The Expressions And Mechanism Of Heat Shock Proteins In Non-Hodgkin's Lymphoma

Lymphoma is one of the very common diseases in hematology department and accounts for 5%of new cancer cases and 3%of cancer deaths.Its morbidity has increase approximately twofold over the last 30 years,the causes are still not clear. non-Hodgkin's lymphoma(NHL) accounts for 80%of all lymphomas and has a more aggressive course than Hodgkin's lymphoma(HL).Combined chemotherapy and immunotherapy like Rituximab had prolonged its survival,but few patients still suffered chemotherapy resistance and relapsed.So it is necessary to explore the pathogenesis of NHL and discovery more effective therapeutic methods.Heat shock proteins(HSPs) are widely expressed in many kinds of cells and elevated under stress conditions such as:heat shock,hypoxia,trauma,oxidative reaction and some kinds of drugs.In cells,HSPs assist the refolding,extension, transport and transmembrane of proteins,the function of HSPs has been named "molecular chaperon".HSPs also can inhibit apoptosis at different stages of apoptosis pathway.In addition,it closely relates to tumor immunity.Studies had indicated that HSPs could increase the tumor immunogenicity,present tumor antigen and contribute tumor immunity reaction.It had been proved that HSPs was abundantly expressed in many tumor cells and was correlated to the progress,biologic behavior and prognosis of tumors.Clinical researches had found HSPs to be a useful marker for the estimation of disease and predication of prognosis.HSPs had also been conducted as a therapy target.Some experiments had tried antisense oligonucleotides and siRNA to inhibit HSPs,and induced apoptosis and chemotherapy sensitivity.The exact relationship between HSPs and lymphoma is still not clear.Our research explored the correlation between HSPs and clinical manifestation of NHL by molecularbiology technology and studied the possible pathways of HSPs activation in Burkitt's lymphoma Raji cell in vitro.All these works may be useful for the understanding of lymphoma and afford us new method for diagnosis and therapy.PartⅠPI3K/AKT Pathway is Needed for Hyperthermia Induced HSP70 Expression in Raji CellPurpose:Hyperthermia has been applied as an adjuvant therapy together with chemotherapy and radiotherapy.However,the full potential of hyperthermia is hindered by few considerations,one of this is heat shock response.Heat shock response is a highly conserved molecular stress response that is involved in the induction of heat shock proteins(HSPs).Hsp70 is one of the most well known and best-studied members of HSPs familiy.It protects cells against a variety of chemical, physical and biological stressors.So the efficiency of anticancer therapy may be diminished by hyperthermia induced HSP70 expression.The activation of HSF1 plays a key role in the induction of HSP70.Recent studies had shown that HSF1 could be activated via PI3K/AKT pathway.After PI3K/AKT pathway was inhibited,the expression of HSP70 also decreased.Therefore PI3K/AKT pathway might be a new target for the inhibiting of HSP70.This study examined the potential relationships between HSP70 expression and PI3K/AKT pathway in Raji cells,and the protection function of hyperthermia pretreatment.Method:According to the different sequences of treatments,the experiment had been divided into 3 groups:1) Raji cells were only treated with chemotherapy agents;2) Raji cells were first treated with hyperthermia and followed by chemotherapy agents; 3) Raji cells were pre-incubated with LY294002,then hyperthermia and chemotherapy agents were followed.The expressions of HSP70,AKT and P-AKT were detected by Western blotting.The apoptosis indexes were analyzed by Annexin V-PI double staining.The cytotoxicity of ADM and DDP were examined by WST-8. Results:1 LY294002 inhibits hyperthermia induced HSP70 expressionHyperthermia rapidly increased HSP70 expression within 2 hours.And HSP70 expression reached a maximum 8 hours after hyperthermia.24 hours later,the expression of HSP70 was still higher than untreated control cells.AKT was also activated after hyperthermia treatment.In the presence of 10umol or 20umol LY294002 before hyperthermia,HSP70 expression and the activation of AKT were drastically attenuated compared with controls(P＜0.05).2 Preheated Raji cells acquire tolerance to DDP induced apoptosis24 hours after hyperthermia,the apoptotic Raji cells increased from 6.44±0.64%to 6.95±0.57%,with no significant difference(P＞0.05).The ratio of apoptotic preheated Raji cells was 9.73±0.28%and 10.50±0.85%after treated with 5ug/ml or 10ug/ml DDP,which was significantly lower than non-preheated cells(14.1±0.5% and 16.9±1.14%)(P＜0.01).3 PI3K inhibitor increases the apoptosis index in preheated Raji cellsLY294002 was used to inhibited HSP70 expression before hyperthermia.After incubated with 10umol LY294002,5ug/ml DDP induced apoptosis index increased from 9.73±0.28%to 15.33±0.45%(P＜0.01) in preheated Raji cells.And 20umol LY294002 elevated the apoptosis index from 9.73±0.28%to 19.5±0.75%(P＜0.01).4 Effects of LY294002 and hyperthermia on cytotoxicity of ADM and DDPRaji cells were more resistant to ADM treatment after hyperthermia treatment,IC50 increased from 5.2ug/ml to 17.0ug/ml.If treated with LY294002 before hyperthermia, Raji cells' sensitivity increased in a dose depended manner.10 umol,20 umol and 40umol LY294002 decreased IC50 to 10.6 ug/ml,8.8ug/ml and 3.6ug/ml.When the cells were treated with DDP,LY294002 also inhibited the protection of hyperthermia.Conclusion:1.Our study primarily demonstrated the existence of PI3K/AKT/HSP70 pathway in Raji cell.The result might afford a new strategy for the inhibition of HSP70 expression in Raji cell. 2.Hyperthermia treatment at 42℃could protect Raji cell against DDP induced apoptosis.The protection of hyperthermia might partly due to the expression of HSP70.PartⅡExpressions of HSP70,90αin non-Hodgkin's lymphoma and its clinical significancePurpose:HSP70 and HSP90 are two of the most important and best studied members of heat shock protein family.Stduies have found HSP70 and HSP90 expressed in many kinds of tumors and their expressions were correlated with stages and prognosis. But HSP70 and HSP90 expressions indicated different significances in different tumors,some of the results even contradicted completely.The possible reason may due to the specificity of tumors.HSP70 and HSP90 were highly expressed in NHL, but their clinical significances were not clearly indicated.Our research detected the expressions of HSP70 and HSP90αin NHL by immunohistochemistry and explored their clinical significances.Methods:NHL tissues were collected from 30 NHL patients with an average age of 43.6 years(6～84years).All the pathology samples were reconfirmed by two pathologists.Staging was according to Ann Arbor classification and pathology typing was refered to American National Cancer Institute Work Group classification(1982). 10 necrotic lymphnoditis and 12 reactive hyperplastic lymphnode were used as controls.The expressions of HSP70 and HSP90αwere detected by immunohistochemistry.If the nucleus or cytoplasma were stained yellow,the cell was considered positive.The percentage of positive cells were calculated as fellows:≤5% (-),6～25%(+),26～50%(++),＞50%(+++);(+++) was also considered as high expression.Results:HSP70 and HSP 90αexpressions located in nucleus and cytoplasma.They were highly expressed in NHL,necrotic lymphnoditis and reactive hyperplastic lymphnode.There were no significant differences in the expressions of HSP 70 and HSP90αbetween NHL,necrotic lymphnoditis and reactive hyperplastic lymphnode (P＞0.05).The positive expression of HSP70 and HSP90αdid not correlate with the degree of malignancy,staging,therapy reaction,B syndrome and extra-lymphonode infestation(P＜0.05).High expression of HSP70(postive cell＞50%) was correlate with the degree of malignancy,staging and therapy reaction(P＜0.05),but was not relate to B syndrome and extra-lymphonode infestation(P＞0.05).None of such relationship was found between the high expression of HSP90αand clinical manifestation(P＞0.05).Conclusion:1.Our study measured HSP70 expression in NHL using immunohistochemistry for the first time and indicated high expression of HSP70 was correlated with the degree of malignancy,stage and therapy reaction.So HSP70 may be a useful indicator for the evaluation of malignancy,stage and therapy reaction of NHL.2.We also primarily demonstrated HSP90αwas highly expressed in china NHL patients,though its expression was not related with clinical manifestation and therapy reaction.3.The high expressions of HSP70 and HSP90αmight indicate them to be good therapy targets for NHL.