Re-induction Of The TRAIL-resistant Kasumi-1 Cell Line And The Role Of LBH589 To Overcome Its Resistance In Vitro

Background:Acute myeloid leukemia (AML) is a group of malignant clonal diseases which is originated from hematopoietic stem/progenitor cells and results from uncontrolled cell proliferation, differentiation disorders and blocked apoptosis of hematopoietic stem/progenitor cells. Among them, AML with recurrent genetic alterations t (8; 21) is a common subtype, accounting for 10-15% of all AML patients and for approximately 40% of patients with FAB classification M2. AML with t (8; 21) was generally considered having better prognosis. Long-term survival was 60%-70% reported by European-American researchers. However, two larger domestic studies reported that the five-year overall survival rates of patients with t (8; 21) positive AML were only 39% and 50%. A South Korea study reported that there was no significant difference in the prognosis between t (8; 21)-positive AML and t (8; 21)-negetive AML. Currently, high-dose cytarabine-based chemotherapy is the mainly treatment to this kind of AML. Relapse and drug resistance are the major factors affecting the prognosis. Therefore, to discover new therapy approaches to improve treatment outcome is of great value. Kasumi-1 cell line is cultured from human patients with t (8; 21)-positive acute myeloid leukemia cell lines, which is a good model for the study.Tumor necrosis factor related apoptosis inducing ligand (TRAIL), also known as Apoptin 2 ligand (Apo-2L), a type II membrane protein, belongs to the tumor necrosis factor superfamily. The interactions between TRAIL and its death receptors can induce apoptosis in a variety of tumor cells and transformed cells instead of normal tissues and cells. TRAIL signal pathway has become one of the hotspots in anti-tumor therapy research. Although TRAIL can kill leukemia cells without affecting normal host cells, the resistance of tumor cells to TRAIL-induced apoptosis obstacles seriously its clinical application.Based on the previous study of our group, rsTRAIL was able to induce apoptosis of Kasumi-1 cell line both in vitro and in vivo, but the viability of Kasumi-1 cell line was no longer decreasing with the increasing TRAIL concentration at above a certain concentration showing some resistance to TRAIL. Then, we established a TRAIL-resistant Kasumi-1 cell line (Kasumi-1 R). After analysis and comparision the mRNA profiles of both Kaumi-1 and Kasumi-1R cells, some resistant genes were screened out. Meanwhile, we realized that the viability of rsTRAIL used in the previous study was poor, even the parental Kasumi-1 cell line was remarkable resistance. Therefore, we chose another more efficient rsTRAIL agent to re-induce the Kasumi-1R cells to establish a more resistance TRAIL-resistant cell line and to explore the possible mechanisms.Orderly regulation of gene transcription is critical to maintain normal cell function. The acetylation and deacetylation of core histones is an important way of transcriptional regulations. Pathogenesis of tumor is considered to be associated with dysfunctions of histone deacetylases and the following inhibition of certain genes transcriptions. Histone deacetylase inhibitors (HDACIs) can restrain HDACs and facilitate acetylation of histone to resist cell cycle, promote differentiation and induce apoptosis through the regulation of chromatin remodeling and gene transcription. HDACIs with various chemotherapy agents show the synergistic effects in anti-tumor therapy, which makes HDACIs become one of the focuses in the therapy of AML, acute lymphocytic leukemia (ALL), and myelodysplastic syndrome (MDS). Panobinostat (LBH589) is a new kind of micromolecule HDACIs, belonging to hydroxamic acids, which can induce apoptosis of tumor cells in lower dose and less toxicities. It is reported that LBH589 can improve acetylation level of histone H3 and H4 and non-histone proteins, such as p53 and Hsp90. And it can attenuate phosphorylation of signal proteins (BCR-ABL, AKT, and ERK1/2) and induce apoptosis in a dose-dependent manner in leukemic cells.In the previous study by our group, LBH589 induced apoptosis of Kasumi-1 cells in a dose- and time-dependent manner, and presented a synergistic effect with rsTRAIL. Thus, to observe whether LBH589 can reverse the TRAIL resistance of TRAIL-resistant Kasumi-1 cell line is significant for further analysis of its clinical value in combination with rsTARIL. This study was designed to re-induce the resistance of TRAIL-resistant Kasumi-1 cell line and explore the effects of LBH589 to TRAIL-resistant Kasumi-1 cell line.Part I Re-induction of the TRAIL-resistant Kasumi-1 cell line and its resistance mechanismsAIMS:To establish a more rsTRAIL-resistant Kasumi-1 cell line and explore its resistance mechanisms.METHODS:1) Kasumi-1 R cells that we established previously were intermittently treated with progressively increasing concentrations of rsTRAIL (high effectiveness) in a continuous gradient way for screening in vitro and a new resistant Kasumi-1 TR2 cell line was expected to be established.2) Proliferation of three cell lines (Kasumi-1, Kasumi-1R and Kasumi-1TR2) were measured by CCK-8 assay and the new IC50 of three cell lines and resistance index were calculated according to viability of three cell lines treated with rsTRAIL of different concentrations.3) The transcription and expression of resistant genes DR4, BCL-2, BCL2A1 and IFNAR1 were detected by real-time PCR and Western blotting.RESULTS:(1) Kasumi-1 TR2 was successively established after re-induction by rsTRAIL of high effectiveness. The proliferation of Kasumi-1 TR2 cell line was faster than Kasumi-1 cell line, with no difference from Kasumi-1 R cell line; (2) The 24 hours IC50 of Kasumi-1TR2 cells was 83133.88ng/ml and the resistance index (RI) for 24h was 14445. The 48 hours IC50 for Kasumi-1TR2 cells was 5402.79ng/ml and the RI for 48h was 255137; (3) the mRNA expression of DR4 was downregulated, and the mRNA expression of BCL-2, BCL2A1 and IFNAR1 were upregulated in Kasumi-1TR2 cells. (4)The total proteins of BCL-2, BCL2A1 and IFNAR were upregulated in Kasumi-1 TR2 cells and Kasumi-1 R cells, but there was no difference of DR4 protein among Kasumi-1, Kasumi-1 R and Kasumi-1 TR2 cells.CONCLUSIONS:After re-induction, a new TRAIL-resistant Kasumi-1 cell line--Kasumi-1TR2 cell line was established. Kasumi-1 TR2 cells were highly resistant to TRAIL. The resistance mechanisms were associated with the up-regulated expression of BCL2, BCL2A1, IFNAR1, and might be associated with the down-regulated expression of DR4.Part ? LBH589 reversed the TRAIL resistance of Kasumi-1TR2 cells in vitroAIMS:To investigate whether LBH589 can reverse TRAIL resistance of Kasumi-1 TR2 cells in vitro.METHODS:Kasumi-1TR2 cells were treated with rsTRAIL alone, LBH589 alone and their combination at variant concentrations. The changes of morphology of Kasumi-1 TR2 cells were observed under microscope after Wrighting staining. The viability of cells was measured by CCK-8 assay and the apoptosis was analyzed through Annexin V/PI double staining by flow cytometry.RESULTS:Kasumi-1TR2 cells were high resistant to rsTRAIL. LBH589 could induce apoptosis of Kasumi-1 TR2 cells, and the resistance of Kasumi-1 TR2 cells could be reversed by LBH589, which was in a time-and dose-dependent manner (P<0.05). The ratios of annexin V positive Kasumi-1TR2 cells were (23.97± 7.77)% and (57.57±6.54)% in 50nM LBH589 group and the combination of 50nM LBH589 plus 50 ng/ml rsTRAIL group, respectively. Both of them were significantly higher than the control group (4.20±2.33)%(P<0.05), and the combination group was significantly higher than the rsTRAIL alone group (13.6±7.05)%(P<0.001).CONCLUSIONS:LBH589 can reverse the TRAIL resistance of Kasumi-1TR2 cells in vitro.Part? The mechanisms of LBH589 to reverse TRAIL resistance of Kasumi-1TR2 cellsAIMS:To investigate the mechanisms of LBH589 to reverse TRAIL resistance of Kasumi-1TR2 cells in vitro.METHODS:The transcription and expression level of resistance genes DR4, BCL2, BCL2A1 and IFNAR1 of Kasumi-1TR2 cells were measured by using real-time quantitative PCR and Western-blot assay after being treated with LBH589 in different concentrations for different time. The transcription and expression level of resistance genes DR4 and BCL2 of Kasumi-1TR2 cells were measured by real-time quantitative PCR and Western-blot assay after being treated with LBH589 alone (50nM), rsTRAIL alone (50ng/ml) and their combination.RESULTS:(1) LBH589 alone and in combination with rsTRAIL could upregulate DR4 mRNA level (P<0.05) and downregulate BCL2 mRNA level (P <0.05). (2) LBH589 alone and in combination with rsTRAIL could upregulate DR4 proteins (P<0.05) and downregulate BCL2 proteins (P<0.05), and the reduction of BCL2 proteins were more significant in the combination group (P<0.05).CONCLUSION:The mechanisms of LBH589 to reverse TRAIL resistance of Kasumi-1TR2 cells include the upregulation of DR4 and downregulation of BCL2.