Combination treatment with sorafenib and wh‑4 additively suppresses the proliferation of liver cancer cells
- Authors:
- Published online on: January 20, 2022 https://doi.org/10.3892/etm.2022.11156
- Article Number: 232
-
Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Hepatocellular carcinoma (HCC) is the leading cause of cancer-associated deaths and is extremely resistant to chemotherapy (1). Due to poor prognosis and lack of effective drugs, the overall survival rate of patients with HCC is below 30% (2,3). Most patients go undiagnosed until the disease has progressed to an advanced stage (4). Molecular therapy targeted against specific molecules in tumor cells or their niche is currently the standard treatment for patients with advanced liver cancer (5). Sorafenib, a multi-kinase inhibitor, is a Food and Drug Administration (FDA)-approved chemical drug for treating patients with HCC (6-8). However, when treated with sorafenib, the average overall survival of patients is only extended by 2.8 months compared with that of untreated patients. Patients treated with sorafenib either suffer from severe side-effects or show disease progression after the initial response (4,9). Previous studies have shown that sorafenib is no longer effective in patients after months of treatment, which suggests that the shortcomings of sorafenib are associated with the development of drug resistance (10-12). ATP-binding cassette (ABC) transporters are involved in tumor cell multidrug resistance (MDR), such as ABCB1 and ABCG2. ABC proteins can transport a wide variety of anticancer drugs, including inhibitors of tyrosine kinases (13). In HCC cells, ABC proteins are upregulated, which is associated with the activation of survival pathways (14,15). ABCB1 has been associated with decreased median survival time in patients with HCC and ABCG2 contributes towards the MDR phenotype in HCC (16,17).
Wh-4, a heat shock protein 90 (Hsp90) inhibitor, was synthesized in the laboratory and was derived from the existing inhibitor, SNX-2112 (18-23). Hsp90 is a member of a highly conserved family of molecular chaperones present in all eukaryotes (24). Although Hsp90 accounts for only 1-2% of total cellular protein content, it is responsible for regulating several activities, including client proteins activity, stability, conformation and function (25,26). Hsp90 facilitates metastasis, rapid cell division, resistance and evasion of apoptosis in cancer cells (27). Many kinases, including PI3K, ERK, vascular endothelial growth factor receptor (VEGFR) and insulin-like growth factor receptor, are Hsp90 client proteins (26). These functionally important kinases depend on Hsp90 to achieve an active conformation or to gain increased stability (25). Cancer cells are dependent on Hsp90, and thus Hsp90 has been successfully used as a target in tumor therapeutics in many clinical trials for Hsp90 inhibitors in multiple tumor types (28). For example, the benzoquinone ansamycin Hsp90 inhibitors, including geldanamycin and its derivative 17-AAG (26,29,30), induced cancer cell apoptosis and disrupted the transcriptional function of HIF1α. Moreover, 17-AAG decreased the colony-formation capacity of lymphoma stem cells (31). In addition, SNX-2112, a novel Hsp90 inhibitor, decreased the cell viability and tumorigenicity of multiple myeloma cells (32).
Although sorafenib, a tyrosine kinase inhibitor, remarkably suppresses the Raf/Ras/MEK/ERK signaling pathway and inhibits receptor tyrosine kinases, including VEGFR, platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor, HCC cells are resistant to sorafenib and its side effects are also severe (33-35). Hsp90 is a molecular chaperone that stabilizes the folding and conformation of proteins in cancer cells. Hsp90 client proteins play an important part in cancer cell proliferation, resistance, and other important cellular processes. According to previous studies, sorafenib interferes with the unfolded protein response (36,37). For example, sorafenib interacts with Hsp90/Hsp70 inhibitors to disrupt the folding of nascent proteins (38,39), which suggests that the combination of sorafenib with wh-4 can effectively inhibit cancer cell proliferation.
Here, we investigated the effect of sorafenib in combination with wh-4 on liver cancer cells. In addition, we examined the anti-tumor efficacy of a novel Hsp90 inhibitor, wh-4, in liver cancer cells.
Materials and methods
Cell lines
Liver cancer cell lines SK-HEP-1, which are liver sinusoidal endothelial cells of tumorigenic origin (cat. no. FH0072), Huh7 (cat. no. FH0873) and HepG2 (cat. no. FH0076) were purchased from the FUHENG Biotechnology in Shanghai. The SK-HEP-1 and HepG2 cell lines were authenticated using short-tandem repeat profiling (FUHENG Biotech). The cells were seeded in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 1% penicillin-streptomycin and incubated in a humidified atmosphere of 5% CO2 at 37˚C. Sorafenib (cat. no. Y0002098), Tris, glycine, SDS and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Merck KGaA). Wh-4 (purity >98.0%) was a kind donation from Professor Huang (Serenex, Inc.; Durham, USA). Wh-4 is a benzamides derivative and was designed to inhibit proteins with purine binding sites, which yielded a novel benzamide hit for Hsp90. Synthetic and modeling analyses of this chemical scaffold prompted effort to combine the benzamide with a 1,2,3,9-tetrahydro-4H-carbazol-4-one moiety. The 1,2,3,9-tetrahydro-4H-carbazol-4-one ring system was established by means of combining 1,3-cyclohexanedione and phenyl hydrazine via the Fischer indole synthesis in a Personal Chemistry microwave apparatus. Use of dimedone or the mono-methyl reagent instead of 1,3-cyclohexanedione yielded the related analogs. The purified tetrahydro-4H-carbazol-4-one was then reacted with the desired 4-fluorobenzonitrile in the presence of sodium hydride (40). Wh-4 was dissolved in DMSO, and a 10-mM stock solution in DMSO was prepared. For further use, the stock was diluted in cell culture medium.
MTT assay
The effect of sorafenib and wh-4 on cell proliferation was determined by the MTT (cat. no. KGT525500; Nanjing KeyGen Biotech Co., Ltd.) uptake method. Approximately 3x103 cells were seeded in each well of a 96-well plate and incubated for 12 h. On the next day, the cells were exposed to the following treatments: Various concentrations of wh-4 only, 10 µM sorafenib only, or a combination of both drugs. The treatment was carried out at 37˚C for 48 h. Finally, MTT (5 mg/ml; cat. no. 96992; Sigma-Aldrich; Merck KGaA) was added to each well and incubated at 37˚C for 4 h. The absorbance was measured using a Shimadzu reader (Thermo Fisher Scientific, Inc.) at 570 nm.
Western blotting
Cells were lysed in ice-cold 1% SDS buffer and centrifuged at 8,000 x g at 4˚C for 10 min. The protein concentration was determined using the BCA method (Beyotime Institute of Biotechnology, Inc.). Then, 20 µg protein was separated by 12% SDS-PAGE and transferred to 0.20-µm polyvinylidene fluoride membranes (cat. no. ISEQ00010; EMD Millipore). The polyvinylidene fluoride membranes were blocked with 5.0% milk in 0.1% TBST (0.1% Tween-20 in Tris-base buffer, pH 7.0) at room temperature for 1.5 h. Then, the membrane was incubated with primary antibodies at 4˚C for 16 h. The primary antibodies used in this study (all purchased from Cell Signaling Technology, Inc.) included anti-Bcl2 (1:1,000; cat. no. 15071), anti-Bax (1:1,000; cat. no. 5023), STAT3 (1:1,000; cat. no. 12640), phosphorylated (p)STAT3Y705 (1:1,000; cat. no. 9145), caspase-3 (1:1,000; cat. no. 9668), caspase-9 (1:1,000; cat. no. 9508), ABCB1 (1:1,000; cat. no. 13342), ABCG2 (1:1,000; cat. no. 42078) and GAPDH (1:1,000; cat. no. 5174;). The membrane was washed with 0.1% TBST buffer three times and subsequently incubated with horseradish peroxidase-conjugated secondary antibody (1:8,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at 37˚C before being treated with the chemiluminescence reagent (EMD Millipore) and exposed to Kodak film.
Reverse transcription-quantitative (RT-q)PCR analysis
Total RNA (tRNA) was extracted using the TRIzol Reagent kit (cat. no. DP424; Tiangen Biotech Co., Ltd.) and treated with RNAse-free DNAase and diethyl pyrocarbonate (Nanjing KeyGen Biotech Co., Ltd.). The extracted tRNA was reverse-transcribed into cDNA using PrimerScript Master mix (Bio-Rad Biotechnology, Inc.) according to the manufacturer's instructions. qPCR was used to evaluate the expression level of genes in the RT-PCR system (CFX96 Real-Time System; Bio-Rad Laboratories, Inc.) using primers (Shanghai GeneChem Co., Ltd.). Primer sequences were as follows: ABCB1, forward, 5'-AGGTGGCGTGGAAGGTCCGGTCC-3', and reverse, 5'-GGTGAGGCCGTGGTAATCGGTGA-3'; ABCG2, forward, 5'-GGTCGGACCTGGTAGGTAATG-3', and reverse, 5'-AATGTTGACCGGTGGCAAGTTA-3'; GAPDH, forward, 5'-AGCCACATCGCTCAGACAC-3, and reverse, 5'-GCCCAATACGACCAAATCC-3. The following PCR conditions were used on the Light Cycler: 95˚C for 5 sec, 60˚C for 5 sec, followed by 40 cycles of 94˚C for 20 sec and 60˚C for 1 min in a 25-µl reaction volume. Relative expression levels were analyzed by the 2-ΔΔCq method with GAPDH as the reference gene (41). All experiments were performed three times.
Colony-formation assay
Approximately 5x103 cells were seeded in each well of a 6-well dish. On the next day, the cells were treated with different concentrations of wh-4 only, sorafenib only, or a combination of both drugs (5 µM sorafenib, 5 µM wh-4) and incubated at 4˚C for another 48 h. The plates were subsequently incubated at 37˚C in a humidified incubator for 21 days. Culture media was replenished every 3 days. The colonies of more than 40 cells was visualized as positive and stained with 0.5% crystal violet for 30 min at 37˚C (cat. no. KGA229; Nanjing KeyGen Biotech Co., Ltd.). The numbers of positive colonies were counted under a light microscope (Nikon Corporation) and calculated. The number of more than 40 cells was divided by 5,000, in order to obtain the colon-forming efficiencies. The experiments were repeated three times.
Scratch wound healing assay
The combined effects of sorafenib and wh-4 treatment on cell migration were examined using a scratch wound healing assay. Cells were counted, and 2x105 cells were seeded into 60-mm cell culture plates (Corning, Inc.) in DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.). Upon reaching 80% confluence, the bottom of the plates was scratched gently and slowly with a sterile pipette tip, and the gap created in the attached monolayer of cells was photographed (Nikon Corporation). Then, the cells were cultured in in DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.). After 48 h, the migration distance of the cells was captured under a light microscope (Nikon Corporation) and calculated by subtracting the gap distance recorded at 0 h from the current gap distance. Data were collected from three independent experiments.
Cell apoptosis analysis
The cells were exposed to the following treatments: Various concentrations of wh-4 only, sorafenib only, or a combination of both drugs. The cells were incubated with the drugs at 37˚C for 48 h. Subsequently, the cells were harvested and washed three times with phosphate buffer, followed by the addition of 0.5 ml binding reagent and 5 µl Annexin V-FITC (cat. no. KGAV116; Nanjing KeyGen Biotech Co., Ltd.). After 30 min, the cells were stained with 5 µl 7-AAD (cat. no. KGAV116; Nanjing KeyGen Biotech Co., Ltd.) for 15 min at room temperature according to the manufacturer's instructions. Apoptosis in the cells was examined using flow cytometry (BD FACSCalibur). All data were analyzed using the FlowJo 10 software (FlowJo, Becton, Dickinson & Company).
Cell Ki-67 analysis
A total of 50,000 cells were seeded in 6-cm plates (Corning, Inc.). They were exposed to the following treatments at 37˚C: wh-4 only, sorafenib only, or a combination treatment of both drugs. After 48 h, the cells were collected, washed, and incubated with Alexa Fluor 488-conjugated Ki-67 antibody (1:100; cat. no. ab197234; Abcam) at room temperature for 1.5 h. Images were captured (Nikon Corporation). Image-Pro Plus was used to calculate the fluorescence intensity of Ki-67 cells (Media Cybernetics).
Plasmid construct, siRNA sequence and transient transfection
STAT3 mRNA was extracted from HepG2 cells and cloned into the pcDNA3.1 vector (Shanghai GenePharma Co., Ltd.). STAT3 DNA sequencing was performed by Sangon Biotech Co., Ltd. The vectors were purified using a plasmid filter maxiprep kit (cat. no. K210027; Thermo Fisher Scientific Inc.). The STAT3 recombinant plasmid (pcDNA3.1-STAT3) was transfected using the 5 µl Lipofectamine® 3000 reagent (cat. no. L300-015; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. pcDNA3.1-STAT3 recombinant was transfected using a serum-free medium, and after 4 h, the medium was replaced with normal medium. Synthetic small interfering RNA (si)-GFP, si-ABCB1 and ABCG2 had the following sequences: GFP sense, 5'-GCAUCAAGGUGAACUUCAA-3'; GFP antisense, 5'-UUGAAGUUCACCUUGAUGC-3'; ABCB1 sense, 5'-GCGGUUAACCAUCGAGUUA-3'; ABCB1 antisense, 5'-UAACUCGAUGGUUAACCGC-3'; ABCG2 sense, 5'-GCAAUCAGACCUGGAACAAUU-3'; ABCG2 antisense, 5'-AAUUGUUCCAGGUCUGAUUGC-3'. Then, 100 pmol siRNA were transfected using the 5 µl Lipofectamine® 3000 reagent (cat. no. L300-015; Thermo Fisher Scientific, Inc.) in 5% CO2 at 37˚C according to the manufacturer's instructions. Also, 8 µg/ml polybrene (cat. no. G04001; Shanghai GenePharma Co., Ltd.) was used to improve the transfection efficiency. After 4 h the medium were replaced with normal medium. 72 h later, the cells were harvested.
Statistical analysis
Statistical analysis was performed using SPSS 19.0 software (SPSS, Inc.). All data are presented as the mean ± SD for at least three independent experiments. Student's t-test was used for two-group comparisons, whilst comparisons among multiple groups were performed using a one-way ANOVA followed by Tukey's test. Results with P<0.05 were considered to indicate a statistically significant difference.
Results
Combination of sorafenib with wh-4 demonstrates an inhibitory effect on the proliferation of liver cancer cells
The chemical structure of wh-4 is shown in Fig. 1A. The MTT assay was used to evaluate the inhibitory effect of the drugs on the proliferation of liver cancer cells. Liver cancer cells were treated with various concentrations of sorafenib or wh-4 for 24 h. It was found that the IC50 values for wh-4 and sorafenib at 24 h were 4.90 and 4.62 µM, respectively, in SK-HEP-1 (Fig. 1B). In addition, the IC50 values for wh-4 and sorafenib at 24 h in Huh7 cells were 4.32 and 5.35 µM, respectively (Fig. 1C). Furthermore, a colony-formation assay was performed to evaluate the anti-tumor effect of the drugs, and a similar outcome was observed (Fig. 2A and B). The colony-formation experiments showed that the combination of 5 µM sorafenib and 5 µM wh-4 significantly inhibited colony formation of liver cancer cells. The combination treatment decreased the efficiency of the colony formation more significantly than with sorafenib or wh-4 alone.
Combination of sorafenib with wh-4 induces apoptosis in liver cancer cells
Flow cytometry results demonstrated that the percentage of Huh7 cells undergoing apoptosis was (54.4±5.64)% when treated with 5 µM wh-4 and (58.7±6.51)% when treated with 5 µM sorafenib (Fig. 3A). The fraction of apoptotic cells after the combination treatment of sorafenib and wh-4 was (66.6±6.22)%, which was higher than that after single-drug therapy (Fig. 3A). Furthermore the effect of combination treatment with the two drugs were examined in SK-HEP-1 cells. The percentage of SK-HEP-1 cells undergoing apoptosis after the combination treatment was (63.5±5.85)%, which was higher than that after single-drug therapy with sorafenib or wh-4. The fraction of apoptotic SK-HEP-1 cells after treatment with sorafenib or wh-4 alone was (54.0±6.34)% and (34.5±4.89)%, respectively (Fig. 3B). The aforementioned results demonstrated that the Bax levels in Huh7 and SK-HEP-1 cells notably increased when subjected to combination treatment compared to those with either drug alone (Fig. 3C and D). The levels of Bcl2 in Huh7 and SK-HEP-1 cells were significantly decreased after combination treatment with the two drugs (Fig. 3C and D). In addition, the caspase-3 and caspase-9 levels were not significantly different (Fig. S1). Collectively, the aforementioned results suggested that combination treatment with sorafenib and wh-4 increased apoptosis in liver cancer cells.
Sorafenib with wh-4 suppresses liver cancer cell proliferation and migration
The scratch wound healing assay demonstrated that both sorafenib and wh-4 inhibited the migration of liver cancer cells. The data suggested that the combination treatment with sorafenib and wh-4 significantly inhibited Huh7 migration (Fig. 4A). A similar additive effect of sorafenib and wh-4 was also observed in SK-HEP-1 cells (Fig. 4B). In addition, the Ki-67 assay demonstrated that sorafenib and wh-4 combination treatment remarkably decreased the proliferation of Huh7 and SK-HEP-1 cells (Fig. 4C and D). The fluorescence intensity level in sorafenib with wh-4 combination treatment was notably decreased (Fig. 4C and D). The aforementioned observations indicated that sorafenib with wh-4 suppressed liver cancer cell proliferation.
Combination treatment with sorafenib and wh-4 decreases the levels of ABC transporter genes
ABCB1 and ABCG2 play an important part in liver cancer cells proliferation (42,43). The si-ABCB1 and ABCG2 silencing efficiency was evaluated (Fig. S2). Knockdown of ABCB1 and ABCG2 inhibited the proliferation of liver cancer cells (Fig. 5A and B). However, the mechanism by which sorafenib leads to the development of resistance remains unclear. After examining the effects of the drugs on cell proliferation, the expression of ABC transporter genes that are responsible for drug resistance were further investigated. The results demonstrated that the combination treatment with sorafenib and wh-4 significantly decreased the expression levels of ABCB1 and ABCG2 in Huh7 cells (Fig. 5C and D). Next, the levels of ABCB1 and ABCG2 were examined in SK-HEP-1 cells. The levels of ABCB1 and ABCG2 were also decreased after combined treatment with sorafenib and wh-4 (Fig. 5E and F). However, the changes in the expression level of P-glycoprotein (P-gp)-encoded and breast cancer resistance protein-encoded ABCG2 were not observed after the liver cancer cells were treated with the chemicals (Fig. S3). The aforementioned results demonstrated that the combination treatment decreased the resistance level in treated cells.
Sorafenib and wh-4 additively inhibit liver cancer cell proliferation by suppressing the STAT3 signaling pathway
The potential molecular mechanism of the additive inhibition of liver cancer cell proliferation by combination treatment with sorafenib and wh-4 was further explored. Individual treatment with sorafenib and wh-4 decreased the phosphorylation level of p-STAT3Y705 in both Huh7 and SK-HEP-1 cells (Fig. 6A and B). To investigate whether STAT3 mediates the proliferation of cells treated with a combination of sorafenib and wh-4, a STAT3 overexpression vector was constructed. The pcDNA3.1-STAT3 vector efficiency was analyzed (Fig. S4). As shown in the soft agar assay in Fig. 6C, STAT3 overexpression remarkably reversed the apoptosis induced by combination treatment with sorafenib and wh-4. Similar effects were observed in SK-HEP-1 cells (Fig. 6D), indicating that this reversal of apoptosis was not a cell-line-specific effect. The aforementioned results suggested that sorafenib with wh-4 may suppress the proliferation of liver cancer cells by the STAT3 pathway.
Discussion
The present study reported that treatment with sorafenib suppressed the proliferation of live cancer cells. This inhibitory effect of sorafenib was significantly enhanced in combination with the Hsp90 inhibitor wh-4, suggesting an additive mechanism of action of these drugs on the inhibition of liver cancer cell proliferation.
Patients with HCC are diagnosed at intermediate or advanced stages when therapies are no longer effective (44). Sorafenib is the first anti-tumor drug approved by the FDA for treating patients with HCC (45). Clinical trials demonstrated that sorafenib prolonged the median overall survival time of patients by about 3-5 months (9,46,47). However, the side effects of this treatment, including anorexia, diarrhea, vomiting and squamous cell carcinoma are apparent (48). In addition, the drug is not effective for all patients with liver cancer, and some patients develop resistance (9). The ABC transporters play a key role in liver cancer cells proliferation (49). In the present study, it was found that the expression of ABC transporter genes was decreased in Huh7 and SK-HEP-1 cells (Fig. 5), suggesting that the chemoresistance was partially limited. In addition, knockdown of ABCB1 and ABCG2 inhibited the proliferation of liver cancer cells in the present study. Combination treatment with sorafenib and wh-4 additively decreased the resistance in liver cancer cells. In Fig. 5A and B, si-GFP was used as a control and the Fig. S2 results demonstrated that the si-ABCB1 partly decreased the expression level of ABCB1. Also, si-ABCG2 demonstrated the same silencing effect.
The development of drug resistance is a major challenge in the treatment of patients with HCC. Thus, a more rational treatment plan should focus on combining two or more therapeutic methods. Wh-4 is a derivative of SNX-2112, and SNX-2112 inhibits target proteins, such as Akt, p38, MAPK and Erk that play a crucial role in regulating cell survival, proliferation, resistance and homeostasis (25). In addition, the anticancer activity of sorafenib is attributed to its multi-kinase inhibitory function on several signaling pathways, such as Raf-1, B-Raf, and the receptor tyrosine kinase activity of VEGFRs and PDGFR-β (50). Induction of apoptosis in HCC cells suggests that sorafenib might promote apoptosis in other cancer cells such as prostate, breast and colorectal cancer cells (51-53). According to previous studies, the anti-tumor activity of sorafenib on cancer cell proliferation and viability may be useful in combination with other therapies or signaling transduction pathway inhibitors (38). Therefore, functional inhibition of Hsp90 target proteins in combination with targets of sorafenib may be an effective cancer treatment strategy. In the present study, it was found that combination treatment with sorafenib and wh-4 additively inhibited the proliferation of liver cancer cells. In addition, it was significant to investigate the additive effect of sorafenib with wh-4 on liver cancer cells. Drug concentration is critical for anti-tumor effects and it was found the effect of one drug was different at different concentration (54,55). It is, therefore, meaningful to adjust the concentration the sorafenib and wh-4.
The changes in the levels of STAT3 were also investigated. Among the STAT family members, STAT3 has received the most attention because it plays a central role in many oncogenic signaling pathways and controls signal transduction pathways in several inflammatory cytokines and growth factors that are implicated in liver damage and repair mechanisms (56). In normal cells, STAT signaling is critical for embryonic development, organogenesis, regulation of cell differentiation, proliferation, growth, and apoptosis, whereas constitutive activation of STAT3 is found in many human types of cancer cell lines and primary tumors including liver, prostate, breast, lung, gastric and head and neck cancer (57-59). STAT3 plays a key role in HCC initiation and progression, and it has been found that its phosphorylation is highly positive in the analysis of HCC biopsies (60-62). Previous more studies demonstrated that STAT3 is an attractive molecular target for the prevention of proliferation and treatment of HCC (56,63). In the present study, the STAT3 pathway was found to mediate apoptosis induced by combination treatment with sorafenib and wh-4. The results demonstrated that STAT3 is implicated in signal transduction that induces apoptosis in liver cancer cells upon combination treatment with sorafenib and wh-4.
In addition, the limitation of the present study was that in vivo experiments in animal were not conducted. Resistance to sorafenib is a major obstacle for clinical treatment. The present in vitro study demonstrated that sorafenib with wh-4 combination treatment significantly inhibited liver cancer cells proliferation and reduced ABCB1 and ABCG2 expression levels which were responsible for liver cancer cells resistance partly. However, it was not known whether sorafenib with wh-4 had the antitumor effect in vivo. It was desirable to investigate the effect of sorafenib with wh-4 treatment on liver cancer cells in vivo.
The present study showed that combination treatment with sorafenib and wh-4 inhibits the proliferation of liver cancer cells and suppresses the development of drug resistance. A novel treatment regimen was also identified to improve the efficacy of sorafenib in patients with liver cancer by targeting the STAT3 pathway. This study demonstrates that combination treatment with sorafenib and wh-4 may present a promising strategy for further clinical therapy of patients with liver cancer.
Supplementary Material
Western blot analysis of Caspase-3 and Caspase-9. Liver cancer cells were treated with 5 μM sorafenib, 5 μM wh-4 and the combination (5 μM sorafenib, 5 μM wh-4, respectively) for 48 h. S, sorafenib; W, wh-4; Ctrl, control.
Silencing efficiency analysis of si-ABCB1 and ABCG2. (A) Western blotting and (B) RT-qPCR assays were used to evaluate the effect of si-ABCB1 and ABCG2 in Huh7. (C) Western blotting and (D) RT-qPCR assays were used to evaluate the effect of si-ABCB1 and ABCG2 in SK-HEP-1. Liver cancer cells were transduced with siRNA, and the cells were collected after 72 h. *P<0.05 and **P<0.01. si-, small interfering RNA; Rel., relative; RT-qPCR, reverse transcription-quantitative PCR.
Western blot analysis of P-gp and BCRP. Liver cancer cells, (A) Huh7 and (B) SK-HEP-1, were treated with 5 μM sorafenib, 5 μM wh-4 and the combination (5 μM sorafenib, 5 μM wh-4, respectively) for 48 h. BCRP, breast cancer resistance protein; P-gp, p-glycoprotein; S, sorafenib; W, wh-4; Ctrl, control.
Western blot and reverse transcription-quantitative PCR analyses of STAT3 in liver cancer cells transduced with pcDNA3.1-STAT3 vector. Liver cancer cells were transduced with pcDNA3.1-STAT3 vector and the cells were collected after 72 h. (A) Huh7 cell protein was analyzed using western blotting and (B) RT-qPCR assay was used to evaluate the gene level. (C) Western blotting was employed to test the protein level in SK-HEP-1 and (D) RT-qPCR assay was used to evaluate the gene level in SK-HEP-1. *P<0.05. Rel., relative; RT-qPCRreverse transcription-quantitative PCR
Acknowledgements
Not applicable.
Funding
Funding: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by grants from the Guangzhou Science and Technology Plan Program (nos. 201904010050 and 202102021276), Medical Scientific Research Fund Project of Guangdong Province (nos. A2018238, A2017312 and A2018540), and Fund of Guangdong Food and Drug Vocational College (nos. 2016YZ001 and 2016YZ023).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
SHC carried out most of the experiments and wrote the manuscript. DDX and PJZ analyzed the data and results. YaW and QYL read and revised the manuscript and contributed to data collection and statistical analysis. ZR and ZL provided technical assistance with several experiments. XW participated in the study design and drafted the paper. HQH conceived the study. YiW and XX participated in the design and coordination of the study. YFW designed the study and revised the manuscript. All authors have read and approved the final manuscript. YFW and SHC have confirmed the authenticity of all the raw data.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
References
Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J and Jemal A: Global cancer statistics, 2012. CA Cancer J Clin. 65:87–108. 2015.PubMed/NCBI View Article : Google Scholar | |
Villanueva A, Minguez B, Forner A, Reig M and Llovet JM: Hepatocellular carcinoma: Novel molecular approaches for diagnosis, prognosis, and therapy. Annu Rev Med. 61:317–328. 2010.PubMed/NCBI View Article : Google Scholar | |
Vitale A, Volk ML, Pastorelli D, Lonardi S, Farinati F, Burra P, Angeli P and Cillo U: Use of sorafenib in patients with hepatocellular carcinoma before liver transplantation: A cost-benefit analysis while awaiting data on sorafenib safety. Hepatology. 51:165–173. 2010.PubMed/NCBI View Article : Google Scholar | |
Reyes R, Wani NA, Ghoshal K, Jacob ST and Motiwala T: Sorafenib and 2-Deoxyglucose synergistically inhibit proliferation of both Sorafenib-Sensitive and -Resistant HCC cells by inhibiting ATP production. Gene Expr. 17:129–140. 2017.PubMed/NCBI View Article : Google Scholar | |
Shen YC, Hsu C and Cheng AL: Molecular targeted therapy for advanced hepatocellular carcinoma: Current status and future perspectives. J Gastroenterol. 45:794–807. 2010.PubMed/NCBI View Article : Google Scholar | |
Palmer DH: Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 359:2498–2499. 2008.PubMed/NCBI | |
Johnson P and Billingham L: Sorafenib for liver cancer: The horizon broadens. Lancet Oncol. 10:4–5. 2009.PubMed/NCBI View Article : Google Scholar | |
Tejeda-Maldonado J, Garcia-Juarez I, Aguirre-Valadez J, González-Aguirre A, Vilatobá-Chapa M, Armengol-Alonso A, Escobar-Penagos F, Torre A, Sánchez-Ávila JF and Carrillo-Pérez DL: Diagnosis and treatment of hepatocellular carcinoma: An update. World J Hepatol. 7:362–376. 2015.PubMed/NCBI View Article : Google Scholar | |
Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, et al: Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 359:378–390. 2008.PubMed/NCBI View Article : Google Scholar | |
Villanueva A and Llovet JM: Second-line therapies in hepatocellular carcinoma: Emergence of resistance to sorafenib. Clin Cancer Res. 18:1824–1826. 2012.PubMed/NCBI View Article : Google Scholar | |
Xin HW, Ambe CM, Hari DM, Wiegand GW, Miller TC, Chen JQ, Anderson AJ, Ray S, Mullinax JE, Koizumi T, et al: Label-retaining liver cancer cells are relatively resistant to sorafenib. Gut. 62:1777–1786. 2013.PubMed/NCBI View Article : Google Scholar | |
Tai WT, Cheng AL, Shiau CW, Liu CY, Ko CH, Lin MW, Chen PJ and Chen KF: Dovitinib induces apoptosis and overcomes sorafenib resistance in hepatocellular carcinoma through SHP-1-mediated inhibition of STAT3. Mol Cancer Ther. 11:452–463. 2012.PubMed/NCBI View Article : Google Scholar | |
Marin JJ, Monte MJ, Blazquez AG, Macias RI, Serrano MA and Briz O: The role of reduced intracellular concentrations of active drugs in the lack of response to anticancer chemotherapy. Acta Pharmacol Sin. 35:1–10. 2014.PubMed/NCBI View Article : Google Scholar | |
Marin JJG, Macias RIR, Monte MJ, Romero MR, Asensio M, Sanchez-Martin A, Cives-Losada C, Temprano AG, Espinosa-Escudero R, Reviejo M, et al: Molecular bases of drug resistance in hepatocellular carcinoma. Cancers. 12(1663)2020.PubMed/NCBI View Article : Google Scholar | |
Wang XQ, Ongkeko WM, Chen L, Yang ZF, Lu P, Chen KK, Lopez JP, Poon RT and Fan ST: Octamer 4 (Oct4) mediates chemotherapeutic drug resistance in liver cancer cells through a potential Oct4-AKT-ATP-binding cassette G2 pathway. Hepatology. 52:528–539. 2010.PubMed/NCBI View Article : Google Scholar | |
Gao B, Yang FM, Yu ZT, Li R, Xie F, Chen J, Luo HJ and Zhang JC: Relationship between the expression of MDR1 in hepatocellular cancer and its biological behaviors. Int J Clin Exp Pathol. 8:6995–7001. 2015.PubMed/NCBI | |
Nies AT, Konig J, Pfannschmidt M, Klar E, Hofmann WJ and Keppler D: Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma. Int J Cancer. 94:492–499. 2001.PubMed/NCBI View Article : Google Scholar | |
Wang S, Du Z, Luo J, Wang X, Li H, Liu Y, Zhang Y, Ma J, Xiao W, Wang Y and Zhong X: Inhibition of heat shock protein 90 suppresses squamous carcinogenic progression in a mouse model of esophageal cancer. J Cancer Res Clin Oncol. 141:1405–1416. 2015.PubMed/NCBI View Article : Google Scholar | |
Liu Y, Wang X, Wang Y, Zhang Y, Zheng K, Yan H, Zhang L, Chen W, Wang X, Liu Q, et al: Combination of SNX-2112 with 5-FU exhibits antagonistic effect in esophageal cancer cells. Int J Oncol. 46:299–307. 2015.PubMed/NCBI View Article : Google Scholar | |
Wang X, Wang S, Liu Y, Ding W, Zheng K, Xiang Y, Liu K, Wang D, Zeng Y, Xia M, et al: The Hsp90 inhibitor SNX-2112 induces apoptosis of human hepatocellular carcinoma cells: The role of ER stress. Biochem Biophys Res Commun. 446:160–166. 2014.PubMed/NCBI View Article : Google Scholar | |
Liu KS, Liu H, Qi JH, Liu QY, Liu Z, Xia M, Xing GW, Wang SX and Wang YF: SNX-2112, an Hsp90 inhibitor, induces apoptosis and autophagy via degradation of Hsp90 client proteins in human melanoma A-375 cells. Cancer Lett. 318:180–188. 2012.PubMed/NCBI View Article : Google Scholar | |
Liu KS, Ding WC, Wang SX, Liu Z, Xing GW, Wang Y and Wang YF: The heat shock protein 90 inhibitor SNX-2112 inhibits B16 melanoma cell growth in vitro and in vivo. Oncol Rep. 27:1904–1910. 2012.PubMed/NCBI View Article : Google Scholar | |
Bachleitner-Hofmann T, Sun MY, Chen CT, Liska D, Zeng Z, Viale A, Olshen AB, Mittlboeck M, Christensen JG, Rosen N, et al: Antitumor activity of SNX-2112, a synthetic heat shock protein-90 inhibitor, in MET-amplified tumor cells with or without resistance to selective MET Inhibition. Clin Cancer Res. 17:122–133. 2011.PubMed/NCBI View Article : Google Scholar | |
Borkovich KA, Farrelly FW, Finkelstein DB, Taulien J and Lindquist S: hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures. Mol Cell Biol. 9:3919–3930. 1989.PubMed/NCBI View Article : Google Scholar | |
Trepel J, Mollapour M, Giaccone G and Neckers L: Targeting the dynamic HSP90 complex in cancer. Nat Rev Cancer. 10:537–549. 2010.PubMed/NCBI View Article : Google Scholar | |
Cheng W, Ainiwaer A, Xiao L, Cao Q, Wu G, Yang Y, Mao R and Bao Y: Role of the novel HSP90 inhibitor AUY922 in hepatocellular carcinoma: Potential for therapy. Mol Med Rep. 12:2451–2456. 2015.PubMed/NCBI View Article : Google Scholar | |
Sarto C, Binz PA and Mocarelli P: Heat shock proteins in human cancer. Electrophoresis. 21:1218–1226. 2000.PubMed/NCBI View Article : Google Scholar | |
McConnell JR and McAlpine SR: Heat shock proteins 27, 40, and 70 as combinational and dual therapeutic cancer targets. Bioorg Med Chem Lett. 23:1923–1928. 2013.PubMed/NCBI View Article : Google Scholar | |
Kamal A, Thao L, Sensintaffar J, Zhang L, Boehm MF, Fritz LC and Burrows FJ: A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature. 425:407–410. 2003.PubMed/NCBI View Article : Google Scholar | |
Solit DB, Zheng FF, Drobnjak M, Münster PN, Higgins B, Verbel D, Heller G, Tong W, Cordon-Cardo C, Agus DB, et al: 17-Allylamino-17-demethoxygeldanamycin induces the degradation of androgen receptor and HER-2/neu and inhibits the growth of prostate cancer xenografts. Clin Cancer Res. 8:986–993. 2002.PubMed/NCBI | |
Newman B, Liu Y, Lee HF, Sun D and Wang Y: HSP90 inhibitor 17-AAG selectively eradicates lymphoma stem cells. Cancer Res. 72:4551–4561. 2012.PubMed/NCBI View Article : Google Scholar | |
Okawa Y, Hideshima T, Steed P, Vallet S, Hall S, Huang K, Rice J, Barabasz A, Foley B, Ikeda H, et al: SNX-2112, a selective Hsp90 inhibitor, potently inhibits tumor cell growth, angiogenesis, and osteoclastogenesis in multiple myeloma and other hematologic tumors by abrogating signaling via Akt and ERK. Blood. 113:846–855. 2009.PubMed/NCBI View Article : Google Scholar | |
Eilard MS, Andersson M, Naredi P, Geronymakis C, Lindnér P, Cahlin C, Bennet W and Rizell M: A prospective clinical trial on sorafenib treatment of hepatocellular carcinoma before liver transplantation. BMC Cancer. 19(568)2019.PubMed/NCBI View Article : Google Scholar | |
Kim JB, Lee M, Park SY, Lee S, Kim HR, Lee HS, Yoon JH and Kim YJ: Sorafenib inhibits cancer side population cells by targeting cJun Nterminal kinase signaling. Mol Med Rep. 12:8247–8252. 2015.PubMed/NCBI View Article : Google Scholar | |
Ha TY, Hwang S, Hong HN, Choi YI, Yoon SY, Won YJ, Song GW, Kim N, Tak E and Ryoo BY: Synergistic effect of sorafenib and vitamin K on suppression of hepatocellular carcinoma cell migration and metastasis. Anticancer Res. 35:1985–1995. 2015.PubMed/NCBI | |
Yi P, Higa A, Taouji S, Bexiga MG, Marza E, Arma D, Castain C, Le Bail B, Simpson JC, Rosenbaum J, et al: Sorafenib-mediated targeting of the AAA+ ATPase p97/VCP leads to disruption of the secretory pathway, endoplasmic reticulum stress, and hepatocellular cancer cell death. Mol Cancer Ther. 11:2610–2620. 2012.PubMed/NCBI View Article : Google Scholar | |
Sauzay C, Louandre C, Bodeau S, Anglade F, Godin C, Saidak Z, Fontaine JX, Usureau C, Martin N, Molinie R, et al: Protein biosynthesis, a target of sorafenib, interferes with the unfolded protein response (UPR) and ferroptosis in hepatocellular carcinoma cells. Oncotarget. 9:8400–8414. 2018.PubMed/NCBI View Article : Google Scholar | |
Vaishampayan UN, Burger AM, Sausville EA, Heilbrun LK, Li J, Horiba MN, Egorin MJ, Ivy P, Pacey S and Lorusso PM: Safety, efficacy, pharmacokinetics, and pharmacodynamics of the combination of sorafenib and tanespimycin. Clin Cancer. 16:3795–3804. 2010.PubMed/NCBI View Article : Google Scholar | |
Booth L, Shuch B, Albers T, Roberts JL, Tavallai M, Proniuk S, Zukiwski A, Wang D, Chen CS, Bottaro D, et al: Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function. Oncotarget. 7:12975–12996. 2016.PubMed/NCBI View Article : Google Scholar | |
Barta TE, Veal JM, Rice JW, Partridge JM, Fadden RP, Ma W, Jenks M, Geng L, Hanson GJ, Huang KH, et al: Discovery of benzamide tetrahydro-4H-carbazol-4-ones as novel small molecule inhibitors of Hsp90. Bioorg Med Chem Lett. 18:3517–3521. 2008.PubMed/NCBI View Article : Google Scholar | |
Xu WW, Li B, Guan XY, Chung SK, Wang Y, Yip YL, Law SY, Chan KT, Lee NP, Chan KW, et al: Cancer cell-secreted IGF2 instigates fibroblasts and bone marrow-derived vascular progenitor cells to promote cancer progression. Nat Commun. 8(14399)2017.PubMed/NCBI View Article : Google Scholar | |
Wang J, Lian Y, Gu Y, Wang H, Gu L and Huang Y, Zhou L and Huang Y: Synergistic effect of farnesyl transferase inhibitor lonafarnib combined with chemotherapeutic agents against the growth of hepatocellular carcinoma cells. Oncotarget. 8:105047–105060. 2017.PubMed/NCBI View Article : Google Scholar | |
Nishanth RP, Ramakrishna BS, Jyotsna RG, Roy KR, Reddy GV, Reddy PK and Reddanna P: C-Phycocyanin inhibits MDR1 through reactive oxygen species and cyclooxygenase-2 mediated pathways in human hepatocellular carcinoma cell line. Eur J Pharmacol. 649:74–83. 2010.PubMed/NCBI View Article : Google Scholar | |
Kuzuya T, Ishigami M, Ito T, Ishizu Y, Honda T, Ishikawa T, Hirooka Y and Fujishiro M: Clinical characteristics and outcomes of candidates for second-line therapy, including regorafenib and ramucirumab, for advanced hepatocellular carcinoma after sorafenib treatment. Hepatol Res. 49:1054–1065. 2019.PubMed/NCBI View Article : Google Scholar | |
Yurdacan B, Egeli U, Guney Eskiler G, Eryilmaz IE, Cecener G and Tunca B: Investigation of new treatment option for hepatocellular carcinoma: A combination of sorafenib with usnic acid. J Pharmacy Pharmacol. 71:1119–1132. 2019.PubMed/NCBI View Article : Google Scholar | |
Cheng AL, Guan Z, Chen Z, Tsao CJ, Qin S, Kim JS, Yang TS, Tak WY, Pan H, Yu S, et al: Efficacy and safety of sorafenib in patients with advanced hepatocellular carcinoma according to baseline status: Subset analyses of the phase III Sorafenib Asia-Pacific trial. Eur J Cancer. 48:1452–1465. 2012.PubMed/NCBI View Article : Google Scholar | |
Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, Kim JS, Luo R, Feng J, Ye S, Yang TS, et al: Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: A phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol. 10:25–34. 2009.PubMed/NCBI View Article : Google Scholar | |
Morisaki T, Umebayashi M, Kiyota A, Koya N, Tanaka H, Onishi H and Katano M: Combining celecoxib with sorafenib synergistically inhibits hepatocellular carcinoma cells in vitro. Anticancer Res. 33:1387–1395. 2013.PubMed/NCBI | |
Ceballos MP, Rigalli JP, Cere LI, Semeniuk M, Catania VA and Ruiz ML: ABC Transporters: Regulation and association with multidrug resistance in hepatocellular carcinoma and colorectal carcinoma. Curr Med Chemistry. 26:1224–1250. 2019.PubMed/NCBI View Article : Google Scholar | |
Wilhelm S, Carter C, Lynch M, Lowinger T, Dumas J, Smith RA, Schwartz B, Simantov R and Kelley S: Discovery and development of sorafenib: A multikinase inhibitor for treating cancer. Nat Rev Drug Discov. 5:835–844. 2006.PubMed/NCBI View Article : Google Scholar | |
Meyer A, Cygan P, Tolzien K, Galvez AG, Bitran JD, Lestingi TM and Nabhan C: Role of sorafenib in overcoming resistance of chemotherapy-failure castration-resistant prostate cancer. Clin Genitourin Cancer. 12:100–105. 2014.PubMed/NCBI View Article : Google Scholar | |
Decker T, Overkamp F, Rösel S, Nusch A, Göhler T, Indorf M, Sahlmann J and Trarbach T: A randomized phase II study of paclitaxel alone versus paclitaxel plus sorafenib in second- and third-line treatment of patients with HER2-negative metastatic breast cancer (PASO). BMC Cancer. 17(499)2017.PubMed/NCBI View Article : Google Scholar | |
Gongora C: Sorafenib inhibits ABCG2 and overcomes irinotecan resistance-response. Mol Cancer Ther. 13(764)2014.PubMed/NCBI View Article : Google Scholar | |
Sun Y, Zhang J, Zhou J, Huang Z, Hu H, Qiao M, Zhao X and Chen D: Synergistic effect of cucurbitacin B in combination with curcumin via enhancing apoptosis induction and reversing multidrug resistance in human hepatoma cells. Eur J Pharmacol. 768:28–40. 2015.PubMed/NCBI View Article : Google Scholar | |
Kim JE, Kim SG, Goo JS, Park DJ, Lee YJ, Hwang IS, Lee HR, Choi SI, Lee YJ, Oh CH, et al: The α-iso-cubebenol compound isolated from Schisandra chinensis induces p53-independent pathway-mediated apoptosis in hepatocellular carcinoma cells. Oncol Rep. 28:1103–1109. 2012.PubMed/NCBI View Article : Google Scholar | |
Subramaniam A, Shanmugam MK, Perumal E, Li F, Nachiyappan A, Dai X, Swamy SN, Ahn KS, Kumar AP, Tan BK, et al: Potential role of signal transducer and activator of transcription (STAT)3 signaling pathway in inflammation, survival, proliferation and invasion of hepatocellular carcinoma. Biochim Biophys Acta. 1835:46–60. 2013.PubMed/NCBI View Article : Google Scholar | |
Schindler C and Darnell JE Jr: Transcriptional responses to polypeptide ligands: The JAK-STAT pathway. Ann Rev Biochem. 64:621–651. 1995.PubMed/NCBI View Article : Google Scholar | |
Zeidler MP, Bach EA and Perrimon N: The roles of the drosophila JAK/STAT pathway. Oncogene. 19:2598–2606. 2000.PubMed/NCBI View Article : Google Scholar | |
Hirano T, Ishihara K and Hibi M: Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene. 19:2548–2556. 2000.PubMed/NCBI View Article : Google Scholar | |
Yoshikawa H, Matsubara K, Qian GS, Jackson P, Groopman JD, Manning JE, Harris CC and Herman JG: SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet. 28:29–35. 2001.PubMed/NCBI View Article : Google Scholar | |
Niwa Y, Kanda H, Shikauchi Y, Saiura A, Matsubara K, Kitagawa T, Yamamoto J, Kubo T and Yoshikawa H: Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma. Oncogene. 24:6406–6417. 2005.PubMed/NCBI View Article : Google Scholar | |
Feng DY, Zheng H, Tan Y and Cheng RX: Effect of phosphorylation of MAPK and Stat3 and expression of c-fos and c-jun proteins on hepatocarcinogenesis and their clinical significance. World J Gastroenterol. 7:33–36. 2001.PubMed/NCBI View Article : Google Scholar | |
Lee C and Cheung ST: STAT3: An emerging therapeutic target for hepatocellular Carcinoma. Cancers (Basel). 11(1646)2019.PubMed/NCBI View Article : Google Scholar |