The Expression Of IL-6 And STAT-3 In SGC-7901 Cell Line And Research Of The Mechanism Of The Antineoplastic Effect Of Bisphosphonates

Gastric carcinoma is one of the most common killer diseases. Proliferation, invasion and metastasis are important processes in the progression of gastric carcinoma. It was found that endogenous Interleukin 6(IL-6) was produced and secreted by immune cells with the stimulation of tumor antigens. Over expression of IL-6 in cancer cells could increase proliferation and differentiation of multiple cells. Endogenous IL-6 is closely related with tumorigenesis in several malignancies.IL-6 can stimulate STAT-3 (Signal Transducer and Activator of Transcription,STAT-3 ) phosphorylation by JAK-STAT pathway. STAT proteins transducer signals for varieties of cytokines. They play important roles in cell growth, living, proliferation, differentiation, anti-apoptotic and transformation. Constitutively activated STAT-3 is found in many cancers; p-STAT-3 may cause abnormal proliferation and apoptosis decrease and result in initiation and progression of tumors.Bisphosphonates(BPs) have been widely used in several bone and metabolic diseases. Recent studies showed that BPs could inhibit cell growth of many cancer cell lines. Nitrogen containing bisphosphonates exert their effects by inhibiting a key enzyme in the mevalonate pathway which is an important target for anti-cancer therapy.To further explore the expression of IL-6 and p-STAT-3 in gastric carcinoma, the effects of BPs on gastric carcinoma cell and to elucidate the putative relationship between IL-6 and p-STAT-3 in gastric carcinoma, we carried out the following studies: immunohistochemical staining and Western blot was used to detect the expression of IL-6 and p-STAT-3 in normal gastric mucosa and gastric carcinoma tissues; flow cytometric analysis , immunocytochemical staining and Western blot methods were employed to evaluate the influence of BPs on the expression of IL-6 , p-STAT-3 protein in human gastric adenocarcinoma cell line (SGC-7901).Materials and Methods1 Cases and Material1.1 Forty two cases of surgically resected gastric carcinoma and control gastric mucosa (>10cm from the border of carcinoma) from January,2006 to December,2007 in Department of Gastrointestinal Surgery, Second Hospital of Hebei Medical University were used. All the cases were pathologically reconfirmed. The patients included in this study did receive any radiotherapy or chemotherapy before surgery.1.2 SGC-7901 human gastric adenocarcinoma cell line.1.3 Bisphosphonates used in this study include Ibandronate(IB) and [3-(6-amio-purin-9-yl)-1-hydroxy-1-hydroxyphosphinoyl-propy l]- phosphonic acid(CP).2 Methods2.1 The expression of IL-6 and p-STAT-3 in the normal gastric mucosa tissue and the gastric carcinoma tissue was detected with immunohistochemical staining.2.2 Total protein was extracted from gastric carcinoma and normal tissues. The expression of IL-6 and p-STAT-3 protein was analyzed with Western blot.2.3 The cell proliferation and IC50 are determined by MTT assay. SGC-7901 cells are randomly divided into 3 groups: control group,solvent group and experimental group. SGC-7901 cell in experimental groups were treated with IB and CP at the concentration of 90,120,150, 180 and 210μmol/L respectively. IC50 is determined 72 hrs after treatment (IB: 220μmol/L;CP: 150μmol/L).2.4 Use IB and CP at concentration of IC50 to treat SGC-7901 cell line for 72h,then collect cells for detecting. Apoptosis rate of the SGC-7901 cells that was treated by IB and CP for 72 hrs was determined with flow cytometric (FCM) DNA content analysis.2.5 Cell cycle distribution of the SGC-7901 ells treated for 24 h,48 h and 72h by IB at the concentration of 160,220,280μmol/L and CP at the concentration of 90,150,210μmol/L was studied with FCM.2.6 The expression of IL-6 and p-STAT-3 in SGC-7901 cell line treated by IB and CP at concentration of IC50 for 24, 48,72h was detected with Western blot methods.2.7 The expression of IL-6 and p-STAT-3 protein in SGC-7901 cell line treated by IB and CP at concentration of IC₅₀ for 72h was observed with immunocytochemical staining.Results:1 The results of immunohistochemical stainingThe positive immunoreaction product of IL-6 is located in the cytoplasma as brown granules, while that of p-STAT-3 is cytoplasma and nuclear stained. The positive expression rate of IL-6 and p-STAT-3 in 42 cases surgically resected normal gastric mucosa tissue was 9.621 % and 0 % respectively. The positive expression rate of IL-6 and p-STAT-3 was 73.81 % (31/42) and 35.71%(15/42) in gastric carcinoma tissue respectively, which was obviously higher than that in constrast tissues (normal gastric mucosa tissue P<0.05). The expression rate of IL-6 and p-STAT-3 was positively correlated (X2=10.227 P<0.05) in gastric carcinoma. The positive IL-6 expression was higher in the cases with lymph node metastasis or with serous layer invasion. No significant correlation was found between the level of IL-6 expression and gender of the patients , histological type and differentiation of gastric carcinoma. The p-STAT-3 expression in gastric carcinoma was closely related with lymph node metastasis and differentiation. The positive expression of p-STAT-3 in the cases with lymph node metastasis was significantly higher than that without. And the positive expression of p-STAT3 in poorly differentiated cases was significantly higher than that in well and moderated differentiated cases. There were no differences in the p-STAT-3 expression between different gender , different histological types and different invasion depth(P>0.05).2 The results of Western blotThe molecular weight ofβ-actin,IL-6 and p-STAT-3 are 42kD,26KD, 90KD respectively. Three bands (42kD,26kD and 90KD) were found.β-actin was used as the standard for semiquantitative analysis of IL-6 and p-STAT-3 protein expression. Western blot results showed that as compared to normal gastric mucosa tissues, 33 cases of the 42 gastric carcinoma cases showed high expression of IL-6 protein (78.57%) , while 27 cases showed high expression of p-STAT-3 (64.28%).3 The effects of IB and CP on SGC-7901 cell proliferation in vitroThe result showed that BPs could significantly inhibit SGC-7901 cell proliferation as compared with control and solvent group. The inhibiting effect was in a dose and time-dependent manner. The inhibit rate reached 76.43% and 51.71% respectively after being treated with 210μmol/L IB and Cp for 72h. The solvent has no effect on cell growth. IC₅₀ of IB and CP was 220μmol/L and 150μmol/L respectively.4 The result of flow cytometric analysesFCM analysis showed that the distribution of cell cycle and proliferation in different groups are significantly changed after IB and CP treatment. The fraction of cells in G0 and G1 phase decreased in treated groups compared with control group. While S fraction cells increased in treated groups in a dose and time-dependent manner. The effect CP was more significant than that of IB.Sub-diploid peak, which is the typical feature of apoptostic cell, was found in the histogram of FCM DNA analysis. Apoptosis rate of SGC-7901 cells in treated group with CP was significantly higher than that in treated group with IB. The result suggested that BPs stimulate apoptosis of SGC-7901 cells. There were no differences between CP and IB.5 The result of immunocytochemistry stainingIL-6-positive and p-STAT-3 positive staining cells in treated groups are decreased than that in control group. The effect of CP is more significant than that of IB.6 The result of Western blotThe relative content of IL-6 protein and p-STAT-3 protein in BPs treated group was lower than that in the control group(P<0.05). Quantitative image analysis indicated that the relative content of IL-6 and p-STAT-3 in treated cells decreased time-dependently(r=-0.627 r=-0.738 P<0.05).The effect of CP is more significant than IB.Conclusions:1 The positive expression rate of IL-6 and p-STAT-3 was obviously higher than that in constrast tissues. The expression rate of IL-6 and p-STAT-3 was positively correlated (X²=10.227 P<0.05) in gastric carcinoma. The positive IL-6 expression was higher in the cases with lymph node metastasis or with serous layer invasion. The positive expression of p-STAT-3 in the cases with lymph node metastasis was significantly higher than that without. And the positive expression of p-STAT3 in poorly differentiated cases was significantly higher than that in well and moderated differentiated cases.2 The anti-tumor mechanism of BPs is down-regulate protein expression of IL-6 and p-STAT-3.3 BPs can inhibit proliferation of tumor cells by inducing apoptosis and prolong cell cycle.4 The effect of Cp on SGC-7901 cell line was more significant than that of IB.