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Overexpression of RhoGDI2 in stage II and III gastric cancer is involved in 5-fluorouracil resistance

Wang Jinguo et. al.

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GTMB Vol 18 2018: 119-130

Overexpression of RhoGDI2 in stage II and III gastric cancer is involved in 5-fluorouracil resistance

Research Article

 

Wang Jinguo*, Hu Minhua, Wu Pei and Wu Jian

1 Department of Gastrointestinal Surgery, Yijishan Hospital, Wannan Medical College, Wuhu, Anhui 241000, PeopleÕs Republic of China

*Correspondence: Wang Jinguo, email: shwangjg@sina.com; Telephone: +86-553-5739558; Fax: +86-553-5738279.

 

Key words: Polymer, Alginate, Chitosan, Dextran Spermine, Pullulan, Nanoparticles

 

Received: 1 June 2018

Accepted: 10 June  2018; electronically published: 10 June  2018

 

Summary

Rho GDP dissociation inhibitor 2 (RhoGDI2) is known to be involved in tumor growth, metastasis and cisplatin resistance. We have further explored RhoGDI2 as a predictor of 5-fluorouracil (5-FU) treatment response in gastric cancer. Here we report that RhoGDI2 promotes tumor progression and 5-FU chemoresistance in gastric cancer. In clinical analysis, RhoGDI2 was overexpressed in 153 of 215 (71.16%) stage II and III patients who received 5-FU-based chemotherapy after radical gastrectomy. Multivariate Cox regression analysis indicated that RhoGDI2 expression was an independent prognostic factor for overall survival (P=0.013) and disease-free survival (P=0.008) of patients with gastric cancer. After being treated with 5-FU, the expression of RhoGDI2 in gastric cancer cells was increased. In RhoGDI2-overexpressing cells treated with 5-FU, cell viability decreased slower than in control cells. These findings demonstrate that RhoGDI2 is an independent prognostic factor for adjuvant 5-FU-based chemotherapy in patients with stage II and stage III gastric cancer. Targeting RhoGDI2 may thus be a useful strategy to enhance chemotherapy efficacy in gastric cancer.

 

 


I. Introduction

Gastric cancer is one of the most common causes of death from cancer worldwide (Rahman et al. 2014). The means of treating advanced gastric cancer are very limited. Many clinical trials have shown that adjuvant therapy, in particular, chemotherapy after radical gastrectomy may improve the survival of gastric cancer patients compared to surgery alone (Wu et al. 2015). The standard chemotherapy regimen used to treat gastric cancer is 5-fluorouracil (5-FU) monotherapy and its combination with other conventional therapeutics (Zhu et al. 2015; Shen et al. 2014). However, there is evidence that gastric cancer resistance to 5-FU, especially stage II and III gastric cancer, is the major cause of therapy failure, leading to disease recurrence and metastasis (Yu et al. 2014). The factors that regulate the resistance of gastric cancer to 5-FU remain poorly understood.

RhoGDI2 belongs to a family of Rho GTPase dissociate inhibitors (GDIs), and is mainly expressed in hematopoietic, endothelial and urothelial cells (Cho et al. 2010; Luo et al. 2013). Previous studies showed that RhoGDI2 inhibits bladder cancer cell metastasis and regulates it progressionin vivoandin vitro (Nitz and Harding 2008). These results suggested that RhoGDI2 might be a novel tumor metastasis suppressor. In contrast, recent studies showed that RhoGDI2 is overexpressed in breast cancer, ovarian carcinoma and pancreatic cancer, and can promote the invasiveness of cancer cells in vivo (Moon et al. 2010; Zhen et al. 2010; Yi et al. 2014). In addition, RhoGDI2 was also reported to be implicated in cisplatin resistance (Cho et al. 2011). Therefore, the role of RhoGDI2incancerremainscontroversial.

In this study, we report the expression pattern of RhoGDI2 in gastric cancer tissues and analyze the correlation between expression levels of RhoGDI2 and the clinicopathologic characteristics of this disease. To evaluate whether RhoGDI2 can influence 5-FU-based chemotherapeutic sensitivity in gastric cancer cells,we upregulated RhoGDI2 expression in gastric cancer cells using plasmid transfection to observe cell viability after treatment with 5-FU. In addition, the relationship between RhoGDI2 expression and disease-free survival (DFS) of stage II and III gastric cancer patients who underwent surgery after 5-FU-based chemotherapy was also analyzed.

Ⅱ. Materials and methods

A. Cell culture

The human gastric cancer cell lines N87 and HTB-103 were obtained from the Chinese Academy of Sciences Cell Bank of Type Culture Collection (Shanghai, China). The gastric cancer cell lines were cultured in RPMI-1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Life Technologies) and 1% penicillin and streptomycin (Life Technologies) in a humidified incubator with an atmosphere of 5% CO2 at 37¡C.

B.Patients and tissues

A total of 215 tumor specimens were obtained from stage II and III gastric cancer patients who underwent radical resection and received 5-FU based chemotherapy after radical gastrectomy at the Department of Gastrointestinal Surgery, Yijishan Hospital, Wannan Medical College (Wuhu, Anhui province, China) from Jan 1 2007 to Dec 31 2009. Patients who were administered neoadjuvant chemotherapy and radiotherapy before surgery were excluded. The pathologic tumor staging was determined according to the American Joint Committee on CancerÕs tumor-node-metastasis classification (2010) (Washington 2010). The patients were followed by history and physical examination every 3 months for 2 years, then every 6 months for up to 5 years. The study was approved by the Ethics Committee for clinical research of Yijishan Hospital, Wannan Medical College. The sections of specimens were fixed with formalin for immunohistochemistry (IHC) tests.

C. Immunohistochemistry (IHC)

The gastric cancer tissues and adjacent normal tissues from the same patient were fixed in formalin and embedded into the same paraffin block. IHC was performed as described by Wang et al. (Wang et al. 2012). RhoGDI2 expression was determined by assessing the intensity of stained tumor cells. The staining intensity was scored as 0 (no staining), 1 (mild staining), 2 (moderate staining), or 3 (intense staining). The stained area was scored as 0 (<5%), 1 (5%–25%), 2 (26%–50%), 3 (51%–75%), or 4 (76%–100%) according to the percentage of positively-stained cells. The final staining scores, calculated by the sum of the intensity and area scores, were divided into three groups as follows: 0–1, negative expression; 2, weak expression; and 3–4, strong expression.

D. Antibodies and western blotting

Anti-RhoGDI2, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Spring Bioscience (Pleasanton, CA, USA). Western blotting was performed as previously described (wang et al. 2012). Briefly, cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. The blot was then probed with anti-RhoGDI2 at a dilution of 1:1000, followed by an incubation with a specific secondary antibody for 1 h at room temperature. Protein bands were visualized by the ECL detection system (Amersham Biosciences AB, Piscataway, NJ, USA). Autoradiogramswere quantified by densitometry (Quantity One software; Bio-Rad, Hercules, CA, USA). As a loading control, GAPDH specific antibody was used. Relative protein levels were calculated by reference to the amount of GAPDH protein.

E. RNA extraction and qPCR

Total RNA was extracted from cultured cells using RNeasy Mini Kit (Qiagen, Hilden, Germany) following manufacturerÕsinstructions. Real-time quantitative polymerase chain reaction (qPCR) analysis was performed as previously described  (wang et al. 2012) on the cells and on 40 frozen tumor tissue and corresponding normal tissue specimens. qPCR analysis of RhoGDI2 expression was conducted using 2 μL of cDNA. The forward primer sequence was 5'-ATGACTGAAAAAGCCCCA-3' and the antisense primer sequence was 5'-TCATTCTGTCCACTCCTT-3'. GAPDH was used as an internal control: 5'-ACAACTTTGGTATCGTGGAAGG-3' (forward) and 5'-GCCATCACGCCACAG TTTC-3' (reverse). All of the experiments were performed at least in triplicate.

F. RhoGDI2-expressing plasmid

Human RhoGDI2 cDNA was amplified by PCR using the primers: forward 5'-CATACTCGAGCGGACAGAGACGTGAAGCAC-3' and reverse 5'-GAAAAGGGTGGGACA GTGCGACTAGGTCAC-3'. PCR products were cloned into pEGFP-C3 (Clontech Laboratories, Mountain View, CA, USA). N87 and HTB-103 cells were transfected with pEGFP-C3‑RhoGDI2 or pEGFP-C3 using Lipofectamine reagent (Life Technologies) according to the manufacturer's instructions. Stable plasmid-transfected clones were selected using 800 μg/ml G418 (Life Technologies) for two weeks, isolated colonies were picked up with tips and the cells were further cultured in the presence of 400 μg/ml G418.

G. Cell viability assay

To analyze the viability of cells, 1×104 cells/well were seeded in 96-well microplates containing 0.2 mL of RPMI-1640. After treatment, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated at 37¡C for 2 h. Then the culture medium was replaced with dimethyl sulfoxide (Sigma-Aldrich). The absorbance at 490 nm (A490) of each well was detected on a microplate reader (Bio-Rad) after shaking at ×2000 rpm for 10 min at room temperature. Cell viability was calculated from the following formula: cell viability (%) = A490 (sample)/A490 (control) ×100.

H. Statistical analysis

The significance of the in vitro data was determined using the StudentÕs t-test. The c2 test and Fisher exact test were used to determine the statistical significance of covariate differences. Survival rates were calculated using the Kaplan–Meier method and the log-rank test was used to compare the survival curves. A P value of less than 0.05 was considered to be statistically significant. All statistical analyses were carried out using the SPSS 15.0 statistical software package (SPSS Inc., Chicago, IL, USA).

Ⅲ. Results

A. Aberrant overexpression of RhoGDI2 in gastric cancer tissues

To detect the expression of RhoGDI2 in normal human gastric mucosa and in gastric cancer tissues, IHC was used. One hundred and fifty-three of 215 (71.16%) stage II and III patientsÕ gastric cancer tissues were shown to be positive for RhoGDI2 (Table 1). Typical immunostainings of RhoGDI2 in normal gastric mucosa and in gastric cancer specimens were shown in Figure.1A. Subsequently, the 40 randomly selected paired cases were used to evaluate RhoGDI2 mRNA and protein expression by qPCR and western blotting respectively. Using qPCR, it was shownthat 27 of 40 (67.5%) cases had at least a 2-fold increase in RhoGDI2 mRNA levels compared to adjacent normal tissues. This difference in RhoGDI2 mRNA expression was significant (P<0.05, Figure.1B). Western blotting also confirmed that the level of RhoGDI2 protein was higher in gastric cancer samples than in the matched adjacent normal tissues (Figure. 1C).

B. Association between RhoGDI2 expression and the clinical features of gastriccancer

To determine the correlation between RhoGDI2 expression level and clinicopathological factors in gastric cancer, the 153 malignant tumors with positive expression (in any degree) of RhoGDI2 from stage II and III patients were analyzed. The clinicopathological characteristics of 153 patients with gastric cancer are shown in Table 1. The data from Table 1 showed that the expression of RhoGDI2 was correlated with tumor differentiation (P=0.000) and recurrence (P=0.019). However, no significant correlation was found between the staining intensity of RhoGDI2 and gender, tumor size and lymphatic invasion. These data indicate that RhoGDI2 may be involved in gastric cancer progression.

C. RhoGDI2 expression is associated with a poor clinical outcome in stage II and III gastric cancer patients

Results from the IHC analysis of 153 stage II and III tissues showed that there was a significant difference between the RhoGDI2-positive and RhoGDI2-negative groups in the number of patients who developed primary gastric cancer recurrence after 5-FU-based chemotherapy (P=0.019). More of the patients with RhoGDI2-positive tumors underwent subsequent recurrence than the patients with RhoGDI2-negative tumors, positive RhoGDI2 expression (weak and strong) was associated with a 5.757-fold increased risk of recurrence (hazard ratio (HR), 5.757; 95% confidence interval (CI), 2.769–11.968; P<0.001). We used the Kaplan–Meier analysis to show that the expression of RhoGDI2 was significantly correlated with the DFS of gastric cancer patients (log-rank test, P<0.05; Figure.2A). Patients with RhoGDI2-positive tumors had significantly lower 5-year overall survival (OS) than those with RhoGDI2-negative tumors (P<0.05; Figure. 2B). In multivariate analysis using clinicopathological variables such as differentiation grade, the expression of RhoGDI2 was an independent prognostic marker to predict patient outcomes (Table 2).This suggests that RhoGDI2 is of clinical significance in the diagnosis and prognosis of patients with stage II and III gastric carcinomas who are treated with 5-FU-based chemotherapy.

D. 5-FU induces upregulation of RhoGDI2 expression

To investigate the role of RhoGDI2 in mediating sensitivity to gastric cancer chemotherapy, we used two human gastric cancer cell lines, N87 and HTB-103. Western blot analysis showed that both untreated cell lines expressed RhoGDI2 protein. Following treatment with 10 μM 5-FU for 24, 48 and 72 h, a gradual increase in the RhoGDI2 mRNA levels was observed in both N87 and HTB-103 cells (Figure. 3A). To examine whether RhoGDI2 protein levels were also altered after 5-FU treatment, western blot analysis was performed. As shown in Figure. 3B, RhoGDI2 levels increased following 5-FU treatment in both cell lines, which was similar to our qPCR data (Figure. 3A).

E. RhoGDI2 increases cell viability

Western blot analysis showed that the expression of RhoGDI2 in N87 and HTB-103 cells was significantly increased after being transfected with aRhoGDI2-overexpressing plasmid (Figure. 3C). To determine whether RhoGDI2 could enhance gastric cancer cell viability after treatment with 5-FU, MTT assays were used. Figure. 4A shows that cell viability was reduced after being incubated with 10 μM 5-FU for 0, 24, 48, and 72 h. However, RhoGDI2-overexpressing cell lines showed a significant enhancement in cell viability with all three incubation times (24, 48, and 72 h) compared to control cells. In addition, cell viability was reduced by 5-FU in a dose-dependent manner (Figure. 4B). In summary, overexpression of RhoGDI2 increased chemoresistance in N87 and HTB-103 cells. These data indicatethat RhoGDI2 mediates chemoresistance in gastric cancer cells.

Ⅳ. Discussion

RhoGDI2 belongs to a family of Rho GDIs which are known to inhibit the activity of Rho GTPase (Griner and Theodorescu 2012). RhoGDI2 has been identified as a metastasis suppressor gene in bladder cancer. Decreased RhoGDI2 expression was associated with a more invasive variant of the HRAS mutation‑positive T24 bladder cancer cell line (Wu et al. 2007). This phenomenon was also observed in ovarian cancer cells. Suppression of RhoGDI2 expression increased ovarian cancer cell growth and invasion in vitro (Stevens et al. 2011). However, Zhang et al. (2009) reported that RhoGDI2 was overexpressed in MDA-MB-231 breast cancer cells and promoted cell migration. In colorectal cancer, RhoGDI2 was found to be overexpressed and increased cancer cells proliferation, motility, and invasion (Li et al. 2012). This is consistent with our trial results. In our study, RhoGDI2 was also found to be overexpressed in gastric cancer tissues compared to their normal counterparts. And RhoGDI2 expression in gastric cancer tissues correlated with tumor differentiation and recurrence. Our data show that RhoGDI2 expression may be implicated in gastric cancer progression.

To further understand how RhoGDI2 contributes to gastric cancer progression, multivariate Cox regression analysis was used. We found that more gastric cancer patients with RhoGDI2-positive tumors underwent subsequent recurrence than patients with RhoGDI2-negative tumors after 5-FU based chemotherapy. Kaplan–Meier analysis also showed that patients with RhoGDI2-positive tumors had a significantly lower 5-year DFS and OS than those with RhoGDI2-negative tumors. This data shows that RhoGDI2 may be a predictor of 5-FU treatment response in gastric cancer.

As a commonly used chemotherapeutic agent 5-FU can interfere with nucleoside metabolism and can be incorporated into RNA and DNA, leading to cytotoxicity and cell apoptosis (Longley et al. 2003). Resistance to 5-FU can result from various causes, including alteration of drug influx and efflux, enhancement of drug inactivation and mutation of the drug target (Longley and Johnston 2005). High expression of thymidylate synthase (Kadota et al. 2011), increased activity of deoxyuridine triphosphatase (Ladner et al. 2000), methylation of the CDKN2A and CCNA1 genes  (Klajic et al. 2014), and overexpression of Bcl-2 (Wu et al. 2015), NF-κB (Uetsuka et al. 2003), ERK (Ahn et al. 2013), and Akt (You et al. 2009) proteins have all been reported to be relevant to 5-FU resistance. Therefore, multiple factors might contribute to 5-FU resistance, and perhapsthe exact mechanism of 5-FU resistance remains to be further elucidated. The present study has shown that the expression of RhoGDI2 is significantly increased in both N87 and HTB-103 cells following 5-FU treatment. Moreover, with upregulated RhoGDI2 expression in gastric cancer cells by plasmid transfection to observe cell viability, cell viability was reduced by 5-FU treatment in a time and dose-dependent manner. However, compared to control cells, the cell viability of RhoGDI2-overexpressing cells decreased more slowly after treatment with 5-FU. Therefore, RhoGDI2 expression in gastric cancer may contribute to 5-FU chemoresistance.

In conclusion, our study indicates that RhoGDI2 decreases 5-FU chemosensitivity in gastric cancer cells and is an independent prognostic factor for adjuvant 5-FU-based chemotherapy in patients with stage II and III gastric cancer. RhoGDI2 could be an effective target for sensitization in gastric cancer although further research is required to confirm our findings.

Acknowledgements

This project was supported by funds from the Natural Science Foundation of Anhui Province (Grant no. 1508085MH147), The Natural Science Foundation of Education Department of Anhui Province (Grant no. 2011KJ193), and The Introduction of Talent Project of Yijishan Hospital (Grant no. YR201002).

Conflict of interest

None.

 


 


Figure legends

Description: E:\拟写论文\clinical and experiment medicine\cancer biologist and therapy\Figure 1.jpg

Figure. 1

RhoGDI2 was overexpressed in gastric cancer.A Immunohistochemical staining for RhoGDI2 expression in normal and gastric cancer tissues (200×, Bar=50 μm). B At least a 2-fold increase in RhoGDI2 mRNA levels compared to noncancerous tissue was observed in 27 (67.5%) of 40 gastric cancer samples. The logarithmic scale 2-ΔΔCt was used to represent the fold change in the qPCR analysis. C Western blotting acknowledged that higher RhoGDI2 expression was observed in gastric cancer samples compared to noncancerous tissues.


 

Description: E:\拟写论文\clinical and experiment medicine\cancer biologist and therapy\Figure 2.jpg

Figure. 2

Kaplan–Meier and log-rank test analysis of survival for 215 stage II and III gastric cancer patients who received 5-FU-based chemotherapy after surgery. A Overall survival (OS) was significantly better in patients with RhoGDI2-negative tumors than in patients with RhoGDI2-positive tumors (P<0.05). B Lower disease-free survival was observed in patients with higher RhoGDI2 expression (P<0.05).


 

Description: E:\拟写论文\clinical and experiment medicine\cancer biologist and therapy\Figure 3.jpg

Figure. 3

5-FU treatment induces RhoGDI2 expression. A qPCR analysis indicated that RhoGDI2 mRNA was increased in both N87 and HTB-103 cells treated with 10 μM of 5-FU for 24, 48, and 72 h. B The level of RhoGDI2 protein was upregulated in N87 and HTB-103 cells following 5-FU treatment. C Western blotting confirmed that RhoGDI2 expression was increased in N87 and HTB-103 cell lines after being transfected with RhoGDI2-expressing plasmid. Values are means ± the standard error of the mean (SEM; * P<0.05).


 

Description: E:\拟写论文\clinical and experiment medicine\cancer biologist and therapy\figure 4.jpg

Figure. 4

RhoGDI2 increases cell viability. A RhoGDI2-overexpressing cell lines show less cell viability reduction at three time points (24, 48, and 72 h) following 10 μM 5-FU treatment (*P<0.05). B Cell viability was reduced by 5-FU treatment in a dose-dependent manner. Cells were treated with six concentrations of 5-FU (0, 2.5, 5, 10, 20, and 40 μM) for 48 h. Half-maximal inhibitory concentrations (IC50s) of 5-FU were determined from threeindependent experiments performed in quadruplicate.


 

Table 1

RhoGDI2 immunohistochemical staining for protein in gastric cancer

variable

n=215

RhoGDI2 expression

p value

Negative

Weak

Positive

Age

0.781

<60

119

32

26

61

³60

96

30

20

46

Gender

0.939

Male

120

34

25

61

Female

95

28

21

46

Differentiation

0.000*

High

77

40

19

18

Moderate or Low

138

22

27

89

T stage

0.839

T1+T2

79

21

18

40

T3+T4

136

41

28

67

N stage

0.069

N0

64

17

20

27

N1+N2+N3

151

45

26

80

TNM stage

0.193

II

85

27

22

36

III

130

35

24

71

Metastasis

0.051

No

132

43

32

57

Yes

83

19

14

50

Tumor Recurrence

0.019*

No

124

37

34

53

 

Yes

91

25

12

54

 

 

*p<0.05 indicates a significant association among the variables.

 

 


 

Table 2

Multivariate Cox proportional hazards models for disease-free survival(DFS) and overall survival (OS)

 

Multivariate analysis (DFS)

Multivariate analysis (OS)

P value

HR (95%CI)

P value

HR (95%CI)

 

Gender (Female vs. Male)

0.708

0.925(0.616,1.391)

0.719

0.928(0.617,1.395)

 

Age (<60 years vs.³60 years)

0.951

1.013(0.664,1.546)

0.973

1.007(0.659,1.539)

 

T stage (T1+T2 vs. T3+T4)

0.307

1.255(0.811,1.942)

0.323

1.246(0.806,1.928)

 

N stage (N0 vs. N1+N2+N3)

0.666

1.148(0.613,2.150)

0.680

1.141(0.610,2.132)

 

TNM stage (II vs. III)

0.178

1.454(0.843,2.508)

0.167

1.467(0.852,2.526)

 

Differentiation(High vs.Low or Moderate)

0.035*

1.653(1.035,2.641)

0.030*

1.685(1.051,2.966)

 

 

RhoGDI2 (negative vs. positive)

0.000*

5.757(2.769,11.968)

0.000*

5.825(2.803,12.105)

 

HR: Hazard ratio; CI: Confidence interval

*p<0.05 indicates that 95% CI of HR was not including 1.

 

 

 

 

 

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