Tumor Biol. DOI 10.1007/s13277-014-1956-3

RESEARCH ARTICLE

AQP5 silencing suppresses p38 MAPK signaling and improves drug resistance in colon cancer cells Xiaoming Shi & Shengchun Wu & Yongbin Yang & Lei Tang & Yüexin Wang & Junjie Dong & Bonan Lü & Guangwei Jiang & Wei Zhao

Received: 27 December 2013 / Accepted: 7 April 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract It is known that aquaporin 5 (AQP5) may represent a novel therapeutic target for treating colon cancer (CC), but whether AQP5 plays a role in the regulation of multidrug resistance (MDR) of colon cancer still remains unclear. In the present study, AQP5 and P-glycoprotein (P-gp), glutathione S-transferase-π (GST-π), topoisomerase II (TOPO II), and thymidylate synthase (TS) were checked in CC and adjacent cancer tissues; AQP5-siRNA was applied to silencing AQP5 in CC cell line HT-29, 5-fluorouracil (5-FU), and cisplatin (DDP) added on cells, and sulforhodamine B (SRB) was used; fluorescence real-time quantitative RT-PCR and Western blot were employed to detect changes in multidrug resistance factor and expression mitogen-activated protein kinase (MAPK) signaling pathway in HT-29. The results showed that AQP5 is significantly induced in cancer tissues than that in adjacent cancer tissues. The expression of AQP5 is positively correlated with drug resistance factors, as demonstrated by the increased expressions of P-gp, GST-π, and TOPO II in CC tissues compared to that in adjacent cancer tissues. Conversely, knockdown of AQP5 in HT-29 human colon cancer cells increased inhibition rates of cancer chemotherapeutic drugs such as 5-FU and DDP. The improved efficacies of chemotherapeutic drugs are associated with the decreased expression of P-gp, GST-π, and TOPO II. In addition, phosphorylation of p38 MAPK was increased by knockdown of X. Shi : S. Wu : Y. Yang : L. Tang : Y. Wang : B. Lü (*) : G. Jiang : W. Zhao Department of General Surgery, Hebei General Hospital, Hebei Shijiazhuang 050051, China e-mail: [email protected] X. Shi e-mail: [email protected] J. Dong Medical Information Institute of Hebei Province, Shijiazhuang, Hebei Shijiazhuang 050071, China

AQP5 in HT-29 cells while phosphorylation and expression of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and Protein kinase B (AKT) were not affected. P38 MAPK inhibitor increased the drug sensitivity of HT-29 cells in a similar way as AQP5-siRNAs do. So these results indicate that AQP5 is associated with drug resistance of colon cancer, and that the AQP5-P38 MAPK pathway may represent a potential drug target to improve drug resistance of colon cancer cells. Keywords Aquaporin 5 . Colonic neoplasms . RNA interference . Multidrug resistance . MAPK signaling pathway Abbreviations CC Colon cancer AQP5 Aquaporin 5 siRNA Small interfering RNA MDR Multidrug resistance P-gp P-glycoprotein GST-π Glutathione S-transferase-π TOPO II Topoisomerase II TS Thymidylate synthase SRB Sulforhodamine B MAPK Mitogen-activated protein kinase ERK Extracellular signal-regulated kinase JNK c-jun N-terminal kinase PI3K Phosphatidylinositol 3-kinase AKT Protein kinase B

Introduction Colon cancer (CC) is a common gastrointestinal malignancy. The mortality rate of colon cancer is ranked the second among the leading causes of mortality from malignancies worldwide

Tumor Biol.

[1]. Clinical treatments for colon cancer include surgery, radiotherapy, chemotherapy, and biological therapy, among which surgery remains the most effective treatment for all stages of colon cancer. However, due to relatively low rates of early detection, systematic chemotherapy treatment may be used to treat colon cancer patients with invasion and metastasis or terminal cancer for the first time [2]. However, the efficacy of chemotherapy is influenced by tumor cell heterogeneity and multidrug resistance (MDR). The development of MDR to chemotherapy remains a major challenge in the treatment of cancer. MDR is induced when tumor cells are resistant to a chemotherapy drug and then become cross-resistant to other drugs. Resistance can develop by numerous complex mechanisms [3–5]. What is worthy of note is that the occurrence MDR could directly lead to failure of chemotherapy. Therefore, identifying genes and mechanisms that is critical to the development of MDR in vivo is of great importance and could help treat colon cancer. Aquaporin (AQP) is a family of membrane transport proteins, which is recognized to regulate movement of water across the hydrophobic cell membranes. AQPs also belong to the family of major intrinsic protein (MIP) [6]. Other than the function of water transportation, the currently identified 14 AQPs play extremely important roles in the physiological processes of different tissues, including kidney, central nervous system, eye, adipose tissue, and exocrine gland. AQPs also regulate pathological processes, such as tumor cell proliferation, invasion, and metastasis [7–11]. Aquaporin 5 (AQP5) is a member of the AQP family and is located on chromosome 12q3 of humans. AQP5 gene encodes a cDNA with length of 1.8 kb, which contains four exons and three introns [12]. AQP5 may play roles in the progress of CC development, progression, and metastasis of humans, as evidenced by its overexpression in CC tissues and association with tumor differentiation, depth of tumor invasion, lymph node metastasis, and TNM stage [13, 14]. However, whether AQP5 plays a role in the regulation of MDR of colon cancer remains unclear. The aim of the present study is to test the hypothesis that AQP5 serves as a critical regulator in the development of multidrug resistance of colon cancer. Here, we collected clinical samples of CC, examined expression levels of AQP5 and multidrug resistance genes, such as P-glycoprotein (P-gp), topoisomerase (TOPO), glutathione S-transferase-π (GST-π), and thymidylate synthase (TS). Furthermore, we applied RNA interfering technology to specifically silence AQP5 expression in HT-29 cell lines of differentiated adenocarcinoma of human colon. Our results demonstrate a sentential role of AQP5 in the MDR development and these findings may provide the basis for a potential beneficial effect of pharmacological inactivation of AQP5 on chemotherapy sensitivity for treating colon cancer.

Materials and methods Clinical specimens After the informed consent was acquired, 45 cases of colon cancer tissues (obtained from patients of 25 males and 20 females, aged 39 to 77, with an average age of 58 years old) were selected from Hebei Provincial People’s Hospital, who had surgical removal and pathological confirmation of colon cancer from January to December in 2011. All patients had no heart disease, diabetes, hypertension, and other underlying diseases and received no radiotherapy, chemotherapy, or immunotherapy before surgical removal of tumors. All of them had adenocarcinoma by histopathological classification, which contains 14 cases of poorly differentiated carcinoma, 19 cases of moderately differentiated carcinoma, and 12 cases of well-differentiated carcinoma. Additional 36 samples were taken from the tissue that is 5 cm away from the tumor in same group of patients (19 males and 17 females, aged 46 to 77 with an average age of 61 years old) and used as adjacent cancer tissues. Tissues are snap frozen in liquid nitrogen after removal, and then transferred to cryogenic refrigerator at −80 °C for storage. Cell lines and reagents Human colon adenocarcinoma cell line HT-29 was purchased from Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cells were cultured in DMEM/F12 medium containing 10 % of fetal bovine serum, 100 U/ml of penicillin, and 100 g /ml of streptomycin, incubated in the incubator at 37 °C with 5 % of CO2 saturated humidity, and treated with 0.25 % of trypsin solution (containing 0.02 % of EDTA) for digestion and passage. Cells at logarithmic growth phase were used in the experiment. AQP5 siRNA transfection and experimental groups AQP5-specific small interfering RNA sequences were designed and synthesized as indicated in reference [15]. The AQP5 siRNA sequences are the following: siRNA#1, 5′-AAAACTCTGCGAACACGGCCCCTGTC TC-3′ (sense) and 5′-AAGGCCGTGTTCGCAGAGTT CCTGTCTC-3′ (antisense); siRNA #2, 5′-AAGAGCAG CCAGTGAAGTAGACCTGTCTC-3′ (sense) and 5′A AT C TA C T T C A C T G G C T G C T C C C T G T C T C - 3 ′ (antisense). In order to increase the inhibition of siRNA on the AQP5 expression, we used the pool of siRNA#1 and siRNA#2 sequences specific to AQP5. The nonspecific control siRNA (NS-siRNA) sequence is: 5′-GGUC UCACUCCCCAUAGAGtt-3'. HT-29 cells were cultured in six-well plates with the cell density of 4× 105/ml

Tumor Biol.

A mRNA expression level

Fig. 1 Expression levels of AQP5 and MDR related genes Pgp, GST-π, TOPO II, and TS in clinical samples. Clinical colon cancer samples with different differentiation level and adjacent noncancerous control tissues were obtained and subjected to a quantitative RT-PCR and b Western blot assays to determine the mRNA or protein expression levels of AQP5, P-gp, GST-π, TOPO II and TS. c Protein expression levels were normalized to internal control gene GAPDH. Values were presented as mean±SD. *P<0.01 versus control group. 1 adjacent noncancerous control tissue group; 2 well-differentiated cancer group; 3 moderate differentiated cancer group; 4 poorly differentiated cancer group

1

6

2

3

4

*

5

*

*

4

*

*

*

* * *

* *

3

*

*

2

*

*

1 0 AQP5

GST-π

P-gp

TOPO2

TS

B AQP5 P-gp GST-π TOPO2 TS GAPDH 1

Protein expression level

C

2.5

1

2

*

1.5

2

2

3

3

4

4

* *

*

*

*

1

* *

* *

* *

* *

*

0.5 0 AQP5

24 h before transfection. DMEM/F12 without serum and antibiotics was used to wash cells before transfection, followed by liposome-mediated siRNA transfection

P-gp

GST-π

TOPO2

TS

(Invitrogen, USA). Specifically, AQP5 siRNA or control NS-siRNA were diluted in DMEM/F12 medium without serum and antibiotics, mixed with Lipofectamine™

Tumor Biol.

2000 transfection reagent, incubated at room temperature to form composites, then the mixture were added into HT-29 cells. The siRNA silencing efficiency and effects on target gene expression were determined 24 h post transfection. The experiments were divided into three groups: the cultured HT-29 cells without transfection (control group), NS-siRNA transfected HT-29 cells (NS-siRNA group), and AQP5 siRNA transfected HT-29 cells (AQP5 siRNA group). Quantitative real-time RT-PCR Quantitative real-time RT-PCR is performed using TRIzol reagent (Invitrogen) according to the manufacturer’s instruction. One-step method was used to extract total RNA in the tissues and cells. Concentration and purity of resulting RNA were quantified, and the integrity of total RNA was confirmed by running on a 1 % agarose gel. Total RNA of 1 μg was used to perform reverse transcription reaction and quantitative realtime PCR according to the instruction of the kit. A 20 μl of PCR reaction system was then utilized, consisting of 1 μl of reverse transcription product, 10 μl of 2×UltraSYBR Mixture, 1 μl of 10 μM upstream and downstream primers, and 8 μl of ddH2O without DNase-RNase. PCR reaction parameters were as follows: initial denaturation at 95 °C for 5 min, denaturation at 95 °C for 30s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, which is repeated for a total of 40 cycles. GAPDH was used as an internal reference gene to calculate relative quantification values (RQ values) of target gene expression, which are used for statistical analysis. The primer sequences used were as follows: AQP5 upstream primer: 5′-CGCCGCAATCCTCTATGG-3′, downstream primer: 5′-GCTGGAATTACCGCGGCT-3′. P-gp upstream primer: 5′-GTGGGGCAAGTCAGTTCATT-3′, downstream primer: 5′-TCTTCACCTCCAGGCTCAGT-3′. GST-π upstream primer: 5′-GGAGACCTCACCCTGTACCA-3′, downstream primer: 5′-GGCTAGGACCTCATGGATCA-3′. TOPO II upstream primer: 5′-CAGGTGGTCGTAATGGTT ATG-3′, downstream primer: 5′-TTTGGACAGATCTGGT TGGA-3′. TS upstream primer: 5′-ACCAACCCTGACGA CAGAAG-3′, downstream primer: 5′-CATGTCTCCCGATC TCTGGT-3′. Proliferating cell nuclear antigen (PCNA) upstream primer: 5′-GTGGAGAACTTGGAAATGGAAAC-3′, Table 1 Immunohistochemical evaluation of AQP5 and MDR proteins in CC tissues with different differentiation

Differentiation

Poorly Moderate Well Adjacent noncancerous

n

14 19 12 36

downstream primer 5′-TTGAAGAGAGTGGAGTGGCT-3′. GAPDH upstream primer: 5′-TGAACGGGAAGCTCAC TGG-3′, downstream primer 5′-GCTTCACCACCTTCTT GATGTC-3′. Western blotting analysis Clinical tissues and cultured cells were added with an appropriate amount of lysis buffer (1 % Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 % NP-40), and incubated on ice for 20 min. The lysates were centrifuged at 8,000 rpm for 10 min at 4 °C. The supernatant was collected and the BCA analysis was used to quantify protein concentration. The same amount of protein lysates were resolved on 10 % SDS-PAGE and then transferred to PVDF membranes, and then 5 % of skim milk powder solution was used to block the membrane at room temperature for 1 h, followed by incubation overnight at 4 °C with antibodies against AQP5, P-gp, GST-π, TOPO II, TS, Protein kinase B (AKT), extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), p38 and their phosphorylation antibodies, and internal reference gene GAPDH at 4 °C (all from Santa Cruz, USA). After rinsed in TBST for three times, the membranes were incubated with secondary antibodies at room temperature for 1 h, followed by a three-time rinse in TBST and one time rinse in TBS. The chemiluminescence method (Santa Cruz) was used for coloration, and strips underwent scanning for integral optical density. GAPDH was used as an internal reference, and the ratio of absorbance values between target proteins and GAPDH were used for comparison. Immunohistochemical staining Pathological sections were prepared, fixed with more than 4 % paraformaldehyde for over 24 h, dehydrated in ethanol, and embedded with paraffin in vertical orientation, and serial sections were performed at the thickness of 4 μm. The SP three-step method was used for immunohistochemical staining. Sections were put in a microwave oven for 20 min for antigen retrieval. After cooling down, the sections were added with drops of 3 % H 2O 2 and then incubated at room

Positive rate (%) AQP5

P-gp

GST-π

TOPO II

TS

11 (78.57) 12 (63.16) 5 (41.67) 4 (11.11)

12 (85.71) 14 (73.68) 6 (50.00) 2 (5.56)

10 (71.43) 12 (63.16) 4 (33.33) 3 (8.33)

11 (78.57) 13 (68.42) 7 (58.33) 5 (13.89)

5 (35.71) 7 (36.84) 4 (33.33) 4 (11.11)

Tumor Biol.

A AQP5 PCNA GAPDH 1

2

3

Pr o t e i n e x p r e s s i o n l e v e l s

B 1

2

1 0.8

*

0.6 0.4 0.2

*

0

PCNA

AQP5

C 1.2 1

OD570

3

1.2

0.8 0.6

*

0.4 0.2 0

1

2

3

D 35 30

Cell number

Fig. 2 Knockdown AQP5 expression inhibited the proliferation activities of HT-29 colon cancer cells. a, b HT-29 cells were transfected with AQP5siRNA or control NS-siRNA for 48 h, and then AQP5 expression level was detected by Western blot. c, d The proliferation activities of HT-29 cells transfected with AQP5-siRNA or control NS-siRNA were determined by SRB assay (c) and cell number counting (d) respectively. Experiments were repeated three times, values were presented as mean±SD. *P<0.01 versus NS-siRNA group. 1 Nontransfected control; 2 NSsiRNA transfected group; 3 AQP5-siRNA transfected group

25

*

20 15 10 5 0

1

2

3

Tumor Biol.

temperature for 10 min to eliminate endogenous peroxidase. PBS was used to rinse the sections, and then added with drops of 10 % goat serum working solution, and incubated at room temperature for 30 min. The primary antibodies against AQP5, P-gp, GST-π, TOPO II, or TS were added and incubated at 4 °C for overnight. Then, the slides were rinsed with PBS at room temperature, added with drops of biotin-labeled secondary antibodies, and incubated at 37 °C for 30 min. The slides were rinsed by PBS, added with drops of HRP-labeled streptavidin, and incubated at 37 °C for 15 min. After being rinsed with PBS at room temperature, 3, 3′-diaminobenzidine (DAB) was used for coloration and then counterstained with hematoxylin. Finally, the slides were dehydrated and sealed into slices. Changes in expression of target proteins were observed and captured by using a microscope.

Sulforhodamine B staining The sulforhodamine B (SRB) staining was performed to determine proliferation inhibition rate of HT-29 cells [16]. The cultured cells were added with pre-cooled trichloroacetic acid (TCA) (the volume fraction was 50 %) with 50 μl/well and fixed at 4 °C for 1 h. The cells were rinsed with ultrapure water for five times and dried. A 100 μl of SRB reagent was added to each well. The cells were stained while being kept away from light for 10 min at room temperature, followed by washing cells with 1 % acetic acid to remove unbound SRB. The cells were dried, and added with 150 μl of 10 mM nonbuffered Tris lye (pH 10.5) to dissolve SRB bound to the protein. The OD values at 545 nm were measured using a microplate reader. Growth inhibition rate (%) is calculated as (1− OD value in experimental group / OD value in control group)×100 %.

Statistical analysis SPSS 11.5 software was used for statistical analysis, and chisquare test was used to compare positive rates; Spearman correlation analysis was used for correlation analysis, and P<0.05 indicates the difference was statistically significant. Table 2 Impact of silencing AQP5 expression on sensitivity to chemotherapeutic drugs for HT29 cells

Results The expression of AQP5 and multidrug resistance genes P-gp, GST-π, TOPO II, and TS in different differentiated human colon cancer tissues Results showed that mRNA and protein levels of AQP5 in CC were significantly higher than that in the adjacent cancer tissue (P<0.05), and its expression was increased with decreased differentiation (Fig. 1a and b). Consistent with the expression level of AQP5, the expression levels of multidrug resistance genes P-gp, GST-π, TOPO II, and TS in CC tissues were induced compared to that in the adjacent cancer tissues (P<0.05), and their expression is inversely correlated with the degree of cancer differentiation (Fig. 1b and c). In agreement with the biochemical analysis results, immunohistochemical analysis revealed that positive staining of AQP5 in CC tissues appeared darker and larger brown particles in colon cancer tissues than that in adjacent tissues. Similarly, drug resistance factors P-gp, GST-π, and TOPO II showed stronger staining in CC tissues and their expression were increased with decreased stage of cancer differentiation. What is worthy of note is that the expression of TS in different differentiated CC tissues seems to show no obvious change (Table 1). Thus, AQP5 was positively correlated with P-gp, GST-π, and TOPO II, but not with TS, according to the statistical analysis of Spearman rank correlation. siRNA-mediated AQP5 silencing inhibits proliferation of human HT-29 colon cancer cells To identify whether AQP5 regulates colon cancer cell proliferation, siRNA-mediated AQP5 silencing was performed. Western blotting analysis showed that AQP5 siRNA significantly inhibited the expression of AQP5 in HT-29 cells, while the control NS-siRNA had no obvious effect. These data indicate that AQP5 siRNA used in the study is sufficient to silence AQP5 gene expression in HT-29 cells (Fig. 2a and b). Next, cell count and SRB staining assays were performed to determine the proliferation activity of HT-29 cells. The results showed that AQP5 siRNA transfection significantly decreased proliferation activity compared to NS-siRNA treated cells (P<0.05). Of note, proliferation inhibition has no

Groups

Inhibition rate of proliferation (%)

Q value

Normal AQP5-siRNA transfection 5-FU (15 μg/ml AQP5-siRNA transfection +5-FU (15 μg/ml)

0 9.11±0.32 25.68±1.71 44.93±2.28

1.38

DDP (2 μg/ml AQP5-siRNA transfection+DDP (2 μg/ml)

18.47±1.25 39.01±1.76

1.51

Tumor Biol.

A

mRNA expression level

Fig. 3 The effects of AQP5 knockdown on the expression of drug resistant genes in HT-29 colon cancer cells. HT-29 cells were transfected with AQP5siRNA or control NS-siRNA, and then, the expression levels of AQP5 and drug resistant genes Pgp, GST-π, TOPO II, and TS expression levels were detected by a quantitative RT-PCR and b, c Western blot assays. Experiments were repeated three times, values were presented as mean±SD. *P<0.01 versus NS-siRNA group. 1 Nontransfected control; 2 NS-siRNA transfected group; 3 AQP5-siRNA transfected group

1

1.8

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1.5 1.2 0.9 *

0.6

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0.3 0

P-gp

GST-π

TOPO2

TS

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P-gp GST-π TOPO2 TS GAPDH 1

Protein expression level

C 1.8

1

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1.5 1.2 0.9

*

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0.3 0

obvious change in NS-siRNA group compared to that of the control group (Fig. 2c and d). In addition, Western blotting results indicated that expression levels of PCNA was significantly decreased by AQP5 siRNA treatment (P < 0.05) (Fig. 2a and b). Collectively, these results demonstrate that AQP5 is required for proliferation of human colon cancer cells.

P-gp

*

GST-π

TOPO2

TS

Effects of AQP5 silencing on chemotherapeutic drug sensitivity in HT-29 cells The SRB staining showed that AQP5 siRNA significantly increased proliferation inhibition rate in response to chemotherapeutic drugs, including 5-fluorouracil (5-FU) and cisplatin (DDP), in HT-29 colon cancer cells compared to cells with

Tumor Biol.

no treatment. Importantly, compared to the treatment with AQP5 siRNA, 5-FU, and DDP alone, AQP5 siRNA significantly increased proliferation inhibition rate in the 5-FU- and DDP-treated cells (P<0.05). The Q values of cells transfected with AQP5 siRNA together with 5-FU or DDP were significantly increased by 1.38 and 1.51 fold respectively, as indicated in Table 2.

What is worthy of note is that compared with no treatment group, expression levels of drug resistance genes did not have significant change in cells transfected with NS-siRNA (P>0.05) (Fig. 3).

Effects of AQP5 silencing on drug resistance gene expression in HT-29 cells

To uncover underlying molecular mechanisms of AQP5 in the regulation of drug resistance in colon cancer cells, western blotting analysis was performed to determine activities of drug resistance mediator, including mitogen-activated protein kinase (MAPK) and PI3K/Akt in HT-29 cells transfected with AQP5 siRNA. Interestingly, AQP5 siRNA significantly decreased phosphorylation levels of p38 (P<0.05), while phosphorylation levels of AKT, ERK, and JNK did not change (Fig. 4). It is remarked that AQP5 siRNA did not change total

Quantitative real-time RT-PCR and Western blotting analysis showed that mRNA and protein levels of multidrug resistance genes P-gp, GST-π, and TOPO II in HT-29 cells were significantly decreased in cells transfected with AQP5 siRNA compared with NS-siRNA treated cells (P<0.05), even though the expression level of TS showed no obvious change (P>0.05).

A

AKT p-AKT ERK p-ERK JNK p-JNK P38 p-P38 GAPDH 1

2

3

B Protein expression level

Fig. 4 Knockdown AQP5 expression affected P38 MAPK pathway in HT-29 colon cancer cells. HT-29 cells were transfected with AQP5-siRNA or control NS-siRNA, and then, the expression and phosphorylation of AKT and MAPK pathway molecules were assayed by Western blot. Experiments were repeated three times, values were presented as mean±SD. *P<0.01 versus NS-siRNA group. 1 Nontransfected control; 2 NSsiRNA transfected group; 3 AQP5-siRNA transfected group

Effects of AQP5 silencing on MAPK and PI3K/Akt pathways in HT-29 cells

4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

1

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* AKT p-AKT ERK p-ERK JNK

p-JNK

P38

p-P38

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protein levels of p38, AKT, ERK, and JNK compared with NS-siRNA group. P38 MAPK inhibitor increased the drug sensitivity of colon cancer cells Knocking down of AQP5 decreased MDR factors expression and increased the drug sensitivity of HT-29 cells, at the same time, reduced phosphorylation of p38-MAPK, thus, inhibition of p38 MAPK signaling may be the molecular mechanism by which AQP5-siRNAs ameliorate drug resistance of colon cancer cells. However, whether p38 MAPK functionally contribute to the drug resistance of HT-29 cells was not clear. We then performed the drug sensitivity experiment using P38 MAPK inhibitor SB203580, the result showed that the P38 MAPK inhibitor could increase the 5-FU and DDP drug sensitivity of colon cancer cells act as AQP5-siRNAs does, as were shown in Fig. 5. The result indicated that p38 MAPK functionally contribute to the drug resistance of HT-29 cells.

Discussion Aquaporins are a family of water channel proteins that transport water into and out of cells. They are widely expressed in a

A

p-P38 P38 GAPDH Control

SB203580

B 80

*

70

IC50 (µg/ml)

Fig. 5 Effects of P38 MAPK on drug sensitivities in HT-29 cells. HT-29 cells were treated with P38 MAPK inhibitor SB203580, then protein expression and phosphorylation of P38 MAPK were detected by Western blot (a), and IC50 (b) of HT-29 cells was determined by SRB assay. GAPDH served as an endogenous reference gene. Values are presented as mean±S.D. (n=3). *P<0.05 compared with nontargeted siRNA group

variety of cells involved in the body fluid secretion and absorption, and participate in a variety of human physiological and pathological processes [17]. The increased water transport is required to fulfill the high metabolic demands of the rapid proliferation in tumor cells. Recently, studies have demonstrated that AQPs are closely associated with many types of human tumors, including gastric carcinoma [18], epithelial ovarian tumors [19], breast cancer [20], biliary tract carcinoma [21], oral squamous cell carcinoma [22], glioblastoma multiforme [23], prostate cancer [24], and primary central nervous system tumors [25]. Other studies have also shown that AQP-1, AQP5, and AQP-8 are all involved in the development and progression of colon cancer [14, 26]. Interestingly, AQP5 promotes tumor progression through promoting proliferation, migration, and invasion of tumor cells, as evidenced by increased expression in malignancies such as lung adenocarcinoma [27], chronic myelogenous leukemia [28], gastric cancer [29], cervical cancer [30], endometrial carcinoma [31], breast cancer [32], and ovarian cancer [33]. This study demonstrated that the expression of AQP5 was increased in colon cancer tissues, and its expression is inversely correlated with tumor cell differentiation, suggesting a role of AQP5 in the progression of colon cancer. The siRNAmediated gene silencing results demonstrate that inhibition of AQP5-p38 MAPK signaling may be the molecular

Control SB203580

60

*

50 40 30 20 10 0

5-FU

DDP

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mechanism by which AQP5 inhibitors such as siRNAs ameliorate drug resistance of colon cancer cells. Chemotherapy is one of the major approaches in CC treatment. It is known that chemotherapy is extremely important for the treatment of CC in the early stage with lymph node metastases and progressive CC. However, the effect of chemotherapy is negatively affected by many factors, and one of the reasons for the failure in chemotherapy is resistance of tumor cells to chemotherapeutic drugs. Multidrug resistance of tumor cells appears to be a critical challenge to effective chemotherapeutic interventions against CC, which is mediated by a combination of cellular factors and complex mechanisms [3–5]. What is worthy of note is that multidrug resistance is mainly divided into two categories. The first one is the classic resistance mechanism involving energy-dependent drug pump, which is mediated by membrane glycoproteins of resistance, including P-gp, multidrug resistance-associated protein (MRP), and lung resistance-related protein (LRP). The other one is the resistance mechanism involving the enzyme of resistance, such as DNA topoisomerase II (TOPO II), TS, and protein kinase C (PKC) [34]. Previous work has shown that other aquaporins, including AQP3 and AQP9, contribute to the chemoresistance of melanoma to arsenite [35]; however, whether AQP5 plays a role in multidrug resistance of CC remains not known. Our experiments with human colon cancer tissues showed that the expression of AQP5 was increased in CC tissues, and was positively correlated with expressions of resistance-associated proteins, including P-gp, GST-π, and TOPO II, suggesting that AQP5 may play a role in the generation of drug resistance of CC cells. In order to investigate the role of AQP5 in the development of drug resistance of CC, we used siRNA-mediated gene silencing to knock down the expression of AQP5 in HT-29 colon cancer cell lines. We have observed an increase of sensitivity to the cancer chemotherapeutic drugs DDP and 5FU in the cells transfected with AQP5-siRNA. More importantly, we also observed a decrease of expression levels of resistance-associated factors P-gp, GST-π, and TOPO II, indicating that AQP5 may regulate drug resistance of CC through P-gp, GST-π, and TOPO II. Others have reported that drug resistance of tumor cells is related to MAPK [36–39] and PI3K/Akt [40] signaling pathways. MAPK is a kind of serine/threonine protein kinase in eukaryotic cells. Upon activation, it transduces extracellular stimulating signals into the cell and cause basic physiological reactions within the cells. There are three MAPK signaling pathways identified in eukaryotes, including ERK, JNK/ stress-activated protein kinase (SPAK), and p38 MAPK [41]. PI3K/Akt is an important signaling pathway for regulating cell growth in eukaryotes, in which the serine/threoninespecific protein kinase Akt is one of the key components [42]. AQP5 is a family of membrane transport protein, and we confirmed it plays a role in the generation of drug resistance

of CC cells through regulating resistance-associated proteins; however, which pathway by which AQP5 regulate MDR genes should be found out. In order to further pinpoint the signaling pathways which mediate AQP5’s effects on drug resistance in colon cancer, we measured MAPK phosphorylation levels in HT-29 cells transfected with siAQP5. We found that silencing of AQP5 expression only reduced phosphorylation and activation of p38 MAPK with other signaling pathways no obvious change. Our study demonstrate that AQP5 is associated with the development of drug resistance of CC by suppressing MDR genes P-gp, GST-π, and TOPO II through inhibiting p38 MAPK pathway. Inactivation of AQP5 may therefore be therapeutically useful for treating drug resistance in colon cancer.

Conflicts of interest None

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