Chemosensitivity And Function Of HSP70 In Drug Resistance In Leukenia

As we all known, the reason of failure, relapse of leukemia is due to multidrug resistance. The abnormal expressions of HSP70mRNA, MDR1mRNA , which regulated by HSF1 in transcription level under several stimulation conditions, often presence in human leukemia. However, the relationship between HSP70 and MDRl is not clear. In order to supply theory reference for experiment study and clinical therapy of children leukemia , several works were explored : (1) chemosensitivity in children leukemia. (2) the significance of the expression of HSP70 and MDRl gene under heat shock and chemotherapy. (3) the relationship of heat shock and chemotherapy resistance.(4) the mechanism of reversing multidrug resistance by Chinese traditional medicine-quercetin in leukemia.First, Methods: MTT assay was used to detect the sensitivity of leukemia cells in peripheral blood or bone marrow to Ara-C, DEX, ADM , DNR, VCR, VP-16, L-ASP, CTX. Results: there was 15 S/S (S, Sensitive), 2 R/R(R, Resistance), 3 S/R, 2 R/S in 22 patients. The general, positive, negative, sensitivity, specificity rates of chemosensitivity in vitro with those of in vivo sensitivity were 77.3%, 83.3%, 33.3%, 78.9%, and 40% respectively.Second, K562 cells were bathed in water under different conditions then gathered. The expression of HSP70 protein was detected byimmunocytochemistry assay. RT-PCR were used to assay the expression of HSP70mRNA, MDRlmRNA . Results: (1) Immunochemistry showed that compared with the control group , HSP70 over expressed in the cells under high temperature and the expression increased and "moved" from cytoplasm to nucleolus after heat shock with the recovery time prolonged. The expression of HSP70 began to decrease at recovery temperature at 3 hours, approaching to control group after 5 hours. The expression of HSP70 in 45 °C group showed no difference compared with the control group. (2) FLT-PCR showed the expression of HSP70mRNA, MDRlmRNA increased following 43°C heat shock, and was 4 and 5.8 times higher than control group at recovery temperature at 2 hours, respectively, then return to normal at 5 hours.Third, MTT assay was used to investigate the growth suppression rate of K562, K562/ADM cells under different ADM and heat shock plus ADM therapy. The function of P-gp was detected by fiowcytometry in K562 cells. RT-PCR was used to detecte the expression of HSP70mRNA and MDRlmRNA under different ADM and heat shock plus ADM in K562 and K562/ADM cells. Results (1) The growth suppression was obvious under high level of ADM in K562 and K562/ADM cells. The growth suppression decreased in heat shock plus ADM than in ADM group. (2) The expression of HSP70mRNA and MDRlmRNA increased in K562 cells under heat shock plus ADM, ADM group compared with control (P<0.01). The expression of HSP70mRNA and MDRlmRNA increased in K562/ADM cells following the increasing of ADM, and higher than control (PO.01).(3) The fluorescence of ADM in cells was decreased in ADM group than in control detected by fiowcytometry, and these cells showed drug resistance.At last, MTT asssy was used to detect the growth suppression ofQuercetin on K562 cells. The expression of HSP70mRNA and MDRlmRNA were detected in ADM plus Quercetin^ Quercetin plus ADM group. The two of them were detected in K562/ADM cells in Quercetin plus ADM group. Results (1) Quercetin can suppress the growth of K562 cells. (2) The expression of HSP70mRNA and MDRlmRNA by RT-PCR decreased in K562 and K562/ADM cells in Quercetin plus ADM group compared with ADM group and control (P<0.01). There is no difference of HSP70mRNA or MDRlmRNA between ADM group and ADM plus Quercetin in K562 cells.Conclusion: The MTT method proves to be a simple, quickly and objective methods in testing tumor cells; The results of MTT is better for selecting reasonable chemotherapy drug and providing a useful value for individual chemotherapy regimen . Then we find heat shock , ADM can induce the over expression of HSP70, MDR1 to maintain stability in K562 and K562/ADM cells. The decrease of chemotherapy sensitivity is due to increasing of MDR1 and P-gp in K562 and K562/ADM cells. HSP70 associates with drug resistance in leukemia. Pre-heating can increase tolerance to stimuli and decrease the sensitivity to ADM in leukemia cells. Quercetin has effect on the growth suppression. Pre-treat with quercetin can down-regulated the expression of HSP70mRNA and MDRlmRNA under other stimuli. Quercetin can increase the sensitivity of ADM and reverse the resistance to ADM in leukemia cells...