Inhibition of apoptosis-regulatory protein Siva-1 reverses multidrug resistance in gastric cancer by targeting PCBP1

Abstract

Introduction: Siva-1, as a pro-apoptotic protein, has been shown to induce extensive apoptosis in a number of different cell lines. In our previous study, we showed that overexpressed Siva-1 decreased the apoptosis of gastric cancer cells. So, we believe that it can also work as an anti-apoptotic protein. The present study aimed to determine the specific role of Siva-1 in anticancer drug resistance in gastric cancer in vivo and in vitro and preliminarily reveal the mechanism.

Materials and methods: A vincristine-resistant MKN-28/VCR gastric cancer cell line with stably downregulated Siva-1 was established. The effect of Siva-1 downregulation on chemotherapeutic drug resistance was assessed by measuring the IC50 and pump rate of doxorubicin. Proliferation, apoptosis of cells, and cell cycle were detected via colony formation assay and flow cytometry, respectively. Additionally, migration and invasion of cells was detected via wound healing and transwell assays. Moreover, we determined in vivo effects of LV-Siva-1-RNAi on tumor size, and apoptotic cells in tumor tissues were detected using TUNEL and hematoxylin and eosin staining.

Results: Siva-1 downregulation reduced the pump rate of doxorubicin and enhanced the response to drug treatment. Siva-1 negatively regulated proliferation and promoted apoptosis of cells by potentiality G2-M phase arresting. Inhibition of Siva-1 expression in MKN-28/VCR cells significantly weakened wound healing ability and decreased invasion ability. Poly(C)-binding protein 1 (PCBP1) was identified as a Siva-1-interacting protein in yeast two-hybrid screening. Semiquantitative RT-PCR and western blotting revealed that Siva-1 downregulation could inhibit expression of PCBP1, Akt, and NF-κB and eventually decrease the expression of MDR1 and MRP1.

Conclusion: he current study demonstrated that the downregulation of Siva-1, which functions as a regulator of MDR1 and MRP1 gene expression in gastric cancer cells by inhibiting PCBP1/Akt/NF-κB signaling pathway expression, enhanced the sensitivity of gastric cancer cells to certain chemotherapies.

Keywords: Gastric cancer; Multidrug resistance; PCBP1; Siva-1.

Figure 1. The effect on cell pump rate of doxorubicin, cell cycle, and apoptotic rate in MKN-28/VCR after Siva-1 was inhibited. (A, B) Pump rate of doxorubicin in MKN-28/VCR after Siva-1 was inhibited, analyzed by flow cytometry. (C, D) Cell cycle in MKN-28/VCR cells after Siva-1 was inhibited, analyzed by flow cytometry. (E, F) Apoptotic rate in MKN-28/VCR cells, analyzed by flow cytometry (*p < 0.05).

Figure 2. The effect on cell colony forming efficiency, the migratory rate, and invasion rate in MKN-28/VCR after Siva-1 was inhibited. (A, B) Proliferation rate of MKN-28/VCR after Siva-1 was inhibited, analyzed by cell colony assay. (C, D) Migratory rate of MKN-28/VCR after Siva-1 was inhibited, analyzed in wound healing assay. (E, F) Invasion rate of MKN-28/VCR after Siva-1 was inhibited, analyzed by transwell assay (*p < 0.05).

Figure 3. Siva-1 interacted with PCBP1. (A) Library quality test showed that the homogenized secondary yeast two-hybrid cDNA library capacity was 9.56 × 10⁶ CFU. (B, C) Yeast two-hybrid assays were performed to search for proteins that interacted with Siva-1. The bait vector with truncated Siva-1 gene was constructed for screening a human gastric cancer yeast two-hybrid cDNA library, and PCBP1 and UXT were identified. Interactions were confirmed based on co-transformants that can grow on histidine-deficient (-His) plates and exhibit β-galactosidase activity. (D) co-IP of Siva-1 with the PCBP1 protein. (E) co-IP to validated Siva-1 and UXT protein interaction in 293 T cells (*p < 0.05; NC: negative control; PC: positive control; IP: immunoprecipitation; IB: immunoblotting; SD: synthetic dropout medium).

Figure 4. Siva-1 regulate PCBP1/Akt/NF-κB signaling pathway expression. (A, B) Relative expression levels of Siva-1 and its density quantification result. (C) Relative expression of Siva-1, PCBP1, Akt, NF-κB, MRP1, and MDR1 mRNA in MKN-28/VCR-shRNA-Siva-1 cells, MKN-28/VCR-shRNA-NC cells, and MKN-28/VCR cells. (D, E) Relative protein expression levels of PCBP1, Akt, NF-κB, MRP1, and MDR1 and their density quantification results. (F, G) Relative protein expression levels of Akt, NF-κB, MRP1, and MDR1 and their density quantification results (*p < 0.05; 1: MKN-28/VCR-shRNA-Siva-1; 2: MKN-28/VCR-shRNA-NC; 3: MKN-28/VCR; 4: MKN-28/VCR with Akt inhibitor).

Figure 5. Inhibition of Siva-1 decreases tumor burden and liver metastases. (A) Volume development of subcutaneous tumor in mouse model. (B) Subcutaneous tumor tissue taken from nude mice when mice were killed on day 13. (C) Mouse body weight was measured when mice were killed on day 13. (D) Representative HE staining, immunohistochemical staining of Siva-1 and PCBP1, and TUNEL staining image in subcutaneous tumor tissue taken from nude mice in three different treatment groups. (E) Percentage of Siva-1-positive cells in three groups. (F) Percentage of PCBP1-positive cells in three groups. (G) Percentage of TUNEL-positive cells in three groups. (H) Representative images of liver metastases in nude mice inoculated with gastric cancer cells in different treatment groups. (I) Hepatic metastases rate of three different treatment groups (*p < 0.05; 1: MKN-28/VCR-shRNA-Siva-1; 2: MKN-28/VCR-shRNA-NC; 3: MKN-28/VCR; HE: hematoxylin and eosin; IHC: immunohistochemistry; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling).