Data are presented seeing that mean??regular deviation (SD) from 3 independent experiments. Debate 3.1. Rhodomyrtone at Low Concentrations DIDN’T Affect Cell Viability in SW1353 Cells Our prior research confirmed that rhodomyrtone inhibited cell development and induced apoptosis in epidermis cancers cells [44]. In this scholarly study, we looked into whether rhodomyrtone suppressed cell viability in human chondrosarcoma SW1353 cells. MTT assay was performed to determine the cell viability and cell proliferation effect of rhodomyrtone on SW1353 cells. Figure 1(a) shows that rhodomyrtone suppressed SW1353 cell viability in a dose- and time-dependent fashion. Rhodomyrtone reduced cell viability of SW1353 cells at the high concentration ( 3? 0.001 vs. untreated control. 3.3. Rhodomyrtone at the Subcytotoxic Concentrations Inhibited SW1353 Cell Invasion and Adhesion To explore the effect of rhodomyrtone on cancer cell metastasis in SW1353?cell, we investigated the inhibition of SW1353 cell invasion by rhodomyrtone using Matrigel-coated Boyden chamber assay. Figures 3(a) and 3(b) show that rhodomyrtone reduced the SW1353 cell invasion in a concentration-dependent manner ( 0.001). The percentage of invaded cells was 48.2??4.4%, 46.4??10.1%, and 43.9??2.9% when treated with 0.5, 1.5, and 3? 0.01 and 0.001 vs. untreated control. 3.4. Rhodomyrtone at the Subcytotoxic Concentrations Inhibited the Expression and Activity of MMP-2 and MMP-9 Previous reports showed MMP-2 and MMP-9 expression was correlated with cancer invasion and the upregulation of MMPs was observed in invasive cancer cells [46C48]. The inhibition of MMP-2 and MMP-9 enzyme activity and protein expression has been shown to inhibit cancer cell migration and invasion in many types of tumor cells [49C52]. In this study, we investigated the expression and activity of MMP-2 and MMP-9 after treatment with rhodomyrtone at low concentrations. Gelatin zymography was performed to determine the activity of MMP-2 and MMP-9. The result demonstrated that rhodomyrtone significantly reduced the activity of MMP-2 and MMP-9 in a concentration-dependent manner as shown in Figures 4(a) and 4(b). The protein expression of MMP-2 and MMP-9 was determined by Western blot analysis. The result showed MMP-2 and MMP-9 protein expression was significantly suppressed by rhodomyrtone as compared to the untreated control as shown in Figures 4(c) and 4(d). These results p-Coumaric acid revealed that the rhodomyrtone inhibited both MMP-2 and MMP-9 activities and protein expression in SW1353 cells. Thus, inhibition of MMPs activities and protein expression is the target for preventing cancer metastases. This is consistent with Mouse monoclonal antibody to LIN28 previous reports, showing that resveratrol attenuated MMP-9 and MMP-2 regulated differentiation of HTB94 cells [52]. Some studies demonstrated that curcumin and curcumin derivative inhibited cancer cell invasion through the downregulation of MMPs in human A549 lung cancer cells [53], MDA-MB-231 human breast cancer cells [54], MCF-7 cells [55], and hepatocellular carcinoma [56]. Open in p-Coumaric acid a separate window Figure 4 Effect of rhodomyrtone on MMP-2 and MMP-9 activities and protein expression. (a) Photograph presented the gelatinolytic activity of MMP-2 and MMP-9. (b) Quantitative analysis of MMP-2 and MMP-9 activities was calculated using NIH ImageJ. (c) Expression of MMP-2 and MMP-9 proteins was detected by using the specific antibodies. (d) Protein levels of MMP-2 and MMP-9 were significantly suppressed by rhodomyrtone in a concentration-dependent manner. Data are presented as mean??standard deviation (SD) from three independent experiments. 0.05, p-Coumaric acid 0.01, 0.001 vs. untreated control. 3.5. Rhodomyrtone at the Subcytotoxic Concentrations Induced the Expression Endogenous Inhibitor of MMP-2 and MMP-9 In this study, we found that the activities of MMP-2 and MMP-9 were inhibited by rhodomyrtone. The activities of MMPs are specifically inhibited by a group of tissue inhibitors of metalloproteinases (TIMPs); TIMP-1 and TIMP-2 have been known to interact with MMP-9 and MMP-2, respectively. Several studies reported that overproduction of TIMPs can reduce metastasis whereas a low level of TIMPs correlates with tumor progression [17, 57, 58]. In this research, the expression of TIMP-1 and TIMP-2 was analyzed by Western blot analysis. We found that rhodomyrtone significantly increased TIMP-1 and TIMP-2 protein p-Coumaric acid expression in a concentration-dependent manner (Figures 5(a) and 5(b)). This is consistent with our previous study which showed that rhodomyrtone reduced A431 cell metastasis by suppressing MMP-2/9 activities and increasing the expression of TIMP-1 and TIMP-2 [43]. Similarly, Ferrari and colleagues showed that the upregulation of TIMP-1 by genipin could inhibit MMP-2 activity and suppressed the metastasis of HepG2 and MHCC97 L cells [57]. Likewise, the inhibition of A549 cell metastasis by increasing TIMP-2 expression [58]. This result indicated that the enzyme activities of MMP-2 and.