The High Expression Of HSP70-2 In Low-metastatic Potential 6-10B Cells Of Nasopharyngeal Carcinoma Regulated By Dinitrosopiperazine

Objective:In this project, we investigated that DNP induced HSP70-2 expression in NPC cell 6-10B and explore the mechanism of DNP involving in NPC metastasis.Methods:1.NPC cell lines,6-10B with low metastasis,5-8F with high metastasis were served as cells model. Cytotoxicity of DNP on 6-10B cells was detected using Methylthiazolyltertrazolium (MTT). non-cytotoxic concentration (NCC) was determined.2.6-10B cells were treated DNP at non-cytotoxic concentration, and untreated 6-10B cells and 5-8F cells served as control groups, and then these cell samples were used to the next experiments.3.After 6-10B-treated with DNP, HSP70-2 expression in 6-10B cells was detected using indirect immunofluorescence, and western -blotting was used to detected dose-course of DNP induced -HSP70-2.4.Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was used to detected gene expression of Hsp70-2.5.SPSS18.0 was used to analyze the data.Results: 1.MTT assay data showed that DNP could inhibit the proliferation of 6-10B cells at the concentrations higher than 100μmol/L (p<0.05), and no effect less than 100μmol/L.0～100μmol/L is the non-cytotoxic concentration (NCC) of DNP.2.The results of indirect immunofluorescence showed HSP70-2 mainly expressed in cytoplasm after DNP treatment at NCC, and DNP-induced cells and 5-8F cells had stronger fluorescence intensity than untreated 6-10B cells.3.Western Blotting was used to detected the dose-course of HSP70-2 expression. The results showed that HSP70-2 expression was higher in DNP-induced 6-10B cells and 5-8F cells than that in untreated-6-10B cells (P<0.05), DNP induced HSP70-2 expression at dose-course.4.Real-time RT PCR was used to detected Hsp70-2 RNA expression. The results showed that Hsp70-2 RNA expression rate was 1.04±0.33 in the untreated,0.81±0.14 in the treatment at 50μmol/L,0.81±0.26 in the treatment at 100μmol/L. There is no significance between treatment and non-treated cells. However, the relative fold gene expression of Hsp70-2 in 5-8F cells was higher than that in 6-10B cells.Conclusions:1.After DNP treatment, HSP70-2 expression dramatically increased. This implies that DNP can upregulate HSP70-2 expression in 6-10B cells. 2.There was no significance between the DNP-treated cells and untreated cells at the mRNA level. This suggested that DNP could not regulate Hsp70-2 expression at RNA, may regulate HSP70-2 protein by Post-transcriptional regulation.3.The higher expression of HSP70-2 in 5-8F cells indicated that HSP70-2 may be associated with the malignant properties (metastatic potential) of tumor.