The Effect Of PLK4 Inhibitor Centrinone Combined With ATRA On Neuroblastoma

Neuroblastoma,a neural crest derived embryonal malignancy,is the most common and deadly extracranial tumor of childhood.It represents 6%-10% of all childhood tumors and accounts for 12%–15% of all childhood cancer related death.High-risk neuroblastoma(HR-NB)accounts for more than 60% of NB.With the improvement of chemotherapy regimen and the development of comprehensive treatment,the prognosis of NB has been significantly improved.However,the five-year survival rate of children with HR-NB is still less than 40%,even though adopt the therapy that included chemotherapy,radiotherapy,surgery,consolidation therapy with high-dose chemotherapy rescued with autologous stem cell transplantation(ASCT),etc.However,despite the intensive multimodal therapy,more than 50% of them will relapsed.During the development of NB,the most important characteristic of NB is that there are different differentiation stages of tumor cells in the same tumor sample at the same time.This characteristic is closely related to differentiation disorder.Most NB at 4S stage have the characteristics of spontaneous regression,that is,neuroblasts differentiate into mature ganglion cells.Children with NB with self-differentiation potential have good prognosis.However,NB patients with extensive metastasis have poor prognosis and are prone to recurrence.The existence of minimal residual disease is one of the reasons for the recurrence,and isotretinoin(13‐cis retinoic acid)can be used for the elimination of minimal residual disease.But for patients receiving isotretinoin treatment,the fiveyear overall survival rate is still only approximately 60%.PLK4 plays an important role in centriole duplication.The deregulation of PLK4 can lead to mitotic defect and contribute to tumorigenesis.Its abnormal expression is associated with poor prognosis in breast cancer,liver cancer,ovarian cancer,acute lymphoma and so on.In addition,PLK4 is endogenously overexpressed in NB,which can promote the migration and invasion of NB cells and enhance the growth and metastasis of implanted tumors in nude mice.Based on this phenomenon,researchers have developed a variety of inhibitors against PLK4 which as a target of clinical treatment,such as CFI-400945,YLT-11,Centrinone etc.Centrinone can inhibit centriole duplication and delay cell mitosis.The purpose of this study was to investigate the effects of the PLK4 inhibitor centrinone and its combination with all-trans retinoic acid(ATRA)on centriole duplication,DNA damage and cell function in NB cells.Methods:1.To analyze the expression of PLK4 in tissuesAdopting immunohistochemistry and western blot to verify the expression of PLK4 in NB tissues and cells as well as in ganglioneuroma tissues.2.To verify the effect of PLK4 inhibitor centrinone on the function of NB cells(1)To detect the IC50 of NB cell lines after adding centrinone by MTT assay.(2)To verify the effects of inhibitor centrinone on proliferation,differentiation and apoptosis of NB cell lines by plate cloning and Western blot.3.To verify the effect of PLK4 inhibitor centrinone on centriole duplication and DNA damage repair by western blot.4.To induce differentiation of NB cell lines by using all-trans retinoic acid,then to detect the expression of Rac1,Rho A,S-100,NSE and other differentiation-related indicators,and after the differentiation model of NB cells was established,test the expression of PLK4、P-PLK4(S305)and γ-tublin.5.Add centrinone and ATRA into NB cells.To verify the effects of the combination on proliferation,migration,differentiation and apoptosis of NB cells by western blot,and test the expression of γ-tublin,53BP1 and BRCA1.Results:1.By examining the patient’s tumor tissue samples,found that the expression of PLK4 in cancer tissues was higher than that in ganglioneuroma and the expression of PLK4 in high-risk NB was higher than that in low-risk NB.2.MTT assay showed that the IC50 value of SK-N-Be(2)cells was 346.5n M and 287.1n M in SH-SY5 Y cells when the inhibitor centrinone was added.After adding centrinone,cell proliferation inhibition,cell differentiation and apoptosis were observed.3.After adding centrinone into NB cells,the expression of γ-tublin decreased,the expression of P53 and BRCA1 increased,and the expression of 53BP1 decreased in NB cells.4.After adding ATRA to establish differentiation model,the expression of γ-tublin decreased.5.After the PLK4 inhibitor centrinone and ATRA were jointly added into NB cells,found that : a,the growth of NB cells was inhibited(P < 0.05),the proliferation of NB cells was significantly decreased compared with that of the single-drug group.b,the migration ability was decreased,but there was no significant difference compared with that of the centrinone group(P > 0.05).c,the expression of Rac1 was increased while the expression of Rho A was decreased.d,the expression of cleaved-casepase 3 and PARP-1 splicing protein was more obvious than that of the single-drug group.e,in addition,the expression of BRCA1 and 53BP1 decreased after combined.Conclusions:1.PLK4 is highly expressed in NB tissues and cells.2.Centrinone can inhibit cell proliferation,centriole duplication,promote cell differentiation and apoptosis,and promote DNA damage.3.ATRA can inhibit centriole duplication.4.Centrinone in combination with ATRA can inhibit cell proliferation,promote cell differentiation and apoptosis,and the combination is more effective than use centrinone and ATRA alone.In addition,they have stronger ability to promote DNA damage and can inhibit the repair ability after DNA damage.