Abstract
Background/Aim: The membrane transporters activated in cancer stem cells (CSCs) are the target of novel cancer therapies for hepatocellular carcinoma (HCC). The present investigation demonstrated the expression profiles of ion channels in CSCs of HCC. Materials and Methods: Cells that highly expressed aldehyde dehydrogenase 1 family member A1 (ALDH1A1) were separated from HepG2 cells, a human HCC cell line, by fluorescence-activated cell sorting, and CSCs were identified based on the formation of tumorspheres. Gene expression profiles in CSCs were investigated using microarray analysis. Results: Among HepG2 cells, ALDH1A1 messenger RNA level was higher in CSCs than in non-CSCs. Furthermore, CSCs exhibited resistance to cisplatin and had the capacity to redifferentiate. The results of the microarray analysis of CSCs showed the up-regulated expression of several genes related to ion channels, such as calcium voltage-gated channel auxiliary subunit gamma 4 (CACNG4). The cytotoxicity of the CACNG4 inhibitor amlodipine was higher at lower concentrations in CSCs than in non-CSCs, and markedly decreased the number of tumorspheres. The cell population among HepG2 cells that highly expressed ALDH1A1 was also significantly reduced by this inhibitor. Conclusion: CACNG4 plays a role in maintaining CSCs, and its inhibitor, amlodipine, could potentially be a targeted therapeutic agent against HCC.
Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related deaths worldwide (1-3). Regardless recent advances in surgery, embolization therapy, chemotherapy, radiotherapy, and molecular targeted therapy, the prognosis of advanced disease remains poor due to its invasiveness and metastatic potential (1-3). Therefore, the molecular mechanisms underlying the malignancy of HCC need to be elucidated in more detail in order to develop effective therapeutics.
Ion channels have been implicated in fundamental mechanisms of cancer cells, suggesting their potential as novel therapeutic targets for cancer (4, 5). Our previous studies have demonstrated that the high expression of various ion channels in esophageal (6), gastric (7), and pancreatic (8) cancer stem cells (CSCs) contributes to resistance against anticancer agents or radiotherapy, and to invasiveness and potential of metastasis (9, 10). Furthermore, we have revealed that the CSCs of gastric cancer exhibit high expression levels of several genes related to voltage-gated calcium channels (VGCCs), and that the VGCCs inhibitor amlodipine, which is widely used to treat hypertension and cardiac angina, has a potent cytotoxic effect at lower concentrations in CSCs (7). These findings highlight an essential role of VGCCs in CSC maintenance and their inhibitors as potential targeted therapeutic agents for various cancers. Nevertheless, the expression profiles of ion channels and their oncogenic functions in the CSCs of HCC remain unknown.
Therefore, this study aimed to investigate the expression and function of ion channels in the CSCs of the HCC cell line HepG2. A microarray analysis showed the up-regulated expression of multiple genes associated with ion channels, including calcium voltage-gated channel auxiliary subunit gamma 4 (CACNG4), in the CSCs of HCC. Moreover, our results also showed that amlodipine, a CACNG4 inhibitor, specifically inhibited the CSCs of HCC.
Materials and Methods
Cell line, antibodies, and other regents. The human HCC cell line HepG2 was obtained from the Japanese Collection of Research Bioresources (JCRB) (Tokyo, Japan). HepG2 cells were cultured in RPMI supplemented with 100 μg/ml of streptomycin, 100 U/ml of penicillin (Nacalai Tesque, Kyoto, Japan), and 10% fetal bovine serum (Sigma-Aldrich Japan, Tokyo, Japan) in an incubator set at 37°C with 5% carbon dioxide. Amlodipine was supplied by FUJI FILM Wako Pure Chemical Corporation (Osaka, Japan) and cisplatin (CDDP) was obtained from Nippon Kayaku (Tokyo, Japan).
Aldeflour assay and cell sorting. ALDH1A1 activity in the HepG2 cell line was assessed using ALDEFLOUR kit according to the manufacturer’s instructions (STEMCELL Technologies, Vancouver, BC, Canada). Flow cytometry using the SH800 cell sorter (Sony, Tokyo, Japan) was performed to separate cells as described previously (6, 8).
Preparation of CSCs. Fluorescence-activated cell sorting was performed on the HepG2 cell population to isolate cells with strong ALDH1A1 activity. The isolated cells were then cultured in RPMI supplemented with 100 U/ml of penicillin, 100 lg/ml of streptomycin, 2% B27 supplement (Gibco, Langley, OK, USA), 10 ng/ml of epidermal growth factor (Invitrogen, Grand Island, NY, USA), and 10 ng/ml of fibroblast growth factor (Invitrogen) for 10 days in ultralow-attachment six-well plates (Corning International, Corning, NY, USA). The formation of spheres in the plates was assessed under an inverted microscope and spheres were collected as described previously (6-8).
Real time reverse transcription polymerase chain reaction (RT PCR). RNA was extracted using RNeasy kit (Qiagen, Valencia, CA, USA). A real-time quantitative PCR analysis was performed using the Step One plus Real-Time PCR System (Applied Biosystems, Foster city, CA, USA) and TaqMan Gene Expression Assay (Applied Biosystems) as described previously (6-8). The expression profiles of the following genes were examined: ALDH1A1: Hs00946916; CACNG4: Hs01061935; CD133: Hs01009259; and EpCAM: Hs00901888 (Applied Biosystems). Gene expression was normalized by the housekeeping gene ACTB (Hs01060665_g1, Applied Biosystems). Assays were conducted in triplicate.
Drug sensitivity assay. Cells from adherent or spheroid HepG2 cells were seeded at a density of 2000 cells per well on 96-well microplates. The viability of cells treated with CDDP or amlodipine for 48 h was assessed using a WST 8 assay (Cell Count Reagent SF; Nacalai Tesque, Inc.) as described previously (6-8).
Microarray analysis. Total RNA was extracted, and RNA quality was monitored using Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Sense-strand cDNA was prepared using the Affymetrix GeneChip WT PLUS Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA). Biotin-labeled cDNA was hybridized to the Human Clariom S Array using Affymetrix GeneChip Hybridization Oven 645 (Thermo Fisher Scientific) as described previously (6-8). Chips were then washed and scanned with Affymetrix GeneChip Scanner 3000 7G (Thermo Fisher Scientific).
Sphere formation assay. CSCs suspended in medium with or without 1 μM amlodipine were seeded at a density of 200 or 400 cells per well on 96-well plates. The cells were cultured in an incubator set at 37°C with 5% carbon dioxide for 1 week without changing or adding medium to prevent disturbances in the formation of tumorspheres. The number of tumorspheres was counted after 1 week as described previously (6-8).
Statistical analysis. The significance of differences was assessed using the Student’s t-test where p-values <0.05 indicated a significant difference. In the drug sensitivity assay, the half maximal inhibitory concentration (IC50) was calculated based on a non-linear regression. Statistical analyses were conducted using JMP software version 10 (JMP, Cary, NC, USA).
Results
Acquisition of CSCs. The ALDEFLOUR assay was conducted on HepG2 cells to identify cells with high ALDH1A1 expression, which were subsequently isolated using fluorescence-activated cell sorting (Figure 1A). CSCs were generated through sphere formation, and sphere formation was verified under an inverted microscope (Figure 1B). RT-PCR analysis of total RNA extracted from spheres showed that CSCs exhibited higher levels of ALDH1A1 mRNA compared to non-CSCs (Figure 1C). Furthermore, the levels of other CSC markers, including CD133, and EpCAM were higher in CSCs than those in non-CSCs (Figure 1C). The Aldefluor assay conducted on CSCs presented approximately 57.5% ALDH1A1 activity (Figure 2A). To assess whether CSCs exhibited redifferentiation capacity, CSCs were cultured on normal culture plates and confirmed to maintain adhesive properties and a typical morphology even after several passages (Figure 2B). To examine resistance to anticancer drugs, treatment of non-CSCs and CSCs with CDDP revealed that the IC50 was approximately 2.18 μM in non-CSCs and 4.10 μM in CSCs (Figure 2C), indicating that non-CSCs were more sensitive to CDDP at lower concentrations compared to CSCs, consistent with the characteristics of CSCs.
Acquisition of cancer stem cells (CSCs) from HepG2 cells. A) Aldefluor assay with HepG2 cells. Cells highly expressing aldehyde dehydrogenase 1 family member A1 (ALDH1A1) were isolated by fluorescence-activated cell sorting (red box). B) Sphere formation assay shows that sorted HepG2 cells highly expressing ALDH1A1 formed spheres. Magnification: ×40 left panel, ×100 right panel. C) Messenger RNA expression levels of ALDH1A1, CD133, and EpCAM were increased in CSCs. The results are presented as mean±standard error of the mean, n=3.
Characteristics of cancer stem cells (CSCs). A) Aldefluor assay with CSCs. High activity of aldehyde dehydrogenase 1 family member A1 (ALDH1A1) was found in CSCs (green box). B) The CSCs obtained had the capacity to redifferentiate. CSCs cultured on normal plates acquired adhesive properties and a typical morphology even after several passages. Magnification: ×40. C) Anticancer drug sensitivity of CSCs. Cisplatin was more cytotoxic at a lower concentration in non-CSCs than in CSCs of HepG2 cells (n=5). IC50: Half maximal inhibitory concentration.
Gene expression profiles of CSCs in HepG2 cells. A microarray analysis was performed to elucidate the gene expression profiles of CSCs in HepG2 cells. We focused on ion channel and transporter genes, which exhibited up-regulated expression in CSCs. The top forty ion channel and transporter-related genes expressed in HepG2 CSCs are listed in Table I. Among these genes, particular attention was given to CACNG4 which is the target of widely used drugs.
Ion channel and transporter genes that are up-regulated in HepG2-CSCs.
Antitumor effects of amlodipine on CSCs. Microarray analysis confirmed significantly higher levels of CACNG4 mRNA in CSCs compared to non-CSCs (Figure 3A). Amlodipine, a specific inhibitor of CACNG4 was employed to assess suppressive effects of the inhibitor on non-CSCs and CSCs of HepG2 cells. The IC50 of amlodipine in non-CSCs and CSCs of HepG2 cells was 19.8 and 10.1 μM, respectively (Figure 3B). These results indicated that the cytotoxicity of amlodipine was greater at a lower concentration in CSCs than in non-CSCs. A sphere formation assay on CSCs treated with or without amlodipine demonstrated that significant reduction in the number of spheres in the presence of amlodipine (Figure 4), indicating its specific inhibitory effects on the CSC activity.
Antitumor effects of amlodipine on cancer stem cells (CSCs). A) Validation of gene expression using real-time quantitative reverse transcription polymerase chain reaction. The expression levels of calcium voltage-gated channel auxiliary subunit gamma 4 (CACNG4) in CSCs were compared with those in non-CSCs among HepG2 cells. The results are presented as mean±standard error of the mean, n=3. Asterisk p<0.05 (significantly different from non-CSCs). B) Sensitivity of CSCs to the CACNG4 inhibitor amlodipine. Amlodipine was more cytotoxic at a lower concentration in CSCs than in non-CSCs among HepG2 cells (n=5).
Effects of amlodipine on sphere formation by CSCs. A treatment with amlodipine (1 μM) decreased the number of spheres in the CSCs of HepG2 cells. The results are presented as mean±standard error of the mean, n=4. Asterisk p<0.05 (significantly different).
Effects of amlodipine on the expression and activity of ALDH1A1. The impact of amlodipine on ALDH1A1 expression was examined in both non-CSCs and CSCs of HepG2 cells. At first, the activity of ALDH1A1 was assessed in non-CSCs treated with amlodipine using the ALDEFLOUR assay, revealing that amlodipine reduced the cell population highly expressing ALDH1A1 in non-CSCs (Figure 5A). Subsequently, the activity of ALDH1A1 was assessed in CSCs treated with amlodipine using the ALDEFLOUR assay, demonstrating a decrease in the population of CSCs with high ALDH1A1 expression (Figure 5B).
Effects of amlodipine on the expression and activity of aldehyde dehydrogenase 1 family member A1 (ALDH1A1). A) Effects of amlodipine on ALDH1A1 activity by non-cancer stem cells (non-CSCs). Amlodipine (1 μM) decreased the cell population highly expressing ALDH1A1 in non-CSCs. B) Effects of amlodipine on ALDH1A1 activity in CSCs. Amlodipine (1 μM) decreased the cell population highly expressing ALDH1A1 in CSCs. The expression of ALDH1A1 was assessed using flow cytometry with Aldefluor staining. The results are presented as mean±standard error of the mean, n=3. Asterisk p<0.05 (significantly different from non-treated HepG2 cells).
Discussion
Previous studies investigated the presence of several CSC-specific markers in HCC, including ALDH1A1, CD44, CD90, CD133, and EpCAM (11-16). High ALDH1A1 expression levels have been associated with tumorigenesis in HCC (11-13). Identification of elevated ALDH1A1 activity and CSC isolation have been successfully achieved using Aldeflour, a substrate for ALDH1A1 (13). We have previously employed Aldefluor to isolate CSCs from esophageal and pancreatic cancer cells (6, 8). In the present study, we also utilized the Aldefluor method to successfully isolate and obtain CSCs from HCC. Our results showed that CSCs expressed high levels of ALDH1A1, had the capacity to redifferentiate, and were resistant to chemotherapy. Gene expression profiling confirmed the up-regulation of ion channel-related genes in the CSCs of HCC, highlighting the potential of selective inhibitors as targeted therapeutic agents against CSCs. Consequently, we focused on investigating the role and function of CACNG4 in CSCs of HCC.
VGCCs play a pivotal role in various physiological mechanisms, such as secretion, nerve conduction, and gene expression in many types of cells by regulating Ca2+ membrane transport (17, 18). VGCCs consist of α, β, δ, and γ subunits, each with multiple isoforms. The γ subunits, encompassing 8 isoforms (CACNG1-8) are unique to L-type channels and are believed to be involved in the activation and inactivation of the channel itself by inhibiting Ca2+ currents (19). CACNG4 is distributed in various cancer cells, and their oncogenic roles have been reported in breast (19, 20), cervical (21), prostate (22), bladder (23), and colon cancers (24). Previous studies have demonstrated that CACNG4 regulates tumor cell growth and dissemination by altering calcium signaling in breast cancer (19). Feng et al. identified CACNG4 as a potential predictor for tumorigenesis in human papillomavirus-related cervical cancer, where Epstein-Bar virus acts as a cofactor or mediator (21). Halatsch et al. identified that CACNG4 was one of the candidate genes contributing to resistance against erlotinib in human glioblastoma multiforme cell lines (25).
Recent studies have investigated the function of VGCCs in various types of CSCs. Zhao et al. demonstrated the potential of calcium voltage-gated channel auxiliary subunit alpha2delta 1 (CACNA2D1) in identifying subpopulations of HCC cancer cells with CSC properties. They showed that treatment with the 1B50-1 monoclonal antibody or the depletion of CACNA2D1 reduced the number of CSCs among HCC cells by inducing apoptosis (26). In our previous study, we found strong expression of CACNA2D1 and calcium voltage-gated channel auxiliary subunit beta 4 (CACNB4) in CSCs from gastric cancer. Furthermore, we observed that amlodipine and verapamil, specific inhibitors of these channels, inhibited the growth of CSCs (7). While these findings indicate the potential of VGCCs as a novel target in CSCs, the relationship between CACNG4 and CSCs remains unknown. To our knowledge, this study is the first to examine CSCs that strongly express CACNG4.
Ca2+ channel blockers have demonstrated effects against various cancer cells. Amlodipine, commonly used for hypertension and cardiac angina, was shown to suppress the proliferation of human breast cancer cells when administered orally (27). Taghizadehghalehjoughi et al. demonstrated that the combination of vincristine and amlodipine was more effective than vincristine alone in inhibiting human neuroblastomas cells (28). Our previous study demonstrated that amlodipine exhibited greater cytotoxicity against VGCCs at lower concentration in CSCs of gastric cancer than in non-CSCs (7). However, it remains unclear whether amlodipine exerts specific inhibitory effects on CSCs of HCC. In the present study, we investigated the effects of amlodipine on CSCs of HCC and were the first to demonstrate its specific inhibitory effects on the formation tumorspheres by these cells. These findings suggest the potential of Ca2+ channel blockers as promising therapeutic agents for drug-resistant HCC.
Conclusion
We herein revealed strong expression of CACNG4 in CSCs of HCC. We observed that the CACNG4 inhibitor amlodipine exerted greater cytotoxicity at lower concentration in CSCs than in non-CSCs. Although further investigations are required to elucidate the role and function of VGCCs in CSCs, Ca2+ channel blockers have potential as novel therapeutic agents against HCC.
Footnotes
Authors’ Contributions
AS, KK, MK, and EO designed the research. AS, KK, and EO wrote the paper. AS, KK, HS, TK, KT, TA, HK, YY, RM, SK, HI, TK, and HF performed cell culture, molecular biology, and several experiments.
Conflicts of Interest
There are no conflicts of interest or financial ties to declare in relation to this study.
Funding
The present study was supported by Grants-in-Aid for Scientific Research (C) (23K08218, 23K08115, 23K06654, 22K08832, 21K08689) and Grants-in-Aid for Young Scientists (22K16518, 20K17659) from the Japan Society for the Promotion of Science.
- Received August 4, 2023.
- Revision received September 5, 2023.
- Accepted September 6, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).