| Section ⅠExpression and clinical significance of Notch/D114and VEGF pathway molecules in human acute myeloid leukemiaBackground:Angiogenesis. the process of new blood vessel formation from an existing network of vasculature. plays a key role in the growth and metastatic spread of solid tumors. Emerging evidence underscores the potential role of bone marrow (BM) vascularization, indicative of angiogenesis, in the progression and chemosensitivity of hematological malignancies, including acute myeloid leukemia (AML). Moreover, the BM microenvironment has been demonstrated to regulate the angiogenic switch and give rise to novel vascularization by controlling the balance between pro-and anti-angiogenic factors. Among them, the vascular endothelial growth factor (VEGF) and Notch/D114are2of the most important families involved in the regulation of angiogenesis.The VEGF family is essential for the initiation of vascular development. Dysregulation of the VEGF families is suggested to have a major impact on leukemic growth and has been established as a crucial step in the development of AML. Indeed, elevated levels of VEGF and VEGFR2have been reported in patients with de novo AML and appear to be negative prognostic factors in AML.Recently, the Notch pathway, particularly the vascular-specific delta-like ligand 4(D114), has been identified as another pivotal pathway in the regulation of vascular development. The Notch signaling pathway is an evolutionarily conserved intercellular pathway affecting a myriad of cellular activities, such as cell survival, proliferation, and differentiation, and thereby may contribute to leukemogenesis.The activation of the Notch signaling pathway is initiated by ligand binding to Notch receptors mainly between adjacent cells, and there seems to be an important Notch cross-signaling between tumor and endothelial cells that promotes angiogenesis. D114-mediated Notch signaling has a unique role in regulating endothelial cell proliferation and differentiation. Studies have demonstrated that the Notch/D114signaling pathway is dysregulated in a wide variety of tumors and displays either increased or decreased angiogenic processes, such as endothelial proliferation, migration and tube formation in vitro, depending on the cell type and context. Furthermore, the prognostic impact of the Notch/D114signaling pathway in different malignancies has demonstrated a diverging pattern, and thus exploring the specific prognostic impact in AML angiogenesis is of substantial interest.Previous studies have shown that the VEGF signaling pathway acts as a potent upstream activating stimulus for angiogenesis, while the Notch/D114signaling pathway contributes to guide cell fate decisions that shape the activation appropriately. Since BM neovascularization depends on the mutual and coordinated interaction of various angiogenic factors from endothelial cells and AML cells, and many of these factors that are present in significant amounts in the BM microenvironment may function in a synchronized fashion, it will be of interest to comprehensively analyze the correlation among these factors and further assess the clinical implications.Objective:The aim of this study was to investigate the expression of6angiogenesis-related factors (Notchl, D114, Hes1, VEGF, VEGFR1, VEGFR2) in peripheral blood (PB) and BM samples of adult patients with untreated AML. Furthermore, we assessed the relationship and clinical significance between the Notch/D114and VEGF signaling pathways in AML angiogenesis. Additionally, we intended to reveal the prognostic impact of the angiogenesis-related Notch ligand Dll4and the receptor Notch1expression levels as well as their association with the VEGF families in AML and provide the basis for anti-angiogenic therapy.Materials and methods:1. Patient samples:PB serum and mononuclear cells from122AML patients and40healthy donors were taken after patients’ consent and approval from the Local Ethics Committee (Qilu Hospital, China).60patients were newly digagnosed and62patients were in complete remission (CR). Moreover, BM mononuclear cells from60untreated AML patients and40healthy controls were obtained. Tissue slides, including eight BM biopsies, were obtained from de novo AML patients. All patients had BM infiltration of AML cells>50%. BM specimens obtained from five healthy donors with no history of cancer were included as controls. PB serum was separated by differential centrifugation method, while AML cells were isolated by density gradient separation from the BM or PB samples of patients with a high percentage of leukemic blasts (>90%) among leucocytes.2. Enzyme-linked immunosorbent assay (ELISA):The serum was centrifuged to remove cell debris and stored at-20℃. VEGF, VEGFR1and VEGFR2protein expression were investigated in serum samples using ELISA.3. Real-time reverse transcription-PCR (RT-PCR) analysis:Total RNA was extracted from cells using TRIzol reagent and reverse transcription was performed with M-MuLV reverse transcriptase. The mRNA expression of Notchl, Dll4, Hes1, VEGF, VEGFR1, and VEGFR2was evaluated in PB or BM mononuclear cells by Real-time RT-PCR, which was performed with SYBR Green PCR Master Mix in a20μl reaction volume.4. Western blot analysis:Cells were solubilized in radio immunoprecipitation assay (RIPA) lysis buffer and cell lysates were separated by10%SDS-polyacrylamide gel electrophoresis. The protein levels of Notchl, Dll4, and Hesl were determined in PB or BM samples as assessed by western blot methods.5. Immunocytochemical analysis:Mononuclear cells from PB samples were placed on polylysine-coated glass slides and fixed with paraformaldehyde. Protein levels were examined by immunocytochemistry using the antibodies against human Notch1, D114, and Hes1.6. Immunohistochemical analysis:The degree of BM angiogenesis was quantifed by measuring the microvessel number using antibody to CD34. The expression of D114and VEGF was detected in human BM biopsies by immunohistochemistry.7. Statistical analysis:The Mann-Whitney U test, analysis of variance (ANOVA) and the Kruskal-Wallis test were used to analyze the differences in the expression of angiogenic factors between the AML and control groups, as well as differences in age, gender, FAB subtype and karyotype subgroups of the AML patients. Spearman’s test was used to evaluate the correlation between the individual expression of the genes studied, and the association of gene expression with clinical features. Median overall survival (OS) within each subgroup was estimated from Kaplan-Meier curves. The log-rank test was used to assess the signicance of univariate survival analyses. Univariate Cox proportional hazards regression models were used to evaluate the predictive effect of each factor alone on survival. Multivariate Cox models estimated the hazard ratio in terms of relative risk (RR) for death, and95%confidence interval (CI) to determine independent risk factors associated with survival. Two-sided P<0.05were considered statistically significant.Results:1. Aberrant expression profile of Notch/D114and VEGF pathway molecules in PB samples of adult AML patients:①ELISA analysis showed that the serum concentration of VEGF in CR group was significantly higher than that in controls, whereas VEGF level in newly diagnosed group was lower than that in CR group. Mean VEGFR2level was12024pg/ml in newly diagnosed group and8972pg/ml in controls. Likewise, mean VEGFR1level was281pg/ml in newly diagnosed group and116pg/ml in controls.②Real-time RT-PCR analysis demonstrated that the mRNA expression of Notchl, D114, Hesl and VEGFR2were significantly elevated in newly diagnosed group compared with controls.③Western blot and immunocytochemistry analyses showed that Notch1, D114and Hesl protein levels were up-regulated in untreated AML patients than in controls.④Spearman correlation analysis showed that Notch1and D114both displayed positive associations with VEGFR2in newly diagnosed group. Conversely, Notch1exhibited a positive correlation with VEGF and a negative correlation with VEGFR2in control group.Together, these data indicate that the Notch/Dll4and VEGF signaling pathways are aberrantly activated in AML.2. Aberrant expression profile of Notch/Dll4and VEGF pathway molecules in BM samples of adult AML patients:①Real-time RT-PCR data demonstrated that median levels of Notchl, Dll4, VEGF and VEGFR2were significantly elevated in the AML patients compared with the controls, while the median levels of VEGFR1were similar in AML and control patients.②Western blot analysis showed that the protein expression levels of Notchl and Dll4were upregulated in the untreated AML patients compared to the controls.③Significantly positive correlations were observed between Notchl and Dll4, VEGF and VEGFR2. Furthermore, Dll4was closely associated with VEGF.Taken together, our results provide evidence that the Notch/Dll4and VEGF signaling pathways are aberrantly activated in AML.3. Degree of BM vascularization correlates with Dll4and VEGF expression in AML patients:①Immunohistochemical analysis revealed increased BM vascularity in newly diagnosed AML patients compared with that in controls. We also observed significant upregulation of Dll4and VEGF expression in newly diagnosed AML patients. Dll4and VEGF protein were upregulated1.27-to4.63-fold and1.49-2.92-fold, respectively, in all AML specimens.②Furthermore, we observed a correlation between the extent of BM vascularization and levels of Dll4and VEGF by Spearman correlation analysis. These results demonstrate that bone marrow vascularity in the newly diagnosed AML patients is increased and correlates with high D114and VEGF expression.4. Correlation of Notch/D114and VEGF pathway molecules expression with clinical features in BM samples of adult AML patients:①Significantly positive associations of the percentage of leukemic blast infiltration were observed with the individual expression of Notchl, D114, and VEGF.②The levels of D114and VEGF were significantly different among the karyotype subgroups.Significant differences between BM levels of these angiogenic factors in AML patients and clinical features were not observed in the gender, age and FAB subtypes.5. Association between Notch/D114and VEGF pathway molecules expression and clinical outcome in BM samples of adult AML patients:①The median OS was25months (range,7.0-45.9months);②Univariate analysis of factors associated with the overall survival showed a significantly shorter survival in patients with the unfavorable karyotype, higher Notchl expression, higher D114expression, or higher VEGF expression. Furthermore, multivariate analysis revealed that the karyotype and expression levels of Notchl, D114, and VEGF were independent prognostic factors for overall survival.③Additionally, the prognostic value of2D114expression was more significant in the subgroup consisting of patients with intermediate-risk cytogenetics. Subgroup analysis showed that Notchl and D114expression levels had a prognostic impact on patients with high VEGF levels.Conclusion:1. Taken together, these results indicate that the Notch/D114and VEGF signaling pathways are aberrantly activated in AML.2. Our results demonstrate that increased D114and VEGF expression correlates with increased in vivo angiogenesis in untreated AML.3. Our data provide evidence that the activation of the Notch/D114signaling pathway may indicate an unfavorable prognosis in AML. In particular, D114may be a relevant prognostic marker in intermediate-risk AML. Section Ⅱ The role of the Notch/D114signaling pathway in the in vitro angiogenesis and its cross-talk with the VEGF signaling pathway in human acute myeloid leukemia cellsBackground:AML is a heterogeneous malignancy of immature and dysfunctional hematopoietic progenitors that proliferate and accumulate in the BM. Interactions between AML blasts and neighboring endothelial cells within BM microenvironment seem important both for disease development and susceptibility to chemotherapy. Our previous studies clearly suggest that angiogenesis plays a crucial rol in AML, including increased BM MVD in de novo AML and the prognostic impact of local angioregulatory mediators as well as serum levels in AML patients receiving intensive therapy. As the toxicities and relative ineffectiveness of traditional cytotoxic therapies become more appreciated, the search for therapeutic advances in AML is increasingly focused on antiangiogenic therapy.BM microenvironment regulates the angiogenic switch and gives rise to novel vascularization by controlling the balance between pro-and antiangiogenic factors. AML cells are demonstrated to secrete angioregulatory mediators to stimulate endothelial cell proliferation and induce angiogenesis. Among them, VEGF is one of the most potent angiogenic factors, and it is overexpressed in several human tumors. Targeting VEGF has shown promise in the treatment of colorectal cancer, demonstrating the potential for cancer therapy based on blocking angiogenesis. However, targeting VEGF has not been successful in a variety of other tumor types, suggesting that other factors or pathways may also play a critical role in tumor angiogenesis. The identication of those factors and pathways may have important implications in human cancer therapy, including AML.Dysregulation of the Notch signaling pathway plays a key role in the pathogenesis and development of numerous tumors, including AML. Recently, the Notch ligand Dll4has been identifed as another critical signaling in regulation of vascular development. Dll4is strongly upregulated in tumor vasculature in mouse models and in human kidney and bladder cancers. Downregulation of Dll4expression by RNA interference is reported to induce renal vascular endothelial cell cycle arrest and thus angiogenesis is reduced, suggesting the promoting effect of Dll4in renal cancer angiogenesis. In contrast, Dll4may play an inhibitory role in tumor angiogenesis. Specifically, Dll4blockage dramatically increased vascular density in a mouse glioma model. However, the possible role of the Notch/Dll4pathway in AML angiogenesis has not been exploredExtensive studies have highlighted the relationship between the Notch/Dll4and VEGF pathways in endothelial cells and solid tumors. Indeed, Dll4expression may be directly upregulated by VEGF. Furthermore, the Notch/Dll4pathway is shown to control vessel sprouting and branching, and restrict angiogenic response to VEGF, resulting in endothelial cell growth inhibition. Notch1is reported to cross-talk with matrix metalloproteinases (MMPs) critically involve in the processes of tumor cell migration and angiogenesis. All of those reports led us to conduct this study to determine whether the Notch/Dll4pathway is associated with AML angiogenesis and how it is related to VEGF pathway or other Notch-responsive genes.Objective:The purpose of the present study was to assess the effect of AML cells on endothelial cell function, explore the role of the Notch/Dll4signaling pathway in AML angiogenesis and further evaluate the cross-talk of the Notch/Dll4and VEGF signaling pathways. It was hoped that the realization of the expected objectives will lay theoretical and laboratory foundation for molecular target therapy treating AMLMaterials and methods:1. In vitro cell coculture model:Human myeloid leukemia cell lines HL60, Kasumi, NB4, and K562were cultured alone or cocultured in Transwell culture plates, where myeloid leukemia cells were placed in the upper chamber and human umbilical vein endothelial cells (HUVECs) in the lower chamber. Alternatively, AML cells and HUVECs were cultured in direct contact in24-well plates but otherwise these cultures were similar to the Transwell coculture.2. Cell proliferation assay:The HUVECs cocultured with AML cells were incubated overnight in96-well plates and subsequently incubated with Cell Counting Kit-8assay (CCK-8) reagent at37℃for2h. CCK-8method was conducted to detect live cells and assess cell proliferation.3. Cell migration assay:HUVECs were added to semi-permeable culture insert membranes in24-well Transwell plates in complete medium, and then AML cell supernatants were added to the lower chamber. Cell migration assay was performed to determine the capacity of HUVECs migration toward AML cell supernatants4. Matrigel in vitro HUVECs tube formation assay:HUVECs were seeded in the solidified Matrigel with basic medium supplemented with cell-free culture supernatants from different treatment (1:1, v/v). In vitro tube formation assay was used to examine the capacity of the in vitro angiogenesis induced by AML.5. Cell transfections:HUVECs, NB4and K562cells were stably transfected with Notch1small interfering RNA (siRNA) or a D114-expressing plasmid (pIRES2-EGFP-D114) using Lipofectamine2000reagent, and maintained under G418selection for at least2weeks. After transfection, AML cells and HUVECs were cultured in direct contact or cocultured in Transwell culture plates. Cell proliferation, migration assay and Matrigel in vitro HUVECs tube formation analysis were performed as described previously.6. ELISA:AML cells were seeded in six-well plates. Then the cell culture supernatant was harvested and cell count was done. Additionally, the cocultured supernatant of AML and HUVECs cells was collected. The above supernatant was centrifuged to remove cell debris and detected for VEGF protein level assay using ELISA kits.7. Real-time RT-PCR analysis:Total RNA was extracted from cells by TRIzol reagent and reverse transcription was conducted with M-MuLV reverse transcriptase. The mRNA expression levels of D114, Notch1, Hes1, Hey1, Hey2, VEGF, VEGFR1, and VEGFR2were investigated in the transfected AML or HUVECs cells by Real-time RT-PCR.8. Western blot analysis:Cells were lysed in radio immunoprecipitation assay (RIPA) and cell lysates were fractionated by10%SDS-polyacrylamide gel electrophoresis. The protein expression levels of D114, Notchl, and Hesl were detected in the transfected AML or HUVECs cells by western blot.9. Gelatin zymography analysis:Protein concentrations of cell-free coculture supernatants were determined with the bicinchoninic acid assay protein reagent kit. An equal amount of protein was seperated on10%SDS-polyacrylamide gel containing1mg/ml gelatin A, The effects of coculture of AML cells with HUVECs on the gelatinolytic activities of MMP2and MMP9were examined by gelatin zymography.Results:1. The effects of AML cells on endothelial cell function:①As determined by ELISA analysis, HL60, Kasumi, NB4and K562cells secreted VEGF protein in culture media.②CCK-8analysis showed that Transwell coculture with AML cells resulted in the increase in HUVECs proliferation, respectively. HUVECs proliferation was also increased in the presence of AML cell supernatants. In contrast with the above Transwell coculture, AML cells did not increase HUVECs proliferation significantly in direct contact coculture.③As assessed by cell migration assay, elevated endothelial cell migration was observed in the supernatants from AML cells.④Matrigel in vitro HUVECs tube formation assay demonstrated that the supernatants from AML cells significantly increased the tube formation by HUVECs.These data suggest that endothelial cell proliferation, migration, and tube formation are enhanced during Transwell coculture with AML cells through VEGF.2. The effects of AML cells on the Notch/D114pathway in endothelial cell:①Real-time RT-PCR analysis demonstrated that Notch1, D114, Hesl and Hey2expressions were increased in HUVECs after Transwell coculture with AML cells. ②Western blot analysis showed that the coculture HUVECs expressed more Notch1and D114at the protein level compared with HUVECs cultured alone.③Gelatin zymography analysis demonstrated that the Notch-responsive genes MMP2and MMP9activities were elevated in HUVECs after Transwell coculture with AML cells.Together, these results indicate that AML cells are able to induce activation of the Notch/D114pathway in HUVECs in Transwell coculture. Furthermore, AML-induced endothelial cell function is associated with concomitant activation of the Notch/D114pathway and upregulation of its downstream genes, such as MMPs in Transwell coculture.3. The effects of Notch1knockdown in endothelial cell on AML-induced endothelial cell function:①As assessed by real-time RT-PCR and western blot analysis, the mRNA and protein expression of Notch1and Hesl showed a remarkable decrease in Notch1siRNA-transfected HUVECs, whereas the transfection of Notch1siRNA resulted in no significant alteration in the D114protein expression, suggesting that Notchl siRNA effectively downregulates the expression level of Notch1in HUVECs.②Transwell coculture of Notch1siRNA-transfected HUVECs only with NB4cells was conducted. Gelatin zymography analysis indicated that MMP2and MMP9activities were significantly reduced in Notchl siRNA-transfected cells compared with controls.③Downregulation of Notchl expression by siRNA resulted in a significant decrease in the proliferation, migration, and tube formation of HUVECs during Transwell coculture with NB4cells.Data provide evidence that downregulation of Notchl expression by siRNA inhibited endothelial cell proliferation, migration, and tube formation during Transwell coculture with AML cells.4. The effects of D114overexpression in AML cells on AML cell proliferation and endothelial cell function: ①NB4and K562cells were transfected with a D114-expressing plasmid to achieve moderate levels of D114expression. As detected by real-time RT-PCR, D114mRNA levels in NB4-D114and K562-D114cells were increased by4.0-and5.6-fold, respectively. Western blot also showed D114protein levels were significantly upregulated after transfection with a D114-expressing plasmid, suggesting that the D114-expressing plasmid effectively upregulates the expression level of D114in AML cells.②D114-tansfected AML cells showed no significant differences in the growth rate as compared with controls. Subsequently, HUVECs coculture with D114-tansfected cells were conducted in direct contact and Transwell cocultures. Upregulation of D114expression by cDNA transfection markedly inhibited the coculture HUVECs proliferation in direct contact coculture, whereas no apparent changes in Transwell coculture occurred.③In vitro angiogenesis analysis demonstrated that D114overexpression partially blunted network formation of endothelial cells in direct contact coculture. However, no marked difference in tube formation of endothelial cells was observed between D114-transfected group and control-transfected group in NB4and K562cells in Transwell coculture.Taken together, our data demonstrate that upregulation of D114expression in AML cells by cDNA transfection suppressed endothelial cell proliferation and tube formation in direct contact coculture.5. The effects of D114overexpression in AML cells on the Notch/D114and VEGF signaling pathways in endothelial cell:②Real-time RT-PCR and western blot analysis showed that upregulation of D114expression in NB4and K562cells was associated with a significantly elevated expression of Notch1, Hes1and Hey2mRNA as well as Notch1and Hesl protein in HUVECs only in direct cell-cell contact.②MMP2and MMP9activities were significantly reduced in D114-transfected AML cells compared with controls only in direct contact coculture. ③By ELISA analysis, VEGF protein levels in the coculture supernatants were markedly decreased in direct contact coculture, whereas no signficant alteration in Transwell coculture occurred.④In agreement with endothelial cell function inhibition, VEGFR2mRNA levels were markedly reduced in HUVECs coculture with D114-transfected AML cells compared with controls in direct contact coculture. Conversely, activation of the Notch pathway by D114led to increased VEGFR1mRNA expression in direct contact coculture.Together, these results indicate that activation of the Notch pathway by D114regulates MMP2and MMP9activities through reduced expression of VEGFR2, whereby VEGF-induced angiogenesis was inhibited in direct contact coculture.6. The effects of VEGF activation on D114-induced endothelial cell function:①Compared with controls, treatment with rhVEGF determined a strong angiogenic response with formation of complex-mesh structures.②This was associated with concomitant upregulation of MMP2and MMP9activities, consistent with the increased angiogenesis.Thus, these data demonstrate that VEGF is responsible for D114-induced endothelial cell function inhibition and the activation of VEGF pathway rescued D114-induced endothelial cell function inhibition in direct contact coculture, as well as provide evidence that D114overexpression could suppress VEGF-induced endothelial cell function in leukemia-endothelium cross-talk.Conclusion:1. Our present study demonstrated that AML cells increased endothelial cell proliferation, migration and tube formation in Transwell coculture through activation of the VEGF and Notch/D114pathways, accompanied by upregulation of MMP2and MMP9.2. Moreover, our data showed that upregulation of D114expression in AML cells by cDNA transfection suppressed VEGF-induced endothelial cell function in direct contact coculture.3. Taken together, we identifed that the Notch/D114pathway activated by VEGF initiated a novel signaling cross-talk between AML and endothelial cells that promoted angiogenesis. Modulation of this pathway may, therefore, hold promise as a novel antiangiogenic strategy for the treatment of AML. |
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