Plantamajoside promotes metformin-induced apoptosis,autophagy and proliferation arrest of liver cancer cells via suppressing Akt/GSK3β signaling

Abstract

Metformin, a well-known antidiabetic drug, exhibits anticancer effect in a variety of cancers, including liver cancer. Plantamajoside (PMS), a phenylethanoid glycoside compound isolated from Plantago asiatica, is proved to possess anticancer effects, too. In our study, we hypothesized that PMS might promote metformin mediated anticancer effects on liver cancer. The half maximal inhibitory concentration (IC₅₀) of metformin was evaluated by cell viability assay. The influence of PMS on proliferation, migration, invasion and apoptosis of metformin-treated cells was evaluated by BrdU incorporation assay, flow cytometry, western blot, wound scratch healing assay, transwell cell migration assay and immunofluorescence. A fasting/feeding mouse model was built to evaluate the influence of PMS on metformin sensitivity in vivo. PMS (2.5, 10 or 40 μg/mL) treatment reduced the IC₅₀ of metformin under different glucose concentrations. PMS (10 μg/mL) promoted metformin (5 mm) induced apoptosis and autophagy, and inhibition on proliferation, migration and invasion of HepG2 and HuH-7 cells. In the fasting/feeding mouse model, PMS (50 mg/kg) promoted metformin (200 mg/kg) induced proliferation arrest and apoptosis in vivo. Meanwhile, PMS reduced the level of pAkt^(ser473) and GSK3β^(ser9) in HepG2 and HuH-7 cells. Restoration of Akt/GSK3β signaling by a constitutively activated myr-Akt1 abrogated the effects of PMS on metformin-treated liver cancer cells. Our results demonstrated that PMS promoted the anticancer effects of metformin on liver cancer in vitro and in vivo.

Keywords

Liver cancer metformin plantamajoside (PMS)Akt/GSK3β signaling

Introduction

Liver cancer is the sixth most common cancer and third cause of cancer-related death all over the world.¹ Risk factors of liver cancer include hepatitis B or C infection, alcohol-related cirrhosis, non-alcoholic steatohepatitis and fatty liver diseases.² Cancer is a genetic disease, and genomic aberrations in oncogenes or tumors suppressors such as CTNNB1, TP53, AXIN1, ARID1A, TERT, CDKN2A, CCND1, RPS6AKA3, FGF3, FGF4 and FGF19 promote the malignant progression of liver cancer.³ Liver cancer cases are mainly distributed in Eastern Asia and sub-Saharan Africa, of which HBV infection is really common.⁴ Though improvement of surgical techniques and treatment options increases the prognosis of liver cancer patients in recent years, the 5-years overall survival remains low at 18%.⁵ This is due to late diagnosis and high recurrence of liver cancer. Therefore, there is an urgent need for early detection and development of novel drug to improve the outcome of liver cancer in patients.

Metformin (1,1-dimethylbiguanide) is a well-known antidiabetic drug used for more than 50 years. It is safe and efficacious as monotherapy or in combination with other drugs, with known pharmacokinetics and pharmacogenomics. The antidiabetic property of metformin is achieved by decreasing hepatic glucose production, reducing intestinal glucose absorption to a lesser extent, and possibly increasing glucose absorbance by peripheral tissues.⁶ The underlying molecular mechanism for metformin is complicated, but it is generally accepted that metformin-induced Adenosine Monophosphate Activated Protein Kinase (AMPK) activation plays a pivotal role .⁷ Apart from being an antidiabetic drug, metformin also shows beneficial effect in the treatment of hepatic diseases, renal disorders, neurodegenerative diseases, bone disorders and cardiocerebral vascular diseases.^8,9 In addition, there is accumulating evidence indicating that metformin has anticancer effect, both in clinical and preclinical studies.^9,10 In hepatocellular carcinoma (HCC), metformin significantly increases the survival of HCC patients with type 2 diabetes (T2D).¹¹ Moreover, the use of metformin is connected with a reducing risk of HCC in diabetic patients.¹² Metformin is reported to suppress cell growth, metastasis, and epithelial-mesenchymal transition (EMT) of HCC cells by activating AMPK signaling.¹³ In metformin treated T2D patient, the peak concentrations of metformin are 20 μM in plasma and 50 μM in hepatic circulations.⁸

Plantamajoside (PMS, C₂₉H₃₆O₁₆) is a unique phenylethanoid glycoside compound isolated from Plantago asiatica.¹⁴ It has been used in food and medicine for many years. PMS has many biological activities, such as antioxidant, anti-inflammatory, antifibrotic, antiviral and immune enhancement.¹⁵ In rat glomerular mesangial cells, PMS suppresses high glucose-induced oxidative stress and inflammation via inactivating Akt/NF-κB pathway.¹⁶ In hepatic stellate cells, PMS exhibits antifibrotic effects by inhibiting proliferation, migration and invasion, and inducing apoptosis of PDGF-BB-treated HSC-T6 cells.¹⁷ PMS is also proved to possess anticancer effect on many cancers, such as melanoma, cervical cancer and liver cancer.^18–21 For example, PMS restrains proliferation and epithelial-mesenchymal transition of HCC cells through downregulating HIF-1α signaling pathway.²⁰ PMS also shows potential inhibitory effect on the development of HCC via affecting cell proliferation, migration, apoptosis and cell cycle distribution.²²

In cancer treatment, a single drug may not cause enough cytotoxicity on cancer cells at its maximal dose in vivo. Besides, cancer cells can quickly adapt to the drug treatment and become resistance. One option to reduce drug resistance and increase cytotoxicity is the combination use of two or more drugs. Though in vitro and in vivo studies in cancer cell lines or mouse models reveal a tumor inhibition effect of metformin, these effects are only observed in high concentrations (>5 mm) .^23–27 Thus, it is necessary to develop new strategies to increase the anticancer effect of metformin. The combination of metformin and other drugs may have enhanced anticancer effects. For instance, galloflavin promotes metformin induced proliferation inhibition and apoptosis in pancreatic cancer.²⁸ Exendin-4 is a GLP-1 receptor agonist. Combined treatment with Exendin-4 increases the inhibition effect of metformin on growth of prostate cancer cells.²⁹ As described above, PMS shows anticancer effect in liver cancer cells. Metformin and PMS are both safe and well-tolerated by human body. Therefore, we wondered if the combined treatment with PMS might increase the anticancer effect of metformin. In our study, we evaluated the effects of PMS on metformin treated liver cancer cells in vitro and a fasting/feeding mouse model in vivo. We found that PMS facilitated the anticancer effect of metformin on liver cancer cells, suggesting a possible use of PMS and metformin together in treating cancer.

Materials and methods

Cell culture and reagents

Liver cancer cell lines HepG2 and HuH-7 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in SILAC RPMI 1640 Medium (no glucose, #A2494201, ThermoFisher Scientific) in combination with 10% fetal bovine serum (Hyclone, USA), 100 U/ml penicillin and 100 μg/mL streptomycin (Hyclone, USA). Normal human liver cell line L-O2 was purchased from the Cell Bank of the Chinese Academy of Sciences and maintained in RPMI-1640 medium (Hyclone, USA). Cells were kept in a humidified atmosphere with 5% CO₂ at 37°C. Glucose concentrations were manipulated by adding extra glucose into the culture medium to a concentration at 2.5, 10 and 25 mm. All cell lines in our study were authenticated by STR profiling. Metformin (#S1950) and PMS (#S9077) were purchased from Selleck chemicals (Huston, USA) and dissolved in phosphate buffered saline (PBS). PBS was used as vehicle control.

Cell viability assay

We tested the viability of cells using CellTiter-Glo Luminescent Cell Viability Assay kit (Promega #G7572, USA) as indicated in previous study.³⁰ At first, cells and CellTiter-Glo agent were equilibrated to room temperature. Next, mixed the cells and agents together at 100 μL per well in 96-well plates. Cell were lysed for 10 min on an orbital shaker, then placed in a dark place for 15 min to stable the luminescence signal. Microplate reader (Luminoskan, Thermofisher, Waltham, USA) was used to record the signal. All samples were repeated in triplicates.

BrdU incorporation assay

BrDu incorporation assay was conducted as previous described.³¹ Cells were stained with 25 μM BrdU (Beyotime #ST1056, China) for 24 h. After that, washed the cells with PBS for once, then fixed the cells with 4% paraformaldehyde (PFA) for 10 min at room temperature. Cells were washed with PBS twice, permeabilized with Triton X-100 for 15 min, denatured by 2N HCL and neutralized by 20 mm Na₂CO₃ for 1 h. Next, cells were blocked by 5% bovine serum albumin at room temperature for 1 h. Washed the cells once with PBS, then stained with BrdU Mouse mAb (Cell signaling #5292, USA, 1: 1000) for 2 h at room temperature. Next, washed the cells with PBS for three times, then incubated with anti-mouse IgG (Alexa Fluor 488 Conjugate) (Cell signaling #4408, USA, 1: 500) for 1 h at room temperature. Cells were stained with DAPI (Invitrogen, USA) for 15 min at room temperature. Images were photographed by LSM880 confocal microscope (Zeiss, Germany).

Flow cytometry

Flow cytometry was performed as previously described.³¹ Cells were dispersed by 0.05% trypsin for 5 min at 37°C. Stop the digesting with FBS, then cells were washed with PBS twice. Cells (1 × 10⁶) were resuspended in PBS, then incubated with 5 μL Annexin V-FITC (Beyotime #C1062, China) for 30 min at 4°C and 10 μL propidium iodide (PI) for 10 min at room temperature avoiding night. Recording the fluorescence signal at 488/530 by Cell Sorter BD FACSAria II (BD Biosciences, USA).

Western blot

Western blot was conducted according to previous study.³² Cultured cells were lysed by RIPA buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1% NP-40, 0.1% SDS) (Beyotime, China) with protease inhibitors (Sigma, USA). Protein concentration was determined by BCA kit (Thermo Fisher #23225, USA). SDS-PAGE (10% or 15%) was used to separate the lysed protein (30 μg). Then protein was transferred to nitrocellulose membranes and incubated with 5% non-fat milk on a shaker for 1 h at room temperature. The expression of specific protein was determined by staining with primary antibody at 4°C overnight and corresponding second antibody at room temperature for 1 h. The primary antibodies were listed below: Phospho-GSK-3β (Ser9) Rabbit mAb (Cell signaling #5558, USA, 1: 1000), Cleaved caspase-3 (Asp175) Rabbit mAb (Cell signaling #9664, USA, 1: 1000), GAPDH Rabbit mAb (Cell signaling #5174, USA, 1: 1000), Cleaved PARP Rabbit mAb (Cell signaling #5625, USA, 1: 1000), Phospho-Akt (Ser473) Antibody (Cell signaling #9271, USA, 1: 1000), Akt Antibody (Cell signaling #9272, USA, 1: 1000), Beclin-1 Rabbit mAb (Cell signaling #3495, USA, 1: 1000), SQSTM1/p62 Rabbit mAb (Cell signaling #8025, USA, 1: 1000), GSK-3β Rabbit mAb (Cell signaling #12456, USA, 1: 1000), and LC3 I/II antibody (Cell signaling #4108, USA, 1: 1000). The corresponding second antibody was anti-rabbit IgG HRP-linked antibody (Cell signaling #7074, USA, 1:4000). The bands were visualized by the enhanced chemiluminescence kit (Amersham, USA) and detected using the Amersham Imager 600 imagers (GE Healthcare, USA). Relative protein quantification was performed with Image Quant TL 8.1 (GE Healthcare) software.

Wound scratch healing assay

Wound healing assay was performed as previous described.³¹ Seeded cells (1 × 10⁵) in 6-well plates for 24–36 h to reach a confluence at 70%. Then a scratch was made by 1 mL pipette right in the middle of the well. Samples were examined and photographed at 0 h and 24 h post-scratching. Relative distance of the wound was measured by Image J. Each sample was carried out in triplicates.

Transwell invasion assay

Transwell invasion assay was performed as previous described.³¹ The transwell chamber (Costar Corp, USA) were pre-coated with 250 μg/mL Matrigel (Corning, USA). Then cells (1 × 10⁵) were seeded in the upper chamber supplemented with 1% FBS. The lower chamber was filled with complete medium containing 10% FBS. Cells were cultured in the transwell chamber for 48 h. After that, the invading cells were fixed by 4% PFA for 10 min at room temperature. Then washed the cells with PBS twice, and stained with 0.5% crystal violet for 30 min at room temperature. Images were obtained by a microscope. Each sample was done in triplicates.

Immunofluorescence

The immunofluorescence analysis was performed as described by previous study.³³ Cells were seeded on glass coverslips for 24 h. Then the coverslips were washed with PBS for once and fixed with 4% PFA for 10 min at room temperature. After that, washed the coverslips with PBS for three times and incubating with Triton X-100 for 15 min at room temperature. Cells were then blocked by 1% albumin at room temperature for 1 h. Next, incubated the cells with LC3I/II antibody (Cell Signaling #4108, USA, 1: 200) at 4°C overnight. Cells were then washed with PBS for three times, and incubated with Anti-rabbit IgG (Alexa Fluor® 488 Conjugate) (Cell Signaling #4412, USA, 1: 1000) at room temperature for 1 h avoiding night. DAPI was used to stain the nuclear. Mounted the cells in LSM880 confocal microscope (Zeiss, Germany).

Fasting/feeding mouse model

The protocols for animal experiment were reviewed and approved by Animal Care and Experimental Committee of Naval Medical University (SYXK (Hu) 2020–0010). We strictly followed our protocols for animal experiment and met the ethnical requirements. The fasting/feeding mouse model was built as previous reported.³⁴ In brief, HepG2 cells (3 × 10⁶) were subcutaneously injected into the 6-week-old female BALB/c nude mice. Tumor xenografts were allowed to growth for 14 days to reach an average volume of 250 mm³. Then mice were randomly divided into Veh, PMS, Met and PMS + Met group (n = 4). PMS (50 mg/kg) and Met (200 mg/kg) were both dissolved in PBS and administered every 48 h by oral gavage. Veh group was treated with equal volume of PBS by oral gavage at the same time. The fasting/feeding cycle were depicted in Figure 5(a). Mice of each group were subjected to a 24 h cycle of fasting/feeding. Namely, mice bearing HepG2 xenografts were treated with either fasting or feeding (6 p.m.–9 a.m. of next day) for 15 h, then the next 9 h were only feeding with water. Drugs were administered at the end of the 15 h fasting period (9 a.m. of next day). Tumor growth was measured by a caliper and calculated by the formula (length × width²)/2. At the end of drug treatment, all mice were anaesthetized by inhalation with 3% isoflurane and sacrificed by broking the neck. Tumors were dissected out and weighed.

Immunohistochemistry

Immunohistochemistry staining was conducted as previous protocol.³⁵ Tissue samples were embedded by paraffin, then cut into 20 μm thick sections. Sections were deparaffinized by xylene and rehydrated by ethanol gradients (100%, 95%, 70% and 50%) and deionized water. The sections were dipped into sodium citrate buffer and heated by microwave, then incubated with Ki-67 Rabbit mAb (Cell Signaling #9027, USA, 1: 400) or Cleaved caspase-3 Antibody (Cell Signaling #9661, USA, 1: 400) at 4°C overnight. Next, sections were incubating with hematoxylin at room temperature for 10 min. ChemMate Envision detection system (Dako Cytomation, USA) were used for color development.

Plasmids and virus transfection

The pCDH-puro-myr-HA-Akt1 plasmid was purchased from Addgene (#46969, USA). The empty pCDH-puro-myr-HA-Akt1 was used as control (EV). Lentivirus particles were produced by co-transfecting with helper plasmids pCMV-VSV-G, pRSV-REV, and pMDL in HEK293 T cells using lipofectamine 3000 (Invitrogen, USA). Collected the virus-containing medium at 24, 48 and 72 h post-transfection. For virus infection, cells (1 × 10⁵) were seeded in 6-well plates for 24 h, then incubated with virus-containing medium and 8 μg/mL polybrene overnight.

Statistical analysis

GraphPad Prism 8.0 software was used for statistical analysis. IC50 of metformin was measured by GraphPad Prism 8.0 using the nonlinear regression model. To compare the difference of two groups, two-tailed Student’s t-test was used. To compare the difference between multiple groups, one-way ANOVA analysis followed by Bonferroni’s post hoc test was used. Data was exhibited as mean ± standard deviation (x ± SD). P ≤ 0.05 was considered statistically significant.

Results

Plantamajoside increases the drug sensitivity of metformin in liver cancer cells

Metformin exerts anti-proliferation and pro-apoptotic effects in various cancers, including liver cancer.^13,24,27 To explore the effect of PMS in metformin-treated liver cancer cells, we first tested the cytotoxicity of PMS on two commonly used liver cancer cell lines by cell viability assay. As shown in Figure 1(a), 1.0 ∼ 10.0 μg/mL PMS exhibited really low cytotoxicity on both HepG2 and HuH-7 cells, with >90% cell viability compared with vehicle control (0 μM PMS group). In contrast, the higher concentrations of PMS (20 and 40 μg/mL) showed an obvious reduce of cell viability on human normal liver cells L-O2 (Figure 1(a)). To determine if PMS had influence on drug sensitivity of metformin, HepG2 and HuH-7 cells were exposed to a series of concentrations of metformin (0–729 mm) in combination with 0, 2.5, 10 or 40 μg/mL PMS. Previous reports indicate that metformin exerts only weak anti-proliferative effects when cells are cultured with high concentration of glucose, but shows strong inhibitory on cell growth when cultured in low glucose concentration.³⁴ Thus, we also evaluated the influence of glucose concentration on PMS and metformin treated liver cancer cells. As shown in Figures 1(b) and (c), we found that PMS significantly reduced the IC50 of metformin in both HepG2 and HuH-7 cells, indicating that PMS enhanced the drug sensitivity of metformin in liver cancer cells. Furthermore, we could observe that glucose concentration showed dramatically impact on the IC₅₀ of metformin in liver cancer cells, suggesting that change in glucose concentration affected the anti-proliferative effect of metformin (Figure 1(b) and (c)). However, we found that PMS reduced the IC₅₀ of metformin under 2.5, 10 and 25 mm glucose concentrations in a same pattern, suggesting that the effect of PMS had no apparent connection with glucose concentration. Above all, our data indicated that PMS increased the drug sensitivity of metformin in liver cancer cells.

Figure 1.

Plantamajoside increases metformin cytotoxicity in liver cancer cells. A, HepG2, HuH-7 and L-O2 cells were seeded in 96-well plates (3000/well), then treated with 0, 1.0, 2.5, 5.0, 10.0, 20.0 and 40.0 μg/mL PMS for 24, 48 or 72 h and evaluated for cell viability. B-C, HepG2 (B) and HuH-7 (C) cells were seeded in 96-well plates (3000/well), then exposed to 0, 0.11, 0.33, 1.00, 3.00, 9.00, 27.00, 81.00, 243.00 and 729.00 mm metformin in combination with 0, 2.5, 10 or 40 μg/mL PMS for 7 days under 2.5, 10 or 25 mm glucose concentrations, and evaluated for cell viability. The IC₅₀ of each group was measured and showed in the figure. *p < 0.05 compared with the 0 μg/mL PMS group.

Plantamajoside promotes metformin induced proliferation arrest and apoptosis in liver cancer cells.

To further evaluate the influence of PMS on the anticancer effect of metformin in liver cancer, HepG2 and HuH-7 cells were treated with 10 μg/mL PMS, 5 mm metformin, 10 μg/mL PMS +5 mm metformin or vehicle control (Veh) under 2.5 mm glucose. We chose 10 μg/mL of PMS because this concentration of PMS had no apparent cytotoxicity on HepG2 and HuH-7 cells, thus this could exclude the influence of PMS cytotoxicity on metformin-treated HepG2 and HuH-7 cells. Moreover, 10 μg/mL PMS reduced the IC₅₀ of metformin to a larger extent, indicating that this concentration of PMS significantly increased the drug sensitivity of metformin in liver cancer cells. Besides, 5 mm metformin for HepG2 and HuH-7 cells showed moderate inhibitory effect on cell viability under 2.5 mm glucose. Thus, these concentrations were selected for the subsequent experiments by balancing the effect and cytotoxicity of PMS on metformin-treated liver cancer cells. In BrdU incorporation assay, we found that PMS had no apparent influence on the percentage of BrdU positive cells, while metformin showed a moderate reduce of BrdU positive cells (Figure 2(a) and (b)). However, the combination of PMS and metformin showed dramatically reduce in BrdU positive cells, indicating that PMS promoted metformin induced proliferation arrest (Figure 2(a) and (b)). In flow cytometry analysis, metformin treatment showed an obvious increase in the percentage of apoptotic cells (Annexin V-FITC and PI double positive subset), and this was further enhanced by combining with PMS (Figure 2(c) and (d)). In western blot analysis, metformin treatment upregulated the levels of cleaved caspase-3 and cleaved PARP, and this was strengthened by combining with PMS (Figure 2(e)). Taken together, our results indicated that PMS increased the anticancer effect of metformin in liver cancer cells, and promoted metformin induced proliferation arrest and apoptosis.

Figure 2.

Plantamajoside promotes metformin induced proliferation arrest and apoptosis of liver cancer cells. A-B, HepG2 and HuH-7 cells were seeded in 6-well plates (5 × 10⁵/well), then incubated with 10 μg/mL PMS, 5 mm metformin (Met), 10 μg/mL PMS +5 mm metformin (PMS + Met) or vehicle control (Veh) for BrdU incorporation assay. Represent images (A) and relative BrdU positive cells (B) were shown. Scale bar = 50 μm. C-D, HepG2 and HuH-7 cells were seeded in 6-well plates (5 × 10⁵/well) and treated with 10 μg/mL PMS, 5 mm metformin (Met), 10 μg/mL PMS +5 mm metformin (PMS + Met) or vehicle control (Veh) for 72 h, then cells were stained with Annexin V-FITC and PI for flow cytometry (C). The Annexin V-FITC and PI double positive subsets were shown (D). E, HepG2 and HuH-7 cells were seeded in 6-well plates (5 × 10⁵/well) and treated with 10 μg/mL PMS, 5 mm metformin (Met), 10 μg/mL PMS +5 mm metformin (PMS + Met) or vehicle control (Veh) for 72 h, then cell lysates were collected for western blot (E). The glucose concentration in above experiments was 2.5 mm. *p < 0.05 compared with the Veh group, #p < 0.05 compared with the PMS group.

Plantamajoside enhances metformin induced migration and invasion inhibition and autophagy of liver cancer cells.

As previous reports suggest that metformin restrains migration and invasion, and induces autophagy in cancer cells including liver cancer,^13,24,36,37 we evaluated the impact of PMS on migration and invasion of metformin treated liver cancer cells. In wound scratch assay, metformin suppressed the wound healing of HepG2 and HuH-7 cells compared with vehicle group, and this was reinforced by combining with PMS (Figure 3(a) and (b)). In transwell invasion assay, metformin decreased the number of invasion cells in both HepG2 and HuH-7, and this was further enhanced by PMS (Figure 3(c) and (d)). To evaluate the influence of PMS on metformin induced autophagy, we detected the expression of autophagy makers Beclin1, p62, LC3I and LC3II by western blot. We found that metformin increased the expression of Beclin1 and the ratio of LC3II/LC3I, and decreased the expression p62 compared with vehicle control, indicating that metformin promoted autophagy of HepG2 and HuH-7 cells (Figures 4(a) and (b)). Furthermore, the effect of metformin on autophagy of liver cancer cells was enhanced by combining with PMS, suggesting that PMS facilitated metformin induced autophagy in liver cancer cells (Figure 4(a) and (b)). In immunofluorescence staining, we found that metformin increased the percentage of cells with LC3 dots, and this was further enhanced by PMS (Figure 4(c) and (d)). Collectively, our results indicated that PMS enhanced the inhibitory effects of metformin on migration and invasion of liver cancer cells, and promoted metformin induced autophagy.

Figure 3.

Plantamajoside enhances the inhibitory effects of metformin on migration and invasion of liver cancer cells. A-B, HepG2 and HuH-7 cells were seeded in 6-well plates (5 × 10⁵/well) and treated with 10 μg/mL PMS, 5 mm metformin (Met), 10 μg/mL PMS +5 mm metformin (PMS + Met) or vehicle control (Veh) for wound scratch healing assay (A). Relative cell migration (B) was shown. Scale bar = 500 μm. C-D, HepG2 and HuH-7 cells were seeded in the upper chamber (1 × 10⁵/well) and incubated with 10 μg/mL PMS, 5 mm metformin (Met), 10 μg/mL PMS +5 m. M metformin (PMS + Met) or vehicle control (Veh) for transwell cell invasion assay (C). Relative cell invasion were shown (D). The glucose concentration in above experiments was 2.5 mm. *p < 0.05 compared with the Veh group, #p < 0.05 compared with the PMS group.

Figure 4.

Plantamajoside promotes metformin induced autophagy of liver cancer cells. A-B, HepG2 and HuH-7 cells were seeded in 6-well plates (5 × 10⁵/well) and treated with 10 μg/mL PMS, 5 mm metformin (Met), 10 μg/mL PMS +5 mm metformin (PMS + Met) or vehicle control (Veh) for 72 h, then collected cell lysates for western blot analysis (A). Relative protein expression normalized to GAPDH were shown (B). C-D, HepG2 and HuH-7 cells were seeded in 6-well plates (5 × 10⁵/well) and treated with 10 μg/mL PMS, 5 mm metformin (Met), 10 μg/mL PMS +5 mm metformin (PMS + Met) or vehicle control (Veh) for 72 h, then cells were stained with LC3 for immunofluorescence. The nuclear was stained with DAPI. Represent images (C) and percentages of cells with LC3 dots (D) were shown. Scale bar = 10 μm. The glucose concentration in above experiments was 2.5 mm. *p < 0.05 compared with the Veh group, #p < 0.05 compared with the PMS group.

Plantamajoside enhances the inhibitory effect of metformin on tumor growth of liver cancer cells in a fasting/feeding mouse model.

To evaluate the influence of PMS on metformin sensitivity in vivo, a fasting/feeding mouse model was established as previous reported .³⁴ In brief, mice bearing HepG2 xenograft were feeding or fasting for 15 h, then giving free drinking water for next 9 h before the fasting or feeding cycle was ended (Figure 5(a)). PMS, metformin or the combination of them were administered at the end of the fasting period, thus there were 9 h for the drugs to act. In our study, we found that metformin (Met) suppressed tumor xenograft growth of HepG2 cells compared with vehicle control group (Veh), with reduced tumor volume and weight (Figure 5(b) to (d)). Furthermore, PMS significantly strengthened the inhibitory effects of metformin on tumor xenograft growth, volume and weight of HepG2 cells (Figure 5(b) to (d)). In IHC staining, metformin treated mice showed reduced Ki67 positive cells and increased Cleaved caspase-3 positive cells, indicating that metformin inhibited proliferation and induced apoptosis in HepG2 xenografts (Figure 5(e) and (f)). In comparison, the PMS + Met group exhibited apparently lower Ki67 positive cells and higher Cleaved caspase-3 positive cells compared with Met group, suggesting that PMS promoted metformin induced proliferation arrest and apoptosis in vivo. Above all, our results indicated that PMS enhanced the inhibitory effect of metformin on tumor growth of liver cancer cells in a fasting/feeding mouse model.

Figure 5.

Plantamajoside enhances the inhibitory effect of metformin on tumor growth of liver cancer cells in a fasting/feeding mouse model. A, the schedule map for the fasting/feeding model. B-D, BALB/c nude mice bearing HepG2 xenografts were treated with PMS (50 mg/kg), Met (200 mg/kg) or the combination of them every 48 h in a 24 h fasting/feeding cycle. Tumor growth curves (B), volume (C) and weight (D) were shown. E-F, tumor sections of HepG2 xenografts were used for IHC staining of Ki67 and Cleaved caspase-3. Represent images (E) and relative positive cells (F) were shown. Scale bar = 50 μm. *p < 0.05 compared with the Veh group, #p < 0.05 compared with the PMS group.

Akt/GSK3β signaling is suppressed by plantamajoside in metformin treated liver cancer cells.

There is growing evidence that PMS affects the activation of several important signaling pathways, such as PI3K/Akt, MAPK and NF-кB.^38–40 To explore the underlying molecular mechanisms of PMS in metformin treated liver cancer cells, we evaluated the possible signaling pathways that regulated by PMS. In western blot analysis, we found that PMS suppressed the phosphorylation of Akt and GSK3β, indicating that PMS suppressed the activating of Akt/GSK3β signaling in HepG2 cells (Figure 6(a) and (b)). Then, HepG2 and HuH-7 cells were treated with PMS, metformin (Met) or the combination of them for western blot analysis. GSK3β plays an important role in controlling cell fate, and phosphorylation at serine nine inactivated GSK3β.⁴¹ In our study, we found that metformin suppressed the phosphorylation of GSK3β at serine nine but did not affect the level of phosphorylated Akt, indicating metformin treatment activated GSK3β independent of Akt in liver cancer cells (Figure 6(c) and (d)). Meanwhile, PMS suppressed the phosphorylation of Akt and GSK3β in metformin treated HepG2 and HuH-7 cells, suggesting that PMS suppressed Akt activation and activated GSK3β further (Figure 6(c) and (d)). These results indicated that Akt/GSK3β signaling was suppressed by PMS in metformin treated liver cancer cells.

Figure 6.

Akt/GSK3β signaling is suppressed by plantamajoside in metformin treated liver cancer cells. A-B, HepG2 cells were seeded in 6-well plates (5 × 10⁵/well) and treated with 2.5, 10 or 40 μg/mL PMS or equal volume of PBS (Veh) for 72 h, then collected cell lysates for western blot (A). Relative protein expression normalized to GAPDH was shown (B). C-D, HepG2 and HuH-7 cells introduced with myr-Akt1 or EV were seeded in 6-well plates (5 × 10⁵/well) and treated with 10 μg/mL PMS, 5 mm metformin, 10 μg/mL PMS +5 mm metformin or vehicle control for 72 h, then collected cell lysates for western blot analysis. Relative protein expression normalized to GAPDH were shown (C–D). The glucose concentration in above experiments was 2.5 mm. *p < 0.05 compared with the Veh group, #p < 0.05 compared with the PMS group.

Restoration of Akt/GSK3β signaling abrogates the effects of plantamajoside in metformin treated liver cancer cells.

To determine if Akt/GSK3β signaling was enrolled in the effects of PMS in metformin treated liver cancer cells, we restored Akt/GSK3β signaling in HepG2 and HuH-7 cells by transducing them with a constitutively activated myr-Akt1. As shown in Figure 7(a), HepG2 and HuH-7 cells transduced with myr-Akt1 showed increased phosphorylation of Akt and GSK3β, indicating the activation of Akt/GSK3β signaling. Moreover, transducing of myr-Akt1 reversed the inhibitory effects of PMS on the phosphorylation of Akt and GSK3β (Figure 6(c) and (d)). To evaluate if restoration of Akt/GSK3β signaling could abrogate the effects of PMS on metformin sensitivity, HepG2 and HuH-7 cells transduced with myr-Akt1 or empty vector control (EV) were exposed to a series of concentrations of metformin (0–729 mm) in combination with or without 10 μg/mL PMS. As shown in Figure 7(b), transducing with myr-Akt1 completely abolished the lowering effects of PMS on the IC50 of metformin, suggesting that restoration of Akt/GSK3β signaling abrogated the effect of PMS on metformin sensitivity. In flow cytometry analysis, PMS showed no effects on metformin induced apoptosis in HepG2 and HuH-7 cells transduced with myr-Akt1 (PMS + Met + myr-Akt1 group) compared with HepG2 and HuH-7 cells transduced with EV control (PMS + Met + EV group) (Figure 7(c) and (d)). In transwell cell invasion assay, PMS showed no influence on metformin induced invasion inhibition in HepG2 and HuH-7 cells transduced with myr-Akt1 (PMS + Met + myr-Akt1 group) compared with HepG2 and HuH-7 cells transduced with EV control (PMS + Met + EV group) (Figure 7(e) and (f)). Taken together, our results indicated that restoration of Akt/GSK3β signaling abrogated the effects of PMS in metformin-treated liver cancer cells.

Figure 7.

Restoration of Akt/GSK3β signaling abrogates the effects of plantamajoside in metformin-treated liver cancer cells. A, HepG2 and HuH-7 cells transduced with myr-Akt1 or EV were used for western blot. B, HepG2 and HuH-7 cells transduced with myr-Akt1 or EV were seeded in 96-well plates (3000/well) and exposed to 0, 0.11, 0.33, 1.00, 3.00, 9.00, 27.00, 81.00, 243.00 and 729.00 mm metformin in combination with 10 μg/mL PMS or equal volume of PBS (EV) for 7 days under 2.5 or 10 mm glucose concentrations, then evaluated for cell viability. C-D, HepG2 and HuH-7 cells transduced with myr-Akt1 or EV were seeded in 6-well plates (5 × 10⁵/well) and treated with 5 mm metformin, 10 μg/mL PMS +5 mm metformin or vehicle control (Veh) for 72 h, then cells were stained with Annexin V-FITC and PI for flow cytometry (C). The Annexin V-FITC and PI double positive subsets were shown (D). E-F, HepG2 and HuH-7 cells transduced with myr-Akt1 or EV were seeded in the upper chamber (1 × 10⁵/well) and incubated with 5 mm metformin, 10 μg/mL PMS +5 mm metformin or vehicle control (Veh) for transwell cell invasion assay. Relative cell invasion (F) was shown. *p < 0.05 compared with the Veh + EV group, #p < 0.05 compared with the PMS + Met + EV group.

Discussion

In the present study, we found that PMS significantly increased the drug sensitivity of metformin in liver cancer cells. PMS promoted the anticancer effects of metformin by increasing metformin-induced inhibition on proliferation, migration, invasion and tumor xenograft growth and promoting metformin-induced apoptosis and autophagy. Our data suggested that the combination of PMS and metformin might show some benefit for liver cancer treatment, and provided therapeutic basis for the combination use of PMS and metformin in liver cancer patients. Indeed, the combination of other drugs may increase the drug sensitivity of metformin in cancer.^28,29 For instance, pioglitazone significantly increases the drug sensitivity of metformin in thyroid cancer cells via downregulating AMPK/mTOR signaling related genes.⁴² In breast cancer, the combination of metformin and everolimus shows additive effect on the inhibition of proliferation and colony formation of breast cancer cells.⁴³ In addition, metformin may increase the drug sensitivity of other anti-cancer drugs. Metformin was found to overcome cisplatin resistance of triple-negative breast cancer via inhibiting RAD51 expression.⁴⁴ These studies and our results suggested that the combination of PMS and metformin might be a feasible strategy to enhance the anticancer effect of metformin in liver cancer.

The anticancer effect of metformin has been validated in liver cancer by us, too. We found that metformin suppressed proliferation, migration and invasion and induced apoptosis and autophagy of liver cancer cells in vitro, especially under low glucose concentration. There are increasing pharmaco-epidemiologic evidences revealing a protective role of metformin in cancer. The first evidence has been published in 2005, which indicates that metformin treatment reduces the risk of cancer in T2D patients.⁴⁵ Soon afterwards, this is demonstrated by many other researchers, including in liver cancer .^10–12 In a rat model of cirrhosis, prolonged metformin treatment reduces fibrosis and inflammation, and leads to a 44% lowering of HCC incidence by suppressing the activation of hepatic progenitor cells.²⁵ In our study, we found that metformin showed enhanced anticancer effects under low glucose concentrations. Previous reports indicate that metformin shows moderate inhibition on proliferation of cancer cells under nutrient rich conditions, but this effect is dramatically enhanced by glucose depletion.^34,46 Cancer cells may accommodate to metabolic challenges by changing between glycolysis and oxidative phosphorylation (OXPHOS). Metformin, an inhibitor of OXPHOS, may destroy the metabolic plasticity of cancer cells under low glucose concentration, thus exhibits enhancing anti-cancer effects.³⁴ Indeed, we found that the IC₅₀ of metformin changed rapidly in different glucose concentrations, and low glucose concentration significantly increased the anticancer effect of metformin. This might due to the inhibition of metabolic plasticity of liver cancer cells.

In our study, low concentration of PMS (10 μg/mL) showed no obvious influence on proliferation, migration, invasion, apoptosis and autophagy of liver cancer cells, but dramatically promoted the inhibitory effects of metformin. Though PMS shows anticancer effects in various cancers including liver cancer, the concentrations of PMS used in these studies are significantly higher than 10 μg/mL.^19–21 We speculated that 10 μg/mL PMS were too low to exert strong anticancer effect in liver cancer cells in our study. Besides, liver cancer cells treated with 20 or 40 μg/mL PMS showed apparently reduce in cell viability, indicating that PMS of these concentrations exhibited stronger anticancer effects. Indeed, there is increasing evidence that PMS exhibits anticancer effect in various cancers. In breast cancer, PMS suppresses proliferation, migration, invasion, tumor xenograft growth and lung metastasis of breast cancer cells by inhibiting MMP9 and MMP2 activities.¹⁹ In esophageal squamous cell carcinoma (ESCC), PMS shows inhibitory effects on viability and LPS-induced EMT of ESCC cells by regulating NF-κB/IL-6 signaling.³⁹ Notably, PMS also shows inhibition effect on proliferation, migration and invasion in liver cancer. PMS inhibits CoCl₂ -induced migration, invasion and EMT of HepG2 cells by reducing hypoxia-inducible factor-1α (HIF-1α) expression .²⁰ Furthermore, PMS shows synergistic effects with sorafenib to overcome drug resistance of liver cancer cells by reprograming the tumor hypoxic microenvironment.⁴⁷

In our study, we found that PMS suppressed Akt/GSK3β signaling in metformin-treated liver cancer cells via reducing the phosphorylation of Akt and GSK3β. It is worth noting that PMS shows inhibitory effect on PI3K/Akt signaling in a variety of cells. In human airway epithelial cells, PMS suppresses the phosphorylation of Akt and p65, thus attenuates LPS-induced inflammation and MUC5AC expression.⁴⁸ PMS is also reported to suppress LPS-induced inflammation in human gingival fibroblasts through reducing the phosphorylation of PI3K and Akt.⁴⁹ Akt/GSK3β signaling is also important for metformin-mediated effects in proliferation, migration, invasion and apoptosis. Metformin is proved to activate GSK3β by reducing the level of phosphorylated GSK3β at serine 9, which is vital for the anticancer effects of metformin under low glucose concentration.³⁴ In our study, PMS suppressed the phosphorylation of Akt and GSK3β in metformin-treated liver cancer cells, suggesting that the effect of PMS on metformin might relate to a greater extent of GSK3β activation.

Conclusion

In conclusion, we found that PMS reduced the IC₅₀ of metformin in liver cancer cells. Moreover, PMS enhanced the inhibitory effects of metformin on proliferation, migration and invasion and tumor xenograft growth, and promoted metformin-induced apoptosis and autophagy. PMS suppressed the phosphorylation of Akt and GSK3β in metformin treated liver cancer cells, and restoration of Akt/GSK3β signaling abrogated the effects of PMS. Our results demonstrated that PMS promoted the anticancer effects of metformin in liver cancer cells.

Footnotes

Authors' contributions

ZHZ and YPW guaranteed the integrity of the entire study. The experiments were conducted by ZW, JLZ and LLZ. Data was analyzed by ZW and YPW. Manuscript was prepared and reviewed by ZHZ and YPW.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This article is supported by the Tenth Peoples Hospital’s cultivation project of Natural Science Foundation of China.

Ethical approval and consent to participate

All procedures performed in studies involving animals were in accordance with the ethical standards of the ethics committee of Naval Medical University.

ORCID iD

Yongpeng Wei

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