Jatrorrhizine inhibits liver cancer cell growth by targeting the expressions of miR-221-3p and miR-15b-5p

Purpose: To investigate the effect of jatrorrhizine on hepatic cancer cell proliferation and its mechanism of action. Methods: Jatrorrhizine-mediated changes in cell viability were measured using (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while apoptosis induction was evaluated by flow cytometry. Transwell assay was used for the measurement of cell invasion, whereas cell migration was assessed by wound healing assay. The protein expression of Axin2 was determined with western blotting assay. Results: Jatrorrhizine significantly (p < 0.049) suppressed the viability of HepG2 and HCCLM3 cells in the concentration range of 0.5 to 16.0 μM. Treatment of HepG2 and HCCLM3 cells with 4.0 μM jatrorrhizine markedly suppressed cell invasion, when compared to untreated cells (p < 0.0493). Jatrorrhizine significantly promoted the apoptosis of HepG2 and HCCLM3 cells at 48 h, relative to untreated cells, but 16.0 μM jatrorrhizine markedly suppressed the expressions of miR-221-3p and miR-15b-5p (p < 0.0493). Moreover, jatrorrhizine significantly up-regulated the protein expressions of Axin2 in HepG2 and HCCLM3 cells at 48 h (p < 0.0493). Conclusion: Jatrorrhizine inhibits the proliferation, and suppressed the invasiveness and migration of HepG2 and HCCLM3 liver cancer cells, but increases their apoptosis. Moreover, it down-regulates the expressions of miR-221-3p and miR15b-5p and promotes Axin2 protein expression in HepG2 and HCCLM3 cells. Therefore, jatrorrhizine is a potential drug candidate for the treatment of liver cancer.


INTRODUCTION
Liver cancer is a frequently diagnosed gastrointestinal tract tumor which ranks third in cancer-related mortality world-wide [1]. The survival of liver cancer patients after hepatic resection is unsatisfactory because of fast tumor recurrence and metastasis [2]. It has been demonstrated that the pathogenesis of liver cancer is highly complicated due to the involvement of multiple genes and proteins [3].
MicroRNAs (miRNAs) which are endogenously present in cells, are non-coding and singlestranded molecules made up of 18 -25 nucleotides. They function in RNA silencing and post-transcriptional regulation of gene expression [4]. It has been reported that the expressions of miR-181 family members were elevated in liver cancer stem cells extracted from α-fetoprotein (AFP)-positive hepatic carcinoma samples [5]. Comparison of miR-21 levels in serum of liver carcinoma patients, chronic hepatitis cases and normal volunteers revealed markedly up-regulated miR-21 expression in hepatic cancer patients [6]. It has been reported that miR-221 expression was abnormally increased in different kinds of cancers such as hepatic, breast and gastric carcinomas [7]. This indicates that miR-221 plays important role in the pathogenesis of liver cancer, and may act as prominent target for the treatment of different cancers. Another member of miR-181 family, miR-15b has also been linked to multiple kinds of cancers, for example, proliferation of glioma cells in the form of miR15b/HOTAIR/p53 regulatory loop [8]. It suppressed the expression of cysteine-rich protein, thereby promoting the proliferation of prostate cancer cells [9]. Jatrorrhizine ( Figure 1) and many other related compounds are present in Tinospora capillipes Gagnep [10][11][12]. Studies have revealed the mitigating effect of jatrorrhizine and its prominent bioactivity profile against several disorders [10][11][12]. Jatrorrhizine and related compounds exert many pharmacological effects such as anticancer, anti-microbial and antiparasitic properties [10][11][12]. The current study was carried out to investigate the effect of jatrorrhizine on hepatic cancer cell proliferation, and the associated mechanism of action. The HepG2 and HCCLM3 cell lines were procured from the American Type Culture Collection (Chinese Academy of Sciences). The cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) containing 10 % FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL) in a 5 % CO2 incubator at 37 ˚C.

Cell proliferation assay
The cell lines were separately seeded in 96-well plates at a density of 2 x 105 cells/well and cultured for 24 h, followed by 48-h incubation with jatrorrhizine at doses of 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 µM at 37 o C. Thereafter, 20 µL of MTT solution (5 mg/mL) was added to each well of the plate, and further incubation of cells was carried out for 4 h. The resultant formazan crystals were solubilized with dimethyl sulfoxide (DMSO, 150 µL). The well plates were shaken, and the absorbance of each well was read at 487 nm using a scanning multi-well spectrophotometer. The viability of each cell line was calculated from the absorbance values.

Flow cytometry
The HepG2 and HCCLM3 cells were plated, each at a density of 2 x 10 6 cells/well, and treated with 16.0 µM jatrorrhizine for 48 h, followed by washing in PBS. For apoptosis determination, the cells were fixed overnight in 70 % methyl alcohol at 20 ˚C prior to washing in PBS. Thereafter, the cells were stained with Annexin V/PI and examined using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Cell invasion assay
The inhibitory effect of jatrorrhizine on invasiveness of cells was determined using 24well Transwell plates (8 mm pore size). Coating of chamber inserts with 200 mg/mL Matrigel was followed by drying overnight under sterile conditions. The HepG2 and HCCLM3 cells were treated with 16.0 µM jatrorrhizine for 48 h, after which 2 x 10 6 cells were put in the top chamber in RPMI-1640 medium. The lower chamber contained RPMI-1640 along with 20 % FBS. The cells were incubated for 48 h, followed by cleaning of the top chamber with cotton swab and subsequent fixing of cells in methyl alcohol. Then, the cells were stained with hematoxylin and eosin for 25 min, and the invaded cells on the underside of the membrane were counted under a light microscope (Olympus Corporation, Tokyo, Japan).

Wound healing assay
The cells were seeded in 6-well plates at a density of 2 x 10 5 cells/well, and were cultured for 24 h to >95 % confluence. A wound was scratched at the middle of the cellular monolayer of each cell line, using the tip of a 100-ml plastic micropipette. Free cells were washed off with PBS, after which the the cells were incubated for 48 h with 16.0 µM jatrorrhizine at 37 ˚C. Wound healing was measured by calculation of width of the original scratch using NIH Image version 1.62 programs. The measurement was made by randomly capturing images of five selected fields.

Western blotting
The HepG2 and HCCLM3 cells seeded at density of 2 x 10 6 cells/well were incubated with 16.0 µM jatrorrhizine for 48 h. Then, total protein content of the cells was extracted with RIPA lysis buffer along with protease inhibitors, in accordance with the instructions from the manufacturer. Each cell lysate was subjected to centrifugation at 12000 x g for 30 min, and the protein content of the supernatant was determined using the bicinchoninic acid method. The proteins were resolved on 12 % SDSpolyacrylamide gel electrophoresis (30-µg protein samples per lane), followed by transfer onto PVDF membrane which was blocked by treatment with 5 % skimmed milk and TBS for 2 h. Then, the membrane was incubated overnight at 4 o C with primary antibodies against Axin2 (catalog no. ab32197 Abcam; dilution 1:1,000) and β-actin (catalog no. ab37168 Abcam; dilution 1:5,000), after which the membrane was washed twice with TBS + Tween-20. Then, the membrane was incubated for 2 h with horseradish peroxidase-conjugated secondary antibody diluted 1:4,500 (Sigma-Aldrich) at room temperature. The bands were visualized using enhanced chemiluminescence system, with GAPDH as loading control.

Statistical analysis
Data are expressed as mean ± standard deviation of independently performed triplicate experiments, and data analysis was done with SPSS 17.0 (SPSS, Inc.). Multiple group data comparisons were made using one-way ANOVA and Student-Newman-Keuls test. Differences were taken as statistically significant at p < 0.05.

Jatrorrhizine inhibited the invasion and migratory potential of HepG2 and HCCLM3 cells
In HepG2 and HCCLM3 cells, treatment with jatrorrhizine at a dose of 4.0 µM significantly suppressed their invasion, when compared to untreated cells (p < 0.0493; Figures 3 A and  3B). Treatment of HepG2 cells with 4.0 µM jatrorrhizine for 48 h suppressed their invasive potential to 32 %, relative to 100 % in untreated cells. Treatment of HCCLM3 cells with 4.0 µM jatrorrhizine suppressed invasion to 36 %, relative to 100 % in control cells. Jatrorrhizine treatment of HepG2 and HCCLM3 cells for 48 h markedly reduced wound healing (p < 0.0493), when compared to control cells (Figures 4A and  4B). Wound healing in 4.0 µM jatrorrhizinetreated HepG2 and HCCLM3 cells decreased to 21 and 26 %, respectively, relative to control cells.

Jatrorrhizine up-regulated Axin2 expression in HepG2 and HCCLM3 cells
In jatrorrhizine-treated HepG2 and HCCLM3 cells, the expression of Axin2 protein was assayed with western blotting (Figure 7). Jatrorrhizine significantly enhanced Axin2 protein expression in HepG2 and HCCLM3 cells at 48 h, relative to untreated cells.

DISCUSSION
Liver cancer is considered the third most common tumor of the gastrointestinal tract, and it is associated with high mortality worldwide [1,14]. It has been demonstrated that miRNAs play crucial role in the pathogenesis of liver cancer and enhancement of its aggressiveness [15]. Studies have reported that in liver cancer tissues, miR-221 and miR-15b levels were abnormally elevated and demonstrated oncogenic characteristics by enhancing tumor growth and progression [16,17]. Indeed, the expressions of miR-221-3p and miR15b-5p have been reported to be associated with several types of cancers. The oncogenic property of miRNA-221-3p in gastric cancers is associated with down-regulation of PTEN expression [18].
Metastatic potential in gastric cancers is promoted by miRNA-221-3p via interaction with progestin and adipoQ receptors, thereby acting as gastric cancer biomarker [19]. Although the mechanism by which miRNAs enhances tumor growth is not clearly understood, miR-221 and miR15b are considered important targets for cancer inhibition and diagnosis. In the present study, the proliferation capacities of HepG2 and HCCLM3 cells were suppressed concentrationdependently by jatrorrhizine. Moreover, in jatrorrhizine-treated HepG2 and HCCLM3 cells, invasiveness and wound healing abilities were inhibited, when compared to control. The present study demonstrated that levels of miR-221-3p and miR-15b-5p were markedly increased in HepG2 and HCCLM3 cancer cells. However, jatrorrhizine treatment markedly suppressed the expressions of miR-221-3p and miR-15b-5p in HepG2 and HCCLM3 cells, when compared to control. These findings indicate that jatrorrhizine mediated inhibition of viabilities of HepG2 and HCCLM3 cells by targeting the expressions of miR-221-3p and miR-15b-5p.
Axin2 acts a negative regulator of Wnt/β-catenin pathway and degrades complexes of β-catenin after de-phosphorylation [20]. It is an antioncogene which regulates tumor growth by interacting with various proteins such as transmembrane receptors, T-cell factors and Wnt [20].
It has been revealed that digestive tract tumors and melanomas in humans are linked to the mutations in the Axin2 gene [21,22]. In the present study, Axin2 protein expressions in HepG2 and HCCLM3 cells were significantly upregulated by jatrorrhizine, relative to untreated cells which showed very weak expression of Axin2 protein.

CONCLUSION
This study has demonstrated that jatrorrhizine inhibits the proliferation of HepG2 and HCCLM3 liver cancer cells, increases their apoptosis and suppresses their invasiveness and migration. Furthermore, it down-regulates the expressions of miR-2213p and miR-15b-5p, but up-regulates protein expression of Axin2 in HepG2 and HCCLM3 cells. Therefore, jatrorrhizine offers a potential therapeutic strategy for the management of liver cancer.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that this work was done by the author(s) named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors. Maolin Wanconceived and designed the study; Bo Deng and Maolin Wan -collected, analyzed the data and wrote the manuscript. Both of the authors read and approved the manuscript for publication.

Open Access
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