[Effect of a novel tyrosine kinase inhibitor HHGV678 on growth inhibition of Bcr-Abl wild type and IM-resistant cell lines in vitro].

This study was aimed to compare HHGV678 with imatinib (IM) in growth inhibition of Bcr-Abl wild type and IM-resistant cell lines, investigate the possibility of replacing IM with HHGV678 in treatment of chronic myeloid leukemia (CML) and IM-resistant CML patients. Viability of two Bcr-Abl wild type cell lines (K562 and 32Dp210) and 16 IM-resistant cell lines (K562R and 15 Bcr-Abl point mutant cell lines) treated with HHGV678 and IM was analyzed by MTT. The apoptosis of those cells was identified by flow cytometry with Annexin V staining and DNA ladder analysis. Western blot was applied for detecting the expression of Bcr-Abl and phosphotyrosine protein levels. The results indicated that HHGV678 significantly inhibited the growth of two Bcr-Abl wild types and IM-resistant cell lines in dose-dependent manner except cell line of T315I point mutant. IC(50) results showed that the growth inhibition of HHGV678 was 15.5 and 28-fold higher than that of IM in K562, 32Dp210 and 1.4 to 124.3-fold higher than that of IM in 15 IM-resistant cell lines respectively. Compared with IM, HHGV678 more significantly inhibited phosphotyrosine kinase protein of the cells mentioned above at different concentrations. With most importance, HHGV678 of 10.0 micromol/L induced cell apoptosis of 40.06% and 33.32% in K562R and 32Dp210(T315I) cell lines, which were much higher than that of IM (19.77% and 10.68%). It is concluded that HHGV678 is more effective than IM in the growth inhibition of Bcr-Abl wild type cell lines and IM-resistant cell lines, especially in strongest IM-resistant cell lines. Further studies are needed to show whether HHGV678 may be a novel targeting drug in treatment of CML and IM-resistant CML patients.

[Growth inhibition and differentiation of imatinib-resistant chronic myeloid leukemia cell induced by cell differentiation agent in vitro].

OBJECTIVE: To investigate the effects of uroacitide (CDA-2), a cell differentiation agent, on the growth inhibition and differentiation of imatinib-(IM) resistant chronic myeloid leukemia (CML) cells. METHODS: IM resistant CML cell line K562R was established from the line K562. K562 and K562R CML cells were cultured with CDA-2 of different concentrations. MTI method was used to detect the survival rates. Bone marrow cells of IM-resistant and non-IM-resistant CML patients were collected and co-incubated with K562 and K562R cells. MTT and colony-forming assays were used to evaluate the efficacy of CDA-2 treatment for cell growth in K562 and K562R cell lines, and IM-resistant or non-IM-resistant bone marrow cells of the CML patients; Annexin-V staining was employed to detect the apoptosis. Cell differentiation was assessed by flow cytometry analysis with CD11b/CD14 markers, reverse transcriptase PCR (RT-PCR) for mRNA levels of NCF-1 and ORM-1 genes and Giemsa staining for the observation in morphology. Cell cycle distribution was detected by stained with propidium iodide and then analyzed by flow cytometer. RT-PCR also was employed for the expression of DNA methyltransferase. RESULTS: Significant cell growth inhibition was found at a dose-dependent manner in the IM-resistant K562R cell line and IM-resistant bone marrow cells of the CML patients compared with the non-resistant K562 cell line and bone marrow cells of the CML patients following 7 days exposure to CDA-2. Although CDA-2 could significantly induce the apoptosis of K562R (15.38%) compared with K562 (5.28%) (P < 0.05), the major reason for the cell growth inhibition of K562R is CDA-2-induced cell differentiation, including the increase of expression of differentiation-related antigens CD11b/CD14, mRNA expression of NCF-1 and ORM-1, and cell cycle arrest in G1-phase at a dose-dependent manner. Because CDA-2 could significantly activate the p21 and p27 gene expression, downregulate the expression of cyclin D1, and down-regulate the expressions of DNMT1 and DNMT(3B) at mRNA level, CDA-2 might be a DNMT inhibitor for restoring some gene function that involved in cell cycle control by demethylation. CONCLUSION: Inhibiting the growth and inducing the differentiation of K562R cells, CDA-2 is very likely to be a potential agent for the treatment of IM resistance CML patients.