Sequential combination therapy of ovarian cancer with cisplatin and γ-secretase inhibitor MK-0752

    Sequential combination therapy of ovarian cancer with cisplatin and γsecretase inhibitor MK-0752 XiuXiu Chen, LiHua Gong, RongYing Ou, ZhenZhen Zheng, JinYan Chen, FengFeng Xie, XiaoXiu Huang, JianGe Qiu, WenJi Zhang, QiWei Jiang, Yang Yang, Hua Zhu, Zhi Shi, XiaoJian Yan PII: DOI: Reference:

S0090-8258(15)30215-8 doi: 10.1016/j.ygyno.2015.12.011 YGYNO 976144

To appear in:

Gynecologic Oncology

Received date: Revised date: Accepted date:

8 October 2015 11 December 2015 14 December 2015

Please cite this article as: XiuXiu Chen, LiHua Gong, RongYing Ou, ZhenZhen Zheng, JinYan Chen, FengFeng Xie, XiaoXiu Huang, JianGe Qiu, WenJi Zhang, QiWei Jiang, Yang Yang, Hua Zhu, Zhi Shi, XiaoJian Yan, Sequential combination therapy of ovarian cancer with cisplatin and γ-secretase inhibitor MK-0752, Gynecologic Oncology (2015), doi: 10.1016/j.ygyno.2015.12.011

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ACCEPTED MANUSCRIPT Sequential combination therapy of ovarian cancer with cisplatin and

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γ-secretase inhibitor MK-0752

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XiuXiu Chen1, LiHua Gong1, RongYing Ou1, ZhenZhen Zheng1, JinYan Chen1, FengFeng Xie1, XiaoXiu Huang1, JianGe Qiu2, WenJi Zhang2, QiWei Jiang2, Yang

1

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Yang2, Hua Zhu1, Zhi Shi2, XiaoJian Yan1

Department of Gynecology, the First Affiliated Hospital of Wenzhou Medical

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University, Wenzhou, Zhejiang 325000, China;

Department of Cell Biology & Institute of Biomedicine, National Engineering

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Research Center of Genetic Medicine, Guangdong Provincial Key Laboratory of

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Bioengineering Medicine, College of Life Science and Technology, Jinan University,

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Guangzhou, Guangdong 510632, China;

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The first two authors contributed equally to the work.

Corresponding authors: Zhi Shi, MD, PhD, Room 708, The 2nd Engineer and Scientific Building, 601 Huangpu Road West, Guangzhou, China, 510632, E-Mail: [email protected], Tel: +86-20-852-245-25, Fax: +86-20-852-259-77; or XiaoJian Yan, MD, PhD, Shangcai village south, Ouhai District, Wenzhou, Zhejiang 325000, China,

E-Mail:

[email protected],

+86-577-555-780-33

1

Tel:

+86-577-555-791-63;

Fax:

ACCEPTED MANUSCRIPT Abstract Objective: Ovarian cancer is one of the most lethal of women cancers and lack potent

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therapeutic options.There have many evidences demonstrate the Notch signaling has

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deregulation in variety of human malignancies.MK-0752 is a novel potent γ-secretase inhibitor and now assessed in clinical trial for treatment of several types of cancer,our objective was to investigate the anticancer effects and mechanisms of MK-0752 alone

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or combined with cisplatin in ovarian cancer.

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Methods: Cell lines used:A2780,OVCAR3,SKOV3,HO8910PM,the effects of MK-0752 and cisplatin on cell proliferation was measured by MTT assay. The effect

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of combination treatment was examined by isobologram analysis.The distribution of

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cell cycle and cell apoptosis were analysed using PI and Annexin V-FITC/PI staining

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by flow cytometric analysis. The mechanism in biochemistry were analysed by using western blot. Mouse xenograft model of A2780 was established to observe the

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anti-ovarian cancer effects in vivo setting, nude mice were randomized into four groups (n=6 per group) and treated every four days with control (solvent) group, MK-0752(25mg/Kg) group, Cisplatin(2mg/Kg )group, combination group(both of MK-0752 and cisplatin). Results: MK-0752 alone actively induced cell growth inhibition,G2/M phase cell cycle arrest and apoptosis with down-regulation of Notch1 and its downstream effectors including Hes1,XIAP,c-Myc and MDM2 in a dose- and time- dependent manner. Moreover, sequential combination of cispaltin prior to MK-0752 significantly promoted cell apoptosis and inhibited the subcutaneous xenograft growth of ovarian 2

ACCEPTED MANUSCRIPT cancer in nude mice. Conclusion: Our data supports the sequential combination of cispaltin prior to

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MK-0752 is a highly promising novel experimental therapeutic strategy against

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ovarian cancer.

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Keywords: Ovarian cancer; MK-0752; Cisplatin; Combination therapy.

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ACCEPTED MANUSCRIPT Introduction Ovarian cancer is the seventh-most common cancer and the eighth-most common

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cause of death from cancer among women [1]. Because most patients with ovarian

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cancer are diagnosed at advanced stage of disease, the prognosis of ovarian cancer is relatively poor with a 5-year survival rate of approximately 44% [1]. Chemotherapy is a general standard treatment for ovarian cancer, typically consisting of platinum and

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taxane based therapy. However, most ovarian cancers recur 6–12 months after last

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treatment [2]. Treatment for recurrent ovarian cancer is currently incurable, prompting the development of new therapeutic strategies.

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The Notch signaling pathway is a highly evolutionally conserved molecular

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pathway that functions in the proliferation and differentiation of many tissues [3]. In

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mammals, there are four Notch receptors (Notch1-4) that are single-pass transmembrane proteins that have rich epidermal growth factor (EGF)-like repeats in

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the extracellular region, and five ligands including three members of Delta-like family (Dll1, 3 and 4) and two members of Serrate-like family (Jag1 and 2) [4]. Binding of the ligand to Notch receptor initiates downstream signaling by a cascade of proteolytic cleavages by α-secretase and γ-secretase to release the intracellular domain of the Notch receptor, which then translocates into the nucleus and associates with the CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factor complex to activate the expression of target genes, such as the Hes-family members and c-Myc [4]. The role of Notch signaling in human cancer was first unraveled in T-cell acute lymphoblastic leukemia [5]. Increasing number of evidence certify that Notch 4

ACCEPTED MANUSCRIPT signaling have deregulation in human hematological malignancies and solid tumors, such as ovarian cancer, breast cancer, head and neck cancer, endometrium cancer and

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so on [6-9]. It has reported that Notch1 and Notch3 play a significant role in ovarian

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cancer [10-14]. The RNA and protein levels of Notch1 and Notch3 are highly expressed in ovarian cancer, and overexpression of Notch proteins were closely relate to poor prognosis and tumor differentiation of ovarian cancer [10, 13]. Due to its

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important roles in cancer, the Notch signaling pathway has been a potential target for

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cancer therapy.

γ-secretase is a key mediator of Notch signaling, and inhibitors for γ-secretase are

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able to prevent Notch receptor activation and therefore against cancer [15]. MK-0752

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is a new potent γ-secretase inhibitor (GSIs), which inhibits γ-secretase to cleave

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substrates such as amyloid precursor protein and Notch with an IC50 of 50 nM [16]. MK-0752 shows promising effects on inhibiting the growth of several types of cancer

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cells and currently investigated in clinical trials [17-20]. However, the effect of MK-0752 on ovarian cancer is still unclear. In this study, we investigated that anticancer effects and mechanisms of MK-0752 alone or combined with cisplatin in preclinical models of ovarian cancer.

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ACCEPTED MANUSCRIPT Material and Methods Cell culture and reagent

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Human ovarian cancer cell lines A2780, OVCAR3, SKOV3 and HO8910PM were

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cultured in Dulbecco’s modified Eagle’s medium (DMEM) including 10% fetal calf serum (FBS), penicillin (100U/ml) and streptomycin (100ng/ml) in a humidified incubator at 37°C with 5% CO2. MK-0752 and csiplatin were ordered from ApeBio

Technologies.

c-Myc

(SC-40)

antibodies

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antibodies were from Cell Signaling

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and Qilu Pharmaceutical, respectively. Anti-PARP (9542) and Anti-XIAP (2045)

were from Santa Cruz Biotechnology. Anti-Cyclin B (610219), Anti-CDK1 (610037)

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and Anti-MDM2 (556353) antibodies were from BD Biosciences. Anti-GAPDH

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(LK9002T) antibodies were from Tianjin Sungene Biotech. Anti-Notch1 (AB61041b)

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and Anti-Hes1 (AB61738a) antibodies were from Shanghai Sangon Biotech. Cell viability MTT assay

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Cells were seeded into 96-well cell culture plates at a density of 5×103 cells per well, and incubated with drugs for 72 hr. Then 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) was added to each well at a final concentration of 0.5 mg/ml, and cells were incubated for an additional 4 h. The resulting formazan crystals were solubilized in 100 μl of DMSO (dimethyl sulfoxide) and absorbance at 570 nm by microplate reader. The concentrations required to inhibit growth by 50% (IC50) were calculated from survival curves using the Bliss method [21]. Cell cycle assay Cells were harvested and washed twice in cold phosphate-buffered saline (PBS), then 6

ACCEPTED MANUSCRIPT fixed with 75% ice-cold ethanol for 30 min at 4°C. After centrifugation at 200 × g for 10 min, cells were washed twice with ice-cold PBS and resuspended with 500 μl of

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PBS containing PI (50 μg/ml), 0.1% Triton X-100, 0.1% sodium citrate, and

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DNase-free RNase (100 μg/ml), and detected by Flow cytometry (FCM) after 15 min incubation at room temperature in the dark. Fluorescence was measured at an excitation wavelength of 480 nm through a FL-2filter (585 nm). Data were analyzed

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using ModFit LT 3.0 software (Becton Dickinson) [22, 23].

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Apoptosis assay

Cell apoptosis was evaluated with FCM assay. Briefly, cells were harvested and

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washed twice with PBS, stained with Annexin V‑FITC and propidium iodide (PI) in

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the binding buffer, and detected by FACSCalibur FCM (BD, CA, USA) after 15 min

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incubation at room temperature in the dark. Fluorescence was measured at an excitation wave length of 480 nm through FL-1 (530 nm) and FL-2 filters (585 nm).

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The early apoptotic cells (Annexin V positive only) and late apoptotic cells (Annexin V and PI positive) were quantified [24]. Western blot analysis Cells were harvested and washed twice with cold PBS, then resuspended and lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 ng/ml PMSF, 0.03% aprotinin, 1μM sodium orthovanadate) at 4 °C for 30 min. Lysates were centrifuged for 10 min at 14,000 × g and supernatants were stored at -80 °C as whole cell extracts. Total protein concentrations were determined with Bradford assay. Proteins were separated on 12% SDS-PAGE gels and transferred to polyvinylidene 7

ACCEPTED MANUSCRIPT difluoride membranes. Membranes were blocked with 5% BSA and incubated with the indicated primary antibodies. Corresponding horseradish peroxidase-conjugated

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secondary antibodies were used against each primary antibody. Proteins were detected

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using the chemiluminescent detection reagents and films [25]. In vivo xenograft studies

Balb/c nude mice were obtained from the Guangdong Medical Laboratory Animal

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center and maintained with sterilized food and water. The experiments have been

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approved by institutional review board. Six female nude mice with 5 weeks old and 15 g weight were used for each group. Each mouse was injected subcutaneously with

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A2780 cells (2 × 106 in 100 μl of medium) under the shoulder. When the

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subcutaneous tumors were approximately 0.3 × 0.3 cm2 (two perpendicular diameters)

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in size, mice were randomized into four groups and treated every 4 days with vehicle alone, MK-0752 (25 mg/kg in 0.5% methylcellulose by oral administration), cisplatin

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(2 mg/kg by intraperitoneal injection), or a sequential combination of cisplatin prior to MK-0752 1 day. The body weights of mice and the two perpendicular diameters (A and B) of tumors were recorded. The tumor volume (V) was calculated according to the formula [26]:

 V=

A+B )3

( 6

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The mice were anaesthetized after experiment, and tumor tissue was excised from the mice and weighted. The rate of inhibition (IR) was calculated according to the 8

ACCEPTED MANUSCRIPT formula: Mean tumor weight of experimental group ×100%

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Mean tumor weight of control group

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IR= 1

Statistical analysis

All results are expressed as mean ± standard deviation (SD). Statistical analysis of the

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difference between treated and untreated groups was performed with Student’s t-test.

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Values of P< 0.05 were considered as significant differences.

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ACCEPTED MANUSCRIPT Results MK-0752 inhibited the growth of ovarian cancer cells in vitro.

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In order to examine the effect of MK-0752 on ovarian cancer cells, cell survival was

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detected by MTT assay. Four human ovarian cancer cell lines A2780, OVCAR3, SKOV3 and HO8910PM were treated with increasing concentrations of MK-0752 for 72 hr. MK-0752 dose-dependently inhibited the growth of ovarian cancer cells with

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the IC50 values range from 129.78 to 152.32 μM. Additionally, cisplatin also

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suppressed the growth of ovarian cancer cells in a dose-dependent manner with the IC50 of 5.64 to 9.39 μM (Figure 1).

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MK-0752 induced cell cycle arrest in ovarian cancer cells.

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To determine whether the growth inhibition of ovarian cancer cells by MK-0752 is as

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a result of cell cycle arrest, cell cycle distribution was assessed after MK-0752 treatment. A2780 and OVCAR3 ovarian cancer cells were treated with different

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concentration of MK-0752 (30, 100, 300 μM) for 24 and 48h, stained with PI and examined by FCM. The cell cycle distribution was analyzed by using ModFit LT 3.0 software. As shown in Figure 2A and 2B, treatment with MK-0752 at 300 μM dramatically resulted in the G2/M phase arrest in both A2780 and OVCAR3 cells with the increased population of subG1 phase. To investigate the molecular mechanism of cell cycle arrest by MK-0752, the cell cycle related proteins were detected by Western blot. After treatment with MK-0752, the protein levels of CDK1 and Cyclin B were decreased in the time- and dose- dependent manners in both A2780 and OVCAR3 cells (Figure 2C). 10

ACCEPTED MANUSCRIPT MK-0752 induced apoptosis in ovarian cancer cells. To explore whether the growth inhibition of ovarian cancer cells by MK-0752 is also

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due to apoptosis, cell apoptosis was assessed after MK-0752 treatment. A2780 and

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OVCAR3 cells were treated with MK-0752 (30,100 and 300 μM) for 48 hr, stained with Annexin V/PI and examined by FCM. As shown in Figure 3A and 3B, treatment with MK-0752 at 300 μM dramatically induced early apoptosis (Annexin V+/PI-) and

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late apoptosis (Annexin V+/PI+) in both A2780 and OVCAR3 cells. To investigate

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the molecular mechanism of cell apoptosis by MK-0752, the apoptotic related proteins were detected by Western blot. After treatment with MK-0752, the cleaved

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C-PARP, which is the marker of apoptosis, was time- and dose-dependently increased

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in both A2780 and OVCAR3 cells. Furthermore, the protein levels of XIAP, MDM2,

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Notch1, Hes1 and c-Myc were significantly decreased in the time- and dose-dependent manners in both cells (Figure 3C).

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Sequential combination of cisplatin and MK-0752 enhanced the growth inhibition of ovarian cancer cells. Cisplatin triggers cancer cells death by crosslinking with DNA, and now is used for first-line therapy of ovarian cancer in clinic [27]. To determine the combined effects of MK-0752 and cisplatin on the growth of ovarian cancer cells, both A2780 and OVCAR3 cells were treated with cisplatin at 3 μM and MK-0752 at 100 μM alone or under three different combinations: added for cisplatin 24 hr followed by MK-0752 for another 48 hr, reversely or at the same time. As shown in Figure 4, the inhibition of cell growth in cisplatin prior to MK-0752 group was significantly higher than those 11

ACCEPTED MANUSCRIPT in other two combinations groups. Sequential combination of cisplatin and MK-0752 increased the apoptosis of

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ovarian cancer cells.

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In order to estimate whether sequential combination of cisplatin and MK-0752 increases the apoptosis of ovarian cancer cells, both A2780 and OVCAR3 cells were treated as same order as Figure 4 and apoptosis was detected. The cell apoptosis in

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cisplatin prior to MK-0752 group was markedly higher than those in other two

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combinations groups (Figure 5A and 5B). Consistently, the results of Western blot showed that the protein levels of apoptosis marker C-PARP were clearly induced in

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cisplatin prior to MK-0752 group (Figure 5C).

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Sequential combination of cisplatin and MK-0752 strengthened the subcutaneous

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xenograft growth inhibition of ovarian cancer in nude mice. To test the antitumor effects of sequential combination of cisplatin and MK-0752 on

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ovarian cancer cells in vivo, we generated the subcutaneous xenograft tumor models by transplanting A2780 cells into nude mice. As shown in Figure 6, compared with MK-0752 or cisplatin alone, sequential combination of cisplatin prior to MK-0752 dramatically inhibited the A2780 tumors growth by reducing their volumes and weights. The inhibition rates of tumor growth in the combined group were 66.9%, which were obviously higher than those in cisplatin (42.8%) or MK-0752 (27.0%) alone group. The net body weights of mice (without tumor) in the four groups were no statistical difference, indicating that sequential combination of cisplatin and MK-0752 at the indicated dose may be safe in mice. 12

ACCEPTED MANUSCRIPT Discussion GSIs are the most widely investigated Notch signaling pathway targeting agents. A

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number of GSIs with distinct chemical structures have been developed to block cell

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growth in a variety of cancers [28,29]. In the present study, our data show that MK-0752 is able to induce growth inhibition, cell cycle arrest and apoptosis with the down-regulation of Notch1 and its downstream effectors including Hes1, XIAP,

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c-Myc and MDM2 in A2780 and OVCAR3 ovarian cancer cells. Sequential

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combination of cisplatin prior to MK-0752 significantly increases cell apoptosis and growth inhibition of ovarian cancer cells in vitro and in vivo. Hes1 is one of the

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effectors of Notch signaling that regulates cell proliferation and differentiation in

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various organs [30]. Notch suppresses apoptosis through direct interference with

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XIAP ubiquitination to increase its stability [31]. Notch also up-regulates of c-Myc and MDM2 to promote cell proliferation [32,33]. Therefore, the down-regualtion of

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these Notch target proteins by MK-0752 suggests the effects of MK-0752 on ovarian cancer cells are due to inhibition of the Notch signal pathway. Several groups have reported the effectiveness of other GSIs in ovarian cancer. The compound GSI-1 could reduce cell proliferation and induce apoptosis in A2780 and OVCAR3 ovarian cancer cells [11,12]. GSI-1 was also able to deplete cancer stem cells and sensitize ovarian cancer to cisplatin [34]. Contrarily, a recent study showed that treatment with the GSIs Compound E, DAPT or dibenzazepine had no effect on the growth of OVCAR3, SKOV3 and several other ovarian cancer cell lines [35]. Inconsistent with our sequential combination findings, Wang and colleges demonstrated that in two 13

ACCEPTED MANUSCRIPT cisplatin-resistant ovarian cancer cells A2780/CP70 and OV2008/C13 pretreatment with another GSI DAPT increased the sensitivity of cisplatin through down-regulation

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of both mRNA and protein levels of Notch1 and Hes1, while cisplatin treatment

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followed by DAPT only presented an additive or antagonistic effects [36]. These different combination results may be due to the diverse experimental conditions, and the combination of GSIs with chemotherapeutic agents need to be further verified.

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Moreover, further study is needed for the efficacy of MK-0752 in more ovarian cancer

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cells, such as drug–resistant cells and patients–derived cells. To date, several clinical trials have provided important data about the efficacy of

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MK-0752 in cancer patients. In a phase I trial, MK-0752 showed clinical benefit in

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patients with high-grade gliomas and significantly inhibited Notch signaling at

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1800-4200 mg weekly dose levels with a half-life of around 15 hr, and the most common dose-limiting toxicities were diarrhea, vomiting, nausea, and fatigue [17].

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MK-0752 was also well-tolerated in a dose-escalation trial administered once daily for 3 consecutive days of every 7 days up to 260 mg/m2 in children with refractory or recurrent CNS malignancies, and although there were no objective responses, two patients experienced prolonged stable disease [18, 20]. Combined 20-30 mg/kg daily mTOR inhibitor ridaforolimus 5 days/week and 1800 mg weekly MK-0752 showed activity in patients with head and neck squamous cell carcinoma [37]. The humanised anti-IGF-1R antibody dalotuzumab 10 mg/kg in combination with MK-0752 1800 mg weekly were tolerable in patients with advanced solid tumors, and seven patients remained on study for more than 4 cycles [38]. However, a recent phase II study 14

ACCEPTED MANUSCRIPT showed that another γ-secretase inhibitor RO4929097 had insufficient activity as a single-agent in patients with recurrent platinum-resistant ovarian cancer [39]. In

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addition, cisplatin is the substrate of ABCC2, ABCC4, ATP7B, SLC22A1, SLC22A2,

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SLC31A1 and SLC47A1 transporters [40]. Whether the pharmacokinetic, pharmacodynamic and clinical outcome of MK-0752 and cisplatin would interfere with each other in patients with ovarian cancer remain to be determined.

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In summary, our study in preclinical models of ovarian cancer not only

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demonstrates that MK-0752 alone can induce cell cycle arrest and apoptosis, but also shows that sequential combination of cisplatin prior to MK-0752 significantly inhibits

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the tumor growth in vitro and in vivo, suggesting this sequential combination may

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make benefits for ovarian cancer treatment.

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ACCEPTED MANUSCRIPT ACKNOWLEDGMENTS This work was supported by funds from the Chinese National Natural Science

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Foundation No. 31271444 and No. 81201726 (Z. S.), the Specialized Research Fund

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for the Doctoral Program of Higher Education No. 20124401120007 (Z. S.), the Guangdong Natural Science Funds for Distinguished Young Scholar No. 2014A030306001 (Z. S), and the Science and Technology Program of Guangzhou No.

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2014J4100009 (Z. S), and the Natural Science Foundation of Zhejiang Province,

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China No.LQ12H16004 (X. J. Y.), the Chinese National Natural Science Foundation No 81503293(X.J.Y) , and the Chinese National Natural Science Foundation No

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81373075 (R.Y.O)

The authors declare that there are no conflicts of interest. 16

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ACCEPTED MANUSCRIPT tumoral and host solute carriers to clinical drug response, Drug Resist Updat, 15

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ACCEPTED MANUSCRIPT Figure legends Figure 1. MK-0752 inhibited the growth of ovarian cancer cells in vitro. (A)

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Chemical structure of MK-0752. (B)Summary of IC50 of MK-0752 and ciaplatin in

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the indicated ovarian cancer cells was sown.（C）Cells were grown in 96-well plates for 24 hr and treated with indicated concentrations of MK-0752 or cisplatin for 72 hr. Cell survival was measured by MTT assay. Data were mean ±SD of three independent

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experiments.

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Figure 2. MK-0752 induced cell cycle arrest in ovarian cancer cells. A2780 (A) and OVCAR3 (B) cells were treated with MK-0752 at the indicated times and

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concentrations. The distribution of cell cycle was detected by FCM with PI staining.

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The percentages of subG1, G1/G0, S, G2/M phase were calculated using ModFit LT

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3.0 software. The protein expression was examined by Western blot after lysing cells, and GAPDH was used as loading control. The representative charts, quantified results

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and Western blot results (C) of three independent experiments were shown. *P<0.05 and **P<0.01 vs. corresponding control. Figure 3. MK-0752 induced apoptosis in ovarian cnacer cells. A2780 (A) and OVCAR3 (B) cells were treated with MK-0752 at the indicated time and concentrations. The apoptosis was detected by FCM Annexin V/PI staining. The proportions of Annexin V+/PI- and Annexin V+/PI+ cells indicated the early and late stage of apoptosis. The protein expression were examined by Western blot after lysing cells, and GAPDH was used as loading control. The representative charts, quantified results and Western blot results (C) of three independent experiments were shown. 24

ACCEPTED MANUSCRIPT *P<0.05 and **P<0.01 vs. corresponding control. Figure 4. Sequential combination of cisplatin and MK-0752 enhanced the growth

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inhibition of ovarian cancer cells. A2780 and OVCAR3 cells were treated with

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cisplatin at 3 μM and MK-0752 at 100 μM alone or under three different combinations: added for cisplatin 24 hr followed by MK-0752 for another 48 hr, reversely or at the same time. The inhibition rate was determined by MTT assay. Data

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were mean ±SD of three independent experiments. *P<0.05 and **P<0.01 vs.

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Figure 5. Sequential combination of cisplatin and MK-0752 increased the

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apoptosis of ovarian cancer cells. A2780 (A) cells and OVCAR3 (B) cells were

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treated with cisplatin at 10 μM 、3 μM respectively and MK-0752 at 100 μM alone or

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under three different combinations: added for cisplatin 24 hr followed by MK-0752 for another 48 hr, reversely or at the same time. The apoptosis was detected by FCM

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Annexin V/PI staining. The proportions of Annexin V+/PI- and Annexin V+/PI+ cells indicated the early and late stage of apoptosis. The protein expression was examined by Western blot after lysing cells, and GAPDH was used as loading control. The representative charts, quantified results and Western blot results (C) of three independent experiments were shown. MK: MK-0752; DDP: Cisplatin. *P<0.05 vs. corresponding control. Figure 6. Sequential combination of cisplatin and MK-0752 increased the apoptosis of ovarian cancer cells. Each mouse was injected subcutaneously with A2780 cells (2 × 106 in 100 μl of medium) under the shoulder. When the 25

ACCEPTED MANUSCRIPT subcutaneous tumors were approximately 0.3 × 0.3 cm2 (two perpendicular diameters) in size, mice were randomized into four groups and treated every 4 days with vehicle

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alone, MK-0752 (25 mg/Kg in 0.5% methylcellulose by oral administration), cisplatin

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(2 mg/kg by intraperitoneal injection), or a sequential combination of cisplatin prior to MK-0752 1 day. The body weights of mice and tumor volume were recorded. The mice were anaesthetized after experiment, and tumor tissue was excised from the mice

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and weighted. The original tumors (A), tumor volume (B), tumor weight (C), net body

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weight (without tumor) (D) and summary data (E) were shown. The values presented are the means ± SD for each group. MK: MK-0752; DDP: Cisplatin. *P<0.05 and

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ACCEPTED MANUSCRIPT Highlights 1 .MK-0752 alone can induce ovarian cancer cell cycle arrest and apoptosis.

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2. Sequential combination of cisplatin prior to MK-0752 significantly inhibits the

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tumor growth in vitro and in vivo.

3. Sequential combination of cisplatin prior to MK-0752 may make benefits for

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ovarian cancer treatment.

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