Experimental Researches Of Bortezomib, A Proteasome Inhibitor, Targeting Treatment In Acute T Lymphoblastic Leukemia

1. The study of induced apoptosis of acute T lymphoblastic leukemia cell line MOLT-4 with BortezomibObjective To investigate the effects of Bortezomib on human acute T lymphoblastic leukemia cell line and its potential mechanisms.Methods MOLT-4 cells were treated with different concentration of Bortezomib, cell proliferation was evaluated by Trypan Blue dye exclusion, cell cycle distribution was analyzed with flow cytometry, biological responses were studied with the methods of CCK-8 and Annexin V/PI staining, NF-κB(p65),Hes1 gene expression were analyzed by RT-PCR.Results Exposure to Bortezomib induced proliferation inhibition and cell cycle arrest in a dose-dependent pattern in MOLT-4 cells. The cell viability was profoundly decreased in Bortezomib-treated group compared with control. confirmed that After 24 hoursincubation with Bortezomib, induced apoptosis was observed in MOLT-4 cells with AnnexinV/PI staining, and the effect was in a temporal and dose dependent fashion. The mRNA expressions of NF-κB and Hesl were decreased significantly after treated with Bortezomib.Conclusions T Bortezomib can induce cell apoptosis and proliferation inhibition in acute T lymphoblastic leukemia cells by down-regulation of NF-κB,Hes1 expression.2.fects of Bortezomib on the growth of acute T lymphoblastic leukemia cell xenograft in nude miceObjective To observe the effects of Bortezomib on the growth of acute T lymphoblastic leukemia cell xenograft in mude mice.Methods Acute T lymphoblastic leukemia cell xenograft was established by subcutaneous injection of MOLT-4 cell into nude mice(1×107 MOLT-4 cells were resuspended in 200ul saline).Nude mice were administrated with Bortezomib by intraperitoneal injection at+1d,+4d,+8d,+11d after MOLT-4 cells injection.Control mice were given equal volumes of normal saline instead of the drug.Tumor incidence was monitored for 63 days after injection.Tumor volume was measured by vernier caliper and weight was made. Observed tissue infiltration of spleen and lymphonode sections prepared 63d after transplantation.At+63days after MOLT-4 cells injection,tumor and tissues were peeled off and fixed in 10% buffered formalin. Hesl,c-Myc,Pre-Tα,and Deltex1 expression was quantified using real-time PCR.NF-κB, Hes1, Caspase3, and Caspase9 expression was detected by western-blotting.Results The xenograft of MOLT-4 cells were developed in all nude mice. While palpable tumors developed in vehicle-treated mice within 3 weeks,Bortezomib treatment delayed palpable tumor formation by-4 weeks.Subsequent tumor size and weight in Bortezomib-treated animals was reduced significantly relative to tumors in vehicle-treated animals. (977.53±475.69mm3 and 2266.23±376.48mg,2850.48±101.84mm3 and 3678.7±1084.15mg, respectively). We found increases in the tissue infiltration of leukemic cells in control mice,compared with Bortezomib-treated mice. The expression of Caspase3 and Caspase9 was up-regulated and the expression of NF-κB,Hes1 was down-regulated by bortezomib. We found Hes1,c-Myc,Pre-Tα, and Deltex1 expression reduced in Bortezomib-treated leukemic mice compared with mice treated with vehicle only by RT-PCR.Conclusions In vivo of nude mice,the growth and proliferation of acute T lymphoblastic leukemia cells can also be inhibited by Bortezomib. Bortezomib decreased the infiltrationg capacity of leukemic cells. Besides by the way of apoptosis induction,Bortezomib can also inhibite the formation and metastasis of leukemic cell xenograft through down regulation of NF-κB and Notch1 expression.3. Influence of Bortezomib on recipient mice survival that transplanted with Notch 1-IC-infected bone marrow progenitors and its potential machanism studyObjective To demonstrate the effect of Bortezomib on prolonging T-ALL-host survival and characterrize the differences in the distribution and behavior of Notch1-IC+ leukemic cells.Methods C57BL/6 mice were transplanted with Notchl-IC (ICN1-GFP)-infected bone marrow progenitors derived from syngeneic mice. Recipient mice were randomized into 2 groups to receive treatment with normal saline, bortezomib.Flow cytometric analyses of CD4+CD8+Notch1-IC+leukemic cells in the peripheral blood of host mice at indicated times after transplantation.Observed tissue infiltration of liver and spleen sections by H-E staining prepared 11d after transplantation.Bone marrow cells were harvested from vehicle-and Bortezomib-treated mice and Hes1,c-Myc,Pre-Tα,Deltex1 expression was quantified using real-time PCR. NF-κB,Hes1,Caspase3,Caspase9 expression was detected by western-blotting.Results Induction of leukemia in control mice of Notch1-IC+(ICN1) progenitors resulted in death in as little as 21d,with cachexia,crouched posture and reduced mobility becoming apparent about 1-2d before death.In contrast in leukemic mice treated with Bortezomib,the disease progressed with significantly(P=0.0172) slower kinetics and reduced severity. We found increases in the tissue infiltration of leukemic cells in control mice,compared with Bortezomib-treated mice.The expression of Caspase3,Caspase9 increased,whereas the expression of Notch 1,NF-κB decreased in livers and spleens of mice treated with Bortezomib by immunohistochemical staining. The same result was confirmed in bone marrow cells of Borrezomib-treated mice by western-blotting.We found Hes1,c-Myc,Pre-Tαand Deltexl expression reduced in Bortezomib-treated leukemic mice compared with mice treated with vehicle only by RT-PCR.Conclusions Bortezomib treatment clearly induced apoptosis and extended the survival of leukemic mice.Bortezomib decreased the infiltrationg capacity of Notch l-IC-transformed T-cell progenitors.Bor induced apoptosis of leukemic cells through down-regulation of NF-κB and Notch 1 expression.