Synergistic anti-tumour activity of ginsenoside Rg3 and doxorubicin on proliferation, metastasis and angiogenesis in osteosarcoma by modulating mTOR/HIF-1α/VEGF and EMT signalling pathways

Abstract

Objectives

The most common cause of osteosarcoma (OS) death is lung metastasis. Currently, doxorubicin is the primary chemotherapy drug used to treat OS, however, it is not effective in inhibiting metastasis, and it has obvious cardiotoxicity. The anticancer activity of ginsenoside Rg3 has been demonstrated in a variety of malignant tumours. The aim of this study was to determine the potential role of ginsenoside Rg3 and doxorubicin in OS and the possible mechanism.

Methods

The potential synergistic effects of ginsenoside Rg3 and doxorubicin on human osteosarcoma cells 143B and U2OS, human umbilical vein endothelial cells, and mice receiving 143B xenografts and lung metastases were investigated.

Key findings

Our study demonstrated that the combination of ginsenoside Rg3 and doxorubicin significantly inhibited cell proliferation, metastasis and angiogenesis in vitro. Mechanically, the anti-tumour activity of ginsenoside Rg3 and doxorubicin by modulating mTOR/HIF-1α/VEGF and EMT signalling pathways. Furthermore, ginsenoside Rg3 combined with doxorubicin inhibits tumour growth and lung metastasis in 143B-derived murine osteosarcoma models. More importantly, ginsenoside Rg3 can effectively ameliorate doxorubicin-induced weight loss and cardiotoxicity in mice.

Conclusions

Consequently, we concluded that the combination of ginsenoside Rg3 and doxorubicin displayed an evidently synergistic effect, which has the potential to be used as an effective and safe therapeutic approach for OS treatment.

Introduction

Osteosarcoma (OS) is the most common primary malignant tumour of bone tissue that originates from mesenchymal cells.^[1] There is an increased risk of OS in children, young adults and people over 60 years of age.^[2] The 5-year survival rate for patients with primary OS is approximately 60%, whereas patients with recurrent or metastatic disease have a survival rate of just 20%, and the lung is the most common site of metastasis.^{[2, 3]} Osteosarcoma is primarily treated with surgery, preoperative and postoperative chemotherapy. Currently, doxorubicin (DOX), methotrexate and cisplatin are the most widely used chemotherapy drugs in the first-line treatment of osteosarcoma.^[4] DOX is a highly effective anti-osteosarcoma drug, patients often suffer from breakthrough tumour growth, local recurrences and distant metastases when receiving chemotherapy,^[5] as well as cardiotoxic side effects that detract from patients’ quality of life and survival time.^[6]

To improve the efficacy and reduce side effects of DOX in the treatment of OS, combined chemotherapy is often used.^[7] For instance, resveratrol can induce apoptosis in OS cells^[8] and reduce the resistance of gastric cancer cells to DOX.^[9] Another research suggested that the combination of oleuropein and DOX can enhance autophagy in OS cells.^[10] These findings indicated that natural compounds can play an important role in the treatment of OS.^[11]

The ginsenosides are an important group of naturally occurring chemicals isolated from ginseng (long-standing herbal medicine used by humans).^[12] These ginsenosides are thought to be responsible for the health-promoting effects of ginseng. Ginsenoside Rg3 (Rg3) is a well-studied ginsenoside with different pharmacological effects, including its anti-inflammatory, antioxidant and immune-boosting properties.^[13] It is also believed to have potential benefits for treating various cancers.^{[14, 15]} In addition, ginsenoside Rg3 enhances the anticancer effect of sorafenib, and doxorubicin in the treatment of hepatocellular carcinoma.^{[16, 17]} According to the State Food and Drug Administration of China in 2000, ginsenoside Rg3, one of the main components of ‘Shenyi capsule’, is an anticancer drug that has been used synergistically in the clinic to treat non-small cell lung cancer or breast cancer.^{[18, 19]} However, no study has investigated the pharmacological role of ginsenoside Rg3 combined with doxorubicin on OS.

Angiogenesis plays a role in normal physiological processes such as wound healing and the female reproductive cycle, as well as meeting the nutrition and oxygen requirements of tumour growth.^[20] Neovascular networks not only promote tumour growth but also allow tumour cells to spread from the primary foci to other organs. The treatment of OS with antiangiogenic drugs, including bevacizumab and sorafenib, has been shown to be effective in clinical studies.^{[21, 22]} This indicates that antiangiogenic therapy is one of the most important tools for treating OS, especially metastatic osteosarcoma.

In this study, the synergistic effects of ginsenoside Rg3 and doxorubicin were validated in 143B, U2OS, and human umbilical vein endothelial cells (HUVECs) and 143B-derived murine osteosarcoma models. Finally, we employed WB to investigate the mechanism of synergistic effect.

Materials and Methods

Drugs and reagents

Ginsenoside Rg3 (≥98%) and doxorubicin (≥98%) were obtained from Aladdin Biochemical Technology (Shanghai, China). The p-mTor (Ser2448) antibody and mTor antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). AKT antibody, p70S6 Kinase antibody, VEGF antibody, N-cadherin antibody, vimentin antibody were purchased from Proteintech (Wuhan, China). The p-AKT(Ser473) antibody, p-p70S6 Kinase (Ser427) antibody was purchased from Signalway Antibody (CollegePark, MD, USA). HIF-1α antibody was obtained from Bioss Antibodies (Beijing, China) and collagen I antibody was purchased from Abcam (Cambridge, UK). GAPDH rabbit polyclonal antibody was obtained from Bioworld Technology (Nanjing, China).

Cell culture and animals

The HUVECs were purchased from Procell Life (Wuhan, China), and all experiments were performed within three passages. The human OS cell line 143B and U2OS cells were obtained from Nanfang Hospital, Southern Medical University (Guangzhou, China). The cells were cultured in a DMEM medium containing 10% FBS in a 37°C incubator containing 5% CO₂.

Male athymic nude mice (6–8 weeks old) were purchased from Bestest (Zhuhai, China). The nude mice were housed in an SPF environment with a temperature of 18–25°C, a 12-h light cycle, and free access to food and water. All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. The Experimental Animal Ethics Committee of South China Hospital, South China University approved all experimental protocols as approval no. 2022-ky-83 (date: 08-31-2022).

Cell proliferation assay

Cells were seeded in 96-well plates at a density of 3 × 10³ cells/well. The cells were then incubated with Rg3 (0–160 μg/ml), DOX (0–20 μg/ml) alone or in combination, respectively. After 48 h, MTT assay was used for cell viability. Measure the absorbance at 490 nm using a microplate reader (Bio-Rad model 680, UK).

Colony formation assay

Cells were inoculated in six-well plates at a density of 1 × 10³ cells/well. After cell attachment, cells were treated with 80 μg/ml Rg3 and 0.3125 μg/ml DOX alone or in combination. Colonies were stained with crystalline violet and photographed for counting 7–10 days after administration.

Transwell migration assay

Cell migration experiments were carried out using transwell plates with a pore size of 8 µm. The density of the cell suspension was 1.5 × 10⁵/ml and serum-free medium was added to the upper chamber of the transwell and 10% FBS medium to the lower chamber. At the same time, drugs were added to the upper chamber, and Rg3 and DOX were treated alone or in combination. After 24 h of treatment, the cells were fixed with 4% paraformaldehyde, stained with crystalline violet and photographed with an inverted microscope (Nikon, ECLIPSE Ti-S, Tokyo, Japan) for counting.

Wound-healing assay

The cells were seeded into six-well plates and when cell confluence reaches 100%, three parallel lines are drawn evenly across the bottom of each well using the tip of the 200 μl tip. The cells were then fed with serum-free medium containing Rg3, DOX, either alone or in combination. Finally, images were taken with an inverted microscope (Nikon, ECLIPSE Ti-S, Tokyo, Japan) at 0 and 24 h, respectively, and the width of the scratches was measured with ImageJ software.

Tube formation assay of HUVECs

HUVECs were inoculated at a density of 2 × 10⁴ per well onto 24-well plates that were pretreated with 250 μl of 1% (w/v) matrigel matrix (Corning, USA) as well as treated with Rg3, DOX alone or in combination for 12 h. The formation of endothelial cell tubes was observed by inverted microscopy (Nikon, ECLIPSE Ti-S, Tokyo, Japan) and the total length was measured with ImageJ software.

Western blot

Proteins were extracted through a cellular RIPA lysis buffer containing a mixture of protease and phosphatase inhibitors and protein concentrations were measured using the BCA method. Proteins were separated using SDS-PAGE gels, then protein samples were transferred to PVDF membranes, closed with 5% skim milk (Solarbio, Beijing), washed three times with TBST, and then treated with appropriate dilutions of primary antibodies (p-AKT, diluted 1:1000; AKT, diluted 1:1000; p-mTor, diluted 1:1000; mTor, diluted 1:1000; p-p70S6 Kinase, diluted 1:1000; p70S6 Kinase, diluted 1:000; HIF-1α, diluted 1:1000; VEGF, diluted 1:4000; N-cadherin, diluted 1:1000; vimentin, diluted 1:1000; collagen I, diluted 1: 1000; GAPAD, diluted 1:1000; tubulin, diluted 1:1000) overnight at 4°C. The next day, the membranes were washed three times with TBST followed by incubation with secondary antibodies for 1 h at room temperature and developed using ECL illumination solution (Millipore, USA).

In-vivo anti-tumour effects

About 100 μl of 7 × 10⁶ 143B cells in serum-free medium mixed with 100 μl of matrigel (Corning, USA) were implanted subcutaneously into the backs of mice. At a mean tumour volume of 100 mm³, the mice were randomly divided into four groups (n = 4). Saline, Rg3 (20 mg/kg) administered once daily by gavage, DOX (2 mg/kg) by intraperitoneal injection twice a week, DOX (2 mg/kg) combined with Rg3 (20 mg/kg). Both medicines were administered continuously for 3 weeks. Tumour volume (V) was determined every 3 days by measuring tumour length (a) and width (b) with vernier callipers, calculated as: V(mm³) = 1/2*ab². Anti-tumour efficacy was determined according to tumour growth and final tumour weight.

In-vivo anti-lung metastatic effects

The stably transfected 143B cells expressing luciferase (143B-LUC; 1 × 10⁶ cells/mice) were injected into mice via the tail vein.^[23] A Xenogen IVIS100 imaging system (Xenogen Corp., Hopkinton, MA, USA) was used to assess lung metastases development in vivo. Three days after injection, mice were randomly divided into four groups (n = 4). Saline, Rg3 (20 mg/kg) administered once daily by gavage, DOX (2 mg/kg) by intraperitoneal injection once weekly, DOX (2 mg/kg) combined with Rg3 (20 mg/kg). Mice were tracked for luciferase readings and body weight and sacrificed 4 weeks after administration. Lung tissue was extracted and luciferase readings were taken. Mice hearts were collected and weighed.

Hematoxylin and eosin (H&E) staining

After the mice were sacrificed, heart, lung and tumour tissues were embedded in paraffin, dewaxed, stained with hematoxylin nuclear staining concentrate and 0.5% eosin staining solution, and sealed with neutral glue. Photographs were examined with an upright microscope (Nikon, Ni-u, Tokyo, Japan) in five random fields.

Immunohistochemistry staining

Paraffin sections are routinely dewaxed in xylene and rehydrated in an alcohol gradient. The tissue antigen was then heated in sodium citrate (pH 6.0) for 15 min and the sections were closed with serum and incubated with primary antibodies anti-ki67 (diluted 1:100); anti-CD31 (diluted 1:100), overnight at 4°C. Samples were examined with an upright microscope (Nikon, Ni-u, Tokyo, Japan) and positive ki-67 cells and CD31 vessel counts were counted in five random fields.

Statistical analysis

GraphPad Prism 9 (GraphPadSoftware, San Diego, CA, USA) was used to plot the images. SPSS 17.0 was used for statistical analysis. The data were analysed by Student’s t-test between two groups and one-way analysis of variance was used to analyse three or more groups. All data are expressed as mean ± standard deviation, with P < 0.05 indicating significance.

Results

Ginsenoside Rg3 enhances the anti-proliferative effect of doxorubicin on osteosarcoma cell lines

The anti-proliferative effects of Rg3 and DOX were measured by MTT and colony formation assay. Rg3 and DOX were administered to OS cells in combination or separately for 48 h. As shown in Figure 1A, B, E and F, Rg3 showed obvious cytotoxicity at 160 μg/ml, while DOX showed dose-dependent inhibition. In particular, DOX toxicity to OS cells was significantly increased when DOX was combined with Rg3 (80 μg/ml). Additionally, the colony formation assay confirmed that Rg3 and DOX inhibited the proliferation of OS cells. The number of clones in the control, Rg3, and DOX groups of 143B cells was 430 ± 14.8, 287 ± 11.4 (P < 0.01, compared with control) and 189 ± 21.1 (P < 0.001, compared with control), respectively (Figure 1C and D). Figure 1H and G shows similar results in U2OS cells. There is a greater inhibitory effect demonstrated with a combination of DOX and Rg3 than with DOX alone (P < 0.05).

Ginsenoside rg3 combined with doxorubicin inhibits the migration of osteosarcoma cells

Transwell assays and wound-healing assays were conducted to examine the effect of Rg3 and DOX on OS cell migration. In Figure 2A and C, Rg3 treatment successfully blocked the passage of OS cells through the pore space into the lower chamber compared with the control group (P < 0.0001, compared with control). DOX alone was not statistically significant in reducing the lower chamber cell count, but DOX used in conjunction with Rg3 further reduced the number of cells in the lower chamber compared with Rg3 alone (P < 0.01, compared with Rg3) (Figure 2B and D). This phenomenon also occurred during the wound-healing assay (Figure 2E and G), when 143B and U2OS cells were treated with Rg3 for 24 h, the wound-healing ratio decreased to 44.4% ± 0.4% (P < 0.001, compared with control) and 47.5% ± 1.5% (P < 0.01, compared with control), respectively, while DOX did not significantly impair wound healing, but DOX combined with Rg3 inhibited wound healing more than Rg3 alone, with wound healing ratio of 19.7% ± 2.4% (P < 0.05, compared with Rg3) and 26.8% ± 0.9% (P < 0.01, compared with Rg3) (Figure 2F and H).

Several studies have suggested that EMT contributes to tumour metastasis. The protein expression of mesenchymal markers N-cadherin, vimentin and collagen I decreased after Rg3 treatment in comparison with the control group. In the DOX-alone treated group, there was no significant difference in protein expression of the EMT pathway. However, the combination of DOX and Rg3 led to the greatest reduction in protein expression (Figure 2I and J and Supplementary Figure S1). The results indicated that the combination of Rg3 and DOX could synergistically inhibit the migration of OS cells by inhibiting the EMT pathway.

Ginsenoside Rg3 combined with doxorubicin inhibits the proliferation, migration and angiogenesis of HUVECs

The angiogenic process requires the proliferation of endothelial cells. The MTT assay showed that Rg3 was cytotoxic to HUVECs only at concentrations starting at 160 μg/ml (P < 0.01, compared with control) (Figure 3A), while DOX had a dose-dependent inhibitory effect on HUVECs, and the inhibitory effect on cell viability was significantly enhanced when DOX was combined with Rg3 (P < 0.01, P < 0.0001, compared with DOX) (Figure 3B).

To study angiogenesis in vitro, HUVECs are a useful tool. HUVECs stimulated with VEGF formed robust tubular structures when inoculated on the stromal gel, while Rg3 inhibited capillary-like networks (Figure 3C and D). Angiogenesis begins with the migration of endothelial cells. The transwell and wound-healing assays showed the same therapeutic effect for HUVECs as OS cells (Figure 3E and G). Fewer HUVECs reach the lower chamber (P < 0.0001) and a slower wound-healing ratio (P < 0.0001) was observed in the Rg3-treated group as compared with the control group. Furthermore, DOX combined with Rg3 produced a better therapeutic effect than Rg3 alone (Figure 3F and H).

In addition, Western blot analysis confirmed that the expression of mesenchymal markers such as N-cadherin, vimentin and collagen I proteins was decreased following Rg3 treatment compared with the control group. Furthermore, combining DOX and Rg3 resulted in the greatest reduction in protein expression (Figure 3I, Supplementary Figure S1). All the results indicated that the combination of Rg3 and DOX could synergistically inhibit the angiogenesis of HUVECs by inhibiting the proliferation, migration and tube formation of HUVECs.

The anti-tumour mechanism of ginsenoside Rg3 in combination with doxorubicin in vitro

To examine how ginsenoside Rg3 combined with DOX inhibits proliferation and angiogenesis in vitro, the expression of proteins related to the mTOR/HIF-1α/VEGF signalling pathway was detected by Western blot. As shown in Figure 4A–C, Rg3 inhibits p-AKT, p-mTOR and p-p70S6K expression in 143B cells, U2OS cells and HUVECs, while the total expression of AKT, mTOR and p70S6K proteins remains unchanged. DOX had no significant effect on p-AKT, p-mTor or p-p70S6K expression. Comparatively, cells treated with Rg3 plus DOX showed even greater reductions in protein expression than those treated with Rg3 alone (Supplementary Figures S2–S4). On the other hand, HIF-1α/VEGF, a downstream protein of mTOR/p70S6K, has an imperative role in angiogenesis. Figure 4D–F show that Rg3 inhibits the expression of HIF-1α and VEGF in 143B cells, U2OS cells and HUVECs compared with controls. Although DOX had no significant effect on the expression of HIF-1α or VEGF, Rg3 combined with DOX produced a more effective therapeutic effect than Rg3 alone (Supplementary Figures S2–S4). As a result, Rg3 synergizes DOX’s anti-tumour effects by inhibiting the mTOR/HIF-1α/VEGF signalling pathway.

Ginsenoside Rg3 enhances doxorubicin’s inhibition of osteosarcoma growth in mice

We investigated the anti-tumour effects of Rg3 and DOX on 143B-cell xenograft mice. Figure 5A and B shows photographs of live animals and tumour tissues extracted from each group. The tumour volumes in the control, Rg3, DOX and combined Rg3 and DOX groups were 1309.4 ± 177.5 mm³, 805.9 ± 78.9 mm³ (P < 0.0001, compared with control), 551.1 ± 55.6 mm³ (P < 0.0001, compared with control) and 390.1 ± 60.5 mm³ (P < 0.05, compared with DOX), respectively (Figure 5C). The tumour weights were 1.05 ± 0.09 g, 0.68 ± 0.10 g (P < 0.01, compared with control), 0.47 ± 0.08 g (P < 0.001, compared with control) and 0.26 ± 0.05 g (P < 0.05, compared with DOX), respectively (Figure 5D). The tumour inhibition rate of the combined Rg3 and DOX group was 70% compared with the control group.

It is evident from the HE staining of tumour tissue (Figure 5G) that the Rg3 combined with the DOX group displayed significantly more foci of necrosis and fibrosis than the other groups. Additionally, anti-proliferation markers ki-67 and CD31-positive vessels (Figure 5G) showed that Rg3 combined with DOX reduced tumour cell proliferation (P < 0.05, compared with DOX) (Figure 5E) and angiogenesis (P < 0.05, compared with Rg3) (Figure 5F). According to the above findings, Rg3 in combination with DOX inhibits tumour growth in vivo by suppressing cell proliferation and angiogenesis.

Ginsenoside Rg3 combined with doxorubicin inhibits lung metastasis of osteosarcoma

Lung metastasis is a major cause of death in OS patients. By in-vivo imaging, we determined whether Rg3 combined with DOX inhibited lung metastasis in mice. In Figure 6A and C, DOX-treated mice did not differ significantly from control mice in luminescence intensity. However, the lung luminescence intensity of mice in the Rg3-treated group was lower than that of the control group (P < 0.05, compared with control). We executed mice and obtained lung tissues, and the luminescence intensity of isolated lung tissues obtained by IVIS assay was consistent with the in-vivo imaging results (P < 0.01) (Figure 6B and D). Meanwhile, fewer and smaller lung metastases were observed on the lung surface in the Rg3 combined with the DOX treatment group compare with DOX (P < 0.001) (Figure 6E).

HE staining of the lung tissue also confirmed the results of the biopsy (Figure 6G). The lung nodules numbers in the control, Rg3, DOX and combined Rg3 and DOX groups were 16.7 ± 1.9, 8 ± 0.8 (P < 0.05, compared with control), 16.3 ± 0.9 and 4.3 ± 0.9 (P < 0.05, compared with DOX), respectively (Figure 6F).

Ginsenoside Rg3 reduces cardiotoxicity of doxorubicin in the treatment of osteosarcoma

To examine whether Rg3 could improve the toxic effects of DOX in mice, we continuously recorded the body weight of mice during the administration of the drug. As shown in Figure 7A and E, DOX-treated mice had lower body weights compared with the control group, whereas the mice in the Rg3 combined with the DOX group exhibited significantly improved body weight than the mice in the DOX group (P < 0.05, P < 0.01, compared with DOX). It is known that DOX has serious cardiotoxic effects. DOX markedly decreased heart weight when compared with control group, whereas Rg3 coupled with DOX increased heart weight (P < 0.05, P < 0.01, compared with DOX) (Figure 7C and G). We also observed that the heart volume was significantly larger than that in the DOX group (Figure 7B and F), which may be due to the fact that ginsenoside Rg3 ameliorated cardiotoxicity. Moreover, cardiac HE staining revealed haemorrhagic foci and neutrophil aggregation in DOX-treated heart tissues, and Rg3 and DOX combined showed significant improvement in myocardial injury over DOX alone (Figure 7D and H).

Discussion

The osteosarcoma is one of the malignant tumours that seriously threaten the health of children and adolescents.^[24] In particular, patients who develop tumour metastasis have a very low 5-year survival rate.^[25] Unfortunately, chemotherapeutic drugs do not effectively treat metastatic osteosarcoma and have some toxic side effects, which reduce patients’ quality of life.^{[26, 27]} Therefore, it is necessary to find a medicine that is capable of inhibiting osteosarcoma growth and metastasis. In our study, we found that the combination of ginsenoside Rg3 and DOX displayed an evidently synergistic effect, which inhibits proliferation, metastasis and angiogenesis, and this process works by inhibiting AKT/mTOR/pS6K, mTOR/HIF-1α/VEGF and EMT signalling pathways. Moreover, DOX toxic effects such as cardiotoxicity and weight loss can be alleviated by ginsenoside Rg3.

Several cytoplasmic proteins contribute to cell proliferation and are associated with cancer, particularly the Akt and rapamycin (mTOR)/p70S6K signalling pathways, which regulate cell survival, growth and proliferation.^{[28, 29]} These pathways are implicated in many human malignancies, including osteosarcoma, breast cancer, prostate cancer and colorectal cancer.^[30–32] The mTOR kinase plays an important role in regulating protein synthesis and cell cycle progression. The p70S6K is an important downstream protein of the Akt/mTOR signalling pathway and mediates mTOR regulation of protein translation. As a result, the Akt/mTOR/p70S6K signalling pathway is essential in the oncogenesis of many cancers, including osteosarcoma.^{[11, 33]} DOX is an anti-tumour antibiotic that slows the growth of tumour cells at every stage of the cell cycle.^[34] We found that ginsenoside Rg3 inhibited the Akt/mTOR/p70S6K signalling pathway and acted synergistically with DOX. This is consistent with the findings of Jin Jiang et al. that ginsenoside Rg3 acts synergistically with erlotinib in the treatment of pancreatic cancer.^[35] Our results show that Rg3 significantly increases the cytotoxicity of DOX to osteosarcoma cells and inhibits cell proliferation. Furthermore, we observed that ginsenoside Rg3 and DOX combined can significantly inhibit ki67 expression in tumour tissue, and that the effect is greater than that of monotherapy.

Most osteosarcoma patients die of metastatic disease, especially lung metastases.^[36] Our results found that DOX did not inhibit the metastasis of osteosarcoma, while the combination of ginsenoside Rg3 and DOX significantly increased the anti-metastatic effect. In-vitro wound-healing, transwell assay and in-vivo lung metastasis model results could confirm this effect. For example, transwell assay results indicate that the inhibition of migration in the combination therapy group was greater than the sum of their individual effects. A further study demonstrated that the inhibition rate of lung metastases in vivo was 74.25% when DOX was combined with Rg3, which was higher than the summation of Rg3 (52.09%) and DOX (0.02%) single drug groups. These results indicate that Rg3 and DOX have a synergistic effect in inhibiting cell migration. The epithelial-mesenchymal transition (EMT) drives tumour progression and is associated with tumour migration, invasion and drug resistance. During EMT, tumour cells change from epithelial to mesenchymal, altering the adhesion molecules on the cell surface and allowing them to migrate and invade.^[37] We found that ginsenoside Rg3 can inhibit EMT by suppressing the expression of mesenchymal markers N-cadherin, vimentin and collagen I.^[38] It has also been shown that ginsenoside Rg3 enhances the expression of the epithelial marker E-cadherin and inhibits the expression of MMP2 and MMP9, resulting in the inhibition of osteosarcoma metastasis.^[14] It is more physiological to inject homozygous osteosarcoma cells in situ or close to bone tissue to assess tumourigenesis, metastasis and angiogenesis. In future experiments, we will construct a model of osteosarcoma in situ.

Angiogenesis plays an important role in the growth and metastasis of osteosarcoma, and VEGF/VEGF-R is essential in the process of angiogenesis.^[39] Based on this, targeting VEGF to inhibit angiogenesis is an effective way to treat osteosarcoma. Nevertheless, some researchers reported that antiangiogenic drugs may increase local invasion and distant metastasis and induce tumour drug resistance. For example, sunitinib exhibited transient tumour suppressive effects in the treatment of pancreatic neuroendocrine carcinoma and glioblastoma, it triggered a significant increase in revascularization and invasiveness.^[40] In our study, we found that ginsenoside Rg3 inhibited osteosarcoma cells proliferation, metastasis, and angiogenesis. We also confirmed that ginsenoside Rg3 confers the antiangiogenic effect by inhibiting the expression of HIF-1α/VEGF. Moreover, previous studies have shown that HIF-1α/VEGF is a downstream protein of the Akt/mTOR signalling pathway^.[41] Furthermore, the immunohistochemistry of CD31 indicates that rg3 can effectively inhibit the formation of microvessels in tumour tissue.

Finally, another benefit of combining ginsenoside Rg3 with DOX is the amelioration of DOX cardiotoxicity and weight loss in mice. A number of studies have demonstrated that ginsenoside Rg3 can alleviate the toxic effects of DOX.^[42] The metabolism of DOX in cardiomyocytes generates reactive oxygen species that can lead to mitochondrial dysregulation, lipid peroxidation and DNA damage, resulting in cardiotoxicity.^[43] Ginsenoside Rg3 can antagonize adriamycin-induced cardiotoxicity by ameliorating endothelial dysfunction induced by oxidative stress through upregulation of the Nrf2-ARE pathway.^{[44, 45]} Notably, the development of nano-delivery systems such as liposomes and micelles has further improved the anti-tumour effects of ginsenoside Rg3 alone and in combination with DOX.^{[17, 46]} Taken together, ginsenoside Rg3 could be used as an adjuvant to DOX to improve the efficacy of osteosarcoma therapy, while minimizing its adverse side effects.

Conclusion

Our research shows that ginsenoside Rg3 combined with doxorubicin not only synergistically inhibits the proliferation and angiogenesis of OS cells and HUVECs by regulating the AKT/mTOR/pS6K, mTOR/HIF-1α/VEGF signalling pathways but also suppresses the metastasis of OS cells by blocking the EMT signalling pathway. Additionally, ginsenoside Rg3 and doxorubicin appeared to be more effective at suppressing tumour growth and lung metastasis in vivo. Furthermore, ginsenoside Rg3 was found to be effective in improving the cardiotoxicity of doxorubicin. In summary, these findings suggest that ginsenoside Rg3 and doxorubicin can be used in combination to improve the treatment of osteosarcoma.

Author contributions

Z.X.F., Y.H.: Methodology, formal analysis, writing—original draft. Z.W.T.: Conceptualization, validation, writing—review and editing. J.M.L., Z.W.: Software, investigation. L.S.Y., L.W.: Funding acquisition, project administration, supervision.

Funding

This work was supported by Hunan Provincial Natural Science Foundation of China (2022JJ30546), the clinical medical technology innovation guided program of Hunan Province Science and Technology Department (2020SK51902), Guangzhou Municipal Science and Technology Project (202102020536), Academician He Lin Scientific Research Fund (2021HLKY07), and Scientific research project of Traditional Chinese Medicine Bureau of Guangdong Province (20221248).

Conflict of Interest

These authors declared that they have no conflict of interest.

Data availability

The data underlying this article are available in the article and in its online supplementary material.

References

Author notes