Rab25 is responsible for phosphoinositide 3-kinase/AKT‑mediated cisplatin resistance in human epithelial ovarian cancer cells

  • Authors:
    • Yang Fan
    • Long Wang
    • Xuechuan Han
    • Xueqin Liu
    • Hongyun Ma
  • View Affiliations

  • Published online on: November 17, 2014     https://doi.org/10.3892/mmr.2014.2963
  • Pages: 2173-2178
Metrics: HTML 0 views | PDF 0 views     Cited By (CrossRef): 0 citations

Abstract

Rab25, a member of the Rab family of small guanosine triphosphatase, was reported to have an essential role in the development of human epithelial ovarian cancer. The present study demonstrated that Rab25 mediated the sensitivity of ovarian cancer to cisplatin, a first‑line chemotherapeutic agent for the treatment of ovarian cancer in the clinic. Overexpression of Rab25 and increased phosphoinositide 3‑kinase (PI3K)/AKT signaling were detected in cisplatin‑resistant SKOV‑3 cells compared with those in cisplatin‑sensitive ES‑2 cells. The results of the present study indicated that cisplatin resistance was primarily due to reduced G1 cell cycle arrest following cisplatin treatment in SKOV‑3 cells. By contrast, the corresponding phenomenon was not observed following treatment with a Rab25‑specific small interfering RNA or treatment with the PI3K/AKT inhibitor LY294002. Of note, inhibition of the PI3K/AKT pathway reduced Rab25 gene expression and sensitized SKOV‑3 cells to cisplatin. Furthermore, knockdown of Rab25 showed an effect comparable with blocking the PI3K/AKT pathway. In conclusion, the results of the present study demonstrated that PI3K/AKT and Rab25 significantly contributed to cisplatin resistance in human epithelial ovarian cancer; in addition, silencing Rab25 or inhibiting the PI3K/AKT pathway markedly increased the sensitivity of these cells to cisplatin.

Introduction

Ovarian cancer is a malignant tumor derived from epithelial and germ cells (1). In humans, ovarian cancer is the second most prevalent gynecological malignancy and has the highest mortality rate among them (2). According to the US Centers for Disease Control and Prevention, there are >22,000 novel cases of ovarian cancer and ~14,000 mortalities each year in the United States (3). Advanced surgical methods and chemotherapeutic agents are available for the treatment of ovarian cancer; however, early diagnosis only occurs in ~25% of cases due to the lack of effective screening programs and non-specific symptoms (46).

Studies from the International Collaborative Ovarian Neoplasm group and the European Organization for Research and Treatment of Cancer have decided that only patients with stages IA or IB (non-clear cell histology and well-differentiated (G1) tumor; according to the International Federation of Gynecology and Obstetrics guidelines) ovarian cancer may avoid chemotherapy (7). The majority of patients with ovarian cancer require chemotherapy to enhance progression-free and overall survival. Cisplatin and analogous platinum derivatives are the front-line chemotherapeutic agents used for the management and treatment of recurrent ovarian cancer (8,9).

The majority of ovarian cancers are initially responsive to chemotherapy; however, in numerous cases patients become cisplatin-resistant due to recurrence and metastasis (10). The mechanism of cisplatin resistance leading to clinical resistance remains to be elucidated. Therefore, understanding the molecular dysregulation underlying chemoresistance in ovarian cancer is essential for developing successful therapeutic strategies.

Rab25 (also known as CATX8) is a Rab11 guanosine triphosphatase (GTPase) family member that belongs to the Rab family. Rab25 was found to contain an unusual amino acid sequence, WDTAGLE, in its guanosine trisphosphate (GTP)-binding domain causing it to be constitutively activated (11). GTPase activity modulates the binding affinities of Rab25, which are critical for its biological functions, including proliferation, signal transduction, apoptosis, microtubule organization, recruitment of H+K+ adenosine triphosphatase, transferring receptor recycling and integrin trafficking (12). Contrary to the function of other Rab11 GTPases, Rab25 overexpression correlated with the aggressiveness of cancers, including ovarian and breast cancer (13,14). Studies have demonstrated that knockdown of RAB25 promoted autophagy and inhibited cell growth in ovarian cancer cells in vitro and in vivo (15,16).

The aim of the present study was to investigate the effect of Rab25 overexpression and phosphoinositide 3-kinase (PI3K)/AKT signaling on cisplatin resistance in ovarian cancer cell lines in order to discover a novel strategy for sensitizing cells to chemotherapeutic agents.

Materials and methods

Cell culture

SKOV-3 and ES-2 ovarian cancer cell lines were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were grown in RPMI 1640 medium (Gibco-BRL, Grand Island, NY, USA) and then supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (FBS) (all HyClone, Logan, UT, USA) in a 5% CO2 atmosphere at 37°C. The cells were routinely subcultured every three days.

RNA extraction and reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total cellular RNA was extracted from cells using the TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Complementary DNA was synthesized using a reverse transcription reagent (Promega Corp., Madison, WI, USA). qPCR was performed using a standard SYBR green PCR kit (Promega Corp.) and PCR-specific amplification was conducted in an Eppendorf 5331 Real-Time PCR machine (Eppendorf, Hamburg, Germany). Gene expression was calculated using the 2−(ΔΔCt) method. The primer sequences were synthesized by Jie Li Biology (Shanghai, China) as follows: RAB25 sense, 5′-GCCCTGGACTCTACCAATGTTGA-3′ and antisense, 5′-GCTGTTCTGTCTCTGCTTGGACAC-3′; GAPDH sense, 5′-GCACCGTCAAGGCTGAGAAC-3′ and antisense, 5′-TGGTGAAGACGCCAGTGGA 3′.

Western blot analysis

The cells were lysed on ice with RIPA lysis buffer [150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris (pH 7.9), 10 mM NaF, PMSF and 1X protease inhibitors], and protein concentrations were measured using the Bicinchoninic Acid Protein Assay reagent (Pierce Biotechnology, Inc., Rockford, IL, USA). Protein extracts (40 μg) were subjected to SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Invitrogen Life Technologies). The mouse anti-human p-AktThr308 antibody (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) was used as the primary antibody, and the horseradish peroxidase-labeled goat anti-mouse immunoglobulin G (1:2,000; Cell Signaling Technology, Inc.) was used as the secondary antibody. The β-actin antibody (1:5,000; Sigma, St. Louis, MO, USA) was used as an internal control. The bands were detected using an enhanced chemiluminescence kit (GE Healthcare, Little Chalfont, UK) and visualized using the ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA). Multi Gauge V3.2 software (Fujifilm, Kanagawa, Japan) was used to quantitatively determinate the gray level of each band (absorbance measured at 570 nm) and the objective band/internal band ratio.

Small interfering RNA (siRNA) transfection

The human Rab25-specific siRNA (siRab25) and control siRNA (siCon) were purchased from Dharmacon, Inc. (Lafayette, CO, USA). DharmaFCET 1 reagent (Dharmacon, Inc.) was used to transfect siRNAs, according to the manufacturer’s instructions. The Rab25 siRNA sense sequence used was 5′-TCCCTCTGGCTGCAGAAGT-3′.

Cell viability assay

Cells were plated into 96-well microplates (104 cells/well) and incubated with siRab25, siCon, LY294002 (PI3K/AKT inhibitor; Sigma) or dimethyl sulfoxide (control; Sigma) for 24 h. In order to investigate the inhibitory effect of cisplatin in cells, a series of cisplatin concentrations (1, 5, 10, 50 and 100 μM; Sigma) was added to the ovarian cancer cells. Cell viability was monitored using an MTT assay (Sigma) as previously described (17). Absorbance was measured at 570 nm using a Bio-Rad MicroPlate Reader, model 450 (Bio-Rad Laboratories). Experiments were performed in triplicate.

Cell cycle analysis

Cells were synchronized in G1-phase by serum starvation for 12 h. Flow cytometric (FCM; BD FACSCalibur; Becton-Dickinson, San Jose, CA, USA) analysis was used to determine the cell cycle phases of cells. In brief, cells were washed with 4°C phosphate-buffered saline (PBS) and fixed with 70% cold ethanol (HyClone, Logan, UT, USA) at 4°C overnight. The fixed cells were then collected, washed with PBS and stained with propidium iodide (PI; Sigma) in the presence of RNAase (Sigma). The phase of cell cycle was analyzed using ModFit software, version 3.2 (Verity Software House, Topsham, ME, USA).

Apoptosis assay

To evaluate cell apoptosis, an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis kit (Cell Signaling Technology, Inc.) was used. In brief, cells were harvested and washed with 4°C PBS. Cells were then resuspended in binding buffer and then incubated with Annexin V-FITC and PI buffers (Invitrogen Life Technologies) for 15 min at 4°C in the dark. Annexin V-FITC and PI signals were then detected using flow cytometry.

Statistical analysis

Values are presented as the mean ± standard error of the mean. Statistical analyses were performed using Student’s t-test. SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was performed for statistical analysis P<0.05 was considered to indicate a statistically significant difference between values.

Results

Rab25 expression and PI3K/AKT pathway activity are increased in cisplatin-resistant cell lines

The cisplatin-resistant SKOV-3 and the cisplatin-sensitive ES-2 ovarian cancer cell lines were used to investigate the role of Rab25 in cisplatin resistance in ovarian cancer. SKOV-3 cells were found to overexpress Rab25 messenger RNA (mRNA), whereas decreased expression of Rab25 mRNA was observed in the ES-2 cells (Fig. 1A). In addition, western blot analysis revealed that Rab25 protein expression levels were significantly elevated in SKOV-3 cells compared to those of ES-2 cells (Fig. 1B and C). Furthermore, AKT activity was markedly increased in the SKOV-3 cell line compared to that in the ES-2 cell line (Fig. 1B and D).

Cisplatin-resistant cell line lacks G1 cell cycle arrest following cisplatin treatment

In order to further elucidate the mechanism of cisplatin resistance in different cells, the cell cycle and apoptotic rate were analyzed. Following treatment with cisplatin for 2 h, there was a significant dose-dependent increase in the percentage of ES-2 cells in G1-phase (Fig. 2A; Table I). However, SKOV-3 cells did not respond to cisplatin treatment and the number of cells in G1-phase was not significantly altered (Fig. 2A; Table I). The apoptotic rates of the two cell lines showed no significant difference from each other (Fig. 2B). These data indicated that deficiency in G1-phase cell cycle arrest was induced by cisplatin rather than apoptosis and therefore, the lack of SKOV-3 cells in G1-pase may be due to cisplatin resistance.

Table I

Cisplatin-induced G1-phase arrest in SKVO 3 cells is lower than that in ES-2 cells.

Table I

Cisplatin-induced G1-phase arrest in SKVO 3 cells is lower than that in ES-2 cells.

SKVO-3ES-2


Phase/Cisplatin0 μM10 μM100 μM0 μM10 μM100 μM
SubG1 (%)1.88±0.21.55±0.351.11±0.761.51±1.3016.7±1.811.75±0.35
G0/G1 (%)26.7±2.03a37.3±0.9a40.7±1.69a39.7±2.5646.7±3.9151.3±1.45
S (%)41.9±1.9528.0±3.930.5±2.1938.7±0.6129.6±1.8831.1±2.16
G2/M (%)31.4±2.7431.4±4.6730.2±1.0719.5±1.4918.4±1.8717.5±0.83

{ label (or @symbol) needed for fn[@id='tfn1-mmr-11-03-2173'] } Mean ± standard deviation (n=3).

a P<0.05 vs. ES-2 cells.

Inhibition of the PI3K/AKT pathway downregulates Rab25 expression levels

In order to investigate whether Rab25 was associated with the PI3K/AKT pathway, SKOV-3 cells were treated with the PI3K/AKT inhibitor LY294002. Following treatment with 10 μM LY294002 for 24 and 48 h, Rab25 mRNA expression in SKOV-3 cells was decreased by ~46.7 and 58.4%, respectively (Fig. 3A). Subsequently, Rab25 protein expression levels were determined using western blot analysis. The results demonstrated that LY294002 treatment markedly suppressed Rab25 protein expression; this therefore indicated that inhibition of the PI3K/AKT pathway downregulated Rab25 protein expression (Fig. 3B and C).

Knockdown of Rab25 or inhibition of the PI3K/AKT pathway increases the sensitivity of SKOV-3 cells to cisplatin

In the present study, a Rab25-specific siRNA was used to block the expression of ubiquitously-produced Rab25 in SKOV-3 cells. Western blot analysis revealed that protein levels of Rab25 were significantly decreased (by 70.2%) following siRab25 transfection as compared with those of the control groups (Fig. 4A). In order to determine whether Rab25 or the PI3K/AKT pathway were involved in the decreased chemosensitivity of cisplatin-resistant cells, the cell growth inhibiting effect of siRab25 or PI3K/AKT signaling inhibitors in combination with cisplatin was measured. An MTT assay demonstrated that siRab25 as well as LY294002 decreased the IC50 values of cisplatin on SKOV-3 cells (Fig. 4B). In addition, Rab25 gene silencing or LY294002 treatment increased the G1-phase cell cycle arrest induced by cisplatin (Fig. 3C). In conclusion, these results indicated that elevation of Rab25, via activation of the PI3K/AKT pathway, may be the mechanism for cisplatin resistance of ovarian cancer cells.

Discussion

Rab25, a Rab11 subfamily protein, was previously reported to be expressed in all eukaryotes, where it shared a conserved mechanism of regulation (12). Rab25 has been suggested to have a comparable function to that of Rab11; in addition, Rab25 was reported to be spatially and functionally associated with the regulation of apical-to-basolateral transcytosis in polarized epithelial cells, indicating that Rab25 was an important regulator of polarized cell surface composition (18). Rab25 was shown to enhance the invasive ability of cells due to its epithelial cell polarity modulatory characteristic, therefore suggesting that Rab25 dysregulation may have a role in tumorigenesis (14). Previous studies have demonstrated that Rab25 controlled tumor progression, aggressiveness and potentially chemosensitivity (13,1921); furthermore, Rab25 was amplified at the DNA level and overexpressed at the RNA level in ovarian cancers (13).

Knockdown of RAB25 promotes autophagy and inhibits cell growth in ovarian cancer (15). The results of the present study were concurrent with the hypothesis that Rab25, as an oncogene, contributed to the aggressiveness of ovarian cancer. In addition, the present study demonstrated that constitutive overexpression of Rab25 due to increased activation of PI3K/AKT signaling in SKOV-3 cells resulted in cisplatin resistance, a severe obstacle for the successful treatment of ovarian cancer. Elucidating the molecular mechanism underlying cisplatin resistance is critical for improving sensitivity to chemotherapeutic agents. It is understood that chemoresistance is due to dysregulation of the balance between the pathways of cellular survival and apoptosis as well as enhanced drug clearance, enhanced detoxification and reduced drug efficacy due to increased DNA repair (22). A previous study has reported that inhibition of the PI3K/AKT pathway may enhance chemosensitivity of resistant ovarian cancers, more prominently in tumors with a high PI3K/AKT activity profile (10). The present study revealed that suppression of the PI3K/AKT pathway by LY294002 reduced Rab25 expression, therefore indicating that Rab25 was a key effector molecule of the PI3K/AKT pathway in cisplatin resistance. Furthermore, knockdown of Rab25 using siRNAs or inhibition of PI3K/AKT signaling increased the sensitivity of SKOV-3 cells to cisplatin via augmentation of G1-phase cell cycle arrest, which was thought to contribute to the sensitization of cells to cisplatin.

In conclusion, the results of the present study confirmed the tumorigenic role of RAB25 in ovarian cancer cells and suggested a novel role of Rab25 in cisplatin resistance. In addition, the results demonstrated that inhibition of Rab25 and the PI3K/AKT pathway sensitized ovarian cancer cells to cisplatin, providing a potential novel adjuvant therapy in combination with cisplatin.

Acknowledgements

The present study was supported by a grant from the National Natural Science Foundation of China (no. 81060215).

References

1 

Kim A, Ueda Y, Naka T and Enomoto T: Therapeutic strategies in epithelial ovarian cancer. J Exp Clin Cancer Res. 31:142012. View Article : Google Scholar : PubMed/NCBI

2 

Colombo N, Van Gorp T, Parma G, et al: Ovarian cancer. Crit Rev Oncol Hematol. 60:159–179. 2006. View Article : Google Scholar : PubMed/NCBI

3 

Wrzeszczynski KO, Varadan V, Byrnes J, et al: Identification of tumor suppressors and oncogenes from genomic and epigenetic features in ovarian cancer. PloS One. 6:e285032011. View Article : Google Scholar : PubMed/NCBI

4 

Holschneider CH and Berek JS: Ovarian cancer: epidemiology, biology, and prognostic factors. Semin Surg Oncol. 19:3–10. 2000. View Article : Google Scholar : PubMed/NCBI

5 

Shih Ie M and Davidson B: Pathogenesis of ovarian cancer: clues from selected overexpressed genes. Future Oncol. 5:1641–1657. 2009. View Article : Google Scholar : PubMed/NCBI

6 

Nelson AE, Francis JE and Zorbas H: Population screening and early detection of ovarian cancer in asymptomatic women. Aust NZ J Obstet Gynaecol. 49:448–450. 2009. View Article : Google Scholar

7 

Trimbos JB, Parmar M, Vergote I, et al; International Collaborative Ovarian Neoplasm 1 and the European Organisation for Research and Treatment of Cancer Collaborators-Adjuvant Chemotherapy in Ovarian Neoplasm. International collaborative ovarian neoplasm trial 1 and adjuvant chemotherapy in ovarian neoplasm trial: two parallel randomized phase III trials of adjuvant chemotherapy in patients with early-stage ovarian carcinoma. J Natl Cancer Inst. 95:105–112. 2003. View Article : Google Scholar : PubMed/NCBI

8 

Aabo K, Adams M, Adnitt P, et al: Chemotherapy in advanced ovarian cancer: four systematic meta-analyses of individual patient data from 37 randomized trials. Advanced ovarian cancer trialists’ group. Br J Cancer. 78:1479–1487. 1998. View Article : Google Scholar : PubMed/NCBI

9 

Markman M: Optimizing primary chemotherapy in ovarian cancer. Hematol Oncol Clin North Am. 17:957–968. 2003. View Article : Google Scholar : PubMed/NCBI

10 

Ali AY, Farrand L, Kim JY, et al: Molecular determinants of ovarian cancer chemoresistance: new insights into an old conundrum. Ann NY Acad Sci. 1271:58–67. 2012. View Article : Google Scholar : PubMed/NCBI

11 

Mitra S, Cheng KW and Mills GB: Rab25 in cancer: a brief update. Biochem Soc Trans. 40:1404–1408. 2012. View Article : Google Scholar : PubMed/NCBI

12 

Agarwal R, Jurisica I, Mills GB and Cheng KW: The emerging role of the RAB25 small GTPase in cancer. Traffic. 10:1561–1568. 2009. View Article : Google Scholar : PubMed/NCBI

13 

Cheng KW, Lahad JP, Kuo WL, et al: The RAB25 small GTPase determines aggressiveness of ovarian and breast cancers. Nat Med. 10:1251–1256. 2004. View Article : Google Scholar : PubMed/NCBI

14 

Caswell PT, Spence HJ, Parsons M, et al: Rab25 associates with alpha5beta1 integrin to promote invasive migration in 3D microenvironments. Dev Cell. 13:496–510. 2007. View Article : Google Scholar : PubMed/NCBI

15 

Liu Y, Tao X, Jia L, et al: Knockdown of RAB25 promotes autophagy and inhibits cell growth in ovarian cancer cells. Mol Med Rep. 6:1006–1012. 2012.PubMed/NCBI

16 

Fan Y, Xin XY, Chen BL and Ma X: Knockdown of RAB25 expression by RNAi inhibits growth of human epithelial ovarian cancer cells in vitro and in vivo. Pathology. 38:561–567. 2006. View Article : Google Scholar

17 

Wang H, Li H, Zuo M, et al: Lx2-32c, a novel taxane and its antitumor activities in vitro and in vivo. Cancer Lett. 268:89–97. 2008. View Article : Google Scholar : PubMed/NCBI

18 

Goldenring JR and Nam KT: Rab25 as a tumour suppressor in colon carcinogenesis. Br J Cancer. 104:33–36. 2011. View Article : Google Scholar :

19 

Cheng KW, Lahad JP, Gray JW and Mills GB: Emerging role of RAB GTPases in cancer and human disease. Cancer Res. 65:2516–2519. 2005. View Article : Google Scholar : PubMed/NCBI

20 

Casanova JE, Wang X, Kumar R, et al: Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells. Mol Biol Cell. 10:47–61. 1999. View Article : Google Scholar : PubMed/NCBI

21 

Wang X, Kumar R, Navarre J, Casanova JE and Goldenring JR: Regulation of vesicle trafficking in madin-darby canine kidney cells by Rab11a and Rab25. J Biol Chem. 275:29138–29146. 2000. View Article : Google Scholar : PubMed/NCBI

22 

Fraser M, Leung B, Jahani-Asl A, Yan X, Thompson WE and Tsang BK: Chemoresistance in human ovarian cancer: the role of apoptotic regulators. Reprod Biol Endocrinol. 1:662003. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

March 2015
Volume 11 Issue 3

Print ISSN: 1791-2997
Online ISSN:1791-3004

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Fan, Y., Wang, L., Han, X., Liu, X., & Ma, H. (2015). Rab25 is responsible for phosphoinositide 3-kinase/AKT‑mediated cisplatin resistance in human epithelial ovarian cancer cells. Molecular Medicine Reports, 11, 2173-2178. https://doi.org/10.3892/mmr.2014.2963
MLA
Fan, Y., Wang, L., Han, X., Liu, X., Ma, H."Rab25 is responsible for phosphoinositide 3-kinase/AKT‑mediated cisplatin resistance in human epithelial ovarian cancer cells". Molecular Medicine Reports 11.3 (2015): 2173-2178.
Chicago
Fan, Y., Wang, L., Han, X., Liu, X., Ma, H."Rab25 is responsible for phosphoinositide 3-kinase/AKT‑mediated cisplatin resistance in human epithelial ovarian cancer cells". Molecular Medicine Reports 11, no. 3 (2015): 2173-2178. https://doi.org/10.3892/mmr.2014.2963