Methanol Extract of Ficus pumila L. Inhibits Proliferation, Induces Apoptosis and Arrests the Cell Cycle in HepG2 Live Cancer Cells

MATERIALS AND METHODS

Collecting leaf samples of medical plants: The study was carried out from April, 2020 to October, 2021. Leaf samples of seven medical plants including Sambucus javanica Blume, Lasia spinosa Thwaites, Hovenia dulcis Thunb, Asarum petelotii (Otto Charles Schmidt). The O.C. Schmidt, Gynura procumbens (Lour.) Merr., Ficus pumila L. and Sabia parviflora Wall. ex Roxb. were collected, classified and stored at the National Institutes of Medicinal Materials, Vietnam. These herbs were used according to the indigenous experience of the Dao people in Sapa Mountain, Lao Cai Province of Vietnam to treat several different liver diseases. An amount of 300 g of each leaf sample were washed and dried at 40°C for 72 hrs and then ground with a mortar and pestle into a fine powder. Weigh 15 g of the obtained powder, add 30 mL of 90% methanol and shake the mixture at 200 revolutions per minute (rpm) for 48 hrs. The extract was filtered with Whatman filter paper and then dried in an oven (WFO-520 EYELA, Tokyo, Japan) at 40°C for 48 hrs. The residue obtained after evaporation will be dissolved in Dimethyl Sulfoxide (DMSO) for further analysis.

Cell culture and 2D cell treatment: The HepG2 cell line was grown under single-layer (2D) culture conditions in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% ampicillin/streptomycin (P/S) at 37°C and 5% CO₂. All these chemicals were supplied by Thermo Fisher.

An amount of 10,000 cells were added to each well of a 96-well culture plates containing 100 μL of RPMI 1640 medium. After 24 hrs, the culture medium was completely replaced with 100 μL of the new culture medium containing extracts at different concentrations. Control samples were cells grown in extract-free RPMI 1640 cell culture medium for 24 and 48 hrs.

Cell culture and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay: The HepG2 live cancer cells were cultured in RPMI 1640 medium supplemented with 1% P/S and 10% FBS in 96-well plates at 37°C, 5% CO₂. After 24 hrs, the cells were treated with FPE at different concentrations from 10 to 300 μg mL^–1. After 24 or 48 hrs of treatment, the cell phenotype was observed and evaluated using an inverted microscope Ts2 Nikon (Tokyo, Japan).

Cell proliferation was measured by using an MTT assay. Briefly, completely remove the old culture medium, add 100 μL of new culture medium containing 10% MTT at 5 mg mL^–1 and incubate the culture dish at 37°C for 4 hrs, protected from light, removed culture medium containing MTT and added 100 μL DMSO per well, measuring the optical density (OD) at a wavelength of 570 nm on a spectrophotometer (Multiskan Sky, Thermo Fisher, Waltham, Massachusetts, USA). The cell proliferation rate was calculated by the formula¹¹:

GraphPad Prism 5.0 software (San Diego, California, USA) was used to determine the IC₅₀ value for each cell line.

Cell culture and 3D cell treatment: An amount of 1,000 HepG2 cells cultured in each well of a 96-well culture plate were anti-adhesion treated with PolyHEMA (Sigma Aldrich, St. Louis, Missouri, USA) containing 100 μL of DMEMF12 medium (Thermo Fisher, Waltham, Massachusetts, USA), which contains 20 nM Epidermal Growth Factor (EGF) and 20nM Fibroblast Growth Factors (FGF) (Sigma Aldrich, St. Louis, Missouri, USA) as reported by Ramcharan et al.²⁰. The tumorsphere culture medium was supplemented with FPE at concentrations of 0, 50, 100, 150 and 300 μg mL^–1. After 5 days of culture, the number of tumorspheres was counted and photographed under an inverted microscope. Tumorsphere size was measured by using the NIS-Elements imaging software version 3.0 and the Eclipse Ts2 microscope (Nikon, Tokyo Japan). A group of cells was defined as tumorspheres if their diameter was >100 nm. The number and size of tumorspheres in the treated sample were compared with the control samples.

Analysis of cell cycle and apoptosis using flow cytometry: The HepG2 hepatocellular carcinoma cells were cultured on 12-well culture plates. After 24 hrs, cells were supplemented with a culture medium containing FPE at concentrations of 100, 200 and 300 μg mL^–1. The control wells were not added FPE. The treatment time was 48 hrs in the 37°C, 5% CO₂ condition. Subsequently, cells were separated from the plate surface by treatment with trypsin/EDTA and collected by centrifugation at 1,500 rpm for 3 min and fixed in 70% ethanol at -20°C overnight. Then, cells were stained with fluorochrome solution (0.1% sodium citrate (w/v), 0.1% Triton X-100 (v/v), 50 μg mL^–1 propidium Iodide (PI) in deionized water) for 2 hrs at 4°C before analyses using the BD-Accuri C6 plus Flow cytometry system (BD Biosciences, Bedford, Massachusetts, USA)²¹.

Total RNA extraction and real-time PCR: The total RNA of HepG2 hepatocellular carcinoma cells was extracted with Trizol according to the manufacturer’s instructions (Invitrogen). Cells cultured on 6-well culture plates were supplemented with 1,000 μL Trizol and mixed with a 1 mL pipette several times to separate cells from the surface of the plate. Incubate cells at room temperature for 5 min, then centrifuge at 12,000 rpm for 10 min at 4°C. Transfer the supernatant to a new Eppendorf tube. Add 2 mL chloroform, vortex the sample for 15 sec and incubate at room temperature for 3 min. Centrifuge at 10,000 rpm for 15 min at 4°C, repeat 2 times. Dry the RNA for 5-10 min and add 100 μL of deionized water. Spectrophotometer at 260/280 nm using a NanoDrop spectrophotometer (Thermo Fisher, Waltham, Massachusetts, USA). Use 1 μg RNA to generate cDNA using SuperScript^™ III Reverse Transcriptase (Invitrogen-Thermo Fisher, Waltham, Massachusetts, USA). The Invitrogen^™ Platinum^™ SYBR^™ Green qPCR SuperMix-UDG w/ROX mix was used for Real-time PCR analysis (Invitrogen-Thermo Fisher, Waltham, Massachusetts, USA) in a qTower3 (Analytik Jena, Jena, Germany) following the manufacturer’s protocol. Amplification was carried out using target primers (Table 1) for 95°C for 2 min and 40 cycles of 15 sec at 95°C and 30 sec at 60°C and their relative expression was determined by normalizing the expression of each target HPRT1. The change in gene expression level was calculated by method 2^{–ΔΔCt 22}.

Statistical analysis: All samples were evaluated at 3 replicates and presented as Mean±Standard Deviation (SD). Study data were analyzed using Graphpad Prism 5.0 software (Graphpad Inc., San Diego, USA), with Mann-Whitney Test (p<0.05).

RESULTS

Inhibitory effect of medical plant extracts on HepG2 liver cancer cells: The inhibitory effect of methanol extracts from seven medical plants on HepG2 liver cancer cells was presented in Table 2. The results showed that the extracts inhibited the proliferation of HepG2 cells in a dose and sample-type-dependent manner. The IC₅₀ values recorded were from 66.46-227.7 μg mL^–1. Ficus pumila L. extract was shown to be a more potent inhibitor of HepG2 cells than the other 6 herbs in the screened group with IC₅₀ value of 66.46 μg mL^–1.

FPE inhibits the proliferation of HepG2 cells: The first objective of this study was to determine the ability of FPE to inhibit the proliferation of HepG2 cells. The HepG2 cells were cultured in FPE-supplemented media at concentrations ranging from 0 to 300 μg mL^–1.

The MTT assay results (Fig. 1a) showed that at the concentration of 50 μg mL^–1, HepG2 liver cancer cells grew well on the surface of the culture medium and did not differ from the control group in both 24 and 48 hrs of processing. However, the number of cells significantly declined at concentrations from 100-300 μg mL^–1 compared to the control group at both 24 hrs (^#p<0.05) and 48 hrs treatment time (*p<0.05). The cell proliferation rate decreased from 81.9±6.6% (at the concentration of 150 μg mL^–1) to 23.8±3.7% (at the concentration of 300 μg mL^–1) after 24 hrs of treatment with the FPE and from 40.4±3.9% (at the concentration of 150 μg mL^–1) to 15.6±2.1% (at the concentration of 300 μg mL^–1) after 48 hrs of treatment with the FPE. The IC₅₀ value determined at 24 hrs was 164 μg mL^–1 and at 48 hrs was 117.8 μg mL^–1. Cell images at 48 hrs of treatment (Fig. 1b) also showed a significant decrease in cell density from the concentration of 150-300 μg mL^–1. On the other hand, at the concentrations of 200 and 300 μg mL^–1, the cells lost their association with the surface of the culture plate and shrank into an oval shape. This image was consistent with the results of the MTT analysis presented above.

FPE inhibits the formation of 3D tumorspheres of HepG2 cells: To evaluate the effect of the FPE on the ability to form tumorspheres in the 3D culture model, cells were incubated in a medium containing different concentrations of the extract. The results (Fig. 2a-b) showed that the FPE at concentrations from 50-150 μg mL^–1 significantly decreased the number of formed tumor spheres, ranging from 57.7±11.4 to 8.8±1.7, compared to the control which is 82.2±19.1, respectively. Meanwhile, at the higher concentrations (200 and 300 μg mL^–1) the tumorspheres were not observed. On the other hand, the size of tumorspheres formed in FPE medium was remarkably reduced in diameter from 65.4±12.9 to 39.6±1.9% compared to the control (100%), respectively.

FPE induces apoptosis of HepG2 cells: Induction of apoptosis is one of the important and common mechanisms of many current anticancer drugs. In this study, the effects of FPE are evaluated at concentrations of 50, 100 and 200 μg mL^–1 after 48 hrs of treatment on apoptosis of HepG2 cells by Flow cytometry and the change in nuclear phenotype with DAPI (4,6-diamidino-2-phenylindole) dye.

The results (Fig. 3a) showed that the percentage of cells with apoptosis was significantly increased in the cells cultured in the medium supplemented with FPE at a concentration of 100 μg mL^–1 (13.2±2.7%) and 200 μg mL^–1 (35.8±5.0%) compared to the control group.

Nuclear staining with DAPI dye (Fig. 3b) indicated that the number of cells where nuclear apoptosis phenotype (arrow) was observed was remarkably increased compared to the control. This result confirmed the above results of Flow cytometry analysis (Fig. 3a). Taken together, these results showed that FPE induced apoptosis in HepG2 cells.

Fig. 1(a-b):

Effect of FPE on cell proliferation and morphology. Cells were cultured in the media supplemented with the FPE of 0-300 μg mL–1 for 24 and 48 hrs, (a) Cell proliferation rate measured by MTT assay were represented as Mean±SD and (b) Morphologic changes of HepG2 cells at 48 hrs of treatment with FPE (a) #p<0.05 vs control at 24 hrs, *p<0.05 vs control at 48 hrs, Mann-Whitney Test and (b) a: 0, b: 10, c: 50, d: 100, e: 200, f: 300 μg mL–1 at 200X magnification and Scale bar = 50 μm

FPE arrests the cell cycle in the G2/M phase: Evaluation of the effect of FPE on the cell cycle was performed at concentrations of 10, 50 and 100 μg mL^–1 for 48 hrs of treatment. The results (Fig. 4a) showed that, at the concentration of 10 μg mL^–1, there was no significant change in the percentage of cells in different cell cycle phases. However, at the concentrations of 50 and 100 μg mL^–1, the percentage of cells in the G2/M phase increased to 24.6±2.5 and 33.6±3.4% compared to 19.7±2.2% in the control. Hence, the FPE arrested the cell division cycle in the G2/M phase. Subsequently, to clarify changes in mRNA expression levels of several cell cycle-related genes, a concentration of 200 μg mL^–1 FPE was used to treat cells for the analyses of cyclin B1, cyclin D1, p53, p21 and GADD45A genes expression by Real-time PCR.

Fig. 2(a-b):

Effect of FPE on tumorsphere formation of HepG2 cells, (a) Cells were cultured under non-adhesive conditions and supplemented with extract concentrations of FPE and (b) Change in the number and size of the tumorspheres was expressed as a percentage compared to the control (a) a: 0, b: 10, c: 50, d: 100, e: 200 and f: 200 μg mL–1, Tumorsphere images at 200X magnification after 5 days of culture, Scale bar = 200 μm and (b) *p<0.05 vs control, Mann-Whitney Test

The results (Fig. 4b) showed that mRNA levels of GADD45A, p53 and p21 genes were increased approximately 4, 1.5 and 3 times, respectively. While the mRNA levels of cyclin B1 and cyclin D1 were increased approximately 10 and 6 times. Thus, the FPE regulated expression of the genes controlling the cell cycle leading to arrest the of cell division.

Fig. 3(a-c):

FPE induces apoptosis in HepG2 cells, (a) Percentage of apoptotic cells measured by flow cytometry were represented as Mean±SD and (b) Cells were treated with FPE. Cell nuclei were stained with DAPI (4 ,6-diamidino-2-phenylindole) and observed under a T2U fluorescence microscope (Nikon) at 200X magnification *p<0.05 vs control, Mann-Whitney Test, a: 0, b: 10, c: 50, d: 100 μg mL–1 for 48 hrs and Scale bar 50 μm

Fig. 4(a-d):

FPE arrests the cell cycle in HepG2 cells, (a) Cells were treated with FPE for 48 hrs, were then stained with PI solution and analyzed by flow cytometry and (b) mRNA expression levels of GADD45A, cyclin B1, cyclin D1, p53 and p21 genes were analyzed by real time PCR (a) a: 0, b: 10, c: 50 and d: 100 μg mL–1, Data represented as cell percentage compared to control, *p<0.05 vs control and (b) *p<0.05 vs control, Mann-Whitney Test

DISCUSSION

Inhibition of proliferation is an important first factor in the evaluation of the anticancer potential of drugs and various compounds. In this study, in a single-layer (2D) culture model, the FPE effectively inhibited HepG2 cell proliferation with an IC₅₀ value defined as 164 and 117.8 μg mL^–1 for 24 and 48 hrs of treatment, respectively. As mentioned above, compared to some other species in the Ficus genus, Ficus pumila L. is less studied on the activity and mechanism of inhibiting cancer cells. Several previous studies have shown that extracts of Ficus pumila L. can inhibit several different types of cancer. However, the effect of Ficus pumila L. on apoptosis, the cell cycle of HepG2 cells or the expression of related genes has not been studied. Cancer cells that were cultured under three-dimensional cell cultures are believed to have more similar properties to cancer cells in vivo than themselves when cultured in a traditional layer (2D)²³. The use of 3D culture models is the current trend in drug research²⁴. In this model, the effect of drugs on spherical cell masses consisting of many layers of cells is compared to the effect on tumors in vivo²⁵. Here, it was shown that the FPE not only effectively inhibited the formation of tumorspheres from HepG2 cells, but also significantly declined the size of tumorsphere cells. This may open the prospect of this extract’s ability to inhibit tumor formation and tumor proliferation in vivo.

Changes in apoptosis can be identified through Flow cytometry analysis or microscopic observation with DAPI dye to detect morphological and structural changes in the cell nucleus²⁶. Meanwhile, genes controlling apoptosis can be evaluated through Real-time PCR, Western blot or immunofluorescence analyses²⁷. In this study, the FPE-induced cell apoptosis was shown. In addition, nuclear staining with DAPI dye confirmed that the FPE produced cells with an apoptosis phenotype. Although there were little data on the apoptosis-inducing ability of Ficus pumila L.in various cancer cell lines, several species of the Ficus genus, such as Ficus virens, have been shown to induce cell apoptosis of breast cancer MCF7²⁸, Ficus septica induces apoptosis on human oral squamous carcinoma cells²⁹, Ficus pumila L. on human prostate cancer cell³⁰. Ficus dubia latex on colorectal cancer cell lines³¹.

Cancer cell proliferation is closely related to the cell cycle and cell cycle arrest can lead to inhibition of cell replication³². This is an important mechanism for the development of current anticancer drugs^33,34. In this study, FPE induced cell cycle arrest in the G2/M phase. There is earlier evidence that some herbal extracts such as Costus speciosus³⁵, Vaccinium vitis-idaea³⁶, Trichosanthes kirilowii tube³⁷ or potential compounds have the ability to halt HepG2 cancer cell division in the G2/M phase.

The P53, P21 is known to be important protein in cell cycle regulation as well as the induction of apoptosis³⁸. Meanwhile, the P53-regulated GADD45A genes have a role in stopping division, promoting nuclear DNA degradation and induction of apoptosis³⁹. An earlier study has shown that GADD45, cyclin B1 and cyclin D1 are all strongly associated with a cell cycle arrest in the G2/M phase and enhanced expression of GADD45 has been shown to inhibit the expression of several key proteins that control the cell cycle such as cyclin B1, cyclin A2, cyclin D1 and eventually halt cell division⁴⁰. Real-time PCR analysis showed that FPE upregulated the expression of p53, p21 and GADD45 genes and remarkably increased the expression of cyclin B1 and cyclin D1 genes. Previously, Choudhari et al.⁴¹ also showed that an aqueous extract of Ficus religiosa induces cell cycle arrest in human cervical cancer cells through the up-regulation of the expression of p53 and p21. Ficus carica was shown to upregulate the expression of p53, p21 in MDA-MB-231 breast cancer cells⁴². Meanwhile, Ficus religiosa halted the Siha cervical cancer cell cycle through enhanced expression of p53 and p21 and pRb genes⁴³.

These results suggested that Ficus pumila L. has the potential as a treatment option for liver cancer. However, this study also has some limitations such as lacking evaluation changes in expression at the protein level as well as identifying chemical components that play an important role in HepG2 cytotoxicity. Further studies are needed to elucidate the inhibitory mechanism of Ficus pumila L. against cancer.

CONCLUSION

This study showed that, under in vitro conditions, the FPE has the ability to inhibit proliferation, tumorsphere formation arrests the cell cycle and induce apoptosis of HepG2 liver cancer cells. Ficus pumila L. has the potential to inhibit liver cancer cells.

SIGNIFICANCE STATEMENT

This study aims to determine the anti-cancer activity of the Ficus pumila L. liver cancer cells. The results showed that the FPE has a strong inhibitory effect on the proliferation of HepG2 liver cancer cells, significantly reducing the number and size of tumorspheres compared to the control. The FPE also increased the apoptosis rate and arrested the cell cycle at the G2/M phase, as well as upregulated the expression of genes involved in cell cycle arrest and apoptosis while downregulating genes associated with tumor growth.