Reversal Of Drug Resistance By Inhibiting Sonic Hedgehog Signal Pathway In Refractory Acute Myeloid Leukemia And The Underlying Mechanism

Background and Objectives:Acute myeloid leukemia (AML) is one of the most common malignant tumors in hematologic system, the proportion is about60%of all leukemias, is due to regulatory factors disorder, blocked cell differentiation, uncontrolled proliferation, during the process of development of hematological stem or progenitor cells, the treatment of the disease is mainly chemotherapy. Although60-80%of patients achieved complete remission and prolong the survival period, but there are still20%-40%of patients with newly diagnosed cannot achieved complete remission, even obtain remission, of which there are40%-60%patients relapsed and become refractory leukemia. The multidrug resistance of leukemia cells (multidrug resistance, MDR) is still a big problem in the treatment of hematological malignancies, the molecular mechanism of MDR was recently thought that geneic abnormalities resulting in signal transduction pathway, the imbalance of cell signaling network, is an important mechanism of drug resistance of leukemia cells. Study found during the process of multi-drug resistance, multiple signal transduction pathway were activated, involved in the mechanism of drug resistance cells, whereas the current pathway more IGF-1R/PI3K/Akt pathway, RAS/RAF/MEK/ERK pathway, the ubiquitin proteasome pathway.HL-60/ADM cells are recognized as a multidrug resistant cell line of acute myeloid leukemia, our preliminary study proved that different concentrations of proteasome inhibitor Bortezomib combined with LBH589in HL-60/ADM cells and refractory AML has synergistic inhibition of proliferation and induction of apoptosis, and expression regulation of MRP1can significantly improve adriamycin uptake, intracellular adriamycin resistance, reversal and reduce the activity of the PI3K/AKT/NF-KB pathway, participate in the reverse AML resistance. Early results showed, primary cell gene of refractory and non refractory AML-M2a expression significantly, and through the analysis of integration found depth cell bioinformatics, refractory AML-M2a biological signal transduction network regulation by the development of a very complex, in which Sonci hedgehog pathway may be the upstream pathway and can regulate other signal pathways.Mammalian Hh protein family has three members, respectively Shh (Sonic hedgehog), Ihh (Indian hedgehog), Dhh (Desert hedgehog), in which Shh expression is the most widely used, and various organs formation, mainly by the signal molecule Shh, membrane receptor patched (PTCH), smoothened (Smo), some intermediate transfer molecules and transcription factor Glis. The pathway plays an important role in embryonic development, in the repair and regeneration of the environment and maintain the stable tissue after injury, and its dysregulation is often lead to the occurrence of tumor and other diseases. The abnormal activation can lead to the generation, many kinds of tumors such as:basal cell cancer, medulloblastoma, pancreatic cancer, prostate cancer and so on. The pathway and chronic myelogenous leukemia stem cells CD34+leukemia cell proliferation, drug resistance and a variety of diseases such as lymphoma, myeloma associated has confirmed the blood system diseases. But the correlation between Shh signal and other signals were not clear.This paper intends to study the expression of Shh pathway in cell lines and primary AML cells, also the correlation between expression and clinical prognosis, using pathway blocker NVP-LDE225on multidrug resistance of acute myeloid leukemia cell line HL-60/ADM, inhibit the proliferation, induction of apoptosis and reversing drug resistance. In order to analyze the Sonic hedgehog pathway in refractory leukemia molecular mechanism in the development of new ways, new therapeutic targets and effective prevention from the search for the early diagnosis of refractory AML index, refractory AML.Objects and methods:1、Detection of Sonic hedeghog pathway level:①acute myeloid leukemia cell line HL60and the corresponding multidrug resistant cell line HL60/ADM as the research object, and mononuclear cells were isolated from35patients with refractory acute myeloid leukemia and38patients with non refractory acute myeloid leukemia patients bone marrow using lymphocyte separation liquid, QRT-PCR detection of cell lines and19cases of refractory AML,23patients with non-refractory AML mononuclear cells in Hedgehog pathway components Gli-1, Ptch, Smo gene expression.②The extraction cell lines and primary cell protein, Western blot method for detection of cell lines and16cases of refractory AML,15patients with non-refractory AML in Gli-1, Ptch, Smo, Shh protein expression, discussion on Shh signaling pathway in the treatment of refractory acute myeloid leukemia in the carcinogenesis and development.2、Doxorubicin (ADM) monotherapy in different effect on HL60cell and HL60/ADM cell proliferation inhibition rate, calculated using the MTT method, the24h IC50value between HL-60/ADM and adriamycin sensitivity of HL-60cells to adriamycin, the drug resistance is30times larger than the defined as cell resistance. After HL60and HL60/ADM cells were cultured in RPMI-1640containing10%serum culture medium with different concentrations of NVP-LDE225, observe the time-dose effect dependent proliferation. Choose the dose of no obvious proliferation inhibition. The HL-60/ADM cells were divided into ADM, Ara-C, DNR, HHT treated-group, NVP-LDE225treatment combined with ADM, Ara-C, DNR, HHT group, HL-60cells were divided into ADM group, NVP-LDE225combined with ADM treatment group, each group of cell number consistent treatment in each group were set up, the blank control group. Groups were detected by MTT cell proliferation, survival curves, calculating the reversal of drug resistance. 3、NVP-LDE225monotherapy in HL60/ADM cells, AnnexinV-FITC/PI staining was used to detect apoptosis, flow cytometry was used to detect the adriamycin uptake rate of HL-60/ADM cells. Total protein in HL60/ADM cells after treatment were extracted and detect changes by Western Blot to detect the protein level of MRP1. To investigate the molecular mechanism of Sonic hedgehog pathway inhibitors reversed the resistance of refractory leukemia cells.Changes in proteinic levels including downstream protein and apoptosis protein were detected by Western Blot method in HL60/ADM cells after treatment of NVP-LDE225monotherapy, detection of proteins including IGF-1R, p-IGF-1R, IRS-1, Akt, p-Akt, Gli-1, Bcl-2, to investigate the effect of the pathway of IGF-1R/PI3K/Akt channel blockers application.4、Mononuclear cells in bone marrow of refractory AML were isolated mononuclear cells under sterile conditions,and divided into NVP-LDE225single treatment group, NVP-LDE225and ADM treatment group, cell proliferation was detected by MTT, draw the proliferation curve, calculate the reversal of drug resistance. For the detection of NVP-LDE225and treatment with different concentration of primary cells apoptosis and adriamycin uptake rate of flow cytometry, changes in cell surface MRP1protein by Western Blot method for detection of drug treatment.5、Choose the strongest synergistic effect of concentration of21nM LBH589combined with12nM Bortezomib(preliminary results) on HL60/ADM cells48h, changes of Western blot detection of Shh channel protein Gli-1and protein IGF-1R.6、Statistical analysis was performed using SPSS13.0software. Two sets of measurement data were compared by statistical analysis using two independent samples t test, non normal data with median said, the normal distribution with mean and standard deviation of data description. The two groups were compared using the chi square test rate. Single factor analysis of variance with more groups, Levene test of homogeneity of variance, Kamo Sai uses the LSD method for multiple comparison, if the variance not neat is using the approximate F test (Welch) instead of variance analysis using Dunnett’s T3method for multiple comparisons. The p<0.05that have the difference of statistics.Results:1、The relative sensitivity of HL60cells, multidrug resistance cell line HL60/ADM in gene level and protein level of Sonic showed high expression of hedgehog pathway. QRT-PCR was detected in19patients with refractory AML and23patients with non refractory AML, mRNA expression showed that, relative expression of Gli-1, Smo, Ptch in refractory AML were respectively0.050(0.011～1.168),0.108(0.011～3.589) and0.130(0.009～2.916), and in the relative expression of non refractory AML were respectively0.025(0.004～0.099),0.039(0.002～0.306) and0.036(0.005～0.367). Two sample t test results suggest that:the P values were0.031,0.018,0.043, there were significant differences. Western blot was detected in16cases of refractory and relapsed AML and15patients with non refractory primary AML specimens, there were13cases and4cases expressed Gli-1, the positive rates were81.3%and26.7%, chi square test results suggest that there were significant differences in P=0.002. Analysis of31cases of primary Gli-1cell AML expression to clinical treatment response and long-term survival, Gli-1high expression group17cases, refractory AML accounted for13cases (76.5%), non refractory AML accounted for4cases (23.5%),10cases were≤2courses of CR were(58.8%),more than7cases2courses of CR or NR (41.2%),7cases of recurrence within one year or more after recurrence of CR (41.2%, CR) after a year recurrence or relapse free accounted for10cases (58.8%);14cases low expression, refractory AML accounted for3cases (21.4%), non refractory AMLin11cases (78.6%),11cases were≤2courses of CR were (78.6%), more than2courses of CR or NR in3cases (21.4%),1cases of recurrence within one year or more after recurrence of CR (7.1%, CR) after a year recurrence orrelapse free accounted for13cases (92.9%); chi square test indicated:the expression of Gli-1in CR patients had no significant effect on the rate(P=0.242), on whether the occurrence of refractory AML and recurrence within one year or has a significant effect on relapse (P=0.002and0.031). The high expression of Gli-1in RFS patients were4.75,6.5months; median RFS, OS patients with lower expression of Gli-1were14,15months. High expression of RFS and OS, there were significant differences between the Gli-1and the low expression of Gli-1(P<0.05).2、MTT method for detection of adriamycin sensitivity:IC50adriamycin to HL60/ADM cell value is about6.833μg/ml, acting on the HL60cells and the value of IC50is0.086μg/ml, HL60/ADM cells resistance multiple of79times HL60, indicating that HL60/ADM cells to adriamycin resistant, can be used as the object of study the next step. NVP-LDE225of low concentration of HL60and HL60/ADM cell inhibitory effect is not significant, dose of cell proliferation rate of0-10μM range is greater than80%, no significant inhibition of cell proliferation. No concentration significantly inhibited the proliferation of cells of10μM combined with different concentrations of ADM, Ara-C, DNR, HHT chemotherapy drug testing whether has the effect of reversing multidrug resistance. IC50doxorubicin alone on HL60/ADM cells is0.061μg/ml, combined with NVP-LDE225-10μM IC500.016μg/ml, reverse multiples of3.75times; IC50daunorubicin alone on HL60/ADM cells is0.213μg/ml, combined with NVP-LDE225-10μM IC500.04μg/ml, reverse a multiple of5.08times. IC50homoharringtonine alone on HL60/ADM cells is0.024μg/ml, combined with NVP-LDE225-10μM IC500.011μg/ml, reverse fold was2.18. IC50cytarabine alone on HL60/ADM cells is40.8μg/ml, combined with NVP-LDE225-10μM IC500.17μg/ml, reverse fold was234. IC50doxorubicin alone on HL60cells is0.00331μg/ml, combined with NVP-LDE225IC500.0031μg/ml. Two sample t test results suggest that:NVP-LDE225-10μM can reverse the multidrug resistance of HL60/ADM cells to ADM, DNR, HHT, Ara-C (P<0.05). Without this phenomenon in HL60cells (P=0.692).3、different concentrations of NVP-LDE225in HL60/ADM cells treated with48h, cell apoptosis was detected by flow cytometry. The control of apoptosis cells group was4.52±1.29%,5μM, lOμM and20μM NVP-LDE225total HL60/ADM cell apoptosis rate were6.17±1.73%,11.82±4.21%,52.41±10.57%. Single factor analysis of variance results show, different concentrations had significant difference between the total HL60/ADM cell apoptosis rate of48h cells (F=46.010; P=0.007). Dunnett T3multiple comparison analysis method, and the control group and the low concentration NVP-LDE225treatment group (5μM,10μM), with the increase of drug concentration, high concentration of NVP-LDE225(20μM) treatment group total apoptosis cells were increased, P values were less than0.05. HL-60/ADM cells with different concentrations of NVP-LDE225after48h treatment, adding doxorubicin concentration is0.5μg/ml, continue to culture1H flow cytometry positive rate of intracellular fluorescence detection, single factor variance analysis showed, there was significant difference between control group, different concentration of adriamycin treatment group the positive rate (F=306.924, P=0.000). LSD multiple comparison analysis showed,5μM NVP-LDE225treatment group and lOμM NVP-LDE225group were65.89±3.93%,70.58±4.71%, significantly higher than the single drug group was3.32±1.93%(P=0.000). Extraction of NVP-LDE225protein after treatment with different concentrations of Western blot detection, can significantly reduce the cell surface expression of MRP1. Display6different protein changes, HL-60/ADM cells to detect Western concentration of blot after NVP-LDE225treatment, the control group and drug treated with different concentrations of IGF-1R, Akt protein levels remained unchanged, and different groups of p-IGF-1R, IRS-1, p-Akt, Bcl-2, Gli-1levels exist significant differences.4、primary cell experiment:doxorubicin alone in refractory AML primary cell line IC50was0.0426±0.0013μg/ml, combined with NVP-LDE225-10μM IC50is0.0119±0.0007μg/ml, reverse fold was3.58. Two sample t test, P value is0, with significant difference. Different concentrations of NVP-LDE225treatment of refractory AML in primary48h cells, flow cytometry showed:the control of apoptotic cells group was21.67±3.06%,5μM, lOμM and20μM NVP-LDE225total apoptosis of primary cells were33.23±1.08%,48.2±2.03%,66.03±4%. Single factor analysis of variance results show, different concentrations of total apoptosis of primary48h cell rate had significant difference (F=144.483; P=0.000). LSD multiple comparison analysis shows that, with the increase of drug concentration, different treatment group total cell apoptosis rate increased, P values were less than0.05. Primary cells cultured with different concentrations of NVP-LDE225after48h treatment, adding doxorubicin concentration is0.5μg/ml, continue to culture1H flow cytometry positive rate of intracellular fluorescence detection, single factor variance analysis showed, there was significant difference between control group, different concentration of adriamycin treatment group the positive rate (F=174.347, P=0.000). LSD multiple comparison analysis showed,5μM NVP-LDE225treatment group,10μM NVP-LDE225,20μM NVP-LDE225group were16.2±1.68%,25.05±1.78%,42.91±2.72%, significantly higher than the single drug group was4.697±2.12%(P=0.000). LSD multiple comparison analysis shows that, with the increase of drug concentration, different cells treated with adriamycin uptake rate was increased, P values were less than0.05. Extraction of NVP-LDE225protein after treatment with different concentrations of Western blot detection, cell surface MRP1expression decreased obviously.5、Bor combined with LBH589could downregulate expression of p-IGF-1R and Gli-1, prove the previous conclusion, indicating the targeted point is Shh pathway.Conclusion:1、Compared to the non-resistant cells and non refractory primary AML cells, the expression of Sonic Hedgehog pathway is higer in drug-resistant cell lines and refractory primary cells. Higher expression has the high incidence of refractory AML and relapse, also low RFS and OS.2、The Sonic hedgehog pathway inhibitor single drug NVP-LDE225-10μM can reverse the drug resistance to ADM, DNR, HHT, Ara-C in HL60/ADM cell line. With the increasing concentration of NVP-LDE225, the apoptosis rate of HL60/ADM cells and adriamycin uptake rate was significantly increased, the cell surface expression of MRP1was significantly lower. NVP-LDE225can decrease the intracellular expression of p-IGF-1R, IRS-1, p-Akt, Gli-1protein, suggesting that Sonic hedgehog pathway can reverse the drug-resistance by regulating IGF-1R/PI3K/Akt pathway. In refractory primary AML cells also confirmed the conclusion.3、LBH589combined with Bortezomib can downregulate the Shh pathway to inhibit the IGF-1R/PI3K/Akt pathway to invese drug-resistance.