Accepted Manuscript Cancer-associated fibroblasts attenuate Cisplatin-induced apoptosis in ovarian cancer cells by promoting STAT3 signaling Huan Yan, Bi-Ying Guo, Shu Zhang PII:

S0006-291X(16)30132-2

DOI:

10.1016/j.bbrc.2016.01.131

Reference:

YBBRC 35248

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 15 January 2016 Accepted Date: 21 January 2016

Please cite this article as: H. Yan, B.-Y. Guo, S. Zhang, Cancer-associated fibroblasts attenuate Cisplatin-induced apoptosis in ovarian cancer cells by promoting STAT3 signaling, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.01.131. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Cancer-associated fibroblasts attenuate Cisplatin-induced apoptosis in ovarian cancer cells by promoting STAT3 signaling Huan Yan, Bi-Ying Guo and Shu Zhang *

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Department of Obstetrics and Gynecology，RenJi Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Key Laboratory of Gynecologic Oncology, Shanghai, China.

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*Corresponding authors at: Department of Obstetrics and Gynecology，RenJi Hospital,

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School of Medicine, Shanghai Jiao Tong University, Shanghai Key Laboratory of Gynecologic Oncology, Shanghai 200127, China. Tel: 86-21-58752345

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E-mail addresses: [email protected] (S. Zhang);

ACCEPTED MANUSCRIPT Abstract One of the main reasons for treatment failure in ovarian cancer is acquired drug resistance.

Cancer in

many

fibroblasts

human

(CAFs)

tumors.

are

known

However,

its

to

enhance

contributions

to

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chemoresistance

associated

chemoresistance acquisition in ovarian cancer are not well understood. Here, we provide the first evidence that the conditioned medium of CAFs (CAFs-CM) could

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attenuate the sensitivity to Cisplatin in A2780 and ES2 ovarian cancer cells and

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protect them from Cisplatin-induced apoptosis. We found the expression level of two anti-apoptotic proteins, Bcl-2 and Survivin, as well as their upstream controller p-STAT3 were significantly increased when ovarian cancer cells were exposed to CAFs-CM. Furthermore, inhibition of STAT3 signaling with Cryptotanshinone could

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down-regulate the expression of Bcl-2 and Survivin, thus weaken the post-target resistance to Cispaltin mediating by CAFs-CM in ovarian cancer cells. In conclusion, our data suggested that CAFs could activate the anti-apoptotic STAT3 signaling,

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thereby decrease the Cisplatin-induced apoptosis and promote chemoresistance in

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ovarian cancer.

Keywords: cancer-associated fibroblasts; epithelial ovarian cancer; chemoresistance; apoptosis; STAT3;

ACCEPTED MANUSCRIPT 1.

Introduction

Ovarian cancer is the leading cause of cancer deaths from gynecological malignancy in women and recurrence, chemotherapy-resistance contributes to the high mortality

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in patients diagnosed with ovarian cancer [1]. Despite significant advances in surgical treatment and Cisplatin-centered chemotherapy of this cancer, 50% to 70% of patients will relapse within one year [2]. When such relapse occurs, in most cases the ovarian

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adenocarcinoma cells have acquired a Cisplatin-resistance phenotype and the

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responsible mechanism remains poorly understood. Several studies have documented that Cisplatin-resistance can arise from increased drug efflux, abnormity of drug target, enhancement of DNA repair，alteration of apoptosis pathways and inactivation of tumor suppressor [2, 3].

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Recently, studies indicated that tumor microenvironment could potentially induce chemoresistance acquisition in tumor cells and the microenvironment has become a major focus in modeling therapeutic response [4]. The cells within the tumor

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microenvironment (stroma) include fibroblasts, endothelial cells, adipocytes,

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mesenchymal cells, mesenchymal stem cells and cells from the immune and inflammatory systems. Fibroblasts within tumors have an activated phenotype, which termed cancer-associated fibroblasts (CAFs). CAFs make up the bulk of cancer stroma of many kinds of cancer, most notably breast, prostate and pancreatic carcinoma [5, 6]. Recent research had discovered that CAFs not only played an important role in tumor growth, angiogenesis and dissemination, but also regulated chemoresistance [7, 8]. In the pancreas, CAFs have been shown to enhance

ACCEPTED MANUSCRIPT chemoresistance in a pancreatic carcinoma model via inhibiting expression of Caspases (8, 9, 7 and 3) [9]. Moreover, CAFs in breast cancer have been correlated with the induction of acquired chemoresistance, which has made CAFs a new target in

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anticancer therapy [10]. Ovarian cancer is one of the most stroma-rich cancers and CAFs are the most abundant. Zhang et al. found a significant association between ovarian CAFs and the

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occurrence of lymph node and omentum metastases in clinical samples,

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demonstrating that ovarian CAFs were related to ovarian cancer progression and patient clinical outcomes [11]. Moreover, Xu et al. and Lai et al. have provided evidence that CAFs isolated from ovarian cancer tissues induced ovarian cancer cells invasion and migration in vitro [12, 13]. In particular, other recent study indicated a

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role of CAFs-secreted cytokines (IL-6, COX-2 and CXCL1) in promoting breast and ovarian carcinoma growth and invasion [14]. CAFs in ovarian carcinoma were shown in those studies to impact tumor

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progression and metastasis, however little research has been done to evaluate the role

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of CAFs in ovarian cancer chemoresistance. The aim of the present study was to investigate the possible role of conditioned medium (CM) of CAFs in Cisplatin resistance of ovarian adenocarcinoma cells. Here, we showed for the first time that a significant decrease of Cisplatin-induced apoptosis in A2780 and ES2 ovarian cancer cells incubated in the presence of CAFs-CM. Our results indicate CAFs-CM could protect ovarian cancer cells from apoptosis by activating the anti-apoptotic STAT3 signaling, including the overexpression of Bcl-2 and Survivin and the potential use of

ACCEPTED MANUSCRIPT STAT3 inhibitors (Cryptotanshinone) could overcome ovarian cancer cells resistance to chemotherapy mediated by tumor microenvironment.

2.1 Primary human CAFs isolation and cell culture

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2. Materials and methods

CAFs were isolated from human ovarian tumor tissues using the method described by

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Lai et al [12]. The human epithelial ovarian cancer (EOC) cells (A2780 and ES2)

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were purchased from the American Type Culture Collection (ATCC, Manassas, VA). CAFs and EOC cells were cultured in RPMI-1640 (Gibco, Foster City, CA, USA) supplemented with 10% (w/v) fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco). All CAFs used for this study were between passage 3

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and 5.

2.2 Preparation of CAFs conditioned medium (CM)

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CAFs-CM was made from the culture of CAFs in RPMI-1640 with 10% of FBS for

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72 h. Then, the CM were collected and centrifuged at 2,000 g to remove cell debris and filtrated with a 0.22-mm filter (Millipore, Bedford, MA, USA). To prepare co-culture CM, CAFs-CM was mixed with fresh RPMI-1640 complete medium at a ratio of 1:1 and 2:1, which were represented by the Mix1:1 and Mix2:1 in the article.

2.3 Immunofluorescence An indirect immunofluorescent labeling technique was used to identify α-SMA and

ACCEPTED MANUSCRIPT Vimentin expression cells using the following primary antibodies: rabbit monoclonal anti-α-SMA (1:100, Abcam, Cambridge, UK) and rabbit monoclonal anti- Vimentin (1:50, Cell Signaling Technology, Beverly, MA, USA) at 4 ℃ overnight, followed

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by incubating with Alexa Fluor® 488 conjugate goat anti-rabbit IgG (H+L) secondary antibody (1:200, Invitrogen, Carlsbad, CA). Cells were counterstained with DAPI (Invitrogen) and examined under the fluorescence microscope (Leica, Wetzlar, Hesse,

2.4 Cell viability assay

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Germany).

Cells were seeded in 96-well plates overnight, and then treated with the indicated concentrations of Cisplatin and Cryptotanshinone for 24h. Then cells were incubated

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in CCK8 reagent (Dojindo, Tokyo, Japan) for 2h. The absorbance value was measured at 450 nm by Thermo Scientific Varioskan Flash (Thermo Fisher Scientific, USA) and

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the IC50 was calculated by GraphPad Prism 5 software.

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2.5 Western blotting

Western blotting was performed as previously described [15]. Antibodies for western blotting are Cytokeratin-8 (1:1000, Abcam), α-SMA (1:1000, Abcam), caspase-3 (1:1000, Cell Signaling Technology), Bcl-2 (1:1000, Cell Signaling Technology); survivin (1:200; Santa Cruz Biotechnology, Inc., CA, USA); STAT3(1:1000, ProteinTech Group, Chicago, IL, USA), phospho-STAT3 (tyr705) (1:1000, ProteinTech Group); and β-actin (1:1000, Sigma-Aldrich). The blots were incubated

ACCEPTED MANUSCRIPT with species-specific secondary antibodies: IRDye 680 anti-mouse and IRDye 800 anti-rabbit (1:10000, LI-COR, Nebraska, USA). The signals were detected by

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Odyssey imaging system (LI-COR).

2.6 Flow cytometry

The apoptosis rate was detected by flow cytometry with apoptosis assay kit

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(Invitrogen) following instructions of the manufacturer. Brifely, cells cultured in the

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presence or absence of CAF CM and exposed to Cisplatin and STAT3 inhibitor for 24 hours were collected and washed in PBS. After re-suspended in 100 µl of 1X binding buffer, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated Annexin V and Propidium Iodide for 15 min at room temperature in the dark. Then

Coulter, USA).

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cells were analyzed by FACS Calibur Flow Cytometer using CXP software (Beckman

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2.7 STAT3 inhibition

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Cryptotanshinone (Selleck Chemicals, Houston, TX, USA) is a selective STAT3 inhibitor, which inhibits the phosphorylation of STAT3 Tyr705. A2780 and ES2 Cells were treated with different concentrations of Cryptotanshinone for 24h. As Cryptotanshinone was dissolved in DMSO, DMSO was chosen as a negative control.

2.8 Statistical analysis All experiments were repeated at least three times. The data were presented as Mean ±

ACCEPTED MANUSCRIPT SD. Statistical analyses were conducted using SPSS 16.0 software (Chicago, IL, USA). The differences between two groups were analyzed using the double-sided Student’s t-test. Comparisons among three or more groups were evaluated using

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one-way analysis of variance (ANOVA). *P < 0.05, **P < 0.01 and***