The Effect Of Hyperthermia Combined With DNAJB1/DNAJA4 Deficiency On Melanoma Cells

Objective:Cutaneous melanoma,a skin cancer deriving from melanocytes,is an incasive cancer with poor survival rates and high metastases rate.Although melanoma is less common among squamous cell carcinoma and basal cell carcinoma,it causes a three-time higher mortality.Melanoma incidence is rising worldwide,with about 160000 new cases per year and 30%deaths.In its very first stages melanoma can be completely cured by surgery,while the prognosis for recurring or advanced disease is sober with a median survival time of less than 9 months and a 5-year survival rate lower than 5%.Recently,the therapeutic scenario has slightly changed thanks to the introduction of biological and immunological response modifiers,the survival rate of melanoma has been improved.Heat shock proteins(HSPs)are a family of highly conserved proteins,induced by stress and ubiquitously expressed across all species.HSPs are involved in the maintenance of normal cellular protein homeostasis by regulating the proper folding of newly synthesized protides and the transport and degradation of mature proteins.Hsps protect against stresses and increase resistance to subsequent stresses.According to their molecular weight and sequence homology,HSPs have been divided into different families,including HSP90,HSP70,HSP40 and small HSPs(HSPB).Many studies reported that the expression of HSP90 and HSP70 was more pronounced in the melanoma than in nevus and mainly located in cell nucleus.With the in-depth study,HSPs are widely expressed in melanoma and related with tumor grade,prognosis and survival time,indicating HSPs are probably considered as a target.Hyperthermia is a procedure by which the temperature of a specific body part or the whole organism is elevated above the standard physiological temperature(range 40-44? for 30-60 min).Treatment at these temperatures is cytotoxic for cells in an environment with a low pO₂ and low pH;both these conditions are found within tumor tissue due to insufficient blood perfusion.Hyperthermia has been used in the treatment of some neoplasms as a single agent or as an adjuvant mehod.Many studies revealed that hyperthermia not only promoted immunogenicity,but also affected the activity of antigen presenting,phagocytic function of NK cells and macrophagocyte and inuced various changes in cytoskeletal organization.Hyperthermia acts as a sensitizer to radiotherapy,chemotherapy and immunotherapy,but it is not always effective,and thus,this has attracted interest in developing effective combination strategies that exploit using hyperthermia in combination with other therapies.Hyperthermia upregulates a family of HSPs.Clinical researches have reported a linical benefit from use of HSPs inhibitors as a treatment of pancreatic cancer,head and neck squamous cell carcinoma,and bladder carcinoma.Preliminary study found HSPB1deficiency sensitizes melanoma cells to hyperthermia induced cell death and PCR array results observed hyperthermia could upregulated DNAJB1 and DNAJA4 expression in murine B16 melanoma cell line.Given the above results,we should detect that the expression of DNAJB1 and DNAJA4 in melanoma and we further detected whether local hyperthermia could affect DNAJB1 and DNAJA4 expression in melanoma tissue and melanoma cell lines.At last we tested the function and mechanism of DNAJB1 and DNAJA4 in melanoma cells,providing another agent in combination of hyperthermia for the treatment of melanoma.Material and Methods:1.Patients:The formalin fixed and paraffin embedded,histologically confirmed melanoma tissue blocks were archived from the Department of Dermatology,No.1 Hospital of China Medical Univerity,dating from 2013 to 2016.The tissues of melanoma subjected to hyperthermia in vitro were also from the Department of Dermatology,No.1 Hospital of China Medical University.The protocol of collecting human samples was approved by Institutional Review Board and was conducted in conform to the Declaration of Helsinki.We chose one murine B16 melanoma cell line and three different human melanoma cell lines(HTB140,A375,and A2058).2.Local hyperthermia treatment:yperthermia in vitro:tissues were cut into 2 equal parts,then fully submerged in the medium and used water bath ot heat at 37? and45? for 30min,respectively.Then they were incubated at 37? with 5%CO₂.Hyperthermia in melanoma cells:we used water bath to heat,and the procedure for hyperthermia was the similar with the above described.3.Immunohistochemical:A standard immunohistochemical(IHC)method was performed on 4?m of sections using the monoclonal antibodies of anti-DNAJB1 and anti-DNAJA4.Image-pro Plus software was used to assess the mean optical density at200×magnification.4.The expression of DNAJB1 and DNAJA4 mRNA:The expression of DNAJB1 and DNAJA4 mRNA was detected by fluorescent real-time quantitative PCR.Total RNA was extracted by using miRNeasy mini kit and reverse-transcribed with GoScript^TM Reverse Transcription System.The cDNA obtained was used as a template for subsequent PCR amplification and relative quantitative result was analyzed with double standard curve method.5.The expression of DNAJB1 and DNAJA4 analyzed by western blot:The expression of DNAJB1 and DNAJA4 were detected by western blot.Total protein was extracted and the concentration was measured with BCA method.The bands were visualized with ECL Western Blotting Substrate.6.The effect of DNAJB1/DNAJA4 on the cell viability:The cells transfected with or without DNAJB1-siRNA/DNAJA4-siRNA gene were seeded in 96-well plates and subjected to hyperthermia at 37? or 45? at day 0,1,2,3,and 4,MTS was added to the medium.The cell viability was analyzed by measuring the absorbance at 490nm using a spectrophotometer.7.The effect of DNAJB1/DNAJA4 on the cell cycle and apoptosis:Flow cytometry was used to analyze the effect of DNAJB1/DNAJA4 on the cell cycle and apoptosis.The cells transfected with or without DNAJB1-siRNA/DNAJA4-siRNA gene were seeded in6-well plates,then subjected to hyperthermia at 37? or 45?,and then APC Annexin?kit and PI were used to detect cell cycle and apoptosis,respectively.8.Statistical analysis:Statistical analysis was performed using GraphPad Prism.For more than two groups of experiments,the differences between each group were analysed by one-way analysis of variance(ANOVA).For experiments with only two groups,quantitative results were expressed as mean±SD,andpaired t-test,or Mann-Whitney test were performed for comparisons of group means.A p value of less than 0.05 was considered statistically significant.Results:1.The expression of DNAJB1 and DNAJA4 in melanoma:By immunohistochemical staining,we observed that DNAJB1 and DNAJA4 were slightly expressed in nevus tissues and melanoma(<1mm);however,a strong expression of DNAJB1 and DNAJA4 were observed in most of melanoma tissues(>1mm)(P<0.01).2.The expression of DNAJB1 and DNAJA4 in melanoma cells and tissues subjected to hyperthermia:One murine B16 melanoma cell line and three different human melanoma cell lines(HTB140,A375,and A2058)were used.Cell lines were cultured with temperatures at 37? and 45? for 30min.The results demonstrated that hyperthermia increased the expression of DNAJB1 and DNAJA4 in melanoma cells in time-dependant manner.The expression of DNAJB1 and DNAJA4 in melanoma tissues treated with hyperthermia was the similar to the results in cell lines,with obvious individual difference.3.Effect of silencing DNAJB1 or DNAJA4 combined with hyperthermia on the biological function in melanoma cell:We established melanoma cells knocked down of DNAJB1/DNAJA4.Compared to transfection with scrambled si RNA,Knockdown of DNAJB1 or DNAJA4 in melanoma cells significantly reduced cell viability at day 2,and persisted to day 4.Meanwhile,there was significant accumulation of the total apoptotic cells and distribution of cell cycle after transfection with DNAJB1-siRNA/DNAJA4-siRNA.Different cell lines presented different sensibility.Silencing of DNAJB1 or DNAJA4 combined with hyperthermia enhanced cell death,and change G0/G1 phase and G2/M phase proportion.Conclusion:1.The expression of DNAJB1 and DNAJA4 is correlated with melanoma which is very useful in clinic and research field.2.Hyperthermia can increase the expression of DNAJB1 and DNAJA4 in melanoma cells in time-dependant manner.3.Hyperthermia combined with silencing of DNAJB1 or DNAJA4 enhanced cell death and proportion of apoptotic cells,implying the potential clinical utility of hyperthermia in combination with DNAJB1 and/or DNAJA4 inhibition in melanoma treatment.