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Quercetin Enhances Phenformin in Inhibition of Cholangiocarcinoma Cell Growth

เควอซิทินเสริมฤทธิ์เฟนฟอร์มินในการยับยั้งการเจริญเติบโตของเซลล์มะเร็งท่อน้ำดี

Rattanaporn Jaidee (รัตนาภรณ์ ใจดี) 1, Sarinya Kongpetch (ศริญญา คงเพชร) 2, Auemduan Prawan (เอื้อมเดือน ประวาฬ) 3, Laddawan Senggunprai (ลัดดาวัลย์ เส็งกันไพร) 4, Upa Kukongviriyapan (ยุพา คู่คงวิริยพันธุ์) 5, Veerapol Kukongviriyapan (วีรพล คู่คงวิริยพันธุ์) 6




หลักการและวัตถุประสงค์: Phenformin และสารฟลาโวนอยด์ quercetin และ myricetin มีฤทธิ์ในการยับยั้งการเจริญเติบโตของเซลล์มะเร็งหลายชนิด งานวิจัยนี้ศึกษาความสามารถในการยับยั้งการเจริญเติบโตของเซลล์มะเร็งท่อน้ำดี เมื่อเซลล์ได้รับ phenformin ร่วมกับ quercetin และ myricetin

วิธีการศึกษา: งานวิจัยใช้เซลล์มะเร็งท่อน้ำดี KKU-M156 ศึกษาความเป็นพิษต่อเซลล์ด้วยเทคนิค sulforhodamine-B assay (SRB) และวิเคราะห์ความต่างศักย์ของเมมเบรนไมโทคอนเดรีย (Mitochondrial membrane potential; Dym) ด้วยใช้เทคนิค JC-1 assay

ผลการศึกษา: Phenformin สามารถยับยั้งการเจริญเติบโตของเซลล์ KKU-M156 และเมื่อให้ร่วมกับ quercetin แต่ไม่ใช่ myricetin สามารถเสริมฤทธิ์ phenformin ในการยับยั้งการเจริญเติบโตของเซลล์มะเร็ง Phenformin และ Quercetin เมื่อให้เดี่ยว ๆหรือให้ร่วมกันสามารถทำให้สูญเสียศักย์ไฟฟ้าระหว่างเมมเบรนของไมโทคอนเดรีย

สรุป: Quercetin สามารถเพิ่ม Phenformin ในการยับยั้งการเจริญของเซลล์มะเร็งท่อน้ำดี โดยกลไกที่เสริมเพิ่มการบาดเจ็บต่อไมโทคอนเดรียนำไปสู่การตายของเซลล์  ผลการศึกษานี้แสดงในเบื้องต้นว่า phenformin ร่วมกับ quercetin มีศักยภาพที่จะเพิ่มสรรถนะการรักษามะเร็งท่อน้ำดี

Background and Objective: Phenformin, and flavonoid compounds, quercetin and myricetin are effective against many types of cancers. This study evaluated the treatments of phenformin in combination with quercetin or myricetin in the inhibition of cholangiocarcinoma (CCA) cells.

Methods: Cholangiocarcinoma cells, KKU-156, were used in this study. Cell cytotoxicity was evaluated by sulforhodamine-B assay (SRB). Mitochondrial membrane potential (Dym) was analyzed by JC-1 assay.

Results: Phenformin significantly inhibited proliferation of cancer cells. Phenformin in combination with quercetin, but not myricetin, could increase the cytotoxicity of cancer cells. Phenformin, quercetin or in the combination induced the loss of mitochondrial transmembrane potential.

Conclusions: Quercetin increased phenformin in inhibition of cancer cell growth. The mechanism may be by the increase of induction of mitochondrial damage leading to cell death.  This finding suggests that phenformin in combination with quercetin has potential to increase efficacy of chemotherapy of cholangiocarcinoma.

 

Introduction

Cholangiocarcinoma (CCA) is an aggressive malignancy, arising from biliary epithelium of the bile duct. The prognosis of CCA is very poor due to very difficult in diagnosis and resistance to chemotherapy.1 The most effective treatment for CCA requires a complete surgical resection of the tumor. However, 5-year survival rate is still low and recurrence of cancer is still common.2 Moreover, combination of gemcitabine and cisplatin, the current standard chemotherapy in CCA, could offer only a small survival benefit.3 New therapeutic strategies are required to improve survival and quality of life in CCA patients.

The use of metformin in diabetes patients has been reportedly associated with reduced risk of CCA in a case-control study.4 Moreover, metformin and its congener, phenformin, have demonstrated antineoplastic in vitro and in vivo.5,6 Phenformin is a more potent antidiabetic agent and more lipophilic than metformin and does not require transporter to get enter into the cells.7 The molecular targets of phenformin is similar to metformin in that they stimulate AMP-activated protein kinase (AMPK) and suppress mTOR signaling pathway which lead to inhibit growth through mitochondria pathway.8,9

Quercetin and myricetin are flavonol compounds from the flavonoid group. Both compounds are commonly found in apples, grapes, broccoli and nuts. Quercetin and myricetin have been demonstrated antitumor activity in various cancers including CCA, breast and bladder cancers.10,11 Two compounds show several effects on cancer cells including the inhibition of glucose transport and glucose utilization.12 This effect may be complementary with effects of phenformin, since phenformin suppresses anabolic processes by decreased glucose utilization via activation of AMPK for the antitumor effect.13 Present study was to investigate whether these flavonols could enhance antitumor activity of phenformin.

Material and Methods

Chemicals and reagents

Phenformin hydrochloride, quercetin, and myricetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ham's F12 nutrient mixture and 0.25% trypsin-EDTA were obtained from Gibco® Life Technoloqies (Grand Island, NY, USA).  Fetal bovine serum (FBS) was purchased from HiMedia Laboratories (Mumbai, India). Sulforhodamine B (SRB) obtained from Sigma-Aldrich (St. Louis, MO, USA). JC-1 mitochondrial membrane potential assay kit was obtained from Cayman Chemical (MI, USA). Cyclosporine was purchased from Novartis Pharmaceutical (Basel, Switzerland).

Cell culture

Human CCA cell line, KKU-M156, were cultured in Ham's F12 medium supplemented with sodium bicarbonate, 10 mM N-2-hydroxyethyl-piperazine-N'-2-ethane­sulfonic acid (HEPES; pH 7.3), gentamycin sulfate (100 µg/L), penicillin G (100 U/L), and 10% (v/v) FBS. Cells were maintained under an atmosphere of 5% CO2 in air at 37°C. The cells were sub-cultured every 3 days using 0.25% trypsin-EDTA.

Cell viability assay

Sulforhodamine B (SRB) was used to determine cell viability. KKU-M156 cells were seeded onto 96-well plate with a density of 7.5x103 cells/well and allowed overnight for adhere. CCA cells were treated with various concentrations of phenformin, 20 µM quercetin, 25 µM myricetin, or the combinations for 24 h. The media was removed and the cultured cells were fixed with cold 10% trichloroacetic acid for 1 h at 4oC and stained with 0.4% SRB in 1% acetic acid 30 minute at room temperature. The excess dye was removed with 1% acetic acid and protein-bound dye was solubilized with 10 mM Tris for determination of absorbance using a microplate reader at a wavelength of 570 nm.

 

Mitochondrial membrane potential assay

Determination of the mitochondrial transmembrane potential (Dym) was performed using 5,5’,6,’-tetrachloro-1,1’,3,3’tetraethylbenzimidazolyl carbocyanine iodide (JC-1) assay, as previously described.14 KKU-M156 cells were seeded onto 96 black-well plate with density of 2x105 cells/well and incubated overnight for adherence. The media was then replaced with fresh media containing phenformin, quercetin 20 µM, and cyclosporine 50 nM and incubated for 6 h.  The culture media was replaced with 5 µM JC-1 staining solution in serum free media (dilution 1:20) and incubated at 37oC for 45 min. The cells were washed with JC-1 buffer and measured fluorescent signals using a fluorescent microplate reader. The excitation and emission wavelengths were set at 485/535 nm (cut-off 515 nm) for measurement of J-monomers and at 535/595 nm (cut-off 590) for J-aggregates.

Statistical analysis

All values were presented as mean ± SEM from three experiments using GraphPad Prism version 6.0 software (GraphPad Software, San Diego, CA, USA). Statistical differences between treatment and control groups were analyzed by one-way analysis of variance (ANOVA) with post-hoc test or Student’s t-test by SigmaStat version 3.11 software (Systat Software, San Jose, CA, USA). Statistical significance was preset at p<0.05.

 

Results

Antitumor activity of phenformin and flavonol compounds

The structure of phenformin, quercetin and myricetin were shown in Fig.1A. Phenformin was tested for cytotoxicity in KKU-156 cells. It was shown that phenformin induced cytotoxicity in concentration-response relation with median inhibitory concentration (IC50) of 1363±427 µM (Fig.2A). When phenformin was used in combination with quercetin or myricetin at varied concentrations, the cytotoxicity was significantly increased in co-treatment with quercetin, when compared with phenformin alone. But the combination of phenformin and myricetin did not increase cytotoxicity (Fig. 2B and C). Quercetin alone at the same concentration used in the combinations induced cytotoxicity by 27.5±3.6%, while myricetin showed cytotoxic effect by 14.8±2.4%.  It is apparent that quercetin has an additive cytotoxic effect to phenformin.

 

Figure 1 The structure of (A) phenformin, (B) quercetin, and (C) myricetin

 

Figure 2 Antiproliferative effect of phenformin in CCA cells. KKU-M156 cells were treated with various concentrations of (A) phenformin (Phen), (B) Phen in combinations with quercetin (QC) or with myricetin (MR) and incubated for 24 h. After treatment, the cell viability was determined using the SRB assay. Each bar represents the mean ± SEM of three independent experiments.  *p < 0.05 versus untreated control group, #p < 0.05 Phen alone versus the corresponding combination of Phen and QC.

 

 

Phenformin and quercetin induced mitochondrial dysfunction

As mitochondria are the powerhouse of energy production by oxidative phosphorylation in mitochondrial matrix and play central role in cell death, disturbance in mitochondrial function may lead to cell dysfunction and cell death.15 Phenformin induced the loss of mitochondria transmembrane potential Dym), as determined by JC-1 assay. In healthy mitochondria, JC-1 is taken up into the mitochondria and forming J-aggregates which emit the red-orange fluorescent signal. The damage mitochondria cannot accumulate JC-1, then JC-1 exists as J-monomer form in cytosol and emits the green fluorescent signal. The ratio of red and green fluorescent signals is an indicative of the status of Dym. It was apparent that phenformin induced decline of the ratio of J-aggregates/J-monomers in concentration response manner (Fig.3A and B). The loss of Dym, was even exaggerated in the presence of quercetin. It is noted that quercetin itself also induced the loss of Dym,.

To examine whether the change in Dym, was due to the opening of the mitochondrial permeability transition pore (MPTP), which plays critical role in maintenance of mitochondrial transmembrane potential. Cyclosporine A, a MPTP inhibitor, was used in the study. Cyclosporine could partial prevent the loss of ym, induced by phenformin or quercetin or the combinations of phenformin and quercetin (Fig. 4 and 5A, B). This indicates that the depolarization of mitochondria is due to the opening of MPTP.

 

Figure 3 Mitochondrial transmembrane potential (ym) in CCA cells treated with phenformin and quercetin. KKU-M156 cells were treated with phenformin (Phen) and combination with quercetin (QC) for 6 h. After treatment, ym was determined by JC-1 assay. (A) Representatives of images of KKU-M156 treated with Phen and QC. (B) Bar graph of fluorescent signal of red/green ratio. Each bar represent the mean ± SEM from three independent experiments. *p < 0.05 versus untreated control group, #p < 0.05 Phen alone versus the combination of Phen and QC.

 

Figure 4 Mitochondrial transmembrane potential (ym) in cells treated with phenformin and cyclosporine. KKU-M156 cells were treated with phenformin (Phen) and cyclosporine (CS) for 6 h. After treatment. ym was determined using the JC-1 assay. (A) Representatives of images of KKU-M156 after treatment. (B) Bar graph of fluorescent signal of red/green ratio. Each bar represents the mean ± SEM from three independent experiments. *p < 0.05 versus untreated control group, #p < 0.05 Phen alone versus the combination of Phen and QS.

 

Figure 5 Mitochondrial transmembrane potential (ym) in cells treated with phenformin, quercetin and cyclosporine. KKU-M156 cells were treated with phenformin (Phen), quercetin (QC), and cyclosporine (CS) for 6 h. After treatment, ym was measured by the JC-1 assay. (A) Representatives image of KKU-M156 after treatment. (B) Bar graph of fluorescent signal of red/green ratio. Each bar represents the mean ± SEM from three independent experiments. *p < 0.05 versus untreated control group, #p < 0.05 combination of Phen and QC versus the combination of Phen, QC and QS.

 

Discussion

Phenformin inhibits several cancer growth including some resistant cancer cells.13,16 The mechanism of action is suggested to be mediated via AMPK-mTOR in the suppression of anabolic process and cause oxidative stress through mitochondrial pathway.13,16 Quercetin and myricetin both potently inhibit glucose transport in rat adipocytes.12 It was then suggested that interference of glucose uptake by these flavonols could enhance phenformin in suppression of tumor cell growth, since tumor cells generally requires high glucose for their proliferation and energy metabolism.17 However, only quercetin shows enhanced cytotoxicity of phenformin in CCA cells. Differential effect between quercetin and myricetin may be that quercetin, but not myricetin could affect glucose uptake in HepG2 cells.18 It is probable that effects on glucose transport is cell type-dependent and needs further investigation. Moreover quercetin is among flavonoid derivatives which show to be a potent AMPK activators and could suppress mTOR pathway.10

The present study showed that only quercetin significantly enhances the cytotoxicity of phenformin. The inhibition of glucose transport may be one among many other effects which mediate antiproliferation of cancer cells. Phenformin causes the loss of Dym, which is due to the opening MPTP and that leads to the outer mitochondrial membrane permeabilization and consequence to cell death.15 The effect via mitochondrial is validated by using cyclosporine, a MPTP inhibitor.19 Cyclosporine can partially inhibit the loss of Dym. The effect of phenformin on mitochondrial function is shown to be similar to metformin which leads to oxidative stress and cell death.20 Moreover, quercetin alone induces the loss of Dym, which the effect is validated by cyclosporine. The enhanced antitumor effect of quercetin may be by enhanced dysfunction of mitochondria with consequence of increased cell death.

It is concluded that quercetin increases phenformin-induced cell death by intensification of mitochondrial damage which leads to cell death. Quercetin in combination with phenformin increase efficacy of antitumor activity and could be a possible effective strategy in cancer chemoprevention.

Acknowledgements

This study was granted by Faculty of Medicine, Khon Kaen University, Thailand (Grant Number IN62302).

References

1.   Khan AS, Dageforde LA. Cholangiocarcinoma. Surg Clin North Am 2019; 99: 315-35.

2.   Banales JM, Cardinale V, Carpino G, Marzioni M, Andersen JB, Invernizzi P, et al. Expert consensus document: Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA). Nat Rev Gastroenterol Hepatol 2016; 13: 261-80.

3.   Valle J, Wasan H, Palmer DH, Cunningham D, Anthoney A, Maraveyas A, et al. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. The New England journal of medicine 2010; 362: 1273-81.

4.   Chaiteerakij R, Yang JD, Harmsen WS, Slettedahl SW, Mettler TA, Fredericksen ZS, et al. Risk factors for intrahepatic cholangiocarcinoma: association between metformin use and reduced cancer risk. Hepatology 2013; 57: 648-55.

5.   Appleyard MV, Murray KE, Coates PJ, Wullschleger S, Bray SE, Kernohan NM, et al. Phenformin as prophylaxis and therapy in breast cancer xenografts. Br J Cancer 2012; 106: 1117-22.

6.   Wandee J, Prawan A, Senggunprai L, Kongpetch S, Tusskorn O, Kukongviriyapan V. Metformin enhances cisplatin induced inhibition of cholangiocarcinoma cells via AMPK-mTOR pathway.  Life Sci 2018;  207: 172-83.

7.   Rajeshkumar NV, Yabuuchi S, Pai SG, De Oliveira E, Kamphorst JJ, Rabinowitz JD, et al. Treatment of Pancreatic Cancer Patient-Derived Xenograft Panel with Metabolic Inhibitors Reveals Efficacy of Phenformin. Clin Cancer Res 2017; 23: 5639-47.

8.   Vancura A, Bu P, Bhagwat M, Zeng J, Vancurova I. Metformin as an Anticancer Agent. Trends Pharmacol Sci 2018; 39: 867-78.

9.   Wang Y, Meng Y, Zhang S, Wu H, Yang D, Nie C, et al. Phenformin and metformin inhibit growth and migration of LN229 glioma cells in vitro and in vivo. Onco Targets Ther 2018;11: 6039-48.

10.  Tao T, He C, Deng J, Huang Y, Su Q, Peng M, et al. A novel synthetic derivative of quercetin, 8-trifluoromethyl-3,5,7,3',4'-O-pentamethyl-quercetin, inhibits bladder cancer growth by targeting the AMPK/mTOR signaling pathway.  Oncotarget 2017; 8: 71657-71.

11.  Tuponchai P, Kukongviriyapan V, Prawan A, Kongpetch S, Senggunprai L. Myricetin ameliorates cytokine-induced migration and invasion of cholangiocarcinoma cells via suppression of STAT3 pathway. J Cancer Res Ther 2019; 15: 157-63.

12.  Strobel P, Allard C, Perez-Acle T, Calderon R, Aldunate R, Leighton F. Myricetin, quercetin and catechin-gallate inhibit glucose uptake in isolated rat adipocytes. Biochem J 2005; 386(Pt 3): 471-8.

13.  Lea MA, Pourat J, Patel R, desBordes C. Growth inhibition of colon cancer cells by compounds affecting AMPK activity. World J Gastrointest Oncol 2014; 6: 244-52.

14.  Suphim B, Prawan A, Kukongviriyapan U, Kongpetch S, Buranrat B, Kukongviriyapan V. Redox modulation and human bile duct cancer inhibition by curcumin. Food Chem Toxicol 2010; 48(8-9): 2265-72.

15.  Halestrap AP. What is the mitochondrial permeability transition pore? J Mol Cell Cardiol 2009;46(6):821–31.

16.  Yuan P, Ito K, Perez-Lorenzo R, Del Guzzo C, Lee JH, Shen CH, et al. Phenformin enhances the therapeutic benefit of BRAF(V600E) inhibition in melanoma. Proc Natl Acad Sci U S A  2013; 110(45): 18226-31.

17.  Dandapani M, Hardie DG. AMPK: opposing the metabolic changes in both tumour cells and inflammatory cells? Biochem Soc Trans 2013; 41: 687-93.

18.  Kerimi A, Jailani F, Williamson G. Modulation of cellular glucose metabolism in human HepG2 cells by combinations of structurally related flavonoids. Mol Nutr Food Res 2015; 59: 894-906.

19.  Bernardi P, Di Lisa F. The mitochondrial permeability transition pore: molecular nature and role as a target in cardioprotection. J Mol Cell Cardiol 2015; 78: 100-6.

20.  Wandee J, Prawan A, Senggunprai L, Kongpetch S, Kukongviriyapan V. Metformin sensitizes cholangiocarcinoma cell to cisplatin-induced cytotoxicity through oxidative stress mediated mitochondrial pathway. Life Sci 2019; 217: 155-63.

 

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