Targeting CML Leukemia Stem Cells By Picropodophyllotoxin And Its Underlying Mechanisms

IntroductionChronic Myeloid Leukemia(,CML) is a lethal hematological malignancy resulting from BCR-ABL oncogene-transformed normal hematopoietic stem cells(HSCs) known as leukemia-initiating cells or leukemia stem cell(LSCs) and seriously threatens the public health.In China, CML ranking the third of leukemia is more than 90% of chronic leukemia and accouts for approximately 18% in all acute and chronic leukemia. Ph chromosome is an important sign of CML derived from the reciprocal translocation No. 9 and No. 22 chromosome. As a result, part of the BCR( "breakpoint cluster region") gene from chromosome 22 is fused with the ABL gene on chromosome 9. This abnormal "fusion" gene generates a protein of BCR-ABLp210, which has a very active tyrosine kinase activity and is believed to be the primary mechanism leading to occurrence of CML. The BCR-ABL tyrosine kinase inhibitor(TKIs) imatinib mesylate(IM) is effective in inducing remissions and improving survival in patients with CML but does not eliminate LSCs because these cells did not depend on BCR-ABL kinase activity for survival. Therefore, ‘‘cure’’ of CML remains elusive following TKIs therapy alone. CML patients currently need to take TKI treatment indefinitely, with risks of toxicity, lack of compliance, drug resistance, relapse, and associated expense. Strategies need to explore to cure CML through eradicating LSCs.Podophyllotoxin, an active component of traditional Chinese medicine D.Versipoellis(Hance)M.Cheng, has a broad anticancer activities. But because of its great toxicity due to inhibition of tubulin makes it failed to clinical application. Picropodophyllin(PPP) is the isomer of podophyllotoxin and has no inhibitory effect on tubulin, so it does not have the cell toxicity. Many studies have shown that PPP also has a wide range of anti-cancer activities, but substantially non-toxic to normal cells. This indicates PPP has a certain specificity to tumor cells. Currently, there are reports found that PPP can effectively induced apoptosis of CML cell lines and primary cells and inhibit its clonogenic ability, but the effects of PPP on CML LSC have not yet been reported. Therefore, this study is divided into three sections elaborate: 1, Effects of PPP on CML LSC apoptosis and colony formation in vitro; 2, Effects of PPP on CML LSCs in vivo; 3, the molecular mechanism underlying of PPP targeting CML LSC. ObjectiveThe objective of this study is to investigate the effect and mechanism of PPP to target CML LSC in vitro and in vivo, thus providing scientific evidence and reference for curing CML through targeting LSCs. Methods1. To investigate the effect of picropodophyllin on human CML LSC in vitro1.1 Mononuclear cells were enriched by Ficoll density gradient centrifugation. CD34+ stem / progenitor cells were then isolated using a positive magnetic bead selection protocol and molecular phenotype of CD34 cells was identified by flow cytometry.1.2 Flow cytometry was employed to detect the apoptosis of CML and healthy human CD34 +stem/progenitor cells induced by PPP; cloning forming assay was employed to detect the colony formation number of CML and healthy human CD34+stem / progenitor cells treated by PPP.2. To investigate the effect of picropodophyllin on SCL-t TA/BCR-ABL transgenic mice2.1 BCR-ABL transgenic mice and SCL-t TA transgenic mice were hybridized to give SCL-t TA / BCR-ABL double transgenic mice(CML mice). HE staining and flow cytometry were employed to identify phenotype of CML mice after induction of BCR-ABL protein.2.2 Investigate the effect of PPP treatment on the body weight, percentage and number of CML LSC in CML mice.3. To identify the molecular mechanism of picropodophyllin on anti-CML LSC3.1 CML CD34+cells were treated with PPP, and then the phosphorylation and acetylation levels of P53 were detected by Western blotting; the expression level of P53 gene and its downstream regulatory genes P21, Puma, Noxa, Bax were detected by q PCR; the expression level of c-Myc and BCL2 were detected by q PCR. The expression level of P21, Noxa and Bax in mice’s bone marrow cells treated with PPP was detected by q PCR.3.2 q PCR was used to detect the IGF1 R gene expression level of CML and healthy human bone marrow mononuclear cells, CD34+CD38-cells, CD34+CD38+cells; flow cytometry was used to detected the IGF1 R protein expression of CML and healthy bone marrow mononuclear cells; Flow cytometry was used to detected the apoptosis of K562 after transfected with si-IGF1R; Western blotting was used to detect the IGF1 R activity and its downstream protein AKT activity of K562 and Ku812 after treated by PPP, OSI-906 and AG-1024; flow cytometry was used to detected the apoptosis of CML CD34+cell after treated by OSI-906 and AG-1024; ELISA was used to detect the expression of IGF1 and IGF2 in CML and healthy human bone marrow supernatant.Results1. To investigate the effect of picropodophyllin on human CML LSC in vitro1.1 Flow Cytometry analysis showed that the purity of CD34 positive cells isolated by MACS was about 89.23%, which reached the need of the following experiments.1.2 The flow cytometry results showed that PPP could induce significant apoptosis in CML LSC, while had a modest effect on healthy hematopoietic stem cells. Also in Cloning forming assay, PPP could inhibit colony formation in CML CD34+ cells, but had no apparent effect on normal CD34+ cells.2. To investigate the effect of picropodophyllin on SCL-t TA/BCR-ABL transgenic mice2.1 Flow cytometry showed in transgenic SCL-t TA/BCR-ABL mice, the percentage of neutrophils was significantly increased in peripheral blood cells and spleen cells compared with FVB mice. The percentage of LSC was also significantly increased in bone marrow and spleen cells compared with FVB mice. HE staining of the spleen cells showed the apparent leukocyte infiltration, bone marrow hyperplasia.2.2 After treatment with PPP, the body weight remained unchanged in both SCL-t TA/BCR-ABL transgenic mice and FVBmice, the percentage and cell number LSC in SCL-t TA/BCR-ABL transgenic mice were significantly decreased compared with vehicle treatment, but remained unchanged in FVB mice.3. To identify the molecular mechanism of picropodophyllin on anti-CML LSC3.1 The treatment of PPP resulted in non-significant influence in P53 phosphorylation and acetylation protein levels in CML CD34+of cells. PPP treatment had nothing to do with P53 gene expression in CML CD34+cells, but resulted in a significant upregulation in P21, Puma, Noxa, Bax gene expression and a significant reduction in c-Myc gene expression. After receiving PPP treatment, P21 and Noxa expression levels were significantly increased in bone marrow cells of CML mice, consistent with the in vitro results; P21 gene expression was also significantly increased in wild-type mice bone marrow cells, but Noxa expression was not significantly increased.3.2 No significant difference of IGF1 R expression between CML and healthy volunteers was observed. Transfection with si IGF1 R in K562 cells did not induce significant apoptosis in K562 cells. ELISA results showed that there was no significant difference of supernatant amount of IGF1 R ligand IGF1 and IGF2 between CML and healthy bone marrow. Compared with IGF1 R specific inhibitor, PPP did not effectively inhibit IGF1 R activity and its downstream signaling AKT pathway. Compared with the PPP, IGF1 R inhibitor induced small apoptosis in CML LSC.4. Conclusion PPP effectively targets CML LSC in vivo and vitro, which is related with activation of P53 signaling pathway and inhibition of anti-apoptotic genes expression.