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Bacteria peptidoglycan promoted breast cancer cell invasiveness and adhesiveness by targeting toll-like receptor 2 in the cancer cells.

Xie W, Huang Y, Xie W, Guo A, Wu W - PLoS ONE (2010)

Bottom Line: All these effects were abrogated by TLR2 blockade.Further investigation showed that the NF-kappaB, STAT3 and Smad3 activities were augmented sequentially in MDA-MB-231 cells after PGN-SA stimulation.NF-kappaB inhibition attenuated STAT3 and Smad3 activities whereas PGN-SA-stimulated cell culture supernatants reversed these inhibitory effects.

View Article: PubMed Central - PubMed

Affiliation: Biology Research Institute of the United Laboratories International Holdings Limited, Zhuhai, China. xiewj75@126.com

ABSTRACT
Chronic bacterial infection increased the risk of many solid malignancies and the underlying mechanism is usually ascribed to bacterial-caused inflammation. However, the direct interaction of infectious bacteria with cancer cells has been largely overlooked. We identified that highly metastatic breast cancer MDA-MB-231 cells expressed high level of Toll-like receptor 2 (TLR2) in contrast to poorly metastatic breast cancer cells and homogenous untransformed breast cells. TLR2 in MDA-MB-231 cells were actively triggered by peptidoglycan (PGN) from infectious bacterium Staphylococcus aureus (PGN-SA), resulting in the promoted invasiveness and adhesiveness of the cancer cells in vitro. PGN-SA induced phosphorylation of TAK1 and IkappaB in the TLR2-NF-kappaB pathway of the cancer cells and stimulated IL-6 and TGF-beta secretion in MDA-MB-231 cells. All these effects were abrogated by TLR2 blockade. Further investigation showed that the NF-kappaB, STAT3 and Smad3 activities were augmented sequentially in MDA-MB-231 cells after PGN-SA stimulation. Phosphorylation of NF-kappaBp65 was initially increased and then followed by phosphorylation of STAT3 and Smad3 in the delayed 4 or 6 hours. NF-kappaB inhibition attenuated STAT3 and Smad3 activities whereas PGN-SA-stimulated cell culture supernatants reversed these inhibitory effects. Our study indicated that TLR2 activation by infectious bacterial PGN played an important role in breast cancer cell invasiveness and illustrated a new link between infectious bacteria and the cancer cells, suggesting the importance of antibiotic therapy to treat cancer with bacterial infection.

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PGN-SA promoted invasiveness and adhesiveness via TLR2 in MDA-MB-231 cells.(A) The bottoms of the top cells in Transwell setting were all coated with a thin layer of matrigel and only the cells with invasive capacity could migrate through the matrigel layer and 8.0 µm pore. IgG (10 µg/ml) treatment was a antibodies control. TLR2 activation by PGN-SA (1 µg/ml) increased the invasive cell number of breast cancer cell lines whereas TLR2 inhibition by anti-TLR2 neutralizing antibody (10 µg/ml) attenuated the invasive cells. (B) The culture dishes were pre-coated with 80 µl of fibronectin (2.5 mg/ml). The adhesive cancer cells stained with 0.05% toluidine blue solution. PGN-SA (1 µg/ml) enhanced MDA-MB-231 cell adhesion to fibronectin layer whereas anti-TLR2 neutralizing antibody (10 µg/ml) abrogated PGN-stimulated adhesive ability. Values were expressed as mean ± SD and in vitro experiments, one representative of six independent is shown (n = 6 *p<0.05; **p<0.01).
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pone-0010850-g002: PGN-SA promoted invasiveness and adhesiveness via TLR2 in MDA-MB-231 cells.(A) The bottoms of the top cells in Transwell setting were all coated with a thin layer of matrigel and only the cells with invasive capacity could migrate through the matrigel layer and 8.0 µm pore. IgG (10 µg/ml) treatment was a antibodies control. TLR2 activation by PGN-SA (1 µg/ml) increased the invasive cell number of breast cancer cell lines whereas TLR2 inhibition by anti-TLR2 neutralizing antibody (10 µg/ml) attenuated the invasive cells. (B) The culture dishes were pre-coated with 80 µl of fibronectin (2.5 mg/ml). The adhesive cancer cells stained with 0.05% toluidine blue solution. PGN-SA (1 µg/ml) enhanced MDA-MB-231 cell adhesion to fibronectin layer whereas anti-TLR2 neutralizing antibody (10 µg/ml) abrogated PGN-stimulated adhesive ability. Values were expressed as mean ± SD and in vitro experiments, one representative of six independent is shown (n = 6 *p<0.05; **p<0.01).

Mentions: Metastatic process involves several critical steps such as invasion and adhesion[23]. To investigate the effects of TLR2 on the metastatic process of the breast cancer cell lines, the Transwell experiment for invasion assay was set up and only highly invasive cells can invade through the matrigel layer and migrate through the 8.0 µm holes[21]. Among the three breast cancer cell lines, MDA-MB-231 cells showed highest capacity in invasion (Fig. 2A). Utilizing TLR2 agonist PGN-SA (1 µg/ml) to activate TLR2 and anti-TLR2 neutralizing antibody to block TLR2, we found that in MDA-MB-231 cells, PGN-SA stimulation promoted more than 2-fold invasive capacity than the unthreatment (Fig. 2A). On the contrary, anti-TLR2 neutralizing antibody notably attenuated the number of invasive breast cancer cells (Fig. 2A). Adhesion to extracellular matrix (ECM) is an important capacity for cancer cell to anchor on the matrix[23]. We deploy fibronectin, an important component of ECM, to investigate the effect of TLR2 activation on MDA-MB-231 cell adhesiveness to this kind matrix. PGN-SA significantly enhanced the adhesion of MDA-MB-231 cells whereas there is no such a similar effect in MCF-7 cells by PGN-SA stimulation (Fig. 2B).


Bacteria peptidoglycan promoted breast cancer cell invasiveness and adhesiveness by targeting toll-like receptor 2 in the cancer cells.

Xie W, Huang Y, Xie W, Guo A, Wu W - PLoS ONE (2010)

PGN-SA promoted invasiveness and adhesiveness via TLR2 in MDA-MB-231 cells.(A) The bottoms of the top cells in Transwell setting were all coated with a thin layer of matrigel and only the cells with invasive capacity could migrate through the matrigel layer and 8.0 µm pore. IgG (10 µg/ml) treatment was a antibodies control. TLR2 activation by PGN-SA (1 µg/ml) increased the invasive cell number of breast cancer cell lines whereas TLR2 inhibition by anti-TLR2 neutralizing antibody (10 µg/ml) attenuated the invasive cells. (B) The culture dishes were pre-coated with 80 µl of fibronectin (2.5 mg/ml). The adhesive cancer cells stained with 0.05% toluidine blue solution. PGN-SA (1 µg/ml) enhanced MDA-MB-231 cell adhesion to fibronectin layer whereas anti-TLR2 neutralizing antibody (10 µg/ml) abrogated PGN-stimulated adhesive ability. Values were expressed as mean ± SD and in vitro experiments, one representative of six independent is shown (n = 6 *p<0.05; **p<0.01).
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pone-0010850-g002: PGN-SA promoted invasiveness and adhesiveness via TLR2 in MDA-MB-231 cells.(A) The bottoms of the top cells in Transwell setting were all coated with a thin layer of matrigel and only the cells with invasive capacity could migrate through the matrigel layer and 8.0 µm pore. IgG (10 µg/ml) treatment was a antibodies control. TLR2 activation by PGN-SA (1 µg/ml) increased the invasive cell number of breast cancer cell lines whereas TLR2 inhibition by anti-TLR2 neutralizing antibody (10 µg/ml) attenuated the invasive cells. (B) The culture dishes were pre-coated with 80 µl of fibronectin (2.5 mg/ml). The adhesive cancer cells stained with 0.05% toluidine blue solution. PGN-SA (1 µg/ml) enhanced MDA-MB-231 cell adhesion to fibronectin layer whereas anti-TLR2 neutralizing antibody (10 µg/ml) abrogated PGN-stimulated adhesive ability. Values were expressed as mean ± SD and in vitro experiments, one representative of six independent is shown (n = 6 *p<0.05; **p<0.01).
Mentions: Metastatic process involves several critical steps such as invasion and adhesion[23]. To investigate the effects of TLR2 on the metastatic process of the breast cancer cell lines, the Transwell experiment for invasion assay was set up and only highly invasive cells can invade through the matrigel layer and migrate through the 8.0 µm holes[21]. Among the three breast cancer cell lines, MDA-MB-231 cells showed highest capacity in invasion (Fig. 2A). Utilizing TLR2 agonist PGN-SA (1 µg/ml) to activate TLR2 and anti-TLR2 neutralizing antibody to block TLR2, we found that in MDA-MB-231 cells, PGN-SA stimulation promoted more than 2-fold invasive capacity than the unthreatment (Fig. 2A). On the contrary, anti-TLR2 neutralizing antibody notably attenuated the number of invasive breast cancer cells (Fig. 2A). Adhesion to extracellular matrix (ECM) is an important capacity for cancer cell to anchor on the matrix[23]. We deploy fibronectin, an important component of ECM, to investigate the effect of TLR2 activation on MDA-MB-231 cell adhesiveness to this kind matrix. PGN-SA significantly enhanced the adhesion of MDA-MB-231 cells whereas there is no such a similar effect in MCF-7 cells by PGN-SA stimulation (Fig. 2B).

Bottom Line: All these effects were abrogated by TLR2 blockade.Further investigation showed that the NF-kappaB, STAT3 and Smad3 activities were augmented sequentially in MDA-MB-231 cells after PGN-SA stimulation.NF-kappaB inhibition attenuated STAT3 and Smad3 activities whereas PGN-SA-stimulated cell culture supernatants reversed these inhibitory effects.

View Article: PubMed Central - PubMed

Affiliation: Biology Research Institute of the United Laboratories International Holdings Limited, Zhuhai, China. xiewj75@126.com

ABSTRACT
Chronic bacterial infection increased the risk of many solid malignancies and the underlying mechanism is usually ascribed to bacterial-caused inflammation. However, the direct interaction of infectious bacteria with cancer cells has been largely overlooked. We identified that highly metastatic breast cancer MDA-MB-231 cells expressed high level of Toll-like receptor 2 (TLR2) in contrast to poorly metastatic breast cancer cells and homogenous untransformed breast cells. TLR2 in MDA-MB-231 cells were actively triggered by peptidoglycan (PGN) from infectious bacterium Staphylococcus aureus (PGN-SA), resulting in the promoted invasiveness and adhesiveness of the cancer cells in vitro. PGN-SA induced phosphorylation of TAK1 and IkappaB in the TLR2-NF-kappaB pathway of the cancer cells and stimulated IL-6 and TGF-beta secretion in MDA-MB-231 cells. All these effects were abrogated by TLR2 blockade. Further investigation showed that the NF-kappaB, STAT3 and Smad3 activities were augmented sequentially in MDA-MB-231 cells after PGN-SA stimulation. Phosphorylation of NF-kappaBp65 was initially increased and then followed by phosphorylation of STAT3 and Smad3 in the delayed 4 or 6 hours. NF-kappaB inhibition attenuated STAT3 and Smad3 activities whereas PGN-SA-stimulated cell culture supernatants reversed these inhibitory effects. Our study indicated that TLR2 activation by infectious bacterial PGN played an important role in breast cancer cell invasiveness and illustrated a new link between infectious bacteria and the cancer cells, suggesting the importance of antibiotic therapy to treat cancer with bacterial infection.

Show MeSH
Related in: MedlinePlus