Ethyl acetate crude fraction of Macaranga subpeltata and silymarin increase Vero cell survival and HepG2 cell death due to oxidative stress

Sample preparation

Plant material was obtained from Ponorogo district, Central Java Province, and was authenticated by taxonomist of Medicinal Plant and Traditional Research Centre of NIHRD-MOH, Republic of Indonesia (ref. no. YK 01.03/2/377/2021). Methanolic leaves extract of M. subpeltata was prepared by maceration according to Widodo et al. (2019); the fractionation to obtain EACFM was performed using liquid–liquid fractionation following the previous method of Widodo et al. (2020).

Cell line preparation

The cell lines (Vero and human hepatocellular carcinoma (HepG2) ATCC HB-8065) were obtained from the Cancer Chemoprevention Research Centre, Faculty of Pharmacy, Universitas Gadjah Mada. The cells were grown in Dulbecco’s Culture Media Modified Eagle Medium (DMEM, Sigma) [consisting of 2% (w/v) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Sigma), 2.35% (w/v) CHNaO₃ (Sigma), 10% (v/v) fetal bovine serum (FBS) (Gibco), 1% (v/v) penicillin-streptomycin (Gibco), Fungizone 0.25% (v/v) (Gibco)] at cell culture plate (Iwaki) in incubator with CO₂ 5% and 37°C.

Radical scavenging assay

In vitro radical scavenging assay was performed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Trolox Equivalent Antioxidant Capacity (TEAC) methods according to a previous study (Widodo et al., 2020).

In vitro assays using cell lines were performed according to CCRC (2019) with little modifications as described in the following.

Cytotoxic test of EACFM on Vero and HepG2 cell lines

Vero and HepG2 8 × 10³ cells/100 µl were grown in DMEM at 96-well cell culture plate (Iwaki); afterwards, the cells were incubated for 24 hours (CO₂ 5%; 37°C). The confluent cells were treated with EACFM and silymarin (Sigma-Aldrich) at a series concentration of 0–1,000 µg/ml (incubated for 24 hours). The media were discarded, and washed with 1× PBS (Sigma-Aldrich) and adding 0.5 mg/ml 3-(4,5-dimethyl azole-2-yl)-2,5-diphenyltetrazolium bromide [50 mg Thiazolyl Blue Tetrazolium Bromide (Sigma) in 10 ml of 1× PBS] in media, and were incubated for 3–4 hours afterward. The reaction was terminated with 10% (w/v) SDS (Sigma) in 0.01 N HCl (Merck) and was kept overnight in the darkroom. The viable metabolically active cells were measured by reading absorbance at λ 595 nm (Multiskan-Thermo Scientific, Finland). Cell viability was calculated from the percentage of living cells.

$\%\mathrm{of}\mathrm{living}\mathrm{cells}=\frac{({\mathit{Ax}}_1-{\mathit{A}}_0)}{({\mathit{Ax}}_0-{\mathit{A}}_0)}\times 100\%$

Note: A₀: absorbance of control media (media only without cell lines)

Ax₀: absorbance of cell with media without EACFM or Silymarin

Ax₁: absorbance of EACFM or silymarin with EACFM or Silymarin x₁ concentration

The inhibition concentration 50% (IC₅₀) was calculated from a linear regression (% of living cells against compound concentration).

Assessment of ROS production

Vero and HepG2 5 × 10⁴ cells in 500 μl DMEM media in each well were grown on 24-well cell culture plates (Iwaki) for 24 hours. The ROS production test was performed using H₂O₂ (Merck): 0, 500 and 1,000 µM, EACFM, and silymarin (Sigma) on Vero and HepG2. The media were discarded and washed with 1× PBS (pH 7.4). Cell lines were treated with EACFM and silymarin (0, 50, 100, and 150 μg/ml) and maintained for 4 hours in a cell culture incubator. The media was discarded and washed with 1× PBS; afterwards, in each cell line in each well, 500 μl of a freshly prepared solution of 20 μM DCFDA (Sigma) in a 10% supplement buffer (2 ml of FBS plus 18 ml of 1× PBS) was added. The DCFDA solution was discarded and replaced with 500 μl H₂O₂ solution in a 10% supplement buffer according to the desired treatment concentration and was incubated for 2 hours afterwards. H₂O₂ solution was discarded and washed with PBS. Cells were harvested using 0.25% (v/v) Trypsin-ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich) without phenol red 100 μl/well and were incubated for 3 minutes; then, 1.0 ml of 10% supplement buffer was added before flow cytometric analysis.

Apoptosis evaluation

Vero and HepG2 cells 5 × 10⁵/ml media were grown for 24 hours in 6-well cell culture plate (Iwaki) until confluence and then were treated with sample tests accordingly. The culture media were discarded and washed with 1× PBS. 1,000 μl of EACFM or silymarin was added according to the concentration applied and was incubated for 4 hours. The cells were treated with H₂O₂ (according to concentrations) and were incubated for 12 hours. The treatment cells were harvested using 150 µl of 0.25% (v/v) trypsin-EDTA and were collected entirely in a separate conical tube each. The remaining cells were taken using 500 µl of PBS and were transferred to the same tube and centrifuged (500 rpm; 4 minutes). The cell pellets were mixed homogeneously with a 100 µl apoptosis reagent (BD Pharmingn^TM) (consisting of 4 μl FITC annexin V, 4 µl propidium iodide (PI), and 92 µl annexin buffer) and were placed in the dark for 15 minutes at room temperature. The cell suspensions were transferred to the microtube and added with 400 µl of annexin buffer before flow cytometric analysis.

Flow cytometric analysis

The ROS production analysis using a flow cytometer (BD Accuri C6, USA) indicated by the fluorescence intensities was measured at an excitation of λ 485 nm and emissions of λ 535 nm. The ROS intracellular was obtained from a minimum of 2,500 cells per treatment. The ROS data were presented as a fold of untreated cells obtained from shifting fluorescence mean of the treatment divided by fluorescence percentage mean of the control without treatments. Cell death evaluation was depicted as a plotting profile from a run limit of 20,000 events per sample treatment.

Data analysis

Flow cytometry analysis data were calculated using BD Accuri C6 software and Statistical Package for the Social Sciences version 16.0 for statistical analysis. All experimental studies were done in triplicate, and the data represented mean ± SD. The effect differences among treatments were subjected to analysis of variance (ANOVA; p = 0.05) followed by the Post-Hoc Duncan Multiple Range Test with a significant level of 95%.

The effect of EACFM and silymarin on cell viability

A recent study revealed that the EACFM has low toxicity (nontoxic) to both the normal cell lines and the cancer cells, especially at low concentrations (<125 µg/ml). When treated with EACFM lower than 125 µg/ml, the Vero cell was more susceptible to cell death than HepG2, whereas silymarin gave a contrary result. Interestingly, via treatment at a low concentration (< 50 µg/ml), silymarin stimulated the viability of Vero cells higher (>100%) than the control. Silymarin demonstrated tissue regenerative properties (Surai, 2015). Wu et al. (2015) reported that silymarin could accelerate the cell cycle, which might enhance hepatocyte proliferation after partial hepatectomy. Moreover, with no lees enticing, silymarin at the same concentration inhibited the growth of HepG2 cells, and the cytotoxic activity of silymarin against HepG2 cells was more prominent than the EACFM (Fig. 2).

Silymarin resulted in a lower IC₅₀ both in Vero and HepG2 cells than those of EACFM, and the selectivity index (SI) of silymarin was higher than EACFM, indicating that silymarin was more selective in killing cancer cells and was relatively safer to the normal cell. SI value exhibits a specific degree of any anticancer drug, and the SI value of more than three indicates that the compound has high selectivity toward cancer cells. Doxorubicin, one of the wide-spectrum tumor medication drugs, shows a high SI value; unfortunately, clinical adverse effects are found in the systemic dosing (Haryanti et al., 2021). Therefore, finding a new anticancer is still a challenging task. IC₅₀ states the concentration of the tested compound that causes cell line lives of 50% during the specified incubation time. The IC₅₀ EACFM on HepG2 cells was lower than on Vero cells implicating that it had an anticancer activity (Table 1). However, EACFM is still a fraction that still contains various compounds that may have diverse pharmacological activities. Further investigation is needed, especially for compound isolation of EACFM, which is responsible for anticancer activity with higher SI.

At the same concentration, silymarin showed better cytoprotective activity on normal cells (Vero) than EACFM; furthermore, silymarin increased the cytotoxicity of H₂O₂ via OS-induced death to enhanced cancerous cells (HepG2) to die. The SI value of silymarin was 2.15, indicating that silymarin has good anticancer activity. Silymarin significantly increases the apoptosis rate of the cancer cells by inhibiting and blocking nuclear factor kappa B activation, inducing cytochrome C release, activating caspase-3 and caspase-9 and cleavage of poly-adenosine diphosphateribose polymerase, and also downregulating p-ERK1/2 and Bcl-2 expression (Fallah et al., 2021).

Figure 1. The IC50 DPPH scavenging activity (A) and TEAC (B). The mean values with the same letter above showed no significant difference according to the DMRT (p = 5%). [Click here to view]

Silymarin at low concentration could promote normal cell (Vero) viability (Fig. 2). Mili? et al. (2013) explained that silymarin has a stimulatory effect on Vero cells similar to fibroblast. Silymarin increases the synthesis of functional and structural proteins, accelerates DNA synthesis, and thus promotes cell proliferation and organ regeneration (Khazaei et al., 2022). According to Surai (2015), the role of silymarin in blocking SO is to protect the integrity of mitochondrial structure and function, trigger prosurvival signals, optimize the electron transport chain, reduce electron leakage, reduce ROS formation, and directly reduce the activity of ROS-producing enzymes in mitochondria.

The ROS production

The administration of EACFM and silymarin alleviated intracellular ROS generation on Vero and HepG2 cell lines. On Vero cells without SO’s induction (H₂O₂-untreated cells), the decreasing capacity of EACFM to ROS production weakened as concentration rose. However, the reduction of ROS production by silymarin on Vero cells did not increase or reduce in line with the elevation of silymarin concentration treatment (Fig. 3).

H₂O₂ up to the concentration of 1.0 mM increased ROS in a dose-dependent manner both in Vero and HepG2 cells. The EACFM could hamper the increase of ROS-causing SO due to H₂O₂ treatment. The ability of EACFM to reduce ROS on Vero cells was in line with the increase of applied concentration, especially for H₂O₂ 1,000 µM (Fig. 4A); on the contrary, ROS production by HepG2 elevated with an increase in EACFM’s concentration (3B). In the negative control (without H₂O₂ treatment), both normal (Vero) and cancerous (HepG2) cell lines, treated with EACFM 50 µg/ml, decreased ROS greater than those treated at higher concentrations which were proportional to silymarin at a concentration of 50–100 µg/ml. Silymarin at 150 _µg/ml gave the best ROS reduction, especially on HepG2 cells, while ROS reduction by EACFM was higher on normal cells than on cancerous cells (Fig. 4).

Figure 2. Viability of Vero and HepG2 cells treated with EACFM and silymarin. [Click here to view]

In various cancer cells, the level of ROS and the expression of AO enzymes are higher than in normal cells as a manifestation of adaptative mechanisms to intrinsic SO. ROS plays a double-edged sword in cancer; moderate elevation of ROS plays a role in the initiation, development, and level of cancer cell metastasis. However, an increase in ROS exceeding the defense mechanism threshold of the AO cancer cells can bring about the death of the cells (Kim et al., 2016; Ndombera, 2017; Perillo et al., 2020). Silymarin suppressed the growth of HepG2 cells (Fig. 2) through a decrease in ROS, and up to 150 µg/ml it still attenuated ROS production (Fig. 4B). A low concentration of EACFM could suppress the growth of HepG2 cells, also indicated by decreasing ROS production. The active cancer cells showed higher basal ROS levels than normal cells as a result of the activation of several oncogenes (Yang et al., 2018). Using a high concentration (150 µg/ml), the EACFM triggered cell death via an elevation of ROS generation levels over a critical threshold of the cancer cells. In normal cells (Vero), the EACFM treatment did not render an increase in ROS; furthermore, EACFM up to 150 µg/ml still had an impact on decreasing ROS (Fig. 4A).

Table 1. IC50 and SI of EACFM and silymarin on Vero and HepG2 cell lines. [Click here to view]

Figure 3. The influence of EACFM and silymarin to ROS production on vero cell line untreated with H2O2. The percentage of the treatment plots followed by the same letters above indicated no significant difference (DMRT test; p = 0.05). [Click here to view]

The effect of EACFM and Silymarin on H₂O₂-induced cell death cell lines

Evaluating cell death using flow cytometry would acquire four states of three occurring cell deaths, i.e., living cell, early apoptosis, late apoptosis, and necrosis. These phases can be determined by measuring the intensity of annexin V and PI. There are various alterations in cell features that encountered apoptosis or necrosis, one of which is phosphatidylserine (PS) translocation from the inner layer to the outside of the plasma membrane. PS is exposed to the outer surface of the cell membrane that subsequently reacts with Annexin V. On the other hand, PI acts as a necrotic cell death marker since it only binds to nucleic acids, the cells with intact plasma membranes, a condition in which PI could not translocate into the cell and bind to RNA and DNA; cells will give very low or absent fluorescence intensity (Vermes et al., 2000).

Under normal conditions, there was a death in Vero cells either via necrosis (2.95% ± 3.17%) or apoptosis (6.67% ± 3.58%), treatment with H₂O₂ 1.0 mM for 12 hours stimulated cell deaths up to 24.41% ± 3.12%. The EACFM could reduce H₂O₂ cytotoxicity, and a concentration of 50 µg/ml had a proportional effect to silymarin 50 µg/ml, which was demonstrated by an increase in living cells of 9.53% while silymarin was 8.09%. A 150 µg/ml of silymarin exhibited a better cytoprotective action in living cell increment (12.54%) than that in EACFM (10.24%) (Fig. 4A).

HepG2 responds to SO in the same mode as primary hepatocyte cells (Liau et al., 2016); therefore, it is representative of an in vitro study. HepG2 cells were more resilient against H₂O₂ regiment than Vero cells, the same treatment with 1.0 mM H₂O₂ for 12 hours to HepG2 still resulted in cell viability of 88.42% ± 4.06% while viability of Vero decreased to 75.58% ± 3.11%. The ROS increased in tone with the increases in H₂O₂ concentration, as seen in the control treatment in Figure 4. Habtemariam and Lentini (2018) explained that ROS generation depended on the type of stimulus and the cell type, and ROS-activated caspase-3 and c-Jun N-terminal kinase as an inductive mechanism to apoptotic cell death. However, at a concentration of 50 µg/ml without H₂O₂ treatment, HepG2 was more sensitive to EACFM than Vero. The EACFM was more prominent in causing HepG2 cell death than silymarin at the same concentration. Using silymarin at 50 µg/ml, the number of living HepG2 was lower than Vero cells, while at the same concentration, there was an elevated viability of normal cells (Vero). On the other hand, HepG2 hepatocarcinoma cell death increased both through apoptosis and necrosis (Fig. 5B). Without H₂O₂ treatment, the EACFM was more pronounced in causing necrotic than apoptotic cell death of HepG2 cells, while silymarin was more likely due to apoptosis.

Silymarin consistently reduced ROS formation in the HepG2 cancerous type cell line even in the existent elevation of H₂O₂; however, it enhanced the HepG2 cell death. Those phenomena implicated silymarin in multiple actions. It might have a synergetic effect with H₂O₂ to modulate cytotoxicity to cancer cells, and it is not via ROS-dependent intoxication; otherwise, it happens through other pathways. Nakamura and Takada (2021) explained that compounds capable of suppressing intracellular ROS, such as deferasirox, would induce apoptosis of multiple myeloma cells.

Figure 4. The influence of EACFM and silymarin to H2O2-induced ROS production on Vero (A) and HepG2 (B) cell lines. The ROS formation represented in fold mean fluorescence compared to untreated cell line. Mean treatments of each cell line type followed by the same letters showed no significant difference (DMRT test; p = 0.05). [Click here to view]

Figure 5. The influence of EACFM and silymarin to cell death of Vero (A) and HepG2 cell lines due to H2O2 treatment. [Click here to view]

In cells treated with H₂O₂, cytoprotective action of EACFM and silymarin only occurred in Vero cells, while in HepG2 they had the opposite effect, namely, cell death escalating. The existence of H₂O₂ enhanced the action of silymarin causing HepG2 death, especially when at a concentration of 150 µg/ml while EACFM was more effective in causing HepG2 cell death at a concentration of 50 µg/ml.