The Experimental Study Of The Effect Of Thermotherapy Combined With Chemotherapy On Human Lung Cancer Cells A549

Preface :The malignant carcinoma is one of the serious diseases that endangering thehuman health, and the number of the patients is nearly up to 1,700,000 every year in our country. The incidence is still constantly rising in recent years, there are 1,500,000 patients dead of malignant carcinoma every year. It has already exceeded the cardiac and cerebrovascular diseases and become the 1st death reason. Though the treatment of the tumor in china has made great progress, the main therapeutic methods remains operation, chemotherapy, radiotherapy and immunotherapy etc, and the tumor thermotherapy (Hyperthermia) is appearing as the fifth kind of tumor therapeutic method following above-mentioned methods. With the further investigation and improvement of thermotherapy technology, thermotherapy has already played an important role in the synthesis therapy and become an important adjuvant therapy method of radiotherapy and chemotherapy. Taxol (TAX) and navelbine (NVB) are the new drugs that have been applied to clinic use in recent years, and their antitumor effectis remarkable. They act on the G2/M period of the tumor cell mainly. TAX is a tubulin-binding agent, it can stabilize microtubules against depolymerization, and inhibit their function; NVB can block tubulin synthesis and make the cell proliferation arrest in the metaphase. At present, most patients are treated by chemotherapeutic option, and they can get better curative effect by great deal of agents, at the same time, the adverse effect is brought out. So it is valuable for clinical application to find out combined therapeutic methods to decrease the amount of chemotherapeutic drugs and improve curative effect, hyperthermia combined chemotherapy is one of the good approachs. For this reason, this study uses lung cancer A549 cell line as the research object to investigate the capacity of inducing apoptosis of thermotherapy, chemotherapy and thermochemotherapy and observes its mechanism for the future clinical application with proliferation assay of human lung cancer cells A549, by Flow cytometry (FCM), and immunohistochemistry. It is expected that the results might provide the theoretical basis for clinical therapy of malignant tumor.Material and methods:1. Human lung cancer cell line A549 was chosen as the trail material, chemotherapy drugs included Taxol (TAX), and Navelbine (NVB). The heating temperature was 42 °C; the heating container was incubator.2. The correct formula of Burgi was used to evaluate the interaction of thermochemotherapy: q=(EA+B/EA+EB-EA>0.05) ; The inhibition rate was 3.39% after heating 2 hours; it showed that thermotherapy could restrain cell growth.(2) Various concentration of TAX and NVB could efficiently inhibit the growth of cell line A549, compared with control group, difference of OD value was significant (P<0.05). The inhibition rates were 88.709￡k 74.86%> 41.81% and 37.01% under the TAX concentration 15.00/ig/mK 3.00jug/mU 0.60/ig/ml and 0.12/ag/ml respectively. After treating the cells by NVB 10.00^g/mK 2.00/ig/mK 0.40/ig/ml and O.OS^g/ml, The inhibition rates were 80.51%, 66.10%, 38.98% and 34.46% respectively. The concentrations inhibiting the growth 5O%(/C5o) of TAX and NVB was 1.057/ag/ml and 0.961/ig/ml. The results suggested that treatment with various concentrations of TAX and NVB can inhibit the growth of lung cancer A549 cells, and show a dose-effect relationship.(3) Compared with single drug group, OD value of TAX combined with hyperthermia had no statistical significance (i^O.OS), the "q" value was between 0.85 to 1.125 which indicated that TAX combined with hyperthermia had the addition jointaction; Compared with the single NVB group, the difference of NVB at 0A0f4.g/ml combined with heating was significant (P<0.05) , the "q" value showed synergistic effect. The IC50 of TAX and NVB was 0.892/ig/ml and 0.544/ig/ml, it indicated that the dose of combined groups was less than TAX or NVB single with the same effect.2. The effect of apoptosis rate:(1) According to the result of flow cytometry, the rate of apoptosis was 0.90%,1.07% and 6.94% after heating 0.5hour,lhour and 2hours respectively. These results indicated that with the increase of heating time, the apoptosis rate had the increasing trend. Compared with the control group, the apoptosis rate was higher, the difference had the statistical significance (P<0.05) .(2) The typical characters of apoptosis in the tested cells were observed after exposed to TAX or NVB. The percent of apoptosis cells was 0.87%, 2.08%, 9.83%, 11.25% and 0.61%, 1.56%,10.29,13.01% respectively, and compared with the control group, the difference had statistical significance CP<0.05) .(3) As to TAX or NVB combined with hyperthermia, the apoptosis rate was increasing and were 19.40% and 19.83% respectively, compared the single drug groups, the difference had statistical significance (/M).O5) .3. The expressions of the 4 proteins:The immunohistochemistry results indicates that the levels of HSP-70, P53, c-Myc proteins increased in hyperthermia group compared with the control group, the difference had the statistical significance (,P<0.01); the expression of Bcl-2 increased, it may be effective to heat tolerance. In chemotherapy and thermochemotherapy group, the expression levels of HPS-70, P53 and c-Myc proteins increased, and Bcl-2 proteins decreased. There was significantly difference compared with control group, respectively (ZM).O1). It showed that the effect to lung cancer cells by thermotherapy, chemotherapy and combined treatment was relative to the expression of above-mentioned proteins.Conclusions:1. Thermotherapy at 42 °C can suppress the growth of the lung cancer A549 cell.Thermotherapy can induce the production of HSP70; thermotherapy also can activate p53 gene and lead to apoptosis of the lung cancer cells; it can upregulate c-Myc gene and improve the hot sensitivity of the cell through regulating cells apoptosis; Apoptosis is one of the mechanisms of thermotherapy.2. Under this experimental condition, the effect of the hyperthermia combined TAX is addition joint action, that is, TAX is not sensitive to heating. But combined with NVB of different concentration, it shows synergistic effect. The results also show that the ability of inhibition is stronger than single chemotherapy, that is, themo-chemotherapy can reduce the doses of drugs and increase the effect.3. After combined with hyperthermia at 42 °C, TAX and NVB can raise the expression levels of HSP70, p53 and c-Myc gene, and can downregulate the expression level of bcl-2 gene, which suggest that it is one of the mechanisms of antitumor that thermochemotherapy can promote apoptosis.