RESUMO
BACKGROUND: Many types of tumors are organized in a hierarchy of heterogeneous cell populations with different molecular signature. Such heterogeneity may be associated with different responsiveness to microenvironment stimuli. In the present study, the effects of lipopolysaccharide (LPS) and lipoteichoic acid (LTA), as well-known mediators of inflammation, on cancerous behavior of three prostate tumor cells, LNCaP, PC3 and DU145, were investigated. METHODS: Expression of TLR1-10, CD14 and MyD88 transcripts was investigated by RT-PCR. Protein expression of TLR2 and 4 was scrutinized by flow cytometry, immunofluorescent staining and Western blotting. Experiments were set up to assess the effects of LPS and LTA at different concentrations and times on cell proliferation, extracellular matrix invasion, adhesion and cytokine production. RESULTS: We showed that prostate cancer cell lines differentially express TLR1-10, MyD88 and CD14 transcripts. DU145 failed to express TLR4 gene. Positively-identified TLR2 protein in all prostate cancer cells and TLR4 protein in PC3 and LNCaP by Western blotting was not accompanied by cell surface expression, as judged by flow cytometry. Immunofluorescent staining clearly demonstrated predominantly perinuclear localization of TLR2 and TLR4. LTA activation of all prostate cancer cells significantly increased cell proliferation. Regardless of lacking TLR4, DU145 cells proliferated in response to LPS treatment. While LPS caused increased invasiveness of LNCaP, invasive capacity of PC3 was significantly reduced after LPS or LTA stimulation. Stimulation of all prostate tumor cells with LTA was associated with increased cell adhesion and IL-8 production. IL-6 production, however, was differentially regulated by LPS stimulation in prostate tumor cells. CONCLUSION: The data shows that cancer cells originated from the same histologically origin exhibit heterogeneous response to the same TLR ligand. Therefore, a thorough and comprehensive judgment on how and to what extent a particular cancer is affected by TLR agonist could not be inferred by studying an individual cell line.
RESUMO
Lipopolysaccharide (LPS) is an important structural component of the outer cell membrane complex of gram negative microorganisms. Its causative role in gram negative bacteria-induced diseases and broad applications in different kinds of cell stimulation experiments provided a conceptual basis for studies directed at the isolation, purification, and detailed chemical characterization of LPS. The main problem with LPS purification protocols is the contamination of the end product with nucleic acids and proteins in variable proportions which could potentially interfere with downstream applications. In this study, a simple procedure for purification of LPS from Escherichia coli (E.coli) and Salmonella typhi (S.typhi) with high purity and very low contaminating nucleic acids and proteins based on the hot phenol-water extraction protocol has been introduced. The purity of extracted LPS was evaluated by silver and coomassie blue staining of SDS-PAGE gels and HPLC analysis. Limulus Amebocyte Lysate (LAL) coagulation activity and rabbit pyrogen assay were exploited to monitor the functionality of purified LPS. The results showed that DNase and RNase treatment of the sample is essential after the sonication step to eliminate nucleic acid contamination in the LPS fraction. Silver staining demonstrated ladder pattern which is characteristic of LPS. No contaminating protein was found as assessed by coomassie blue staining. HPLC fractionation revealed high degree of purity comparable with commercial LPS. Parenteral administration of purified LPS resulted in substantial increase of rabbits' body temperature (mean: 1.45°C). LAL coagulation assay confirmed the functional activity of the purified LPS. In conclusion, the protocol presented here could be employed for isolation of LPS with high purity and functional activity.
