(B) A histogram representing TUNEL assay results. with untreated settings or EGCG treatment. Immunohistochemistry revealed improved caspase 3 and SHP-1 activity and decreased phosphorylation of BCR-ABL in the EGCG-MP-treated group relative to that in the EGCG-treated group. Conclusions and Implications EGCG-MP induced SHP-1-mediated inhibition of BCR-ABL and STAT3 signalling and more effectively than EGCG. This derivative may be a potent chemotherapeutic agent for CML treatment. Furniture of Links and (Liedtke = 4 per group) and injected i.p. with 15?mgkg?1 of EGCG or EGCG-MP dissolved in 4% Tween-80 five occasions per week. The tumour quantities (size width2 0.5236) and tumour weights were RAF1 measured and body weights monitored twice per week for 3 weeks. Immunohistochemistry (IHC) IHC was performed in tumour sections using the indirect avidin/biotin-enhanced HRP Ascomycin (FK520) method. Antigen retrieval was performed after deparaffinization and dehydration of the cells sections by microwave for 10?min in 10?mM citrate buffer. Tumour sections were cooled within the bench for 30?min, treated with 3% hydrogen peroxide in methanol for 10?min, and blocked with 6% horse serum for 30?min at room temperature. Sections were then incubated with the primary antibodies of cleaved caspase3, SHP-1 and p-BCR-ABL at 4C over night inside a humidified chamber. Ascomycin (FK520) Sections were washed in PBS and incubated with secondary antibody biotinylated goat anti-rabbit (1:150, Vector Laboratories, Burlingame, CA, USA) or biotinylated rabbit anti-rat IgG (1:150, Abcam, Boston, MA, USA) for 40?min inside a humidified chamber. After washing the antibodies were detected with the Vector ABC complex/HRP kit (Vector Laboratories), and developed with 3,3-diaminobenzidine tetrahydrochloride. For semiquantitation, 10 photomicrographs (200) were obtained having a CCD video camera, avoiding gross necrotic areas. Data analysis All data are offered as means SD. Statistical analysis of the data was performed using the SigmaPlot version 12 software (Systat Software, Inc., San Jose, CA, USA). One-way anova was utilized for comparisons of multiple organizations. Student’s < 0.05 between the control and EGCG or EGCG derivatives-treated organizations. All experiments were repeated at least three times. Results Stability of EGCG, EGCG-MO, EGCG-ML and EGCG-MP and their cytotoxic effects in K562 and KBM5 CML cells To evaluate the stability of the EGCG derivatives EGCG-MO, Ascomycin (FK520) EGCG-ML and EGCG-MP (Number?1A) in cultured cells, we performed HPLC analysis in DMEM tradition medium. We found that 50% of EGCG-MP remained after 23?min in tradition medium, while 50% of EGCG-ML, EGCG-MO and EGCG remained after 8 and 3?min respectively. Similarly, 20% of EGCG-MP remained after 116?min, while 20% of EGCG-ML, EGCG-MO and EGCG remained after 73.5, 16.5 and 4.5?min respectively. These results indicated that EGCG-MP was more stable than the additional EGCG derivatives, including EGCG (Number?1B). Also, we checked the intracellular levels of EGCG and EGCG-MP in K562 cells by HPLC analysis. As demonstrated in Number?1C and ?and1D,1D, approximately 1.5% of EGCG-MP was recognized in K562 cells after exposure to 40?M EGCG-MP, while there was no maximum of EGCG in K562 cells, incubated under the same conditions. In contrast, we observed approximately 0.39% of EGCG only when increasing its concentration up to 200?M. Next, the cytotoxic effect of EGCG and its derivatives was evaluated by MTT assay in K562 and KBM5 cells exposed to 0, 10, 20, 40 or 80?M for 24?h. As demonstrated in Number?1C, EGCG-ML and EGCG-MP exhibited higher dose-dependent cytotoxicity in K562 and KBM5 cells, compared with EGCG.