HOXB4 knockdown enhances the cytotoxic effect of paclitaxel and cisplatin by downregulating ABC transporters in ovarian cancer cells

Accepted Manuscript HOXB4 knockdown enhances the cytotoxic effect of paclitaxel and cisplatin by downregulating ABC transporters in ovarian cancer cells

Ying Li, Jingli Sun, Shaofeng Gao, Heping Hu, Pengmu Xie PII: DOI: Reference:

S0378-1119(18)30401-3 doi:10.1016/j.gene.2018.04.033 GENE 42757

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Gene

Received date: Revised date: Accepted date:

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Please cite this article as: Ying Li, Jingli Sun, Shaofeng Gao, Heping Hu, Pengmu Xie , HOXB4 knockdown enhances the cytotoxic effect of paclitaxel and cisplatin by downregulating ABC transporters in ovarian cancer cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2018.04.033

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ACCEPTED MANUSCRIPT HOXB4 knockdown enhances the cytotoxic effect of paclitaxel and cisplatin by downregulating ABC transporters in ovarian cancer cells

Ying Li1, Jingli Sun1, Shaofeng Gao1, Heping Hu2, Pengmu Xie3,* 1

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Department of Obstetrics and Gynecology, Shanxian Central Hospital of Shandong Province,

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Heze 274300, PR China. 2

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Department of Oncology, Shanxian Central Hospital of Shandong Province, Heze 274300,

PR China.

Department of gynaecology, Jining No. 1 People’s Hospital, Jining 272011, PR China.

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* Corresponding to Pengmu Xie, from the Department of gynaecology, Jining No. 1 People’s

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Hospital, No. 6 Jiankang Road, Jining 272011, PR China. E-mail: [email protected].

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Abstract Therapeutic effects of anti-cancer drugs for ovarian cancer were limited due to the rapid development of chemotherapy resistance. The aim of this study was to test whether knockdown of Homeobox B4 (HOXB4) enhanced the cytotoxic effect of paclitaxel and

in Taxol-resistant

A2780 (A2780/Taxol)

and

DDP-resistant

SKOV-3

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upregulated

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cisplatin in ovarian cancer cells. HOXB4 expressions at mRNA and protein levels were

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(SKOV-3/DDP) cells. HOXB4 knockdown enhanced the cytotoxic effects of Taxol and DDP in A2780/Taxol and SKOV-3/DDP cells, respectively. HOXB4 silencing suppressed the

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phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and reduced the expression of

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ABCB1, ABCC1 and ABCG2 in ovarian cancer cells. PI3K inhibitor LY294002 or siRNA targeting Akt (si-Akt) treatment inhibited cell viability, decreased protein levels of ABCB1,

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ABCC1 and ABCG2, and increased LDH release in A2780/Taxol and SKOV-3/DDP cells.

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These findings revealed that HOXB4 knockdown enhanced the cytotoxic effects of Taxol and DDP by downregulating ABC transporters via inhibiting the PI3K/Akt pathway in ovarian

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cancer cells.

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Keywords: HOXB4, paclitaxel, cisplatin, ovarian cancer, ATP-binding cassette transporters, PI3K/Akt pathway

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1. Introduction Ovarian cancer is the fifth leading cause of cancer-related female death in the United States, with an estimated 22,280 new diagnosed cases and 14,240 deaths in 2016 (Siegel et al., 2016). Although considerable advances have been made in early diagnose and therapy of

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ovarian cancer over the past decades, its overall 5-year survival rate and overall cure rate still

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remain approximately 46% and 30%, respectively (Jr et al., 2009; Siegel et al., 2016).

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Cisplatin (DDP) and paclitaxel (Taxol) are the common chemotherapeutic drugs for the treatment of various cancers, including ovarian cancer (Basenengquist, 2006). However, the

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emergence of drug resistance strikingly weakened the therapeutic effects of DDP and Taxol in

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ovarian cancer (Sherman-Baust et al., 2011). Hence, it is imperative to better understand the molecular basis of drug resistance in ovarian cancer and identify more effective therapy

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strategies.

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Homeobox (HOX) genes, a family of transcription factors containing a highly conserved homeodomain region, are key regulators in the development and progression of multiple

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cancers including ovarian cancer (Abateshen, 2002; Kelly et al., 2011). Increasing studies

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have identified the critical roles of multiple HOX genes in the oncogenesis, diagnosis and prognosis of ovarian cancer (Kelly et al., 2011). For instance, Homeobox B13 (HOXB13) was highly expressed in ovarian cancer and promoted cell proliferation and invasion of ovarian cancer cells (Miao et al., 2007; Yamashita et al., 2006). HOXB4 has been demonstrated to be implicated in the self-renewal of hematopoietic stem cells (Zheng et al., 2007), and identified as a cancer-related gene in various malignancies, such as breast cancer (Zhou et al., 2017) and medulloblastoma (Bonfim-Silva et al., 2017). Hong et al. demonstrated that HOXB4 was 3

ACCEPTED MANUSCRIPT highly expressed in ovarian cancer tissues and cells, indicating that HOXB4 might be implicated in carcinogenesis of ovarian cancer (Hong et al., 2010). Wang et al. found that HOXB4 depletion partly abrogated resistance of human myelogenous leukemia K562/ADM cells to multiple drugs (Adriamycin, Etoposide, Vindesine and Cytarabine) (Wang et al.,

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2016). However, the roles and molecular mechanisms of HOXB4 underlying the development

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of multi-drug resistance in ovarian cancer are still undefined till now.

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In the present study, we firstly demonstrated that HOXB4 expression was upregulated in ovarian cancer cells, as well as Taxol-resistant A2780 (A2780/Taxol) and DDP-resistant

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SKOV-3 (SKOV-3/DDP) cells. Function studies suggested that depletion of HOXB4

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enhanced the cytotoxic effects of Taxol and DDP in ovarian cancer cells by downregulating ATP-binding cassette (ABC) transporters via phosphatidylinositol 3’-kinase (PI3K)/protein

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2. Materials and methods

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kinase B (Akt) signaling pathway.

2.1. Cell culture and transfection

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Human ovarian cancer cells A2780 were obtained from Institute of Biochemistry and

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Cells Biology of Chinese Academy of Sciences (Shanghai, China) and A2780/Taxol cells were purchased from Bogoo Biotechnology (Shanghai, China). SKOV-3 and SKOV-3/DDP were purchased from Shanghai Cellular Research Institute (Shanghai, China). Human normal ovarian epithelial cells (HOEC) were purchased from Pricells (Wuhan, China). All cells were grown in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% Fetal Bovine Serum (FBS; Gibco) and 1% penicillin-streptomycin solution (Gibco) in a 5% CO2 humidified atmosphere at 37°C. Taxol, DDP and LY294002 4

ACCEPTED MANUSCRIPT were purchased form Sigma-Aldrich Co. ltd (Louis, MO, USA). siRNAs targeting HOXB4 (siHOXB4-1 and siHOXB4-2) and Akt (siAkt), and the scramble control (siControl) were synthesized from GenePharma Co., Ltd. (Shanghai, China). siRNAs were transfected into ovarian cancer cells by Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA)

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according to the manufacturer’s instructions.

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2.2. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

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MTT assay was performed to explore the effects of Taxol, DDP, HOXB4 and LY294002 on cell viability. A2780/Taxol and SKOV-3/DDP cells were treated with Taxol (0, 0.5, 1, 2, 5,

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10, 20, 30, 40, and 50 M) or DDP (0, 1, 5, 10, 20, 30, 40, 50, 60, 70, and 80 M) for 48 h.

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Transfected A2780/Taxol and SKOV-3/DDP cells were treated with Taxol (10 M) or DDP (30 M) for 48 h. Additionally, A2780/Taxol and SKOV-3/DDP cells were treated with

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LY294002 (20 μM) for 48 h. Then, 10 μl MTT solution (5 mg/ml, Sigma-Aldrich) were added

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into each well of 96-well plates and incubated for another 4 h. Next, 150 μl DMSO (Sigma-Aldrich) was added into each well. Optical densities were measured at 490 nm

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wavelength using a Bio-Rad microplate reader (Bio-Rad, Hercules, CA, USA).

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2.3. Lactate dehydrogenase (LDH) release assay Cells treated as described in MTT assay were seeded into 96-well plates at a cell density of 4 × 103 cells per well and cultured overnight. LDH release was examined by LDH cytotoxicity assay kit (Beyotime, Nantong, China). The absorbance at 450 nm was determined using a Bio-Rad microplate reader (Bio-Rad). 2.4. Caspase-3 activity assay Caspase-3 activity was determined using a Caspase 3 Assay Kit (ab39401, Abcam) 5

ACCEPTED MANUSCRIPT following the instructions of manufacturer. Briefly, cells were re-suspended in 50 μl of chilled cells lysis buffer and then incubated for 10 min on ice, followed by protein extraction and concentration determination. Next, 50 μl of 2 × reaction buffer containing 10 mM DTT and 5 μl of 4 mM DEVD-p-NA substrate was added into each sample. The absorbance was

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monitored at 405 nm wavelength using a Bio-Rad microplate reader (Bio-Rad).

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2.5. Real time-quantitative PCR (RT-qPCR) assay

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Total RNA from ovarian cells were extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the instructions of manufacturer, followed by the quantification by

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Nanodrop 2000 (Thermo Fisher Scientific, Rockford, IL, USA). Then M-MLV reverse

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transcriptase (Invitrogen) and random primers were employed to synthesize cDNA from total RNA sample (1 μg/sample). Fast SYBR Green Master Mix (Thermo Fisher Scientific) was

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used to detect the expressions of HOXB4 and β-actin mRNAs, with β-actin as an internal

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reference. The primer sequences of HOXB4 and β-actin were as follows: HOXB4, 5’-GCA AAG AGC CCG TCG TCT-3’ (forward) and 5’-GAA ATT CCT TCT CCA GCT-3’

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(reverse); β-actin, 5’-AGT GTG ACG TGG ACA TCC GCA AAG-3’ (forward) and 5’-ATC

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CAC ATC TGC TGG AAG GTG GAC-3’ (reverse). 2.6. Western blot assay The treated cells were lysed using RIPA buffer (Thermal Fisher Scientific) containing cocktail (Roche, Mannheim, Germany) for 45 min on ice. Proteins in the supernatant were obtained by high-speed centrifugation (15,000 g) for 20 min at 4°C and quantified using BCA Protein Assay Kit (Thermo Fisher Scientific). Next, protein samples (50 µg/lane) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 6

ACCEPTED MANUSCRIPT and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After treated with 5% skimmed milk for 1 h at room temperature, the members were incubated overnight at 4°C with primary antibodies against HOXB4 (Abcam), Akt (Cells Signaling Technology, Inc, Danvers, MA, USA), cleaved caspase-3 (Abcam), Bax

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(Cells Signaling Technology), phospholyrated-Akt (p-Akt; Cells Signaling Technology),

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phospholyrated-p70S6K (p-p70S6K; Cells Signaling Technology), ATP Binding Cassette

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Subfamily C Member 1 (ABCC1; Abcam), ATP Binding Cassette Subfamily B Member 1 (ABCB1; Abcam), ATP Binding Cassette Subfamily G Member 2 (ABCG2; Abcam), and

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β-actin (Abcam). Then the membranes were incubated with HRP-conjugated goat anti-rabbit

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IgG secondary antibodies (Abcam) for 2 h at room temperature. The protein signals were visualized with Pierce™ ECL western blotting substrate (Thermo Fisher Scientific) and

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2.7. Statistical analysis

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quantified by Scion Image software (Version 4.02; Scion Corporation, Frederick, MD, USA).

Results were presented as mean ± standard deviation (SD) from 3 independent

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experiments. Student’s t-test or one-way variance analysis (ANOVA) was employed to

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perform statistical analysis using GraphPad software (Version 3.0; GraphPad, San Diego, CA, USA). P < 0.05 was defined as statistically significant. 3. Results

3.1. HOXB4 expression was upregulated in ovarian cancer cells RT-qPCR and western blot assays were performed to explore HOXB4 expression status in ovarian cancer cells. The results showed that HOXB4 expressions at both mRNA (Fig. 1A and Fig. 1B) and protein (Fig. 1C and Fig. 1D) levels were increased in ovarian cancer cells 7

ACCEPTED MANUSCRIPT (A2780 and SKOV-3) compared with human normal ovarian epithelial cells (HOEC). Previous studies showed that HOXB4 was associated with multiple-drug resistance in human myelogenous leukemia K562/ADM cells (Wang et al., 2016). Hence, we used Taxol-resistant A2780 cell line (A2780/Taxol) and DDP-resistant SKOV-3 cell line (SKOV-3/DDP) to

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determine the roles of HOXB4 in the drug resistance of ovarian cancer cells. As shown in Fig.

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1A and 1C, HOXB4 expressions at mRNA and protein levels were upregulated in

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A2780/Taxol cells compared with A2780 parental cells. Similarly, an increase of HOXB4 expressions at mRNA (Fig. 1B) and protein (Fig. 1D) levels was also observed in

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SKOV-3/DDP cells when compared with that in SKOV-3 parental cells. These results

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indicated that HOXB4 might be associated with the drug resistance of ovarian cancer. 3.2. HOXB4 knockdown enhanced the cytotoxic effects of Taxol and DDP in drug-resistant

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ovarian cancer cells

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MTT assay was used to determine the viability of A2780, A2780/Taxol, SKOV-3, and SKOV-3/DDP cells treated with designated concentrations of Taxol or DDP. As shown in Fig.

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2A and Fig. 2B, Taxol and DDP inhibited the viability of A2780, A2780/Taxol, SKOV-3, or

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SKOV-3/DDP cells in a concentration-depended manner. To investigate the roles of HOXB4 in the drug resistance of ovarian cancer, siControl, siHOXB4-1 and siHOXB4-2 were transfected into A2780/Taxol and SKOV-3/DDP cells, followed by the detection of knockdown efficiency by western blot assay. The results showed that HOXB4 expression was decreased in A2780/Taxol (Fig. 2C) and SKOV-3/DDP cells (Fig. 2D) after transfection with siHOXB4-1 and siHOXB4-2 compared with siControl group. siHOXB4-1 (siHOXB4) was used for the following experiments due to its higher knockdown efficiency. LDH as a 8

ACCEPTED MANUSCRIPT cytosolic enzyme can release into soluble cell fraction when cells were damaged and dead, therefore, LDH release was used as an assessment indicator of cell cytotoxicity (Choksakulnimitr et al., 1995; Fischer et al., 2003). Hence, the effect of siHOXB4 on the cytotoxic effects of Taxol and DDP in A2780/Taxol and SKOV-3/DDP cells were assessed by

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detection of cells viability, LDH release, apoptosis-related protein caspase-3 activity, and the

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expression of cleaved caspase-3 and Bax. Results revealed that Taxol treatment and HOXB4

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silencing both reduced cells viability, increased LDH release, improved caspase-3 activity, and elevated the expression levels of cleaved caspase-3 and Bax in A2780/Taxol cells

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compared with control groups. HOXB4 knockdown enhanced the cytotoxic effects of Taxol

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on A2780/Taxol cells (Fig. 2E, 2G, 3A, and 3C). Similarly, DDP administration or HOXB4 knockdown resulted in suppression of cells viability, improvement of LDH release and

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caspase-3 activity, and increase in the expression levels of cleaved caspase-3 and Bax in

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SKOV-3/DDP cells. HOXB4 knockdown enhanced the cytotoxic effects of DDP on SKOV-3/DDP cells (Fig. 2F, 2H, 3B, and 3D). Therefore, we concluded that HOXB4

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knockdown enhanced the cytotoxic effects of Taxol and DDP in drug-resistant ovarian cancer

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cells.

3.3. HOXB4 knockdown suppressed PI3K/Akt signaling pathway in drug-resistant ovarian cancer cells

Previous studies showed that inhibition of the PI3K pathway could enhance the cytotoxic effect of Taxol and DDP in ovarian cancer cells (Hu et al., 2002; Ohta et al., 2006). Hence, we further investigated whether HOXB4 exerted its function via the PI3K/Akt signaling pathway. The results showed that knockdown of HOXB4 reduced the protein levels of p-Akt and 9

ACCEPTED MANUSCRIPT p-p70S6K in A2780/Taxol cells compared with siControl group. Moreover, the overall expression of Akt was not changed in ovarian cancer cells transfected with siControl or siHOXB4. Therefore, we concluded that HOXB4 knockdown suppressed the PI3K/Akt signaling pathway in drug-resistant ovarian cancer cells.

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3.4. Downregulation of HOXB4 suppressed the expressions of ABC transporters (ABCB1,

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ABCC1 and ABCG2) in drug-resistant ovarian cancer cells

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It is well known that ABC transporters (ABCB1, ABCC1 and ABCG2) are closely associated with Taxol and DDP resistance in various tumors (Chen et al., 2014; Schinkel and

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Jonker, 2003). Hence, the effects of HOXB4 knockdown on the expressions of ABCB1,

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ABCC1 and ABCG2 in A2780/Taxol cells were detected by western blot assay. Results showed that the expressions of ABCB1, ABCC1 and ABCG2 were reduced in

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HOXB4-knockdown A2780/Taxol cells than that in siControl-transfected cells.

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3.5. Inhibition of the PI3K/Akt pathway suppressed cell viability, increased LDH release, and inhibited the expressions of ABC transporters in drug-resistant ovarian cancer cells

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PI3K signaling inhibitor (LY294002) was used to confirm inhibition of the PI3K/Akt

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signaling could affect the cytotoxicity in A2780/Taxol and SKOV-3/DDP cells. As displayed in Fig. 6A and Fig. 6B, cell viability was decreased and LDL release was increased in A2780/Taxol and SKOV-3/DDP cells following the treatment of LY294002 for 48 h. Moreover, we demonstrated that LY294002 suppressed the expressions of ABCB1, ABCC1 and ABCG2 in A2780/Taxol and SKOV-3/DDP cells treated with LY294002 for 48 h (Fig. 6C and 6D). To further confirm the effect of the PI3K/Akt signaling, we employed si-Akt to silence Akt. As shown in Fig. 6E and 6F, treatment with si-Akt decreased the viability and 10

ACCEPTED MANUSCRIPT increased LDL release in A2780/Taxol and SKOV-3/DDP cells. The expression levels of Akt, p-Akt, ABCB1, ABCC1 and ABCG2 were all reduced in A2780/Taxol and SKOV-3/DDP cells after transfection with si-Akt for 48 h (Fig. 6G and 6H). Taken together, all results demonstrated HOXB4 knockdown enhanced the cytotoxic effects by downregulating ABC

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transporters via inhibition of the PI3K/Akt signaling pathway in ovarian cancer cells.

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4. Discussion

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Ovarian cancer accounts for 2.4% of cancer-associated female death and chemotherapy is a common therapeutic approach for ovarian cancer (Agarwal and Kaye, 2003; Siegel et al.,

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2016). However, chemotherapy resistance strikingly impaired chemotherapeutic efficiency

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and triggered a higher relapse rate in ovarian cancer (Agarwal and Kaye, 2003). Hence, it is necessary to better understand the molecular mechanism underlying the drug resistance to

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optimize treatment strategies.

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HOXB4 has been demonstrated to be implicated in drug resistance in some cancers and reports about this are contradictory. For instance, HOXB4 suppressed cell migration and

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potentiated the cytotoxic effect of doxorubicin by targeting STARD13 in breast cancer cell

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(Zhou et al., 2017). HOXB4 knockdown enhanced chemosensitivity and decreased drug efflux in adriamycin-resistant chronic myelogenous leukemia cells by downregulating P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) expression via PI3K/Akt signaling pathway (Wang et al., 2016). Hence, in the present study, we aimed to explore the roles and molecular mechanisms of HOXB4 underlying Taxol and DDP resistance in ovarian cancer. Firstly, our study demonstrated that HOXB4 expressions at mRNA and protein levels were higher in ovarian 11

ACCEPTED MANUSCRIPT cancer cells, which was in accordance with a previous study (Hong et al., 2010). We further explored the association between HOXB4 and drug resistance in A2780/Taxol and SKOV-3/DDP cell lines. The results showed that HOXB4 expression was increased in A2780/Taxol and SKOV-3/DDP cells compared to that in corresponding parental cells,

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indicating that HOXB4 may be implicated in Taxol and DDP resistance in ovarian cancer.

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Subsequent functional analyses indicated that HOXB4 downregulation potentiated cytotoxic

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effect of Taxol and DDP in A2780/Taxol and SKOV-3/DDP cells.

The PI3K/Akt signaling pathway has been demonstrated to be implicated in various

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biological processes such as proliferation, apoptosis, metabolism and angiogenesis (Engelman

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et al., 2006; Hu et al., 2005). Moreover, the PI3K/Akt signaling pathway was closely associated with the development of ovarian cancer and chemotherapy resistance, and could

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act as a therapeutic target for ovarian cancer (Mabuchi et al., 2015). Inhibition of the PI3K

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pathway could potentiate the cytotoxic effect of Taxol and DDP in ovarian cancer (Hu et al., 2002; Ohta et al., 2006). Hence, we further investigated whether HOXB4 exerted its function

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via the PI3K/Akt signaling pathway. The results showed that HOXB4 knockdown suppressed

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the PI3K/Akt signaling, as demonstrated by the reduction of p-Akt and p-p70S6K expression in drug-resistant ovarian cancer cells. Mounting evidence has suggested that ABC transporters (including ABCB1, ABCC1 and ABCG2) could induce active drug efflux, resulting in cancer occurrence, multidrug resistance (MDR) and chemotherapeutic failure in cancer therapy (Kathawala et al., 2015). ABCB1, also named as MDR1 or P-gp, has been demonstrated to be implicated in the resistance of ovarian cancer cells to Taxol and DDP (Duan et al., 2004; Sun et al., 2015). Chen et al. also validated 12

ACCEPTED MANUSCRIPT that Hedgehog signaling regulated drug (Taxol, DDP and doxorubicin) resistance of epithelial ovarian cancer cells by targeting ABCB1 and ABCG2 (Chen et al., 2014). ABCC1, also known as MDR-associated protein-1 (MRP1), also can acted as a transporter of anticancer drug Taxanes (Taxol, docetaxel) and doxorubicin (Schinkel and Jonker, 2003). Thus, we

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supposed that HOXB4-meidated drug (Taxol and DDP) resistance might be associated with

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abnormal expressions of ABCB1, ABCC1 and ABCG2. Our study showed that HOXB4

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knockdown suppressed expressions of ABCB1, ABCC1 and ABCG2 in drug-resistant ovarian cancer cells, suggesting that HOXB4 silencing reinforced cytotoxic effect of Taxol and DDP

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by downregulating expressions of ABCB1, ABCC1 and ABCG2 in drug-resistant ovarian

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cancer cells. Then, we further demonstrated that LY294002 (a potent and selective PI3K inhibitor) or siRNA targeting Akt (si-Akt) suppressed cell viability and promoted LDH release

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in A2780/Taxol and SKOV-3/DDP cells. Moreover, we further demonstrated that ABCB1,

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ABCC1 and ABCG2 were downstream targets of PI3K/Akt pathway and LY294002 or si-Akt downregulated expressions of ABCB1, ABCC1 and ABCG2, which is in accordance with a

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previous study (Zou et al., 2014).

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Taken together, our study revealed that HOXB4 was highly expressed in ovarian cancer cells. HOXB4 silencing enhanced the cytotoxic effect of Taxol and DDP by downregulating ABC transporters via inhibition of the PI3K/Akt signaling pathway. Our study for the first time elucidated the critical roles and molecular basis of HOXB4 underlying drug resistance in ovarian cancer cells, providing a potential therapeutic target for relieving drug resistance of ovarian cancer. Conflict of interest 13

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ACCEPTED MANUSCRIPT Ishikawa, M., 2006. Suppression of invasive characteristics by antisense introduction of overexpressed HOX genes in ovarian cancer cells. Int. J. Oncol. 28, 931-938. Zheng, C.L., Zhou, B., Lu, M., 2007. Regulatory role of HOXB4 in self-renewal of hematopoietic stem cells - review. J. Exp. Hematol. 15, 647-651.

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Zhou, G., Liu, X., Xiong, B., Sun, Y., 2017. Homeobox B4 inhibits breast cancer cell

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2014. 3-Methyladenine can depress drug efflux transporters via blocking the

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PI3K-AKT-mTOR pathway thus sensitizing MDR cancer to chemotherapy. J. Drug

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Target. 22, 839-848.

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Figure legends Fig. 1 HOXB4 was highly expressed in ovarian cancer cells. HOXB4 expressions at mRNA (A and B) and protein (C and D) levels were measured in human normal ovarian epithelial cells (HOEC), ovarian cancer cells (A2780 and SKOV-3), Taxol-resistant A2780

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cells (A2780/Taxol) and DDP-resistant SKOV-3 cells (SKOV-3/DDP). *P < 0.05, n = 3.

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Fig. 2 HOXB4 silencing enhanced the cytotoxic effect of Taxol and DDP in

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drug-resistant ovarian cancer cells. (A and B) The cell viability was determined by MTT. A2780 and A2780/Taxol cells were treated with Taxol (0, 0.5, 1, 2, 5, 10, 20, 30, 40, and 50

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M) for 48 h. SKOV-3 and SKOV-3/DDP cells were treated with DDP (0, 1, 5, 10, 20, 30, 40,

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50, 60, 70, and 80 M) for 48 h. (C and D) HOXB4 expressions were detected in untreated cells, A2780/Taxol and SKOV-3/DDP cells transfected with siControl, siHOXB4-1, or

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siHOXB4-2. Transfected A2780/Taxol and SKOV-3/DDP cells were treated with Taxol (10

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M) or DDP (30 M) for 48 h. The cell viability (E and F) and LDL release (G and H) were determined by MTT and LDH release assays. *P < 0.05, n = 3.

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Fig. 3 HOXB4 knockdown increased caspase-3 activity and the levels of cleaved

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caspase-3 and Bax in drug-resistant ovarian cancer cells. (A and B) Transfected A2780/Taxol and SKOV-3/DDP cells were treated with Taxol (10 M) or DDP (30 M) for 48 h. The caspase-3 activity was measured by caspase-3 activity assay. (C and D) The expression levels of cleaved caspase-3 and Bax were determined by western bolt. *P < 0.05, n = 3. Fig. 4 HOXB4 knockdown suppressed the activation of PI3K/Akt signaling pathway in A2780/Taxol cells. The protein expressions of p-Akt, p-p70S6K and Akt were detected by 18

ACCEPTED MANUSCRIPT western blot assay in untreated cells or A2780/Taxol cells transfected with siControl or siHOXB4 for 48 h. *P < 0.05 vs siControl, n = 3. Fig. 5 Downregulation of HOXB4 suppressed expressions of ABC transporter proteins (ABCB1, ABCC1 and ABCG2) in A2780/Taxol cells. The protein expressions of ABCB1,

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siControl or siHOXB4 for 48 h. *P < 0.05 vs siControl, n = 3.

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ABCC1 and ABCG2 were detected in untreated cells or A2780/Taxol cells transfected with

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Fig. 6 Inhibition of the PI3K/Akt pathway suppressed cell viability, increased LDH release, and inhibited the expressions of ABC transporters in drug-resistant ovarian

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cancer cells. A2780/Taxol and SKOV-3/DDP cells were treated with 20 μM LY294002 or

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transfected with si-Akt for 48 h. (A) Cell viability was detected by MTT assay in treated A2780/Taxol and SKOV-3/DDP cells. (B) LDH release was measured by LDH release assay

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in A2780/Taxol and SKOV-3/DDP cells. (C and D) The expression levels of ABC

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transporters (ABCB1, ABCC1 and ABCG2) were determined by western blot in A2780/Taxol and SKOV-3/DDP cells. (E and F) Cell viability and LDH release were

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detected by MTT and LDH release assay, respectively. (G and H) The expression levels of

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Akt, p-Akt, ABCB1, ABCC1 and ABCG2 were determined by western blot. *P < 0.05 vs Control, n = 3.

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ACCEPTED MANUSCRIPT Abbreviations ABCB1, ATP Binding Cassette Subfamily B Member 1; ABCC1, ATP Binding Cassette Subfamily C Member 1; ABCG2, ATP Binding Cassette Subfamily G Member 2; HOXB13, Homeobox B13; HOXB4, Homeobox B4; LDH, lactate

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time-quantitative

PCR;

SDS-PAGE,

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RT-qPCR,

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dehydrogenase; PI3K, phosphatidylinositol 3-kinase; PVDF, polyvinylidene fluoride;

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sulfate-polyacrylamide gel electrophoresis.

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sodium

dodecyl

ACCEPTED MANUSCRIPT Highlights

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1. HOXB4 expression was upregulated in ovarian cancer cells 2. HOXB4 knockdown enhanced the cytotoxic effects 3. HOXB4 knockdown suppressed PI3K/Akt signaling pathway 4. HOXB4 downregulation suppressed the expressions of ABC transporters

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Graphics Abstract

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