Materials and methods Cells and reagents MCF 7, MDA MB 157, and BT 549 human br

We also tested the effect of SPARC on the invasive potential of other prostate cancer cells, finding that recombinant 価格 INNO-406 human SPARC caused an enhanced invasiveness of the androgen independent Du 145 and 22Rv1 cells, but not the androgen dependent LNCaP cells. Taken together, these results indicate that SPARC is the major factor respon sible for the enhanced invasiveness of M cells stimulated by S CM, and that it can stimulate the invasiveness of other androgen independent prostate cancer cells. Of additional interest, knock down of SPARC in S cells was accompanied with a decrease in PAI 1 expression at the mRNA and protein levels. It has been reported that the expression of PAI 1 is modulated by changes in the expression levels of SPARC.<br><br> Therefore, although our immunodepletion experiments suggest that PAI 1 may not play a major role in the enhanced invasiveness of M cells by S CM, we cannot completely rule out the involvement Lapatinib 臨床試験 of PAI 1 in the cooperative interaction between our tumor cell sub populations, albeit secondary to modulation of SPARC levels. In order to determine the relative importance of SPARC in the M and S cell interactions through diffus ible factors or direct cell to cell contact, we performed a co culture experiment, in which M cells were mixed and cultured together, in a 1 1 proportion, with S cells bear ing a construct for the doxycycline inducible expression of a SPARC specific shRNA. Under control conditions, S cells significantly enhanced the invasiveness of M cells. Importantly, doxycycline induced SPARC knock down abrogated the capacity of S cells to enhance the invasive behavior of M cells.<br><br> These re sults indicate that the expression of SPARC by S cells explains not only their ability to enhance the invasive ness of M cells through secreted factors, but also the overall pro invasive properties of S cells on M cells, encompassing effects mediated Lonafarnib ic50 by diffusible factors and by putative direct cell to cell contact. SPARC produced by non CSC S cells boosts the in vivo growth and metastatic dissemination of CSC enriched M cells Our previous studies had indicated that the meta static potential of CSC enriched M cells is strongly boosted by co implantation in immunodeficient mice together with non CSC S cells. To determine the im portance of SPARC expressed by S cells in their pro metastatic cooperation with M cells, we proceeded to the implantation of M cells together with S.<br><br> sh3399 cells in the dorsal prostates of male SCID beige mice. Tumor growth at the site of implantation and spread to distant organs was monitored by bioluminescent imaging, with or without doxycycline in the drinking water of mice. Without doxycycline administration, co implantation of M cells and S. sh3399 cells strongly accelerated tumor growth at the orthotopic implantation site as compared to the growth rate of M cells alone. Remarkably, administration of doxycycline, which suppresses SPARC expression in S. sh3399 cells, significantly inhibited the tumor stimulating effect of S. sh3399 cells on M cells. In control experiments, S. sh3399 cells alone did not grow detectable tumors or metastases 24 days after implantation in the prostate, and doxycycline alone did not inhibit the growth of M cells.