Mangiferin suppresses human metastatic osteosarcoma cell growth by down-regulating the expression of metalloproteinases-1/2 and parathyroid hormone receptor 1

The study evaluates the protective effect of mangiferin on osteosarcoma cell proliferation and metastasis. Saos-2 and U2OS cells were treated with mangiferin (25, 50, 75 and 100 µM) for 72 h. Mangiferin reduced the cell viability, invasion, and cell adhesion and migration rate. Matrix metalloproteinases-2/9 (MMP-2/9) mRNA expression was reduced significantly, while the levels of tissue inhibitors of metalloproteinases-1/2 (TIMP-1/2) were elevated in Saos-2 and U2OS cells. Mangiferin treatment significantly reduced parathyroid hormone receptor 1 (PTHR1) mRNA and protein expression by more than 0.5-fold in both osteosarcoma cells. In addition, the immunofluorescent analysis also showed decreased PTHR1 expression following treatment with mangiferin. In summary, we have demonstrated that treatment with mangiferin reduces cell viability, proliferation, invasion, adhesion and migration, and induces apoptosis of osteosarcoma cells. Therefore, treatment with mangiferin can be effective agent in inhibiting growth and inducing apoptosis in osteosarcoma cells. Our experimental results provide evidence for the therapeutic effect of mangiferin in osteosarcoma cells.


Introduction
Osteosarcoma is severe malignant bone tumor (Li et al. 2018), and teenagers and adults are affected mostly by osteosarcoma (Luetke et al. 2014). Although water fluoridation is believed to be the main cause of osteosarcoma without clear research data to conclude this (Iida and Kumar 2009). Ottaviani and Jaffe (2009) have reported the increased mortality rate in childhood due to malignant bone and joint tumor. Chemotherapy and surgical resection have improved the survival rate of osteosarcoma up to 70%. However, osteosarcoma recurrence and metastasis leads to an increased mortality rate (Moreno et al. 2017;Berner et al. 2015). Parathyroid hormone receptor 1 (PTHR1) plays a major role in the pathophysiology of osteosarcoma (Lupp et al. 2010) and expressed in metastatic cells and tissues (Ho et al. 2014). Ho et al. (2015) have reported that the PTHR1 knockdown in osteosarcoma cells decreases the growth and invasion, and enhances tumor differentiation. Overexpression of PTHR1 in osteosarcoma increases motility and proliferation. Furthermore, it delays upregulation of genes which are responsible for the extra cellular matrix (ECM) production and osteoblastic differentiation (Ho et al. 2015). The putative mechanism suggested for PTHR1 is parathyroid hormone (PTH) is known to downregulate the expression of PTHrP receptor in osteoblast-like cells through a cAMP-dependent and PKA-independent pathway (Kawane et al. 2003).
Mangiferin is well-known xanthone found in several mango fruits such as barks, peel, leaves, stone, stalks and kernel, and in higher plants (Imran et al. 2017). Dar et al. (2005) have reported the several pharmacological effects of mangiferin such as antioxidant, anticancer, antiaging, antiviral, hepatoprotective, analgesic and immunomodulatory potential. Thus, the study analyzed the ability of mangiferin suppresses human metastatic osteosarcoma cell growth by down-regulating the expression of MMP-2/9 and PTHR1.

Cell culture
Saos-2 and U2OS cells were obtained from ATCC to cultured in M199 medium containing heparin, antibiotics (1%) and FBS (10%) at room temperature with 5% CO 2 . The preliminary investigation was carried out with different concentration of mangiferin from 25 to 200 µM. However, we noted the optimum and significant effect between 25 and 100 µM of mangiferin. Thus, we selected these concentrations in this study.

Clonogenic assays
Saos-2 and U2OS cells were seeded (1.1 × 10 4 cells/well) in growth medium treated with mangiferin (25, 50, 75 and 100 µM) for 72 h and crystal violet (0.2%) was used for staining for 30 min at 37 °C. Cells were washed with water and the cell survival rate was compared to the appropriate controls (Chaudhary et al. 2015).

Cell adhesion assays
Saos-2 and U2OS cells were seeded (1.5 × 10 4 cells/ well) and treated with mangiferin (25, 50, 75 and 100 µM) for 72 h, washed repeatedly with PBS, and stained with hematoxylin and eosin (H&E). The stained image was analyzed under a microscope and the cell adhesion rate was calculated (Do Thi and Hwang 2014).

Cell invasion assays
Saos-2 and U2OS cells were seeded (1.5 × 10 4 cells/ well) in growth medium and treated with mangiferin (25, 50, 75 and 100 µM) for 72 h and percent cell invasion was determined as described previously (Do Thi and Hwang 2014).

Western blot analysis
Western blot experiment was used to measure the cellular effect of mangiferin on PTHR1 protein expression. Saos-2 and U2OS cells were seeded (2.5 × 10 4 cells/ well) in growth medium and incubated with mangiferin (50, 75 and 100 µM) for 72 h. Protease inhibitor was used to lyse the cells in cell lysis buffer, and cell extract was collected by centrifugation and protein content in the extract was determined by Bradford assay. Proteins in the extract were separated membrane, and non-fat (5%) was incubated with the immunoblots to block non-specific sites and then incubated with primary antibodies (1:300 dilution ratio) overnight at cold temperature. Then, blots were incubated with HRP-conjugated antibodies for 60 min at cold temperature. The protein levels were visualized by Dmitriev et al. (2005).

Immunofluorescence
Saos-2 and U2OS cells were seeded (2 × 10 4 cells/well) in growth medium and treated with mangiferin (75 and 100 µM) for 72 h, Then, cell was incubated with BSA for 60 min and treated with PTHR1 antibody for 12 h and followed by fluorescein isothiocyanate (FITC)-conjugated antibody for 60 min (Balic et al. 2011).

Statistical analysis
Results are given as mean ± standard error of the mean (SEM). Student's t-test was used to compare the groups, and analysis of variance (ANOVA). The P < 0.05 was considered as significant.  -CTC TTT GGC GTC CAC TAC ATTG-3′  5′-TTG AGG AAC CCA TCG TCC TTG-3′   2

Discussion
In our study, we showed that the mangiferin inhibited cell proliferation of Saos-2 and U2OS cells. We speculate that the inhibition of proliferation, invasion and migration are associated with the inhibition of PTHR1. In addition, mangiferin treatment inhibited the subcuta-neous osteosarcoma tumor growth and proliferation (data not shown). These results indicate the antitumor activity of mangiferin against osteosarcoma. Tsai et al. (2014) have showed killing effect of paclitaxel against osteosarcoma cells. However, toxic side effects and drug resistance are main disadvantage of paclitaxel which leads to major problem for their clinical application. Mangiferin is wellknown xanthone found in several mango fruits such as barks, peel, leaves, stone, stalks and kernel, and in higher plants (Imran et al. 2017). Dar et al. (2005) have reported the several pharmacological effects of mangiferin such as antioxidant, anticancer, antiaging, antiviral, hepatoprotective, analgesic and immunomodulatory potential. An acidic environment is mandatory for tumor cell proliferation and invasion (Rofstad et al. 2006). In the current study, the reduced adhesion, cell proliferation, invasion, and migration confirmed that tumor cell metastasis occurred following treatment with mangiferin. Do Thi et al. (2014) reported that degradation of the extracellular matrix (ECM) is a key step in metastasis. Matrix metalloproteases (MMPs) play key role in degradation of the ECM, which leads to increased acceleration and migration of metastatic tumor (Libra et al. 2009). Guo et al. (2015) also reported that the inhibition of invasive and migratory potential of A549 cells through reduced expression of MMP-2/9. In this study, MMP-2/9 mRNA and protein expression was reduced significantly following treatment with mangiferin. These results indicate that the anti-metastatic effect of mangiferin. We speculate that the inhibition of MMP-2/9 is key mechanism for the inhibition of osteosarcoma. Therefore, the inhibition of PTHR1 expression could be useful therapeutic approach for the treatment of osteosarcoma. In this study, mangiferin reduced the mRNA and protein expression of PTHR1, which further confirms the anti-metastatic effect of mangiferin. In summary, we have demonstrated that treatment with mangiferin reduces cell viability, invasion, adhesion, proliferation, and migration, and induces apoptosis osteosarcoma cells. Therefore, treatment with mangiferin can be effective agent in inhibiting growth and inducing apoptosis in osteosarcoma cells. Our experimental data provide evidence for the therapeutic effect of mangiferin in osteosarcoma cells.