Molecular and Cellular Pharmacology
Ginsenoside Rg3 attenuates cell migration via inhibition of aquaporin 1 expression in PC-3M prostate cancer cells

https://doi.org/10.1016/j.ejphar.2012.02.040Get rights and content

Abstract

Ginsenoside Rg3 (Rg3), one of the bioactive extracts found in ginseng root, was reported to have anti-cancer activity in various cancer models. The anti-proliferation effect of Rg3 on prostate cancer cells has been well reported. To test whether Rg3 has an anti-metastatic effect on prostate cancer, we treated a highly metastatic PC-3M prostate cancer cell line with Rg3. We found that Rg3 (10 μM) led to remarkable inhibition of PC-3M cell migration. Simultaneously, exposure to Rg3 suppressed expression of the aquaporin 1 (AQP1) water channel protein, which has previously been reported to be involved in cell migration. Overexpression of AQP1 attenuated Rg3-induced inhibition of cell migration, and introduction of a shRNA targeting AQP1 abrogated the inhibitory effect of Rg3, although the basal level of cell migration was decreased by RNA interference. In mechanism study, estrogen receptor- and glucocorticoid receptor-dependent pathways are proved uninvolved in the AQP1 regulation by Rg3. However, Rg3 treatment triggered the activation of p38 MAPK; and SB202190, a specific inhibitor of p38 MAPK, antagonized the Rg3-induced regulation of AQP1 and cell migration, suggesting a crucial role for p38 in the regulation process. Deletion analysis of the promoter region of AQP1 was also conducted using dual-luciferase assay, which indicated that the − 1000 bp to − 200 bp promoter region was involved in the AQP1 regulation by Rg3. In all, we conclude that Rg3 effectively suppresses migration of PC-3M cells by down-regulating AQP1 expression through p38 MAPK pathway and some transcription factors acting on the AQP1 promoter.

Introduction

Prostate carcinoma is among the most common malignant cancers worldwide (Jemal et al., 2010). Whereas localized prostate carcinoma can be treated by radical prostatectomy or radiation therapy, patients are at increased risk of cancer metastasis (Cooperberg et al., 2009). Hormone manipulation has been developed as an effective resolution for advanced metastatic prostate carcinoma, however, the occurrence of loss or mutation of the androgen receptor in cancer cells results in failure of androgen deprivation therapy (Devlin and Mudryj, 2009). Many other therapeutic agents and protocols have been developed and utilized clinically, however, few of them have been shown to be dramatically effective. Moreover, drug resistance and cytotoxic side effects also impede the use of alternative therapies (Mahon et al., 2011).

Today, natural products have become a valuable resource for the development of new drugs (Cragg et al., 2009). Ginseng, which refers to the root of Panax ginseng and its related species, has been used for thousands of years in Asian countries and been reported to exhibit a wide range of medicinal effects. Ginsenoside Rg3, which has been posited to be one of the active ingredients in ginseng, has been shown to have various biological effects including anti-cancer activities (Jia and Zhao, 2009, Jia et al., 2009). Many studies have demonstrated that Rg3 has an anti-proliferation effect in various cancer cell lines including prostate cancer (Chen et al., 2008, Kim et al., 2004). An anti-metastasis activity for Rg3 has also been reported in several models (Iishi et al., 1997, Mochizuki et al., 1995). In this report, we sought to elucidate the effect of Rg3 treatment on a highly metastatic androgen receptor-negative prostate cancer cell line, PC-3M.

Aquaporin (AQP) is a water channel protein family eliciting fundamental functions in water transportation and osmotic homeostasis. The first member to be discovered, Aquaporin 1 (AQP1) is ubiquitously expressed in the human body (Borgnia et al., 1999). In addition to its basic function, AQP1 facilitates cell migration in a variety of cell types (Papadopoulos et al., 2008). Overexpression of AQP1 is common to malignancies from various organs and tissues, and several cancer cell lines expressing high levels of AQP1 exhibit enhanced migration in vitro and greater metastatic potential in vivo (Verkman et al., 2008). In addition, AQP1 promotes endothelial cell migration and angiogenesis, which is another important factor in tumor progression (Clapp and Martinez de la Escalera, 2006).

Cellular AQP1 expression is controlled through a number of mechanisms. Osmotic change is a principal factor in AQP1 regulation and can be elicited through transcription (Jenq et al., 1998, Jenq et al., 1999) or proteasome-mediated degradation (Leitch et al., 2001). Additionally, tonicity-independent mechanisms, such as glucocorticoid triggered AQP1 expression, have been described (Moon et al., 1997). Because previous studies have identified Rg3 as having glucocorticoid-like actions in cancer cells (Hien et al., 2010) and glucocorticoid is thought to modulate AQP1 expression, which is important in cell migration, we hypothesized that Rg3 may affect cell migration through AQP1 in PC-3M cells.

We found that Rg3 significantly suppresses cell migration and AQP1 expression in PC-3M cells. Furthermore, we showed that Rg3 treatment activated p38 MAPK, whereby PC-3M cell migration was mediated. We also offer possible mechanisms on the regulatory pattern of AQP1 through deletion analysis of the promoter of AQP1 using luciferase assay.

Section snippets

Drugs and reagents

Ginsenoside Rg3 was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and the purity was at least 95% as determined by HPLC. Rg3 was dissolved in dimethyl sulfoxide (DMSO) in a 100 mM stock solution and stored at − 20 °C. Aliquots of stock solution were added directly to the culture media.

The proteasome inhibitor MG132 (z-Leu-Leu-Leu-al, no. C2211) and dexamethasone (no. D1756) were purchased from Sigma-Aldrich. SB202190

Ginsenoside Rg3 inhibits cell migration but does not influence the proliferation of PC-3M cells

To evaluate the anti-metastatic effect of Rg3, we treated the metastatic prostate PC-3M cell line with various concentrations of Rg3 (up to 10 μM). Results from the MTT proliferation assay showed similar cell viability between the control group and the groups treated with various concentrations of Rg3 (Fig. 1B). However, in wound healing assays, the groups treated with 1 μM and 10 μM of Rg3 migrated much slower compared with the control group (Fig. 1C, D), suggesting anti-migration effects of Rg3.

Discussion

Ginsenoside Rg3, a principal bioactive component of ginseng extract, has been reported to exhibit anti-proliferative effects in cancer cell lines derived from various tissues (Wang et al., 2007). Previous reports have shown that Ginsenoside Rg3 and Rh2, its metabolite found in the human intestine, suppress proliferation and cell attachment of prostate cancer cells (Kim et al., 2004, Liu et al., 2000). Similar to other cancer subtypes, patients suffering from prostatic carcinoma are at risk for

Acknowledgments

This work was supported by the National Natural Science Foundation of China (no. 81020108031, 30572202, 30973558, 30772571, 30901815, 30901803), the Major Specialized Research Fund from the Ministry of Science and Technology in China (no. 2009ZX09103-144) and Research Fund from Ministry of Education of China (111 projects no. B07001).

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