Hedgehog Pathway Mediates Resistance To Radiation And Is A Target For Reversion Of Responses In Refractory Acute Myeloid Leukemia Cells

BackgroundAcute myeloid leukemia (AML) is one of the most prevalent malignant tumors with shorter survival, and caused by uncontrolled proliferation of hematological stem or progenitor cells. Stem cell transplantation (SCT) continues to be an effective treatment. Total body irradiation (TBI) combined with chemotherapy is currently the most common procedure of traditional preparative myeloablative regimen. However, there are still high failure rate in patients who receive TBI before SCT, especially in refractory AML. One of the causes of failure to treatment which is still unclear is attributable to radio-resistant leukemia cell clones remain. Therefore, understanding the mechanisms of resistance to radiotherapy and increasing the therapeutic efficacy are significant for devising novel therapies in AML, such as using targeted drug as a radiation sensitizer.The hedgehog (Hh) signaling pathway plays a key role for embryonic development, and is necessary to support tumorigenesis, proliferation and metastasis in numerous tumor types. Many previous studies have also demonstrated that overexpression of Hh signaling genes have been link to radiation resistance, and down-regulation can enhance radiation response in many tumor types, including pancreatic cancer, anaplastic thyroid cancer, esophageal cancer, and non-small cell lung cancers. In addition, several clinical studies showed a positive correlation between overexpression of Hh signaling genes and poorer outcomes in various kind of cancer. Since many genes involved in controlling cell cycle, signal transduction, apoptosis, and repair of DNA damage were regulated by Hh signaling, the Hh inhibitors were considered as potential target to improve radiation response. For example, targeted inhibition of Hh as induction treatment followed by radiation was reported as a new therapeutic strategy and promising treatment option in basal cell carcinoma.Crosstalk between hedgehog and other signaling pathways may partly explain the mechanism for radiosensitivity enhancement. One possible is PI3K/AKT pathway, which is well known to be implicated in radioresistance. Another possible reason is Nuclear factor kappaB (NF-kB), which could be considered as an essencial factor for DNA repair induced by radiation. Deregulated PI3K/AKT and NF-kB activity are the central mediators of cancer cell responsiveness to radiation. Although the detailed mechanisms of Hh signaling effect on PI3K/AKT and NF-kB pathway is still unclear, several studies have shown that Hh protein could promote cell proliferating, migration and VEGF production through PI3K/AKT pathway, and another study demonstrated that PI3K/AKT signaling are essential for Hh pathway.On the contrary, the activity of PI3K/AKT and NF-kB can be reduced by using Hh inhibitors.Previous study showed that Rad51 is an HR-specific protein, which could be decrease by PI3K/AKT inhibitor, and high levels of Rad51 could enhance resistance to DNA damage. In addition, Rad51-mediated HR is necessary for cell survival in radioresistance HL60 cell line, and down-regulation of Rad51 expression could overcome radiation resistance potentially.In particular, a recent study has indicated that aberrant Hh pathway is a negative prognostic factor in acute myeloid leukemia (AML). Moreover, other studies have shown that Hh signaling is essential for the survival and drug resistance of leukemia cells. Interestingly, a recent report demonstrated that inhibition of the Hh pathway with LDE225 could sensitize to 5-azacytidine in AML cells, and a clinical trial based on these results is ongoing. However, the effects and mechanisms of Hh pathway signaling on radiation resistance, and the application of inhibitor in acute myeloid leukemia have not been reported. In the present study, we hypothesized that disruption of Hh could increase sensitivity to ionizing radiation of radiation-resistant leukemia cells.Materials and Methods1. Cell establishment of HL60/RX cells:HL60 cells were irradiated 12 times over 3 months to produce the radiation-resistant HL60(HL60/RX) subline. In the present study, we used 2 cell lines including HL60/ADR (a adriamycin-resistant cells), and HL60 cells as control models to investigate the mechanism of Hedgehog signaling pathway resulting in resistance.2. Radiosensitivity of HL-60, HL-60/RX and HL-60/ADR cells:Sensitivity parameters (DO, Dq, SF2), y-H2AX foci, and apoptosis rate were analyzed to investigate their response to radiation on HL-60, HL-60/RX and HL-60/ADR cells by clonogenic assays, flow cytometry, and confocal fluorescent microscope respectively.3. Hedgehog signal pathway significantly enhances the radioresistance on HL60/RX and HL60/ADR cells:To investigate the role of Hh signaling pathway in radiation resistance of leukemia cells, we detected the expression of Smoothened (Smo), a key transducer of the Hh signaling pathway, and Hh target protein Glioma-associated oncogene family zinc finger 1 (Gli-1) in three cell lines by Western blot. On the contrary, HL60/ADR and HL60/RX cells were pretreated with NVP-LDE225(a novel synthetic Smo antagonist) or DMSO for control followed by radiation. Sensitivity parameters were calculated subsequently by Colony assay and sensitizer enhancement ratio (SER) was calculated as the DO value of radiation plus vehicle divided by the DO value of radiation plus LDE225. Furthermore, y-H2AX foci, and apoptosis rate were analyzed to evaluate the sensitivity to radiation.4. NVP-LDE225 pretreatment sensitizes HL60/RX and HL60/ADR cells to radiation via inhibition of the PI3K/AKT/NF-kB pathway:To further explore the mechanism of NVP-LDE225 on enhancing radiosensitivity in HL60/RX and HL60/ADR cells, cells were treated in four ways respectively:the control group), NVP-LDE225 alone group(10μmol/L), radiation alone group(4.8Gy), and combined NVP-LDE225 (10μmol/L) plus radiation(4.8Gy) group. We measured the expression of Gli-1, pAKT, NF-kB, Rad51, and BAK in the cells by western blot analysis after treatment. Furthermore, we observed the nucleus translocation of NF-kB by confocal fluorescent microscope-5. In vivo studies:Female BALB/c nude mice (6 weeks of age) were maintained in caged housing in specific pathogen-free facilities. Approximately 1×107 HL-60/ADR or HL-60/RX cells were injected subcutaneously into the right posterior flank of each nude mouse. Mice bearing HL-60/ADR or HL-60/RX tumors of 75-150 mm3 in volume were assigned randomly to four groups respectively (6 mice per group):HL-60/ADR cells+vehicle; HL-60/RX cells+vehicle; HL-60/ADR cells+ RT; HL-60/RX cells+RT; HL-60/ADR cells+NVP-LDE225; HL-60/RX cells+ NVP-LDE225; HL-60/ADR cells+NVP-LDE225+RT; HL-60/RX cells+ NVP-LDE225+RT. Tumor sizes was calculated as:V=0.5×axb2, where a is the longest and b is the shortest diameter of the tumor mass using caliper respectively. At 2 weeks, the mice were sacrificed and the tumors were fixed in 10% neutralized formalin overnight after weighting.6. Histopathological and immunohistochemical staining examinations:The tumor tissues were dehydrated, paraffin fixed, and cut into 4μm sections for histopathological and immunohistochemical (IHC) examination. Subsequently, slides were stained for Gli-1, NF-kB, p-AKT, and BAK protein expression using standard techniques. Hematoxylin and eosin staining were also performed and observed by light microscope.7.Statistical analysis:Results are presented as mean±standard deviation. Statistical analysis was performed by SPSS 22.0 using a Student’s t-test, one-way analysis of variance (ANOVA) followed by the Bonferroni test when equal variances were assumed, while Dunnett’s T3 test were used when equal variances were not assumed. P value<0.05 was considered statistically significant.Results1. HL60 cells were irradiated 12 times over 3 months to produce the radiation-resistant HL60(HL60/RX) subline successfully with the last dose 4.8Gy. The survival curve of cell lines after radiation showed that the Dq,D0, andSF2 values were 1.134±0.456 Gy,1.282±0.271Gy and 0.345±0.06 for HL60 cells,4.513±0.804 Gy,3.033±0.29 Gy, and 0.814±0.04for HL-60/RX cells,3.310±0.677 Gy, 2.437±0.259 Gy, and 0.730±0.04 for HL-60/ADR cells respectively. These results demonstrated that HL-60/RX and HL60/ADR cells have remarkable resistance to radiation compared with HL/60 cells (P<0.001). After 4.8Gy radiation, apoptosis ratio was significantly higher in HL60 (61.0%) than in HL60/RX (9.7%) and HL60/ADR (10.8%) cell lines (P<0.001), as well as the the y-H2AX positive level in HL60 exhibited significantly increases compared with HL60/RX and HL60/ADR (P<-.001). This data supported that the enhanced radiation resistance in radioresistant sublines is linked to a decrease in apoptosis and y-H2AX express.2. Compared with the parental cell, the radiation resistant cell line HL60/RX and HL60/ADR cells showed an overexpression of Hh signaling molecules including SMO and Gli-1. On the contrary, the results of clonogenic assays showed a significant decrease in clonogenic survival fraction at different radiation dosages both in HL60/ADR and HL60/RX cells after exposing to NVP-LDE225 with the SER were 1.283 for HL60/ADR, and 1.245 for HL60/RX. Moreover, the two radiation-resistant cells with combination of inhibitor and radiation could significantly increase radiation-induced apoptosis and exhibit higher expression of γ-H2AX foci than control or single agent. The results suggested that Hh signaling pathway activation may be associated with the resistant phenotype.3. After 48 hours of pretreatment with NVP-LDE225, significante decrease of Gli-1 were observed both in HL60/ADR and HL60/RX cells. Subsequently, the decreased in expression of pAKT and NF-kB (nucleus) were seen in cells treated with NVP-LDE225. In accordance with the PI3K/AKT/NF-κB pathway contributes to radiation resistance, overexpress of BAK (a key role in mitochondrial apoptosis) was observed in combined group, consistent with the results of apoptosis analyzed by flow cytometry. However, overexpress of pAKT and NF-kB (nucleus) with low express of BAK were observed in the radiation alone group. Correspondingly, using confocal fluorescent microscope, we observed that the NF-kB was translocated from cytoplasm into nucleus after radiation, but it become dysfunctional after induction treated with inhibitor. Therefore, these results supported that Hh pathway inhibitor sensitize cells exposed to radiation maybe associated with the decreasing expression of PI3K/AKT/NF-kB pathway, and inhibition of Hh pathway could reduce the DNA homologous recombination repair capativity.Rad51 is a critical player in homologous recombination repair, which could regulate by PI3K/AKT.Compared to control group, the expression of Rad51 was significantly increase after radiation both in HL60/ADR and HL60/RX. However, both HL60/ADR and HL60/RX cell lines treated with NVP-LDE225 and radiation had no increased level of Rad51. These findings revealed that HL60/ADR and HL60/RX cells exhibit higher DNA repair capacity by which become resistant to radiation.4. The two radioresistant leukemia cells bearing mice were established after inoculating into the right posterior flank of each nude mouse. The results showed that tumors grew rapidly in control group, NVP-LDE225 alone group, and radiation alone group, although monotherapy was able to retard growth slightly. However, combined radiation and NVP-LDE225 treatment resulted in a significant reduction in the growth of HL60/ADR and HL60/RX xenografts. Consistent with the results of tumor growth, the tumor weight of combining treatment group greatly lower than any other groups.Histopathological examination showed that tumors from the combining treatment group have more multifocal areas of necrosis than other groups. Consistent with the results in vitro, both HL60/RX and HL60/ADR tumor cells exhibit high expression of Gli-1 protein, which was reduced after treatment with NVP-LDE225 as expected. Additionally, the expression of pAKT and NF-kB were also reduced after NVP-LDE225 treatment, which is similar to the data obtained in vitro. Finally, combining radiation and NVP-LDE225 increases BAK expression than other treatment, and there were no difference among control group, NVP-LDE225 group, and radiation group. These experiments reveal that the protocol of pretreated with LDE225 followed by radiation could efficiently inhibit HL60/ADR and HL60/RX tumor growth in vivo, and the mechanism which LDE225 sensitizes cells to radiation may be through inhibition of Gli-1/pAKT/NF-kB pathway.Conclusion1. The expression of Hedgehog pathway activity in HL60/RX and HL60/ADR were significantly higher than that observed in HL60, on the contrary, the inhibition of Hedgehog pathway by the use of NVP-LDE225 significantly sensitizes HL60/ADR and HL60/RX cells to radiation. Hh pathway may play an important role in radiation resistance both in radio-resistant leukemia and drug-resistant leukemia cells.2. The enhanced radiation resistance in HL60/RX and HL60/ADR were linked to a decrease in apoptosis and increase in radiation DNA damage repair, which characters could be reversed by using Hedgehog pathway inhibitor.3. The effect of Hedgehog pathway inhibitor mediating radioresistance may through up-regulating Gli-1/PI3K/AKT/NF-kB pathway in vitro and in vivo.