Effective Delivery of siRNA-Loaded Nanoparticles for Overcoming Oxaliplatin Resistance in Colorectal Cancer

Abstract

Chemotherapy resistance represents a formidable obstacle in advanced or metastatic colorectal cancer (CRC) patients. It is reported that ATPase copper transporting alpha (ATP7A) plays an important role in chemotherapy resistance in CRC. Here, we identified ATP7A as a potentially key gene of OXA resistance in CRC. The patients with higher expression of ATP7A tended to have platinum drug resistance. While the lower expression of ATP7A by siRNA knockdown resulted in enhancement of OXA sensitivity and increased OXA-induced apoptosis. Further, we demonstrated a novel and safe strategy to increase CRC chemosensitivity by delivering siRNA into tumor cells via a novel nanoparticle, DAN. In summary, our study provided a novel nanocarrier-based delivery of ATP7A to interfere in a key gene of chemo-resistance in CRC, which may be a novel therapeutic strategy to overcome chemotherapy resistance in CRC.

Keywords: ATP7A; chemoresistance; colorectal cancer; oxaliplatin; siRNA delivery.

Figure 1

Implications of ATP7A expression and clinical characteristics. (A) Expression of ATP7A in OXA resistance and OXA responder of CRC patients. (B) IHC staining scores of chemotherapy resistance patients and chemotherapy responders. **P < 0.01 and ***P < 0.001 when compared to the control group.

Figure 2

ATP7A inhibition reverse OXA resistance in vitro. (A) ATP7A protein and ATP7A mRNA expression were inhibited by the siATP7A in HCT116 and LOVO cell lines. (B) OXA sensitivity in HCT116 and LOVO cell lines treated with siATP7A was determined using the CCK-8 test after treatment with various concentrations of OXA for 48 h. (C) The IC50 of OXA for siNC and siRNA transfected HCT116 or LOVO cells. (D) Together with OXA, decreased expression of ATP7A significantly suppressed cancer cell lines proliferation. **P < 0.01 and ***P < 0.001 when compared to the control group.

Figure 3

Knockdown of ATP7A expression may inhibit cancer tumorigenesis in vitro. (A) Colony formation assays in HCT116 and LOVO cells. (B) A flow cytometer was performed to assess the apoptosis rate of HCT116 and LOVO cells. (C) Wound healing assay of HCT116 and LOVO cells. (D) Transwell assay was performed to evaluate the invasion of HCT116 and LOVO cells. *P < 0.05; **P < 0.01 and ***P < 0.001 when compared to the control group.

Figure 4

Construction of DAN nanoparticle. (A) The schematic illustration of the nanoparticle DAN-siATP7A construction. The size (B) and zeta potential (C) distribution of DAN-siATP7A. (D) The size measurement of DANsiATP7A in the RPMI-1640 supplemented with 10% FBS. The protein expression (E) and mRNA expression (F) of ATP7A after being transfected with DAN-siATP7A in HCT116 and LOVO cell lines. (G) The in Virto cellular uptake of Cy5-labeled DAN-siNC and free Cy5-labeled siNC in vitro. The confocal image was captured 8 hours and 24hours after the transfection of HCT116 and LOVO cell lines. (H) The scheme of medication on tumor-bearing mice. (I) The IHC analysis of ATP7A and the PCR analysis of ATP7A mRNA in tumor tissue implied effective knockdown of ATP7A via DAN-siATP7A nanoparticles. ***P < 0.001 and ns, not significance, when compared to the control group.

Figure 5

siRNA-loaded nanoparticle reverses chemoresistance in HCT116-bearing mice. (A) The scheme of medication on tumor-bearing mice. (B) The image of the tumor was dissected from the nude mice at the end of the in vivo study. Tumor weight (C), tumor volume (D) and mice weight (E) after the treatment. (F) The HE staining and TUNEL analysis of the tumor tissue of the nude mice. ***P < 0.001 when compared to the control group.

Figure 6

In vivo toxicity analysis. (A) The image of organs including heart, liver, spleen, lung, kidney, and brain from the nude mice at the end of the in vivo study and the organ index (organ index = the weight of the organ/the weight of the mice) of the nude mice. Data were expressed as means ± SD. Group A stands for PBS; Group B stands for OXA; Group C stands for DAN-siATP7A and Group D stands for DAN-ATP7A + OXA. (B) The HE staining of organs. (C) The blood test of ALT, AST, BUN, Cr, UC of the nude mice, and no toxicity were found in our in vivo experiment.