Research ArticleExperimental Studies
Open Access

Flavokawain B Inhibits Growth of Cholangiocarcinoma Cells by Suppressing the Akt Pathway

JUN HYUK SON, YOUNG HOON CHOI, SANG HYUB LEE, WOO HYUN PAIK, JI KON RYU and YONG-TAE KIM
In Vivo May 2023, 37 (3) 1077-1084; DOI: https://doi.org/10.21873/invivo.13182

Abstract

Background/Aim: Cholangiocarcinoma is a devastating malignancy with limited treatment options and poor prognosis. Natural products have gained considerable attention for showing antitumor effects with less toxicities. Flavokawain B (FKB), a natural product, has been studied for its antitumor effects on various cancer cells. However, the anti-tumor effect of FKB on cholangiocarcinoma cells remains unknown. This study aimed at investigating the antitumor effect of FKB on cholangiocarcinoma cells in vitro and in vivo. Materials and Methods: SNU-478, a human cholangiocarcinoma cell line, was used in this study. Effects of FKB on cell growth inhibition and apoptosis were investigated. The synergistic anti-tumor effect of FKB and cisplatin in combination was also evaluated. Western blotting was performed to examine the underlying molecular mechanisms of the effect of FKB. A xenograft mouse model study was performed to investigate the effect of FKB in vivo. Results: FKB inhibited cell proliferation of cholangiocarcinoma cells in a concentration- and time-dependent manner. FKB also induced cellular apoptosis additively in combination with cisplatin. Akt pathway was suppressed by FKB either alone or in combination with cisplatin. In the xenograft model, FKB treatment in combination with cisplatin/gemcitabine significantly inhibited tumor growth of SNU-478 cells. Conclusion: FKB showed an antitumor effect through the induction of apoptosis, which was mediated by suppressing the Akt pathway in cholangiocarcinoma cells. However, the synergistic effect of FKB and cisplatin was not definite.

Key Words:

Cholangiocarcinoma is a cancer of the bile ducts, including intrahepatic, perihilar, and extrahepatic cholangiocarcinoma and gallbladder carcinoma. The overall incidence of cholangiocarcinoma in Korea was 14.4 per 100,000 people per year and the 5-year survival rate was only 28.5%, which was hardly improved in the past 20 years, referring to the 2019 Korea Central Cancer Registry data (1). Surgery is the only treatment that can provide a chance for a cure. However, only 20-30% of patients are candidates for curative surgery, because most patients are diagnosed at an advanced stage (2). Even after curative resection, most patients develop recurrence. Therefore, systemic chemotherapy is a very important treatment modality for cholangiocarcinoma. Currently, palliative systemic chemotherapy with gemcitabine and cisplatin is the standard of care for advanced cholangiocarcinoma, but the survival benefit is modest at about 3 months (3). Some target-oriented agents have been tried to achieve better outcomes; however, significant survival benefits have not been reported from many clinical trials (4). The underlying genetic variability and chemoresistance are major obstacles in the development of effective targeted agents (5).

Some natural dietary compounds have been known for possessing chemopreventive effects and for their ability to sensitize tumor cells. Many studies have attempted to overcome chemoresistance and induce apoptosis with less toxicities using chemopreventive natural compounds and some studies have shown beneficial effects (6).

Flavokawain B (FKB), known as 6′-hydroxy-2′,4′-dimethoxychalcone, is a natural chalcone which has been shown to have antioxidant, anti-inflammatory, and anticancer properties (7, 8). Many studies have shown that FKB has an cytocidal action against various cancer cell lines (9-21). However, the anticancer effect and molecular mechanism of FKB on cholangiocarcinoma cells are not well studied.

Therefore, the present study investigated the effect of FKB on cholangiocarcinoma cells in vitro and in vivo. The effect of the combination treatment of FKB with conventional cytotoxic chemotherapeutic agents was also examined and the mechanism of its action explored.

Materials and Methods

Cell culture and reagents. SNU-478, a human cholangiocarcinoma cell line, was purchased from Korean Cell Line Bank (Seoul, Republic of Korea). SNU-478 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640; Welgene, Gyeongsan, Republic of Korea) containing 10% fetal bovine serum at 37°C and 5% CO2. The culture medium was supplemented with 1% streptomycin and penicillin (Corning, Corning, NY, USA).

FKB (Abcam, Cambridge, MA, USA) was dissolved in DMSO, aliquoted and stored at −80°C. Antibodies against AKR mouse thymoma kinase (Akt), phosphor-Akt, cleaved Poly (ADP-ribose) polymerase (PARP), and beta-actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Cisplatin (Dong-A ST, Seoul, Korea) and gemcitabine (Yuhan, Seoul, Republic of Korea) were dissolved in sterile phosphate-buffered saline to produce 100-μmol/l stock solutions.

Cell viability MTT assay. SNU-478 cells were plated at a density of 4×103 cells/well in 96-well culture plates. After 24 h, cells were treated with DMSO 0.1% vehicle control, FKB, and/or cisplatin for 24, 48, and 72 h. After the treatment, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Promega, Madison, WI, USA) reagent was added to the wells and incubated at 37°C for 3 h. Thereafter, sodium dodecyl sulfate (SDS) solution was added to dissolve the formazan crystals. The absorbance was determined at 570 nm using Sunrise™ (Tecan, Mannedorf, Switzerland) microplate reader. The number of viable cells was calculated by the uptake and reduction of MTT comparing the treated cells to the control group.

Fluorescence-activated cell sorting (FACS) analysis of apoptosis. Apoptosis was analyzed using an annexin-V (AV) and propidium iodide (PI) (BD Biosciences, Franklin Lakes, NJ, USA) assay. SNU-478 cells were treated with 0.1% DMSO vehicle control, FKB, and/or cisplatin. Cells were suspended with 100 μl of Annexin-binding buffer (BD Biosciences). Following treatment, cells were stained with AV and PI solution at room temperature in the dark, for 15 min. Treated samples were analyzed with a flow cytometer (FACScanto II; BD Biosciences). The percentage of apoptotic cells was determined using FlowJo software (FlowJo LLC, Ashland, OR, USA).

Western blot analysis. After treatment, cells were harvested and lysed in lysis buffer containing protease inhibitors (Roche, Basel, Switzerland). Cell lysates were kept on ice for 15 min and then centrifuged at 16,000×g for 15 min. The BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine protein concentration. Volumes of clarified protein lysates containing the same amount of protein (30 μg) were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred onto polyvinylidene difluoride membrane (Amersham Biosciences, Amersham, UK). Blots were then blocked with 3% bovine serum albumin in PBS containing 1% Tween-20 (PBS-T) for 1 h at room temperature and incubated with primary antibodies overnight. Proteins were revealed using horseradish peroxidase-conjugated secondary antibody. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (ImageQuant LAS 4000; GE Healthcare, Uppsala, Sweden).

Mouse xenograft studies. All protocols for the animal studies were approved by the Institutional Animal Care and Use Committee at Seoul University Hospital (No.15-0116-S1A0). All animal procedures were consistent with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).

The mouse xenograft experiments were performed using four-week-old male BALB/c nude mice (Orient Co, Ltd, Gyeonggi-do, Republic of Korea). All mice were allowed to acclimate to a vivarium environment for 1 week prior to tumor cell inoculation. Subcutaneous inoculation with 1×106 SNU-478 cells suspended in Matrigel was performed to generate tumors. Treatment was initiated when the subcutaneous tumors reached a minimum of 100 mm3 in size.

Successfully generated tumor-bearing mice were assigned into the following four treatment groups and each group consisted of five mice: 1) control (vehicle alone); 2) FKB (intraperitoneal injection at 25 mg/kg, twice-a-week for 2 weeks); 3) cisplatin and gemcitabine (intraperitoneal injection of 5 mg/kg of cisplatin and 100 mg/kg of gemcitabine, twice-a-week for 2 weeks); and 4) cisplatin, gemcitabine and FKB (intraperitoneal injection of 5 mg/kg of cisplatin and 100 mg/kg of gemcitabine, and 25 mg/kg of FKB, twice-a-week for 2 weeks).

Tumor volumes were calculated twice-a-week using the ellipsoid volume formula (π/6×(length×width2) based on the tumor size, which was measured with calipers. One week following the last treatment, mice were sacrificed. Tumors were excised and their weight was measured with an electronic scale and tumor volume was calculated.

Statistical analysis. Data are expressed as mean±SEM. Differences between mean values of groups were analyzed using the Student’s t-test and regarded as significant when p<0.05. All analyses were performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA).

Results

FKB reduced cell viability of cholangiocarcinoma cells in combination with cisplatin. MTT assay was conducted to evaluate the antiproliferative effect of different concentrations (0-100 μmol/l) of FKB on SNU-478 cells for 72 h. FKB inhibited the growth of SNU-478 cells in a concentration-dependent manner and the half maximal inhibitory concentration (IC50) of FKB towards SNU-478 cells was estimated to be 69.4 μmol/l (Figure 1).

Figure 1.
Figure 1.

Flavokawain B (FKB) inhibited the growth of SNU-478 cells in a concentration-dependent manner as shown in MTT assay. The graph represents the means±SEM of three independent experiments.

To examine the synergistic effect of FKB with cisplatin on cell growth inhibition, SNU-478 cells were examined by using light microscopy at 72 h after treatment with cisplatin, FKB, or their combination. MTT assay was also performed at 24, 48, and 72 h after treatment (Figure 2). The concentration of each agent was chosen to induce moderate cell death. The combination treatment significantly reduced cell viability compared with the control (DMSO) at 24, 48, and 72 h (p<0.05). Survival of the cells treated with the combination of FKB and cisplatin was reduced by 52.4%, 34.7%, and 23.6% at 24, 48, and 72 h compared with the vehicle control, respectively. However, significant differences in cell viability among cells treated with FKB, cisplatin, and cisplatin+FKB at 24, 48, and 72 h were not observed.

Figure 2.
Figure 2.

Flavokawain B (FKB) and/or cisplatin inhibited the growth and colony formation of SNU-478 cells. (A) Morphological changes in cells treated for 72 h with FKB and/or cisplatin or DMSO (the vehicle control) were examined through light microscopy under 200× magnification. (B) MTT assays were used to evaluate cell viability at different incubation times (24, 48, and 72 h). The viability was calculated as the percentage viability relative to that of the vehicle control (DMSO)-treated cells. The graph represents the means±SEM of three independent experiments. *p<0.05 compared with DMSO treatment.

FKB in combination with cisplatin induced apoptosis of cholangiocarcinoma cells. The apoptotic effect of FKB on SNU-478 cells was further evaluated by flow cytometry after annexin V and PI staining. FKB and cisplatin alone significantly increased the proportion of early apoptotic (PI−/AV+) and late apoptotic/necrotic cells (PI+/AV+) compared with the control. Cisplatin induced early apoptosis more than late apoptosis. FKB induced late apoptosis more than early apoptosis. The combination treatment of FKB and cisplatin induced apoptosis more efficiently than cisplatin (p<0.05) or FKB alone (p=0.626). The mean rates of apoptosis in SNU-478 cells treated with DMSO, cisplatin, FKB and the combination treatment were 5.0%, 13.3%, 20.6%, and 21.8%, respectively at 24 h (Figure 3).

Figure 3.
Figure 3.

Flavokawain B (FKB) treatment caused apoptotic cell death in SNU-478 cells. (A) SNU-478 cells were stained with annexin V and propidium iodide and analyzed by flow cytometry after FKB/cisplatin treatment for 24 h. The results are representative of three independent experiments. (B) Percentage of early apoptotic cells (PI−/AV+), late apoptotic/necrotic cells PI+/AV+), and total apoptotic cells are displayed in the graph. Data represent the means±SEM of three independent experiments. *p<0.05 compared with DMSO treatment.

Combination treatment of FKB with cisplatin inhibited the growth of SNU-478 cells synergistically by inducing apoptosis through modulating the Akt pathway. In order to determine apoptosis in a different way, immunoblotting for PARP and cleaved PARP was performed. Moreover, immunoblotting for Akt and P-Akt was conducted to investigate whether FKB treatment affected the PI3K-Akt signaling pathway (Figure 4).

Figure 4.
Figure 4.

Combination treatment with Flavokawain B (FKB) and cisplatin inhibited the growth of SNU-478 cells synergistically by inducing apoptosis through modulating the Akt pathway. Cells were treated with FKB and/or cisplatin. The protein levels of PARP, cleaved PARP, Akt, and p-Akt were examined by western blotting.

The expression of cleaved PARP was increased with cisplatin or FKB single treatment, and was more noticeable with cisplatin. With the combination treatment, the increased expression of cleaved PARP was more prominent compared with that with FKB or cisplatin single treatment.

The immunoblotting of Akt and P-Akt showed that Akt activation was suppressed with FKB single treatment and combination treatment with FKB and cisplatin. Single-agent treatment with cisplatin also appeared to inhibit Akt activation, but not as much as FKB single treatment or the combination treatment of FKB and cisplatin.

These findings suggest that the combination treatment with FKB and cisplatin inhibited the growth of SNU-478 cells synergistically by inducing apoptosis through modulating the Akt pathway.

FKB showed antitumor effect on a xenograft model of cholangiocarcinoma. To determine the in vivo effect of FKB on cholangiocarcinoma, a SUN-478 subcutaneous xenograft model was established. Subcutaneous xenografts were excised and tumor volume and weight were measured at the end of the experiment (Figure 5A). The FKB single treatment group showed tumor growth inhibition (Figure 5B). The mean final tumor volume of the FKB treatment group was 347.5 mm3, whereas the mean tumor volume of the untreated group was 522.1 mm3, and there was no significant difference between the two groups (p=0.405). The cisplatin/gemcitabine treatment group showed significantly reduced final tumor volume compared with that of the untreated group (191.7 mm3 vs. 522.1 mm3, p<0.05). The combination treatment of cisplatin/gemcitabine with FKB exhibited tumor growth inhibition and resulted in a significant difference in the mean final tumor volume compared with that of the untreated group (159.5 mm3 vs. 522.1 mm3, p<0.05). However, a significant difference in the mean final tumor volume between the cisplatin/gemcitabine treatment group and the combination treatment of cisplatin/gemcitabine with FKB group was not observed (191.7 mm3 vs. 159.5 mm3, p=0.324).

Figure 5.
Figure 5.

Flavokawain B (FKB) showed an antitumor effect on a xenograft model of cholangiocarcinoma. Mice were sacrificed and tumors were excised (A). Tumor weight was measured and tumor volume calculated. The mean tumor volume over time (B) and the final mean tumor weight (C) are displayed in the graphs. *p<0.05.

The mean weights of the tumors in the cisplatin/gemcitabine treatment group and the combination treatment of cisplatin/gemcitabine with FKB group were significantly lower than that of the untreated group (p<0.05) (Figure 5C). However, a significant difference in the mean weight of the tumor between the cisplatin/gemcitabine treatment group and the combination treatment of cisplatin/gemcitabine with FKB group was not observed (p=0.170).

Discussion

FKB is a natural chalcone found in kava plant and many studies have shown that it has anti-cancer properties. In the current study, we evaluated the effect of FKB on SNU-478 cholangiocarcinoma cells in vitro and in vivo. The current standard of care for advanced cholangiocarcinoma is a palliative systemic cytotoxic chemotherapy with gemcitabine and cisplatin (22, 23). In our preliminary study, we showed that treatment of SNU-308, SNU-478, and SNU-1196 cholangiocarcinoma cell lines with gemcitabine and fluorouracil (5-FU) did not cause significant cell death, whereas cisplatin led to moderate cell death. Therefore, we selected cisplatin in this study.

In the current study, FKB was found to inhibit cholangiocarcinoma cell proliferation and induce apoptosis. However, significant differences in cell viability among cisplatin, FKB, and the combination of cisplatin and FKB treatment were not observed in the MTT assay. In the apoptosis assay, more apoptosis occurred with the combination of cisplatin and FKB treatment compared that with cisplatin or FKB single treatments. Taken together, the combination treatment induced more apoptosis compared with single-agent therapy; however, the difference was not statistically significant. We used 50 μmol/l of FKB in this study; however, the IC50 of FKB towards SNU-478 cells was about 70 μmol/l. The low concentration of FKB used in our study might have resulted in the unclear synergistic effect of cisplatin with FKB towards SNU-478 cells.

The PI3K/Akt signaling cascade is one of the most frequently dysregulated pathways in cancer. Dysregulation of the PI3K/Akt pathway has been implicated in the pathogenesis of many cancers including cholangiocarcinoma by promoting cell proliferation, tumorigenesis, and metastasis (24, 25). Akt is a serine-threonine kinase, which is activated by phosphorylation at two sites, threonine 308 and serine 473. Previous studies have shown that P-Akt expression was increased in >80% of extrahepatic cholangiocarcinoma (26) and >60% of intrahepatic cholangiocarcinoma (27, 28). Furthermore, treatment targeting Akt inhibited tumor cell growth by inducing apoptosis in cholangiocarcinoma (26, 28-30). FKB has been shown to have anti-tumor effects by affecting the PI3K/Akt pathway in several cancer cells, such as oral carcinoma (13), gastric cancer (18), and lung cancer cells (31).

In the current study, FKB was found to inhibit cholangiocarcinoma cell proliferation either alone or in combination with cisplatin by suppressing Akt activation. As far as we know, this is the first report that shows the antitumor effect of FKB in cholangiocarcinoma cells by modulating the Akt pathway.

Furthermore, we performed an in vivo study to verify the antitumor effects of FKB using SNU-478 subcutaneous xenograft model. The FKB treatment alone group showed a tendency to inhibit tumor growth compared to the control group, but there was no significant difference in the mean final volume and weight of tumors between the two groups. The combination treatment of cisplatin/gemcitabine with FKB group showed significant tumor growth inhibition and resulted in a significant reduction in tumor volume and weight compared with those of the control group. However, no significant differences were found in the mean final volume and weight of the tumors between the cisplatin/gemcitabine treatment group and the cisplatin/gemcitabine with FKB treatment group. Putting these results together, it could be inferred that FKB showed tendency to inhibit tumor growth in the SNU-478 xenograft model, but it did not show a definite antitumor effect. This may have occurred because the dose of FKB (25 mg/kg, 4 times/wk for 2 weeks) used in this study was low.

There have been few in vivo studies using FKB. In these, FKB was administered orally or via the intraperitoneal route. With the intraperitoneal route, FKB was injected up to 200 mg/kg daily for 28 days (32). Thus, the dose of FKB used in our study could have been low and could be related with the slight antitumor effect of FKB shown in our study. Furthermore, we sacrificed the mice at 2 weeks after the initiation of treatment. In the previous in vivo studies using FKB, treatment was continued for about 3-4 weeks (10, 32, 33). Therefore, our treatment duration may have been short. Meanwhile, we performed the in vivo study using cisplatin/gemcitabine with FKB as combination treatment; however, gemcitabine was not used in our in vitro study. This might have caused the inconsistent results of the effect of FKB between our in vitro and in vivo studies. Although we did not examine the mechanism of FKB action in our in vivo study, several previous studies demonstrated tumor growth inhibition in various cancer cell xenograft models with FKB treatment via various molecular mechanisms (8, 10, 18, 33, 34).

In conclusion, the results of our study suggest that FKB has an antitumor effect through the induction of apoptosis, which is mediated by suppressing the PI3K/Akt signaling pathway in cholangiocarcinoma cells. However, the synergistic effect of FKB and cisplatin was not definite. Further research is needed to verify the antitumor effect and mechanism of action of FKB in cholangiocarcinoma.

Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1A2A3075631).

Footnotes

  • Authors’ Contributions

    JHS and SHL designed the study. JHS conducted the experiments. JHS, YHC, SHL, WHP, JKR, and YK analyzed the experimental data. JHS, SHL, and WHP wrote the manuscript. All Authors read and approved the final manuscript.

  • Conflicts of Interest

    The Authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence this work.

  • Received December 26, 2022.
  • Revision received February 23, 2023.
  • Accepted February 28, 2023.

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).

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Flavokawain B Inhibits Growth of Cholangiocarcinoma Cells by Suppressing the Akt Pathway
JUN HYUK SON, YOUNG HOON CHOI, SANG HYUB LEE, WOO HYUN PAIK, JI KON RYU, YONG-TAE KIM
In Vivo May 2023, 37 (3) 1077-1084; DOI: 10.21873/invivo.13182
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