Ginnalin A and SB203580 show additive effect on Hep-3B hepatocellular carcinoma cell line Ginnalin A ve SB203580 Hep-3B hepatosellüler karsinoma hücre hattında additif etki gösterir

Objective: Investigation of the anticarcinogenic effects of natural products with low toxicity is very important in the development of new therapeutic strategies against cancer. Ginnalin A (GA) is one of the most important phenolic compounds of Acer genus and its anticancer effect has been shown that in various cancer cell lines. SB203580, a p38 MAPK inhibitor, can inhibit cell proliferation independently of p38 MAPK. The objective of this study was to investigate combination effect of GA and SB203580 on Hep-3B cell line. Material and methods: Cell viability was determined by using XTT method after the treatment with GA, SB203580 and combination of both. Anticarcinogenic effects of GA and SB203580 both in single and in combination have been analyzed with Caspase-3 activity assay and expression levels of important genes involved in cell cycle and apoptosis were evaluated by qPCR. Results: GA and SB203580 have shown additive effect on Hep-3B cells in the combination inhibited 50% of cell viability. And, SB203580 increased the effect of GA on activation of Caspase-3 and expressions of genes important in apoptosis and cell cycle. Conclusion: This study indicates that GA and SB203580 can be an effective for development of new therapeutic strategies in hepatocellular carcinoma.


Introduction
Hepatocellular carcinoma (HCC), the third leading cause of cancer-related death worldwide, is the most common type of primary liver cancer with a rate of 80-90%. It is the fifth most frequent cancer type in men, while is ranked seventh in women [1,2]. However, the incidence of HCC varies in different geographical regions depending on the risk factors. The most important risk factors for HCC are chronic infection with Hepatitis B, C and D viruses, alcohol intake and aflatoxin exposure. Accordingly, the incidence of HCC is high in the East, Southeast and Central Asia and the sub-Saharan Africa where the prevalence of hepatitis infections is high. In a similar manner, HCC cases are frequently encountered in South China and sub-Saharan Africa where foods are contaminating with aflatoxin [3][4][5]. Also, non-alcoholic steatohepatitis, obesity, metabolic diseases such as diabetes and genetic susceptibility can be associated with development of HCC [6].
There are various treatment approaches for HCC: liver transplantation, surgical resection, percutaneous ablation, trans-arterial chemoembolization, radioembolization and chemotherapy [7,8]. Especially, liver transplantation, liver resection and percutaneous ablation are the most effective treatment options, but these are effective for HCC only in case of early stage [9]. In addition, because of the late diagnosis of HCC due to the slow development of the disease and lack of biological markers that can detect the onset of the disease, HCC patients are diagnosed at advanced stage [10,11]. Thereby, chemotherapeutic approaches are applied in treatment for advanced HCC [12]. Especially, sorafenib, a multiple kinase inhibitor, is used as an antiangiogenic agent in advanced HCC patients and can increase the survival time [13]. However, drug resistance is observed in patients who initially responded to sorafenib [14]. Therefore, novel therapeutic strategies are needed for the HCC.
One of the therapeutic strategies developed against cancer is to take advantage of potential anticancer activities of natural products. It has been reported that phenolic compounds, found in natural products such as plants, fruits and vegetables, have antibacterial, antiviral, antiatherosclerotic, anti-inflammatory and anticarcinogenic effects as well as antioxidant properties [15,16]. Phenolic compounds, secondary metabolites with one or more aromatic rings possessing one or more hydroxyl groups, are classified as phenolic acids and analogs, flavonoids, tannins, stilbenes, curcuminoids, coumarins, lignans, quinines and others [17]. Gallotannins are the hydrolyzable tannins and Chinese tannin, Turkish tannin, Tara tanin, Acer tanin and Hamamelis tanin are the important examples of gallotannins. Acer tannin extracted from Acer genus commonly known as maple [18,19]. Ginnalins are found in several members of the Acer genus and it has been shown that Ginnalin A (GA), one of the most abundant phenolic compound in Acer genus, has various pharmacological properties such as antioxidant, antibacterial, a-glucosidase enzyme inhibition [20,21]. In studies with human breast cancer and human colorectal cancer cell lines it has been shown that GA has anticarcinogenic effects on these cells and is the more effective than Ginnalin B and C [22,23]. One of the most important strategies in the treatment of cancer is the targeting of signaling pathways, particularly those associated with cancer development. For this purpose, various inhibitor molecules are used to determine the role of proteins involved in signaling pathways. SB203580, a pyridinyl imidazole, is used to elucidate the role of p38 mitogen-activated protein kinase (MAPK) in various biological processes [24]. p38 MAPK participates in regulation of apoptosis, growth inhibition, differentiation, cell cycle arrest, and acts as a tumor suppressor by virtue of these roles [25,26]. Although SB203580 is a p38 MAPK inhibitor, it has been reported that SB203580 suppresses cell proliferation independently of p38 MAPK. This antiproliferative activity of SB203580 has been described by inhibiting the phosphorylation of protein kinase B (PKB), a protooncogen [27]. In a study with hepatocellular carcinoma cells, it has been indicated that SB203580 induces autophagy which may be responsible for the antiproliferative effect of SB203580 [24].
There is no study investigating the anticarcinogenic effect of GA on HCC cells. Also, as a natural product, the effectiveness of GA in the combination strategies is unknown. On the other hand, little is known about the antiproliferative activity of SB203580 on HCC cells. Therapeutic agents developed against cancer affect not only the cancer cells but also non-malignant cells. The use of these agents with high doses also causes side effects. Therefore, combined application of them can reduce side effects, may also cause similar effects with lower doses depending on the synergistic or additive effect. In this study, we aimed to investigate the possible synergistic or additive effect of GA and SB203580 on Hep-3B human HCC cells. For this, primarily the combination activity of GA and SB203580 on the cell viability was assessed, then, expression levels of genes, important in apoptosis and cell cycle, were determined and Caspase-3 activity assay was performed to show anticarcinogenic effect.

Cytotoxicity assay
The XTT method was used to assess the cytotoxic effects of GA, SB203580 and combination of them in Hep-3B cells and each experiment was carried out in triplicate. The cells (5 × 10 3 cells/well) were seeded into 96-well plates and incubated for 24 h. After the incubation, the cells were treated with 0-500 μM GA and 0-100 μM SB203580 for 24, 48 and 72 h. Then, XTT solution was added to the each well and absorbance values of the working groups were read at 450 nm (reference wavelength 630 nM) in a microplate reader after the 4 h. The concentrations of GA and SB203580 inhibited 50% of cell viability (IC 50 ) was determined with CompuSyn Version 1.0 software and the concentrations of combination applied to the cells were determined considering the IC 50 values of GA and SB203580. Accordingly, the cells were treated with GA (0-300 μM) and SB203580 (0-60 μM) in a ratio of 5:1 for 72 h followed by XTT analysis.
The effect sizes of combination treatment of GA and SB203580 on the Hep-3B cells were determined by calculating the combination index (CI) with the CompuSyn Version 1.0 software by Ting-Chao Chou and Nick Martin. This program is based on the median-effect analysis according to the Chou-Talalay method and synergy, additivity, and antagonism are defined as CI < 1, CI = 1, and CI > 1, respectively [28,29]. According to the obtained results, GA and SB203580 were applied to the cells at IC 50 or in combination that shown additive effect.

Analysis of Caspase-3 activity
The activity of Caspase-3 was measured using the Caspase 3 Assay Kit (BioVision;Cat # K106-25) according to the manufacturer's instructions. Briefly, cytosolic extracts of cells, treated with GA, SB203580 and combination of them, were obtained with cell lysis buffer and the protein concentrations were measured using the Bradford reagent. Analysis was performed with 100 μg protein sample for each group. Caspase-3 activity was determined by detection of chromophore p-nitroaniline (p-NA) after cleavage from the labeled substrate DEVD-p-NA by active Caspase-3 in a microplate reader at a wavelength of 400 nm. Each experiment was carried out in triplicate.

RNA isolation and qPCR analysis
For the evaluation of gene expression levels, total RNAs were extracted from control and treatment groups using TRIzol Reagent and reverse transcription was performed using iScript™ cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instructions.

Gene
Primer sequence performed using EvaGreen qPCR Mastermix (Mastermix-S) and qPCR protocol was applied as denaturation at 95°C for 10 min, followed by 35 cycles consisting of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The primers suitable for the target genes were designed using IDT PrimerQuest (https://eu.idtdna. com/site) and primer sequences were shown in Table 1.
ACTB and CYPA were used in the normalization of the results as housekeeping genes.

Statistical analysis
Data of Caspase-3 activity were analyzed statistically using IBM SPSS 21.0 (SPSS Inc., Chicago, IL, USA) and differences between the groups were analyzed by one-way ANOVA test. The gene expressions analysis were performed by using the 2 −ΔΔCT method with RT 2 Profiles™ PCR Array Data Analysis. p-value <0.05 was considered to be statistically significant.

Effect of GA, SB203580 and combination of them on cell viability
The cytotoxic effect of GA, SB203580 and combination of them was determined using the XTT assay. GA and SB203580 inhibited the cell viability of Hep-3B cells in a concentration and time manner as shown in Figures 1  and 2. The IC 50 of GA and SB203580 were found to be 155 μM and 32.5 μM in of Hep-3B cells for 72 h, respectively. After the determitation of IC 50 values, GA and SB203580 applied to the cells in a ratio of 5:1 for 72 h. According to the results of CI, GA and SB203580 have shown additive effect on Hep-3B cells at concentrations of combination which inhibited 50% of cell viability (CI = 1). These concentrations were 86.22 μM and 17.24 μM for GA and SB203580, respectively. However, it has been found that the combination of GA and SB203580 exerts an antagonistic effect when the cell inhibition exceeds 50% (Figure 3). Considering these results, the cells were treated with GA and SB203580 at the concentrations which they showed additive effect on Hep-3B cells for 72 h as well as IC 50 of GA or SB203580 for subsequent experiments.

Effect of GA, SB203580 and combination of them on Caspase-3 activity
After the treatment with SB203580 and combined with GA, the Caspase-3 activity of cells increased to 7.8 and 8.2 folds, respectively, compared to the control group (p < 0.05). These increases were also found to be significant compared to the group which GA was treated with IC 50 (p < 0.05). On the other hand, there was no significant difference in Caspase-3 activity between the group in which the SB203580 was applied with IC 50 and the combination group (p > 0.05) (Figure 4).

Effects of GA, SB203580 and combination of them on expressions of genes associated with apoptosis and cell cycle
The effects of GA, SB203580 and combination of them on expressions of genes are important in apoptosis and cell cycle were determined using qPCR analysis, after total RNA isolation and cDNA synthesis from control and treatment groups cells were performed.
According to the qPCR results, after the treatment with IC 50 of GA, the expression of CASP3, CASP8, CASP9, CYCS and P53 genes significantly increased to 12.09, 10.14, 3.37, 16.15 and 4.15 folds, respectively, compared with the control group. On the other hand, the expression of CCND1 and CDK6 genes was significantly decreased to 4.16 and 6.78 folds. When the cells were treated with IC 50 of SB203580 the expression of CASP3, CASP7, CASP8 and BAX genes significantly increased to 15.7, 4, 12.14 and 4.2 folds, respectively, and the expression of CCND1 and CDK4 genes was significantly decreased to 6.75 and 3.15 folds, respectively. After the combined treatment, a significant increase was observed in the expression of CASP3, CASP9, BAX and CYCS genes as 18.38, 15.37, 14 and 13.82 folds, respectively. A significant decrease was observed in the expression of BCL2, CCND1, CDK4 and CDK6 genes as 2. 19, 8.96, 9.78 and 7.65 folds, respectively, compared with the

Discussion
Hepatocellular carcinoma (HCC), is a large proportion of the total liver cancer burden in the world, has an everincreasing incidence [30,31]. Despite the increasing incidence, limited efficacy of current treatment options requires the development of new treatment strategies [32]. Recent studies have shown that natural products can modulate different signaling mechanisms involved in the carcinogenesis process [33]. Accordingly, the investigation of anticarcinogenic activities of natural products in various cancer cells contributes to the development of new therapeutic strategies against cancer.
GA is a phenolic compound found in Acer sp., especially Acer ginnala. It has been reported that the extracts of Acer sp. have antiproliferative effect on colon cancer cells and arrest the cell cycle in S phase [22]. In another study, González-Sarrías reported that GA arrested the cell cycle in the S-and G2/M-phases and decreased cyclins A and D1 protein levels in breast and colon cancer cells [23]. SB203580, investigated for its combination effect with GA on HCC cells in present study, is a p38 MAPK inhibitor and it is frequently used to clarify the role of p38 MAPK. On the other hand, several studies have shown that SB203580 inhibits cell proliferation independent of p38 MAPK. Lali et al. reported that SB203580 can inhibit phosphorylation of retinoblastoma protein, a cell cycle regulatory molecule, in interleukin-2-stimulated T cells. Researchers have shown that SB203580 suppresses PI 3-kinase/protein kinase B (PKB) which is involved in the regulation of retinoblastoma phosphorylation [27]. It is known that retinoblastoma is a tumor suppressor and it inhibits the activity of E2F transcription factors in hypophosphorylated state, thus, cell cycle progression is suppressed. The phosphorylation of retinoblastoma via cyclin-dependent kinases leads to the release of E2F which causes the progression of the cell cycle [34].
In another study, it has reported that SB203580 induces autophagy via activating adenosine monophosphate-activated protein kinase (AMPK) and death-associated protein kinase (DAPK) in HCC cells. The researchers have suggested that it can be associated with antiproliferative effect of SB203580 [24].
In this study, the combination effect of GA, has anticarcinogenic effect in various cancer cell lines, and SB203580 which can suppress the proliferation of cancer cells has been investigated in the Hep-3B human HCC cell line. Firstly, the effect of GA and SB203580 on cell viability in HCC cells was analyzed and IC 50 values for 72 h were found to be 155 μM and 32.5 μM, respectively. Then, GA and SB203580 were treated to the cells in a ratio of 5:1 for 72 h.
The results of CI analysis showed that GA and SB203580 have additive effect with low concentrations (Fa ≥ 0.5) on HCC cells. For further analysis, GA and SB203580 were treated to the cells with IC 50 of them and in combination (86.22 μM GA and 17.24 μM SB203580) that shown additive effect with leading to 50% inhibition (CI = 1).
The one of the most important features of cancer cells is evasion from apoptosis. For this reason, the induction of apoptosis is the most important step for maintenance of the number of healthy cells and clearance of tumor [35]. It is known that the cell death pathways have an important place in therapeutic strategies. One of the best indicators in evaluating the efficacy of a new therapeutic agent is the induction of apoptosis in cancer cells. For this purpose, in this study, apoptotic effect of GA, SB203580 and combination of both was evaluated with Caspase-3 activity assay and gene expression analysis.
Apoptosis is induced through two major pathways; intrinsic and extrinsic. Activation of Caspase-3 which occurs through proteolytic cleavage of procaspase-3 is one of the most important steps for the induction of apoptosis in both pathways [36]. In this study, SB203580 and combined with GA increased the Caspase-3 activity in HCC cells. However, GA did not cause a significant increase in Caspase-3 activity. Although they were applied to the cells with lower concentrations in combination, it has been observed that a significant increase in Caspase-3 activity as 8.2 fold. It is thought that this result confirms the additive effect observed in cytotoxicity analysis. After the combined treatment, a significant increase was observed in the expression of CASP3, CASP9, BAX and CYCS genes and a significant decrease was observed in the expression of BCL2 gene. The release of cytochrome c into cytosol from mitochondria is mediated by the apoptotic protein Bax, while the antiapoptotic protein Bcl-2 blocks the release of cytochrome c in intrinsic pathway of apoptosis. In addition, activation of Caspase-9 and Caspase-3 occurs with release of cytochrome c [37]. Caspase-3, encoded by the CASP3 gene, is the most important effector proteinase in the caspase cascade because when it is activated, apoptosis process begins irreversibly [38]. According to the qPCR results, a significant increase was determined in expression of CYCS gene which encodes cytochrome c protein. It is thought that this result correlates with the reduction in the BCL2 gene and increase in the BAX gene. Also, when the increase in CASP3 and CASP9 genes expression is considered, it can be suggested that GA and SB induce intrinsic pathway of apoptosis. However, more comprehensive and advanced analyzes are needed for this. Furthermore, when GA and SB203580 were applied alone to HCC cells, expression of important genes, responsible for apoptosis induction, was also affected. However, it is observed that the combination of GA and SB203580 has a greater effect on the expression of genes important in apoptosis.
On the other hand, expressions of genes encoding key proteins of cell cycle were also analyzed in this study. A significant decrease was observed in the expression of CCND1, CDK4 and CDK6 genes in combination group. Furthermore, when GA and SB203580 were applied alone to HCC cells, expression of these genes was also affected. CCND1,2,3/CDK4,6 complex is responsible for the progression of the G1 phase in the cell cycle [39]. The reductions in expression of CCND1, CDK4 and CDK6 genes which are encode these members of the complex can considered an important data indicating that GA and SB203580 can block the cell cycle in the G1 phase.
The use of therapeutic agents with high doses causes side effects in cancer treatment. Therefore, synergistic or additive effects of possible chemotherapeutic agents in combination treatments are very important because they are effective together with lower doses. In this study, it has been shown that GA and SB203580 have an additive activity in HCC cells. Our findings indicated that GA and SB203580 can be an effective for development of new therapeutic strategies in HCC.