Stevenleaf from Gynostemma Pentaphyllum inhibits human hepatoma cell(HepG2)through cell cycle arrest and apoptotic induction

Syed Sjid Hussin,Fn Zhng,Yunyun Zhng,Kirn Thkur,Mhrukh Nudhni,Crlos L.Cespedes-Acu,Zhojun Wei,c,d,*

a School of Food Science and Biological Engineering,Hefei University of Technology,Hefei,230009,China

b Department of Basic Sciences,Research Group in Chemistry and Biotechnology of Bioactive Natural Products,Faculty of Sciences,University of Bio-Bío,Andrés Bello Avenue,Chillan,Chile

c College of Biological Science and Technology,Fuzhou University,Fuzhou,350108,China

d Biological Science and Engineering College,North Minzu University,Yinchuan,750021,China

ABSTRACT The anticancer activity of stevenleaf(SV)on the basis of cell viability,cell cycle,and apoptosis induction in HepG2 cancer cells were evaluated.SV controlled the growth of HepG2 cells with IC50 of 139.82 μmol/L for 24 h,IC50 of 119.12 μmol/L for 48 h and cell cycle arrested at G0/G1 phase,induced cell apoptosis and enhanced intracellular ROS generation.For cell cycle arrest,the mRNA expression levels of p21,p27 and p53 were up-regulated,while the expression levels of Cyclin A,Cyclin D1,Cyclin E and CDK1/2 were downregulated.SV efficiently up-regulated TNF R1, TRADD1 and FADD and down-regulated Caspase8 for cell death receptors;similarly,up-regulated Bax,Bak,Cyt c,Apaf1,Caspase3 and Caspase9,and down-regulated Bcl2, Bcl xl and Bad for mitochondrial signal pathway.SV induced the mTOR-mediated cell apoptosis in HepG2 cells via activation of Akt and AMPK.The mechanistic explanation for the anticancer activity of SV as functional food can be derived from above results.

Keywords:Gynostemma Pentaphyllum Stevenleaf HepG2 cell Cell cycle Apoptosis

1.Introduction

Cancer is invasive and destructive to human body and becoming a growingly important health problem globally,which leads to high number of mortalities[1,2].Among various cancer types,liver cancer is known as a leading causes of mortality[3].Asia-pacific region such as China is known for the high incidence of liver cancer,which is 1.5 times higher than the global cases[4,5].

Apoptosis characterized with physical deformation of cells can lead to cell death.[6].A caspase family (cysteine aspartic specific proteases) is involved in mediating apoptosis through mitochondrial pathway[7]via membrane transition and permeability.Cyclin D, Cyclin E, and their corresponding cyclin-dependent kinases(CDK2 and CDK4) play a critical role in the growth of the cells at the first gap phase(G1),and the initiation of DNA synthesis phase(S)in cell cycle arrest.The second gap phase(G2)and mitotic phase(M)are regulated by Cyclin B1 and CDK1[2,8].

Reactive oxygen species (ROS) is a kind of important inducers for apoptosis, thereby blocking cancer cell migration and seizing cell cycle arrest[9].Because of the poor prognosis and severe side effect of traditional therapies such as radiotherapy,chemotherapy and surgery, researchers have explored different phytochemicals and clinical methods to treat liver cancer with significant efficacy.Cell cycle arrest and tumor cell apoptosis are the best strategies,which combat cell growth abnormality with the help of numerous natural phytochemicals[2,10,11].

Gynostemma pentaphyllum Makino is a folk in Asia,especially in China also known as“Jiaogulan”that has been used as an energizing agent by people from mountainous regions in southern China.With the passage of time, G.pentaphyllum leaves, herb and stem have also been used by the Chinese people to treat common cold and infectious diseases, cardiovascular disease, hepatitis, hyperlipo-proteinemia and cancer for centuries[12].Stevenleaf(SV)is a triterpenoid in the form of saponins also called gypenoside derived from G.pentaphyllum Makino.Gypenoside has many pharmacological properties such as the anti-inflammatory[13],lipid metabolism regulatory[14,15],anti-oxidative[6,15],anti-proliferative,neuroprotective [16,17], and anxiolytic activities [18,19].Gypenoside also plays an essential role in health supplements in beverages such as biscuits, noodles, face washes and bath oil [20].The people of southwestern province of China regularly consume G.pentaphyllum as a tea and revealed the longevity and cancer-free survival[21].

Although there are many types of gypenoside,which exhibit cell cycle arrest,and apoptosis in several human cancer cell lines,there is no available information,which explains whether SV can induce the cell cycle arrest, cell apoptosis and ROS generation in HepG2 cells.Present research is the first ever report which provides the evidence that SV has an effect on cell cycle arrest, cell apoptosis,and ROS generation in HepG2 cells in-vitro.

2.Materials and methods

2.1.Chemicals and reagents

Stevenleaf (SV) was obtained from Chengdu Must Bio-Technology Co., Ltd (Chengdu, China) and dissolved in dimethyl sulfoxide(DMSO)to get a stock solution.Cell growth medium and antibodies were procured from Invitrogen(Carlsbad,CA,USA)and Cell Signaling Technology(Danvers,MA,USA)respectively.

2.2.Cell culture condition

For cell experiments, human hepatocellular cell line (HepG2)was obtained from Shanghai wei atlas biological company co.LTD(Shanghai,China)and cultured under a humidified 5%CO2and 95%air atmosphere at 37°C[22].

2.3.MTT assay

MTT assay was used to measure the effects of SV on HepG2 cells [22,23].Cultured HepG2 cells were revived after every two days to maintain the density of 90%confluence,followed by washing with PBS.Then the cells were detached by addition of 0.25%trypsin-EDTA and incubation for 2 min; afterward, centrifuged at 1000 r/min for 5 min,and resuspended with fresh medium for the cells collection.For MTT assay,prior to SV treatments(Untreated,50, 100, 150, 200, and 250 μmol/L), about 5×104cells were cultured in 96 well plates for 24 and 48 h, respectively.100 μmol/L of 5-fluorouracil (5-Fu) was used as a positive control.The 20 μL of 5 g/ml MTT reagent was added into each well of the plate and incubated for 4 h.Subsequently, after pouring 150 μL of DMSO to each well,the absorbance of each samples was measured at 570 nm.For cell viability,the relative cell survival was calculated based on the ratio of absorbance of extraction treatment over that of control treatment[22,23].

2.4.Analysis of cell cycle by flow cytometry

Flow cytometry procedure was followed the descriptions by Zhang et al.[24].Approximately, 2.5×105cells/mL in each plate was treated with different concentrations of SV (Untreated, 30,60, 90, 120, and 150 μmol/L) for 24 h at 37°C.For harvesting and dispersion of the treated cells, the cells were washed with cold PBS twice, followed by fixation with 70% ice-cold ethanol at 4°C overnight.Afterwards, the cells were suspended with propidium iodide solution and then incubated for 30 min at dark.Flow cytometry(BD FACS Calibur,NJ,USA)was used to determine the percentage of the cells in G0/G1 phase,S phase and G2/M phase using cell cycle analysis kit(BD Bioscience,NJ,USA).Flowjo software version 7.6.1(BD Bioscience,NJ,USA)was used to analyze the data.

2.5.Detection of apoptosis by flow cytometry

Briefly,the Annexin V-FITC-PI double staining assay(Beyotime,Shanghai,China)was applied to determine the apoptotic cell based on cell cytometry[11,25].About 1×106cells/well were cultured in each plate,incubated for overnight,and treated with different doses of SV (Untreated, 90, 120, and 150 μmol/L).After 24 h, the cells were collected with 0.5%trypsin without EDTA followed by twice washing with cold PBS and centrifuged at 1000 r/min for 5 min.Then after, 5 μL of Annexin-V and 10 μL of PI were added for 15 and 5 min under dark, respectively; and then, the flow cytometry was performed within 1 h.

2.6.Measurement of intracellular ROS generation

2′,7-dichlorofluorescein-diacetate(DCFH-DA)is a highly sensitive fluorescent probe and is widely used to determine the change of intracellular ROS generation.Briefly,HepG2cells(5×105cells/mL)treated with SV at different concentrations were cultured in sixwell plates.After incubation for 24 h, cells were collected with 0.5 mL trypsin and washed twice with washing buffer.The cells were incubated with DCFH-DA(Sigma Aldrich,MO,USA)for 30 min at 37°C in the dark[26].Flow cytometer was used to detect the fluorescence of each samples,and the level of positive ROS generation is expressed as percentage of the control[26].

2.7.Real time PCR

For this purpose, HepG2 cells were treated with different concentrations of SV(Untreated, 90, 120, and 150 μmol/L).After 24 h incubation, the treated cells were washed twice with PBS, and subsequently, total RNA was isolated using RNA ISO plus reagent(TaKaRa,Dalian Company,LTD,Liaoning,China).Further,reversetranscription reaction was performed to obtain the first strand cDNA for real time PCR reactions.β-actin was used as the internal control for normalization of the mRNA expression levels and calculated by 2-ΔΔCtmethod [23].Table 1 shows the nucleotide sequences of the primers.

2.8.Western blot analysis

Total protein was extracted from HepG2 cells treated with SV at different concentration(Untreated,90,120,and 150 μmol/L)for 24 h[27,28].In brief,protein samples were boiled for 10 min after adding 2×loading buffer and separated via 10×SDS-PAGE,transferred to polyvinylidene difluoride (PVDF) membrane (Millipore,Darmstadt,Germany).PVDF membrane was blocked with blocking buffer(150 mmol/L NaCl,20 mmol/L Tris-HCl,0.1%Tween 20,and 5% skim milk) at room temperature for 2 h.The primary antibodies were incubated with the PVDF overnight at 4°C.Subsequently,the PVDF was washed with TBST (150 mmol/L NaCl, 20 mmol/L Tris-HCl, and 0.1% Tween 20), and incubated with appropriate secondary antibodies containing HRP at 37°C for 1 h.Gel imager(Thermo Scientific, MA, USA) was used photograph the western blotting results and detect the optical density value of specific proteins.The relative protein level of each protein was expressed as the ratio of optical density values of specific protein and β-actin[27,28].

2.9.Statistical analysis

For analyzing the obtained results, SPSS 22.0 software (SPSS,Inc., Chicago, IL, USA) was used.All the data were expressed asmean±SD.One-way ANOVA of variance was performed via Origin lab (Origin Pro 8.0) software (MA, USA), at a significance level of P ＜0.05.

Table 1 Primers for RT-PCR.

3.Results

3.1.Inhibitory effect of SV on HepG2 cell proliferation

To date; more than 230 compounds such as saponins, sterols,flavonoids, and polysaccharides have been identified and purified from G.pentaphyllum.Among them, saponins, also known as gypenoside,are the largest groups consisting of 189 forms[29],and are fully characterized using spectroscopic methods[30,31].

After exposure the cells to different concentrations of SV for 24 and 48 h, MTT assay was performed to examine the inhibitory action of SV on HepG2 cells.It was observed that the cellular inhibition of HepG2 cells treated by SV was time and dose-dependent.At the concentration of 100 μmol/L,5-Fu maintained the inhibition rate of 52.53%.The inhibitory effects on the proliferation of HefG2 cells were significantly increased with the increase concentration of SV via dose and time-dependently(Fig.1A).The IC50value of SV on HepG2 cell was 139.81 μmol/L for 24 h and 119.12 μmol/L for 48 h incubation,respectively(Fig.1B).

3.2.Effect of SV on the cell cycle arrest in HepG2 cells

Significant changes in G0/G1 phase were observed in SV treated cells as compare with the untreated cells.The proportion of cells at G0/G1 phase were increased to 64.4%, 65.1%, 67.8%, 75.6%, and 83.7%after treatment with SV with the concentration at 30,60,90,120,and 150 μmol/L,respectively(Fig.2).Our study revealed that SV could induce cell cycle of HepG2 cells arrest in the G0/G1 phase.

Table 2 Ratio of cells in apoptotic phase.

The mRNA expression levels of Cyclin D1,Cyclin E,CDK1,CDK2,Survivin, CHK2, and p16 were down regulated, while those of p21,p27, p53, Wee1, and p62 were up regulated in HepG2 cells after treatment with SV(Fig.3A).Furthermore,the similar effects on the expression levels of corresponding proteins responsible for G0/G1 arrest were also observed as detected by western blotting(Fig.3B and 3C).

3.3.Effect of SV on cell apoptosis in HepG2 cells

The flow cytometry analysis was performed after incubation with Annexin-V and PI to assess the ratio of apoptotic cells.After treatment with SV for 24 h,the total ratio of apoptotic cells including in early apoptotic and late apoptotic phase were increased significantly (Table 2 and Fig.4), which indicated that SV could induce the cell apoptosis in HepG2 cells effectively.

3.4.Effect of SV on ROS generation in HepG2 cells

ROS generation was analyzed by DCFH-DA, which is a fluorogenic probe through FCM.Our results demonstrated that after treatment with SV at various concentrations, ROS generation was enhanced in a dose- and time-dependent manner in HepG2 cells(Fig.5).The ROS levels in HepG2 cells were increased from 51.3%of untreated group to 69.0%,74.3%,95.8%,and 99.0%in the groups treated with 60,90,120 and 150 μmol/L of SV,respectively(Fig.5).

Fig.1.The inhibition reaction on the proliferation in SV treated HepG2 cells.A.Rate of inhibition; B.Curve of inhibition.All the data are expressed as means±SD of the replicates.Statistical analysis at P ＜0.05 was analyzed by One-way ANOVA.

Fig.2.G0/G1 phase cell cycle arrest in SV treated HepG2 cells.A,Distribution of cell cycle in HepG2 cells after SV-treatment;B,the numbers of cells in different phases of the cell cycle,after treatment of different concentrations of SV for 24 h.Statistical analysis was performed using One-way ANOVA at P ＜0.05.

3.5.Effect of SV on the expression of genes in death receptor pathway

Death receptor pathway also known as external mitochondrial pathway is a critical pathway which induces cell apoptosis.The mRNA expression level of PARP,TNF R1,TRADD,and FADD were up regulated; while that of Caspase8 down regulated in HepG2 cells after SV exposure(Fig.6A).The expression level of corresponding protein (Fig.6B and 6C) displayed the same trend of the mRNA expression levels of these genes.

3.6.Effects of SV on the expression of genes in mitochondrial pathway

Mitochondria pathway is considered as one of the main intrinsic pathways,which exerts cell apoptosis.For examining the effect of SV on HepG2 cell, the expression levels of Bcl2, Bcl xl, Bax, Bak,Cyt c,Smac,Apaf1,Bad,Caspases3,and Caspases9 genes were investigated by the RT-PCR and western blotting.After treatment with SV,the mRNA expression levels of Bad, Bcl2 and Bcl xl (anti-apoptotic molecules)were down regulated;while the mRNA expression levels of Bax,Bak,Smac,Cyt c,Caspase3,and Caspase9 were up regulated in HepG2 cells (Fig.7A).The decreased protein levels of Bcl2 and Bcl xl and the increased protein levels of Bak, Bax, Smac, Caspase3 and Caspase9 (Fig.7B and 7C) in HepG2 cells clearly indicated the involvement of mitochondrial pathway in SV induced apoptosis.

3.7.Effects of SV on the expression of genes in other apoptosis-related pathways

To explore whether other genes in apoptosis related pathways are involved in SV-induced apoptosis, we examined the effects of SV on the expression levels of PKCε, Akt, AMPK, mTOR, and JNK1.As shown in Fig.8,the expression levels of these apoptotic related genes were also dramatically increased.

4.Discussion

Though there is a recent upsurge in modern interventions with improved efficacy,but to combat the cancer burden across the globe is still a challenging issue.According to latest global trend,development of new drugs with higher selectivity and less toxicity in cancer prevention is adopted to customize the therapy.Among the natural products,the stevenleaf(SV)from G.Pentaphyllum has received a prominent interest.To further extend its application in cancer treatment,we explored the inhibitory mechanism of SV on HepG2 cancer cells.

Fig.4.Induction of apoptosis in SV treated HepG2 cells.A,Distribution of apoptotic cell in four quadrants after Annexin V-PI double staining(a,Untreated;b,90 μmol/L;c,120 μmol/L;d,150 μmol/L).B,Rate of apoptotic cells in early and late apoptosis;All the data are presented as a mean±SD and statistical analysis was performed by using One-way ANOVA(P ＜0.05),and nominated as a superscript(a,b,c,d).

Fig.5.Effect of SV treatment on ROS generation in HepG2 cells.A,untreated cells;B,60 μmol/L;C,90 μmol/L;D,120 μmol/L;E,150 μmol/L;F,ROS generation profiles under treatment with different concentrations of SV.

Fig.6.The expression level of death receptor pathway related genes in SV treated HepG2.A,the mRNA expression levels of death receptor pathway related genes.B and C,the protein expression levels of death receptor pathway related genes.β-actin was indicated the internal control.All the data are presented as a mean±SD and statistical analysis was performed by using One-way ANOVA(P ＜0.05),and nominated as a superscript(a,b,c,d).

Several mechanisms have been reported earlier which emphasize that the cell cycle arrest is the key regulator in cancer related studies.After treatment with SV, the cell number in the G0/G1 stage cells was increased while cell number in S and G2/M phase was decreased.The similar effects were also detected by RT-PCR and western blotting on the assessment of mRNA expression of genes as well as the corresponding proteins expression (CHKs,Cyclin,and Cyclin-dependent kinases)responsible for G0/G1 arrest,respectively.When the cells undergo apoptosis or necrosis, the DNA is released, which indicates the low number of nuclear DNA content within the cells.These findings illustrated the molecular mechanism of SV induced apoptosis in HepG2 cells.In various organisms, most of the differentiated cells exit at G0/G1 phase of the active cell cycle where they are metabolically active for days or even years,performing specific functions.Nevertheless,by regulating cyclin-dependent kinases (CDK2) together with Cyclin E,the cells frequently migrate from G1 phase to S phase [32].It is well understood that the higher cell ratio in G1 phase is attributed to the expression levels of Cyclin D1, which has binding affinity to CDK4 and CDK6 [33,34].Previous studies also reported the consequent activation of protein kinase at c-mediated intervals after consequent up-regulation of p53.Hence, it induces the G1 phase arrest and supports the cellular repair mechanisms in mock cell lines [35,36].Previously, it was described that p21, p27, and p57 participated in controlling proliferation during development,differentiation,and response to cellular stress[37].CHK2 was regulated via p53 dependent pathways,which further activated the p21 protein expression during cell cycle progression and acted as CDK1 inhibitor similar to Wee1.Due to binding of p21 with CDK, Cyclin and B1 complex,the kinase activation is reduced and it ultimately induces the cell cycle arrest[38].

Fig.7.The expression level of mitochondria pathway related genes in SV treated HepG2.A,the mRNA expression levels of death receptor pathway related genes.B and C,the protein expression levels of death receptor pathway related genes.β-actin was indicated the internal control.All the data are presented as a mean±SD and statistical analysis was performed by using One-way ANOVA(P ＜0.05),and nominated as a superscript(a,b,c,d).

Fig.8.The mRNA expression level of other apoptosis related genes in SV treated HepG2 cells.All the data are presented as a mean±SD and statistical analysis was performed by using One-way ANOVA(P ＜0.05),and nominated as a superscript(a,b,c,d).

Some other genes such as Survivin and Wee1 are also known as regulatory factors in cell cycle arrest.Survivin is an inhibitor of apoptosis protein family [39].When over expressed, it serves as an anti-apoptotic gene in most human tumors by inhibiting apoptosis and regulating cell division [38].Our results were in accordance with the previous studies,which stated that SV induced the G0/G1 cell cycle arrest in colorectal cancer and human colon cancer[40,41].

Apoptosis is very important phenomenon during cell progression.During the cell apoptosis,the movement of phosphatidylserine from the inner plasma membrane leaflet to the outer one causes the delocalization with Annexin-V which depicts the Annexin-V positive phenomenon in the early stage of apoptosis [42].In contrast, plasma membrane rapture stimulates the direct connection between PI and DNA, which indicates the Annexin-V/PI positive phenomenon in the later stage of apoptosis.Previously, it has been documented that gypenoside triggers the progression of many tumor cells such as lung carcinoma[43],human colorectal cancer[44],human colon cancer[41],and prostate cancer[6].

To further confirm the apoptosis mechanism of SV treatment in HepG2 cells,generation of the intracellular reactive oxygen species(ROS) was detected.Our result showed the significant increase in ROS,which indicated that SV treatment could induce apoptosis in HepG2 cells.Until now, many experimental studies have demonstrated that high level of ROS production is involved in cancer cell death,as high levels of ROS induce apoptosis[45].

In current study, we also investigated the expression levels of genes related to apoptosis pathways including mitochondria pathway,death receptor pathway and other apoptosis related pathway after SV treatment.In death receptor pathway,SV treatment led to up-regulation of TNF R1 which was further bound to death domain receptor TRADD1(TNF receptor associated death domain protein).Inside the cells,TRADD1 recruits some other signaling molecules to activate FADD receptor.The FADD receptors activate the Caspase8 and hence induce the apoptosis similar to Fas-initiated apoptosis[46,47].

In mitochondrial pathway,the Bcl2 family consists of two major groups as it can regulate the mitochondrial membrane permeability via PT pore.The Bcl2 family genes are also known as intracellular suppressor of apoptosis,perform the cytoprotective function in cells[48],and cooperate with Bax(pro-apoptotic protein)[49].Our results reported that after SV treatment, the mRNA expression of Bax was in upward trend whereas,the expression of Bcl2 followed the downward trend.The expression levels of corresponding proteins also showed the same tendency as of the mRNA expression levels.Besides,our data also revealed the over expression of mRNA levels of p53 gene.Therefore, it was speculated that mutual cooperation between Bcl2 and p53 mediated mechanism might affect the cell survival and death in SV treated cells.

Fig.9.The plausible molecular mechanism of SV treated HepG2 cells.

Cytochrome c (Cyt c) is a small hemeprotein complex found within the inner membrane of mitochondria.Various studies have showed the critical role of Cyt c during apoptosis.Upon apoptotic stimuli, the Cyt c is executed from mitochondria to cytosol where it binds with apoptotic protease activating factor 1(Apaf1)and as a result,apoptosome is formed.Furthermore,it recruits multiple procaspase9 molecules and promotes their cleavage to an active form caspase9,which further initiate caspase3,and induce the apoptosis in cells [24,50].The parallel effects on the changes of expression levels of mRNA as well as corresponding proteins were seen in our study which indicated the SV induced apoptosis.

The Akt/mTOR is a significant survival pathway, which shows numerous molecular alterations in cancerous cells [51].Recently,the inhibiting effects of many agents with Akt/mTOR pathway are linked with metabolic disorders[52]and many of them have been validated in clinical development.JNKS are basically the proteins kinase also known as pro-apoptotic factors,which regulated the cell growth and cell apoptosis [53].JNKs can also alienate the activity of Bcl2 protein(pro-apoptotic factors),which is a significant factor involved in the initiation of caspase8 activation[54].Our study indicated that the up-regulation of mRNA expression of JNK1 exerted a possible affirmative effect on SV induced apoptosis in HepG2 cells.

To summarize the anticipated anticancer effect of SV by triggering the cell cycle arrest and induction of apoptosis,a flow chart was arranged to depict the whole phenomenon in Fig.9.

5.Conclusion

The present study highlighted the importance of SV by inhibiting the cell apoptosis;stimulating the cell cycle arrest at G0/G1 phase,ROS generation, induction of the apoptosis, activation of cellular death receptor pathway,and the promotion of mitochondrial pathway in HepG2 cancer cells.Our results provides an advantageous and edifying model system for cytotoxic or apoptotic effects of SV.To recapitulate our findings, we contemplate that SV as a plantbased natural compound can be used as functional food.Through the further studies,it would emerge as a promising anti cancerous drug,which will extend its current relevance for the treatment of hepatocellular carcinoma cancer.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The National Natural Science Foundation of China(31850410476),and the Major Projects of Science and Technology in Anhui Province(18030701144,1804b06020347,18030701142,18030701158,201903a06020021).