCB1 gene in HCT116/L-OHP cells treated with ZJW. The binding sites of MALAT1 and miR-200s were predicted by bioinformatics tools and confirmed by qRT-PCR. qRT-PCR was used to detect the expression of miR-200s and MALAT1 in HCT116/L-OHP cells treated with ZJW. A xenograft model of CRC in nude mice was established to observe the effect of ZJW combined with L-OHP on the growth of subcutaneously transplanted tumors. Apoptosis in tumor cells was detected by TUNEL staining. The activation of the JNK signaling pathway and the expression of drug resistance-related proteins were detected by immunohistochemistry and immunofluorescence. qRT-PCR was used to detect the expression of miR-200s and the MALAT1 gene in the tumors.

Results: Our study showed that ZJW could significantly decrease the proliferation and promote apoptosis of HCT116/L-OHP cells treated with L-OHP. We further proved that ZJW could reverse the drug resistance of HCT116/L-OHP cells by reducing MALAT1, indirectly upregulating miR-200s, alleviating the activation of the JNK signaling axis, and downregulating the expression of resistance proteins such as MDR1/ABCB1 and ABCG2. ZJW combined with L-OHP inhibited the growth of subcutaneously transplanted tumors and induced apoptosis in nude mice. ZJW reduced the expression of MALAT1 and upregulated the expression of miR-200s in transplanted tumors. In addition, ZJW also alleviated the activation of the JNK signaling pathway while reducing the expression of MDR1/ABCB1 and ABCG2.

Conclusions: Our study identified that MALAT1 promotes colorectal cancer resistance to oxaliplatin by reducing the miR-200s expression. ZJW may reverse chemoresistance by inhibiting the expression of MALAT1 and regulating the miR-200s/JNK pathway, providing an experimental basis for the clinical application of ZJW in relieving chemotherapy resistance.

Figure 1

Effect of ZJW on the proliferation and apoptosis of HCT116/L-OHP cells. (a) CCK-8 results show that HCT116/L-OHP cells were more resistant to oxaliplatin than sensitive HCT116 cells. (b) The results show that the IC10 dosage in HCT116/L-OHP cells was 20 μg/mL. (c) HCT116/L-OHP cells were treated with different concentrations of oxaliplatin for 48 hours, and the inhibitory effect of ZJW combined with L-OHP on HCT116/L-OHP cells was stronger than that of L-OHP alone. (d and e) Flow cytometry results show the apoptosis rates of HCT116/L-OHP cells treated with ZJW, L-OHP, or L-OHP combined with different concentrations of ZJW. ^∗∗, P < 0.01; ^∗∗∗, P < 0.001 compared to HCT116/L-OHP.

Figure 2

ZJW mediates the miR-200s/JNK signaling pathway to regulate drug resistance in colorectal cancer. (a) Real-time PCR assay of miR-200s levels in HCT116 and HCT116/L-OHP cells treated with L-OHP combined with different concentrations of ZJW. (b and c) Western blot assay of JNK and p-JNK protein in HCT116 cells and HCT116/L-OHP cells treated with L-OHP combined with different concentrations of ZJW. ^∗, P < 0.05; ^∗∗, P < 0.01; ^∗∗∗, P < 0.001 compared to HCT116/L.

Figure 3

Effect of ZJW on the expression of the drug resistance-related proteins MDR1/ABCB1, MRP4/ABCC4, and ABCG2 in HCT116/L-OHP cells. (a and b) Western blot assay of ABCG2, MDR1/ABCB1, and MRP4/ABCC4 proteins in HCT116 cells and HCT116/L-OHP cells treated with L-OHP combined with different concentrations of ZJW. (c) Real-time PCR assay of ABCG2, MDR1/ABCB1, and MRP4/ABCC4 levels in HCT116 and HCT116/L-OHP cells treated with L-OHP combined with different concentrations of ZJW. ^∗, P < 0.05; ^∗∗, P < 0.01; ^∗∗∗, P < 0.001 compared to HCT116/L.

Figure 4

ZJW mediated the MALAT1/miR-200s/JNK signaling pathway regulating drug resistance in colorectal cancer. (a and b) Bioinformatics analysis predicted the existence of multiple complementary binding sites between MALAT1 and miR-200s. (c) Real-time PCR assay of MALAT1 levels in HCT116 and HCT116/L-OHP cells. ^∗∗∗∗, P < 0.0001 compared to HCT116 cells. (d) Real-time PCR assay of MALAT1 levels in HCT116/L-OHP cells treated with L-OHP combined with different concentrations of ZJW. ^∗∗∗, P < 0.001; ^∗∗∗∗, P < 0.0001 compared to HCT116/L. (e) MALAT1 expression in control or MALAT1 siRNA-transfected HCT116/L-OHP cells. ^∗∗∗∗, P < 0.0001 compared to HCT116/L cells. (f) miR-200s expression in control or MALAT1 siRNA-transfected HCT116/L-OHP cells. ^∗∗∗∗, P < 0.0001 compared to HCT116/L cells.

Figure 5

ZJW reverses MDR and affects apoptosis in a nude mouse xenograft model. (a) Photographs of nude mouse tumors in the xenograft model. (b and c) Change in tumor weight in nude mice at 28 days. (d and e) ZJW affected apoptosis in a nude mouse xenograft model. ^∗, P < 0.05; ^∗∗, P < 0.01; ^∗∗∗, P < 0.001; ^∗∗∗∗, P < 0.0001 compared to MODEL.

Figure 6

Effects of ZJW on the JNK signaling pathway, miR-200s, and MALAT1 expression in vivo. (a and d) Immunohistochemistry detection of MRP4 protein in vivo. (b and c) Western blot and real-time PCR assays of JNK and p-JNK levels in vivo. (e and f) Real-time PCR assay of miR-200s levels in vivo. (g) Real-time PCR assay of MALAT1 levels in vivo. ^∗, P < 0.05; ^∗∗, P < 0.01; ^∗∗∗, P < 0.001; ^∗∗∗∗, P < 0.0001; ns, no significance compared to MODEL.

Figure 7

Effect of ZJW on the expression of drug resistance-related proteins in vivo. (a and b) Immunofluorescence detection of MDR1/ABCB1 and ABCG2 proteins in vivo. (c and d) Immunohistochemistry detection of MRP4/ABCC4 protein in vivo. (e and f) Western blot assay of ABCG2, MDR1/ABCB1, and MRP4/ABCC4 levels in vivo. (g) Real-time PCR assay of ABCG2, MDR1/ABCB1, and MRP4/ABCC4 levels in vivo. ^∗, P < 0.05; ^∗∗, P < 0.01; ^∗∗∗, P < 0.001; ^∗∗∗∗, P < 0.0001; ns, no significance compared to MODEL.