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The cytoplasmic translocation of Cx32 mediates cisplatin resistance in ovarian cancer cells
Accepted Manuscript The cytoplasmic translocation of Cx32 mediates cisplatin resistance in ovarian cancer cells Weili Wu, Lixia Fan, Zeqing Bao, Yu Zhang, Yuexia Peng, Min Shao, Yuke Xiang, Xiaomin Zhang, Qin Wang, Liang Tao PII:
S0006-291X(17)30719-2
DOI:
10.1016/j.bbrc.2017.04.053
Reference:
YBBRC 37611
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 10 April 2017 Accepted Date: 11 April 2017
Please cite this article as: W. Wu, L. Fan, Z. Bao, Y. Zhang, Y. Peng, M. Shao, Y. Xiang, X. Zhang, Q. Wang, L. Tao, The cytoplasmic translocation of Cx32 mediates cisplatin resistance in ovarian cancer cells, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.04.053. 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 The cytoplasmic translocation of Cx32 mediates cisplatin resistance in ovarian cancer cells Authors
Xiang1, Xiaomin Zhang1, Qin Wang1, Liang Tao1 1
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Weili Wu1, Lixia Fan1, Zeqing Bao2, Yu Zhang1, Yuexia Peng1, Min Shao1, Yuke
Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-Sen
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University, Guangzhou 510080, People’ s Republic of China; 2Zhaoqing Medical
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college Pharmacology teaching and research section, Zhaoqing 526020, People’ s Republic of China. Correspondence
Liang Tao, Department of Pharmacology, Zhongshan School of Medicine, Sun
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Yat-Sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China. Tel: +86-20-8733-5468, Email: [email protected]. Qin Wang, Department of Pharmacology, Zhongshan School of Medicine, Sun
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Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China.
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Authorship Note: Weili Wu and Lixia Fan made equal contribution to this paper.
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ACCEPTED MANUSCRIPT Abstract Ovarian cancer is the most lethal gynecologic malignancy, and cisplatin is one of the first-line chemotherapeutic agents. However, acquired cisplatin resistance
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prevents the successful treatment of patients with ovarian cancer. Gap junction (GJ) and connexin (Cx) are closely related to tumor formation, but the relationship between cisplatin resistance and GJ or Cx are undetermined. In this study, we
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established the cisplatin-resistant human ovarian cancer cell line A2780-CDDP.
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Here we showed that cisplatin resistance was correlated to the loss of GJ and the upregulation of Cx32 expression. Enhancing GJ in A2780-CDDP cells could increase the apoptotic response to cisplatin treatment. Furthermore, although Cx32 expression was increased in A2780-CDDP cells, it was more localized to the
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cytoplasm rather than in the membrane, and knockdown of Cx32 in A2780-CDDP cells sensitized them to cisplatin treatment. In summary, Cx32 is involved in cisplatin resistance, and cytoplasmic Cx32 plays an important role in
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chemoresistance.
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Keywords: Cisplatin; Resistance; Ovarian cancer; Cx32; Gap junction
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ACCEPTED MANUSCRIPT Introduction Cisplatin is a chemotherapeutic agent that is widely used in the treatment of several malignancies, such as ovarian, testicular, breast, and bladder cancer [1]. It is the
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standard first-line agent for ovarian cancer [2,3]. However, resistance can quickly emerge after treatment, leading to tumor treatment failure [4,5]. Thus, understanding the underlying mechanism of drug resistance to cisplatin and increasing the sensitivity
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to therapeutic drugs are key steps towards the improved treatment of patients with
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ovarian cancer.
Gap junctions (GJs) are plasma membrane channels that directly connect the neighboring cells [6]. GJs comprise of two hemichannels known as connexons, and each connexon contains six transmembrane proteins known as connexins (Cxs) [7,8].
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These GJs provide a channel for immediate intercellular communication and allow for the direct intercellular exchange of ions, small metabolites and second messengers such as sodium, potassium, cAMP/cGMP and glutathione [9,10,11]. Many
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physiological processes are controlled by regulatory molecules that are exchanged
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through GJs, and thus, GJs are crucial in diverse processes. Likewise, gap junction intercellular communication (GJIC) has been shown to be actively involved in signal transduction in tumor cells [12,13]. Cxs are a group of homologous proteins that form the inter-membrane channels of GJs [14]. Abnormal expression and distribution of Cxs are closely related to tumor formation. They can regulate cell proliferation, migration, and apoptosis in a GJIC-independent manner [15,16]. The mechanisms of resistance to platinum-based compounds are very complex. 3
ACCEPTED MANUSCRIPT These mechanisms include decreased accumulation of platinum compounds by either active efflux or impaired influx[17]; detoxification via GSH conjugates; increased DNA damage repair[18] ; aberrant DNA methylation[19]; up- or downregulated
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expression of microRNAs[20,21]. In addition, increasing observations have indicated that Cxs and GJ also contribute to cancer progression and chemoresistance. For example, Paul R. Gielen et al. reported that the increased levels of Cx43 in human
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glioma cells enhanced resistance to TMZ treatment [22]. J Yang et al. showed that
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gefitinib resistance was correlated with upregulation of Cx26 in the HCC827 and PC9 cell lines [23]. In contrast, Meiling Yu et al. reported that Cx43 reversed the resistance of A549 cells to cisplatin [24]. However, the relationship between Cx32 and chemoresistance is rarely reported.
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In our previous studies, we found that GJIC showed a rapid, dose-dependent decrease when exposed to cisplatin, and inhibition of GJIC compromises the effectiveness of these compounds [25]. These results remind us that GJ and Cxs may
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be linked to chemoresistance. However, the above conclusion was drawn from
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drug-sensitive cancer cells rather than drug-resistant cells. Therefore, in the present study, we established the cisplatin-resistant human ovarian cancer cell line A2780-CDDP and directly investigated the connection between GJ/Cxs and chemoresistance.
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ACCEPTED MANUSCRIPT Materials and Methods Materials: Cell culture-related reagents, Lipofectamine™ 2000, CM-DiI and calcein AM were obtained from Invitrogen (Carlsbad, CA, USA). Cisplatin, retinoic acid
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(RA), and anti-Cx32, anti-Cx43 and secondary antibodies for Western blotting were obtained from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor®488 Phalloidin was obtained from Cell Signaling Technology (Danvers, Massachusetts, USA). Cx32
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siRNAs was constructed by Ribbon (Guangzhou, China). Other reagents were
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obtained from Sigma (St. Louis, MO, USA) unless otherwise stated. Methods: Cell lines
The A2780 cell line was obtained from the American Type Culture Collection
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(Manassas, VA, USA). Both paternal A2780 (cisplatin-sensitive) and A2780-CDDP (cisplatin-resistant) cell lines were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in an atmosphere containing 5% CO2.
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Induction of cisplatin resistance
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The cisplatin-resistant cell line A2780-CDDP was generated by exposing the parental cell line A2780 to cisplatin over an 11-month period, with the concentration of cisplatin gradually increasing from 0.5 µg/ml to 16 µg/ml. Cell Viability Assay
A Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to examine cell viability according to the manufacturers’ instructions. Parachute dye-coupling assay 5
ACCEPTED MANUSCRIPT GJ function was determined as described by Goldberg et al[26]. Donor cells were double-labeled with calcein-AM and CM-Dil, and then loaded onto the recipient cells. The two cell types attached to the monolayer for 4 h. The signal intensity was
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observed using a fluorescence microscope (Olympus IX71, Tokyo, Japan). The average number of receiver cells containing calcein per donor cell was regarded as a measure of the degree of GJIC.
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Drug Treatment
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RA was used for the pharmacological regulation of GJIC. Cells were incubated with 40 µM RA for 24 h to enhance GJIC.
RNA Isolation and Real-time quantitative PCR Analysis
Total RNA was isolated using a HiPure Total RNA Mini Kit (Megan, Guangzhou,
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China). Reverse transcription was performed with 2 µg of RNA using the Reverse Transcription System (TRANS, Beijing, China). Cx43, Cx32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected with the
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following respective primers: 5’-GGTCTGAGTGCCTGAACTTGCCT-3' (Forward)
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and 5'-AGCCACACCTTCCCTCCAGCA-3' (Reverse); 5'-GGTGGACCTATGTCATCAGCG-3' (Forward) and 5'-GCCAGGGTAGAGCAGATAAAAGA-3' (Reverse); 5'-GGAGCGAGATCCCTCCAAAAT-3' (Forward) and 5'-GGCTGTTGTCATACTTCTCATGG-3' (Reverse). Real-time quantitative PCR (qRT-PCR) was performed using the ABI StepOneTM quantitative real-time PCR system (Foster City, CA, USA). The comparative CT method was used to quantify the 6
ACCEPTED MANUSCRIPT expression for each gene relative to that of in the A2780 cells using GAPDH as a normalization control. Western blot
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Cells for Western blot analysis were washed with PBS, scraped into lysis buffer, sonicated and centrifuged. The protein concentration was determined with a BCA protein assay kit (Bio-Rad Co., Hercules, CA, USA). A total of 20 µg of protein from
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each sample was separated using SDS-PAGE and transferred to a nitrocellulose
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membrane. Membranes were blocked and then incubated with specific antibodies overnight at 4°C. Monoclonal antibodies targeting Cx32 (1:2000), Cx43 (1:8000) and tubulin (1:10000) were used. Membranes were incubated with the relevant secondary antibody. Immuno-positive bands were scanned by ImageQuant LAS 4000TM.
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siRNA transfection experiments
siRNAs targeting the human Cx32 gene as well as nonspecific sequence siRNAs were added to cells with Lipofectamine™ 2000 transfection reagent according to the
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manufacturer’s protocol. The sequences for the synthesized siRNAs targeting Cx32
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(siCx32) were as follows:
siCx32_1: 5'-CCGGCATTCTACTGCCATT-3'; siCx32_2: 5'-GGCTCACCAGCAACACATA-3'; and siCx32_3: 5'-GCAACAGCGTTTGCTATGA-3'. Membrane protein extraction Membrane-bound and cytoplasmic proteins were extracted by using a Mem-PERTM Plus Membrane Protein Extraction kit (ThermoFisher Scientific™, USA) according to 7
ACCEPTED MANUSCRIPT the manufacturers’ instructions. Immunofluorescence assay Cells were fixed with 4% paraformaldehyde and then blocked with 2% bovine serum
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albumin. The cells were incubated with anti-Cx32 primary antibodies (1:200) overnight at 4°C. The cells were then incubated with FITC-conjugated anti-mouse secondary antibodies (1:400) for 1 h. Alexa Fluor®488 Phalloidin (1:20) was applied
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for 15 min to stain the cytoskeleton of the cells. Hoechst 33258 was used for the
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nuclear staining of the cells. Then, the cells were visualized using a confocal laser scanning fluorescence microscope (ZEISS LSM710). Statistical analysis
The data were statistically analyzed using unpaired Student’s t-test with GraphPad
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considered significant.
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Prism 6.0 software. The results are expressed as the mean ± SD, and p < 0.05 was
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ACCEPTED MANUSCRIPT Results 1. The establishment of a cisplatin-resistant cell line. To investigate the mechanism of acquired cisplatin resistance, we established the
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cisplatin -resistant cell line A2780-CDDP by intermittently exposing A2780 cells to gradually increasing concentrations of cisplatin. To monitor drug resistance, the resistance index (RI) was measured during the cisplatin resistance process, and the RI
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was 1.2, 1.7, 2.9, 4.3 and 7.2 respectively (Figure 1A and B). For the remainder of this
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article, the cells at these various stages (when RI are 1.2, 1.7, 2.9, 4.3 and 7.2) are denoted as A2780-RI(1.2), A2780-RI(1.7), A2780-RI(2.9), A2780-RI(4.3) and A2780-CDDP.
Cisplatin is known to induce apoptosis in cancer cells to inhibit tumor growth.
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Therefore, we detected the levels of cleaved caspase-3 and found that cleaved caspase-3 was activated during the cisplatin treatment in A2780 cells. In A2780-CDDP cells, the induction of cleaved caspase-3 was significantly abrogated
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(Figure 1D and E). These results indicated that A2780-CDDP cells were resistant to
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cisplatin by escaping apoptosis. To confirm A2780-CDDP was a stable genetic variant, we cultivated
A2780-CDDP cells without cisplatin for 20 days, and tested RI on the 10th and 20th day. The levels of cleaved caspase-3 were also detected after the cisplatin treatment. Our results showed that the RI on these two stages had no significant difference (RI was 7.2 and 7.16 respectively, Figure 1F and G), and the apoptosis pathways in
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ACCEPTED MANUSCRIPT A2780-CDDP cells were not activated (Figure 1H and I). These results indicated that A2780-CDDP cells were stably resistant to cisplatin. 2. GJ is involved in the development of cisplatin resistance.
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GJ has been frequently reported to correlate with cancer progression, but the relationship between GJ and cisplatin resistance remains poorly studied. To explore the effect of GJ in the process of cisplatin resistance, we investigated GJ using a
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parachute dye-coupling assay in A2780-RI(1.2), A2780-RI(1.7), A2780-RI(2.9),
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A2780-RI(4.3) and A2780-CDDP cells. During the process of cisplatin resistance, GJIC was gradually reduced (Figure 2A and B). This result revealed that GJ may be involved in the cisplatin resistance of ovarian cancer.
To demonstrate the role of GJ in cisplatin resistance, GJ function was activated
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by RA. The RA treatment alone did not affect cell survival (Figure 2C). The parachute dye-coupling assay was used to confirm that RA activated the GJ function (Figure 2D and E). A2780-CDDP cells were treated with RA (40 µM) for 24 h and then exposed
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to cisplatin prior to measuring the cell viability. The results showed that enhanced GJ
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by RA treatment resulted in sensitivity to cisplatin (Figure 2F). Therefore, our results confirmed a significant role of GJ in cisplatin resistance in ovarian cancer cells. 3. The expression of Cxs in the development of cisplatin resistance. The present results indicated that GJ was involved in cisplatin resistance. We further explored whether the Cxs responsible for GJ were related to cisplatin resistance. In A2780-RI(1.2), A2780-RI(1.7), A2780-RI(2.9), A2780-RI(4.3) and A2780-CDDP, we used q-PCR to analyze the levels of Cx43, Cx40, Cx37, and Cx32, which have been 10
ACCEPTED MANUSCRIPT detected in the ovaries [27]. The results showed that Cx40 and Cx37 had a low mRNA expression levels in A2780 and A2780-CDDP cells (data not shown), and the Cx43 mRNA levels rarely changed in A2780-CDDP cells compared to A2780 cells (Figure
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3B). However, Cx32 mRNA levels were elevated in A2780-CDDP cells nearly fourfold compared to A2780 cells (Figure 3A). To further confirm the expression of Cx32 and Cx43, protein levels were examined by Western blotting. Our results
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showed that Cx32 protein expression was highly elevated in A2780-CDDP cells
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(Figure 4 C and D), while Cx43 showed no significant change in expression (Figure 4 E and F). This result corresponded to that of the mRNA levels. To confirm the effect of Cx32 on cisplatin resistance, we knocked down Cx32 in A2780-CDDP cells with siRNA-Cx32. After Cx32 was knocked down, cells were
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treated with ciaplatin for 48 hours. Cx32, cleaved caspase-3 and cell inhibition were detected. The results showed that the reduced Cx32 expression increased the apoptotic response to cisplatin treatment in A2780-CDDP cells (Figure 3G), and sensitized
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A2780-CDDP cells to cisplatin treatment (Figure 3H). Meanwhile, Cx32 knockdown
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did not affect GJ function in A2780-CDDP cells (Figure 3I). These results suggest that Cx32 protein confers cisplatin resistance in ovarian cancer cells by modulating the apoptosis pathway, and this effect is independent of GJ. 4.Cx32 translocates from the membrane to the cytoplasm in A2780-CDDP cells As GJ was decreased whereas Cx32 expression was elevated during the cisplatin resistance process, it drove us to explore the underlying mechanism. In recent years, it has been reported that Cxs may have abnormally localize to subcellular compartments 11
ACCEPTED MANUSCRIPT [28]. Therefore, we hypothesized that the increased number of Cx32 proteins abnormally
localize
intracellularly.
To
resolve
this
issue,
we
extracted
membrane-bound and cytoplasmic proteins from A2780 and A2780-CDDP cells.
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Western blotting was used to confirm the expression of Cx32 in membrane and cytoplasm. The results showed that the amount of Cx32 in membranes of A2780-CDDP cells was lower than that in A2780 (Figure 4A and B). In contrast,
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cytoplasmic Cx32 was highly elevated in A2780-CDDP cells (Figure 4C and D).
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Immunofluorescence was used to validate the localization of Cx32 in A2780 and A2780-CDDP cells. The results confirmed that the increased number of Cx32 proteins was expressed in cytoplasm in A2780-CDDP cells, and the amount of membrane-bound Cx32 was much lower (Figure 4E). Therefore, these results
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indicated that the reduced Cx32 expression in membrane resulted in the loss of GJIC
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and that the increased cytoplasmic Cx32 played a role in chemoresistance.
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ACCEPTED MANUSCRIPT Discussion Cisplatin is one of the standard first-line chemotherapeutic drugs for ovarian cancer, but prolonged exposure to cisplatin could lead to acquired resistance, which hampers
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the successful treatment of patients. Therefore, it is essential to explore the mechanism of cisplatin resistance in ovarian cancer cells. Prior work by our group identified that the toxicities of cisplatin was reduced when there was no GJ between
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the cells, and cisplatin could inhibit the activity of connexin channels [25]. These
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previous results imply that GJ/Cx is likely associated with chemoresistance. In searching for the direct relationship between GJ/Cx and the cisplatin resistance, we established the cisplatin-resistant cell line A2780-CDDP to investigate the relationship between chemoresistance and GJ/Cx. Our results showed that GJ was
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reduced during the process of acquired cisplatin resistance. Enhancing GJ could reverse the cisplatin resistance of A2780-CDDP. GJs have long been believed to suppress tumor progression during carcinogenesis. One of the mechanisms is that GJs implicated
in
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have been
the bystander
effect,
which
is
beneficial
for
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chemotherapeutics [29,30]. The bystander effect is a phenomenon by which cytotoxic molecules or signals can be transferred from targeted cells to adjacent cells. By this process, cisplatin-induced death signals can be transmitted to neighboring cells via GJ. Therefore, reduced GJ during the cisplatin resistance process may lead to the reduced transfer of cytotoxic molecules and signals, leading to decreased responsiveness to cisplatin. Developing drugs and therapies that can increase GJ may be a new potent strategy to enhance the chemotherapeutic effects of drugs. 13
ACCEPTED MANUSCRIPT Further investigation revealed that the decrease of GJ function was due to reduced membrane localization of Cx32, whereas the levels of cytoplasmic Cx32 were elevated. The Cxs protein accumulated in the cytoplasm fail to constitute GJ.
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Therefore, cytoplasmic Cx32 is involved in chemoresistance in a GJ-independent maner. Other than its role as a constituent of GJ, Cxs play many other roles independent of GJIC. For example, Cx43, Cx40 and Cx32 have very significant
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impacts on cellular injury resistance via processes independent of GJ coupling [31].
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Susan F. Murphy et al. reported that Cx43 levels were inversely correlated with temozolomide sensitivity of glioblastoma cells [32]. Here, we found that Cx32 protein conferred chemoresistance in A2780-CDDP cells and not relied on GJ formation. Some studies support the role of Cx32 protein independent of GJ in the development
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of cancer. For example, cytoplasmic accumulation of Cx32, which failed to facilitate GJ, could enhance the motility and metastatic ability of human hepatoma cells in vitro and in vivo [33]. As Cx32 konckdown increased cleave caspase-3 expression, we
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reason that Cx32 is involved in cisplatin resistance by modulating the apoptosis
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pathway. Here, the results of our study show that Cx32 is involved in chemoresistance in two ways. The first is that decreased Cx32 in the membrane results in the loss of GJ, thereby leading to cisplatin resistance; the other is that increased cytoplasmic Cx32 plays a role in chemoresistance independent of GJ. The phenomenon of cytoplasmic Cx32 accumulation is intriguing, and Cx32 may be a potential novel target for overcoming cisplatin resistance in ovarian cancer.
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ACCEPTED MANUSCRIPT Acknowledgements The present study was supported by the Joint Fund of the National Nature Science Foundation of China (grant No. U1303221), the National Natural Science Foundation
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of China (grant No. 81373439 and 81473234), and the Fundamental Research Funds
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for the Central Universities (grant No.16ykjc01 and 16ykzd11).
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ACCEPTED MANUSCRIPT [18] D.W. Shen, L.M. Pouliot, M.D. Hall, M.M. Gottesman, Cisplatin resistance: a cellular self-defense mechanism resulting from multiple epigenetic and genetic changes, Pharmacol Rev 64 (2012) 706-721. [19] C.T. Viet, D. Dang, S. Achdjian, Y. Ye, S.G. Katz, B.L. Schmidt, Decitabine rescues cisplatin resistance in head and neck squamous cell carcinoma, PLoS One 9 (2014) e112880. [20] D. Cheng, Y. Xu, C. Sun, Z. He, MicroRNA-451 sensitizes lung cancer cells to cisplatin through regulation of Mcl-1, Mol Cell Biochem 423 (2016) 85-91.
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ACCEPTED MANUSCRIPT Figure Legends Fig. 1 The establishment of a cisplatin-resistant cell line (A2780-CDDP). (A) At different cisplatin-resistant stages, cells were treated with 0.3 µg/ml, 1 µg/ml, 3 µg/ml,
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10 µg/ml, 30 µg/ml, or 100 µg/ml of cisplatin for 48 h, and the inhibition of cell growth was determined with the CCK-8 assay. (B) During the process of drug resistance, the RI was measured. (C) Representative images from the assay described
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in (A). The scale bars represent 25 µm. (D) Cisplatin induced cleaved caspase-3
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expression in A2780 cells but not A2780-CDDP cells. Cells were treated with cisplatin (5 µg/ml) for 24, 36, or 48 hours, and protein expression was determined using Western blot. (E) Cleaved caspase-3 quantification. (*, P < 0.05, **, P < 0.01, the results represent the mean ± SD of 3 independent experiments.) . (F, G) The
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inhibition of cell growth at the indicated concentration of cisplatin (10 and 20 days respectively after A2780-CDDP cells were cultivated without cisplatin). Error bars represent the SD (n = 3). (H, I) Cisplatin induced cleaved caspase-3 expression in
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A2780 cells but not A2780-CDDP cells. Cells were treated with cisplatin (5 µg/ml)
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for 48 h after A2780-CDDP cells were cultivated without cisplatin for 10 and 20 days respectively.
Fig. 2 GJ is involved in the development of cisplatin resistance in A2780-CDDP cells. (A) A parachute dye-coupling assay was used to detect GJ. The scale bars represent 25 µm. (B) Quantitative results from A. (C) Cell viability assays after RA treatment. Cells were treated with RA at concentration of 20 µM, 40 µM or 60 µM, 18
ACCEPTED MANUSCRIPT and cell viability was determined using CCK-8. (D) A parachute dye-coupling assay was performed after RA (40 µM) treatment. The scale bars represent 25 µm. (E) Quantification of communicative cells. (F) RA treatment reversed the cisplatin
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resistance in A2780-CDDP cells. Cells were pretreated with RA (40 µM) and then treated with cisplatin at the indicated concentration. Cell viability was determined 48 hours later using the CCK-8 assay. (*, P < 0.05, **, P < 0.01, ***, P < 0.001, the
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results represent the mean ± SD of 3 independent experiments.)
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Fig. 3 Expression of connexins during the development of cisplatin resistance in A2780 cells. (A) Cx32 was upregulated during the development of resistance to cisplatin treatment. qPCR was used to detect the expression of Cx32. (B) Cx43 expression showed no significant differences between the A2780 and A2780-CDDP
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cells. qPCR was used to detect the expression of Cx43. (C, D) A2780-CDDP cells showed increased expression of Cx32. Quantitative results are shown in D. (E, F) A2780-CDDP and A2780 cells showed no significant difference in the expression of
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Cx43. Western blotting was used to detect the expression of Cx32 and Cx43 in A2780
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and A2780-CDDP cells. Bar graphs were derived from densitometric scanning of the blots. (G) Cisplatin (10 µg/ml) induced cleaved caspase-3 expression in Cx32 knockdown cells in A2780-CDDP. (H) After Cx32 knockdown, cells were treated with cisplatin at the indicated concentration. Cell viability was determined 48 hours later using the CCK-8 assay. (*, P < 0.05, **, P < 0.01, the results represent the mean ± SD of 3 independent experiments). (I) Cx32 knockdown does not affect GJ in
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ACCEPTED MANUSCRIPT A2780-CDDP. GJ function was detected by parachute dye-coupling assay. The scale bars represent 25 µm. Fig. 4 Translocation of Cx32 from the membrane to the cytoplasm in
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A2780-CDDP cells. Membrane protein (A) and cytoplasmic protein (C) were separated. Na/K-ATPase was used as a loading control for membrane protein, and tubulin was used for cytoplasmic proteins. Quantification of protein expression is
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shown in (B) and (D). (*, P < 0.05, **, P < 0.01, the results represent the mean ± SD
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of 3 independent experiments.) (E) Cx32 translocated into the cytoplasm in A2780-CDDP cells based on immunofluorescence staining. The scale bars represent
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1. The gradual changing processes of gap junction and connexins were investigated during the process of acquired cisplatin resistance. 2. The loss of gap junction meditates cisplatin resistance in ovarian cancer cells. 3. Cx32 confers cisplatin resistance in a channel-independent manner in ovarian cancer cells. 4. The translocation of Cx32 from membrane to cytoplasm contributes to the cisplatin resistance.
S0006-291X(17)30719-2
DOI:
10.1016/j.bbrc.2017.04.053
Reference:
YBBRC 37611
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 10 April 2017 Accepted Date: 11 April 2017
Please cite this article as: W. Wu, L. Fan, Z. Bao, Y. Zhang, Y. Peng, M. Shao, Y. Xiang, X. Zhang, Q. Wang, L. Tao, The cytoplasmic translocation of Cx32 mediates cisplatin resistance in ovarian cancer cells, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.04.053. 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 The cytoplasmic translocation of Cx32 mediates cisplatin resistance in ovarian cancer cells Authors
Xiang1, Xiaomin Zhang1, Qin Wang1, Liang Tao1 1
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Weili Wu1, Lixia Fan1, Zeqing Bao2, Yu Zhang1, Yuexia Peng1, Min Shao1, Yuke
Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-Sen
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University, Guangzhou 510080, People’ s Republic of China; 2Zhaoqing Medical
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college Pharmacology teaching and research section, Zhaoqing 526020, People’ s Republic of China. Correspondence
Liang Tao, Department of Pharmacology, Zhongshan School of Medicine, Sun
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Yat-Sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China. Tel: +86-20-8733-5468, Email: [email protected]. Qin Wang, Department of Pharmacology, Zhongshan School of Medicine, Sun
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Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China.
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Authorship Note: Weili Wu and Lixia Fan made equal contribution to this paper.
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ACCEPTED MANUSCRIPT Abstract Ovarian cancer is the most lethal gynecologic malignancy, and cisplatin is one of the first-line chemotherapeutic agents. However, acquired cisplatin resistance
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prevents the successful treatment of patients with ovarian cancer. Gap junction (GJ) and connexin (Cx) are closely related to tumor formation, but the relationship between cisplatin resistance and GJ or Cx are undetermined. In this study, we
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established the cisplatin-resistant human ovarian cancer cell line A2780-CDDP.
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Here we showed that cisplatin resistance was correlated to the loss of GJ and the upregulation of Cx32 expression. Enhancing GJ in A2780-CDDP cells could increase the apoptotic response to cisplatin treatment. Furthermore, although Cx32 expression was increased in A2780-CDDP cells, it was more localized to the
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cytoplasm rather than in the membrane, and knockdown of Cx32 in A2780-CDDP cells sensitized them to cisplatin treatment. In summary, Cx32 is involved in cisplatin resistance, and cytoplasmic Cx32 plays an important role in
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chemoresistance.
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Keywords: Cisplatin; Resistance; Ovarian cancer; Cx32; Gap junction
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ACCEPTED MANUSCRIPT Introduction Cisplatin is a chemotherapeutic agent that is widely used in the treatment of several malignancies, such as ovarian, testicular, breast, and bladder cancer [1]. It is the
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standard first-line agent for ovarian cancer [2,3]. However, resistance can quickly emerge after treatment, leading to tumor treatment failure [4,5]. Thus, understanding the underlying mechanism of drug resistance to cisplatin and increasing the sensitivity
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to therapeutic drugs are key steps towards the improved treatment of patients with
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ovarian cancer.
Gap junctions (GJs) are plasma membrane channels that directly connect the neighboring cells [6]. GJs comprise of two hemichannels known as connexons, and each connexon contains six transmembrane proteins known as connexins (Cxs) [7,8].
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These GJs provide a channel for immediate intercellular communication and allow for the direct intercellular exchange of ions, small metabolites and second messengers such as sodium, potassium, cAMP/cGMP and glutathione [9,10,11]. Many
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physiological processes are controlled by regulatory molecules that are exchanged
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through GJs, and thus, GJs are crucial in diverse processes. Likewise, gap junction intercellular communication (GJIC) has been shown to be actively involved in signal transduction in tumor cells [12,13]. Cxs are a group of homologous proteins that form the inter-membrane channels of GJs [14]. Abnormal expression and distribution of Cxs are closely related to tumor formation. They can regulate cell proliferation, migration, and apoptosis in a GJIC-independent manner [15,16]. The mechanisms of resistance to platinum-based compounds are very complex. 3
ACCEPTED MANUSCRIPT These mechanisms include decreased accumulation of platinum compounds by either active efflux or impaired influx[17]; detoxification via GSH conjugates; increased DNA damage repair[18] ; aberrant DNA methylation[19]; up- or downregulated
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expression of microRNAs[20,21]. In addition, increasing observations have indicated that Cxs and GJ also contribute to cancer progression and chemoresistance. For example, Paul R. Gielen et al. reported that the increased levels of Cx43 in human
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glioma cells enhanced resistance to TMZ treatment [22]. J Yang et al. showed that
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gefitinib resistance was correlated with upregulation of Cx26 in the HCC827 and PC9 cell lines [23]. In contrast, Meiling Yu et al. reported that Cx43 reversed the resistance of A549 cells to cisplatin [24]. However, the relationship between Cx32 and chemoresistance is rarely reported.
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In our previous studies, we found that GJIC showed a rapid, dose-dependent decrease when exposed to cisplatin, and inhibition of GJIC compromises the effectiveness of these compounds [25]. These results remind us that GJ and Cxs may
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be linked to chemoresistance. However, the above conclusion was drawn from
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drug-sensitive cancer cells rather than drug-resistant cells. Therefore, in the present study, we established the cisplatin-resistant human ovarian cancer cell line A2780-CDDP and directly investigated the connection between GJ/Cxs and chemoresistance.
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ACCEPTED MANUSCRIPT Materials and Methods Materials: Cell culture-related reagents, Lipofectamine™ 2000, CM-DiI and calcein AM were obtained from Invitrogen (Carlsbad, CA, USA). Cisplatin, retinoic acid
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(RA), and anti-Cx32, anti-Cx43 and secondary antibodies for Western blotting were obtained from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor®488 Phalloidin was obtained from Cell Signaling Technology (Danvers, Massachusetts, USA). Cx32
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siRNAs was constructed by Ribbon (Guangzhou, China). Other reagents were
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obtained from Sigma (St. Louis, MO, USA) unless otherwise stated. Methods: Cell lines
The A2780 cell line was obtained from the American Type Culture Collection
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(Manassas, VA, USA). Both paternal A2780 (cisplatin-sensitive) and A2780-CDDP (cisplatin-resistant) cell lines were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in an atmosphere containing 5% CO2.
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Induction of cisplatin resistance
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The cisplatin-resistant cell line A2780-CDDP was generated by exposing the parental cell line A2780 to cisplatin over an 11-month period, with the concentration of cisplatin gradually increasing from 0.5 µg/ml to 16 µg/ml. Cell Viability Assay
A Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to examine cell viability according to the manufacturers’ instructions. Parachute dye-coupling assay 5
ACCEPTED MANUSCRIPT GJ function was determined as described by Goldberg et al[26]. Donor cells were double-labeled with calcein-AM and CM-Dil, and then loaded onto the recipient cells. The two cell types attached to the monolayer for 4 h. The signal intensity was
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observed using a fluorescence microscope (Olympus IX71, Tokyo, Japan). The average number of receiver cells containing calcein per donor cell was regarded as a measure of the degree of GJIC.
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Drug Treatment
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RA was used for the pharmacological regulation of GJIC. Cells were incubated with 40 µM RA for 24 h to enhance GJIC.
RNA Isolation and Real-time quantitative PCR Analysis
Total RNA was isolated using a HiPure Total RNA Mini Kit (Megan, Guangzhou,
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China). Reverse transcription was performed with 2 µg of RNA using the Reverse Transcription System (TRANS, Beijing, China). Cx43, Cx32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected with the
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following respective primers: 5’-GGTCTGAGTGCCTGAACTTGCCT-3' (Forward)
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and 5'-AGCCACACCTTCCCTCCAGCA-3' (Reverse); 5'-GGTGGACCTATGTCATCAGCG-3' (Forward) and 5'-GCCAGGGTAGAGCAGATAAAAGA-3' (Reverse); 5'-GGAGCGAGATCCCTCCAAAAT-3' (Forward) and 5'-GGCTGTTGTCATACTTCTCATGG-3' (Reverse). Real-time quantitative PCR (qRT-PCR) was performed using the ABI StepOneTM quantitative real-time PCR system (Foster City, CA, USA). The comparative CT method was used to quantify the 6
ACCEPTED MANUSCRIPT expression for each gene relative to that of in the A2780 cells using GAPDH as a normalization control. Western blot
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Cells for Western blot analysis were washed with PBS, scraped into lysis buffer, sonicated and centrifuged. The protein concentration was determined with a BCA protein assay kit (Bio-Rad Co., Hercules, CA, USA). A total of 20 µg of protein from
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each sample was separated using SDS-PAGE and transferred to a nitrocellulose
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membrane. Membranes were blocked and then incubated with specific antibodies overnight at 4°C. Monoclonal antibodies targeting Cx32 (1:2000), Cx43 (1:8000) and tubulin (1:10000) were used. Membranes were incubated with the relevant secondary antibody. Immuno-positive bands were scanned by ImageQuant LAS 4000TM.
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siRNA transfection experiments
siRNAs targeting the human Cx32 gene as well as nonspecific sequence siRNAs were added to cells with Lipofectamine™ 2000 transfection reagent according to the
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manufacturer’s protocol. The sequences for the synthesized siRNAs targeting Cx32
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(siCx32) were as follows:
siCx32_1: 5'-CCGGCATTCTACTGCCATT-3'; siCx32_2: 5'-GGCTCACCAGCAACACATA-3'; and siCx32_3: 5'-GCAACAGCGTTTGCTATGA-3'. Membrane protein extraction Membrane-bound and cytoplasmic proteins were extracted by using a Mem-PERTM Plus Membrane Protein Extraction kit (ThermoFisher Scientific™, USA) according to 7
ACCEPTED MANUSCRIPT the manufacturers’ instructions. Immunofluorescence assay Cells were fixed with 4% paraformaldehyde and then blocked with 2% bovine serum
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albumin. The cells were incubated with anti-Cx32 primary antibodies (1:200) overnight at 4°C. The cells were then incubated with FITC-conjugated anti-mouse secondary antibodies (1:400) for 1 h. Alexa Fluor®488 Phalloidin (1:20) was applied
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for 15 min to stain the cytoskeleton of the cells. Hoechst 33258 was used for the
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nuclear staining of the cells. Then, the cells were visualized using a confocal laser scanning fluorescence microscope (ZEISS LSM710). Statistical analysis
The data were statistically analyzed using unpaired Student’s t-test with GraphPad
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considered significant.
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Prism 6.0 software. The results are expressed as the mean ± SD, and p < 0.05 was
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ACCEPTED MANUSCRIPT Results 1. The establishment of a cisplatin-resistant cell line. To investigate the mechanism of acquired cisplatin resistance, we established the
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cisplatin -resistant cell line A2780-CDDP by intermittently exposing A2780 cells to gradually increasing concentrations of cisplatin. To monitor drug resistance, the resistance index (RI) was measured during the cisplatin resistance process, and the RI
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was 1.2, 1.7, 2.9, 4.3 and 7.2 respectively (Figure 1A and B). For the remainder of this
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article, the cells at these various stages (when RI are 1.2, 1.7, 2.9, 4.3 and 7.2) are denoted as A2780-RI(1.2), A2780-RI(1.7), A2780-RI(2.9), A2780-RI(4.3) and A2780-CDDP.
Cisplatin is known to induce apoptosis in cancer cells to inhibit tumor growth.
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Therefore, we detected the levels of cleaved caspase-3 and found that cleaved caspase-3 was activated during the cisplatin treatment in A2780 cells. In A2780-CDDP cells, the induction of cleaved caspase-3 was significantly abrogated
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(Figure 1D and E). These results indicated that A2780-CDDP cells were resistant to
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cisplatin by escaping apoptosis. To confirm A2780-CDDP was a stable genetic variant, we cultivated
A2780-CDDP cells without cisplatin for 20 days, and tested RI on the 10th and 20th day. The levels of cleaved caspase-3 were also detected after the cisplatin treatment. Our results showed that the RI on these two stages had no significant difference (RI was 7.2 and 7.16 respectively, Figure 1F and G), and the apoptosis pathways in
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ACCEPTED MANUSCRIPT A2780-CDDP cells were not activated (Figure 1H and I). These results indicated that A2780-CDDP cells were stably resistant to cisplatin. 2. GJ is involved in the development of cisplatin resistance.
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GJ has been frequently reported to correlate with cancer progression, but the relationship between GJ and cisplatin resistance remains poorly studied. To explore the effect of GJ in the process of cisplatin resistance, we investigated GJ using a
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parachute dye-coupling assay in A2780-RI(1.2), A2780-RI(1.7), A2780-RI(2.9),
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A2780-RI(4.3) and A2780-CDDP cells. During the process of cisplatin resistance, GJIC was gradually reduced (Figure 2A and B). This result revealed that GJ may be involved in the cisplatin resistance of ovarian cancer.
To demonstrate the role of GJ in cisplatin resistance, GJ function was activated
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by RA. The RA treatment alone did not affect cell survival (Figure 2C). The parachute dye-coupling assay was used to confirm that RA activated the GJ function (Figure 2D and E). A2780-CDDP cells were treated with RA (40 µM) for 24 h and then exposed
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to cisplatin prior to measuring the cell viability. The results showed that enhanced GJ
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by RA treatment resulted in sensitivity to cisplatin (Figure 2F). Therefore, our results confirmed a significant role of GJ in cisplatin resistance in ovarian cancer cells. 3. The expression of Cxs in the development of cisplatin resistance. The present results indicated that GJ was involved in cisplatin resistance. We further explored whether the Cxs responsible for GJ were related to cisplatin resistance. In A2780-RI(1.2), A2780-RI(1.7), A2780-RI(2.9), A2780-RI(4.3) and A2780-CDDP, we used q-PCR to analyze the levels of Cx43, Cx40, Cx37, and Cx32, which have been 10
ACCEPTED MANUSCRIPT detected in the ovaries [27]. The results showed that Cx40 and Cx37 had a low mRNA expression levels in A2780 and A2780-CDDP cells (data not shown), and the Cx43 mRNA levels rarely changed in A2780-CDDP cells compared to A2780 cells (Figure
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3B). However, Cx32 mRNA levels were elevated in A2780-CDDP cells nearly fourfold compared to A2780 cells (Figure 3A). To further confirm the expression of Cx32 and Cx43, protein levels were examined by Western blotting. Our results
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showed that Cx32 protein expression was highly elevated in A2780-CDDP cells
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(Figure 4 C and D), while Cx43 showed no significant change in expression (Figure 4 E and F). This result corresponded to that of the mRNA levels. To confirm the effect of Cx32 on cisplatin resistance, we knocked down Cx32 in A2780-CDDP cells with siRNA-Cx32. After Cx32 was knocked down, cells were
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treated with ciaplatin for 48 hours. Cx32, cleaved caspase-3 and cell inhibition were detected. The results showed that the reduced Cx32 expression increased the apoptotic response to cisplatin treatment in A2780-CDDP cells (Figure 3G), and sensitized
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A2780-CDDP cells to cisplatin treatment (Figure 3H). Meanwhile, Cx32 knockdown
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did not affect GJ function in A2780-CDDP cells (Figure 3I). These results suggest that Cx32 protein confers cisplatin resistance in ovarian cancer cells by modulating the apoptosis pathway, and this effect is independent of GJ. 4.Cx32 translocates from the membrane to the cytoplasm in A2780-CDDP cells As GJ was decreased whereas Cx32 expression was elevated during the cisplatin resistance process, it drove us to explore the underlying mechanism. In recent years, it has been reported that Cxs may have abnormally localize to subcellular compartments 11
ACCEPTED MANUSCRIPT [28]. Therefore, we hypothesized that the increased number of Cx32 proteins abnormally
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To
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membrane-bound and cytoplasmic proteins from A2780 and A2780-CDDP cells.
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Western blotting was used to confirm the expression of Cx32 in membrane and cytoplasm. The results showed that the amount of Cx32 in membranes of A2780-CDDP cells was lower than that in A2780 (Figure 4A and B). In contrast,
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cytoplasmic Cx32 was highly elevated in A2780-CDDP cells (Figure 4C and D).
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Immunofluorescence was used to validate the localization of Cx32 in A2780 and A2780-CDDP cells. The results confirmed that the increased number of Cx32 proteins was expressed in cytoplasm in A2780-CDDP cells, and the amount of membrane-bound Cx32 was much lower (Figure 4E). Therefore, these results
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indicated that the reduced Cx32 expression in membrane resulted in the loss of GJIC
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ACCEPTED MANUSCRIPT Discussion Cisplatin is one of the standard first-line chemotherapeutic drugs for ovarian cancer, but prolonged exposure to cisplatin could lead to acquired resistance, which hampers
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the successful treatment of patients. Therefore, it is essential to explore the mechanism of cisplatin resistance in ovarian cancer cells. Prior work by our group identified that the toxicities of cisplatin was reduced when there was no GJ between
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the cells, and cisplatin could inhibit the activity of connexin channels [25]. These
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previous results imply that GJ/Cx is likely associated with chemoresistance. In searching for the direct relationship between GJ/Cx and the cisplatin resistance, we established the cisplatin-resistant cell line A2780-CDDP to investigate the relationship between chemoresistance and GJ/Cx. Our results showed that GJ was
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reduced during the process of acquired cisplatin resistance. Enhancing GJ could reverse the cisplatin resistance of A2780-CDDP. GJs have long been believed to suppress tumor progression during carcinogenesis. One of the mechanisms is that GJs implicated
in
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chemotherapeutics [29,30]. The bystander effect is a phenomenon by which cytotoxic molecules or signals can be transferred from targeted cells to adjacent cells. By this process, cisplatin-induced death signals can be transmitted to neighboring cells via GJ. Therefore, reduced GJ during the cisplatin resistance process may lead to the reduced transfer of cytotoxic molecules and signals, leading to decreased responsiveness to cisplatin. Developing drugs and therapies that can increase GJ may be a new potent strategy to enhance the chemotherapeutic effects of drugs. 13
ACCEPTED MANUSCRIPT Further investigation revealed that the decrease of GJ function was due to reduced membrane localization of Cx32, whereas the levels of cytoplasmic Cx32 were elevated. The Cxs protein accumulated in the cytoplasm fail to constitute GJ.
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Therefore, cytoplasmic Cx32 is involved in chemoresistance in a GJ-independent maner. Other than its role as a constituent of GJ, Cxs play many other roles independent of GJIC. For example, Cx43, Cx40 and Cx32 have very significant
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impacts on cellular injury resistance via processes independent of GJ coupling [31].
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Susan F. Murphy et al. reported that Cx43 levels were inversely correlated with temozolomide sensitivity of glioblastoma cells [32]. Here, we found that Cx32 protein conferred chemoresistance in A2780-CDDP cells and not relied on GJ formation. Some studies support the role of Cx32 protein independent of GJ in the development
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of cancer. For example, cytoplasmic accumulation of Cx32, which failed to facilitate GJ, could enhance the motility and metastatic ability of human hepatoma cells in vitro and in vivo [33]. As Cx32 konckdown increased cleave caspase-3 expression, we
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reason that Cx32 is involved in cisplatin resistance by modulating the apoptosis
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pathway. Here, the results of our study show that Cx32 is involved in chemoresistance in two ways. The first is that decreased Cx32 in the membrane results in the loss of GJ, thereby leading to cisplatin resistance; the other is that increased cytoplasmic Cx32 plays a role in chemoresistance independent of GJ. The phenomenon of cytoplasmic Cx32 accumulation is intriguing, and Cx32 may be a potential novel target for overcoming cisplatin resistance in ovarian cancer.
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ACCEPTED MANUSCRIPT Acknowledgements The present study was supported by the Joint Fund of the National Nature Science Foundation of China (grant No. U1303221), the National Natural Science Foundation
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of China (grant No. 81373439 and 81473234), and the Fundamental Research Funds
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for the Central Universities (grant No.16ykjc01 and 16ykzd11).
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ACCEPTED MANUSCRIPT [18] D.W. Shen, L.M. Pouliot, M.D. Hall, M.M. Gottesman, Cisplatin resistance: a cellular self-defense mechanism resulting from multiple epigenetic and genetic changes, Pharmacol Rev 64 (2012) 706-721. [19] C.T. Viet, D. Dang, S. Achdjian, Y. Ye, S.G. Katz, B.L. Schmidt, Decitabine rescues cisplatin resistance in head and neck squamous cell carcinoma, PLoS One 9 (2014) e112880. [20] D. Cheng, Y. Xu, C. Sun, Z. He, MicroRNA-451 sensitizes lung cancer cells to cisplatin through regulation of Mcl-1, Mol Cell Biochem 423 (2016) 85-91.
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[21] M. Li, W. Chen, H. Zhang, Y. Zhang, F. Ke, X. Wu, Y. Zhang, M. Weng, Y. Liu, W. Gong, MiR-31 regulates the cisplatin resistance by targeting Src in gallbladder cancer, Oncotarget 7 (2016) 83060-83070.
[22] P.R. Gielen, Q. Aftab, N. Ma, V.C. Chen, X. Hong, S. Lozinsky, C.C. Naus, W.C. Sin, Connexin43 confers Temozolomide resistance in human glioma cells by modulating the mitochondrial apoptosis pathway, Neuropharmacology 75 (2013) 539-548.
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[23] J. Yang, G. Qin, M. Luo, J. Chen, Q. Zhang, L. Li, L. Pan, S. Qin, Reciprocal positive regulation between Cx26 and PI3K/Akt pathway confers acquired gefitinib resistance in NSCLC cells via GJIC-independent induction of EMT, Cell Death Dis 6 (2015) e1829.
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[24] M. Yu, C. Zhang, L. Li, S. Dong, N. Zhang, X. Tong, Cx43 reverses the resistance of A549 lung adenocarcinoma cells to cisplatin by inhibiting EMT, Oncol Rep 31 (2014) 2751-2758. [25] Q. Wang, T. You, D. Yuan, X. Han, X. Hong, B. He, L. Wang, X. Tong, L. Tao, A.L. Harris, Cisplatin and oxaliplatin inhibit gap junctional communication by direct action and by reduction of connexin expression, thereby counteracting cytotoxic efficacy, J Pharmacol Exp Ther 333 (2010) 903-911. [26] G.S. Goldberg, J.F. Bechberger, C.C. Naus, A pre-loading method of evaluating gap junctional communication by fluorescent dye transfer, Biotechniques 18 (1995) 490-497.
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[30] K. Kranz, F. Paquet-Durand, R. Weiler, U. Janssen-Bienhold, K. Dedek, Testing for a gap junction-mediated bystander effect in retinitis pigmentosa: secondary cone death is not altered by deletion of connexin36 from cones, PLoS One 8 (2013) e57163. [31] J.H. Lin, J. Yang, S. Liu, T. Takano, X. Wang, Q. Gao, K. Willecke, M. Nedergaard, Connexin mediates gap junction-independent resistance to cellular injury, J Neurosci 23 (2003) 430-441. [32] S.F. Murphy, R.T. Varghese, S. Lamouille, S. Guo, K.J. Pridham, P. Kanabur, A.M. Osimani, S. Sharma, J. Jourdan, C.M. Rodgers, G.R. Simonds, R.G. Gourdie, Z. Sheng, Connexin 43 Inhibition Sensitizes Chemoresistant Glioblastoma Cells to Temozolomide, Cancer Res 76 (2016) 139-149. [33] Q. Li, Y. Omori, Y. Nishikawa, T. Yoshioka, Y. Yamamoto, K. Enomoto, Cytoplasmic accumulation of connexin32 protein enhances motility and metastatic ability of human hepatoma cells in vitro and in vivo, Int J Cancer 121 (2007) 536-546.
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ACCEPTED MANUSCRIPT Figure Legends Fig. 1 The establishment of a cisplatin-resistant cell line (A2780-CDDP). (A) At different cisplatin-resistant stages, cells were treated with 0.3 µg/ml, 1 µg/ml, 3 µg/ml,
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10 µg/ml, 30 µg/ml, or 100 µg/ml of cisplatin for 48 h, and the inhibition of cell growth was determined with the CCK-8 assay. (B) During the process of drug resistance, the RI was measured. (C) Representative images from the assay described
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in (A). The scale bars represent 25 µm. (D) Cisplatin induced cleaved caspase-3
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expression in A2780 cells but not A2780-CDDP cells. Cells were treated with cisplatin (5 µg/ml) for 24, 36, or 48 hours, and protein expression was determined using Western blot. (E) Cleaved caspase-3 quantification. (*, P < 0.05, **, P < 0.01, the results represent the mean ± SD of 3 independent experiments.) . (F, G) The
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inhibition of cell growth at the indicated concentration of cisplatin (10 and 20 days respectively after A2780-CDDP cells were cultivated without cisplatin). Error bars represent the SD (n = 3). (H, I) Cisplatin induced cleaved caspase-3 expression in
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A2780 cells but not A2780-CDDP cells. Cells were treated with cisplatin (5 µg/ml)
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for 48 h after A2780-CDDP cells were cultivated without cisplatin for 10 and 20 days respectively.
Fig. 2 GJ is involved in the development of cisplatin resistance in A2780-CDDP cells. (A) A parachute dye-coupling assay was used to detect GJ. The scale bars represent 25 µm. (B) Quantitative results from A. (C) Cell viability assays after RA treatment. Cells were treated with RA at concentration of 20 µM, 40 µM or 60 µM, 18
ACCEPTED MANUSCRIPT and cell viability was determined using CCK-8. (D) A parachute dye-coupling assay was performed after RA (40 µM) treatment. The scale bars represent 25 µm. (E) Quantification of communicative cells. (F) RA treatment reversed the cisplatin
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resistance in A2780-CDDP cells. Cells were pretreated with RA (40 µM) and then treated with cisplatin at the indicated concentration. Cell viability was determined 48 hours later using the CCK-8 assay. (*, P < 0.05, **, P < 0.01, ***, P < 0.001, the
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results represent the mean ± SD of 3 independent experiments.)
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Fig. 3 Expression of connexins during the development of cisplatin resistance in A2780 cells. (A) Cx32 was upregulated during the development of resistance to cisplatin treatment. qPCR was used to detect the expression of Cx32. (B) Cx43 expression showed no significant differences between the A2780 and A2780-CDDP
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cells. qPCR was used to detect the expression of Cx43. (C, D) A2780-CDDP cells showed increased expression of Cx32. Quantitative results are shown in D. (E, F) A2780-CDDP and A2780 cells showed no significant difference in the expression of
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Cx43. Western blotting was used to detect the expression of Cx32 and Cx43 in A2780
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and A2780-CDDP cells. Bar graphs were derived from densitometric scanning of the blots. (G) Cisplatin (10 µg/ml) induced cleaved caspase-3 expression in Cx32 knockdown cells in A2780-CDDP. (H) After Cx32 knockdown, cells were treated with cisplatin at the indicated concentration. Cell viability was determined 48 hours later using the CCK-8 assay. (*, P < 0.05, **, P < 0.01, the results represent the mean ± SD of 3 independent experiments). (I) Cx32 knockdown does not affect GJ in
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ACCEPTED MANUSCRIPT A2780-CDDP. GJ function was detected by parachute dye-coupling assay. The scale bars represent 25 µm. Fig. 4 Translocation of Cx32 from the membrane to the cytoplasm in
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A2780-CDDP cells. Membrane protein (A) and cytoplasmic protein (C) were separated. Na/K-ATPase was used as a loading control for membrane protein, and tubulin was used for cytoplasmic proteins. Quantification of protein expression is
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shown in (B) and (D). (*, P < 0.05, **, P < 0.01, the results represent the mean ± SD
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of 3 independent experiments.) (E) Cx32 translocated into the cytoplasm in A2780-CDDP cells based on immunofluorescence staining. The scale bars represent
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1. The gradual changing processes of gap junction and connexins were investigated during the process of acquired cisplatin resistance. 2. The loss of gap junction meditates cisplatin resistance in ovarian cancer cells. 3. Cx32 confers cisplatin resistance in a channel-independent manner in ovarian cancer cells. 4. The translocation of Cx32 from membrane to cytoplasm contributes to the cisplatin resistance.