Methyl Protodioscin Induces Apoptosis in Human Osteosarcoma Cells

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Methyl Protodioscin induces Apoptosis in Human Osteosarcoma Cells by Caspase-Dependent and MAPK Signaling Pathways Shun-Cheng TSENG, Tai-shan Shen, Chia-Chieh Wu, Ing-Lin Chang, HsinYao Chen, Chen-Pu Hsieh, Chun-Hsiang CHENG, and Chiu-Liang Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04800 • Publication Date (Web): 16 Mar 2017 Downloaded from http://pubs.acs.org on March 17, 2017

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Journal of Agricultural and Food Chemistry

Figure 1. Effects of methyl protodioscin on viability of MG-63. 153x62mm (300 x 300 DPI)

ACS Paragon Plus Environment


Figure 2. Methyl protodioscin triggers apoptotic death in MG-63 cells. 167x127mm (300 x 300 DPI)


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Figure 3. Methyl protodioscin induces the generation of ROS in MG-63 cells. 162x92mm (300 x 300 DPI)



Figure 4. Methyl protodioscin induces the depolarization of mitochondrial membrane in MG-63 cells. 131x106mm (300 x 300 DPI)


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Figure 5. MAPK and caspase signaling pathways are involved in Methyl protodioscin-induced apoptosis in MG-63 cells. 167x120mm (300 x 300 DPI)



Figure 6. MPD inhibits the expression of Bcl-2 and IAPs in MG-63 cells. 60x72mm (300 x 300 DPI)


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Table of contents 81x40mm (300 x 300 DPI)



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Methyl Protodioscin induces Apoptosis in Human Osteosarcoma Cells by Caspase-Dependent and MAPK Signaling Pathways

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⊥ Shun-Cheng Tsenga, †, Tai-Shan Shena, †, Chia-Chieh Wu†,‡,§, , Ing-Lin Chang†,

4

Hsin-Yao Chen†, Chen-Pu Hsieh†,‡, Chun-Hsiang Cheng‡*, Chiu-Liang Chen†, * ∥

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†

Department of Orthopedic Surgery, Changhua Christian Hospital, Changhua 50006, Taiwan, R.O.C ‡

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Orthopedics & Sports Medicine Laboratory, Changhua Christian Hospital, Changhua 50006, Taiwan, R.O.C. § Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan, R.O.C. ⊥ School of Medicine, Kaohsiung Medical University, Kaohsiung 80761, Taiwan,

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R.O.C. ∥ Department of Nursing, Da Yeh University, Changhua 51591, Taiwan, R.O.C.

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a.

The first two authors contributed equally to this work, and each should be considered first author.

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*Corresponding authors: 1. Chun-Hsiang Cheng

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Orthopedics & Sports Medicine Laboratory, Changhua Christian Hospital 135 Nansiao St.Changhua 500-06, Taiwan, R.O.C. Telephone number: 886-(4)-7238595; Fax number: 886-(4)-7228289 E-mail: [email protected] 2. Chiu-Liang Chen, Professor.

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Department of Orthopedic Surgery, Changhua Christian Hospital 135 Nansiao St.Changhua 500-06, Taiwan, R.O.C. Telephone number: 886-(4)-7238595; Fax number: 886-(4)-7228289 E-mail: [email protected]

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Running title: Tseng et al: METHYL PROTODIOSCIN TRIGGERS APOPTOSIS IN MG-63 CELLS

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Abstract

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Methyl protodioscin (MPD), a furostanol saponin derived from the rhizomes of

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Dioscorea collettii var. hypoglauca (Dioscoreaceae), has been shown to exhibit a

36

broad bioactivities such as anti-inflammation and anti-tumor activity. Here, we

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explored the molecular mechanisms by which MPD-induced apoptosis in MG-63 cells.

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The data shown that MPD significantly suppressed cell growth (cell viabilities: 22.5 ±

39

1.9 % for 8 µM of MPD versus 100 ± 1.4% for control, p < 0.01) and enhanced cell

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apoptosis. The exposure of MPD resulted in a significant induction of reactive oxygen

41

species, loss of mitochondrial membrane potential, and activation of caspase-9 and

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caspase-3 (P < 0.01, all cases). Furthermore, the treatment with MPD increased the

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levels of phosphorylated JNK and p38 MAPK and markedly decreased the levels of

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phosphorylated ERK in MG-63 cells. Co-administration of the JNK-specific

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antagonist, or the p38-specific antagonist, or the caspase antagonist (P < 0.05, all

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cases) have reversed the apoptotic effects in MPD treatment. We also found that

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exposure to MPD resulted in a significant reduction in protein level of anti-apoptotic

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proteins Bcl-2, survivin, and XIAP (P < 0.05, all cases). In conclusion, our results

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indicate that MPD induces apoptosis of human osteosarcoma MG-63 cells, at least in

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part, by caspase-dependent and MAPK signaling pathways.

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Key words: Methyl protodioscin, MG-63 cells, apoptosis, caspase, MAPK pathways

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Introduction The major forms of primary bone cancer are osteosarcoma, Ewing’s sarcoma and

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chondrosarcoma. Osteosarcoma is the most common subtype in children and

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adolescents. 1It is an aggressively malignant neoplasm correlated with a high

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occurrences of distant metastasis, recurrence, and death.2, 3

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Surgery and chemotherapy are effective treatments in patients with early-stage

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osteosarcoma.4 Nevertheless, these therapies are less effective in those with late-stage

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osteosarcoma. Therefore, novel strategies and effective drugs are urgently required,

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especially for patients with advanced disease.

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A number of plant-based drugs have been shown to be effective in anti-tumor

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therapies including vinblastine, vincristine (both from Madagascar periwinkle,

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Catharanthus roseus), Taxanes (from the bark of the pacific yew tree Taxus

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brevifolia), and Camptothecin (from the Camptotheca acuminate).5, 6 Furthermore,

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many herbal compounds have also been found to have anti-neoplastic functions and

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may exhibit less side effects such as ursolic acid and Methyl protodioscin.7, 8

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Methyl protodioscin (MPD; NSC-698790) is a naturally occurring furostanol

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biglycoside steroid found in the rhizomes of Dioscorea collettii var. hypoglauca

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(Dioscoreaceae) and in tubers of Dioscorea pseudojaponica Yamamoto.9 Importantly,

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Cheng et al have reported that MPD can be synthesized.10 Cao et al., have showed 3


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when an intravenous dose of 40 mg/kg of MPD are administered to rats, the maximum

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concentration of MPD was 218.6 ± 23 µg/ml at 2 min postdose in plasma and the

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half-life of MPD was 29.91 ± 1.3 min in plasma. Besides, this dose does not result in

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serious adverse effects in an animal model.11 In addition, previous studies have found

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that MPD is potent against many human cancers including colon cancer, non-small

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cell lung cancer, melanoma, ovarian cancer, breast cancer, renal cancer, and prostate

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cancer.12, 13

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Therefore, in this study we evaluated the potential anti-cancer effect of MPD and

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elucidated the possible signaling pathways governing its apoptotic effects in human

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osteosarcoma MG-63 cells.

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Materials and Methods

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Chemicals

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Methyl protodioscin (purity ≧96%; HPLC #CFN99585, ChemFaces; Wuhan

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ChemFaces Biochemical Co.,Ltd., Wuhan, Hubei, PRC) (10.63 mg) was dissolved in

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1ml DMSO (#D2650; Sigma-Aldrich) to obtain a 10 mM stock solution. Propium

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iodine, Triton X-100, Ribonuclease A and Tween-20 were obtained from

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Sigma-Aldrich.

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Cell culture

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Human osteosarcoma MG-63 cells (Bioresource Collection and Research Center ,

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Hsinchu, Taiwan) were grown in Minimum Essential Medium (Gibco, Carlsbad, CA,

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USA) containing 10% fetal bovine serum, 1% penicillin/streptomycin, 0.1 mM

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non-essential amino acids, and 1.0 mM sodium pyruvate (Gibco).

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MTT assay for cell viability

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The effect of MPD on cell viability was determined by the MTT assay (#0793;

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Amresco, St. Louis, MO, USA) as previously described.14 Briefly, 5 x 104 cells/well

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were cultured in 24-well plates and exposed to different concentrations of MPD (0, 1,

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2, 4, 8 µM) in culture medium for 24 h. After MPD treatment, 100 µl of MTT reagent

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(1mg/ml) was added to each well and incubated for 2h. Absorbance at 590 nm was 5


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read with an ELISA reader (Synergy H1 Multi-Mode Reader, BioTek Instruments,

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Winooski, VT, USA). The half maximal inhibitory concentration (IC50) of MPD was

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calculated by the "Forecast" function in Microsoft Excel.

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Cell cycle analysis

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MG-63 cells were seeded in 12-well plates at a density of 1 x 105 cells/well

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overnight. Cells were treated with various concentrations of MPD (0, 2, 4, 8 µM) for

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24 h. Floating and adherent cells were collected by centrifugating at 1500 rpm for 10

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min. Cell pellets were washed by phosphate-buffered saline (PBS), and fixed with

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cold ethanol at -20°C for 24 h. Cells were rehydrated with cold PBS, and then

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resuspended in PBS with propium iodine (40 µg/ml), Triton X-100 (0.1%), and

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Ribonuclease A (0.2 µg/ml) at 370C for 1 h in the dark and subjected to flow

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cytometer. The cell cycle distribution were analyzed using CXP Analysis software

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(Beckman Coulter; Brea, CA, USA).

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Detection of intracellular ROS

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24 h. At the end of treatment, cell pellets were collected by centrifugation at 1500 rpm

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for 10 minutes. The measurement of ROS was performed by the Muse™ Oxidative

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Stress kit (#MCH100111; Merck Millipore) according to the manufacturer’s 6



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guidelines using a Muse™ Cell Analyzer (Merck Millipore).

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Apoptosis Assay

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24 h. After incubation, the cells were harvested, and cell apoptosis was analyzed by

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the Muse™ Cell Analyzer (Merck Millipore) using the Muse™ Annexin V & Dead

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Cell kit (#MCH100105; Merck Millipore) according to the manufacturer’s protocol.

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Mitochondrial membrane potential Assay

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In order to determine mitochondrial membrane potential, the cells were seeded in

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12-well plates at a density of 1 x 105 cells/well overnight. Cells were treated with

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various concentrations of MPD (0, 2, 4, 8 µM) for 24 h. After incubation, the cells

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were harvested and the mitochondrial membrane potential was analyzed by the

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Muse™ Cell Analyzer (Merck Millipore) using the Muse™ MitoPotential kit

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(#MCH100110; Merck Millipore) according to the manufacturer’s protocol.

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Immunoblotting

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Cell lysates were separated on 10% or 12% polyacrylamide gels and

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electrophoretically transferred to PVDF membranes (Millipore, Bedford, MA), which

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were blocked in 0.1% BSA (Bovine serum albumin; #A8531 Sigma) to prevent

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non-specific binding. Membranes were then washed in PBST (0.1% Tween-20 in PBS) 7


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for 5 min at room temperature (RT). Each membrane was then incubated with primary

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antibodies against a specific protein overnight at 4°C. After washing the membranes

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three times with PBST, the membranes were incubated with an appropriate

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peroxidase-conjugated secondary antibody (Cell Signaling Technology) for 1 h at RT.

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For image quantification, Image J program was used.

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The primary antibodies used in this study included cleaved caspase-3 (#9661),

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Caspase-9 (#9508), Bcl-2 (#9258), XIAP (#2042), Survivin (#2808), GAPDH

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(#2118), PARP (#9542), p-ERK (#4370), ERK (#4695), p-SAPK/JNK (#4668),

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SAPK/JNK (#9258), p-P38 (#9211), P38 (#9212) (all from Cell Signaling

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Technology), cleaved Caspase-8 (#NB100-56116; Novus Biologicals, Littleton, CO,

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USA) and bax (#sc-493; Santa Cruz Biotechnology, CA, USA).

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Statistical analysis

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Values are mean ± SD from at least three independent experiments. Statistical

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comparisons of differences between groups were analyzed using the two-way analysis

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of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons. A

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P value less than 0.05 was considered significant. Statistical analyses were performed

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using the software package GraphPad Prism (Version 4.0, GraphPad Software; San

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Diego, CA, USA). 8



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Results

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Effect of methyl protodioscin on viability of MG-63 cells

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The viability of MG-63 cells after exposure to different doses of MPD (0-8µM) was

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tested by the MTT assay. We found that exposure to 2, 4, or 8 µM MPD resulted in

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significant the growth repression in MG-63 cells in a concentration- and

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time-dependent manner (Figure 1A and B). The half maximal inhibitory concentration

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(IC50) of MPD was 5.30 ± 0.2 µM at 24 h. These data show that MPD has the

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potential to reduce the growth of MG-63 cells.

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Effects of Methyl protodioscin on G2/M phase arrest and

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apoptosis in MG-63 cells

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We used flow cytometry to investigate whether MPD inhibited cell viability by the

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induction of apoptosis. After treating MG-63 cells with a series of different MPD

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concentrations for 24 h, we found that exposure to 2 or 4 µM of MPD resulted in a

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increase of cells in the G2/M phase. In addition, we found that exposure to 4 or 8 µM

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of MPD for 24 hours caused in an upregulation of cells in apoptotic sub-G1

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population (Figure 2A). Moreover, the results of the Muse™ Annexin V & Dead Cell

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Assay showed a significant increase in percentage of apoptotic cells (early- and

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late-stages of apoptosis, and cell death) after exposure to MPD (2, 4, 8 µM) for 24 h 10



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(Figure 2B). These data show that MPD has the potential to induce apoptosis in

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osteosarcoma cells.

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Methyl protodioscin triggers reactive oxygen species

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generation in MG-63 cells

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To investigate whether MPD triggered cell death via generation of intracellular

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reactive oxygen species (ROS), we analyzed ROS levels by Muse™ Oxidative Stress

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kit in MPD-treated MG-63 cells. The data showed that intracellular ROS levels were

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significantly upregulated in MG-63 cells after exposure to MPD (0-8 µM) (Figure 3).

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This result indicates that ROS-mediated pathway may be involved in apoptosis

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induced by MPD.

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Methyl protodioscin induces loss of mitochondrial

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membrane potential

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To check mitochondria-mediated pathways were involved in MPD-triggered apoptosis,

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we analyzed mitochondrial membrane potential by Muse™ MitoPotential kit in

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MPD-treated MG-63 cells. The data showed that MPD treatment (0-8 µM) resulted in

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a dose-dependent increase in percentage of depolarized cells (Figure 4), indicating

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that MPD induces apoptosis, at least in part, by inducing loss of mitochondrial

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membrane potential.

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Methyl protodioscin induces apoptosis through regulation of

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MAPK signaling pathways in osteosarcoma

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MAPK signaling plays an important role in many cellular processes including cell

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division, differentiation, proliferation and apoptosis.15,

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Western blot analysis to investigate whether MAPK signaling pathways were

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involved in MPD-inducing apoptosis. As shown in Figure 5A, treatment of MPD (0-8

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µM) for 24 h resulted in upregulations of phospho-JNK and phospho-p38 MAPK and

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decreased expression of phospho-ERK. To confirm that those kinases played a role in

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MPD-triggered apoptosis, we pretreated MG-63 cells with the JNK-specific

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antagonist (SP600125) (40 µM), the p38 MAPK-specific antagonist (SB203580) (40

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µM) for 2 h, and then co-treated cells with MPD (8 µM) for 24 h. As shown in Figure

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5B, administration of SP600125 and SB2023580 resulted in a significant decrease in

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cleaved caspase-3, indicating that MPD-triggered apoptosis is governed, at least in

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part, by the JNK and p38 MAPK pathway.

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Methyl protodioscin induces apoptosis via a

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caspase-dependent pathway in MG-63 cells

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In our study, we used


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In order to confirm whether caspase activation was involved in MPD-induced

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apoptosis, we measured the protein levels of the activated forms of caspase-8, -9, -3,

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and PARP in MG-63 cells after exposure to MPD by Western blot analysis. As shown

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in Figure 5C, the treatment with MPD (8 µM) for 24 h resulted in up-regulated

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caspase-related proteins such as activated forms of caspase-3, -9 and PARP in MG-63

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cells. To evaluate the role of caspase in MDP-induced apoptosis, the caspase

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antagonist ZVAD-FMK was administered in MPD-treated MG-63 cells. As shown in

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Figure 5B and 5D, the pan-caspase antagonist resulted in a significant reduction in

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percentage of apoptotic cells and reversed MPD-induced cell toxicity, indicating that

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MPD induces apoptosis, at least in part, by activating the intrinsic apoptotic pathway.

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Methyl protodioscin induces apoptosis by altering the

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expression of Bcl-2 and XIAP

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To gain insight into the involvement of the mitochondria-mediated pathway in

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MPD-induced apoptosis, we performed Western blot analysis to determine changes in

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expression of Bcl-2 family members. We found that the treatment with MPD for 24 h

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resulted in downregulation of the pro-survival protein Bcl-2 in MG-63 cells (Figure 6).

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In addition, we measured the protein level of inhibitors of apoptosis proteins (IAP)

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family such as XIAP and survivin.. Our data showed that MPD treatment resulted in a 13


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dose-dependent decrease in expression of XIAP and survivin. Those findings indicate

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that MPD triggers apoptosis, at least in part, by suppressing the protein expression of

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Bcl-2, XIAP and survivin.

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Discussion

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A number of bioactive compounds extracted from plants have been shown to be

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candidate for chemotherapeutic drugs. However, new chemotherapeutic drugs should

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be both highly effective against neoplastic cells and have minimal cytotoxic effects in

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noncancerous cells.17 Our data show that exposure to MPD for 24 h significantly

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inhibited cell proliferation at a dose of 5.30 ±0.2 µM (IC50 value) and that this drug

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induced cytotoxicity in a dose- and time- manner (Figure 1A, and B). Consistent with

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our findings, Yin et al., have showed that oral administration of MPD at 50mg/kg/day

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significantly inhibits bone loss without side effects in an animal model of

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osteoporosis.18 In addition, He et al., have found that oral administration of high dose

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(80mg/kg) MPD does not result in serious adverse effects in an animal model.19 Thus

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MPD is a potentially useful anti-cancer drug.

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Previous studies have shown that many natural herbal compounds can inhibit cell

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growth, arrest cells at crucial points in the cell cycle, and significantly induce

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apoptosis in tumorous cells. Therefore, they are considered candidates for anti-tumor

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drugs.20, 21 Many studies have demonstrated that treatment with MPD inhibits

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proliferation of cancer cells by arresting cells in G2/M phase and increasing cell

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population of sub-G1 phase.8, 12, 22 In this study, we found that exposure to MPD

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resulted in a significant increase in cell populations of sub-G1 and G2/M phases in 15


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osteosarcoma MG-63 cells (Figure 2A).

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Apoptosis plays an critical role in maintaining the balance between cell death and

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cell growth.23 There are two major apoptotic pathways. One is the extrinsic pathway

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which is initiated by extracellular death receptor ligand signaling. The other is the

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intrinsic or mitochondrial pathway which is regulated by the activation of a series of

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caspases.24 The results from our study indicated that MPD triggered apoptosis, at least

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in part, by activating caspase-9 and caspase-3, resulting in PARP cleavage (Figure

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5C). Treatment with ZVAD-FMK (a pan-caspase-specific inhibitor) resulted in a

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significant decrease of apoptotic cells and increased cell viability after exposure to

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MPD (Figure 5B, and D). These findings indicate that activation of caspases is

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involved in MPD-triggered apoptosis.

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Studies have demonstrated that natural plant compounds such as curcumin derived

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from Curcumin Longa and ursolic acid derived from Vaccinium oxycoccus promote

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apoptosis of human osteosarcoma cells via the mitochondrial apoptotic pathway.5, 25

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Induced ROS causes oxidative damage and depolarization of the mitochondrial

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membrane, which initiate the mitochondrial mediated pathway.26 Here, we showed

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that exposure to MPD triggered the generation of ROS and resulted in the collapses of

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mitochondrial membrane potential, and thereby activated the mitochondrial apoptotic

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pathway in osteosarcoma MG-63 cells (Figure 3 and 4). 16



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The Bcl-2 family of proteins plays a critical role in regulating the mitochondrial

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apoptotic pathway.27 Mitochondrial membrane depolarization is associated with

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suppression of Bcl-2. Inhibitors of apoptosis proteins (IAPs) also play an important

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role in suppressing apoptosis by binding to cleaved forms of caspases such as caspase

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-9 and -3.28 Our data showed that exposure of MPD resulted in downregulation of

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anti-apoptotic proteins Bcl-2, XIAP (X-linked inhibitor of apoptosis protein) and

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survivin (Figure 6) in osteosarcoma cells. Thus, these findings suggest that

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MPD-mediated apoptosis is associated with the suppression of pro-survival protein

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expression.

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The members of MAPK family such as c-Jun N-terminal kinase (JNKs),

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extracellular signal-regulated kinase (ERK1/2), and p38 MAPK play critical roles in

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maintaining cell homeostasis condition such as survival and apoptosis.29 Our results

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showed that the treatment with MPD activated phospho-JNKs and phospho-p38

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MAPK, and decreased the expression of phospho-ERK (Figure 5A). In addition, we

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found that exposure to the JNK-specific inhibitor (SP600125), the p38

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MAPK-specific inhibitor (SB203580), and the caspase inhibitor (ZVAD-FMK)

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significantly attenuated the apoptotic effects of MPD (Figure 5B). Taken together,

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these data show that MPD triggers apoptosis, at least in part, by activating JNK, p38

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MAPK, and caspase signaling pathways. 17


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In conclusion, our findings demonstrate that MPD induces caspase-dependent

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apoptosis, ROS-mediated disruption of mitochondrial membrane potential, and the

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down-regulation of Bcl-2, XIAP and survivin proteins. In addition, our data show that

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MPD mediates caspase-dependent apoptosis at least partially by activating JNK and

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p38 MAPK pathways in human osteosarcoma MG-63 cells.

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Acknowledgments: This present study was partially supported by research grants (105-CCH-IRP-028 and 105-CCH-IRP-029) from Changhua Christian Hospital.

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Author Contributions: S.-C.T. and T.-S.S performed the experiments and wrote the paper; C.-H.C. and

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C.-L.C designed the experiments and revised the manuscript; I.-L.C., H.-Y.C., C.-P.H

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and C.-C. W analyzed the data and drafting of the paper.

310 311 312

Conflicts of Interest: The authors of the manuscript do not have any potential conflict of interest.

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Figure legends

314 315

Figure 1. Effects of methyl protodioscin on viability of MG-63. MTT assays were

316 317 318 319

performed with MG-63 cells treated with methyl protodioscin (MPD) at the indicated concentrations. The results showed that MPD signiﬁcant growth repression of MG-63 cells in a dose- (A) and time- (B) dependent manner. Experiments were conducted with three biological replicates per treatment and the values represent the mean ± SD.

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**P < 0.001 compared with the untreated cells.

322 323 324 325 326 327 328 329

Figure 2. Methyl protodioscin triggers apoptotic death in MG-63 cells. (A) Cells were exposed to the indicated doses of MPD for 24 h. Samples were stained with PI and assayed by flow cytometry. Quantitative results of cell-cycle distribution in each phase are shown in the lower panel. (B) Cells were exposed to the indicated doses of MPD for 24 h. Apoptosis was assayed by Muse™ Annexin V & Dead Cell kit. Quantitative analysis of apoptotic cells are shown in the lower panel. Experiments were conducted with three biological replicates per treatment and the values represent the mean ± SD. **P˂0.001 as compared with the untreated group.

330 331 332 333

Figure 3. Methyl protodioscin induces the generation of ROS in MG-63 cells. Cells were exposed to the indicated concentrations of MPD for 24 h. ROS levels were

334 335 336 337

assayed by Muse Oxidative Stress kit. Quantitative analysis of ROS-positive cells are shown in the lower panel. The Symbol "M1 and M2" represents ROS-negative or positive, respectively. Experiments were conducted with three biological replicates per treatment and the values represent the mean ± SD.**P < 0.001 as compared with

338 339

the untreated group.

340 341 342 343 344 345

Figure 4. Methyl protodioscin induces the depolarization of mitochondrial membrane in MG-63 cells. (A) Cells were exposed to the indicated concentrations of MPD for 24 h. Changes of mitochondrial membrane potential were assayed by Muse™ MitoPotential kit. (B) Quantitative analysis shows depolarized cells for the indicated concentrations of MPD and are shown in histogram. Experiments were conducted with three biological replicates per treatment and the values represent the mean ± SD.

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**P < 0.001 as compared with the untreated group.

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Figure 5. MAPK and caspase signaling pathways are involved in Methyl protodioscin-induced apoptosis in MG-63 cells. (A) The activity of JNK and p38 19


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MAPK are upregulated by the treatment with MPD. Cells were treated with MPD at

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the indicated concentrations for 24 h. The harvested cells were analyzed by Western

352 353 354

blotting using indicating antibodies. Quantitative results of phosphorylated ERK1/2, p-JNK, and p-p38 were normalized to their total protein level, and shown in histogram. GAPDH was used as a loading control. (B) MAPK inhibitor and caspase inhibitor

355 356

decrease the formation of cleaved caspase-3 induced by treatment with MPD. MG-63 cells were pretreated with the JNK-specific inhibitor (SP600125) (40 µM), or the

357

p38-specific inhibitor (SB203580) (40 µM), or the caspase inhibitor (ZVAD-FMK)

358 359 360

(40 µM) for 2 hours, and incubated with or without MPD (8 µM ) for another 24 hours. The collected cells were assayed by Western blotting using anti-cleaved caspase-3 antibody. The levels of cleaved caspase-3 were quantified and normalized to GAPDH

361 362 363 364 365

levels and are shown in histogram. (C) The cleaved forms of caspases are induced by treatment with MPD. The cell lysates were prepared as described in (A), and assayed by Western blotting using indicated antibodies. The cleaved forms of caspase-9, -8, -3, and PARP were quantified and normalized to GAPDH levels and are shown in histogram. (D) Caspase antagonist blocks the anti-proliferative effect caused by the

366 367 368

treatment with MPD. MG-63 cells were pretreated with the caspase inhibitor (ZVAD-FMK) (40 µM) for 2 hours, and incubated with or without MPD (8 µM) for another 24 hours. MTT assays were performed to assay cell viability. Experiments

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were conducted with three biological replicates per treatment and the values represent the mean ± SD. Data were analyzed using a two-way ANOVA; *P < 0.05, **P

Methyl Protodioscin Induces Apoptosis in Human Osteosarcoma Cells

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