Introduction
Melanoma is a type of skin cancer that develops from melanin-containing cells (melanocytes) located in the basal layer of the epidermis (1). The incidence of melanoma is the lowest among all skin cancers, but it is the most malignant and aggressive type of skin cancer, accounting for 75% of skin cancer-related deaths (2). Advanced melanoma frequently metastasizes to the lymph nodes (stage III) or distant organs (stage IV) (2,3). In 2017, ~87,100 individuals were diagnosed with advanced melanoma and 9,730 succumbed to the disease in the United States (4). At an early stage, melanoma may be curable by surgical excision, with a 5-year survival rate of >95% (5). However, at the advanced or metastatic stage there are fewer treatment options for controlling melanoma progression; as a result, the 5-year survival rate markedly decreases to 16% (6). Over the past decade, a large number of studies and clinical trials have been conducted to assess the potential molecular pathogenesis and treatment strategies for controlling advanced melanoma (7-18). Traditional chemotherapy was first used to inhibit tumor cell division, but with little survival benefit (7). Several intracellular signaling pathways have been identified, such as the mitogen-activated protein kinase (MAPK) (8-10) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathways (11-13), while a number of mutated oncogenes have also been identified, including BRAF (14,15), c-KIT (16,17) and RAS (18,19). These findings have led to novel approaches to controlling melanoma progression biologically, which have indeed achieved some improvement (20-22). More recently, tumor immunotherapy was applied to treat metastatic melanoma, such as pembrolizumab and nivolumab, which are programmed cell death protein 1 (PD-1) inhibitors that act by blocking the interaction of PD-1 with PD-L1 and restore immune function to eliminate tumor cells (23-25). Unfortunately, all these therapies have exhibited only limited effectiveness, as tumor resistance eventually develops. Thus, further study of new agents and the molecular mechanisms underlying melanoma development and progression may help identify novel therapeutic targets or drugs for controlling advanced melanoma.
Ginsenoside Rg3, a type of steroidal saponin component, is extracted from steamed Panax ginseng and has been shown to promote immune response and possess antitumor activity (26). Rg3 is the most active extract of steroidal saponins (27) and has been shown to inhibit the growth of different types of human cancers, such as colon (28,29), lung (30,31), breast (32), ovarian (33,34) and gallbladder cancers (35), glioma (36,37), leukemia (38) and hepatocellular carcinoma (39,40). Mechanistically, Rg3 was able to induce tumor cell apoptosis, thereby inhibiting tumor cell proliferation and metastasis (28-40) by suppression of nuclear factor (NF)-κB (29,41), AP-1 (activator protein 1) (42), vascular endothelial growth factor (VEGF) (43,44) and PI3K/Akt signaling (33,38), and activation of AMP-activated protein kinase (AMPK) pathway-related apoptosis (45). Considering these antitumor effects, Rg3 warrants further investigation as a novel and promising agent in the treatment of advanced melanoma.
In the present study, the role of Rg3 in melanoma was further assessed by evaluating its in vitro and in vivo inhibitory effects on melanoma cell growth and metastasis and melanoma-induced angiogenesis, aiming to provide useful insight into the effects of Rg3 on B16 melanoma cells and elucidate the underlying molecular events.
Materials and methods
Animals and cell lines
This animal use protocol was approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences. A total of 200, 6-week-old, male C57BL/6 mice (weighing ~21 g) were purchased from Beijing Experimental Animal Technical Co., Ltd., and housed at the Animal Center of Jilin University under the following conditions: Housing, 5 mice per cage; temperature, 22-25°C; humidity, 50-60%; 12 h light/dark cycle. The mice had access to food and water ad libitum.
A highly metastatic subline of murine B16 melanoma cells were kindly provided by Dr Xiaochun Xu of UT M.D. Anderson Cancer Center and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1 mM pyruvate, 4 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 µg/ml), and 100 µg/ml hygromycin B (all from Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Tumor cell viability determination using the MTT assay
To assess the effects of ginsenoside Rg3 on tumor cells in vitro, B16 cells were plated in 96-well plates at a density of 4×103 cells/well and incubated overnight, and were then treated for 72 h with a series of concentrations (1, 2.5, 5, 7.5, 10, 12.5 or 15 µg/ml) of Rg3 (Shanghai Haoran Biological Technology Co., Ltd., Shanghai, China). Rg3 was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and filtered through a 0.2-µm membrane before use. At the end of the experiments, cell proliferation was assessed by 10% MTT (Sigma-Aldrich; Merck KGaA), as described previously (46).
Cell counting assay
B16 cells (1.5×104) were seeded into 24-well plates and grown for 24 h, followed by treatment with different concentrations of Rg3 (2.5, 5 and 7.5 µg/ml) or 1% DMSO (control) for up to 6 days. The cells were then stained with 1% trypan blue (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the number of living cells was determined using a Bio-Rad TC10 Automated Cell Counter.
Flow cytometric cell cycle distribution assay
After 48 h of treatment with DMSO (control) and 2.5 or 5 µg/ml Rg3, B16 cells were trypsinized, washed with ice-cold phosphate-buffered saline (PBS), and fixed in 70% ethanol overnight. On the following day, the cells were stained with 0.5 mg/ml propidium iodide (PI; Sigma-Aldrich; Merck KGaA) in PBS containing 50 µg/ml RNase A (Sigma-Aldrich; Merck KGaA) and then analyzed using a flow cytometer with ModiFit LT™ software version 4.0 (BD Biosciences, Franklin Lakes, NJ, USA).
Immunofluorescence and immunohistochemistry
B16 cells were plated onto coverslips in 24-well plates at a density of 1×105 cells/well and grown overnight, followed by treatment with 5 µg/ml Rg3 for 24 h. The cells were then fixed with 4% freshly made paraformaldehyde and assessed with immunofluorescence, as described previously (47). A primary antibody against mouse PCNA (BioLegend, Inc., San Diego, CA, USA) and a fluorochrome-conjugated secondary antibody (1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used in this study.
B16-formed tumors from animal experiments were fixed in 4% paraformaldehyde solution and embedded in paraffin to prepare 5-μm sections. The tissue sections were then evaluated using immunohistochemistry, as described previously (47). A mouse anti-PCNA (1:500; BioLegend, Inc.) or rat anti-mouse VEGF antibody (1:500, BioLegend, Inc.) was used in the present study.
Tumor cell invasion assay
B16 cells were grown and treated with 0, 2.5 and 5 µg/ml Rg3 for 24 h, and then detached with 0.25% trypsin/EDTA solution (Sigma-Aldrich; Merck KGaA) for 5 min. Following inactivation of trypsin by addition of an equal volume of serum-containing medium, the cells were counted with a hemocytometer and placed into the top wells of Boyden chambers (BD Biosciences) at a density of 2×104, while the filters (8-µm pore size) were pre-coated with Matrigel (65 µl/filter; Osmonics, Inc., Minnetonka, MN, USA; the bottom wells were filled with 20% FBS-containing medium and the cells were incubated for 5 h. Subsequently, cells that had invaded into the lower surface of the filters were fixed with methanol for 10 min, stained with Harris’ hematoxylin (Sigma-Aldrich; Merck KGaA) for 10 min, and then counted under an inverted Olympus IMT-2 microscope (Olympus Corporation, Tokyo, Japan) at a magnification of ×400. Cell numbers from 18 microscopic fields were summed for each filter.
Vascular endothelial cell proliferation and migration assays
B16 cells were grown in RPMI-1640 medium containing 0 (control) or 5 µg/ml Rg3 for 24 h, and the conditioned medium was used to culture vascular endothelial cells (Cell Bank of Chinese Academy of Sciences, Beijing, China) for an additional 24 h, followed by cell viability MTT assay, PCNA staining and Boyden chamber invasion assay, as described above.
Western blot analysis
B16 cells were grown and treated with 0 (control) or 5 µg/ml Rg3 for 0, 24, 48 or 72 h and lysed for western blotting. Mouse tumor xenografts were also lysed for western blotting. The protocol was performed as described previously (47). Rabbit polyclonal antibodies against ERK (cat. no. sc-514302) and p-ERK (cat. no. sc-13073), Akt (cat. no. sc-8312) and p-Akt (cat. no. sc-7985-R), mammalian target of rapamycin (mTOR) (cat. no. sc-8319) and p-mTOR (cat. no. sc-101738), hypoxia-inducible factor (HIF)-1α (cat. no. sc-10790) and β-actin (cat. no. sc-130656) were used according to the manufacturer’s suggested dilutions (all from Santa Cruz Biotechnology, Inc.).
Mouse tumor cell xenograft assay
C57BL/6 mice were subcutaneously injected in the right flank with 2×106 B16 cells, and then randomly divided into five groups (n=10) and intraperitoneally injected with DMSO (control) and 0.3, 1.0 or 3.0 mg/kg Rg3 and/or 20 mg/kg 5-fluorouracil (5-FU; Sigma-Aldrich; Merck KGaA) for 10 consecutive days. Two days after drug withdrawal, the mice were sacrificed and the tumors were resected, weighed and examined for PCNA expression.
Mouse tumor cell metastasis assay
C57BL/6 mice were subcutaneously injected in the right hind footpad with 5×105 B16 cells, and then randomly divided into 5 groups (n=10) and intraperitoneally injected with DMSO (control) and 0.3, 1.0 or 3.0 mg/kg Rg3 and/or 20 mg/kg 5-FU (Sigma-Aldrich; Merck KGaA) for 35 consecutive days. The primary tumors were excised 21 days after the subcutaneous injection of B16 cells. At 14 days after resection, the mice were sacrificed and their lungs were fixed in Bouin’s solution for analysis of tumor cell metastasis.
B16 cell-induced mouse angiogenesis assay
C57BL/6 mice were inoculated intradermally with 5×105 B16 cells on the dorsal flank flap and then randomly divided into four groups (n=3) and intraperitoneally injected with 0 (control), 0.3, 1.0 or 3.0 mg/kg Rg3 for 5 consecutive days. Seven days after the last injection, the mice were sacrificed and the skin was separated from the underlying tissues for quantification of angiogenesis by counting the number of vessels oriented toward the tumor mass under a dissecting microscope. The tumor size was approximated by averaging the diameters of the short and long axes of the residual inoculated cells.
Determination of microvascular density (MVD)
Tumor tissues from the angiogenesis assay were stained with antibodies against the endothelial marker CD31, and MVD was determined according to the method of Weidner et al (48).
Statistical analysis
Data are expressed as the mean ± standard deviation and were statistically analyzed using analysis of variance (ANOVA) or the unpaired t-test for two-group comparisons, while the ANOVA Tukey’s multiple comparison test was performed for analysis of differences among three or more groups. All experiments were performed in triplicate and repeated at least three times. P≤0.05 was considered to indicate statistically significant differences.
Results
Rg3 inhibits the growth of B16 melanoma cells
In the present study, the effect of Rg3 on the growth of B16 melanoma cells was first evaluated in vitro, and Rg3 was found to significantly inhibit B16 cell growth in a dose-dependent manner (Fig. 1A); the IC50 was 7.76±0.46 µg/ml. The cell counting assay revealed that Rg3 also reduced the numbers of living B16 melanoma cells in a dose-dependent manner (Fig. 1B). Moreover, the cell cycle analysis demonstrated that 48 h of treatment with Rg3 led to an arrest of the cell cycle at the S phase (0.11 vs. 12.68 and 24.37% after 2.5 and 5 µg/ml of Rg3 treatment, respectively; Fig. 1C). As shown in Fig. 1D, Rg3 significantly downregulated PCNA expression in B16 melanoma cells.
Furthermore, our in vivo data on B16 cell xenografts and Rg3 treatment demonstrated that tumor growth (Fig. 2A and C) and PCNA expression (Fig. 2B and D) in Rg3-treated mouse tumor cell xenografts were inhibited in a dose-dependent manner (1 or 3 mg/kg Rg3 exerted a stronger inhibitory effect compared with 0.3 mg/kg Rg3, P<0.05; however, there was no significant difference between 1 or 3 mg/kg Rg3 and 20 mg/kg 5-FU).
Rg3 inhibits the metastasis of B16 melanoma cells
We then assessed the effect of Rg3 on tumor cell invasion and metastasis, and found that the in vitro invasion capacity of B16 cells treated with 2.5 or 5 µg/ml Rg3 was significantly reduced compared with that of controls (P<0.01; Fig. 3A and C). Trypan blue staining demonstrated similar numbers of living cells among these three groups of cells before being placed into Boyden chambers (data not shown), ruling out the possibility of Rg3 cytotoxicity.
Furthermore, the in vivo tumor cell metastasis assay demonstrated that mice injected intraperitoneally with 0.3, 1.0 or 3.0 mg/kg Rg3, or 20 mg/kg 5-FU daily for 7 weeks exhibited markedly reduced numbers of lung metastatic nodules (Fig. 3B and D). As the 3.0 mg/kg Rg3 group had a lower number of tumor cell lung metastatic nodules compared with the 0.3 mg/kg Rg3 group (P<0.05), the inhibitory effect was dose-dependent. Moreover, since MMP-2 and MMP-9 were highly associated with tumor invasion and metastasis (49,50), their expression was assessed in Rg3-treated tumor tissues using immunohistochemistry and was found to be downregulated (Fig. 3E).
Rg3 inhibits B16 melanoma-induced angiogenesis by reducing VEGF expression
Angiogenesis is an important characteristic of tumor lesions, as melanoma growth and metastasis are dependent on angiogenesis (51). We hereby investigated the effect of Rg3 on B16 melanoma-induced angiogenesis. Seven days after intradermal injection of B16 cells, tumor lesions had formed in the skin and the number of vessels oriented toward the tumor lesions was counted. We found that intraperitoneal injection of Rg3 was associated with a slightly smaller tumor size, but the difference relative to the control was not statistically significant (data not shown). However, the number of blood vessels in the Rg3 groups were markedly reduced compared with the control group (Fig. 4A and C). In addition, CD31 staining revealed that MVD was significantly reduced following treatment with Rg3 (Fig. 4B and D). Since VEGF is a key factor in the regulation of angiogenesis (52), VEGF expression was evaluated in these tumor lesions and was found to be downregulated in Rg3 groups (Fig. 4E and F). Reduced expression of VEGF may be caused by downregulation of the main transcription factor, HIF-1a (Fig. 4G). Furthermore, Rg3 also inhibited the expression of VEGF in B16 cells cultured on coverslips (Fig. 5A) and the effects of B16 cell-conditioned medium on regulation of proliferation and migration of vascular endothelial cells was then assessed. Our data demonstrated that Rg3-treated vascular endothelial cells exhibited weaker staining for PCNA and lower OD value, indicating that Rg3 reduced vascular endothelial cell proliferation (Fig. 5B and C) and migration (Fig. 5D and E).
To summarize, Rg3 inhibited the expression of VEGF in B16 cells, and VEGF downregulation further decreased angiogenesis by attenuating proliferation and migration of vascular endothelial cells.
The antitumor effects of Rg3 are mediated by regulation of ERK and Akt signaling
Finally, we attempted to explore the molecular pathways underlying the antitumor role of Rg3. Since ERK and Akt signaling are two major pathways implicated in melanoma progression, the expression of their pathway proteins was assessed in B16 cells and tumor tissues. Our data demonstrated that Rg3 treatment decreased the levels of phosphorylated ERK, Akt and mTOR in B16 cells and tumor cell xenografts (Fig. 6). However, there was no significant change in the expression of total ERK, Akt and mTOR (Fig. 6).
Discussion
Advanced melanoma is difficult to control clinically; thus, it is important to develop novel and effective agents for melanoma patients. Rg3, an extract obtained from Panax ginseng, has shown antitumor activity in various types of human cancers (28-40). However, the number of studies investigating its role in melanoma is very limited. It was recently demonstrated that Rg3 can inhibit the proliferation of melanoma cells by decreasing HDAC3 and increasing acetylation of p53 (53). However, there are no more reported data on its role in metastasis, melanoma-induced angiogenesis, or other underlying molecular pathways. The aim of the present study was to investigate the antitumor role of Rg3 in melanoma, and determine whether Rg3 can inhibit melanoma growth, lung metastasis and melanoma-induced angiogenesis in vitro and in vivo. Our data demonstrated that the antitumor effects of Rg3 in melanoma were mediated through downregulation of the ERK and Akt signaling pathways.
Cell cycle progression comprises a series of events that take place during cell proliferation, including the G0, G1, S, G2 and M phases of the cell cycle (54). Our data revealed that exposure of B16 cells to Rg3 arrested tumor cells at the S phase of the cell cycle. Since DNA is synthesized in the S phase, this result indicates that Rg3 can prevent DNA synthesis in melanoma cells, which was reflected by the reduction of PCNA immunostaining, a marker of DNA replication (55). However, a previous study on gallbladder cancer revealed that Rg3 induced tumor cell cycle arrest at the G0/G1 phase, the major checkpoint for cell division or apoptosis (35), indicating that Rg3 may regulate different checkpoints of the cell cycle in different types of tumor cells.
MMPs are a family of enzymes responsible for the degradation of various extracellular matrix components (49). Among the 20 identified MMPs, MMP-2 and MMP-9 are highly associated with tumor dissemination and invasiveness (49,50). Numerous studies have demonstrated that upregulation of these two proteins is closely associated with tumor metastasis (56,57). The findings of the present study indicated that Rg3 may suppress melanoma cell metastasis through downregulation of MMP-2 and MMP-9 (Fig. 3E). Moreover, MMP-2 and MMP-9 have also been shown to stimulate angiogenesis and promote VEGF release (58,59). VEGF is a potent and selective endothelial mitogen, inducing rapid and complete angiogenic response by binding to its receptor (60). Consistent with these findings, our data are, to the best of our knowledge, the first to demonstrate that Rg3 decreased melanoma-induced angiogenesis by inhibiting the expression of VEGF, which may be attributed to the reduced expression of MMP-2 and MMP-9.
The ERK and Akt signaling pathways are constitutively activated in melanoma, and play a key role in melanoma development and progression (11). Various molecules, such as MMP-2, MMP-9 and VEGF, are regulated by these two pathways (59,61). It has been reported that Rg3 inhibits breast cancer cells by deactivating NF-ĸB signaling, with ERK and Akt serving as the potential upstream targets (62). Pharmacological inhibitors of the two kinases abrogate the antitumor effect of Rg3 (62). Consistent with these data, our study demonstrated that ERK and Akt signaling was deactivated following Rg3 treatment of B16 cells, supporting the antimelanoma role of Rg3. The downregulation of the two pathways may also explain the reduced expression of MMP-2, MMP-9 and VEGF.
However, in addition to the ERK and Akt pathways, other mechanisms must still be investigated to determine whether they could mediate the effects of Rg3 on melanoma cells in vitro and in vivo. For example, Rg3 has been shown to induce apoptosis in colon cancer cells by activating AMPK signaling (45). It is worth investigating whether Rg3 exerts a similar effect on melanoma. Furthermore, immune response should be examined to assess whether it is activated by Rg3 in melanoma. As reported previously, Rg3 can activate ovalbumin-induced immune response (63), thereby causing tumor cell elimination (64,65). Therefore, it may be hypothesized that Rg3 may also exert antitumor effects by activating the immune system.
In conclusion, Rg3 effectively reduced melanoma cell growth, metastasis and melanoma-induced angiogenesis in vitro and in vivo through suppression of DNA synthesis and expression of MMP-2, MMP-9 and VEGF. The underlying molecular events may include downregulation of the ERK and Akt pathways. Based on the findings of the present study, Rg3 may be a promising novel agent for the treatment of melanoma. However, the effectiveness of Rg3 must be further assessed in a clinical trial of metastatic melanoma.