Gastric cancer shows the highest invasive and metastasis features, especially lymph metastasis, which is closely associated with poor prognosis of gastric cancer. Although there is evidence that interleukin-6 (IL-6) can promote gastric cancer progression, the underlying specific mechanisms and the mechanisms of gastric cancer lymphangiogenesis are largely unknown. In the present study, we explore whether IL-6 could promote the proliferation and invasion activity of gastric cancer cells, and whether IL-6 mediating VEGF-C production affected the lymphangiogenesis in gastric cancer cells. Our results revealed that IL-6 and its receptors (IL-6 and gp130) are broadly expressed in various gastric cancer cell lines including SGC-7901, MGC, MKN-28 and AGS. Exogenous IL-6 increased the ability of gastric cancer cell proliferation and invasion, which could be weakened by AG490. In addition, exogenous IL-6 promoted the VEGF-C production of gastric cancer cells and the lymphangiogenesis of HDLECs. As we expected, AG490 was able to reduce these effects. Western blot analysis showed that IL-6 increased JKA, STAT3, p-STAT3 and VEGF-C protein levels in the gastric cancer cells. However, the JKA, STAT3, p-STAT3 and VEGF-C protein expression levels were inhibited by AG490. Our data suggested that IL-6 mediates the singnal pathway of JAK-STAT3-VEGF-C promoting the growth, invasion and lymphangiogenesis in gastric cancer. Thus, IL-6 and its related signal pathways may be a promising target for treatment of gastric cancer growth and lymphangiogenesis.
Gastric cancer is a relatively common and second leading cause of cancer death in the world. The Agency for Research on Cancer (IARC) GLOBOCAN reported more than 700,000 deaths of gastric cancer cases and nearly one million new gastric cancer patients occurred globally in 2012 (
The characteristics of invasive, aggressive and lymph node metastatic gastric cancer are determined by various factors. One of these factors is the tumor microenvironment. Many studies have confirmed that interleukin-6 (IL-6) is a very important cytokine amongst all cytokines of the tumor microenvironment, which promote tumor cell proliferation and invasion. In a previous study, patients with high serum IL-6 levels responded poorly to chemotherapy and was asssociated with an inferior survival outcome in non-small cell lung cancer cases (
However, the specific mechanisms responsible for IL-6 promotion of the progression of malignant tumors have not been elucidated. The biological effects of IL-6 are mediated through a membrance receptor complex that contains the receptor of IL-6 (IL-6R) and a regulating receptor gp130. Briefly, IL-6 binds to IL-6R then combines the receptor gp130, causing the activation of Janus tyrosine kinase (JAK) (
Although, previous studies have shown that an increased levels of serum IL-6 was found to be negatively correlated with survival in patients with gastric carcinoma (
Therefore, we performed the present study to investigate various gastric cancer cell lines expressing IL-6R and gp130. Exogenous IL-6 stimulation of the gastric cancer cells was used in order to determine the effects of IL-6 on gastric cancer proliferation, invasion and metastasis, as well as the underlying mechanisms involved.
The gastric cancer cell lines SGC-7901, MGC, MKN-28 and AGS were provided by the Key Laboratory of the Ministry of Eduction for Gastrointestinal Cancer, Fujian Medical Universtiy, Fuzhou, Fujian, China. All the cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (both from Gibco, Carlsbad, CA, USA). Human dermal lymphatic endothelial cells (HDLECs, purchased from Sciencell, San Diego, CA, USA) were incubated in endothelial cell medium (Sciencell). All the cells were incubated at 37°C under 95% air and 5% CO2.
Total RNA was extracted from the gastric cancer cells grown in 6-well plates using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's. The RNA was quantified by its absorption at 260 nm and stored at −80°C. cDNA was synthesized using the AVM First Strand cDNA synthesis kit (Invitrogen). The primers for IL-6R, gp130 and β-actin were synthesized according to primer design priciples. The sequences of the primers of IL-6R were: forward, 5′-AGACAGCGTGACTCTGACCT-3′ and reverse, 5′-ACTACTGGCGACGCACAT-3′ and yielded a 472-bp product. Gp130 yielded a 460-bp product, and the sequences of the primers were as follows: forward, 5′-TCGTGCCTGTTTGCTTGA-3′ and reverse, 5′-TGGACTGACGGAACTTGG-3′; β-actin yielded a 254-bp product, as an internal control, and the sequences of the primers were as follows: forward, 5′-CTGTCTGGCGGCACCACCAT-3′ and reverse, 5′-GCAACTAAGTCATAGTCCGC-3′. Briefly, RT-PCR was performed under the following conditions: 30 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1 min followed by 10 min for final extension at 72°C. The data of IL-6R and gp130 were normalized relative to the expression of β-actin mRNA expression in the respective samples.
Cells were washed three times with ice cold PBS and then incubated on ice with 250
The effects of IL-6 on gastric cell proliferation were assessed using an MTT assay. Cells were seeded in 96-well plates at a density of 2.5×103 cells/well and allowed to attach for 12 h. The cells were then treated by different concentrations of IL-6 (1, 10, 50 and 100 ng/ml) with or without AG490 (50
The invasion assay was performed using Transwell 24-well plates with 8-
To analyze IL-6 promoting VEGF-C secretion in the gastric cancer cells, the gastric cancer cells were seeded into plates at a density of 3×106 cells/well with 4 ml of RPMI-1640 medium supplemented with 10% FBS. The gastric cancer cells were treated with 100 ng IL-6 or 100 ng IL-6+AG490 (50
To test the gastric cancer cell culture supernatants promoting HDLEC cells the ability of tube-formation, we used the IL-6 (100 ng) and IL-6 (100 ng+AG490) to treat the gastric cancer cells and collected the gastric cancer cell culture supernatants for 24, 48 and 72 h. Subsequently, we used the supernatants to culture the HDLECs and observe the role of IL-6 and IL-6+AG490 in the tube formation of HDLECs. The phenomenon of tube formation was observed 1, 3, 5, 8 and 24 h after culture. Images were digitally captured at 6 h after plating. All tube formation experiments were observed by a microscope. Each small tube was counted as 1, 1 branch as 2, 2 branches as 3, by parity of reasoning. Experiments were repeated 3 times, calculating the average of the tube numbers.
Data were analyzed using GraphPad Prism 5 software. Analysis of variance was conducted followed by one-way ANOVA or an unparied t-test. The data are expressed as the means ± standard deviation (SD). A P-value <0.05 was considered to indicate a statistically significant difference.
We analyzed the mRNA expression of IL-6R and gp130 in the gastric cancer cell lines AGS, SGC-7901, MKN-28 and MGC-803. Using RT-PCR, the mRNA expression was detected of IL-6R and gp130 in all gastric cancer cell lines (
We tested the IL-6R and gp130 protein expression in the gastric cancer cell lines (AGS, SGC-7901, MKN-28 and MGC-803) by western blot analysis. As expected, the protein expression of IL-6R and gp130 was observed in all cell lines (
To determine whether the exogenous IL-6 promotes gastric cancer cell proliferation, we first treated the AGS and SGC-7901 cells with different concentrations of IL-6 (1, 10, 50 and 100 ng/ml) with or without AG490 (50
To examine the effect of IL-6 on AGS and SGC-7901 cell invasion, we used Transwell assay to determine the effects of exogenous IL-6 treated gastric cancer cells. We counted the number of invaded cells. The total number of cells in the AGS+IL-6 group that invaded through the Transwell polycarbonate filter was significantly higher than that of cells in the AGS group, which was lower than the number of cells in the AGS+IL-6+AG490 group (
To investigate the mechanisms of IL-6 responsible for the increase in proliferation and invasion, we assessed the changes in JAK, STAT3, p-STAT3 and VEGF-C levels. The JAK-STAT3 signal pathway is vital to the survival of gastric cancer cells. The western blot analysis results indicated that the JAK, STAT3, p-STAT3 and VEGF-C protein levels in the AGS+IL-6 cells increased in comparison to the AGS+IL-6+AG490 cells, which was higher than the protein levels in the AGS cells (
We analyzed the IL-6 effect of VEGF-C secretion levels in the AGS cells and SGC-7901 cell culture supernatants. ELISA analysis revealed that IL-6 marked increased VEGF-C secretion levels in the AGS cells and SGC-7901 cell culture supernatants, when compared to the contorl group. Moreover, AG490 significantly suppressed the VEGF-C secretion levels in the AGS cells and SGC-7901 cell culture supernatants, when compared to the IL-6 group (
After confirming IL-6 induced secretion of VEGF-C in the AGS cells and SGC-7901 cell culture supernatants, we further analyzed the role of the IL-6 in the tube formation of HDLECs. The number of tubes was observed by a microscope. In the tube formation experiment, the HDLECs cultured had formed a few tubes 2–6 h after seeding. Thus, we decided to observe the tube formation for 6 h in our experiment. In the treatment with IL-6, the tube number of HDLECs increased obviously, and adding to the inhibition by AG490 of JAK-STAT3 signal pathway, the tube number of IL-6+AG490 groups were suppressed in the AGS and SGC-7901 cells (
IL-6 has been described as a factor causing differentiation of B cells to plasma cells, and is an important inflammation cytokine (
The present study demonstrated the role of exogenous IL-6 in promoting the proliferation, invasion and metastasis of human gastric cancer cells. We further investigated the possible mechanisms underlying this process. We determined whether IL-6R and gp130 were expressed in the gastric cancer cell lines AGS, SGC-7901, MKN-28 and MGC-803. We found the mRNA and protein of IL-6R and gp130 expression in all gastric cancer cell lines, and the mRNA and protein of IL-6R and gp130 expression in the AGS and SGC-7901 cell lines were higher compared to the levels in the MKN-28 and MGC-803 cell lines. The different mRNA and protein of IL-6R and gp130 expression in different gastric cancer cell lines have also been reported by Matsuo
We examined cell proliferation at 24, 48 and 72 h by MTT assay after IL-6 was added to the gastric cancer cell culture plate. Compared with the Control and IL-6+AG490 groups, cell proliferation in the IL-6 group was more rapid, and the cell proliferation in the IL-6+AG490 group was slower than that in the IL-6 group. The data from the
Many studies show that IL-6 promotes tumor cell proliferation, growth (
In view of the foregoing, we speculated that IL-6 could promote VEGF-C production in gastric cancer cells and the JAK-STAT3 plays an important role in the process. In this study, we assessed the changes in JKA, STAT3, p-STAT3 and VEGF-C protein expression. The JAK-STAT3 signal pathway and VEGF-C are vital to the survival of cancer cells (
In the present study, we verified the biological functions of IL-6 on various gastric cancer cell lines. We provided evidence that IL-6 can promote the gastric cancer cell proliferation, invasion and lymphangiogenesis
The present study was supported by the Key Project of Science and Technology Reserch Program in Fujian Province (no. 2012B002), the Fujian Provincial Natrual Science Foundation (no. 2014J01309), the Backbone Teacher Project of Fujian Medical University (no. JGG200716), the China Non intervention Gastric Cancer Registration Survey Clinical Research Projects (no. QT-201403), the Ministry of Health Medicine Science and Technology Development and Research (no. W2013FZ08) and the National Clinical Key Specialty Construction Project (General Surgery) of China.
mRNA expression of IL-6R and gp130 in the gastric cancer cell lines SGC-7901, MGC, MKN-28 and AGS cells. (A and B) The IL-6R and gp130 mRNA expression levels in the AGS and SGC-7901 cells was higher than that in the MKN-28 and MGC-803 cells. The mRNA levels of IL-6R and gp130 expression were similar between the AGS and SGC-7901 cells. The mRNA expression of β-actin was used as an internal control. (B and D) The densitometric value for both groups were normalized to the internal control and relative expression with the following equation: the mRNA expression of IL-6R and gp130 in MKN-28 cells were taken as 1. Data represent the mean ± standard deviation (SD), n=3; *P<0.05.
Protein expression of (A) IL-6R and gp130 in the gastric cancer cell lines SGC-7901, MGC, MKN-28 and AGS cells detected by western blot analysis. The IL-6R and gp130 protein expression was semiquantified by western blot analysis. The protein expression of β-actin was used as an internal control. (B and C) The densitometric value for both groups were normalized to the internal control and relative expression with the following equation: the protein expression of IL-6R and gp130 in AGS cells were taken as 1. Data represent the mean ± standard deviation (SD), n=3; *P<0.05.
Effects of IL-6 and AG490 which is the specific inhibitor of JAK-STAT3 signal pathway on the proliferation of AGS and SGC-7901 cells. The effects of IL-6 and AG490 on the ability of proliferation of AGS and SGC-7901 cells were analyzed by MTT assay. The proliferation of AGS and SGC-7901 cells were significantly increased in a concentration- and time-dependent manner after IL-6 stimulation (A and B). The proliferative effects of IL-6 were abolished by AG490 treatment in the AGS and SGC-7901 cells (C and D). *P<0.05.
Effects of IL-6 and AG490 which is the specific inhibitor of JAK-STAT3 signal pathway on the invasive ability of AGS and SGC-7901 cells. The effects of IL-6 and AG490 on the invasive ability of AGS and SGC-7901 cells were analyzed by Transwell assay. (A) Invasion ability of AGS cells were significantly increased with IL-6 stimulation. The invasion effects of IL-6 were abolished by AG490 treatment in the AGS cells. (C) Number of invaded cells in AGS-control, AGS+IL-6 and AGS-IL-6+AG490 groups. (B and D) The SGC-7901 cells, and the effects of IL-6 and AG490 on the invasive ability of SGC-7901 is similar to AGS cells (magnification, ×400). *P<0.05.
AG490 inhibits the activity of the IL-6-JAK-STAT3-VEGF-C signal pathway. (A) Western blot analysis suggested that the IL-6 markedly promoted the expression of JAK, STAT3, p-STAT3 and VEGF-C protein. The promoting effects of IL-6 were abolished by AG490 treatment in the AGS cells. β-actin protein was used as an internal control. The densitometric value for all groups were normalized to the internal control and relative expression with the protein expression of JAK (B), STAT3 (C), p-STAT3 (D) and VEGF-C (E) in AGS group cells were taken as 1. Results are expressed as mean ± standard deviation (SD), n=3; *P<0.05.
ELISA analysis of the IL-6 effect of VEGF-C secretion levels in the AGS cells and SGC-7901 cell culture supernatants. The analysis revealed that IL-6 markedly increased VEGF-C secretion levels in the AGS cells and SGC-7901 cell culture supernatants, when compared to the control group (A and B). AG490 significantly suppressed the VEGF-C secretion levels in the AGS and SGC-7901 cell culture supernatants, when compared to the IL-6 group (A and B). *P<0.05.
Analysis of the role of IL-6 in the tube formation of HDLECs. The number of tubes was observed by a microscope for 6 h. In the treatment with IL-6, the tube number of HDLECs increased obviously, adding to the inhibition (AG490) of JAK-STAT3 signal pathway, the tube number of IL-6+AG490 groups were suppressed in the AGS cells and SGC-7901 cells (A and C refer to AGS cells; B and D refer to SGC-7901 cells). (Original magnification, ×400). *P<0.05.