The cycling conditions were 95C for 5 min; 95C for 30 sec, 52C for 1 min, and 65C for 2 min for 40 cycles; and an extension step at 65C for 5 min. samples, we used the affinity probe biotin-labeled HA, which only binds to CD44 in its active conformation . The results demonstrated increased HA binding on cancerous tissues with a median MSI of 56834. 5 compared with minimal or low binding on the corresponding normal tissue with a median MSI of 1 1.9795 (Fig. 1A and 1C), indicating the presence of two distinct states of CD44 activity determined by microenvironments. Furthermore, we examined the relationship between activated CD44 and tumor grade, histological type, tumor size, lymph node metastasis stage, age at diagnosis, estrogen receptor status, progesterone receptor, Her 2 receptor, and Ki 67. Despite the obvious differences between cancerous and normal breast tissues, no significant association was observed between activated CD44 expression and these tumor characteristics (Table ?(Table1),1), indicating no correlation between CD44 activation and clinical tumor parameters. In addition, further examination revealed that alterations of CD44 states can occur within the breast tumor microenvironment (Fig. S1). HA binding of CD44 on normal cells was observed when normal cells were co-cultured with breast cancer cells, indicating that the conversion of CD44 from an inactive state to an active state (Fig. S1). This result provides evidence for the specific location of active CD44 within the tumor microenvironment. Two CD44 activation states in normal and breast cancer cell lines To further confirm that the two activation states of CD44 are differentially located, we assessed CD44 expression and fl-HA binding activity on four breast cancer cell lines (MDA-MB-231, MDA-MB-468, BT-549, and Hs578T) and four normal cell lines (PBMCs, NIH3T3, CV-1, and NFs). The peripheral blood mononuclear cells (PBMCs) from 10 donors and NFs derived from 3 adults were assessed. The results from flow cytometry analysis indicated that CD44 expression was abundant in all four breast cancer cell lines and four normal cell lines; no significant difference between normal and cancer cells was noted (Fig. ?(Fig.2A).2A). However, significantly increased fl-HA binding was observed in cancer cell lines compared with normal cells (Fig. ?(Fig.2B).2B). These results indicated that the CD44 activation state differs in normal and cancer cells, revealing striking similarities to the observations made in clinical tissues. Open in a separate window Figure 2 Two activation states of CD44 in normal and breast cancer cell lines(A) CD44 expression on four breast cancer Rabbit Polyclonal to RPS20 cell lines (MDA-MB-231, MDA-MB-468, BT-549, and Hs578T) and four normal cells (PBMC, Ebselen NIH3T3, CV-1, and NFs) were determined by Ebselen flow cytometry. (B) The binding activity of HA by CD44 on four breast cancer cell lines (MDA-MB-231, MDA-MB-468, BT-549, and Hs578T) and four normal cells (PBMC, NIH3T3, CV-1, and NFs) were determined and analyzed. (C) The CD44 isoform expression pattern was detected by agarose gel electrophoretograms in normal cells and cancer cells. (D) The CD44 variants expression was detected by western blot in normal cells and cancer cells. To determine whether the CD44 expression pattern is altered in these four breast cancer cell lines compared with the four normal cells, we next determined the expression of CD44 variants. Using a primer pair spanning the entire variant region in a reverse transcription polymerase chain reaction (RT-PCR) assay, each transcribed CD44 variant isoform is theoretically amplified as described previously . Our results indicate that the CD44 expression pattern differed between normal cells and cancer cells (Fig. ?(Fig.2C).2C). Human breast cancer cell lines (MDA-MB-468, BT-549, and Hs578T) were characterized by the appearance of several bands on agarose gel electrophoretograms when examining the CD44 variant region with a primer pair spanning the entire variant region. These results were consistent with previous reports . By contrast, almost no expression of the CD44v gene was observed in normal cells (Fig. ?(Fig.2C).2C). Similarly, changes in expression patterns were detected in normal cells and cancer cells by western blotting using an immunoblotting antibody against the standard region of CD44 (Fig. ?(Fig.2D).2D). Although altered CD44 mRNA and protein expression patterns were detected in three of four cancer cell lines tested, which might be attributed to the heterogeneity of cancer cell lines, no obvious difference was noted regarding the fl-HA binding activity of the four cancer cell lines as analyzed in Fig. ?Fig.2B,2B, suggesting that factors other than Ebselen CD44v expression affect the different CD44 activation states noted between normal and cancer cells. Engineering and synthesis of HA-coated nanoparticles To develop HA-coated nanoparticles containing drugs, a nanotechnology engineering approach was employed based on structural and functional studies of HA as described previously [21, 27]. Paclitaxel (PTX) was mixed with the lipid molecules and assembled into lipid clusters, which were subsequently covalently coated with HA to form HA-Lipid-PTX nanoparticles. HA-free Lipid-PTX nanoparticles were prepared similarly Ebselen with the omission of HA. The particle size.