1,25(OH)2D3 Promotes The Apoptotic Effect Of Adriamycin On Papillary Thyroid Cancer Cells

Objective:Papillary thyroid carcinoma?PTC?is the most common malignant tumor in the endocrine system.The incidence of growth rate that become the fastest rising entity tumor over the past 10 years.Although with the continuous development of the multi disciplinary cooperative diagnosis and treatment model,the prognosis of most PTC is better and the 10 year survival rate is about>90%.However,for stage IVB and IVC PTC patients often appear to be difficult to control the traditional treatment of local invasion and distant metastasis,and some lost the opportunity for surgical treatment of patients who are not sensitive to radioactive iodine therapy,the prognosis is poor.It is of great theoretical and practical significance to find new targets related to treatment and to treat refractory PTC.In 2012,China issued the"guidelines for the diagnosis and treatment of thyroid nodules and differentiated thyroid cancer"and in 2015 the American Thyroid Association?ATA?released the"adult thyroid nodules and differentiated thyroid cancer management guidelines"are mentioned in?Adriamycin,ADM?adriamycin can be used for the treatment of IVB and IVC PTC patients.ADM can play an antitumor effect in a variety of ways,Including inhibiting DNA topoisomerase II,increasing intracellular reactive oxygen species,activating p53,increasing cyclin G2 expression and inhibiting heat shock protein to regulate the proliferation,apoptosis and invasion of tumor cells.However,there are many side effects and dose dependence in the clinical application of ADM.A large number of experimental studies have proved that the synergistic effect or synergistic effect of drugs can improve the efficacy of drugs combined with many different anticancer mechanisms and it can reduce the adverse effects of the drug in recent years.Phosphorylated signal transduction and activator of transcription 3?p-STAT3?is a potential intracellular transcription factor that functions in the tumor cells to transmit the cytoplasmic signal and to initiate the double function of gene transcription in the nucleus.It can accelerate the formation of tumor by regulating cell cycle,apoptosis and invasion,and can accelerate the progression of tumor,and it is reported that ADM activates p-STAT3 while inducing cell apoptosis,which leads to the effect of apoptosis.Currently,we need a chemotherapy that requires a good effect and less side effects in clinic.It is possible to improve ADM apoptosis by inhibiting the activation of p-STAT3 while inducing apoptosis.Basing on the result of the study shows that 1,25-dihydroxy-vitamin D₃[1,25?OH?2D3]was used as a candidate drug for enhances the treatment of ADM in other types of malignant tumors.However,there is a lack of research on 1,25?OH?2D3 on ADM-induced apoptosis of PTC cells.The study is to investigate the changes of ADM induced apoptosis in PTC cells pretreating with1,25?OH?2D3.Therefore,we are going to explore its potential mechanism,meanwhile providing the basis for the clinical application of 1,25?OH?2D3 as ADM on PTC chemosensitizer.Methods:Since Dec.2014 to Apr.2016,there were 80 cases of PTC in thyroid surgery,and 80 cases of control with the same period of hospitalization.?1?Enzyme-linked immunosorbent assay?ELISA?detects serum level of 1,25?OH?2D3.?2?Vitamin D3receptor?VDR?and p-STAT3 expression were detected by streptavidin-peroxidase immunohistochemical staining?IHC?in PTC specimens and clinical significance were analyzed.?3?PTC cell line K1 and IHH4 for cell experiment.CCK-8 detects the effect on the proliferation.?4?Flow cytometry?FCM?detects the influence on apoptosis.?5?Western blot detects the protein expression changes such as cleaved caspase-3,p-STAT3,VDR,Bcl-xL and PTPN2.?6?Lentiviral vector was used to overexpress p-STAT3 in K1and IHH4 cell lines.?7?Small interfering RNA?siRNA?were used to knock down VDR and PTPN2 in K1 and IHH4 cell lines.Results:?1?ELISA:The level of serum 1,25?OH?2D3 in the PTC group was lower than that in control group?P=0.028?.?2?IHC:p-STAT3 had a positive expression in majority PTC tissue[up to 63.8%],and low expression in paired adjacent non-cancerous tissue?26.2%?.?3?The positive expression rate of VDR in PTC tissue cellar nucler was 32.5%,and that has a significant difference in the nuclear expression of p-STAT3?56.2%??p<0.05?.There was a negative correlation between the nuclear expression of p-STAT3and VDR in PTC tissue?R²=0.227?.?4?The level of serum 1,25?OH?2D3 in the PTC was irrelevant in clinical parameter.The expression of p-STAT3 in PTC tissues was not related to the age,sex,clinical stage and risk of recurrence of PTC,but it was associated with tumor size and lymph node metastasis?P=0.012,P=0.039?.?5?CCK-8:1,25?OH?2D3 could increase ADM inhibting the proliferation of PTC cells.?6?FCM:1,25?OH?2D3 could increase ADM induced apoptosis in PTC cells,and the change trend of apoptosis inhibitory protein Bcl-xL was consistent with the expression of p-STAT3.?7?Up-regualted H19 inhibited tumorigenesis of PTC in vivo.?8?Overexpression of p-STAT3 can rescue 1,25?OH?2D3 to increase the inhibitory effect of ADM on PTC cells,apoptosis and the expression of apoptosis related protein Bcl-x L,cleaved caspase-3.?9?1,25?OH?2D3 can promote the increase in the expression of VDR in the nucleus of PTC,and increase the expression of PTPN2 in PTC cells.Knock down the expression of PTPN2 can rescue the inhibition of 1,25?OH?2D3 on p-STAT3.?10?Knock down the expression of VDR can rescue the expression of 1,25?OH?2D3 on PTPN2 and p-STAT3.Conclusions:?1?The nuclear expression of p-STAT3 has negative correlation with expression of VDR in the PTC tissue,and t is associated with tumor size and lymph node metastasis.?2?1,25?OH?2D3 could induce the expression of PTPN2,and it could inhibit ADM induced the expression of p-STAT3-Bcl-x L that increase the apoptosis effect of ADM on PTC cells.