Study Of Effect Of Hyperthermia Combined With Adriamycin On Hematologic Malignancies

BackgroundCancer is one of the malignant diseases that threatens human health and the incidence and mortality kept on rising in recent years. Despite the high cure rate for patients treated by combination of radiotherapy and chemotherapy at early stage, the prognosis of patients with distant metastases or at late stage is still disappointing. With the emergence of cancer cells with multi-drug resistance,thus it is necessary for us to seek effective methods for the treatment of tumor patients,especially for the multi-drug resistant patients.Thermotherapy as a new therapeutic strategy has drawn many researchers' attention to tumor therapy, but the laggard theoretical research has been being the obstacle of the progression in clinical practice. With further investigation into biological function to high temperature(hyperthermia) and improvement of thermotherapy technique, thermotherapy has played an important role in the complex treatments of tumor as an auxiliary method of radiotherapy and chemotherapy.The tumor thermotherapy (hyperthermia) has become a fifth treatment method for tumor.Thermal sensitivity of different tumor cells and possible molecular mechanisms were worth investigating. Adriamycin is an effective chemotherapeutic agent for tumor treatment,however the effects of combining with hyperthermia is not known. Adriamycin is a kind of peculiar medicine independing on cell cycle and has been applied into clinic with remarkable antitumor effect,and is one of the most commonly used and effective chemotherapeutic drugs and exhibits a wide spectrum of activity agaist many solid tumors,leukaemias,lymphomas and multiple myeloma in system chemotherapy in recent years.At present, adriamycin is applicated to mainly treat solid tumors in combination with other therapeutic modalities,but it was less reported that it was used for treating the tumors in the hematopoietic system in combination with hyperthermia.For the reason,in this study, tumors of the hematologic malignancies cell lines such as human chronic myelocytic leukaemia cell line K562, adriamycin-resistant subline K562/ADM, human B-cell lymphoma cell line Raji and human multiple myeloma cell line ARH-77 were investigated for the in vitro and in vivo antitumor activity and capacity of adriamycin and hyperthermia in inducing apoptosis and the sensitivities of these cell lines to hyperthermia and thermochemotherapy, as well as the possible mechanism by proliferation assay of cells and FCM and so on.Furthermore the combined effects could be evaluated.We expect to provide an experimental basis for clinical therapeutic alliance of the hematologic malignancies,a theoretical basis for clinical practice and a novel therapeutic approach for malignant hematopoietic system tumors.ChapterⅠThe experimental study of the inhibitory effect of adriamycin combined with hyperthermia on human the hematologic malignancies cell lines in vitroObjectiveTo investigate the inhibitory effects of adriamycin in combination with hyperthermia on proliferation of human cell lines K562, K562/ADM,Raji and ARH-77 and the influence on apoptosis of these cell lines in vitro.Methods 1. K562, K562/ADM, Raji and ARH-77 cell lines were cultivated using RPMI-1640 culture medium containing 10% fetal cattle serum.2. The working concentrations of adriamycin against K562, K562/ADM, Raji and ARH-77 cell lines were determined by MTT assay.3. Based on the different temperature of heating(40℃,41℃,42℃)and adriamycin, the tests were assigned to 8 groups, and 37~C hyperthermia was used as the control group.4. After K562, K562/ADM, Raji and ARH-77 cells were treated, the survival rate of cell lines were analysed with trypan blue exclusion method and the growth inhibitory rates were analysed with MTT assay. The numbers of the cells and colonies were counted, the rate of the colony formation and inhibition calculated and drawing of the cell survival curve of different groups.5. The cell apoptosis was determined by flow cytometry(FCM) in the different management groups.6. The correct formula of Veleriote was used to evaluate the interaction of thermochemotherapy.7. The data were processed with SPSS 11.0 software package. Results were analyzed with one-way ANOVA and expressed as mean + standard deviation. LSD and Tamhane's T2 were used for multiple comparisons.ChapterⅡExperimental study in vitro sensitization effect of thermochemotherapy on human the hematologic malignancies cell linesObjectiveTo observe the sensitization effect of treatment with adriamycin in combination with hyperthermia in vitro on human cell lines K562, K562/ADM, Raji and ARH-77, the expressions of Bcl-2 and P-gp,and the intracellular concentration of adriamycin.Methods1. K562, K562/ADM, Raji and ARH-77 cell lines were cultivated using RPMI-1640 culture medium containing 10% fetal cattle serum.2. The working concentrations of adriamycin against K562, K562/ADM, Raji and ARH-77 cell lines were determined by MTT assay.3. The expressions of Bcl-2, P-gp and the intracellular concentration of adriamycin were determined by flow cytometric.4. The data were processed with SPSS 11.0 software package. Results were analyzed with one-way ANOVA and expressed as mean±standard deviation. LSD and Tamhane's T2 were used for multiple comparisons.ChapterⅢAn initial study of adriamycin combined with hyperthermia in nude miceObjectiveTo study the influence of adriamycin in combination with hyperthermia on tumor formation and tumor growth of human B lymphoma cell line(Raji) in nude mice in vivo.Methods24 BALB/C nude mice were divided into four groups:the control group (37℃), the chemotherapy group (37℃+ADM), the hyperthermia group (42℃) and the thermochemotherapy group (42℃+ADM). Every nude mouse was injected 5×10~6 Raji cells subcutaneously.The time and the rate of tumor formation were observed,the perpencicular tumor diameter was measured with sliding caliper at a 3-day intervals and the tumor volume was calculated,then the growth curves of tumors were plotted. Three weeks after tumor formation,all the mice were killed and the histopathological changes of the tumor were observed by HE staining. The data were processed with SPSS 11.0 software package. Results were analyzed with one-way ANOVA and expressed as mean±standard deviation. LSD and Tamhane's T2 were used for multiple comparisons. ChapterⅣWhole body hyperthermia in the treatment of 15 patients with malignant hematopathy: Observation of the safety, accustomization and efficacy in warming, temperature-measuring and temperature-controllingObjectiveTo probe into the toleration of patients with malignant hematopathy under whole-body hyperthermia(WBH),and test the compliance,safety and efficacy of WBH.MethodsThe experiment was conducted in the Department of Hematology, Zhujiang Hospital of Southern Medical University from April to July 2005. Fifteen patients with malignant hematopathy were selected, and all of them took part in the experiment voluntarily.Patients received WBH with WELLRAY photon thermotherapy intelligent system manufactured by Guangdong Weier Medicine and Sciences Co.Ltd. Retention catheterization and peripheral venous cannula were done before treatment, eletrocardio-monitoring was connected and the temperature receptor of body surface was fixed and so on. Then we warmed the intrarectal temperature until it reached 40～41℃, kept the homoiothermism for 2 hours,meanwhile,monitored the temperature changes of body surface,air and treatment couch, and electrocardio-monitoring was performed at the same time. Blood routine examination and biochemiscal indexes etc.were detected before treatment and 24 hours after treatment. The qualify of life in patients before and 24 hours after WBH were evaluated with Karnofsky(KPS) scale, the scale range was 0～100 points, 100 points as normal without symptoms and physical signs; 10 points as dangerous, close to death; Zero as death.ResultsChapterⅠThe experimental study of the inhibitory effect of adriamycin combined with hyperthermia on human the hematologic malignancies cell lines in vitro1. The concentration of adriamycin in the experiment was defined as its IC50.Then hyperthermia and chemotherapy were used singly or concurrently.2. The effect on cell proliferation and apoptosis rate: Compared with the control group, chemotherapy group, and hyperthermia group, the thermochemotherapy group showed suppression of cell growth, and the apoptosis rate was higher with statistical significance in the difference.3. Compared with the control group, the chemotherapy group and the hyperthermia group, the rates of colony formation and inhibition were decreased and the difference was of statistical significance in the thermochemotherapy group.ChapterⅡExperimental study in vitro sensitization effect of thermochemotherapy on human the hematologic malignancies cell lines1. The concentration of adriamycin in the experiment was defined as its IC50. Then hyperthermia and chemotherapy were used singly or concurrently.2. The FCM results indicated that the expression rates of Bcl-2 and P-gp proteins in hyperthermia, adriamycin chemotherapy and thermochemotherapy groups were down-regulated. Compared with the control group, the difference had statistical significance(P＜0.01).3. Thermochemotherapy remarkably increased the intracellular concentration of adriamycin(P＜0.01).ChapterⅢAn initial study of adriamycin combined with hyperthermia in nude miceThe time of tumor formation in the control group (37℃), the chemotherapy group (37℃+ADM), the hyperthermia group (42℃) and the thermochemotherapy group (42℃+ADM) group were (11.2+1.7), (20.2+2.3), (15.3+1.6) and (23.8+1.7) days. Three weeks after tumor formation all the mice were killed, the average weight of tumors were (6.04+1.06)g, (2.75+0.32)g, (3.90+0.32)g and (1.48+0.21)g, the tumor growth inhibition rate was 75.5% and HE staining showed that there were more necrosis regions in the tumor tissue in the thermochemotherapy group.ChapterⅣWhole body hyperthermia in the treatment of 15 patients with malignant hematopathy:Observation of the safety, accustomization and efficacy in warming,temperature-measuring and temperature-controllingFifteen patients entered the final analysis and no subject withdrew from the experiment.1. All of the 15 patients with malignant hematopathy could endure being warmed to 40℃for 120 minutes, and had a good compliance.2. Clinical symptoms and physical signs of patients turned better 24 hours after WBH with an average increase of 10 points.3. There were no significant differences in blood routine and biochemical indexes etc. before WBH and 24 hours after WBH.Conclusion1. The effect on cell proliferation and inhibition rate: Thermotherapy could restrain the growth of both the drug-sensitive and drug-resistant cells with indexes of the clone formation, survival rate, and inhibition rate and so on. In the thermochemotherapy group, the ability of inhibition is stronger to the sensitive cells.2. The effect of apoptosis rate: These results indicated that with the increase of temperature, the apoptosis rate had the increasing trend. Compared with the control group,the chemotherapy group and the hyperthermia group, the apoptosis rate of the thermochemotherapy was higher.3. The correct formula of Veleriote was used to evaluate the interaction of thermochemotherapy: Under this experimental condition, the effect of the hyperthermia combined ADM is joint-action. The results also showed that themochemotherapy can reduce the doses of drugs and improve the effect.4. Thermochemotherapy remarkably increased the intracellular concentration of adriamycin both in the sensitive cells and in the drug resistant cells, and down-regulate the expression rates of Bcl-2 and P-gp proteins.5. The animal experiment showed that the time of tumor formation obviously prolonged, the tumor volume diminished, and the growth inhibition rate raised up in the thermochemotherapy group compared with the control group, the chemotherapy group and the hyperthermia group.7. WBH system can be used in whole body heating agaist malignant hematopathy, and the process of treatment is safe and worthy to be spread clinically. It can markedly increase the qualify of life as well as the clinical symptoms and physical signs of patients with malignant hematopathy, and has shown its effectiveness initially, but the evaluation of long-term curative effect is still in observation.