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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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TLR2 was differentially expressed in breast cancer cells and untransformed breast cells.(A and B) Surface expression of TLR2 in four cell lines was analyzed by direct immunofluorescence using flow cytometer and IgG was used as control antibodies. The differences of TLR2 expression levels in breast cancer MCF-7, MDA-MB-468 and MDA-MB-231 cells were significant and TLR2 in untransformed MCF-10A cells was nearly undetectable. (C) TLR2 mRNA levels in the cell lines were measured by real-time PCR. RNA yield differences were calculated using GAPDH as an endogenous control and the comparative threshold cycle (Ct value). MDA-MB-231 cells showed the lowest Ct value among these cell lines. (D) TLR2 protein levels were analyzed by Western-blotting. Values were expressed as mean ± SD (n = 6 *p<0.05; **p<0.01).
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pone-0010850-g001: TLR2 was differentially expressed in breast cancer cells and untransformed breast cells.(A and B) Surface expression of TLR2 in four cell lines was analyzed by direct immunofluorescence using flow cytometer and IgG was used as control antibodies. The differences of TLR2 expression levels in breast cancer MCF-7, MDA-MB-468 and MDA-MB-231 cells were significant and TLR2 in untransformed MCF-10A cells was nearly undetectable. (C) TLR2 mRNA levels in the cell lines were measured by real-time PCR. RNA yield differences were calculated using GAPDH as an endogenous control and the comparative threshold cycle (Ct value). MDA-MB-231 cells showed the lowest Ct value among these cell lines. (D) TLR2 protein levels were analyzed by Western-blotting. Values were expressed as mean ± SD (n = 6 *p<0.05; **p<0.01).

Mentions: Previous report pointed that mouse hepatoma H22 cells expressed TLR2 receptor involved in tumor growth by listeria monocytogenes stimulation[8]. We sought to determine the effect of TLR2 stimulation with infectious bacteria on breast tumor invasiveness and adhesiveness. Therefore, we initially detected TLR2 expression levels of selected breast cancer cell lines. By flow cytometer detection, we found TLR2 was differentially expressed in MCF-10A, MCF-7, MDA-MB-468 and MDA-MB-231. Qualitative PCR analysis indicated the similar result. TLR2 protein level in MDA-MB-231 cells was much higher than the other cell lines (Fig. 1A and B). Real-time PCR analysis showed the number of threshold cycles (Ct) of MDA-MB-231 was the least among those of other three cell lines (Fig. 1C). These results were confirmed by western blot analysis (Fig. 1D). Since each of these cell lines is distinguished in invasive and metastatic capacities, differential expression of TLR2 in those cell lines might be related to their metastatic capacity.


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)

TLR2 was differentially expressed in breast cancer cells and untransformed breast cells.(A and B) Surface expression of TLR2 in four cell lines was analyzed by direct immunofluorescence using flow cytometer and IgG was used as control antibodies. The differences of TLR2 expression levels in breast cancer MCF-7, MDA-MB-468 and MDA-MB-231 cells were significant and TLR2 in untransformed MCF-10A cells was nearly undetectable. (C) TLR2 mRNA levels in the cell lines were measured by real-time PCR. RNA yield differences were calculated using GAPDH as an endogenous control and the comparative threshold cycle (Ct value). MDA-MB-231 cells showed the lowest Ct value among these cell lines. (D) TLR2 protein levels were analyzed by Western-blotting. Values were expressed as mean ± SD (n = 6 *p<0.05; **p<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2877101&req=5

pone-0010850-g001: TLR2 was differentially expressed in breast cancer cells and untransformed breast cells.(A and B) Surface expression of TLR2 in four cell lines was analyzed by direct immunofluorescence using flow cytometer and IgG was used as control antibodies. The differences of TLR2 expression levels in breast cancer MCF-7, MDA-MB-468 and MDA-MB-231 cells were significant and TLR2 in untransformed MCF-10A cells was nearly undetectable. (C) TLR2 mRNA levels in the cell lines were measured by real-time PCR. RNA yield differences were calculated using GAPDH as an endogenous control and the comparative threshold cycle (Ct value). MDA-MB-231 cells showed the lowest Ct value among these cell lines. (D) TLR2 protein levels were analyzed by Western-blotting. Values were expressed as mean ± SD (n = 6 *p<0.05; **p<0.01).
Mentions: Previous report pointed that mouse hepatoma H22 cells expressed TLR2 receptor involved in tumor growth by listeria monocytogenes stimulation[8]. We sought to determine the effect of TLR2 stimulation with infectious bacteria on breast tumor invasiveness and adhesiveness. Therefore, we initially detected TLR2 expression levels of selected breast cancer cell lines. By flow cytometer detection, we found TLR2 was differentially expressed in MCF-10A, MCF-7, MDA-MB-468 and MDA-MB-231. Qualitative PCR analysis indicated the similar result. TLR2 protein level in MDA-MB-231 cells was much higher than the other cell lines (Fig. 1A and B). Real-time PCR analysis showed the number of threshold cycles (Ct) of MDA-MB-231 was the least among those of other three cell lines (Fig. 1C). These results were confirmed by western blot analysis (Fig. 1D). Since each of these cell lines is distinguished in invasive and metastatic capacities, differential expression of TLR2 in those cell lines might be related to their metastatic capacity.

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