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Cite This: Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
Orlistat Displays Antitumor Activity and Enhances the Efficacy of Paclitaxel in Human Hepatoma Hep3B Cells Bang-Jau You,†,§ Li-Yun Chen,‡,§ Po-Hsiang Hsu,‡ Pei-Hsuan Sung,‡ Yu-Ching Hung,‡ and Hong-Zin Lee*,‡ †
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Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan ‡ School of Pharmacy, China Medical University, Taichung 404, Taiwan ABSTRACT: Orlistat has been proved to be an effective fatty acid synthase inhibitor that is able to inhibit the proliferation and induce apoptosis in many cancer cell types. However, the anticancer effects of orlistat on hepatocellular carcinoma are undefined. We found that orlistat inhibited cell growth and induced G0/G1 cell cycle arrest with increased cyclin D, cyclin E, and p21 expression in human hepatoma Hep3B cells. Furthermore, protein expression of cyclin A, cyclin B, Cdk1, Cdk2, and Cdk4 was reduced by orlistat. This study investigated the role of lipid metabolism on orlistat-induced human hepatoma Hep3B cell death. The decrease in the expression of key enzymes in fatty acid metabolism, including FASN, ACOT8, PPT1, FABP1, CPT1 and CPT2, was observed after orlistat treatment. We also demonstrated that peroxisomal activity was involved in the orlistat-induced Hep3B cell death. In this study, we established an in vitro model to investigate the effect of orlistat on lipid accumulation. We found that orlistat significantly inhibited the cellular lipid content when administered in fatty acid overload conditions in Hep3B cells. Combination treatment of orlistat and paclitaxel was able to induce a synergistic effect on growth inhibition and cell apoptosis in Hep3B cells. Our data suggested that orlistat displays antitumor activity and enhances the efficacy of paclitaxel in Hep3B cells.
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INTRODUCTION Orlistat was initially approved by the U.S. Food and Drug Administration in 1999 as a prescription drug to treat obesity. Orlistat has also been shown to have an irreversible inhibitory effect on the thioesterase domain of fatty acid synthase (FASN) that has prompted researchers to explore its anticancer activity.1−3 Moreover, a number of reports have demonstrated the anticancer activity of orlistat in a variety of malignant cell lines, including prostate cancer, glioma, breast carcinoma, etc.1,4,5 These studies indicated that orlistat inhibited the proliferation and induced cell death of cancer cell lines by inhibiting fatty acid synthesis and causing apoptosis.1,4,5 However, the anticancer effect of orlistat on hepatocellular carcinoma has not yet been explored. Many cancer cells show high rates of de novo lipid synthesis to promote rapid division and growth. Therefore, it has been indicated that alteration of lipid metabolic pathways or blocking fatty acid de novo synthesis could offer new therapeutic strategies in cancer therapy.6−8 FASN is a key enzyme involved in lipid biosynthesis that is upregulated in many cancer cells, and it is generally accepted that the overexpression of FASN correlates with higher metastatic potential, poorer prognosis, and resistance toward cancer chemotherapeutics in human cancer.9−12 FASN synthesizes © XXXX American Chemical Society
palmitate, a long-chain saturated fatty acids, from malonyl CoA and acetyl CoA. The thioesterase domain is one of seven domains of FASN, which is essential in regulating the length of the fatty acid chain.13 It has also been demonstrated that FASN inhibition reduces the proliferation in vitro and induces caspase-dependent apoptosis by blocking lipogenic pathway in cancer cell lines.14−16 Paclitaxel (taxol) is a tubulin-binding agent and widely used chemotherapeutic drug in the treatment of breast cancer, lung cancer, and ovarian cancer. The development of drug resistance after prolonged use and the consequence of the toxicity of paclitaxel at a higher dosage limit its use in patients.17−19 Many reports have indicated that paclitaxel may disturb G2/M transition and induce cell cycle arrest and apoptosis in tumor cells.20,21 However, few studies show that paclitaxel is effective against liver cancer. Combinations of antitumor drugs with different or similar mechanisms of action have been found to exhibit a synergistic effect that is greater than the sum of the individual drugs.22,23 It has been demonstrated that fatty acid de novo synthesis and metabolism for cellular energy production were involved in orlistat-induced Received: September 19, 2018
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DOI: 10.1021/acs.chemrestox.8b00269 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
Article
Chemical Research in Toxicology cancer cell death.4,24 Previous studies demonstrated that orlistat has strong effects on the cell cycle of cancer cell lines by inducing G1-S stability.24,25 Furthermore, β-tubulin was found to be another target of orlistat.26 It has also shown that FASN inhibitors enhance the antitumor effect of paclitaxel through disruption of tubulin palmitoylation and microtubule organization.27 Orlistat has already been used for many years as an antiobesity drug and its safety has been validated. Based on the above reasons, this study investigates whether a combination of orlistat and paclitaxel may have a synergistic anticancer effect in human hepatoma Hep3B cells. In the current study, the hepatoma Hep3B cells were treated with orlistat, and cell cycle and cell apoptosis were evaluated. The role of peroxisome and the expression of proteins involved in lipid metabolism were also examined. We hypothesized that two anticancer drugs with different or similar mechanisms of action would yield a synergistic effect and it is possible to significantly enhance the sensitivity of cancer cells to anticancer drugs. Therefore, we examined whether a combination of orlistat and paclitaxel may have a synergistic anticancer effect.
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SDS. For detecting catalase activity, the separated gel was incubated in 0.03% H2O2 for 5 min and then incubated in 1% ferric chloride and 1% potassium ferricyanide solution. FFA/BSA Complex Solution Preparation. To induce free fatty acid (FFA) overloading, Hep3B cells at 70% confluence were exposed to a long-chain mixture of FFAs (containing oleic acid, palmitic acid, linolic acid, linoleic acid, and arachidonic acid in proportions of 25:40:15:15:5). Stock solution of FFA/BSA complex was prepared as reported previously.30 Briefly, a 100 mM FFA stock solution was prepared in 0.1 M NaOH by heating at 70 °C. A 1% (w/v) bovine serum albumin (BSA) solution was prepared in ddH2O at 55 °C. Various concentrations of FFA/BSA complex solution were prepared by adding the appropriate amount of FFA stock solution to 1% BSA. The FFA/BSA complexed solution is cooled to room temperature, sterile filtered (0.45-μm pore size membrane filter), and stored at −20 °C until use. Oil Red O Staining. Oil Red O staining was performed as described previously.31 After treatments, cells were fixed with 3.7% formalin for 60 min and then stained with Oil Red O working solution (Oil Red O stock solution (3 mg/mL in isopropanol): ddH2O = 3:2) for 60 min. To quantitate the dye, isopropanol was added to each sample for 5 min, and samples were measured in duplicate by using a Multiskan Ascent 96/384 Plate Reader (Helsinki, Finland) at 510 nm. Cell Cycle analysis. Cell cycle analysis was performed as described previously.28 The cells were stained with propidium iodide (50 μg/mL) and analyzed using a FACScan flow cytometer (Becton Dickinson Instruments). Analysis of Cytotoxic Synergy. Hep3B cells were exposed to orlistat and paclitaxel in combination in different ratios for 24 h, and the effect on cell growth was examined by the Trypan blue exclusion assay method as described above. This effect was further calculated for synergism using the following equation: combination index (CI) = CA,x/ICx,A+ CB,x/ICx,B, where CA,x and CB,x are the concentrations of drug A and B used in combination to produce an effect x% and ICx,A and ICx,B are the theoretical individual drug concentrations to achieve the same effect x%. A CI of less than, equal to, or more than 1 indicates synergic, additive, or antagonistic effects, respectively. Annexin V-FITC/PI Double Staining Assay. After treatments, cells were incubated with FITC-conjugated annexin V and propidium iodide as previously described.28 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) Staining. Nuclear morphology was detected by 4′,6-diamidino-2phenylindole dihydrochloride (DAPI) staining as previously described.32 Real-Time Quantitative Polymerase Chain Reactions (RTPCR) Analysis. For the quantitative RT-PCR, total RNA from Hep3B cells was isolated using AllPure Total RNA Isolation Kit (AllBio Science Inc., Taiwan). Total RNA (1 μg) was used for reverse-transcription by AllScript First-Strand cDNA Synthesis SuperMix Kit (AllBio Science Inc., Taiwan) according to the manufacturer’s instruction. Quantitative PCR was performed using AllScript Green qPCR SuperMix UDG (AllBio Science Inc., Taiwan) following the manufacturer’s protocol and detected by StepOnePlus Real-Time PCR System (Applied Biosystems, USA). The PCR amplification conditions were used as below: 94 °C for 10 min, followed by 40 cycles of 94 °C for 15 s and 60 °C for 1 min. The primers used in this study were as follows: ACOT8: forward 5′AGTGGAGCGGACACGAACAG-3′, reverse 5′TGTTGCACGGCCTTCACA-3′; CPT1A: forward 5′TTTCCATTCCTTCCCATTCGTA-3′, reverse 5′TGGGCTCGTGCGACATT-3′; CPT2: forward 5′-CAGGGCTTTGACCGACACTT-3′, reverse 5′-TCAGGCAAGATGATCCCTTTG-3′; FABP1: forward 5′-CACCGCTGGGTCCAAAGT-3′, reverse 5′-CATTGTCTCCAGCTCACATTCC-3′; FASN: forward 5′-CGCTCGGCATGGCTATCT-3′, reverse 5′CTCGTTGAAGAACGCATCCA-3′; PPT1: forward 5′CTCGTGCAAGCCGAATACTG-3′, reverse 5′TGCTGTGGTTGCGATACACA-3′; β-actin: forward 5′-GACTTAGTTGCGTTACACCCTTTCTTG-3′, reverse 5′GACTGCTGTCACCTTCACCGTTC-3′. All qPCR assays were
MATERIALS AND METHODS
Materials. Orlistat, palmitic acid, propidium iodide, and RNase A were purchased from Sigma Chemical Company (St. Louis, MO, USA). Annexin V-FITC apoptosis detection kit was purchased from BioVision (Mountain view, CA, USA). Antibodies to various proteins were obtained from the following sources: Cdk1, Cdk2, Cdk4, cyclin B, and p21 were purchased from BD Biosciences (San Diego, CA, USA). Cyclin D1 was purchased from Abcam (Cambridge, MA, USA). β-Actin, ACOT8, CPT1A, CPT2, cyclin A, cyclin E, FABP1, FASN, and PPT1 were from GeneTex Inc. (Irvine, CA, USA). Horseradish peroxidase (HRP)-conjugated goat antimouse and -rabbit IgG were from Abcam. Human Hepatocellular Carcinoma Cell Line Hep3B Cells. Hep3B cells were obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan) and cultured in monolayer culture in Dulbecco’s modified Eagle’s medium (Life Technologies, Rockville, MD, USA) with 5% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine at 37 °C in a humidified incubator containing 5% CO2. Trypan Blue Exclusion Assay. Cells were seeded at a density of 5 × 104 cells per well onto 12-well plate 48 h before being treated with drugs. After 24 h of drug treatment, the number of viable cells was determined when the cell population was stained with Trypan blue. Protein Preparation and Western Blot Analysis. Protein preparation and Western blot analysis were performed as previously described.28 The proteins (50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred onto Immobilon-P Transfer Membranes (Millipore, Bedford, MA, USA). Membranes were probed with antibodies to βactin (1:20000, the detection of β-actin was used as an internal control in the data of Western blotting analysis), ACOT8 (1:500), Cdk1 (1:2500), Cdk2 (1:2500), Cdk4 (1:500), CPT1A (1:500), CPT2 (1:500), cyclin A (1:500), cyclin B (1:500), cyclin D1 (1:500), cyclin E (1:500), FABP1 (1:1000), FASN (1:1000), p21 (1:500), and PPT1 (1:500). Secondary antibody consisted of a 1:20000 dilution of HRP-conjugated goat antimouse IgG (for β-actin, Cdk1, Cdk2, Cdk4, cyclin B, and p21) or HRP-conjugated goat antirabbit IgG (for ACOT8, CPT1A, CPT2, cyclin A, cyclin D1, cyclin E, FABP1, FASN, and PPT1). Evaluation of Catalase Activity. Catalase activity assay was performed as previously described.29 Cells were collected and sonicated in cold phosphate buffer (50 mM, pH 7.8). The proteins (100 μg) were separated by 9% polyacrylamide gels without 0.1% B
DOI: 10.1021/acs.chemrestox.8b00269 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
Article
Chemical Research in Toxicology run in triplicate, and the fold change of the target gene expression between treatment and control is calculated by using the comparative CT method. Data Analysis and Statistics. One-way ANOVA followed by Bonferroni post hoc test was used for analysis of difference between each experimental group. A P value
