Oleanolic Acid Induces Metabolic Adaptation in Cancer Cells by

May 23, 2014 - School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China. ⊥. Department of Pathophysiology, Norman Bethune ...
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Oleanolic acid induces metabolic adaptation in cancer cells by activating AMP-activated protein kinase pathway Jia Liu, Lanhong Zheng, Ning Wu, Leina Ma, Jiateng Zhong, Ge Liu, and Xiukun Lin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf500622p • Publication Date (Web): 23 May 2014 Downloaded from http://pubs.acs.org on June 2, 2014

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Oleanolic acid induces metabolic adaptation in cancer cells by activating AMPactivated protein kinase pathway Jia Liu 1, 3, *, Lanhong Zheng *, Ning Wu 1, *, Leina Ma 4, Jiateng Zhong 5, Ge Liu 1, 3, Xiukun Lin, **, 1, 6 1. Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China 2. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China 3. Graduate School, University of Chinese Academy of Sciences, Beijing 100049, China 4. School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China 5. Department of Pathophysiology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China 6. Department of Pharmacology, Capital Medical University, Beijing 100069, China

AUTHOR INFORMATION * These authors contributed equally to this work. Corresponding Author ** (X. L.) Phone: 86-10-83911837. Fix: 86-10-83911837. Email: [email protected] Funding This work was supported by National innovative drug development projects of (2014ZX09102043-001) and 863 High Technology Project (No. 2014AA093503). The study was also supported in part by National Foundation of Natural Sci. of China (81302906,

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81273550 and 41306157) and Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences (2013B01YQ01). Notes The authors declare no competing financial interest Key words Aerobic glycolysis; AMPK; Metabolism; Oleanolic acid 1

Abstract

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Cancer cells are well known to require constant supply of protein, lipid, RNA and DNA

3

via altered metabolism for accelerated cell proliferation. Targeting metabolic pathway is,

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therefore, a promising therapeutic strategy for cancers. Oleanolic acid (OA) is widely

5

distributed in dietary and medicinal plants and displays a selective cytotoxicity to cancer

6

cells, primarily by inducing apoptosis and cell cycle arrest. In this study, we investigated

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if OA inhibited growth of tumor cells by affecting their metabolism. OA was found to

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activate AMP-activated protein kinase (AMPK), the master regulator of metabolism, in

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prostate cancer cell line, PC-3, and breast cancer cells line, MCF-7. AMPK activation is

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required for the anti-tumor activity of OA on cancer cells. Lipogenesis, protein synthesis

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and aerobic glycolysis were inhibited in cancer cells treated with OA, in an AMPK

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activation-dependent manner. The metabolic alteration was shown to mediate the tumor

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suppressor activity of OA on cancer cells. Collectively, we provided evidence that OA, as

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a widely distributed nutritional component, is able to exert anti-tumor function by

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interfering metabolic pathway in cancer cells. This finding encouraged researchers to

16

study if affecting cancer metabolism is a common mechanism by which nutritional

17

compounds suppress cancers.

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INTRODUCTION

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Rapid and unlimited growth of cancer cells requires a constant supply of both energy

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and structural materials that is essential for cell division. Transformed cells usually adjust

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their metabolic machinery to these needs by modifying the activity and expression of key

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metabolic enzymes in a concerted fashion.1 Metabolic switch is not only an adaptive

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response of pre-existing malignant cells, but also is a driver of tumorigenicity.2 The

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population that suffers from metabolic syndromes, such as diabetes, is more prone to

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cancers,2 although the detailed mechanisms underlying increased tumor risk from altered

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metabolism is still unknown. Interfering with metabolism has been shown to retard the

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growth of tumor cells,2 indicating it as a promising therapeutic target for cancer treatment.

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Multiple kinases have been shown to participate in the regulation of metabolic activity.

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AMP-activated protein kinase (AMPK) is one of the most important kinases that trigger

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metabolic adaption in cells. As an energy sensor, AMPK is an enzyme consisting of three

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subunits (α, β and γ). α subunit is responsible for its catalytic activity and the other two

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ones serves as regulatory parts. AMPK senses the increase in intracellular AMP/ATP

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ratio and responds to the changes by phosphorylating and activating/inactivating some

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key metabolic enzymes, thereby inducing metabolic switch from glycolysis and

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anabolism to oxidative phosphorylation and catabolism. This AMPK-mediated metabolic

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alteration compromises the generation of molecular intermediates that is essential for

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macromolecule synthesis, and hence, slows down cell proliferation. AMPK, evidenced by

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recent studies, is closely implicated in the correlation between metabolism and cancer.

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AMPK suppresses aerobic glycolysis in malignant cells and also hampers the growth of

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tumors in vivo.3 In line with the tumor suppressor function of AMPK, the risk of cancer

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has been found to be lowered in Type 2 diabetes patients who received the administration

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of metformin, an AMPK activator.4 This observation raises the possibility that AMPK is

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likely to be a promising pharmacological target for cancer therapy.

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Activated AMPK suppresses the activity of acetyl-CoA carboxylase (ACC) by

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phosphorylation at Ser218 and negatively regulated the expression of fatty acid synthase

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(FASN), finally inhibiting lipid synthesis in cancer cells. In parallel, mammalian target of

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the rapamycin (mTOR) is a downstream effector of AMPK, which promotes protein

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synthesis by activating p70S6K and eIF-4E. Through phosphorylation-dependent

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activation of TSC1/TSC2, a major upstream negative regulator of mTOR activity, AMPK

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can abrogate the stimulatory effect of mTOR on mRNA translation in transformed cells.

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AMPK also promotes glycolysis by phosphorylating and activating 6-phosphofructo-2-

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kinase/fructose-2,6-biphosphatase 2 (PFKFB2), a key enzyme in glycolytic pathway.

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Accumulated

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intermediates required for the synthesis of macromolecules. Above all, the activation

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status of AMPK determines the metabolism in cancer cells.1 Biosynthesis of

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macromolecules, such as lipid, protein and nucleic acids, may be braked by reactivating

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the kinase function of AMPK in cancer cells that has no or low AMPK activity.

evidences

have

documented

that

enhanced

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glycolysis

produces

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Oleanolic acid (OA) belongs to naturally occurring triterpenoid that is widely

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distributed in edible and medicinal plants. OA has been known, for a long time, to exert a

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variety of bioactivity, including tumor-suppressing function.5 However, the molecular

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mechanism by which OA was able to inhibit tumor growth has been not completely

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explored. In this study, we found that OA was able to induce AMPK activation in cancer

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cells. Sequentially, AMPK activation led to the metabolic alteration in OA-treated cancer

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cells. Finally, we demonstrated that AMPK-dependent alteration in metabolic pattern is

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associated with the anti-tumor function of OA.

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MATERIALS and METHODS

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Cell culture. Human prostate carcinoma cell line, PC-3, and human breast cancer cell line,

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MCF-7, were purchased from American Type Culture Collection C (ATCC). Human normal

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liver cell, L-02, was obtained from Shanghai Cell Collection (Shanghai, China). PC-3 was

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cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS; GIBCO-BRL) at

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37 ○C under a humidified 5% CO2 condition. MCF-7 cells were cultured in DMEM.

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Chemical agents. Oleanolic acid (OA) was purchased from Sigma Aldrich (O5504). An

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AMPK inhibitor, Compound C, was purchased from Millipore (#171260). The cells were

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incubated with Compound C (20 µM) for 1 hr prior to OA treatment. An mTOR activator,

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MHY1485, was purchased from Millipore (#5.00554.0001). The cells were incubated with

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MHY1485 (2 µM) for 1 hr prior to OA treatment. The above substances were all dissolved

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with DMSO for storing. PBS or culture media was added to dilute the agents when needed.

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Immunoblotting assays. Total proteins were extracted from cells with M-PER®

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Mammalian Protein Extraction Reagent (Thermo Scientific, IL), separated with

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electrophoresis with 10-12 % SDS-PAGE gel and transferred onto 0.45 µm nitrocellulose

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membranes. Membranes were incubated in blocking solution (PBS, 0.1 % Tween-20, and

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5 % nonfat dry milk powder) for 2 hr at room temperature, and then incubated with

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primary antibodies (1:1000, listed below). After overnight incubation, the membranes

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were washed with PBS containing 0.1 % Tween-20 for three times, and then were

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incubated with horseradish peroxidase-conjugated secondary antibody for 1 hr at room

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temperature. The involved antibodies were described as follows: Phospho-AMPKα

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(Thr172), (Santa Cruz, sc-33524); AMPKα (CST, #2532); Phospho-HMG-CoA reductase

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(Ser872) (Millipore, #09-356); HMG-CoA reductase (Millipore, #ABS229); Phospho-

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Acetyl-CoA Carboxylase (Ser79) (CST, #3661); Acetyl-CoA Carboxylase, (CST, #3676);

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Fatty Acid Synthase, (CST, #3180). The intensity of blots in some figure was determined

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with ImageJ software.

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Immunofluorescence. PC-3 and MCF-7 cells were incubated with or without OA

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(100 µg/ml) for 6 hr. The cells were fixed with 4% Polyoxymethylene for 30 minutes and

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then incubated with phospho-AMPKα antibody (1:200) for 2 hr at 4 ○C. Subsequently,

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the cells were stained with FITC-conjugated secondary antibody and 4´,6´-diamidino-2-

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phenylindole (DAPI). A confocal microscope (CLS-2SS, Thorlabs) was used to observe

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the fluorescence.

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Detection of fatty acid synthesis activity. A radioactive assay was employed to

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detect the effect of OA on lipid synthesis in PC-3 and MCF-7 cancer cells. The cells were

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incubated with [3H]-acetyl-CoA (2µCi) (Perkin Elmer, #NET290050UC) for 12 hr as

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well as OA (50 or 100 µg/ml) for 6 or 12 hr. Lipid was isolated with a mixture of

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Water/Methanol/Chloroform (1:1:1) according to a procedure previously described 6.

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Organic fraction that contains radioactive lipid was evaporated with N2, followed by

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detection with a MicroBeta Liquid Scintillation Counter (Perkin Elmer).

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Protein synthesis detection assay. The effect of OA on nascent protein synthesis in

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cancer cells were detected using Click-iT® HPG Alexa Fluor® Protein Synthesis Assay

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Kit (Life technologies, #C10428). 5×103 PC-3 and MCF-7 cells were treated with 50 or

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100 µg/ml of OA for 6 or 12 hr (Fig. 3C). For the experiments in Fig. 3E, 100 µg/ml of

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OA was added to the culture of 5×103 PC-3 cells with or without 1 hr compound C (20

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µM) pretreatment, and then, the cells were incubated for another 12 hr. The subsequent

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experiments were performed according to the procedures provided by the manufacturer.

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The fluorescence was detected and record with a confocal microscope (CLS-2SS,

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Thorlabs). The signal intensities were determined with ImageJ and the relative values

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normalized by those in untreated control groups were presented in Fig. 3C and 3E.

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Glycolytic activity detection assays. Glucose consumption of cancer cell treated with

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OA was assessed with Glucose Uptake Colorimetric Assay Kit (BioVision, #K676).

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Briefly, 5×103 PC-3 and MCF-7 cells were treated with 50 or 100 µg/ml of OA for 6 or

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12 hr (Fig. 4A). For the experiments shown in Fig. 4D, 100 µg/ml of OA was added to

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the culture of 5×103 PC-3 cells with or without compound C (20 µM) pretreatment for 1

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hr and the cells were incubated for 12 hr. Then, the cells were starved for glucose with

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100 µl of Krebs-Ringer-Phosphate-HEPES (KRPH) buffer containing 2 % BSA for 30

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min, followed by 20 min exposure with 100 nmol of 2-DG. Subsequent steps were done

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according to the manufacturer’s instructions. The absorbance in each sample was

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measured with a microplate reader at 412 nm. Glucose consumption was determined

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based on the established standard curves.

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Lactate production was determined with Lactate Colorimetric Assay Kit II (BioVision,

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#K627) following the manufacturer’s instructions. Briefly, 5 µl of culture media were

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harvested from PC-3 and MCF-7 cells incubated with 50 or 100 µg/ml of OA for 6 or 12

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hr (Fig. 4B) and mixed with 45 µl of Lactate Assay Buffer. Compound C (20 µM) was

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simultaneously added to PC-3 cells in some experiments (Fig. 4E). A 10kDa MW spin

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filter (BioVision, #1997-25) was used to remove the lactate dehydrogenase in serum.

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After 50 µl of Reaction Mix was added, the reaction was performed for 30 min at room

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temperature. The absorbance in each sample was measured with a microplate reader at

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450nm. The concentration of lactic acid in each samples were determined with the

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established standard curve. The values in each group relative to that untreated control

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group were shown in Fig. 4B and Fig. 4E.

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Oxygen consumption rate was determined with Oxygen Consumption Rate Assay Kit

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(Cayman Chemical Company, #600800). PC-3 and MCF7 cells were treated with OA

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and/or compound C according to the procedures used to evaluate the glucose uptake and

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lactic acid production. After 12 hr OA stimulation, the cells were processed following the

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manufacturer’s instruction. The signals in each sample were read at 650 nm with

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VICTOR™ X3 Multilabel Plate Reader (Perkin Elmer). The oxygen consumption rate

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was presented as lifetime versus time (µs/hr). Their values was normalized by untreated

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control samples and shown in Fig. 4C and 4F.

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Cell cycle analysis. Cell cycle analysis was performed by fluorescence-activated cell

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sorter (FACS)-based evaluation of DNA content with Propidium Iodide (PI) staining.

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2×105 PC-3 cells were treated with OA for 12 hr (indicated groups were pretreated with

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20 µM compound c for 1 hr prior to OA stimulation). The cells were fixed with cold 70%

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ethanol for 1 hr. Subsequently, the cells were processed with RNase (20 µg/ml) at 37○C

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for 1 hr and then stained with PI (50 µg/ml), immediately followed by FACS-based cell

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cycle analysis (Aria II, BD Biosciences). 5×104 cells were counted for each sample.

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Colony formation assay. 3×103 PC-3 cells were plated in a 6-well plate and then cultured

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in media with or without 100 µg/ml OA at 37 ○C under a humidified 5 % CO2 condition for

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10 days. Colonies consisting of more than 50 cells were counted after staining with crystal

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violet solution.

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Animal experiments. The animal experimental procedures were approved by

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Committee on Animal Care in Jilin University (Permission No. 2013032, Changchun,

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China). 5×106 PC-3 cells were injected into the flanks of 4-6 week old male nude

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BALB/c mice (n=18). OA (40mg/kg, low dose or 120 mg/kg, high dose) was orally

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administrated every day for 4 weeks. So the mice were divided into three groups: Control

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(n=6), low dose OA (n=6) and high dose OA (n=6). The diameters of tumors were

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periodically determined with calipers. The volumes were calculated using the following

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formula. Volume (mm3) = length × (width)2 / 2. The expression of indicated proteins was

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detected in the dissected tumors after the mice were sacrificed by immunoblot assay.

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Statistical analysis. The experiments except immunoblot assays were performed for

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at least three times. All values were reported as means ± SD, and compared at a given

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time point by unpaired, two-tailed t test. Data were considered to be statistically

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significant when p < 0.05 (*) and p < 0.01 (**).

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RESULTS AND DISCUSSION

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OA induces AMPK activation in cancer cells in a dose- and time-dependent fashion.

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The chemical structure of OA was shown here (Fig. 1A). First of all, AMPK activation

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was examined in OA-treated cancer cells by immunoblotting analysis of its

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phosphorylation at Thr172. AMPK phosphorylation was found to be increased in all the

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tested cells (Fig.1B). The increase in phosphorylated AMPK expression was correlated

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with the dose of OA administered to cancer cells (Fig.1B). Also, the phosphorylation of

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AMPK was progressively elevated during the time when cancer cells were exposed with

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OA (Fig.1C). OA-induced phosphorylation of AMPK was also further confirmed in

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cancer cells by immunofluorescent staining (Fig. 1D). These data showed that OA

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induced the phosphorylation and activation of AMPK in cancer cells in a concentration-

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and time-dependent manner.

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OA has been demonstrated to exert multiple bioactivities, including anti-inflammation,

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anti-cancer, anticoagulation7 and hepatoprotection,8 by affecting specific molecular

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pathways in cells. These signaling pathways were associated with apoptosis, cell cycle

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regulation, metastasis, angiogenesis and inflammation. AMPK, the central modulator of

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metabolism, has been reported to be activated in mouse cardiomyocytes under OA

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treatment. In turn, AMPK activation protected cardiomyocytes from contractile

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dysfunction induced by hypoxia in a FOXO3-dependent manner.9 This finding shed light

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on the mechanism of OA cardioprotective activity. However, it is still unclear that OA is

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able to induce AMPK activation in cancer cells. In this study, we revealed that OA

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treatment resulted in the phosphorylation and activation of AMPK in cancer cells both in

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dose- and time-dependent fashion.

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AMPK activation mediated the anti-tumor activity of OA on cancer cells.

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Subsequently, we aimed to establish the role of AMPK activation in tumor suppressor

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property of OA on cancer cells. Cell proliferation was determined by counting the cells at

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the different time points. An AMPK inhibitor, Compound C, was used to block the

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AMPK activation in PC-3 cancer cells treated with OA (Fig. 2A). OA was able to

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decelerate the proliferation of PC-3 cells, whereas Compound C-mediated AMPK

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inactivation resulted in a partial recovery of the growth of PC-3 cells (Fig. 2B). Cell cycle

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progression was also retarded by OA treatment. However, this effect was reversed by

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compound C (Fig. 2C). Furthermore, compound C was also able to abolish the inhibitory

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activity of OA on colony forming ability of PC-3 cells (Fig. 2D).

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Subsequently, we aimed to establish the role of AMPK activation in anti-tumor activity

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of OA. We found that AMPK inhibitor, compound C, was able to rescue the effect of OA

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on PC-3 cells, suggesting that AMPK activation is required for OA to suppress tumor

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growth. AMPK activation has been also found to be essential for anti-tumor activity of

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other natural compounds. Curcumin induced apoptosis in CaOV3 ovarian cancer cells in

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an AMPK-dependent way. Blocking AMPK activation with compound c was able to

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abolish curcumin induction of apoptotic event in cancer cells.10 However, compound C-

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dependent AMPK inactivation in OA-treated cancer cells partially, not completely,

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abolished OA suppression of the proliferation, cell cycle progression and colonigenecity

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of PC-3 cells, implying some molecular mechanisms other than AMPK activation are

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responsible for OA anti-tumor activity.

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OA-stimulated AMPK activation suppressed lipid biosynthesis in cancer cells.

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AMPK is a master kinase that regulates the cellular lipid metabolism. Its activation

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leads to a metabolic switch from anabolism to catabolism by phosphorylating several

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enzymes that play key roles in the biosynthesis of macromolecule, such as lipids.

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As a rate-limiting enzyme for fatty acid synthesis, acetyl-CoA carboxylase 1 (ACC1)

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produces malonyl-CoA, an intermediate essential for fatty acid elongation, by catalyzing

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the carboxylation of acetyl-CoA. ACC1 is overexpressed in a variety of human cancers

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and highly contributes to the enhanced lipogenesis in cancer cells.11 AMPK inhibits

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ACC1 activity by phosphorylating it at Ser79.12 In OA-stimulated cancer cells, ACC1

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phosphorylation was found to be augmented in both dose- and time-dependent manners

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(Fig. 3A and 3B) and appeared to be closely correlated with OA-induced AMPK

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activation (Fig. 1B and 1C). Compound C abolished ACC1 phosphorylation at Ser79

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(Fig. 3C), indicating the requirement of AMPK activation in OA-stimulated ACC1

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phosphorylation.

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Fatty acid synthase (FASN) is also a critical enzyme in lipogenic pathway and is

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frequently found to be overexpressed in cancer cells.13 Its transcription level is negatively

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regulated by AMPK-dependent SREBP1c phosphorylation.14 As shown in Fig. 3A and

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3B, OA reduced the expression of FASN protein in cancer cells. This effect was

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associated with both OA concentration and the time of OA treatment (Fig. 3A and 3B).

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After the treatment of OA, the protein levels of FASN were decreased. However, the

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protein levels of FASN were restored in the presence of compound C (Fig. 3C).

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In addition to the fatty acid, sterol is also required for the growth of cancer cells. 3-

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hydroxy-3-methylglutaryl-CoA reductase (HMGR) is the enzyme responsible for

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generating mevalonic acid, an essential intermediate for the synthesis of cholesterol and

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isoprenoids. AMPK has been reported to phosphorylate and inactivate HMGR.1 OA was

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found to increase the phosphorylation of HMGR in cancer cells, both in dose- and time-

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dependent fashion (Fig. 3A and 3B). Compound C treatment was able to block the OA-

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induced phosphorylation of HMGR at Ser872 (Fig. 3C).

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Reactivation of AMPK has been documented to suppress the production of lipid in

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cancer cells. Radioactive assays revealed that OA was able to diminish the de novo lipid

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synthesis, shown as a reduced incorporation rate of [3H] acetyl-CoA into lipid product of

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PC-3 cells (Fig. 3D). OA suppression of lipid synthesis was rescued by compound C (Fig.

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3E), implying that AMPK activation is essential for its effect against fatty acid

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production.

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Next, we investigated if OA was able to affect the lipogenesis of cancer cells by

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detecting the phosphorylation or expression of some lipid synthesis-associated genes,

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which have been confirmed as AMPK downstream effectors, and performing [3H] acetyl-

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CoA incorporation assay. The results showed that OA was able to suppress the lipid

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synthesis in a dose- and time-dependent manner. Compound C abolished the inhibitory

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effect of OA on lipogenic process in cancer cells, supporting the role of AMPK activation

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in OA suppression of lipid synthesis (Fig. 3). Pomolic acid, a natural anti-tumor

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compound, was also found to suppress the lipid synthesis in MCF-7 breast cancer cells in

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an AMPK-dependent manner.15 There is a possibility that all the compounds identified as

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AMPK activators may exert an inhibitory activity on lipogenic pathways.

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Next, we investigated if OA was able to affect the lipogenesis of cancer cells by

299

detecting the phosphorylation or expression of some lipid synthesis-associated genes,

300

which have been confirmed as AMPK downstream effectors, and performing [3H] acetyl-

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CoA incorporation assay. The results showed that OA was able to suppress the lipid

302

synthesis in a dose- and time-dependent manner. Compound C abolished the inhibitory

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effect of OA on lipogenic process in cancer cells, supporting the role of AMPK activation

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in OA suppression of lipid synthesis. Pomolic acid, a natural anti-tumor compound, was

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also found to suppress the lipid synthesis in MCF-7 breast cancer cells in an AMPK-

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dependent manner.15 There is a possibility that all the compounds identified as AMPK

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activators may exert an inhibitory activity on lipogenic pathways.

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Protein synthesis was inhibited in cancer cells treated with OA in an AMPK-

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activation-dependent way.

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AMPK reduces the activity of the mammalian target of rapamycin complex 1

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(mTORC1) by phosphorylating its upstream inhibitory regulator, TSC2,16 and its

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regulatory subunit, Raptor.17 In OA-treated cancer cells, mTOR was shown to be less

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phosphorylated at Ser2448 (Fig. 4A and 4B). The expression of phosphorylated p70S6K,

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a downstream effector of mTOR that hampers protein synthesis by inactivating S6

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ribosomal protein, was consequently reduced in cancer cells under OA treatment (Fig. 4A

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and 4B). This mTOR-dependent phosphorylation is required for the kinase activity of

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p70S6K.18

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eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), was also found to

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be decreased in OA-stimulated cancer cells (Fig. 4A and 4B). The Thr37 and Thr46

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phosphorylations prime 4E-BP1 for subsequent phosphorylations that abolish the

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inhibitory activity of 4E-BP1 on mRNA translation.19 Therefore, OA-induced

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dephosphorylation of 4E-BP1 probably suppresses protein synthesis in cancer cells. The

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changes in the phosphorylation level of these protein synthesis-related factors were all

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abolished by Compound C (Fig. 4C), indicating that AMPK activation is required for OA

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inhibition of mTOR signaling pathway.

The phosphorylation of another important mTOR downstream substrate,

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A fluorescence-based protein synthesis assay kit was used to determine the effect of

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OA on protein synthesis in cancer cells. Microscopic observation revealed that the

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intensity of fluorescence was reduced in cells 3 and 12 hr after the treatment of different

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dose of OA (Fig. 4D), suggesting that OA compromised the nascent synthesis of protein.

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Compound C suppression of AMPK was able to partially prevent the reduction in protein

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synthesis in cancer cells treated with OA (Fig. 4E).

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We also studied the changes in protein synthesis in cancer cells treated with OA. The

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data showed that protein synthesis in PC-3 and MCF-7 cells was significantly

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compromised under OA stimulation. Consistently, the promoters of protein synthesis

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were also inactivated under OA treatment, evidenced by decreased phosphorylation levels.

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Compound C-mediated AMPK inhibition revealed that AMPK activation is required for

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the inhibitory effect of OA on protein synthesis in cancer cells.

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OA suppressed aerobic glycolysis in cancer cells in an AMPK-dependent manner.

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AMPK has been recently demonstrated as a negative regulator of the Warburg effect in

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cancer cells 3. The inhibitory effect of AMPK on glycolysis may be associated with its

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suppression of PKM2 activity via affecting mTOR activation.20 Herein, we investigated if

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OA treatment affected the glycolytic metabolism in cancer cells. Glucose uptake was

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significantly reduced in cancer cells treated with OA (Fig. 5A). Furthermore, cancer cells

344

produced less lactic acid, which is the end product of glycolysis, under the treatment of

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OA (Fig. 5B). The changes in glucose consumption and lactate generation indicated that

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OA suppressed the glycolytic metabolism in cancer cells.

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Cancer cells

are also featured

with compromised mitochondrial oxidative

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phosphorylation that contributes to the maintenance of malignant phenotypes. Oxygen

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consumption, an indicator for the efficiency of oxidative phosphorylation, was

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subsequently measured in OA-stimulated cancer cells. An increase in O2 consumption

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was observed in cancer cells exposed with OA (Fig. 5C).

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Similar to the role of AMPK activation in lipid and protein synthesis, compound C was

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also able to partially inhibit OA-induced metabolic switch in cancer cells, evidenced by

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increases in glucose uptake, lactate production, and O2 consumption in the cells treated

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with both OA and compound C (Fig. 5D, 5E and 5F).

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Increasing evidences indicated that Warburg effect is critical for the unlimited growth

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of cancers and targeting it seems to be an effective strategy for cancer treatment. We

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therefore examined if OA interfere with the metabolic pathways in cancer cells. The

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results on glucose uptake, lactate production and oxygen consumption rates showed that

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OA switch metabolic patterns from aerobic glycolysis to mitochondrial oxidative

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phosphorylaion. Similarly, AMPK inhibition protected PC-3 cells from this metabolic

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change. However, it is unknown how OA-induced AMPK lead to the reversion of

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Warburg effect.

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AMPK-dependent metabolic adaptation contributes to the tumor suppressor

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activity of OA on cancer cells.

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Finally, we aimed to study the contribution of metabolic alteration to the anti-tumor

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activity of OA on cancer cells. Therefore, we used a small molecule activator, MHY1485,

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to interfere with the changes in metabolic pathway without affecting AMPK activation.21

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mTOR inhibition was rescued by MHY1485 in OA-stimulated cancer cells and AMPK

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activation was not affected under this treatment (Fig. 6A). MHY1485 was able to

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counteract the inhibitory effect of OA on protein synthesis in cancer cells and to stop the

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metabolic switch from aerobic glycolysis to oxidative phosphorylation (data not known),

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through mTOR pathway. Cell proliferation assay showed that MHY1485 could speed up

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the growth of PC-3 cells under the treatment of OA (Fig. 6B). The number of colonies in

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PC-3 cells was also increased by MHY1485 (Fig. 6C).

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We also determined the contribution of metabolic alteration to the anti-tumor activity of

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OA. Restoring protein synthesis and aerobic glycolysis in cancer cells partially abolish

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AMPK effect on cancer cells (Fig. 6), confirming the change in metabolic pattern is, at

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least partially, responsible for anti-tumor activity of OA. The reason mTOR activators

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recovered proliferation and colony formation ability of PC-3 cells only in a partial

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manner may be that lipid synthesis was still not restored in OA-treated cells, or that there

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are still some mechanisms underlying OA anti-tumor function, other than interfering

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metabolism. For example, there is a possibility that OA may induce G0/G1 cell cycle

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arrest via AMPK/p53/p21 pathway. Previous studies showed that AMPK phosphorylates

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p53 at Ser15, facilitates its accumulation and enhances its transcription activity of p21CIP,

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a CDK inhibitor, in both normal and cancer cells.22, 23 p27KIP is also reported to be

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substrate of AMPK kinase and phosphyralted at Thr-198. This phosphorylation event

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enhances the inhibitory function of p27KIP on cell cycle progression.24 In addition,

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AMPK activation-induced FASN downregulation may directly affect cell cycle

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progression in tumor cells.25 Furthermore, p53-associated apoptosis is another possible

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mechanism by which OA exerts AMPK-dependent anti-tumor activity, based on a recent

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publication reporting AMPK overexpression lead to increased p53 nuclear accumulation

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and apoptosis. 26

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Another question about OA induction of AMPK pathway is the mechanism by which

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OA initiates AMPK activation. Recently, H2O2 was found to induce AMPK activation in

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insulin-secreting cells.27 AMPK signaling can be also activated in a p38 MAPK-

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dependent manner in cancer cells treated with anti-tumor compound.10 Thus, further

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studies should be focused on these molecular pathways.

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Another question about OA induction of AMPK pathway is the mechanism by which

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OA initiates AMPK activation. Recently, H2O2 was found to induce AMPK activation in

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insulin-secreting cells.27 AMPK signaling can be also activated in a p38 MAPK-

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dependent manner in cancer cells treated with anti-tumor compound.10 Thus, further

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studies should be focused on these molecular pathways.

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OA reduced tumor growth and activated AMPK pathway in mice.

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To further confirm the antitumor activity of OA and its stimulatory effect on AMPK

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pathway, we established a tumor xenograft mouse model by injecting PC-3 cells into the

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flanks of mice. OA was orally administrated every day at two different doses, followed

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by periodically measuring the diameters of tumors in mice. The results showed that tumor

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growth was significantly retarded by the administration of high dose OA (P