Antiproliferative activity of rosamultic acid is associated with induction of apoptosis, cell cycle arrest, inhibition of cell migration and caspase activation in human gastric cancer (SGC-7901) cells

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Antiproliferative activity of rosamultic acid is associated with induction of apoptosis, cell cycle arrest, inhibition of cell migration and caspase activation in human gastric cancer (SGC-7901) cells Cheng-Guang Sui , Fan-Dong Meng , Yan Li , You-hong Jiang PII: DOI: Reference:

S0944-7113(15)00116-6 10.1016/j.phymed.2015.05.004 PHYMED 51828

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Phytomedicine

Received date: Revised date: Accepted date:

5 February 2015 28 April 2015 11 May 2015

Please cite this article as: Cheng-Guang Sui , Fan-Dong Meng , Yan Li , You-hong Jiang , Antiproliferative activity of rosamultic acid is associated with induction of apoptosis, cell cycle arrest, inhibition of cell migration and caspase activation in human gastric cancer (SGC-7901) cells, Phytomedicine (2015), doi: 10.1016/j.phymed.2015.05.004

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Antiproliferative activity of rosamultic acid is associated with induction of apoptosis, cell cycle arrest, inhibition of cell migration and caspase activation in human gastric cancer (SGC-7901) cells Cheng-Guang Suia,*, Fan-Dong Menga, Yan Lia, You-hong Jianga

Molecular Oncology Department of Cancer Research Institution, The First Hospital of China

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Medical University, Shengyang City 110001, China

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a

Corresponding author:

Guang-Shui Jiang,

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Molecular Oncology Department of Cancer Research Institution, The First Hospital of China Medical University, No. 155 North of Nanjing Street, Heping District, Shenyang, Liaoning

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110001, China. Tel: 0086-024-83232354. fax: 0086-024-83282473.

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Abstract

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E-mail address: [email protected]

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Background: Gastric cancer is the second leading cause of cancer related deaths after lung cancer globally. Among natural products, natural triterpenes represent a structurally diverse group of organic compounds with potent antitumor activity. Purpose: The objective of the present research work was demonstrate the antiproliferative and apoptotic activity of rosamultic acid, a natural triterpenoid, in human gastric cancer (SGC-7901)

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cells. Its effect on cellular morphology, cell cycle arrest, DNA fragmentation and expression levels of caspase-3, caspase-8 and caspase-9 were also determined. Methods: Antiproliferative activity of rosamultic acid was evaluated by MTT assay. Phase

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contrast, fluorescence microscopy as well as flow cytometry using Hoechst 33342, acridine orange/ethidium bromide and Annexin V-FITC as cellular probes were used to evaluate induction of apoptosis by rosamultic acid. Protein level expressions were analyzed by western blot analysis.

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Results: The results revealed that rosamultic acid induced dose-dependent as well as time dependent cytotoxic effects in SGC-7901 gastric cancer cells. It also led to a reduction in clonogenic activity along with inhibiting the cell migration. Characteristic features of apoptosis induced by rosamultic acid were observed and quantified. Cell cycle arrest at sub-G1 phase was

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induced by rosamultic acid along with downregulation of expression levels of CDK4, CDK6 and

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cyclin D1. Rosamultic acid also significantly led to the activation of caspase-3, -8 and -9 during the 48 h treatment along with cleaving PARP in a dose-dependent manner. DNA fragmentation

The current study strongly reveal that rosamultic acid inhibits gastric cancer

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Conclusion:

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following rosamultic acid treatment was also observed in these cells.

proliferation by inducing apoptosis mediated through cell cycle arrest, downregulation of cell

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cycle related protein expressions, inhibition of cell migration, DNA damage, and activation of caspases.

Keywords: Gastric cancer, Apoptosis, Rosamultic acid, Cell cycle, Caspases

Introduction

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Gastric cancer is the second leading cause of cancer related deaths after lung cancer, although the global incidence rate has now decreased. Despite the decreased global incidence of gastric cancer, it remains a prevalent burden in many Asian countries as compared to the western countries. China represents the country with the highest prevalence of gastric cancer, with an

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estimated 380,000 new cases each year, accounting for more than 40% of the worldwide annual cancer incidence.In china, gastric cancer represents the third leading cause of cancer related deaths (Jiang, 2012; Kamangar et al., 2006; Chinese Ministry of Health, 2012). Gastric cancer represents the major cancer type in Asia, accounting for around 21% of malignant cancers. More

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than 70% of the gastric cancer patients are considered to be incurable at the time of cancer diagnosis due to the already advanced stage and spreading of the tumor to other parts of the body. The recurrence rate is also very high in patients with resectable cancers resulting in

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majority of the patients finally left with advanced cancer (Clarke et al., 1961). The etiology of gastric cancer remains to be defined as is the case with other cancers. The

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susceptibility of the people to gastric cancer depends on a number of factors including lifestyle,

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age, environmental and genetic factors. For example, vitamin C, carotenoids and green tea have been implied to have preventive effects in gastric cancer (Yoshida et al., 2010; Hwang et al.,

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1994). Gastric cancer is treated bythe surgical resection of the operable tumor, complemented by localized radiotherapy and chemotherapy with conventional chemotherapeutic drugs (Liu et al.,

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2013). Systemic chemotherapy is widely accepted as palliative treatment, leading to good responses, improved life quality, and increased survival rates. Chemotherapy has been vigorously investigated in gastric cancer. Several drugs have shown potent anticancer activity; however, single-drug chemotherapy has been unsuccessfulin increasing survival rates. Several combination treatments have been established with high activity in locally advanced and

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metastatic disease. Among these combinations are eplrubicln plus claptaUn plus 5-FU (ECF), etoposide plus leucovorin plus 5-FU (ELF), 5-fluorouracll (5-FU) plus high dose methotrexate plus doxorublcln (FAMTX), etoposide plus doxorubicin plus clsplatln (EAP) etc (Schipper and Wagener, 1996). Although the response rates of these combination regimens are encouraging,

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the toxicity is immense. This is due to the non-specific toxicity of these drugs which also target normal cells in addition to cancer cells. This non-specific toxicity of these drugs necessitates the development of novel therapeutic agents to treat this deadly cancer (Cervanteset al., 2008;

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Yoong et al., 2011).

A number of studies have drawn attention to natural products extracted from Chinese medicinal herbs as anticancer agents in gastric cancer therapy (Li et al., 2010; Chen et al., 2012). Over 60% of the current anticancer drugs have their origin in one way or another from natural sources.

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Nature continues to be the most prolific source of biologically active and diverse chemotypes. Among natural products, natural triterpenes represent a structurally diverse group of organic

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compounds with potent bioactivity including antitumor effects. Several triterpenoids, including

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ursolic and oleanolic acid, betulinic acid, celastrol, pristimerin, lupeol, and avicins have been reported to possess anticancer activity (Petronelli et al., 2009).

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In the present research work, we report the anticancer and apoptotic inducing effects of

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rosamultic acid (A-ring contracted triterpenoid) isolated from the roots of Rosa multiflora, in human gastric cancer (SGC-7901) cells. We also demonstrated that these effects of rosamultic acid were mediated through cell cycle arrest, downregulation of cell cycle related protein expressions, inhibition of cell migration, DNA damage, and activation of caspases in these cells. The current report constitutes the first such report on this naturally occurring triterpene. Materials and methods

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Chemicals and other reagents MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide], DMSO, propidium iodide (PI) and trypsin were purchased from Sigma (St. Louis, MO, USA). RPMI-1640,

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penicillin, streptomycin and other cell culture supplies were from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum was from Hyclone (Logan, UT, USA).Acridine orange/ethidium bromide (AO/EtBr) and Hoechst 33342were obtained from Fluka (Ronkonkoma, NY, USA). Annexin V-FITC-Propidium Iodide Apoptosis Detection Kit was purchased from (Beyotime

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Institute of Biotechnology, Shanghai, China). The cell-permeable pan-caspase inhibitor, the caspase-3 specific inhibitor, the caspase-9 specific inhibitor, the caspase-8 specific inhibitor, and the caspase-2 specific inhibitorwere purchased from (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Therabbit polyclonal antibodies (Abs) against caspase-3, caspase-8, the mouse

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monoclonal Abs (mAbs) againstpoly (ADP-ribose) polymerase (PARP), and the rabbit mAb against the active form ofcaspase-3 were purchased form PharMingen (Cell Signaling

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Technology, Inc.). All other chemicals and solvents used were of the highest purity grade.

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Plant material

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The roots of Rosa multiflora were collected in June 2014 from Guangzhou Province, China and identified by Prof. Liu-Xian Jiang, a voucher specimen (Voucher specimen number: 14-18-776-

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08) was deposited in the Herbarium of Southeast University, Nanjing, China. Extraction and isolation The root parts of the plant were thoroughly washed with water, shade dried and then grinded into small pieces. Dichloromethane/n-butanol (1:1) was used for cold extraction which was carried out for 72 hours in an extraction apparatus. The extract was then concentrated under reduced

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pressure in a rotary evaporator at 45 oC and was then kept in a refrigerator at 4 oC prior to use. Repeated chromatography of the extract on silica gel (50 g, 100–200mesh, Merck China) with chloroform: methanol (3:1) yielded rosamultic acid. White powder, ESI-MS m/z 509 (M+Na)+ , C30H46O5, 1 H-NMR (300 MHz, C5D5N): d 6.10 (1H, s, H-3), 5.57 (1H, s, H-12), 4.62 (1H, d,

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J = 15 Hz, H-1b), 4.46 (1H, d, J = 15 Hz, H-1a), 4.03 (1H, d, J = 12 Hz, H-24b), 3.79 (1H, d, J = 12 Hz, H-24a), 3.02 (1H, s, H-18), 1.68 (3H, s, H-27), 1.45 (3H, s, H-23), 1.40 (3H, s, H-29), 1.21 (3H, s, H-25), 1.10 (3H, s, H-26), 1.09 (3H, d, J = 6 Hz, H- 30), 13C-NMR (75 MHz, C5D5N): d 61.3 (C-1), 158.4 (C-2), 131.3 (C-3), 49.4 (C-4), 63.9 (C-5), 18.3 (C-6), 35.3 (C-7),

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42.2 (C-8), 44.2 (C-9), 51.2 (C-10), 27.4 (C-11), 128.3 (C-12), 140.5 (C-13), 42.6 (C-14), 30.0 (C-15), 26.6 (C-16), 48.6 (C-17), 55.1 (C-18), 72.9 (C-19), 42.6 (C-20), 27.2 (C-21), 38.8 (C22), 25.4 (C-23), 66.8 (C-24), 19.8 (C-25), 19.1 (C-26), 25.7 (C-27), 181.2 (C-28), 27.4 (C-29),

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Cell line and culture conditions

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17.0 (C-30). It was characterized as rosamultic acid.

Human gastric cancer (SGC-7901) cancer cells were obtained from Cancer Research

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Institute of Beijing, China.These cells were cultivated in in RPMI-1640 medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 μg/ml streptomycin. Cells were cultured in

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CO2 incubator (New Brunswick, Galaxy 170R, eppendroff) with an internal atmosphere of 95%

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air and 5% CO2 gas and the cell lines were maintained at 37 oC. MTT assay for cell viability evaluation Cell viability was measured using MTT assay. Gastric cancer (6 × 102) cells were seeded

in 200 μl of RPMI-1640 medium into 96-well plates, and cultured overnight. Then the medium was replaced with fresh RPMI-1640 or the same media containing different concentrations (0,

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2.5, 5, 25, 50, 75 and 100 µM) of rosamultic acid. After a further incubation for 24 or 48 h, 30 μl of MTT (2 mg/ml) was added to each well followed by 3 h incubation. The medium was discarded and 170 μl of dimethyl sulfoxide was added into each well, and incubated for 30 min.

(experimental OD value/control OD value) × 100%.

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The OD490 nm was measured. The cell viability index was calculated according to the formula:

Cytotoxicity was expressed as the concentration of bergamottin inhibiting cell growth by 50% (IC50 value).

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Colony formation assay

This assay was done as follows. Five ml complete medium containing 400 cells was added to a 60-mm dish. After cell culture with 5% CO2 for 14 days at 37 oC, the supernatant was

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thrown away, and the cells were washed with PBS 3 times. The cells were then fixed with 5% paraformaldehyde for 20 min, and stained with GIMSA (Solarbio, Beijing, China) for 30 min.

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Colonies were numbered under an inverted microscope (Nikon, Tokyo, Japan). The whole assay

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was carried out in triplicate.

In vitro wound healing/migration assay

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Human gastric cancer (SGC-7901) cancer cells (1x 105 cells/ml) were seeded in a 6-well

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plate and incubated at 37 oC and 95 % full confluent monolayer was obtained. Upon 12 h of serum starvation, a 50 ml pipette tip was used to create a straight cell‑ free wound. Each well was washed 3 times with PBS and then subjected to various concentrations of rosamultic acid (0, 25, 50 and 100 µM for 48 and 72 h) in a medium. After incubation, cells were fixed and stainedwith 2.5% ethanol containing 0.3% crystal violet powder for 15 min, and

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arbitrarilyselected fields were photographed under a light microscope. The number of cells that migrated into the scratched area were counted. Morphological changes of gastric cancer cells observed by light microscopy

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Human gastric cancer (SGC-7901) cancer cells were seeded in 96-well plates (2 × 105 cells/well) and incubated overnight to allow adhesion. The cells were treated with rosamultic acid at concentrations of 0, 25, 50 and 100 µM for 48 h. The morphological changes were observed under an inverted light microscope (Olympus, Center Valley, PA, USA) after 48 hours.

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Morphological changes of gastric cancer cells observedby fluorescence microscopy using Hoechst 33342 and Acridine orange/ethidium bromide staining

Human gastric cancer (SGC-7901) cancer cells which were grown on coverslips in 12-

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well plates were exposed to different concentrations (0, 25, 50 and 100 µM) of rosamultic acid for 48 hours, then the cells were washed twice with PBS and stained with Hoechst 33342

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(Hoechst Staining Kit, Beyotime, China) for 1 h at room temperature. Fluorescence microscopy was used to detect and measure cell shape captured from different random visual fields. The ratio

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of apoptotic cells to total cell number was calculated. The rosamultic acid-treated cells were also

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stained with acridine orange and ethidium bromide and visualized under the fluorescence microscope.

V-FITC/propidium iodide (PI) double-staining

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Annexin

assay for

quantification of

apoptotic/necrotic cells. Annexin V-FITC/propidium iodide (PI) double-staining was performed with an Annexin V-FITC Kit (BDBioscience, USA). Human gastric cancer (SGC-7901) cancer cells were treated with different concentrations (0, 25, 50 and 100 µM) of rosamultic acid for 48 hours. The cells

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were trypsinized, rinsed twice with PBS, and resuspended in 1× binding buffer. The cells were labeled with 10 μl of FITC-conjugated annexin V and 10 μl of propidium iodide. The cells were incubated for 20 min in dark at 37 oC and then 450 μl of binding buffer was added and the samples were immediately analyzed with a flow cytometer (Becton Dickinson, San Jose,

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CA).The annexin V-FITC−/PI− cell population was considered as normal, while the annexin VFITC+/PI− and Annexin V-FITC+/PI+ cell populations were considered as indicators of early and late apoptotic cells, respectively.

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DNA fragmentation analysis by gel electrophoresis

After subjecting human gastric cancer cells (SGC-7901) to rosamultic acid treatment for 48 h at various concentrations (0, 25, 50, and 100 µM), both adherent and floating cells were collected and washed with PBS. Pellets were then lysed with DNA lysis buffer (40 mM EDTA,

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150 mM Tris, pH7.6, 0.8% SDS) at room temperature for 30 min. After centrifugation for 15

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min at 15 000 × g, the supernatants were collected and treated with RNase A (final concentration, 500 μg/ml) for 30 min at 37 oC, followed by digestion with proteinase K (final o

C. The DNA was extracted using the

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concentration 500 μg/ml) for 2.0 h at 55

phenol/chloroform/isoamylol (25:24:1), precipitated with ethanol, dissolved in TE buffer (10

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mM Tris, pH 8.0, 1 mM EDTA), and subjected to 2% agarose gel electrophoresis for DNA

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fragmentation analysis.

Cell cycle analysis by flow cytometry Briefly, human gastric cancer cells (1 × 106) were seeded into each well of 6-well plates

and incubated for 24 h for cell attachment and recovery. The cells were treated with different concentrations (0, 25, 50 and 100 µM) of rosamultic acid. Untreated cells (control) were also

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incorporated. After incubation for 24 h, the cells were harvested and fixed with ice-cold 70% ethanol (5 mL) at −20 °C for 2 h. Prior to analysis, the cells were washed with cold PBS and resuspended in 400 μl of PBS, 20 μl PI and 20 μl RNase A. The DNA contents were recorded by a

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flow cytometer (Becton Dickinson, San Jose, CA) equipped with Cell Quest software.

Western Blot Analysis

Western blot assay was done as previously reported (Zhang et al., 2009) with slight

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modifications. Bradford assay (Bio-Rad) was used to determine the protein content. After electrophoresing a total of 20-40 Ag ofprotein on 15% SDS-PAGE gels, it was transferred to nitrocellulose membranes. Membranes wereblocked, incubated with primary Abs at the

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suitabledose, and consequently incubated withprimary antibody, washed and incubated with horseradish peroxidase conjugated secondary antibody (1:2500 dilution; Bio-Rad). Detection

Danvers, MA, USA).

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Statistical analysis

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was performed using a chemiluminescent western detection kit (Cell Signaling Technology, Inc.,

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Data are expressed as mean ± SD of three independent experiments. SPSS.13.0 software was used to perform statistical analysis. Differences were analyzed using one-way analysis of

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variance (ANOVA) or two-way ANOVA. P<0.05 was considered statistically significant. Results

Characterization of rosamultic acid isolated from the root extract of Rosa multiflora

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Rosamultic acid was isolated as white powder, White powder, ESI-MS m/z 509 (M+Na)+ , corresponding to the molecular formula C30H46O5.1H-NMR, and 13C-NMR data revealed that the data corresponds to rosamultic acid molecular architecture. HPLC chromatogram, chemical structure and

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C-NMR of rosamultic acid are shown in Fig. 1A, Fig. 1B and Fig. 1C

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respectively. The spectroscopic data was also compared with the literature data available.

Antiproliferative activity of rosamultic acidand its effect on colony formation in human gastric cancer cells (SGC-7901)

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To examine the inhibitory effect of rosamultic acid on the proliferation of human gastric cancer cells, MTT assay was conducted. SGC-7901 cells were treated with different concentrations (0, 2.5, 5, 25, 50, 75 and 100 µM) of rosamultic acid dissolved in DMSO and the same volume of solvent was used as a control. The results showed that rosamultic acid exerted

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potent and dose-dependent as well as time dependent antiproliferative effects on SGC-7901

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cancer cells (Fig. 2) after treatment for 24, 48 and 72 h. Further, we also evaluated the anticlonogenic effects of this triterpenoid which indicated that rosamultic acid decreased the number

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of colonies comprising of SGC-7901 cancer cells which also showed concentration-dependence. These results when taken together indicate that rosamultic acid suppresses both anchorage-

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dependent (antiproliferative) as well as anchorage-independent (colony formation) growth of

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SGC-7901 cancer cells (Fig. 3 and Fig. 4 A to D).

Effects of rosamultic acid on the migration of SGC-7901 gastric cancer cells

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We also evaluated the effect of rosamultic acid on the cell migration of SGC-7901 gastric cancer cells using wound-healing (scratch motility) assay. Confluent monolayers of cells were scratched to form wounds, then cultured in the absence or presence of various concentrations of rosamultic acid (25, 50 and 100 µM), and examined at different time intervals (48 and 72 h) after

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cell monolayers had been wounded. Rosamultic acid exhibited potent inhibitory effects against cell migration (Fig. 5). As shown in Fig. 5, rosamultic acid-induced cells moved slowly compared with thecontrol group in a dose-dependent manner.The number of cells migrated into the scratched area were photographed and calculated as a percentage (%) of migration.

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Rosamultic acid showed time-dependent as well as dose-dependent inhibitory effect against cell migration. Fig. 5 represents untreated (0 µM) control cells while Fig. 4 B, C and D represent effect of 25, 50 and 100 µM dose of rosamultic acid respectively.

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Morphological evaluation of rosamultic acid-induced apoptosis in SGC-7901 cancer cells using

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phase contrast and fluorescence microscopy

Further, we studied the effect of rosamultic acid on the cellular morphology and

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apoptosis induction in SGC-7901 cancer cells using phase contrast and fluorescence microscopy. Rosamultic acid-treated cells showed characteristic morphological changes of apoptosis

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including detachment of the cells from substratum and cell shrinkage. As can be seen in Fig. 6

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A-D, Untreated SGC-7901 cells appeared as densely packed and disorganized multilayers, whereas after incubation with various concentrations of rosamultic acid for 48 h, several of the cells became irregularly shaped, some cells were broken with no content, some were rounded and shrunken, and detached from each other or floated in the medium. Further fluorescence microscopy using Hoechst 33342 and PI double staining and Acridine orange/ethidium bromide staining also revealed morphological features including

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chromatin condensation, fragmented nuclei and nuclear shrinkage which increased with the increasing dose of rosamultic acid (Fig. 7 A-D and Fig. 8 A-D) after 48 h. Bright nuclear condensation was noted by Hoechst 33342 and PI staining, and apoptotic cells were observed as masses in the cells (Fig. 7). The numbers of broken and necrotic/apoptotic cells increased in

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conjunction with increasing concentrations of rosamultic acid. Similarly, in case of acridine orange/ethidium bromide staining, green intact nuclei indicated normal cells, while as red staining indicated apoptosis. The number of cells with red staining increased with increasing

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dose of rosamultic acid (Fig. 8). Apoptosis quantification by Annexin V-FITC/PI assay

Annexin V/PI double staining was used to detect apoptosis in human gastric cancer cells (SGC-7901) (Fig. 9A-D). SGC-7901 cells were treated with different concentration (0, 25, 50

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and 100 µM) of rosamultic acid for 48 h. Rosamultic acid induced both early and late apoptosis

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in a concentration-dependent manner (Fig. 9B-D) as compared to the untreated control cells (Fig. 9A). When the cells were treated with 25, 50 and 100 µM for 48 h, the average proportion of

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Annexin V-staining positive cells (total apoptotic cells) significantly increased from 3.2% in control to 34.3%, 46.8% and 65.9% respectively. Fig. 10 shows the graphical representation of

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the increase in the apoptotic cells with increase in the dose of rosamultic acid.

Effect of Rosamultic acid on DNA fragmentation in SGC-7901 human gastric cancer cells Besides the morphological changes of apoptosis in Rosamultic acid-treated SGC-7901 cells, DNA fragmentation of wasalso examined by observation of the formation of DNA ladder. As shown in Fig. 11, DNA ladder appeared to be more evident with the increasing Rosamultic

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acid concentration, however, no DNA fragments were observed in the control groups (Fig. 11, 0 µM). However, 25, 50 and 100 µM doses of rosamultic acid after 48 h exposure led to a substantial increase in DNA fragmentation (Fig. 11, right panel).

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Rosamultic acid induced sub-G1 (G0/G1) cell cycle arrest in SGC-7901 gastric cancer cells. To determine the distribution of rosamultic acid -treated SGC-7901 cells in different phases of the cell cycle, DNA content in cells was detected by propidium iodide (PI) staining and flow cytometry. The results showed that treatment with different concentrations of rosamultic

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acid for 48 h led to an increase in the population of cells in the sub-G0/G1 phase (apoptotic population) (P < 0.01) (Fig. 12A-D). This increase in sub-G1 population was also accompanied by a corresponding increase of the cells in G2/M phase of the cell cycle. As compared to the control (Fig. 12A), where only 1.5% of the cells were in sub-G1 phase, 25, 50 and 100 µM

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rosamultic acid-treated cells showed 6.2% (Fig. 12B) , 32.6% (Fig. 12C) and 45.8% (Fig. 12D)

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of the cells in the sub-G1 phase (apoptotic phase) of the cell cycle. Rosamultic acid-induced downregulation of the cell cycle related protein expressions

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Since rosamultic acid induced cell cycle arrest in sub-G1 (G0/G1) phase, therefore we

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also evaluated the effect of this triterpenes on the expression levels of the cell cycle related proteins including CDK4, CDK6 and cyclin D1 in SGC-7901 gastric cancer cells. The results

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showed that rosamultic acid induced a considerable downregulation in the expression levels of proteins such as CDK4, CDK6 and cyclin D1that are known to control the cell cycle (Fig. 13 A). This decrease in the expression level of these proteins showed concentration dependence of rosamultic acid (Fig. 13 B). Thus we can safely conclude that rosamultic acid induced sub-G1 arrest due to downregulation of these proteins.

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Rosamultic acid-induced apoptosis was mediated through caspase-3, caspase-9 andcaspase-8 activation To demonstrate the relation between caspase activation and apoptosis induction by

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rosamultic acid in SGC-7901 cancer cells, we evaluated the activities of caspase-3, -9 and -8 using the colorimetric assay. The results revealed that rosamultic acid significantly led to the activation of caspase-3, -8 and -9 during the 48 h treatment (Fig. 14). Western blot analysis showed that full-length procaspase-9, procaspase-8 and procaspase-3 decreased with the

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increased dose of rosamultic acid, while as their cleaved form increased especially at higher doses. The results also showed that PARP, which is a hallmark of caspase-3 activation during apoptosis, was also cleaved at 50 and 100 µM dose of rosamultic acid, but not at lower doses.

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Discussion

Rosa multiflora, commonly known as multiflora rose is a species of rose native to China, eastern

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Asia, Korea and Japan. The plant is a scrambling shrub climbing over other plants and is grown as an ornamental plant (Roger and Martyn, 2004). The plant contains a diverse kind of

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phytochemicals including pentacyclic triterpenes (tormentic acid and its glucoside known as

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rosamultin), flavonoids, phenolic acids, volatile fragrance molecules, carotenoids, tannins etc (Bhandari et al., 2007; Pang et al., 2009). Different parts of the plant are used as herbal medicine

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for the treatment of a variety of diseases including trauma, cold, flu, inflammation, management of pain. Recent studies have shown that Rosa multiflora extracts also possess high antioxidant and antimutagenic effects in all tested assays (Chrubasik et al., 2006; Chrubasik et al., 2007; Shan et al., 2005).

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Apoptosis is a cellular suicide program that exterminates unwanted, faulty and potentially dangerous cells during the development and maintenance of cell homeostasis. Inducing apoptosis is a key tactic to eliminate cancer cells without stimulating an inflammatory reaction. Regulation of apoptotic signaling pathways encompasses a complicated system consisting of several

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elements. Several conventional drugs are currently used in anticancer chemotherapy which are believed to induce cell apoptosis via activation of these elements (Earnshaw, 1995; Kundu et al., 2005). Therefore, the ability of cancer cells to induce the apoptotic program has been recognized as one of the major mechanisms which might serve for the development of novel approaches to

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treat cancer.Caspases are the vital machineries in the implementation of apoptosis (Kerr et al., 1972; Chiarugi and Giannoni, 2008).

Usually, caspases associated with apoptosis can be divided into the initiator caspases and

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the executioner caspases. Caspase-8 and -9 are the initiator caspases in the death receptor and the mitochondrial pathways, respectively. Caspase-3, is the crucialexecutioner caspase in apoptosis

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pathway (Li and Yuan, 2008; Kantari and Walczak, 2011). Our results showed that rosamultic

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acid induces apoptosis which is mediated through the activation of caspase-3, -8 and -9. Procaspase-9, procaspase-8 and procaspase-3 decreased with the increased dose of rosamultic

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acid, while as their cleaved form increased especially at higher doses. PARP was also cleaved at

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50 and 100 µM dose of rosamultic acid, but not at lower doses. Dysregulation in the cell division and apoptosis are connected to the development of

most cancers. Many anticancer drugs function primarily to induce apoptosis in cancer cells and prevent tumor development (Li et al., 2011; Khoo et al., 2010). The morphological changes of apoptosis observed in most cell types initially start with a reduction in cell volume and condensation of the nucleus (Moongkarndi et al., 2004). In many cases extensive DNA damage

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leads to activation of cell cycle check points and results in cell cycle arrest and apoptosis (Pietenpol and Stewart, 2002). In the present study, we found that rosamultic acid induced apoptosis in SGC-7901 gastric cancer cells as revealed by fluorescence microscopy as well as Annexin V-FITC assay. When the cells were treated with 25, 50 and 100 µM for 48 h, the

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average proportion of Annexin V-staining positive cells (total apoptotic cells) significantly increased from 3.2% in control to 34.3%, 46.8% and 65.9% respectively. Further, we evaluated the effect of rosamultic acid on cell cycle phase distribution using flow cytometry. It was observed that rosamultic acid induced cell cycle arrest in the sub-G1 phase. The expression

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levels of the cell cycle related proteins includingCDK4, CDK6 and cyclin D1 in SGC-7901 gastric cancer cells were also demonstrated. Rosamultic acid induced a considerable downregulation in the expression levels of proteins such as CDK4, CDK6 and cyclin D1that are

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known to control the cell cycle. In addition, rosamultic acid also led to a significant DNA fragmentation. Higher doses of (50 and 100 µM) rosamultic acid after 48 h exposure led to a

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substantial increase in DNA fragmentation.

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Tumor invasion is a collective feature of many malignant and deadly tumors resulting in high morbidity and death on account of their high growth rate, invasive potential, and their

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resistance towards the drug treatment. Migration and invasion are the key features of cancer progression and metastasis (Lee et al., 2008; Neudauer and McCarthy, 2003; Goncharova et al.,

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2002). As a result, therapeutic approaches for preventing or suppressing cancer invasion, migration and metastasis can significantly improve the survival of the patients.We also evaluated the effect of rosamultic acid on the cell migration of SGC-7901 cancer cells. The wound healing assay revealed that rosamultic acid significantly reduced the migration of SGC-7901 cells in a

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dose-dependent manner. The compound also induced anticlonogenic effects against the colony formation tendency in these gastric cancer cells. Various plant triterpenes have been reported to induce cell cycle arrest including the pentacyclic

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triterpenes. These include asiatic acid, odoratol, ursolic acid, oleanolic acid, boswellic acid etc Triterpenoids are highly multifunctional compounds and as a result have promise as agents in the treatment of cancer because of their ability to block the NF-κB activation, induce apoptosis, and prevent proliferation, invasion, metastasis and angiogenesis (Hsu et al., 2005; Cazal et al., 2010).

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As far as rosamultic acid is concerned, we could not find reports of its anticancer action or its effects on the cell cycle arrest. To the best of our knowledge, the current research work on this molecule has not been reported earlier and as such constitutes the first such report. In conclusion, we can summarize that rosamultic acid exhibits antiproliferative effects in

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SGC-7901 cancer cells by inducing apoptosis which is mediated by activation of caspase-3,

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caspase-8 and caspase-9. Rosamultic acid also induced cell cycle arrest at sub-G1 phase along with downregulation of expression levels of proteins such as CDK4, CDK6 and cyclin D1 which

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are key controllers of cell cycle. The triterpene also inhibited cell migration as well as colony

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formation tendency of SGC-7901 cancer cells. Conflict of interest

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The authors declare that there is no conflict of interest to reveal.

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Fig. 1. (A) HPLC chromatogram, (B) molecular structure and (C) 13C- NMR of rosamultic acid isolated from the roots of Rosa multiflora.

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Fig. 2. Dose-dependent and time-dependent antiproliferative effect of rosamultic acid in human gastric cancer cells (SGC-7901). Data are shown as the mean ± SD of three independent experiments. *, P < 0.05, **, P < 0.01, vs 0 µM (control).

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Fig. 3. Anticlonogenic activity of rosamultic acid on human gastric cancer cells (SGC-7901). The anti-colony formation effect showed strong dose-dependence. Data are shown as the mean ± SD of three independent experiments. *, P < 0.05, **, P < 0.01, vs 0 µM (control). Fig. 4. Effect of rosamultic acid on colony formation in SGC-7901 cancer cells. The cells were treated with 0 µM (control, A), 25 µM (B), 50 µM (C) and 100 µM (D) concentration of rosamultic acid respectively. Fig. 5. Effect of rosamultic acid on human gastric cancer (SGC-7901) cell migration in vitro. Photographs of wound of cells treated with 0, 25, 50 and 100 μM of rosamultic acid for 48 and

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72 h. The number of cells migrated into the scratched area was calculated as a percentage of migration. Fig. 6. Rosamultic acid-induced morphological changes in SGC-7901 gastric cancer cells as detected by inverted phase contrast microscopy (magnification 200X). Rounded and shrunk cells were observed in rosamultic acid-treated cells (Red colored arrows). A, represents control (untreated cells), B,C and D represent effect of 25, 50 and 100 µM of rosamultic acid on cell morphology of SGC-7901 cells.

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Fig. 7. Rosamultic acid-induced apoptosis in SGC-7901 gastric cancer cells. (A) Untreated SGC7901 control cells, (B) SGC-7901 cells treated with 25 μM, (C) 50 μM and (D) 100 μM concentration of Rosamultic acid respectively. The cells were treated with Rosamultic acid for 48 h, stained with Hoechst 33258/PI and observed by fluorescence microcopy at a magnification of × 200. Bright nuclear condensation/apoptotic cells is shown by orange arrows.

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Fig. 8. Rosamultic acid-induced apoptosis in SGC-7901 gastric cancer cells. (A) Untreated SGC7901 control cells, (B) SGC-7901 cells treated with 25 μM, (C) 50 μM and (D) 100 μM concentration of Rosamultic acid respectively. The cells were treated with Rosamultic acid for 48 h, stained with acridine orange/ethidium bromide and observed by fluorescence microcopy at a magnification of × 200. Red staining (orange arrows) shows apoptotic cells.

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Fig. 9. Quantification of rosamultic acid-induced apoptosis in human gastric cancer cells (SGC7901). The cells were subjected to different doses of rosamultic acid (0, 25, 50 and 100 µM) for 48 h and analyzed by flow cytometry with annexin V-FITC/PI staining. Percentage of apoptotic cells increases from 3.2% in control cells (A), to 34.3%, 46.8% and 65.9% in 25 µM (B), 50 µM (C) and 100 µM (D) rosamultic acid-treated cells respectively.

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Fig. 10. Increase in the number of Annexin V positive cells with increase in the dose of rosamultic acid. The results are mean ± SEM and mean values of three independent experiments. * p < 0.05, ** p < 0.01 versus the 0 μM of rosamultic acid-treated cells.

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Fig. 11. Rosamultic acid induces DNA fragmentation in human gastric cancer cells (SGC-7901). The cells were treated with 0, 25, 50 and 100 μM Rosamultic acid for 48 h. Cells from each sample were harvested for DNA gel electrophoresis as described in Materials and methods.

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Fig. 12. Effect of Rosamultic acid on the cell cycle arrest in human gastric cancer (SGC-7901) cells. Cells were treated with (B) 25 μM, (C) 50 μM and (D) 100 μM of rosamultic acid for 48 h. (A) shows the control (untreated) group. The percentage of cells in the sub-G1 phase increased significantly with increase in the rosamultic acid dose from 0 μM (A) to 25, 50 and finally to 100 μM. The DNA histogram shows the distribution and the percentage of cells in phases of the cell cycle. Results are the mean ± SD of 3 independent experiments. Fig. 13 A and B. Effect of Rosamultic acid on the expression of proteins that regulate the cell cycle (Fig. 13A) of gastric cancer cells. Following treatment of SGC-7901 cells with different doses of Rosamultic acid (0, 25, 50 and 100 µM) for 48 h, (A) western blot analysis was used to analyze changes in the expression levels of CDK4, CDK6 and cyclin D1 proteins. (Fig. 13B) The histogram shows the quantification (%) of the western blot presented in A. * P < 0.05, **P < 0.01, when compared with the control group. Fig. 14. Effects of Rosamultic acid treatment on caspases activation and PARP cleavage in SGC7901 cancer cells. Caspase activities in Rosamultic acid-treated SGC-7901 cells. The lysates of rosamultic acid treated cells were adjusted to equal protein amounts and the enzymatic activities

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of caspases-8, -9, and -3 were measured using the colorimetric assay kits. The cells were treated with 0, 25, 50 and 100 µM concentration of rosamultic acid then lysed and subjected to western blot analysis to detect total and activated caspase-8, -9, -3 and PARP. The data shown are representative for three independent experiments

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