Bay K 8644 | Inhibitor Screening

The calcium channel agonist Bay K 8644 promotes the growth of human liver cancer HepG2 cells in vitro: suppression
with overexpressed regucalcin

ImageMasayoshi Yamaguchi1 · Tomiyasu Murata2 · Joe W. Ramos1

Received: 27 April 2020 / Accepted: 13 June 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
Hepatocellular carcinoma is one of the most prevalent malignant diseases and causes a third of cancer-related death. The consequences of altered calcium homeostasis in cancer cells may contribute to tumor progression. Regucalcin plays an inhibitory role in calcium signaling linked to transcription regulation. Regucalcin gene expression is downregulated in the tumor tissues of liver cancer patients, suggesting an involvement as a suppressor in hepatocarcinogenesis. We investigated whether Bay K 8644, an agonist of the L-type Ca2+ channel, promotes the growth of human liver cancer and if the effect of Bay K 8644 is suppressed by overexpressed regucalcin using the HepG2 cell model. The colony formation and growth of HepG2 cells were promoted by culturing with Bay K 8644 (0.1–10 nM). This effect was suppressed by inhibitors of signal- ing processes linked to cell proliferation, including PD98059 and wortmannin. Death of HepG2 cells was stimulated by Bay K 8644 with higher concentrations (25 and 100 nM). The effects of Bay K 8644 on cell growth and death were abolished by verapamil, an antagonist of calcium channel. Mechanistically, culturing with Bay K 8644 increased levels of mitogen- activated protein kinase (MAPK) and phospho-MAPK. Notably, overexpressed regucalcin suppressed Bay K 8644-promoted growth and death of HepG2 cells. Furthermore, overexpressed regucalcin prevented growth and increased death induced by thapsigargin, which induces the release of intracellular stored calcium. Thus, higher regucalcin expression suppresses cal- cium signaling linked to the growth of liver cancer cells, providing a novel strategy in treatment of hepatocellular carcinoma with delivery of the regucalcin gene.
Keywords Regucalcin · Bay K 8644 · Cell proliferation · Liver cancer · HepG2 cells · Calcium signaling · Thapsigargin · Carcinogenesis

Introduction
Liver cancer is one of the leading causes of cancer-related mortality around worldwide [1, 2]. Hepatocellular carci- noma (HCC) is the primary cancer of the liver. The risk factors associated with HCC are related to cirrhosis from a chronic and diffuse hepatic disease, which result from continuous regeneration with liver injury and chronic viral

* Masayoshi Yamaguchi [email protected]
1 Cancer Biology Program, University of Hawaii Cancer Center, University of Hawaii at Manoa, 701 Ilalo Street, Honolulu, HI 96813, USA
2 Laboratory of Analytical Neurobiology, Faculty
of Pharmacy, Meijo University, Yagotoyama 150, Tempaku, Nagoya 468-8503, Japan

infections, including hepatitis B virus or hepatitis C virus [2–6]. Hepatocarcinogenesis is a multistep process initiated by genetic changes in hepatocytes or stem cells, leading to proliferation, apoptosis, dysplasia and neoplasia [1–6]. The prognosis of advanced HCC remains poor in spite of the development of novel therapeutic strategies [6–8], including multi-kinase inhibitors [9] and microRNAs, a novel class of noncoding small RNAs [10].
ImageCalcium signal participates in key processes of cancer, including proliferation and migration. The consequences of altered calcium transport in cancer cells may be significant and contribute to tumor progression [11]. Activated calcium signaling may play a role in the promotion of carcinogenesis [11–14]. Characterizing such changes may help to identify new therapeutic targets. Notably, regucalcin, a suppressor of calcium signaling, plays a multifunctional role in cell regula- tion as a suppressor in various signaling processes linked totranscription in various types of cells and tissues [15–19]. Regucalcin plays a role in maintenance of intracellular cal- cium homeostasis, inhibition of calcium signaling-depend- ent protein kinase and phosphatase activities, suppression of nuclear DNA and RNA synthesis, protein output, cell proliferation, and apoptotic cell death, which are mediated through diverse signaling processes [20–23].

In recent studies, regucalcin has been demonstrated to be involved in suppression of human cancers, including liver cancer [24]. The gene expression and protein levels of regucalcin are downregulated in the tumor tissues of various types of human cancers [25–30]. Survival of these cancer patients is prolonged with a higher regucalcin expression in the tumor tissues [25–30]. Translational studies, further- more, demonstrated that overexpression of regucalcin sup- presses the growth of various types of human cancer cells in vitro [25–32]. Downregulated expression of the regucal- cin gene may contribute to malignancies in various tissues of human subjects [33]. Regucalcin may play a crucial role as a suppressor in human cancers. Regucalcin, which was found as an inhibitor of calcium signaling [15, 34], is therefore proposed to be a novel target molecule in the diagnosis and therapy of human cancer.

Bay K 8644 is an agonist of the L-type Ca2+ channel in
various types of cells [35, 36]. In the present study, there- fore, we investigate whether or not Bay K 8644 impacts on the growth of HepG2 cells of the human liver cancer model in vitro. Bay K 8644 was found to stimulate colony formation and growth of HepG2 cells, suggesting that activated calcium signaling contributes to development of hepatocarcinogenesis. Notably, the stimulatory effects of Bay K 8644 on the growth of HepG2 cells were suppressed by overexpression of regucalcin. Thus, higher regucalcin expression may contribute to repression of development of hepatocarcinogenesis, which is mediated via a calcium signaling-dependent mechanism. A system for delivery of the regucalcin gene may therefore provide a new therapeutic strategy for HCC.

Materials and methods
Reagents

Dulbecco’s Modification of Eagle’s Medium (DMEM) with
4.5 g/L glucose, L-glutamine and sodium pyruvate, and anti- biotics (100 μg/ml penicillin and 100 μg/ml streptomycin; P/S) were purchased from Gibco Life Technologies Corpo- ration (Grand Island, NY, USA). Fetal bovine serum (FBS) was from Omega Scientific Inc. (Tarzana, CA, USA). Lipo- fectamine reagent was obtained from Promega (Madison, WI, USA). Geneticin (G418), Bay K 8644, wortmannin, PD98059, staurosporine, dibucaine, caspase-3 inhibitor,
thapsigargin, and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise spec- ified. Caspase-3 inhibitor was diluted in phosphate buffered saline (PBS) and other reagents were dissolved in 100% ethanol before use.
Human liver cancer cells

We used human liver cancer HepG2 cells, which were obtained from the American Type Culture Collection (Rock- ville, MD, USA). The HepG2 cell line was derived from a 15-year-old child with primary hepatoblastoma [37]. HepG2 cells were not derived from HCC [37]. This cell line, how- ever, was reported to be genetically the best model for hepa- tocellular carcinoma tumor studies [38]. HepG2 cells were suitable as a transfection host. The cells were cultured in a DMEM containing 10% FBS and 1% P/S.
Transfection of regucalcin cDNA

HepG2 cells were transfected with the pCXN2 vector (Addgene, Inc., Cambridge, MA, USA; 600 µg/ml) that expresses cDNA encoding human full length (900 bp) regu- calcin (regucalcin cDNA/pCXN2) [27, 39]. For transient transfection assays, HepG2 cells (2 × 105 cells/well) were grown on 24-well plates to approximately 70–80% conflu- ence. The regucalcin cDNA/pCXN2 or empty pCXN2 vector were transfected into HepG2 cells using the synthetic cati- onic lipid, a Lipofectamine reagent, according to the manu- facturer’s instructions (Promega, Madison, WI, USA) [39]. This efficiency was in the range of 60–80%. After incuba- tion for overnight, Geneticin (G418, 500 μg/ml) was added to culture wells for selection, and the cells were cultured for 3 weeks. Surviving cells were plated at limiting dilu- tion to isolate transfectants. Multiple surviving clones were isolated, transferred to 35-mm dishes, and grown in medium with G418 (100 μg/ml). We obtained transfectant clones 1 and 2, which exhibit stable expression of regucalcin. The regucalcin levels in these clones were markedly (p < 0.001) increased as compared with that of wild-type cells, as shown in Fig. 5A and B. Clone 1 with higher levels of regucalcin was used in the following experiments.
Assay of colony formation

HepG2 wild-type cells or transfectants (1 × 103 cells/well) were seeded into 6-well plates and cultured in DMEM con- taining 10% FBS, 1% P/S and 1% fungizone (Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of either vehicle (1% ethanol as a final concentration) or Bay K 8644 (1 nM) under conditions of 5% CO2 and 37 °C for 14 days when visible clones were formed on the plates [40]. After culture, the colonies were washed with PBS and fixed withmethanol (95%, 0.5 ml per well) for 20 min at room tem- perature, and then washed 3 times with PBS. The colonies were then stained with 0.5% crystal violet (Sigma-Aldrich, St. Louis) for 30 min at room temperature. Stained cells were washed 6 times with PBS. The plates were air-dried for 2 h at room temperature. The colonies containing > 50 cells were counted under a microscope (Olympus MTV-3; Olympus Corporation, Tokyo, Japan).

Assay of cell growth

To determine alteration of cell growth, HepG2 wild-type cells or transfectant (1 × 105/ml per well) were cultured using 24-well plates in DMEM containing 10% FBS, 1% P/S and 1% fungisone for 1, 2, 3, or 5 days in a water-saturated atmosphere containing 5% CO2 and 95% air at 37 °C in the presence or absence of either vehicle (1% ethanol as a final concentration) or Bay K 8644 (0.1, 1, 10, 25 or 100 nM) [39, 41]. In other experiments, HepG2 wild-type cells or transfectants (1 × 105/ml per well) were cultured for 3 days in DMEM containing 10% FBS and 1% P/S in the pres- ence or absence of either vehicle (1% ethanol), wortmannin (0.1 or 1 μM), PD98059 (1 or 10 μM), staurosporine (10 or
100 nM), dibucaine (10 or 100 nM), thapasigagin (1, 10,
100, 500 or 1000 nM) with or without Bay K 8644 (1 or 10 nM). After culture, the cells were detached from each well by adding a sterile solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca2+/Mg2+-free PBS (Thermo Fisher Scientific, Waltham, MA, USA) with incubation for 2 min at 37 ℃. Each well was then added 0.9 ml of DMEM contain- ing 10% FBS and 1% P/S. The number of cells in the cell suspension was counted as described below in the section “Cell counting”.

Assay of cell death

HepG2 wild-type cells or transfectant (1 × 105/ml per well) were cultured using 24-well plates in DMEM containing 10% FBS, 1% P/S and 1% fungisone for 3 days. On reach- ing subconfluence, they were cultured for an additional 24 h in the presence or absence of either vehicle (PBS or 1% ethanol as a final concentration), Bay K 8644 (25 or 100 nM), or thapasigagin (1, 10, 100, 500 or 1000 nM) [42]. In other experiments, HepG2 wild-type cells or transfectants (1 × 105/ml per well) were cultured for 3 days reaching sub- confluence, the cells were cultured for an additional 24 h in the presence or absence of either vehicle (1% ethanol), Bay K 8644 (25 or 100 nM), or thapasigagin (1, 10, 100, 500 or 1000 nM) with or without caspase-3 inhibitor (10 μM) for 24 h [42]. After culture, the cells were detached by addition of sterile solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca2+/Mg2+-free PBS per well as described in the
section of “Cell growth assay”, and the cell number was counted as described below in the section “Cell counting”.
Cell counting

To detach cells on each well, the culture dishes were incu- bated for 2 min at 37 ℃ after addition of a solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca2+/Mg2+-free PBS, and the cells were detached through pipetting after addition of DMEM (0.9 ml) containing 10% FBS and 1% P/S [39, 41, 42]. Aliquot (0.1 ml) of 0.5% trypan blue stain- ing solution was added into the medium containing the sus- pended cells (0.1 ml). After mixing, the number of viable cells was counted under a microscope (Olympus MTV-3) with a Hemocytometer (Sigma-Aldrich) using a cell coun- ter (Line Seiki H-102P, Tokyo, Japan). For each dish, we took the average of two counts. Cell numbers are shown as number per well. Data are presented as the mean ± standard deviation (SD) obtained from eight wells of two replicate plates per data set using different cell preparations.
Assay of western blotting

HepG2 wild-type cells or transfectants (1 × 106 cells/well) were plated in 100 mm dishes added 10 ml of DMEM containing 10% FBS, 1% P/S and 1% fungizone [25]. After culture for 3 days, the cells were washed three times with cold PBS and removed from the dish by scrap- ing using cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) with addition of protease and pro- tein phosphatase inhibitors (Roche Diagnostics, Indian- apolis, IN, USA). The lysates were then centrifuged at 17,000×g, at 4 ℃ for 2 min. The protein concentration of the supernatant was determined for Western blotting using the Bio-Rad Protein Assay Dye (Bio-Rad Labora- tories, Inc., Hercules, CA, USA) with bovine serum albu- min as a standard. The supernatant was stored at – 80 ℃ until used. Samples of forty micrograms of supernatant protein per lane were separated by SDS polyacrylamide gel electrophoresis (12% SDS-PAGE) and transferred to nylon membranes for immunoblotting using specific antibodies against various proteins obtained from Cell Signaling Technology (Danvers, MA, USA), including Ras [catalog number (cat. no.) 14429 rabbit], PI3 kinase p1100 α (cat. no. 4255, rabbit), Akt (cat. no. 9272, rab- bit), mitogen-activated protein kinase (MAPK; cat. no. 4695, rabbit), phosphorylated-MAPK (cat. no. 4370, rab- bit), and β-actin (cat. no. 3700, mouse), and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), including NF- κ B p65 (cat. no. sc-109, rabbit) snd β-catenin (cat. no. sc-39350, mouse). Rabbit anti-regucalcin antibody was obtained from Sigma-Aldrich (cat. no. HPA029103, rabbit). Target proteins were incubated with one of theprimary antibodies (1:1,000) overnight at 4 ℃, followed by horseradish peroxidase-conjugated secondary anti- bodies (Santa Cruz Biotechnology Inc, mouse cat. no. sc-2005 or rabbit cat. no. sc-2305; diluted 1:2000). The immunoreactive blots were visualized with a SuperSignal West Pico Chemiluminescent Substrate detection system (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Three blots from independ- ent experiments were scanned on an Epson Perfection 1660 Photo scanner, and bands quantified using Image J2 software.

Statistical analysis

Data are presented as the mean ± standard deviation (SD). Statistical significance was determined using GraphPad InStat version 3 for Windows XP (GraphPad Software Inc. La Jolla, CA). Multiple comparisons were per- formed by one-way analysis of variance (ANOVA) with Tukey–Kramer multiple comparisons post test for para- metric data as indicated. A p value of < 0.05 was consid- ered statistically significant.

Result
Bay K 8644 stimulates colony formation and growth of HepG2 cells

Bay K 8644 is an agonist of L-type calcium channel and stimulates calcium influx into the cells [35, 36]. To deter- mine the effects of Bay K 8644 on the colony formation of HepG2 wild-type cells, the cells were cultured in the pres- ence of either vehicle (1% ethanol as a final concentration) or Bay K 8644 (1 nM) for 14 days (Fig. 1A and B). The colony formation of HepG2 cells was promoted by culturing with Bay K 8644 (Fig. 1A and B). Furthermore, we deter- mined the effect of Bay K 8644 on the growth of HepG2 cells in vitro. Cells were cultured in the presence of Bay K 8644 (0.1, 1, 10, 25 and 100 nM) for 1 (Fig. 1C), 2 (Fig. 1D),
3 (Fig. 1E), and 5 (Fig. 1F) days. The growth of HepG2 cells was promoted by culturing with Bay K 8644 (0.1, 1 and 10 nM). Stimulatory effects of Bay K 8644 on cell growth were revealed with higher concentrations (25 and 100 nM) for longer culture periods (3 and 5 days).
We next investigated whether the stimulatory effects of Bay K 8644 (10 nM) on the growth of HepG2 cells are

Bay K 8644 stimulates colony formation and growth in human liver cancer HepG2 cells in vitro. A HepG2 cells (1 × 103 cells/2 ml per well in 6-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 14 days in the presence or absence of either vehicle (1% ethanol as a final concentration) or Bay K 8644 (1 nM). After culture, the colonies were stained with 0.5% crystal violet and counted. A Representative photo. B The numbers of colonies contain- ing more than 50 cells were counted under a microscope. Data are shown as the mean ± SD of two plates (6 wells) per data set usingdifferent cell preparations. In separate experiments (C, D, E and F), HepG2 cells (1 × 105 cells/ml per well in 24-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 1, 2, 3, or 5 days in the presence or absence of either vehicle (1% ethanol) or Bay K 8644 (0.1, 1, 10, 25 or 100 nM). After culture, the number of attached cells was counted. Data are presented as the mean ± SD obtained from 8 wells of two replicate plates per data set using differ-ent cell preparations. *p < 0.001 versus control (control; white bar or gray bars). 1-way ANOVA, Tukey–Kramer post-test
linked to cell-signaling process. Stimulatory effects of Bay K 8644 on cell growth were suppressed by culturing with vari- ous inhibitors of intracellular signaling pathways, includ- ing staurosporine (Fig. 2A), an inhibitor of protein kinase C [43], dibucaine (Fig. 2B), an inhibitor of calcium/calm- odulin-dependent protein kinases [44], PD98059 (Fig. 2C), an inhibitor of MAPK [45], and wortmannin (Fig. 2D), an inhibitor of PI3K [46]. These results suggest that Bay K 8644-promoted growth of HepG2 cells is the result of the activation of various signaling pathways, including calcium signaling processes.

Stimulatory effects of Bay K 8644 on the growth are independent of the death of HepG2 cells

The number of HepG2 cells was reduced by culturing with Bay K 8644 at comparatively higher concentrations (25 or 100 nM), indicating that cell death is partly induced by Bay K 8644 (Fig. 3A). Such effects were abolished by the pres- ence of an inhibitor of caspase-3, which induces apoptotic
cell death [42] (Fig. 3B). This result suggests that the stimu- latory effects of Bay K 8644 on the growth of HepG2 cells are independent of effects on cell death.
Stimulatory effects of Bay K 8644 on the growth of HepG2 cells are blocked by calcium channel antagonists

Verapamil is an antagonist of calcium channels related to Bay K 8644 [47]. To determine the effects of verapamil on the growth of HepG2 cells, the cells were cultured in the presence of verapamil (1, 10, 50, or 100 μM) (Fig. 4A). Cul- ture with verapamil (50 or 100 μM) inhibited cell growth. Interestingly, the stimulatory effects of Bay K 8644 (10 nM) on cell growth were not exhibited in the presence of vera- pamil (50 μM). Furthermore, to determine the effects of verapamil on cell death, HepG2 cells reaching subconflu- ence were cultured with verapamil (1, 10, 50 or 100 μM) (Fig. 4B). Verapamil stimulated cell death (Fig. 4B). Stimu- latory effect of Bay K 8644 (100 nM) on cell death was

Stimulatory effects of Bay K 8644 on the growth of human liver cancer HepG2 cells are blocked by inhibitors of various sign- aling pathways in vitro. HepG2 cells (1 × 105 cells/ml per well in 24-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 3 days in the presence or absence of either vehicle (1% ethanol as a final concentration) or Bay K 8644 (10 nM) with or with- out staurosporine (1 or 10 nM) (A), dibucaine (10 or 100 nM) (B),PD98059 (0.1 or 1 μM) (C) or wortmannin (10 or 100 nM) (D). After culture, the number of attached cells was counted. Data are presented as the mean ± SD obtained from 8 wells of two replicate plates per data set using different cell preparations. *p < 0.001 versus control (gray bar without inhibitor). #p < 0.001 versus control (gray bar with Bay K 8644 alone).1-way ANOVA, Tukey–Kramer post-test

Effects of Bay K 8644 on the death of human liver can- cer HepG2 cells in vitro. HepG2 cells (1 × 105 cells/ml per well in 24-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 3 days, and the cells reaching subconfluence were then cul- tured for additional 24 h in the presence of vehicle (1% ethanol as a final concentration) or Bay K 8644 (0.1, 1, 10, 25 or 100 nM) without
(A) or with (B) caspase-3 inhibitor (10 µM) plus Bay K 8644 (25 or 100 nM). After culture, the number of attached cells counted. Data are presented as the mean ± SD obtained from 8 wells of two replicate plates per data set using different cell preparations. *p < 0.001 versus control (gray bar). 1-way ANOVA, Tukey–Kramer post-test

Effects of verapamil, a calcium channel blocker, on the growth and death of human liver cancer HepG2 cells cultured with Bay K 8644 in vitro. A HepG2 cells (1 × 105 cells/ml per well in 24-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 3 days in the presence or absence of either vehicle (1% ethanol as a final concentration) or verapamil (1, 10, 50 or 100 μM) with or with- out Bay K 8644 (10 nM). B HepG2 cells (1 × 105 cells/ml per well in 24-well plates) were cultured in DMEM containing 10% FBS and 1%
P/S for 3 days without verapamil, and they were additionally cultured for 24 h in the presence of either vehicle (1% ethanol) or verapamil (1, 10, 50 or 100 μM) with or without Bay K 8644 (100 nM). After culture, the number of attached cells counted. Data are presented as the mean ± SD obtained from 8 wells of two replicate plates per data set using different cell preparations. *p < 0.001 versus control (gray bar). #p < 0.001 versus control (gray bar with Bay K 8644 alone). 1-way ANOVA, Tukey–Kramer post-testnot further potentiated in the present of verapamil (50 μM) (Fig. 4B). Thus, the effects of Bay K 8644 on the growth and death of HepG2 cells were blocked by verapamil, an antagonist of calcium channel, supporting the view that Bay K 8644 stimulates calcium entry mediated by calcium chan- nels in the cells.

Effects of Bay K 8644 on levels of proteins involved in cell signaling

Mechanistically, we investigated whether or not Bay K 8644 impacts on the levels of proteins linked to cell
growth of HepG2 cells (Fig. 5A and B). Culture with Bay K 8644 (10 nM) decreased the levels of Ras, PI3K and Akt, while it increased the levels of MAPK and phospho- MAPK in HepG2 cells. Meanwhile, the levels of NF-κB and β-catenin were not changed by culturing with Bay K 8644 (10 nM) (Fig. 5). In addition, levels of p21, an inhibi- tor of cell cycle [21, 23], were not altered by culturing with Bay K 8644 (10 nM) (data not shown). Changes in the levels of various proteins linked to cell sign- aling and transcription processes in human liver cancer HepG2 cells cultured with Bay K 8644 in vitro. HepG2 cells (1 × 106 cells/10 ml of medium per dish) were cultured in DMEM containing 10% FBS and 1% P/S for 3 days in the presence of either vehicle (1% ethanol as a final concentration) or Bay K 8644 (10 nM). After culture, the cells were removed from the dish with a cell scraper in cell lysis buffer containing protease inhibitors. Forty micrograms of supernatant pro-
tein per lane were separated by SDS-PAGE and transferred to nylon membranes for Western blotting using antibodies against various pro- teins. A Representative data are presented. B Immunoblot band inten- sity was quantitated and is presented as fold of control (without Bay K 8644). Data are presented as the mean ± SD obtained from four dishes per data set using different cell preparations. *p < 0.001 versus control (white bar). Student’s t-test

Overexpressed regucalcin suppresses the Bay K 8644‑promoted growth of HepG2 cells

Regucalcin, a calcium-binding protein, is well known as an inhibitor of intracellular calcium signaling [18, 19]. We therefore investigated whether overexpression of regucal- cin suppresses the stimulatory effects of Bay K 8644 on the growth of HepG2 cells. To generate the regucalcin- overexpressing HepG2 cells, the cells were transiently transfected with the empty pCXN2 vector or the vector containing full length (33 kDa protein) human regucalcin by using lipofection methods. We obtained transfectant clones 1 and 2 with higher stable expression of regucalcin. The regucalcin levels in these clones were increased about six- or four-fold as compared with wild-type cells, respec- tively (Fig. 6A and B). We used clone 1 in the following experiments. Overexpression of regucalcin was found to decrease the expression of Ras, PI3K, Akt, MAPK, and phospho-MAPK, which link to cell signaling and tran- scriptional activity (Fig. 6C and D). Then, to determine the effects of overexpressed regucalcin on colony formation, HepG2 wild-type cells and transfectants were cultured for 14 days when colony formation clearly appeared (Fig. 6E). The number of colonies was decreased in the regucalcin- overexpressing HepG2 cells as compared with that of the wild-type cells (Fig. 6E and F). Furthermore, the growth of HepG2 wild-type cells was enhanced with increasing days in culture (Fig. 6G). This enhancement was depressed in the transfectants (Fig. 6G). Thus, overexpression of regucalcin was found to suppress the colony formation and growth of human liver cancer HepG2 cells in vitro.
We next investigated whether overexpressed regucalcin suppresses the stimulatory effects of Bay K 8644 on the growth and death of HepG2 cells in vitro (Fig. 7). Stimula- tory effects of Bay K 8644 (1 or 10 nM) on the growth of HepG2 cells were not caused in the transfectants (Fig. 7A). Also, the effects of Bay K 8644 (25 and 100 nM) on the death of HepG2 cells were blocked in the transfectants (Fig. 7B). These results suggest that the suppressive effects of overexpressed regucalcin on the growth of HepG2 cells are independent of cell death. Thus, higher regucalcin expression was shown to suppress the stimulatory effects of Bay K 8644, an agonist of calcium entry into the cells, on the growth and death of HepG2 cells.

Overexpressed regucalcin prevents the effects
of thapsigargin on the growth and death of HepG2 cells

Thapsigargin is an inhibitor of the calcium-pumping enzyme (Ca2+-ATPase) in the endoplasmic reticulum of cells and causes an elevation of cytoplasmic Ca2+ levels by induc- ing release of Ca2+ from intracellular store, including the endoplasmic reticulum [48, 49]. We therefore investigated whether or not overexpressed regucalcin prevents the effects of thapsigargin on the growth and death of HepG2 cells in vitro. Culture with thapsigargin (100, 500, and 1000 nM) suppressed the growth of HepG2 cells (Fig. 8A) and stimu- lated the death of the cells (Fig. 8C). These effects of thap- sigargin were not revealed in regucalcin-overexpressing HepG2 cells (Fig. 8B and D). This result suggests that

Overexpression of regucalcin suppresses colony formation and growth in human liver cancer HepG2 cells in vitro. Cells were trans- fected with regucalcin cDNA vector. A Regucalcin content in HepG2 cells cultured in DMEM containing 10% FBS and 1% P/S for 3 days was analyzed by immunoblotting with an anti-regucalcin antibody. Lane 2 and 3; the cells transfected with the human regucalcin cDNA / pCXN2 (designated as clone 1 and 2). B The levels of regucalcin are shown as fold of wild-type cells. Data are presented as the mean ± SD obtained from four dishes per data set using different cell prepara- tions. C Representative data of the levels of various proteins linked to cell signaling are shown. D The levels of various proteins are shown as fold of wild-type cells. Data are presented as the mean ± SD obtained from four dishes per data set using different cell prepara- tions. E To determine colony formation, wild-type cells and transfectants (1 × 103 cells/2 ml per well in 6-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 18 days. After culture, the colonies were stained with 0.5% crystal violet, and counted. Pho- tos of plates with crystal violet staining are presented. F Number of colonies is presented. Data are shown as the mean ± SD of two plates (6 wells) per data set using different cell preparations. G To deter- mine alteration of cell growth, HepG2 wild-type cells and transfect- ants (1 × 105 cells/ml per well in 24-well plates) were cultured inDMEM for 1, 2, 3 or 5 days. After culture, the number of attached cells per dish was counted. Data are presented as the mean ± SD obtained from 8 wells of two replicate plates per data set using differ- ent cell preparations. *p < 0.001 versus wild type (white bar) or con- trol vector (gray bar). 1-way ANOVA, Tukey–Kramer post-test

7 Overexpression of regucalcin blocks the stimulatory effects of Bay K 8644 on the proliferation and suppresses the effects on death of human liver cancer HepG2 cells in vitro. A To determine the effects of overexpressed regucalcin on cell proliferation, HepG2 wild-type cells or transfectants (1 × 105 cells/ml per well in 24-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 3 days in the presence or absence of either vehicle (1% ethanol
as a final concentration) or Bay K 8644 (1 or 10 nM). B To deter- mine the effects of overexpressed regucalcin on cell death, HepG2
wild-type cells or transfectants (1 × 105 cells/ml per well in 24-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 3 days, and the cells reaching subconfluence were then cultured for additional 24 h in the presence of vehicle (1% ethanol) or Bay K 8644 (25 or 100 nM). After culture, the number of attached cells was counted. Data are presented as the mean ± SD obtained from 8 wells of two replicate wells per data set using different dishes and cell preparations. *p < 0.001 versus control (without Bay K 8644). 1-way ANOVA, Tukey–Kramer post-test

Overexpression of regucalcin blocks thapsigargin mediated proliferation and death of human liver cancer HepG2 cells in vitro. A, B To determine the effects of thapsigargin on cell proliferation, HepG2 wild-type cells or transfectants (1 × 105 cells/ml per well in 24-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 3 days in the presence or absence of either vehicle (1% ethanol as a final concentration) or thapsigargin (1, 10, 100, 500or 1000 nM). C, D To determine the effects of thapsigargin on cell death, HepG2 wild-type cells or transfectants (1 × 105 cells/ml per

overexpressed regucalcin protects disturbance of intracel- lular calcium homeostasis.

Discussion
Hepatocarcinogenesis is a multistep process initiated by external stimuli that leads to genetic changes in hepatocytes or stem cells, resulting in proliferation, apoptosis, dyspla- sia and neoplasia. The prognosis of advanced HCC remains poor in spite of the development of novel therapeutic strat- egies, including multi-kinase inhibitors and microRNAs, a novel class of noncoding small RNAs [6–10]. Analysis of established microarray databases demonstrated that the prolonged survival in HCC patients was associated with increased regucalcin gene expression [27]. Translational studies showed that overexpression of regucalcin sup- presses the proliferation, increased cell death, and migra- tion in the HepG2 cells of human cancer model in vitro [27]. Regucalcin may play a crucial role as a suppressor in hepatocarcinogenesis.
well in 24-well plates) were cultured in DMEM containing 10% FBS and 1% P/S for 3 days, and the cells reaching on subconfluence were then cultured for additional 24 h in the presence of vehicle (1% ethanol) or thapsigargin (1, 10, 100, 500 or 1000 nM). After culture, the number of attached cells was counted. Data are presented as the mean ± SD obtained from 8 wells of two replicate plates per data set using different cell preparations. *p < 0.001 versus control (without thapsigargin). 1-way ANOVA, Tukey–Kramer post-test

Regucalcin inhibits calcium signaling in various types of normal and cancer cells [18, 19, 34]. Therefore, we inves- tigated whether or not overexpressed regucalcin suppresses promotion of the growth of HepG2 cells linked to activation of calcium signaling, leading to development of hepatocar- cinogenesis [50]. Bay K 8644, an agonist of the L-type Ca2+ channel, promoted the growth of HepG2 cells in vitro, and this effect of the agonist was found to be repressed by over- expression of regucalcin. Calcium signaling may therefore be mechanistically important in the suppressive effect of regucalcin in human liver cancer.
Verapamil is an antagonist of the L-type Ca2+ channel
[47]. Culturing with verapamil repressed the growth of HepG2 cells promoted by Bay K 8644, supporting the view that Bay K 8644 effects are due to L-type Ca2+ channel acti- vation in the cells. Furthermore, the stimulatory effects of Bay K 8644 on the growth of HepG2 cells were found to be suppressed by culturing with various inhibitors of intracellu- lar signaling pathways, including staurosporine, an inhibitor of protein kinase C [43], dibucaine, an inhibitor of calcium- dependent protein kinases [44], PD98059, an inhibitor of

MAPK [45], and wortmannin, an inhibitor of PI3K [46]. These results suggest that Bay K 8644-promoted growth of HepG2 cells is mediated by the activation of various signal- ing pathways linked to calcium-signaling processes. Over- expressed regucalcin suppressed the stimulatory effects of Bay K 8644 on the growth of HepG2 cells. Regucalcin may suppress activation of various signaling pathways, including calcium signaling.
Intracellular calcium with higher levels is known to cause apoptotic cell death [23, 34, 42]. Death of HepG2 cells was induced by using higher concentrations of Bay K 8644. This cell death was prevented by culturing with an inhibitor of caspase-3, a protein that activates apoptosis and nuclear DNA fragmentation [51]. High levels of calcium activate DNA fragmentation in isolated rat nucleus [51]. Interest- ingly, overexpressed regucalcin suppressed the death of HepG2 cells cultured in the presence of high concentrations of Bay K 8644, suggesting that this cell death is related to calcium increased in the cells.

Stimulation with Bay K 8644 increased the levels of MAPK and phospho-MAPK, which lead to activation of transcription and cell proliferation. Meanwhile, culturing with Bay K 8644 decreased levels of Ras, PI3K, and Akt in HepG2 cells. However, Bay K 8644-promoted cell growth was suppressed by culturing with inhibitors of calcium/
calmodulin-dependent protein kinases [44], MAPK [45], and PI3K [46]. These signaling processes may be enhanced by Bay K 8644 stimulation. Bay K 8644 may potently acti- vate MAP kinase-linked signaling process to promote cell proliferation. Meanwhile, levels of p21, an inhibitor of cell cycle [22, 24], were not changed by culturing with Bay K 8644 (data not shown). Further studies remain to elucidate molecular mechanism by which Bay K 8644 promotes cell growth, using other HCC cell lines and normal hepatocytes. Overexpression of regucalcin decreased the levels of
MAPK and phosphor-MAPK in HepG2 cells. Previous studies showed that overexpressed regucalcin increased the levels of p21, p53 and retinoblastoma (Rb) in HepG2 cells [27], which leads to depression of cell proliferation. Regucalcin plays a crucial role in the regulation of various gene expressions by binding to DNA [20, 52]. Further- more, regucalcin has been shown to directly inhibit the activities of Ca2+/calmodulin-dependent protein kinase
[53] and protein kinase C [54], which plays a pivotal role in process of calcium signaling in liver cells. Regucalcin may therefore suppress both processes of intracellular cal- cium signaling and nuclear transcription activation, which leads to suppression of the Bay K 8644-promoted growth of HepG2 cells. Regucalcin gene expression is enhanced by activation of Ca2+ signaling in liver cells [55, 56]. In Hypothetic mechanism by which overexpressed regucalcin suppresses the proliferation of cancer cells promoted by Bay K 8644, an agonist of Ca2+ channel. Bay K 8644 stimulates entry of Ca2+ into the cells, and activates intracellular signaling processes linked to Ca2+-dependent protein kinases, MAP kinase, and Ras/PI3K/Akt. Activation of these signaling processes may promote cell growth. Bay K 8644 may potently activate MAP kinase-linked signaling process to
promote cell proliferation. Activated Ca2+ signaling enhances expres- sion of the regucalcin gene. Cytoplasmic regucalcin is localized into the nucleus and regulates the expression of various genes. Further- more, overexpressed regucalcin suppresses diverse signaling path- ways implicated to cell proliferation. Abbreviation: RGN regucalcin

, we summarized a hypothetic mechanism by which regucalcin suppresses the growth of HepG2 cells promoted by stimulation of calcium agonist.
Thapsigargin is an inhibitor of the calcium-pumping enzyme (Ca2+-ATPase) in the endoplasmic reticulum of cells and induces an elevation of cytoplasmic Ca2+ lev- els [48, 49]. Furthermore, we investigated whether higher regucalcin expression suppresses the effects of thapsigar- gin on HepG2 cells in vitro. Culturing with thapsigargin suppressed the growth of HepG2 cells and stimulated the death of the cells in vitro. Interestingly, such effects were not revealed in regucalcin-overexpressing HepG2 cells. There- fore, overexpressed regucalcin may protect cells from the increased growth and the stimulated death of HepG2 cells due to excess calcium that is released from the endoplas- mic reticulum of intracellular calcium store [48, 49]. Regu- calcin has been shown to activate Ca2+-pumping enzymes (Ca2+-ATPase) in the endoplasmic reticulum [57], mito- chondria [58] and plasma membranes [59] in liver cells to maintain intracellular calcium homeostasis. Current result may further support the view that regucalcin suppresses pro- cess of calcium signaling in the cells.

In conclusion, the present study demonstrates that Bay K 8644, an agonist of the L-type Ca2+ channel, promotes the growth of HepG2 cells in vitro, and that this action is suppressed by overexpression of regucalcin, which is a potent inhibitor of calcium signaling [33]. The remod- eling of calcium signaling linked to calcium channels and pumps plays a pivotal role in the stimulation of growth and metastasis of cancer cells [10–13, 49]. Overexpressed regucalcin may inhibit activation of calcium signaling and its related activation of transcription in liver cancer cells. This may be significant as a mechanism by which regucalcin suppresses the growth and metastasis of cancer cells. Downregulated regucalcin gene expression may in this way contribute to development of malignancies [32]. Delivery of the regucalcin gene, which is overexpressed in tumor tissues, may constitute a novel therapeutic approach to treatment of hepatocarcinogenesis by controlling these calcium signaling processes.

Author contributions MY conceived the study. MY designed, and MY, TM and JWR performed the experiments. All authors discussed the findings. MY wrote the manuscript, and all authors edited the manu- script. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Funding This study was supported in part by funds provided by the University of Hawaii Cancer Center and the B.H. and Alice C. Beams Endowed Professorship in Cancer Research from the John A. Burns School of Medicine (J.W.R.), and the Foundation for Biomedical Research on Regucalcin, Japan (M.Y.).Data availability The datasets used during the present study are avail- able from the corresponding author upon reasonable request.

Compliance with ethical standards

Conflict of interest All authors declare that they have no conflicts of interest.
Research involving human participants and/or animals This article does not contain any studies with human participants or animals per- formed by any of the authors. All experimental protocols used data- bases or cell culture in vitro.

References
1. Sayiner M, Golabl P, Younossi Z (2019) Disease burden of hepato- cellular carcinoma: a global perspective. Dig Dis Sci 64:910–917
2. Mak LY, Cruz-Ramon V, Chinchilla-Lopez P, Torres HA, LoConte NK, Rice JP, Foxhall LE, Sturgis EM, Merrill JK, Bai- ley HH, Mendez-Sanchez N, Yuen MF, Hwang JP (2018) Global epidemiology, ptrevention, and management of hepatocellularcar- cinoma. Am Soc Clin Oncol Educ Book 38:262–279
3. El-Serag HB (2011) Hepatocellular carcinoma. N Engl J Med 365:1118–1127
4. Dragani TA (2010) Risk of HCC: genetic heterogeneity and com- plex genetics. J Hepatol 52:252–257
5. Cha MY, Kim CM, Park YM, Ryu WS (2004) Hepatitis B virus X protein is essential for the activation of Wnt/beta-catenin signaling in hepatoma cells. Hepatology 39:1683–1693
6. Shin JW, Chung Y-H (2013) Molecular targeted therapy for hepa- tocellular carcinoma: current and future. World J Gatroenterol 19:6144–6155
7. Meena AS, Sharma A, Kumari R, Mohammad N, Singh VS, Bhat MK (2013) Inherent and acquired resistance to paclitaxel in hepatocellular carcinoma: molecular events involved. PLoS ONE 8:e61524. https://doi.org/10.1371/journal.pone.0061524
8. Mohammad N, Singh SV, Malvi P, Chaube B, Athavale D, Vanuopadath M, Nair SS, Nair B, Bhat MK (2015) Strategy to enhance efficancy of doxorubicin in solid tumor cells by methyl- β-cyclodextrin: involvement of p53 and Fas receptor ligand com- plex. Sci Rep 5:11853. https://doi.org/10.1038/srep11853
9. Wilhelm SM, Adnane L, Newell P, Villanueva A, Llovet JM, Lynch M (2008) Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling. Mol Cancer Ther 7:3129–3140
10. Callegari E, Elamin BK, Sabbioni S, Gramantieri NM (2013) Role of microRNAs in hepatocellular carcinoma: a clinical perspective. Onco Targ Ther 6:1167–1178
11. Monteith GR, Davis FM, Roberts-Thompson SJ (2012) Calcium channels and pumps in cancer: changes and consequences. J Biol Chem 287:31666–33167
12. Tennakoon S, Aggarwal A, Kallay E (2016) The calcium-sensing receptor and the Hallmarks of cancer. Biochim Biophys Acta 1863:1398–1407
13. Varghese E, Samuel SM, Sadiq Z, Kubatka P, Liskova A, Benacka J, Pazinka P, Kruzliak P, Busselberg P (2019) Anti-cancer agents in proliferation and cell death: the calcium connection. Int J Mol Sci 20:3017. https://doi.org/10.3390/ijms20123017
14. Resende RR, Andrade LM, Oliveira AG, Guimaraes ES, Guati- mosim S, Leite MF (2013) Nucleoplasmic calcium signaling and cell proliferation: calcium signaling in the nucleus. Cell Commun Signal 11:14. https://doi.org/10.3390/ijms20123017

15. Yamaguchi M, Yamamoto T (1978) Purification of calcium bind- ing substance from soluble fraction of normal rat liver. Chem Pharm Bull (Tokyo) 26:1915–1918
16. Shimokawa N, Yamaguchi M (1993) Molecular cloning and sequencing of the cDNA coding for a calcium-binding protein regucalcin from rat liver. FEBS Lett 327:251–255
17. Shimokawa N, Matsuda Y, Yamaguchi M (1995) Genomic clon- ing and chromosomal assignment of rat regucalcin gene. Mol Cell Biochem 151:157–163
18. Yamaguchi M (2005) Role of regucalcin in maintaining cell home- ostasis and function. Int J Mol Med 15:372–389
19. Yamaguchi M (2011) Regucalcin and cell regulation: role as a suppressor in cell signaling. Mol Cell Biochem 353:101–137
20. Yamaguchi M (2011) The transcriptional regulation of regucalcin gene expression. Mol Cell Biochem 346:147–171
21. Yamaguchi M (2013) Role of regucalcin in cell nuclear regu- lation: involvement as a transcription factor. Cell Tissue Res 354:331–341
22. Yamaguchi M (2013) Suppressive role of regucalcin in liver cell proliferation: Involvement in carcinogenesis. Cell Prolif 46:243–253
23. Yamaguchi M (2013) The anti-apoptotic effect of regucal- cin is mediated through multisignaling pathways. Apoptosis 18:1145–1153
24. Yamaguchi M (2015) Involvement of regucalcin as a suppressor protein in carcinogenesis. Insight into the gene therapy. J Cancer Res Clin Oncol 141:1333–1341
25. Yamaguchi M, Osuka S, Weitzmann MN, El-Rayes BF, Shoji S, Murata T (2016) Prolonged survival in pancreatic cancer patients with increased regucalcin gene expression: overexpression of regucalcin suppresses the proliferation in human pancreatic can- cer MIA PaCa-2 cells in vitro. Int J Oncol 48:1955–1964
26. Yamaguchi M, Osuka S, Weitzmann MN, Shoji S, Murata T (2016) Increased regucalcin gene expression extends survival in breast cancer patients: overexpression of regucalcin suppresses the proliferation and metastatic bone activity in MDA-MB-231 human breast cancer cells in vitro. Int J Oncol 49:812–822
27. Yamaguchi M, Osuka S, Weitzmann MN, Shoji S, Murata T (2016) Prolonged survival in hepatocarcinoma patients with increased regucalcin gene expression: HepG2 cell proliferation is suppressed by overexpression of regucalcin in vitro. Int J Oncol 49:1686–1694
28. Yamaguchi M, Osuka S, Shoji S, Weitzmann MN, Murata T (2017) Survival of lung cancer patients is prolonged with higher regucalcin gene expression: suppressed proliferation of lung ade- nocarcinoma A549 cells in vitro. Mol Cell Biochem 430:37–46
29. Yamaguchi M, Osuka S, Murata T (2018) Prolonged survival of colorectal cancer patients is associated with higher regu- calcin gene expression: overexpressed regucalcin suppresses growth of human colorectal carcinoma cells in vitro. Int J Oncol 53:1313–1322
30. Yamaguchi M, Osuka S, Hankinson O, Murata T (2019) Pro- longed survival of renal cancer patients is concomitant with a higher regucalcin gene expression in the tumor tissues: overex- pression of regucalcin depresses the growth of human renal cell carcinoma cells in vitro. Int J Oncol 54:188–198
31. Li X, Huang Y, Guo S, Xie M, Bin X, Shi M, Chen A, Chen S, Wu F, Hu Q, Zhou S (2019) Exogenous regucalcin negatively regulates the progression of cervical adenocarcinoma. Oncol Lett 18:609–616
32. Yamaguchi M, Murata T (2020) Overexpression of regucalcin supresses the growth of human osteosarcoma cells in vitro: repres- sive effect of extracellular regucalcin. Cancer Invest 38:37–51
33. Nitschkowski D, Marwitz S, Kotanidou SA, Reck M, Kugler C, Rabe KF, Ammerpohi O, Goldmann T (2019) Live and let die: epigenetic modifications of survivin and regucalcin in non-small
cell lung cancer tissues contribute to malignancy. Clin Epigenet 11:157. https://doi.org/10.1186/s13148-019-0770-6
34. Yamaguchi M (2000) Role of regucalcin in calcium signaling. Life Sci 66:1769–1780
35. Panner A, Wurster RD (2006) T-type calcium channels and tumor proliferation. Cell Calcium 40:253–259
36. Monge L, Silvestre RA, Miralles P, Peiro E, Villanueva ML, Marco J (1988) In vitro effects of Bay K 8644, a dihydropyri- dine derivative with hypoglycaemic properties, on hepatic glu- cose production and pancreatic hormone secretion. Biochem Pharmacol 37:2933–2937
37. Knowles BB, Howe CC, Aden DP (1980) Human hepatocel- lular carcinoma cell lines secrete the major plasma protein and hepatitis B surface antigen. Science 209:497–499
38. Ao L, Guo Y, Song X, Guan Q, Zheng W, Zhang J, Huang H, Guo Z, Wang X (2017) Evaluating hepatocellular carcinoma cell lines for tumour samples using within-sample relative expression orderings of genes. Liver Int 37:1688–1696
39. Misawa H, Inagaki S, Yamaguchi M (2002) Suppression of cell proliferation and deoxyribonucleic acid synthesis in cloned rat hepatoma H4-II-E cells overexpressing regucalcin. J Cell Bio- chem 84:143–149
40. Fang Z, Tang Y, Fang J, Zhou Z, Xing Z, Guo Z, Guo X, Wang W, Jiao W, Xu Z, Liu Z (2013) Simvastatin inhibits renal cancer cell growth and metastasis via AKT/mTOR, ERK and JAK2/ STAT3 pathway. PLoS ONE 17(8):e62823
41. Yamaguchi M, Daimon Y (2005) Overexpression of regucalcin suppresses cell proliferation in cloned rat hepatoma H4-II-E cells: Involvement of intracellular signaling factors and cell cycle-related genes. J Cell Biochem 95:1169–1177
42. Izumi T, Yamaguchi M (2004) Overexpression of regucalcin suppresses cell death in cloned rat hepatoma H4-II-E cells induced by tumor necrosis factor-α or thapsigargin. J Cell Bio- chem 92:296–306
43. Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tom- ita E (1986) Staurosporine, a potent inhibitor of phospholipid/ Ca2+ dependent protein kinase. Biochem Biophys Res Commun 135:397–402
44. Nakagawa T, Sawada N, Yamaguchi M (2005) Overexpression of regucalcin suppresses cell proliferation of cloned normal rat kidney proximal tubular epithelial NRK52E cells. Int J Mol Med 16:637–643
45. Peleck SL, Charest DL, Mordret GP, Siow YL, Palaty C, Camp- bell D, Chaslton L, Samiei M, Sanghera JS (1993) Network- ing with mitogen-activated protein kinases. Mol Cell Biochem 127:157–169
46. Serrano-Nascimento C, da Silva TS, Nicola JP, Nachbar RT, Masini-Repiso AM, Nunes MT (2014) The acute inhibitory effect of iodide excess on sodium/iodide symporter expression and activity involves the PI3K/Akt signaling pathway. Endocri- nology 155:1145–1156
47. Vohra J (1982) Verapamil in cardiac arrhythmias: an overview. Clin Exp Pharmacol Physiol Suppl 6:129–134
48. Kaneko Y, Tsukamoto A (1994) Thapsigargin-induced persis- tent intracellular calcium pool depletion and apoptosis in human hepatoma cells. Cancer Lett 79:147–155
49. Rosado JA, Rosenzweig I, Harding S, Sage SO (2001) Tumor necrosis factor-α inhibits store-mediated Ca2+ entry in the human hepatocellular carcinoma cell line. Am J Physiol Cell Physiol 280:C1636–C1644
50. Ali ES, Rychkov GY, Barritt GJ (2019) Deranged hepatocyte intracellular Ca2+ homeostasis and the progression of non- alcoholic fatty liver disease to hepatocellular carcinoma. Cell Calcium 82:102057. https://doi.org/10.1016/j.ceca.2019.10205 7
51. Yamaguchi M, Sakurai T (1991) Inhibitory effect of calcium-bind- ing protein regucalcin on Ca2+-activated DNA fragmentation in rat liver nuclei. FEBS Lett 279:281–284
52. Tsurusaki Y, Yamaguchi M (2004) Role of regucalcin in liver nuclear function: Binding of regucalcin to nuclear protein or DNA and modulation of tumor-related gene expression. Int J Mol Med 14:277–281
53. Mori S, Yamaguchi M (1990) Hepatic calcium-binding pro- tein regucalcin decreases Ca2+/calmodulin-dependent protein kinase activity in rat liver cytosol. Chem Pharm Bull (Tokyo) 38:2216–2218
54. Yamaguchi M, Mori S (1990) Inhibitory effect of calcium-binding protein regucalcin on protein kinase C activity in rat liver cytosol. Biochem Med Metab Biol 43:140–146
55. Murata T, Yamaguchi M (1997) Molecular cloning of the cDNA coding for regucalcin and its mRNA expression in mouse liver: The expression is stimulated by calcium administration. Mol Cell Biochem 173:127–133
56. Murata T, Yamaguchi M (1998) Ca2+ administration stimulates the binding of AP-1 factor to the 5’-flanking region of the rat gene for the Ca2+-binding protein regucalcin. Biochem J 329:157–163
57. Takahashi H, Yamaguchi M (1999) Role of regucalcin as an acti- vator of Ca2+-ATPase activity in rat liver microsomes. J Cell Bio- chem 74:663–669
58. Takahashi H, Yamaguchi M (2000) Stimulatory effect of regucal- cin on ATP-dependent Ca2+ uptake activity in rat liver mitochon- dria. J Cell Biochem 78:121–130
59. Yamaguchi M, Mori S, Kato S (1988) Calcium-binding protein regucalcin is an activator (Ca2+–Mg2+)-adenosine triphosphatase in the plasma membranes of rat liver. Chem Pharm Bull (Tokyo) 36:3532–3539
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