17β-estradiol inhibits mesenchymal stem cells-induced human AGS gastric cancer cell mobility via suppression of CCL5- Src/Cas/Paxillin signaling pathway

Abstract

Gender differences in terms of mortality among many solid organ malignancies have been proved by epidemiological data. Estrogen has been suspected to cast a protective effect against cancer because of the lower mortality of gastric cancer in females and the benefits of hormone replacement therapy (HRT) in gastric cancer. Hence, it suggests that 17β-estradiol (E2) may affect the behavior of cancer cells. One of the key features of cancer-related mortality is metastasis. Accumulating evidences suggest that human bone marrow mesenchymal stem cells (HBMMSCs) and its secreted CCL-5 have a role in enhancing the metastatic potential of breast cancer cells. However, it is not clear whether E2 would affect HBMMSCs-induced mobility in gastric cancer cells. In this report, we show that CCL-5 secreted by HBMMSCs enhanced mobility in human AGS gastric cancer cells via activation of Src/Cas/Paxillin signaling pathway. Treatment with specific neutralizing antibody of CCL-5 significantly inhibited HBMMSCs-enhanced mobility in human AGS gastric cancer cells. We further observe that 17β-estradiol suppressed HBMMSCs-enhanced mobility by down-regulating CCL5-Src/Cas/paxillin signaling pathway in AGS cells. Collectively, these results suggest that 17β-estradiol treatment significantly inhibits HBMMSCS-induced mobility in human AGS gastric cancer cells.

Keywords: CCL-5; Cell mobility; Estrogen; Human gastric cancer cell; Mesenchymal stem cell.

Fig 1

Inhibitory effect of CCL-5 neutralizing antibody on HBMMSCs-induced human AGS cell mobility. HBMMSCs (5x10⁴) and human AGS cells (5x10⁴) were co-cultured with/without CCL-5 neutralizing antibody. The effect of CCL-5 secreted from HBMMSCs on mobility of AGS gastric cancer cells was measured. The responses to different concentration of CCL-5 neutralizing antibody treatment were measured by the mobility assay. **, p<0.01 versus control (line 1); #, p<0.05 and ##, p<0.01 versus only HBMMSCs co-culture (line 2) (mean ± SD, n = 3).

Fig 2

Increased CCL-5 expression by HBMMSCs in AGS/HBMMSC co-culture system. (A) Enzyme-linked immunosorbent assay for CCL-5 concentration in AGS cells (5x10⁴) alone, AGS cells (5x10⁴)/HBMMSCs(5x10⁴), and HBMMSCs(5x10⁴). (B) Quantitative reverse transcription PCR for relative CCL-5 mRNA level to beta-actin in AGS cells alone, AGS cells in co-culture, HBMMSCs in co-culture, and HBMMSCs alone. (C) The effect of CCL-5 from supernatant of HBMMSCs on mobility of AGS gastric cancer cells was measured. **, p<0.01 versus control; #, p<0.05 and ##, p<0.01 versus only HBMMSCs co-culture (line 2) (mean ± SD, n = 3).

Fig 2

Increased CCL-5 expression by HBMMSCs in AGS/HBMMSC co-culture system. (A) Enzyme-linked immunosorbent assay for CCL-5 concentration in AGS cells (5x10⁴) alone, AGS cells (5x10⁴)/HBMMSCs(5x10⁴), and HBMMSCs(5x10⁴). (B) Quantitative reverse transcription PCR for relative CCL-5 mRNA level to beta-actin in AGS cells alone, AGS cells in co-culture, HBMMSCs in co-culture, and HBMMSCs alone. (C) The effect of CCL-5 from supernatant of HBMMSCs on mobility of AGS gastric cancer cells was measured. **, p<0.01 versus control; #, p<0.05 and ##, p<0.01 versus only HBMMSCs co-culture (line 2) (mean ± SD, n = 3).

Fig 2

Increased CCL-5 expression by HBMMSCs in AGS/HBMMSC co-culture system. (A) Enzyme-linked immunosorbent assay for CCL-5 concentration in AGS cells (5x10⁴) alone, AGS cells (5x10⁴)/HBMMSCs(5x10⁴), and HBMMSCs(5x10⁴). (B) Quantitative reverse transcription PCR for relative CCL-5 mRNA level to beta-actin in AGS cells alone, AGS cells in co-culture, HBMMSCs in co-culture, and HBMMSCs alone. (C) The effect of CCL-5 from supernatant of HBMMSCs on mobility of AGS gastric cancer cells was measured. **, p<0.01 versus control; #, p<0.05 and ##, p<0.01 versus only HBMMSCs co-culture (line 2) (mean ± SD, n = 3).

Fig 3

Recombinant CCL-5 increases human AGS cell mobility. Human AGS cells were treated with recombinant CCL-5 (1, 10, 20, 50 and 100 ng/ml) for 24h, and subsequently measured the capacity of cell mobility. The responses to different concentration of CCL-5 treatment were measured by mobility assay. **, p<0.01 versus control (line 1) (mean ± SD, n = 3).

Fig 4

CCL-5 induces AGS cell mobility via Src signaling pathway. (A) AGS cells were pretreated with vehicle, LY294002 (Akt activation inhibitor, 1μM), U0126 (ERK1/2 activation inhibitor, 1μM), SB203580 (p38 MAPK inhibitor, 1μM), SP600125 (JNK1/2 inhibitor, 1μM) or PP2 (Src inhibitor, 1μM) for 1h, and followed by recombinant CCL-5 (10 ng/ml) administration for 24h. (B) AGS cells were treated with these inhibitors, respectively. AGS cells were harvested and measured for cellular mobility capacity. **, p<0.01 versus control (line 1); ##, p<0.01 versus CCL-5 treatment (line 2) (mean ± SD, n = 3).

Fig 4

CCL-5 induces AGS cell mobility via Src signaling pathway. (A) AGS cells were pretreated with vehicle, LY294002 (Akt activation inhibitor, 1μM), U0126 (ERK1/2 activation inhibitor, 1μM), SB203580 (p38 MAPK inhibitor, 1μM), SP600125 (JNK1/2 inhibitor, 1μM) or PP2 (Src inhibitor, 1μM) for 1h, and followed by recombinant CCL-5 (10 ng/ml) administration for 24h. (B) AGS cells were treated with these inhibitors, respectively. AGS cells were harvested and measured for cellular mobility capacity. **, p<0.01 versus control (line 1); ##, p<0.01 versus CCL-5 treatment (line 2) (mean ± SD, n = 3).

Fig 5

Inhibition of HBMMSCs-induced cellular mobility by 17β-estradiol in human AGS gastric cancer cells. HBMMSCs (5x10⁴) and human AGS cells (5x10⁴) were co-culture with/without 17β-estradiol (10^-8M) treatment for 24h and 48h. The effect of 17β-estradiol on HBMMSCs-induced cellular mobility in human AGS gastric cancer cells was measured. The effect of 17β-estradiol (10^-8M) alone on mobility and CCR5 protein expression in AGS cells was measured by cell migration assay and immunoblotting. **, p<0.01 versus control; ##, p<0.01 versus only HBMMSCs co-culture (mean ± SD, n = 3).

Fig 6

17β-estradiol down-regulates CCL-5-induced mobility activity by suppressing Src/Cas/Paxillin activation in human AGS cells. Human AGS cells were pre-treated with 17β-estradiol (10^-8 and 10^-9 M) for 1h, and followed by CCL-5 (1 ng/ml) treatment for 24h. AGS cells were then harvested for immunoblotting assay (A), and measuring the capacity of cell mobility (B). **, p<0.01 versus control (line 1); ##, p<0.01 versus CCL-5 treatment (line 2) (mean ± SD, n = 3).

Fig 7

A schematic representation showing 17β-estradiol inhibition of cell mobility via suppression of CCL5-Src/Cas/Paxillin pathways in human AGS gastric cancer cells. CCL-5 is mainly secreted from HBMMSCs, which is great enhanced when co-culturing with AGS cells. Soluble CCL-5 induces the activation of Src, Cas and paxillin, then contributing to the changes in organization of cellular actin cytoskeleton, which thus promotes cell mobility events in human AGS gastric cancer cells. Treatment of 17β-estradiol significantly inhibits HBMMSCs-induced cell mobility by suppressing activation of CCL5-Src/Cas/paxillin signaling pathway.