Background
Hepatocellular carcinoma (HCC) is identified as the fifth most common cause of cancer related mortality in worldwide and the prognosis for HCC remains very poor [
1‐
6]. Sorafenib, a chemotherapeutic agent, is an approved drug available for liver cancer treatment. The reduced sensitivity of cancer cells and drug resistance are becoming more common, because sorafenib is used continuously. However, the efficacy of targeted therapies is limited due to drug resistance and acute cytotoxicity [
7,
8]. Therefore, novel therapeutic strategies for HCC to address drug resistance and toxicity are urgently needed.
The development of drug resistance restricts the efficacy of single therapeutic agents [
9]. Thus, new approaches that inhibit tumour growth have major clinical significance. Combinations of different therapeutic agents to inhibit several pathways could be a more effective strategy for suppressing cancer and could be more effective for treating cancer patients than single therapeutic agents. The natural iso–flavonoid (biochanin A) is found in red clover, chick peas, and several other plant sourcesis catego-rized as a phytoestrogen and has been demonstrated to exhibit various pharmacological properties [
10,
11]. Biochanin A is known to exhibit an anticancer effect against various cancer types. Previous studies have shown that biochanin A inhibits endothelial cell functions such as cell viability, migration, and invasion through inhibition of the ERK/AKT/mTOR signalling pathway [
10]. Recent studies have demonstrated that biochanin A treatment reduced cancer progression by inhibiting cancer cell proliferation, cellular signalling, invasion, and antioxidant systems [
12‐
14] in colon cancer [
15], prostate cancer [
16], hepatoma [
17] and human pharynx squamous carcinoma cells [
18]. The possibility of MAPK pathway inhibition would have therapeutic benefits in patients with oncological diseases who have continous activation of this signalling pathway [
19,
20]. RAF inhibitors generally exhibit greater response rates in clinical trials than MEK inhibitors which may be related to ERK activity suppression. SB590885 is a novel triarylimidazole that selectively inhibits RAF kinases with more potency towards the BRAF active conformation than the inactive conformation [
19,
21]. Previous studies have shown that the combination of SB590885 and the AKT inhibitor ZSTK474 impacted the proliferation of papillary thyroid cancer cell lines by inhibiting the ERK MAPK and PI3K/AKT signalling pathways [
19,
22]. However, whether the combination of biochanin A and SB590885 inhibits hepatocellular carcinoma (HCC) growth remains a blank box.
Therefore, to develop a therapeutic strategy that could provide an improvement in the treatment of advanced HCC without increased toxicity, we investigated the effects of combina-tions of biochanin A with the BRAF inhibitor SB590885 on anti-proliferative and survival pathways in HCC cells in vitro and in vivo.
Materials and methods
Cell culture
The HCC cell lines Bel-7402 and SK-Hep-1 were obtained from Shanghai Genechem Co. (Shanghai, China). The STR genotyping reports of the HCC cell lines Bel‑7402 and SK-Hep-1 are listed in Additional file
1: Tables S3 and S4. The cell lines were cultured in RPMI-1640 medium supplemented with 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) and 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and were maintained at 37 °C in a humidified incubator containing 5% CO
2.
Cell viability assay
Biochanin A and SB590885 were purchased from Targetmol (Shanghai, China). Cell viability was tested using the CCK‑8 assay according to the manufacturer’s instructions. Cells (7 × 10
3) were seeded into a 96‑well plate and cultured in regular growth medium containing 10% foetal bovine serum. After 24 h, the cells were exposed to serial dilutions of biochanin A (12.5 μM, 25 μM, 50 μM, 75 μM, and 100 μM), SB590885 (3 μM, 6 μM, 9 μM, 12 μM, and 15 μM). After the cells were incubated at 37 °C for 48 h, the medium was removed and 100 μl of RPMI‑1640 and 10 μl of CCK‑8 reagent were added. The cells were incubated for 2 h at 37 °C. Finally, the absorbance of each well was measured at 450 nm using SpectraMaxi3X (Molecular Devices, Silicon Valley, CA, USA). All cell viability assays were performed at least three times. The combination index (CI), which was calculated by the Chou–Talalay equation, indicates synergistic effects at CI < 1, additive effects at CI = 1, and antagonism at CI > 1 [
23].
The HCC cell lines Bel‑7402 and SK-Hep-1 were incubated in a six-well plate with 75 μM biochanin A and 12 μM SB590885 for 10 days as previously described [
22,
24]. The colonies were stained with crystal violet. The visible colonies were photographed by digital single lens reflex (Nikon D5600) and counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Cell cycle and apoptosis analysis
The HCC cell lines Bel‑7402 and SK-Hep-1 were incubated in complete cell culture medium with 75 μM biochanin A and 12 μM SB590885 for 48 h. The cells were collected in cold PBS and then incubated with150 μl of RNase A (10 μg/ml) for 30 min at 37 °C in the dark, stained with 400 μl propidium iodide (PI) (50 μg/ml) and placed at 4 °C in the dark for 30 min [
24,
25]. The stained cells were analysed using a flow cytometer (BD Bioscience, CA, US) [
25]. For apoptosis analyses, high-affinity Annexin-V (AV) and PI (BD Biosciences, CA, USA) were used as previously described [
26].
Western blot analysis
SK-Hep1 and Bel-7402 cells were plated into 6-well plates for 24 h, and then treated with 75 µM biochaninA,12 µM SB590885 or the combination for 48 h. Total protein from the cells was extracted using RIPA buffer with a proteinase inhibitor (Beyotime, China). Protein samples were subjected to SDS–PAGE and then transferred to PVDF membranes (Millipore, US). The membranes were blocked with 5% BSA for 1 h, and incubated with primary antibodies such as Bcl-2 (1:1000), Bax (1:1000), cleaved PARP (1:1000), cleaved caspase-9 (1:1000), P21 (1:1000), P27 (1:1000), CyclinD1 (1:1000), p-MEK (1:1000), MEK (1:1000), p-ERK (1:1000), ERK (1:1000), AKT (1:1000), p-AKT (1:1000), P70S6K (1:1000), p-P70S6K (1:1000), S6 (1:1000), p-S6 (1:1000), β-actin (1:1000) (Cell Signaling Technology, Inc. (Danvers, MA, USA)) and GAPDH (1:1000)(Qianchen Biotech, Shanghai) at 4 °C overnight. The membranes were washed three times and incubated with the secondary anti-rabbit or anti-mouse antibody for 1.5 h at room temperature. Protein bands were visualized by Bio-Rad ChemiDoc Imaging System.
Xenograft model analysis and groups of treatment
The HCC cell xenograft model was established as previously described [
27]. Briefly, 5 × 10
6 Bel-7402 cells were injected subcutaneously into the right flank near the hind leg of each nude mouse until the tumor volume was ~ 100 mm
3. Biochanin A was dissolved in dimethyl sulfoxide (DMSO) at stock concentration of 100 mM, and stored in the dark at − 20 °C. SB590885 was dissolved in a 10 mM stock solution in DMSO and stored in the dark at − 20 °C. Then, we treated male athymic nude mice bearing palpable Bel-7402 xenografts tumours (~ 100 mm
3) with control (vehicle-treated mice) 50 μl 0.5% dimethyl sulfoxide (DMSO), biochanin A (25 mg/kg/day), SB590885 (7.5 mg/kg/day) and the combination by intraperitoneal injection for 5 weeks, 7 times/week. The tumour volume was measured every week and was calculated by the following formula: volume = 1/2 (length × width
2). After 5 weeks, the mice were euthanized and the tumours were isolated.
Immunohistochemistry
Tumor xenografts were formalin-fixed, paraffin-embedded, and sectioned in preadherent slides. The slides were subjected to the indicated primary antibody against PCNA (1:200) (Cell Signalling Technology, Inc. (Danvers, MA, USA)) overnight. The sections were incubated with secondary antibody and then developed with 3,3′-diaminobenzidine chromogen according to the protocol. All samples were visualized, and images were captured using a DM2500 fluorescence microscope (Danaher, Wetzlar, Germany). Image‑Pro Plus 4.5 software was used to analyse the staining data.
Liver and kidney toxicity analysis
Serum and plasma were tested for liver and kidney safety markers [
28] (ALP, ALT, AST, total bilirubin, creatinine, and urea nitrogen; Additional file
1: Table S2) by using a SpectraMaxi3X(Molecular Devices, Silicon Valley, CA, USA).
Statistical analysis
Statistical analyses were performed using SPSS 17.0 software (IBM, Armonk,New York, USA). Graphs were generated with GraphPad Prism 7.0 software (GraphPad, San Diego, CA, USA). All experiments were performed three independent times. Statistical significance was determined by Student’s t-test, and a two-sided P-value< 0.05 was considered statistically significant.
Discussion
The heterogeneity of HCC is characterized by both heritability [
29] and phenotype/morphology [
30]. Tumours are characterized by complex patterns such as location and timing. This complex and multivariable tumour network constantly responds to and affects the liver environment,which is the main reason for the limited success of different targeted single-therapy trials for HCC [
31]. Therefore, combining multiple anticancer drugs seems to be a reasonable way to prevent tumour resistance. In this study, we provide evidence that combined treatments play a crucial role in the chemoprevention of HCC.
Chemotherapy is an important treatment option for various clinical factors. Among the chemotherapeutic agents, sorafenib is administered and is significant in the treatment of HCC. However, sorafenib only improves median overall survival by ~ 3 months [
32‐
34]. Single–agent treatments are usually partial and relatively transient, and toxicity to normal tissues is one of the major obstacles to successful cancer chemotherapy [
7,
35]. Several studies have shown that natural products play an important role in the inhibition and treatment of cancers [
36,
37]. Thus, there has been increasing focus on the application of combined treatments using natural products for HCC. For example, a previous study showed that biochanin A combined with ginsenoside Rh2 exhibited synergistic effects against MDA-MB-231 and MCF-7 cell proliferation [
37]. Biochanin A, an isoflavone, has been shown to exert anticancer effects against various cancers. SB590885 is a serine/threonine-protein kinase B-Raf (BRAF) inhibitor. Our results indicated that the combination of biochanin A and (the BRAF) inhibitor SB590885 significantly inhibited the proliferation and viability of HCC in vitro and in vivo. Apoptosis is a tightly regulated signalling process that involves the coordination of anti-apoptotic and pro-apoptotic proteins [
38]. In this study, our results demonstrated that biochanin A combined with SB590885 induced apoptosis in HCC cells more markedly than either biochanin A or SB590885 alone. Intriguingly, a recent study showed that biochanin A induced S phase arrest in lung cancer cells [
39]. However, we found that the combination of biochanin A and SB590885 induced apoptosis, along with G0/G1 phase cell cycle arrest in HCC cells. These results revealed that drugs acting on different cells might cause cell cycle arrest at different phases.
Several studies have suggested that HCC cell activation by different factors is known to increase both Ras/Raf/ERK MAPK and PI3K/AKT/mTOR signalling [
1,
40]. Sorafenib, the drug available for the treatment of patients with advanced HCC, inhibits the Ras/Raf/MAPK pathway [
32], but does not directly inhibit the PI3K/AKT/mTOR pathway, which also plays an important role in HCC proliferation. The PI3K pathway is known to be activated in 30% to 50% of HCC cases [
41]. Somatic mutation of PIK3CA, enhancement of Akt expression and phosphorylation ribosomal protein S6, and a decrease in PTEN expression have been reported in HCC [
42‐
45]. These studies suggest that combined targeting of the PI3K/AKT/mTOR and Ras/Raf/MAPK pathways might provide benefits in the treatment of HCC. Previous studies have shown that biochanin A inhibited endothelial cell functions such as cell viability, migration, and invasion through inhibition of the ERK/AKT/mTOR signalling pathway [
10]. SB590885 is a serine/threonine-protein kinase B-Raf (BRAF)inhibitor. Previous studies have shown that the combination of SB590885 and the AKT inhibitor ZSTK474 impacted the proliferation of papillary thyroid cancer cell lines via inhibition of the ERK MAPK and PI3K/AKT signalling pathways [
19,
22]. Our results indicated that the combination of biochanin A and SB590885 suppressed the growth of hepatocellular carcinoma cells via inhibition of the ERK MAPK and PI3K/AKT/mTOR signalling pathways. Furthermore, a hepatorenal toxicity test showed that the combination biochanin A and SB590885 did not induce any significant hepatorenal toxicity. The safety and efficacy of this combination strategy provides the possibility of improving of therapeutic outcomes for advanced HCC patients.
Conclusion
In conclusion, our results show that combination of the natural product Biochanin A with the BRAF inhibitor SB590885 synergistically suppressed proliferation, and promoted cell cycle arrest and apoptosis in HCC cells. The combination of biochanin A and SB590885 inhibited the HCC cells proliferation through ERK MAPK and PI3K/AKT pathways. We found that there was no significant hepatorenal toxicity with the drug combination, as indicated by the hepatorenal toxicity test. These results indicate that combination Biochanin A and SB590885 is a potential treatment for HCC.
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