Leptin and HER-2 are associated with gastric cancer progression and prognosis of patients

Biomedicine & Pharmacotherapy 66 (2012) 419–424

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Original article

Leptin and HER-2 are associated with gastric cancer progression and prognosis of patients Yiting Geng a,b,1, Jian Wang a,1, Rong Wang a, Kai Wang a, Yanjie Xu a, Guoxin Song c, Changping Wu b,*,2, Yongmei Yin a,*,3 a b c

Department of Oncology, the First Affiliated Hospital of Nanjing Medical University, Guangzhou Road, #300, 210029 Nanjing, PR China Department of Oncology, the Third Affiliated Hospital of Soochow University, Juqian Road, #185, 213003 Changzhou, PR China Department of Pathology, the First Affiliated Hospital of Nanjing Medical University, Guangzhou Road, #300, 210029 Nanjing, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 February 2012 Accepted 1 March 2012

We conducted this study to evaluate the expression of leptin and its receptor, OB-Rb in gastric cancer and their relationship to clinicopathological features, VEGF and HER-2 expression, as well as the prognostic value. One hundred and ten gastric cancer specimens were detected for leptin, OB-Rb, VEGF and HER-2 by immunohistochemistry (IHC), and 96 specimens of normal gastric mucosa served as the control. The expression level of leptin, OB-Rb and HER-2 in gastric tissues were significantly higher than normal tissues (49.1% vs. 34.0%, 60.9% vs. 46.0%, 19.1% vs. 8.0%, P < 0.05). There was a correlation between the expression of leptin and HER-2, both of which were significantly associated with invasion depth, lymph node metastasis, AJCC stage and VEGF expression. However, there was no correlation between OB-Rb expression and all clinicopathological features. Cox regression analyses showed that age, tumor size, histological grade, serosa invasion, AJCC stage, chemotherapy, leptin and HER-2 overexpression were prognostic factors. The survival of patients with leptin positive expression was significantly poorer than those with negative expression (OS: 20.0 months vs. 32.5 months, FPS: 12.0 months vs. 18.0 months, P < 0.01). Leptin, rather than OB-Rb, played an important role in the progression and angiogenesis of gastric cancer, and was a prognostic factor for poor outcome. ß 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Leptin HER-2 Gastric cancer Vascular endothelial growth factor Immunohistochemistry

1. Introduction Leptin, a kind of polypeptide hormone encoded by OB gene, is secreted by adipocytes and other tissues such as placenta, gastric mucosa and skeletal muscle. It controls food intake and energy balance by binding with OB-receptor (OB-R) located in hypothalamus [1,2]. Leptin participates in the regulation of multiple physiological and pathological process, including

Abbreviations: CACs, Circulating angiogenic cells; EGFR, epidermal growth factor receptor; ERK, extracellular signal regulated protein kinase; HER-2, human epidermal growth factor receptor-2; IGF-1, insulin-like growth factor-1; IHC, immunohistochemistry; JAK, Janus kinase; MMPs, Matrix metalloproteases; NR, lymph nodes ratio; OB-R, Obesity-receptor; OS, overall survival; PFS, progressionfree survival; PI3K, Phosphatidylinositol 3 kinase; STAT, signal transducer and activator of transcription; AJCC, American Journal of Critical Care; VEGF, vascular endothelial growth factor. * Corresponding authors. E-mail addresses: [email protected] (C. Wu), [email protected] (Y. Yin). 1 These authors contributed equally to this work. 2 Tel. +86 13 9 01 50 30 29. 3 Tel.: +86 13 9 51 84 27 27; fax: +86 25 83 71 00 40. 0753-3322/$ – see front matter ß 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2012.03.002

reproduction, hematopoiesis, and wound healing by acting on human organs or tissues through neurohumoral mechanism [3]. OB-R, a member of type I chemokine receptor family which having six subtypes (OB-Ra–OB-Rf), is distributed in multiple normal and tumor cells [4–7]. In gastric cancer, leptin can promote proliferation and inhibit apoptosis of tumor cells [8]. It plays an important role in tumor invasion and progression by activating Jak/STAT3, ERK1/2, and PI3K signal pathways [9], and exerts a variety of biological effects by transactivating JAK2 and ERK1/2 through phosphorylation of Epidermal Growth Factor Receptor (EGFR) [10]. Researchers reported that the expression level of leptin was significantly higher in cases with lymph node metastasis, lymphovascular invasion and late stage [11,12]. This study investigated the expression of leptin and OB-Rb in gastric cancer tissues, and estimated its effect on tumor progression by analyzing the correlation between leptin expression and various clinicopathological characteristics. Meanwhile, we evaluated the role of leptin in tumor angiogenesis by detecting vascular endothelial growth factor (VEGF) expression in order to provide more evidence for molecular-targeted therapy of gastric cancer.

Y. Geng et al. / Biomedicine & Pharmacotherapy 66 (2012) 419–424

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The human epidermal growth factor receptor-2 (HER-2) gene, an oncogene located in chromosome 17 (17q12-q21), encodes a 185-kd transmembrane tyrosine kinase receptor (p185). It is a member of EGFR family. The amplification and overexpression of HER-2 have been observed in various human malignant tumors [13–15]. HER-2 overexpression is associated with invasion and metastasis of gastric cancer and has independent prognostic value [16,17]. Recently, the ToGA trial conduced in advanced gastric cancer patients verified that HER-2 positive patients could benefit from trastuzumab (a monoclonal antibody of HER-2) treatment [18]. It has been reported that leptin transactivated HER-2 through both EGFR and JAK2 activation, which can cause the growth of breast cancer cells with HER2 overexpression [19]. Because of the similar effects between Leptin and HER-2 on tumor proliferation, invasion, metastasis, and patients’ survival in breast cancer, we suppose that there may be somewhat relationship between leptin and HER-2 in gastric cancer. Leptin probably influence the expression level or activity of HER-2 in a certain way. In view of this problem, we also performed a correlation analysis.

2005 were involved in this study. Besides, 96 normal gastric mucosa tissues achieved by endoscopy in healthy physical examinees were served as negative control. All the specimens were histologically examined according to the Japanese Classification of Gastric Carcinoma [20] by one pathologist. There were 79 males (71.8%) and 31 females (28.2%) in the postoperative patients, whose age ranged from 28 to 85 years (median: 63.5 years). Tumor localization was cardia in 43 (39.1%), corpus in 40 (36.4%), antrum in 27 (24.5%). According to the AJCC standard, there were one in stage IB, seven in stage IIA, five in stage IIB, 19 in stage IIIA, 20 in stage IIIB, 53 in stage IIIC and five in stage IV respectively. Other clinicopathological features were showed in Table 1. All the patients were followed up for 74 months to obtain the outcome. The experimental protocols were approved by the appropriate institutional review committee and meet the guidelines of the responsible governmental agency.

2. Patients and methods

The Hercep Test (DAKO, Glostrup, Denmark) was used according to the protocol of the manufacturer. The primary antibodies were used in this study at a dilution of 1:100 as below: for leptin, A20 (Santa Cruz, CA, USA), for OB-Rb, M-18 (Santa Cruz Biotech, CA, USA), for HER-2, A0485 (DAKO, Carpinteria, CA) and for VEGF antibody (Maxim, Fuzhou, China). All the samples were fixed

2.2. Immunohistochemistry

2.1. Patients One hundred and ten gastric cancer tissues which were surgically resected in Jiangsu province hospital from 2003 to

Table 1 Expression of leptin and obesity-receptor (OB-R)-b in gastric cancer tissues and the correlation with clinicopathological features. Leptin n Gender Male Female Age > 50 50–69 7 0 Location Fundus Corpus Antrum Histological grade Well differentiated Moderately differentiated Poorly or undifferentiated Size  5 cm > 5 cm pT T1 + T2 T3 + T4 pN 1 2 3a 3b NR < 20% 20–39% 40–59% 6 0% pM 0 1 AJCC stage I + II IIIA + IIIB IIIC IV

OB-Rb +

P

+

P

79 31

41 15

38 16

0.905

32 11

47 20

0.788

15 62 33

7 31 18

8 31 15

0.859

7 23 13

8 39 67

0.792

43 40 27

21 19 16

22 21 11

0.603

16 15 12

27 25 15

0.806

8 56 46

7 26 23

1 30 23

0.093

4 22 17

4 34 29

0.783

53 57

29 27

24 30

0.562

20 23

33 34

0.932

16 94

13 43

3 51

0.018

7 36

9 58

0.892

24 29 52 5

17 16 20 3

7 13 32 2

0.060

11 14 17 1

13 15 35 4

0.370

16 19 16 59

11 14 6 25

5 5 10 34

0.031

8 6 5 24

8 13 11 35

0.631

105 5

55 1

50 4

0.202

43 0

62 5

0.154

13 39 53 5

12 21 22 1

1 18 31 4

0.005

7 18 18 0

6 21 35 5

0.122

The bold characters denote that the P value is less than 0.05, which has the statistic significance.

Y. Geng et al. / Biomedicine & Pharmacotherapy 66 (2012) 419–424

in formalin solution and embedded in paraffin. 3–4 mm sections were dewaxed in xylene, dehydrated in ethanol, and incubated in 3% H2O2 for 15 minutes to destroy endogenous labeled activity. After incubation in 10% normal bovine serum for 10 min, each slide was incubated overnight at 4 8C with the primary antibodies. Biotin labeled mouse-rabbit immunoglobulin was chosen as a second antibody. The sections as positive and negative control were provided by the manufacturer. 2.3. Evaluation of staining HER-2 immunostaining was scored according to Hercep Test TM (DAKO) by two experienced pathologists who were blinded to the outcomes. Because of the inherent biological differences between breast and gastric tumors and notably tumor heterogeneity, a new set of immunohistochemistry scoring criteria were developed specifically for gastric cancer: 0, no staining or less than 10% tumor cells with membrane staining; 1 +, more than 10% tumor cells with faint staining in partial membrane; 2 +, more than 10% tumor cells with weak to moderate staining in partial membrane; 3 +, more than 10% tumor cells with strong staining in partial membrane. Specimens with scores 0 or 1 + were considered HER-2 negative and with score 2 + or 3 + were considered HER-2 positive [21]. Leptin, OB-Rb and VEGF immunostaining was scored as follows: intensity (I) of staining was graded on a scale of 0–3, with 0 representing no detectable stain and 3 representing the strongest stain. The staining area (R), defined as the total amount of positively staining cells was recorded as 0 (none positive staining), 1 (positive staining in less than 25% of the cells), 2 (positive staining in 25–50%), or 3 (positive staining more than 50%). The staining index was obtained by multiplying the scores for I with R. Specimens with index 0–4 were considered negative staining and with index more than 4 were considered positive staining.

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2.4. Statistical analysis The correlations between leptin, OB-Rb expression and clinicopathological characteristics of patients were checked by x2 test. Correlation among the expression of leptin, OB-Rb, HER-2 and VEGF was examined by Spearman coefficients. The influence of leptin and OB-Rb on survival was assessed by the Cox proportional hazards model using backward-LR methods. Survival curves were plotted using Kaplan-Meier method and the statistical differences were analyzed by the log-rank test. For all statistical analyses, SPSS 17.0 computer software (SPSS, Chicago, IL, USA) was used, with significance defined as a P-value less than 0.05. 3. Results 3.1. Leptin and obesity-receptor (OB-R)-b expression in gastric cancer and the correlation with clinicopathological features The IHC staining of leptin and OB-Rb in tumor tissue specimens are showed in Fig. 1. Leptin positive staining was detected in 49.1% gastric cancers, and the positive rate was significantly higher in gastric cancer tissues than normal gastric mucosa (49.1% versus 34.0%, P = 0.038). The expression level of leptin in gastric cancer was associated with tumor infiltration depth, lymph nodes ratio (NR, the proportion between metastatic and examined lymph nodes) and stage. Leptin expression was higher in those with T3 or T4, high NR and late stage than in those with T1 or T2, low NR and early stage (T1 + T2: 18.8%, T3 + T4: 54.3%, P = 0.018. LNR < 20%: 31.3%, 20– 39%: 26.3%, 40–59%: 62.5%,  60%: 57.6%, P = 0.031. Stage I + stage II: 7.7%, stage IIIA + stage IIIB: 20.5%, stage IIIC: 58.5%, stage IV: 80%, P = 0.005). However, no significant difference was observed in gender, age, location, histological grade, size, or distal metastasis (P > 0.05). The expression of OB-Rb was also higher in gastric cancer tissues than normal tissues (60.9% versus 46.0%, P = 0.043), and was not associated with factors aforementioned (P > 0.05), as Table 1

Fig. 1. Leptin and obesity-receptor (OB-R)-b immunohistochemistry (IHC) in gastric cancer tissues (magnification 200). Leptin and OB-Rb were stained by brown at the surface of the cells. 1. Negative expression of leptin. 2. Weak positive expression of leptin. 3. Strong positive expression of leptin. 4. Negative expression of OB-Rb. 5. Weak positive expression of OB-Rb. 6. Strong positive expression of OB-Rb.

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showed. In addition, there was a correlation between leptin and OBRb expression in gastric cancer tissues (r = 0.1900, P = 0.046).

positive rate was significantly lower in HER-2 negative specimens (42.7%).

3.2. Human epidermal growth factor receptor-2 (HER-2) expression in gastric cancer and the correlation with clinicopathological features

3.5. Correlation between leptin and human epidermal growth factor receptor-2 (HER-2) expression in gastric cancer

HER-2 positive staining was detected in 21 of 110 gastric cancers (19.1%), which was significantly higher than in normal tissues (19.1% vs. 8.0%, P = 0.033). The expression level of HER-2 in gastric cancer was higher in cases with high NR, late AJCC stage and distal metastasis than in those with low NR, early AJCC stage, and without distal metastasis (P < 0.05).

In this study, we analyzed the relationship between leptin and HER-2 expression in gastric cancer, and the result indicated that leptin had a positive correlation with HER-2 expression (Spearman coefficient = 0.217, P = 0.023). The positive expression rate of leptin was 71.4% in HER-2 positive cases and 43.8% in HER-2 negative cases, respectively.

3.3. Correlation between leptin/obesity-receptor (OB-R)-b expression and vascular endothelial growth factor (VEGF) in gastric cancer

3.6. Survival analysis

The correlation between expression of leptin and VEGF in gastric cancer was analyzed by Spearman correlation. In this research, the positive expression rate of VEGF was 49.1% (59.3% in leptin positive cases and 39.3% in leptin negative cases, respectively). Leptin expression was positively correlated with VEGF expression in gastric cancer tissues (Spearman coefficient = 0.200, P = 0.036). However, the relationship between OB-Rb and VEGF expression was not significant in statistics (P = 0.464). 3.4. Correlation between human epidermal growth factor receptor-2 (HER-2) and vascular endothelial growth factor (VEGF) expression in gastric cancer A positive correlation between HER-2 and VEGF was also confirmed (Spearman coefficient = 0.263, P = 0.005). VEGF positive staining was detected in 76.2% HER-2 positive specimens, and the

Cox regression univariate analyses showed that age, tumor size, histological grade, serosa invasion, lymph node metastasis, high NR, carcinoma cell embolus, late stage, chemotherapy absence, leptin and HER-2 overexpression were all prognostic factors, both for overall survival (OS) and progression free survival (PFS), P < 0.05. Taken a step further, Cox regression multivariate analyses confirmed tumor size, histological grade, high NR, late stage, chemotherapy absence, leptin and HER-2 overexpression as independent poor prognostic factors (P < 0.05), as Table 2 showed. The Kaplan-Meier curves were showed in Fig. 2. The median OS and PFS of patients with leptin positive expression was significantly worse than negative cases, (OS: 20.0 months versus 32.5 months, P < 0.01, PFS: 12.0 months versus 18.0 months, P < 0.01), which showed in Fig. 2-1 and Fig. 2-2. However, both OS and PFS of patients were not influenced by OB-Rb expression (P > 0.05), as Fig. 2-3 and Fig. 2-4 showed.

Fig. 2. Survival curves for patients with different expression level of leptin and obesity-receptor (OB-R)-b. 1. Overall survival (OS) of patients with leptin positive expressed was significantly shorter than that of leptin negative ones (20.0 months versus 32.5 months, P < 0.01). 2. Progression-free survival (PFS) of patients with leptin positive expressed was also significantly shorter than that of negative ones (12.0 months versus 18.0 months, P < 0.01). 3. OS of patients with positive and negative expression of OBRb (20.3 months versus 21.5 months, P > 0.05). 4. PFS of patients with positive and negative expression of OB-Rb (12.5 months versus 12.8 months, P > 0.05).

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Table 2 Association of various factors with survival determined by the Cox proportional hazards model. Factors

Code

P

HR (95.0% CI)

Histological grade Size NR Stage Chemotherapy HER-2 Leptin

1 (8) or 2 (56) or 3 (46)  5 cm (53) or > 5 cm (57) < 20% (16) or 20–39% (19) or 40–59% (16) or 60% (59) I + II (13) or IIIA + IIIB (39) or IIIC (53) or IV (5) Yes (30) or no (80) Negative (89) or positive (21) Negative (56) or positive (54)

0.001 0.021 0.008 0.002 0.025 0.000 0.035

2.090 1.715 1.432 1.826 0.576 2.976 1.664

4. Discussion and conclusion In this study, leptin and OB-Rb were both detected in gastric cancer tissues and normal gastric mucosa, and the positive staining rates were much higher in cancer tissues than controls. In addition, the expression level of leptin was significantly higher in samples with serosa invasion, higher NR and later stage. Leptin expression was not associated with other clinicopathological features such as gender, age, tumor location, histological grade, and size. However, there was no significant correlation between OB-Rb expression and all the clinicopathological features aforementioned. To be compatible with previous reports, these results suggested that leptin, but not OB-Rb, played an important role in tumor invasion and progression. Recently, it has been confirmed that leptin overexpression is a poor prognostic factor for patients with gastric cancer, with a hazard ratio of 1.7 [12]. In this study, we also confirmed a significant prognostic value of leptin in gastric cancer by Cox regression multivariate analysis. The outcome of patients with leptin positive expression was significantly poorer than leptin negative ones. Thus, detecting leptin expression in gastric cancer tissues could be helpful to predict the prognosis of patients. Up to now, the mechanism of tumor cell proliferation and metastasis induced by leptin is not well understood. Because OBRb has a similar structure to EGFR, leptin may promote tumor growth by transactivation of EGFR [10], and the activation of JAKSTAT and ERK2 pathways is also involved [22]. Additionally, Saxena, et al. have found a bidirectional crosstalk between leptin and insulin-like growth factor-I (IGF-1) signaling which can promote invasion and migration of breast cancer cells via transactivation of EGFR [23]. Recently, it is reported that leptin can upregulate the expression of plasminogen activator inhibitor-1 in human vascular endothelial cell, and contribute to hypercoagulation [24]. Hence it is not difficult to conclude that hypercoagulation of gastric cancer patients induced by leptin is benefit to the adhesion of tumor cells to vascular endothelium and the formation of cancerous embolus, which causes invasion and metastasis. Several studies have suggested that lymph nodes ratio (NR), the proportion between metastatic and examined lymph nodes, had a better prognostic value than the number of metastatic lymph nodes [25,26]. Thus, we added analyses relating to NR in this study, and confirmed that NR was an independent prognostic factor for patients with gastric cancer. Leptin expression was significantly associated with NR instead of the number of metastatic lymph nodes. Avoiding from the interference of operation, the results were more accurate and convincing. It is well known that the growth and metastasis of tumor were determined by the formation of blood vessels. In present study, we concluded a significant positive correlation between leptin and vascular endothelial growth factor (VEGF) expression, which was compatible with previous report [12]. It is reported that leptin has a dose- and time-dependent angiogenic effect in vivo, which is equivalent to the angiogenic effect of VEGF [27]. The impairment of ulcer healing was observed in leptin-deficient mice owing to

(1.364–3.204) (1.083–2.715) (1.099–1.867) (1.243–2.683) (0.355–0.934) (1.688–5.244) (1.036–2.672)

deficiency of VEGF expression and angiogenesis, and systemic administration of leptin could reverse the impairment of gastric ulcer healing by increasing VEGF expression [28]. The mechanism of angiogenesis induced by leptin is not well understood. Upregulating the expression of matrix metalloproteinases (MMPs) may be an important factor [29]. While, extracellular matrix is degraded by MMPs, a variety of growth factors such as bFGF, TGFb, et al., which can increase VEGF expression are released. In addition, leptin could specifically promote circulating angiogenic cells (CACs) adhesion to tubular endothelial structures and migration along outgrowing sprouts of endothelial cells, and stimulation of CACs with leptin increased their capacity to promote new vessel formation, which depended on the activation of src kinase and integrin alphavbeta5 [30]. Recently, Huang, et al. suggested that leptin could induce vascular smooth muscle cell proliferation via promoting transition from G1 to S phase and ERK1/2 and NF-kappaB pathway might contribute to this procession [31]. All these results showed that leptin played an important role in tumor angiogenesis. Crosstalk between different members of receptor families has become a well-established theory in signal transduction. EGFR family has structural similarity to OB-R, which suggests the possible transactivation of HER-2 by leptin. In MCF-7 cells, the administration of leptin increased HER-2 phosphorylation on Tyr 1248 [32]. Soma, et al. revealed that leptin could transactivate HER-2 through both EGFR and JAK 2 activation, causing the proliferation of cancer cells with HER-2 overexpression [19]. In this study, leptin expression was positively associated with HER-2, both of which were significantly correlated with VEGF expression. Leptin and HER-2 played a concordant role in tumor metastasis, progression, angiogenesis and patients’ outcome, which suggests a certain connection between them. Targeting leptin signaling systems might be beneficial to overcome the resistance to chemotherapy. So far, the regulation mechanism of leptin to HER-2 in gastric cancer has not been well explored, which requires further researches. In conclusion, the expression of leptin in gastric cancer tissues is significantly higher than normal tissues, and is associated with the degree of tumor invasion and metastasis. The significant correlation between leptin and HER-2 suggested that they synergistic participated in tumor progression, metastasis and angiogenesis. Leptin can be seemed as a poor prognostic factor, meanwhile, it also can be a new target for the therapy of gastric cancer. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Authors’ contributions Yongmei Yin contributed to conception and design, and gave final approval of the version to be published. Yiting Geng acquired

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data, revised it critically for important intellectual content, and drafted the manuscript. Rong Wang acquired data and revised the manuscript. Kai Wang and Yanjie Xu contributed to statistic analysis. Guoxing Song contributed to IHC. Acknowledgements This work was supported by grants from the Jiangsu Provincial Natural Science Foundation (NO.BK2008477), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry 2009 (IA09), the open project program of the health bureau of Jiangsu province (XK18 200904), the Jiangsu Province Foundation for Key Medical Persons (RC2011053) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References [1] Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425–32 [PubMed: 10509783]. [2] Schwartz MW, Baskin DG, Kaiyala KJ, Woods SC. Model for the regulation of energy balance and adiposity by the central nervous system. Am J Clin Nutr 1999;69:584–96 [PubMed: 10197558]. [3] Ahima RS, Flier JS. Adipose tissue as an endocrine organ. Trends Endocrinol Metab 2000;11:327–32 [PubMed: 10996528]. [4] Tsuchiya T, Shimizu H, Horie T, Mori M. Expression of leptin receptor in lung: leptin as a growth factor. Eur J Pharmacol 1999;365:273–9 [PubMed: 9988112]. [5] Hino M, Nakao T, Yamane T, Ohta K, Takubo T, Tatsumi N. Leptin receptor and leukemia. Leuk Lymphoma 2000;36:457–61 [PubMed: 10784389]. [6] Mix H, Widjaja A, Jandl O, et al. Expression of leptin and leptin receptor isoforms in the human stomach. Gut 2000;47:481–6 [PubMed: 10986207]. [7] O’brien SN, Welter BH, Price TM. Presence of leptin in breast cell lines and breast tumors. Biochem Biophys Res Commun 1999;259:695–8 [PubMed: 10364481]. [8] Zhao XL, Zhu ZH, Huang KH, Xia ZS. Effect of leptin on proliferation and apoptosis in human gastric cancer cell line SGC7901. Fudan university. J Med Sci 2005;32(1):47–50. [9] Cirillo D, Rachiglio AM, la Montagna R, Giordano A, Normanno N. Leptin signaling in breast cancer: an overview. J Cell Biochem 2008;105:956–64 [PubMed: 18821585]. [10] Shida D, Kitayama J, Mori K, Watanabe T, Nagawa H. Transactivation of epidermal growth factor receptor is involved in leptin-induced activation of janus-activated kinase 2 and extracellular signal regulated kinase 1/2 in human gastric cancer cells. Cancer Res 2005;65:9159–63 [PubMed: 16230373]. [11] Ishikawa M, Kitayama J, Nagawa H. Expression pattern of leptin and leptin receptor (OB-R) in human gastric cancer. World J Gastroenterol 2006;12(34):5517–22 [PubMed: 17006991]. [12] Zhao X, Huang K, Zhu Z, Chen S, Hu R. Correlation between expression of leptin and clinicopathological features and prognosis in patients with gastric cancer. J Gastroenterol Hepatol 2007;22:1317–21 [PubMed: 17559372]. [13] Rabindran SK. Antitumor activity of HER-2 inhibitors. Cancer Lett 2005;227:9– 23 [PubMed: 16051028].

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