Background
Hepatocellular carcinoma (HCC) is the seventh most common cancer and the third leading cause of cancer death worldwide, with few therapeutic options [
1]. The American Cancer Society indicates that there are more than 600,000 deaths and over 700,000 new cases of primary liver cancer in the world each year [
2]. The treatment of HCC continues to be a challenge; the outcome of traditional surgical treatment is poor with 20% survival at 1 year, 5% at 3 years, and a median survival of 8 months [
3]. Although chemotherapy is of considerable benefit to patients with HCC, it is associated with significant side-effects; hence, highlighting the need for therapeutic strategies that target tumor cells without causing cytotoxicity in healthy hepatocytes [
4,
5].
Berberine (BBR) is an isoquinoline alkaloid, which can be isolated from a variety of naturally occurring plants such as
Coptidis rhizoma,
Phellodendron chinense schneid, and
Phellodendron amurense [
6]. BBR has an anti-tumor effect on many cancers including melanoma, neuroblastoma, lung cancer, colonic carcinoma, breast cancer, and HCC [
7‐
12]. BBR acts both in vitro and in vivo to suppress human cancer cell growth via suppression of tumor cell proliferation, induction of tumor cell apoptosis, and inhibition of both invasion and metastasis [
6,
13]. In HCC, BBR inhibits proliferation and migration as well as induces cell cycle arrest and apoptosis [
8,
14‐
19]. However, BBR demonstrates very low to no cytotoxic effect on healthy liver tissue [
18]. In addition, BBR appears to have a protective effect on healthy liver tissue specifically protective against chemically-induced hepatotoxicity [
20].
Many tumor suppressor genes and oncogenes are related to HCC tumorigenesis. Expression of tumor suppressor genes including Kruppel-like factor 6 (KLF6) [
21,
22], activating transcription factor 3 (ATF3) [
23], and the cyclin-dependent kinase inhibitor protein
p21 [
24] have been found to be reduced in HCC. KLF6 is down-regulated in several types of cancers, and overexpression of wild-type KLF6 inhibits HCC cells proliferation and migration [
21,
25,
26], while KLF6 down-regulation by siRNA increases HepG2 proliferation [
22]. ATF3 promotes cell death, cell arrest and suppresses Ras-mediated tumorigenesis [
27]. In HCC, Niclosamide induces cell apoptosis via upregulation of ATF3 and activation of pERK [
28]. p21 has been found to inhibit DNA synthesis and proliferation in human liver cancer cells [
24,
29]. Alternatively, oncogenes pituitary tumor transforming gene 1 (PTTG1) [
22,
30,
31] and E2F transcription factor 1 (E2F1) [
32] are overexpressed in HCC. Reduced expression of PTTG1 decreases cell proliferation and induces apoptosis in HCC cells [
22,
31], while overexpression of E2F1 promotes HCC cell growth and invasion [
33].
The above findings suggest that BBR is a promising candidate for the treatment of HCC. However, the molecular mechanisms underlying the anti-neoplastic action of BBR in HCC are not fully understood. It is possible that BBR acts by modulating these tumor suppressor genes and oncogenes known to play a role in HCC, a hypothesis tested in the present study.
Methods
HCC cell lines
HepG2 (Cat# HB-8065), Hep3B (Cat# HB-8064) and SNU-182 (Cat# CRL-2235) were purchased from ATCC (Manassas, VA, USA). HepG2 and Hep3B cells were cultured in medium Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). SNU-182 cells were cultured in medium RPMI 1640 with 10% FBS. All cells were cultured at 37 °C in a humidified chamber with 95% air and 5% CO2.
Chemicals
BBR (Cat# B3251) and PD98059 (Cat# sc-3532) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Santa Cruz Biotechnology (Dallas, TX, USA). The concentration of stocks are 10 mM in water for BBR and 25 mM in DMSO (Sigma-Aldrich, St. Louis, MO, USA) for PD98059.
Western blots
Cells were seeded in 12 wells plate with cell number 1 × 105 per well overnight and then treated PD98059, BBR or both for 24 h. After treated, samples were lysed with a lysis buffer, and protein concentrations were determined by using coomassie blue method. Forty µg of total protein were separated on SDS-PAGE gels and then transferred to PVDF membranes. Membranes were immunoblotted with the appropriate primary antibodies (KLF6, ATF3, p21, E2F1, PTTG1, total ERK1/2, and phosphor-ERK1/2) (Santa Cruz, Dallas, TX, USA) at 4 °C overnight. After washing, membranes were incubated with a secondary antibody (Jackson ImmunoResearch Laboratory, West Grove, PA, USA), detected with chemiluminescence reagent (Thermoscientific, Hampton, NH, USA) and exposed by autoradiography.
Cell viability assay
Cell viability was determined by using the CellTiter Assay (MTS) kit (Promega, Madison, WI, USA). Cells were trypsinized and seeded 5000 cell/well into 96-well plates and incubated overnight in DMEM with 10% FBS in CO2 incubator. After overnight incubation, cells were treated with or without BBR for 24 or 72 h in DMEM with 5% FBS. Prior to conducting the cell viability assay, cells were washed with PBS twice and incubated in PBS 100 μl/well. Twenty microliters of CellTiter solution was added to each well. Cells were incubated in CO2 incubator for 2 h. Absorbance was determined with a microplate reader at 490 nm.
Cell number count
Cells were trypsinized and seeded 40,000 cells/well into 12-well plates and incubated overnight in DMEM with 10% FBS in CO2 incubator. After overnight incubation, cells were treated with or without BBR for 72 h in DMEM with 5% FBS. After 72 h treatment, cells were trypsinized, and Cell number were counted by the hemocytometer under a microscope. Dead cells were excluded by Trypan Blue stain.
ERK1/2 stimulation and PD98059 experiments
Cells were pretreated with dimethyl sulfoxide (DMSO, solvent of PD98059 used as a control) or PD98059 25 µM for 30 min then treated with or without BBR 100 µM for 24 h. Cell viability was analyzed by MTS assay. ERK1/2 phosphorylation, and BBR- regulated protein expression was analyzed by western blot.
Statistical analysis
Comparisons of multiple groups were carried out by analysis of variance (ANOVA), followed by a post-test using the Fisher (among groups) or Dunnett (compared with control group) tests (XLSTAT Software, New York, NY, USA). A p < 0.05 was considered statistically significant. All experiments were repeated 3 times (n = 3). All values are presented as mean ± SEM.
Discussion
Overall, we confirmed that BBR inhibited HepG2 and Hep3B cell proliferation [
17,
18,
34]. In addition, we demonstrated that BBR inhibited cell proliferation of SNU-182 cells. However, HepG2 cells appeared to be the most sensitive, while SNU-182 was the least sensitive to BBR treatment. For example, 100 µM of BBR treatment for 24 h inhibited cell viability by approximately 80% in HepG2 cells, while the same concentration and duration of BBR inhibited cell viability by about 40 and 50% in Hep3B and SNU-182 cells, respectively. In literature, cancer cell line with p53 gene deleted was reported to be more resistant to drug treatment [
35]. Hep3B is a p53 deficient cell line, thus it is not surprised that Hep3B is more resistance to BBR treatment than HepG2. This finding is consistent with previously reported results [
34]. In addition, HepG2 and Hep3B are “well-differentiated”, while SNU-182 is a “poorly-differentiated” HCC cell line [
36]. Our results suggest that “poorly-differentiated” HCC cells is less sensitive to BBR treatment.
In addition to cell proliferation, BBR also regulates gene expression differently between these three HCC cell lines. BBR stimulated expression of three tumor suppressor genes, KLF6, ATF3 and p21, and reduced two oncogenes E2F1 and PTTG1 in HepG2 cells, while BBR just induced ATF3 and reduced E2F1 expression in Hep3B cells. As HepG2 expresses wild type p53 and Hep3B is a p53-deficient HCC cell line, these results suggest that BBR regulation of KLF6, p21 and PTTG1 expression is possibly p53 dependent. Indeed, BBR has been found to up-regulate miR-23a via regulation of p53 [
37]. In contrast, BBR reduced expression of ATF3 tumor suppressor genes and also oncogenes E2F1 and PTTG1 in SNU-182 cells. BBR regulated different genes in SNU-182 as compared to HepG2 and Hep3B cells. This difference may be explained by SNU-182’s poorly-differentiated cell line. However, this discordance i.e., the effect of BBR on tumor suppressor genes and oncogenes expression, may also explain the observed differences in the response of cell proliferation to BBR between cell lines.
The ERK1/2-specific inhibitor, PD98059 partially blocked BBR-induced inhibition of cell proliferation in HCC cell lines, suggesting that activation of the ERK1/2 pathway is involved in BBR-inhibited cell proliferation. Indeed, Aspafilioside B, a steroidal saponin extracted from Asparagus filicinus and a known active cytotoxic component, has been shown to induce apoptosis via ERK1/2 activation in HepG2 cells [
38]. In addition, PD98059 completely blocked BBR-induced KLF6 and ATF3 expression in HepG2 and Hep3B cells, respectively suggesting that activation of the ERK1/2 pathway is involved in BBR’s-regulation of gene expression in the HCC cell line. However, PD98059 did not block BBR-reduced E2F1 and PTTG1 expression, suggesting that BBR mediated regulation of E2F1 and PTTG1 are independent of ERK1/2 pathway. These results also indicate that ERK1/2 is not the only signaling pathway under BBR regulation.
DMSO, the solvent for PD98059, has been discovered to induce p21 expression in B cell lines [
39]. When we did PD98059 experiments, we noticed that DMSO alone also increased p21 protein expression in HepG2. Furthermore, our data indicated that DMSO reversed the effects of BBR on p21 protein expression from stimulation became inhibition. Our data suggest that DMSO effects and interaction with BBR may need to be considerate when doing experiments that are involved DMSO and BBR.
KLF6 has been reported to upregulate p21 [
40] and ATF3 [
41], but suppress PTTG1 [
22] expression in cancer cells. However, in our experiments, PD98059 completely blocked BBR-induced KLF6 expression, but did not block BBR-regulated ATF3 and PTTG1 expression in HepG2 cells. These results suggest that BBR-regulated ATF3 and PTTG1expression was not through KLF6 regulation.
Authors’ contributions
TYC and YHC designed and performed the study, analyzed the data, and wrote the manuscript; HLW and JM performed the study; MD and RA reviewed and edited the manuscript. All authors read and approved the final manuscript.