Ilex hainanensis Merr targets ITGAV to suppress the proliferation and metastasis of osteosarcoma cells

Abstract

Background: Osteosarcoma (OS) is the most common primary malignant bone tumor. Hence, there is an urgent need to identify effective and safe therapeutic agents against OS. It has been reported that Ilex hainanensis Merr (IME) possesses antitumor properties. Integrin subunit alpha V (ITGAV) is important for the diagnosis, treatment, and prognosis of tumors. Purpose: The objective of this study was to whether IME can play a role in the treatment of osteosarcoma by regulating ITGAV. Methods: Western blot and real-time PCR were used to detect the expression of ITGAV in non-tumorous tissues, osteosarcoma tissues, and metastatic tumors. The expression of ITGAV in MG63, U2OS, and hFOB1. A total of 19 cells was determined through Western blotting and real-time PCR. The expression of ITGAV in OS cells treated with different concentrations of DDP was determined through Western blotting. Agter transfecting with control or si-ITGAV, and subsequently treated with control or 5 μmol/L DDP, MTT assay and transwell assay were used to detect the proliferaion and migration of cells. Western blot was used to detect the expression of ITGAV in cells treated with different concentrations of IME and MTT assay and transwell assay were used to detect the proliferaion and migration of cells. MG63 and U2OS cells were treated with control, 5 μmol/L DDP, 25 μmol/L IME, or 5 μmol/L DDP combined with 25 μmol/L IME, the expression of ITGAV was determined through Western blotting and real-time PCR. MTT assay and transwell assay were used to detect the proliferation and migration of cells. Inhibitory effect of IME on lung metastasis of osteosarcoma in vivo. Results: ITGAV was highly expressed in tumors, with the highest expression found in metastatic tumors and higher in OS cells. A low concentration of DDP (5 μmol/L) inhibited the expression of ITGAV. However, ITGAV may be related to the development of resistance to DDP. Silencing of ITGAV downregulates the proliferation and migration of OS cells as the effect of low-concentration DDP (5 μmol/L). IME inhibited the proliferation and migration of MG63 and U2OS cells in a concentration-dependent manner and decreased the expression of ITGAV. MTT and Transwell assays showed that 25 μmol/L IME and 5 μmol/L DDP exhibited similar inhibitory effects on the proliferation and migration of OS cells. The combination of IME with DDP resulted in the amplification of these inhibitory effects. Both DDP and IME downregulated the expression of ITGAV, and the inhibition of ITGAV was amplified by the combination of IME with DDP. In-vivo studies have shown that IME and DDP, independently or in combination, may significantly inhibit the metastasis of OS to the lungs. Conclusion: IME may reduce the resistance of OS cells to DDP to some extent.

Keywords: Ilex hainanensis Merr; cisplatin; integrin subunit alpha V; osteosarcoma.

Figure 1

The expression of ITGAV in osteosarcoma. (A) The structure of IME. (B, C) The expression of ITGAV in nontumorous tissues, osteosarcoma tissues, and metastatic tumors obtained from patients with osteosarcoma was determined through Western blotting and real-time PCR. **P<0.05, vs adjacent normal tissues. (D, E) The expression of ITGAV in MG63, U2OS, and hFOB1.19 cells was determined through Western blotting and real-time PCR. **P<0.05, vs hFOB1.19 cells. Abbreviations: IME, Ilex hainanensis Merr; ITGAV, integrin subunit alpha V; nor, non-tumorous tissues; os,  osteosarcoma tissues;  me, metastatic tumors.

Figure 2

ITGAV is involved in the development of resistance to DDP in osteosarcoma. (A, B) The expression of ITGAV in MG63 and U2OS cells treated with different concentrations of DDP was determined through Western blotting. (C) MG63 and U2OS cells were transfected with control or si-ITGAV and subsequently treated with control or 5 μmol/L DDP. MTT assay was performed, and the results represent the mean ± SD of three independent experiments. **P<0.05, vs control. ## P<0.05, vs si-ITGAV. (D, E) MG63 and U2OS cells were transfected with control or si-ITGAV, and subsequently treated with control or 5 μmol/L DDP. The migration of MG63 and U2OS cells was determined using the Transwell assay. The cells were counted, and the results represent the mean ± SD of three independent experiments. **P<0.05, vs control. ## P<0.05, vs si-ITGAV. Abbreviations: DDP, cisplatin; ITGAV, integrin subunit alpha V.

Figure 3

IME inhibits the proliferation and migration of osteosarcoma cells. (A) hFOB1.19, MG63, and U2OS cells were treated with different concentrations of IME. MTT assay was performed, and the results represent the mean ± SD of three independent experiments. **P<0.05, vs control. ##P<0.05, vs 5 μmol/L DDP.  (B, C) MG63 and U2OS cells were treated with different concentrations of IME. The migration of MG63 and U2OS cells was detected using the Transwell assay. The cells were counted, and the results represent the mean ± SD of three independent experiments. **P<0.05, vs control. (D, E) The expression of ITGAV in MG63 and U2OS cells treated with different concentrations of IME was determined through Western blotting. Abbreviations: IME, Ilex hainanensis Merr; ITGAV., integrin subunit alpha V.

Figure 4

IME enhances the inhibitory effect of DDP on osteosarcoma cells. (A) MG63 and U2OS cells were treated with control, 5 μmol/L DDP, 25 μmol/L IME, or 5 μmol/L DDP combined with 25 μmol/L IME. MTT assay was performed, and the results represent the mean ± SD of three independent experiments. **P<0.05, vs control. ## P<0.05, vs 5 μmol/L DDP. (B, C) MG63 and U2OS cells were treated with control, 5 μmol/L DDP, 25 μmol/L IME, or 5 μmol/L DDP combined with 25 μmol/L IME. The migration of MG63 and U2OS cells was detected using the Transwell assay. The cells were counted, and the results represent the mean ± SD of three independent experiments. **P<0.05, vs control. ## P<0.05, vs 5 μmol/L DDP. (D, E) MG63 and U2OS cells were treated with control, 5 μmol/L DDP, 25 μmol/L IME, or 5 μmol/L DDP combined with 25 μmol/L IME. The expression of ITGAV in MG63 and U2OS cells was determined through Western blotting and real-time PCR. **P<0.05, vs control. ## P<0.05, vs 5 μmol/L DDP. (F) The metastasis of osteosarcoma after treatment with control, DDP, IME, or DDP combined with IME. **P<0.05, vs control. ## P<0.05, vs DDP. Abbreviations: IME, Ilex hainanensis Merr; DDP, cisplatin; ITGAV, integrin subunit alpha V.