Novel ent-Kaurane Diterpenoid from Rubus corchorifolius L. f. Inhibits

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A Novel Ent-kaurane Diterpenoid from Rubus corchorifolius L.f Inhibits Human Colon Cancer Cell Growth via Inducing Cell Cycle Arrest and Apoptosis Xuexiang Chen, Xian Wu, Wen Ouyang, Min Gu, Zili Gao, Mingyue Song, Yunjiao Chen, Yanyin Lin, Yong Cao, and Hang Xiao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05376 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017

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Journal of Agricultural and Food Chemistry

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A Novel Ent-kaurane Diterpenoid from Rubus corchorifolius L.f

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Inhibits Human Colon Cancer Cell Growth via Inducing Cell Cycle

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Arrest and Apoptosis

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Short Title: Inhibitory Effects of a Novel Diterpenoid on Human Colon Cancer

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Cells

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Xuexiang Chen, a,b,c Xian Wu, b Wen Ouyang,e Min Gu, b Zili Gao, b Mingyue

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Song,b Yunjiao Chen,a,d Yanyin Lin, a,d Yong Cao,a,d * Hang Xiao b*

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a) College of Food Science, South China Agricultural University, Guangzhou 510642, P. R. China b) Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States c) College of Public Health, Guangzhou Medical University, Guangzhou 511436, P. R. China d) Guangdong Province Engineering Research Center for Bioactive Natural Products Guangzhou 510642, P. R. China e) Pharmacy College, Hunan University of Traditional Chinese Medicine, Changsha 410007, P. R. China

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*Corresponding authors:

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Yong Cao, College of Food Science, South China Agricultural University, Guangzhou

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510642, P. R. China, Tel: +8620 85286234; Fax: +8620 85286234.

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E-mail address: [email protected] (C. Yong)

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Hang Xiao, Department of Food Science, University of Massachusetts, 100 Holds

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worth Way, Amherst, MA 01003, USA, Tel: (413) 545-2281; Fax: (413) 545-1262.

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E-mail address: [email protected].(H. Xiao) 1

ACS Paragon Plus Environment


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Abstract: The tender leaves of Rubus corchorifolius L. f. have been consumed as tea

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for drinking in China since ancient times. In this study, a novel ent-kaurane

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diterpenoid was isolated and identified from Rubus corchorifolius L. f. leaves as

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ent-kaur-2-one-16β,17-dihydroxy-acetone-ketal (DEK). DEK suppressed the growth

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of HCT116 human colon cancer cells with the IC50 value of 40 ± 0.21 µM, while it

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did not cause significant growth inhibition on CCD-18Co human colonic

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myofibroblasts at up to100 µM. Moreover, DEK induced extensive apoptosis and S

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phase cell cycle arrest in the colon cancer cells. Accordingly, DEK caused profound

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effects on multiple signaling proteins associated with cell proliferation, cell death

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and inflammation. DEK significantly up-regulated the expression levels of

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pro-apoptotic proteins such as cleaved caspase-3, cleaved caspase-9, cleaved PARP,

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p53, Bax and tumor suppressor p21Cip1/Waf1; down-regulated the levels of cell cycle

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regulating proteins such as cyclinD1, CDK2, and CDK4, and carcinogenic proteins

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such as EGFR and COX-2; and suppressed the activation of Akt. Overall, our results

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provided a basis for using DEK as a potential chemopreventive agent against colon

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carcinogenesis.

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Keywords: Rubus corchorifolius L.f.,

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ent-kaur-2-one-16β,17-dihydroxy-acetone-ketal, colon cancer, cell cycle, apoptosis

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1. Introduction Colon cancer is the second most common cancer in both developed and

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developing countries. More than a million cases are diagnosed every year, making it

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one of the largest burdens on healthcare systems worldwide 1. The most prevalent

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treatments for colon cancer are surgery, chemotherapy and radiation therapy 2.

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Chemotherapy is widely used to restrain tumor grow and relieve symptoms after

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surgery 3. However, long term usage of anticancer drugs is usually associated with

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toxicity and undesirable side effects 4. Therefore, the development of novel

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anticancer agents with high efficiency and low toxicity is great importance for

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preventing and treating colon cancer. Epidemiological evidence suggests that

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dietary intake of various vegetables and fruits is associated with reduced risk of

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colon cancer. The beneficial effects of vegetables and fruits can be at least partially

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ascribed to the bioactive components found in these foods 5. There are some

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important hallmarks of cancer cells that distinguish them from normal cells, such as

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insensitivity to the cell signaling that regulate cell cycle, resisting programed cell

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death (apoptosis), evading the immune system and formation of new blood vessels

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(angiogenesis). Induction of cellular apoptosis and cell cycle arrest is among the

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most important approaches to control the growth and progression of cancer cells2-3.

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Many mechanisms have been reported to be involved in the cancer

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chemopreventive effects of dietary bioactive components, such as antioxidant and

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anti-inflammatory activity, induction of cell cycle arrest and apoptosis in cancerous

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cells, and activation of phase II detoxifying enzymes8 . 3



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Rubus corchorifolius L. f. is also known as raspberry, march bubble, milk bubble,

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etc. The tender leaves of Rubus corchorifolius L. f. contains coumarin, tea

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polyphenols, tannins, alkaloids, flavonoids and volatile oil compounds diterpene and

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triterpen9–12. Previous studies have demonstrated various health promoting effects of

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Rubus corchorifolius L. f., including antioxidant, hypoglycemic, antibacterial,

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analgesic, anti-inflammatory, antiasthmatic, antiviral, and anti-cancer effects12–14.

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The fruits of Rubus corchorifolius L. f. can be consumed directly, and its tender

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leaves have been used as tea due to its unique flavor and various health promoting

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functions 15. Moreover, the fruits, roots, and leaves of R. corchorifolius L. f. have

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been utilized to treat diarrhea, extravagated blood, and alcoholism 16. In our previous studies, we found that Rubus corchorifolius L. f. contained

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significant amount of ent-kaurane 17–19, which are chemical markers in some plants

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20

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including antibacterial, antitumor, and anti-inflammatory effects17,21–23. Some

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ent-kaurane diterpenoids, such as epieriocalyxin A and oridonin have recently been

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used as anticancer agents 23-24. However, information on the anticancer properties of

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the ent-kaurane diterpenoids found in R. corchorifolius L. f. and the underlying

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molecular mechanisms remain unavailable. In this study, we utilized 80% ethanol to

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extract terpenoids from R. corchorifolius L. f. leaves. Chemical examination of the

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extract of R. corchorifolius L. f. leaves led to the isolation of a novel ent-kaurane

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diterpenoid compound ent-kaur-2-one-16β,17-dihydroxy-acetone-ketal (DEK).

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Furthermore, we determined its inhibitory effects and mechanisms of action on

. Ent-kaurane diterpenoids have been reported to exert many biological activities

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human colon cancer cells.

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2.

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2.1. Chemicals

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Materials and methods

Dulbecco’s modified Eagle’s medium (DMEM, glutamine, high glucose) fetal calf

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serum, penicillin and streptomycin were purchased from Gibco (Invitrogen

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Corporation, Carlsbad, CA, USA). RNase, propidium iodine (PI) and 3-(4,

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5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) were purchased

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from Sigma-Aldrich (Natick, MA, USA). Apoptosis detection kit of Annexing V/PI

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double staining was purchased from Bio vision (Mountain View, CA, USA). BCA

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protein assay kit purchased from Thermos Scientific (Rockford, IL, USA). Antibody

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for β-Actin, cleaved PARP, cleaved caspase-3, cleaved caspase-9, p21 Cip1/Waf1, p27,

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cyclinD1, CDK4, p53, Bax, COX-2, EFGR, Akt, and phospho-Akt were obtained

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from Cell Signaling Technology (Beverly, MA, USA). All chemical reagents used in

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this study were of analytical grade.

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2.2. Plant sampling

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R. corchorifolius L. f. leaves were collected in July 2013 in Zhangjiajie, Hunan, P.

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R. China, and identified by Prof. Bo-Ru Liao. Leaves were shade-dried and stored in

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a 4°C at the College of Food Science, South China Agricultural University

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(Guangzhou, China).

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2.3. Equipment

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Thin-layer chromatography (TLC) was performed on percolated silica gel plate G 5



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(Qingdao Haiyang Chemical, Qingdao, China). 1H NMR and 13C NMR spectra were

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obtained using a 600 MHz NMR spectrometer (Bruker Vance, Switzerland). Mass

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spectra were obtained using the EI-mass spectrometer (Thermo Fisher Scientific,

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Waltham, MA, USA). All reagents and chemicals were purchased from commercial

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sources and were not modified prior to use.

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2.4. Preparation of plant extracts

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Air-dried R. corchorifolius leaves (10.0 kg) were crushed and extracted three

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times with 10 equivalent volumes of 80 % ethanol. Filtrates were evaporated to

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dryness in an evaporator under a water bath and then weighed (1.5 kg). Filtrates

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were then further extracted with ethyl acetate (ethyl acetate extract, 0.536 kg). The

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ethyl acetate extract was subjected to column chromatography on a 17 L column

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containing 6.00 kg silica gel (100-200 mesh). Gradient elution of the column was

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performed using petroleum ether-ethyl acetate and methanol (100:0, 100:50, 100:150,

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100:400, and 0:100, v/v). The solution of separation was collected 500 mL per bottle,

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then the 1885 bottles were obtained, the 1885 bottles solution of separation were

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determined by TLC using vanillin-sulfuric acid as the detection reagent, and

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fractions with similar TLC profiles were merged. The similar components were

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merged, then total of 40 fractions were detected, which were respectively labeled

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from Fr.1 to 40. One of the Fr. 29 (1.89 g) underwent gradient elution with different

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concentrations of chloroform and methanol (50:1 to 0:1) by using silica gel column

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chromatography (600 mesh). The solution of separation was collected 50 mL per

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bottle and 53 bottles were collected. Some fractions of Fr. 29 were isolated using 6


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TLC analysis, including Fr. 29-9, which was recrystallized with methanol to acquire

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the unknown compound (180.00 mg), referred to as compound 29-9.

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2.5 Analysis of cell viability, cell cycle and apoptosis

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Cell viability (MTT) assay, apoptosis and cell cycle analysis were conducted as

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we previously described 25. In brief, HCT116 human colon cancer cells (3000

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cells/well) and CCD-18Co normal human colon cells (1×104 cells/well) were

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purchased from American Type Cell Collection (ATCC, Manassas, VA, USA) were

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seeded in 96-well plates. After 24 hours, cells were treated with a serial

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concentrations of compound 29-9 (purity ≥ 98%), and the cell viability was

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determined by a colorimetric assay 25. HCT116 cells were seeded in six-well plates

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for cell cycle and apoptosis analysis. After 24 hours of incubation, cells were treated

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with a serial concentrations of compound 29-9 in serum complete DMEM media.

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After 48 hours of treatment, serum complete DMEM media containing floating cells

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were harvested and combined with adherent cells. Cell pellets were then rinsed with

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1 mL of ice-cold PBS, and subject to cell cycle and apoptosis analysis using flow

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cytometry as we described previously 25. DMSO was used as vehicle to deliver

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compound 29-9 to the cells. The final concentration of DMSO in all experiments

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was 0.1% v/v in cell culture media.

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2.6. Colony formation assay

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Assay for colony formation was conducted as we previously described 26. HCT116

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cells (400 cells/well) were seeded in 6-well tissue culture plates. After 24 hours, cells

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were treated with several concentrations of compound 29-9 in 2 mL of complete 7



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medium. Medium was changed every four days. After 12 days of incubation,

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HCT116 cell colonies were rinsed with PBS, and then stained with 0.2 % crystal

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violet for 10 min. After being rinsed with ddH2O to remove residual dye, plates were

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imaged with a high-resolution scanner (HP Inc. Palo Alto, CA, USA) and colonies

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were counted.

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2.7. Effect of DEK on mitochondrial membrane potential (MMP)

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HCT116 cells (5×104 cells/well) were seeded in 6-well tissue culture plates.

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Gentle mixing was performed to prevent accumulation of cells in the center of each

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well. After 24 hours of incubation for attachment, HCT116 cells were treated with

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the compound 29-9 in 2mL of serum complete DMEM media. After another 72

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hours of incubation in the presence of the compound, cells floating in the media were

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collected and combined with adherent cells. After centrifugation (1600×g, 1 min),

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the supernatant was removed. PBS (1mL) was added and cells were centrifuged once

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more (1600×g, 1 min). The supernatant was removed and the cells were incubated

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with 500 µL PBS containing 0.1 µM Rhodamine-123 solution for15 min at room

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temperature. Cells were then centrifuged (1600×g, 1min) and the supernatant was

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removed. Cells were washed twice with PBS at 4 °C. Cells were then suspended in

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300 µL PBS and analyzed using an EPICS XL-MC cell analyzer (analytical

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cytometry facility) set at an excitation wavelength of 480 nm and an emission

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wavelength of 520 nm.

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2.8. Ca2+ release detection

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HCT116 cells (5×104 cells/well) were seeded in 6-well tissue culture plates. Gentle 8


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mixing was performed to prevent accumulation of cells in the center of each well.

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After 24 hours of incubation to allow cell attachment, cells were treated with the

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compound in 2 mL of serum complete medium. After another 72 hours of incubation

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with the compound, cells floating in the medium were collected and combined with

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adherent cells. Cells were then centrifuged (1600×g, 1 min) and the supernatant was

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removed. 1mL of PBS was added, and the cells were centrifuged again (1600×g, 1

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min). After removal of the supernatant, cells were incubated in 0.5 mL of basic

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medium containing 0.5 µM Fluo3-AM (Sigma-Aldrich) solution for 40 min at 37°C.

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After centrifugation (1600×g, 1 min), the supernatant was removed and two PBS

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washes were performed. The cells were then suspended in 300 µL of PBS and

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analyzed using an EPICS XL-MC cell analyzer at the analytical cytometry facility.

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2.9. Immunoblotting analysis

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Human colon cancer HCT116 cells (5×104 cells /mL) 12 mL were seeded in

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15-cm cell culture dishes. After 24 hours of incubation for cell attachment, cells were

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treated with compound 29-9 (60 µM) for 72 hours. Cells were washed with ice-cold

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PBS, and collected with cell scrapers. Whole cell lysates were then subjected to

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Western blot analysis as we previously reported 27–30. Antibody for β-Actin, cleaved

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PARP, cleaved caspase-3, cleaved caspase-9, p21 Cip1/Waf1, p27, cyclinD1, CDK4, p53,

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Bax, COX-2, EFGR, cleaved Akt, and phospho-Akt were purchased from Cell

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Signaling Technology (Beverly, MA, USA).

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3. 10. Statistical analysis

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All results are expressed as mean ± standard deviation (SD). Statistical 9



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significance between groups was determined by a Student’s two-tailed t test (two

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groups) or an analysis of variance (ANOVA) (more than two groups). P-value

Novel ent-Kaurane Diterpenoid from Rubus corchorifolius L. f. Inhibits

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