Enhancement Of Therapeutic Effect Of TAX On Lung Tumor Cells By Thermotherapy And Its Underlying Mechanisms

The incidence rate of lung cancer was increasing rapidly and in many developed countries,it had already been in the first place in male malignant tumor and the second or third place in female. The histological types of lung cancer changed significantly and the incidence rate of adenocarcinoma went on increasing. Though treatments of lung cancer developed very well, the survival rate of lung cancer was only 15% in USA, 10% in European countries and no more than 8.9% in developing countries. TAX had notable anti-tumor effect. Thermo-therapyplayed an important role in comprehensive treatments of cancer and could not increase cell tocoxic effect, so sensitivity of tumor cells to chemotherapy enhanced by thermotherapy gained more and more attention. This research aimed at enhancement of therapeutic effect of TAX on lung tumor cells and its underlying mechanisms.Methods1. Influence of Thermotherapy with TAX on Biological Characteristcs of Lung CellsA549, H446 and BEAS-2B cells were used and treated by three methods, which were control group, TAX group and 43℃+TAX group (Table 1), then they were observed and recorded by microscope. MTT assay was adopted to check proliferation rates of cells. LDH checking and wounding-healing assay were carried out. Apoptosis was detected by Hoechst33342/PI and DNA ladder. 2. Function of Important Signal Transducting Pathways in Therapeutic Effect of Thermotherapy with TAX on Lung Tumor Cells and their RelationshipsA549 and H446 cells were used and treated by six methods, which were control group, TAX group, 43℃+TAX group (Table 1), SP600125 group (43℃+TAX group with SP600-125), wortmannin group (43℃+TAX group with wortmannin) and NAC group (43℃+TAX group with NAC), and then ROS, Phosphorylation of JNK and Akt, Caspase-3 and MKK4 of each group were checked. FCM was used to check changes of apoptosis and cell cycle. Tests in vivo were carried out.3. Function of HSP70 in Therapeutic Effect of Thermotherapy with TAX on Lung Tumor CellsA549, H446 and BEAS-2B cells were used and treated by four methods, which were control group, TAX group, 43°C+TAX group and 43℃group (Table 1). Western Blotting was used to check changes of HSP70 and tests in vivo were carried out at the same time.4. Function of GTSP1 in Therapeutic Effect of Thermotherapy with TAX on Lung Tumor CellsThe Recombinant Plasmid pcDNA3.0-GSTP1 was constructed and transfected into A549 cells. Transfected cells and non-transfected cells were treated by thermotherapy with TAX, and then observed and recorded by microscope. MTT assay was adopted to check proliferation rates of cells. FCM was used to check cell cycles.5. Statistics AnalysisSPSS 12.0 was adopted. T-Test, ANOVA, Chi-Square Test and LSD were used. P value less than 0.05 was considered statistically significant.Results1. Influence of Thermotherapy with TAX on Biological Characteristcs of Lung Cells1.1 Thermotherapy with TAX can reduce injury of TAX on BEAS-2B cells (P＜0.05) and enhance the therapeutic effect of TAX on A549 cells and H446 cells (P＜0.05);1.2 Thermotherapy can injure cell membrane, which made LDH leak, so LDH in 43℃+TAX group was lower than that in control group and TAX group (P＜0.05);1.3 24h after nicking, cells in control group began to grow to the nicking area while other groups'cells began to die. 48h after nicking, cells in control group filled in the nicking area, but the nicking areas in other groups'were still very large and cells had almostly died;1.4 Cells in control group showed dark green and dark red, cells in TAX group had more green, which told us that apoptosis happened, but in 43℃+TAX group, cells showed bright green to testify obvious apoptosis;1.5 DNA electrophoresis showed that DNA in 43℃+TAX group had classical DNA ladder and did not happen in control or in TAX group, which told us thermotherapy can induce cell apoptpsis.2. Function of Important Signal Transducting Pathways in Therapeutic Effect ofThermotherapy with TAX on Lung Tumor Cells and their Relationships2.1 Changes of ROSThe expression of ROS in the cells of 43℃+TAX group was higher than the others' (P＜0.05), which can change cell inner environment, injure cell brane and transduct as signal in cells to change signal trnasducting pathways in cells.2.2 Changes of MKK4 and p-JNKJNK signal transducting pathway was activated in 43℃+TAX group, so the expression of MKK4 and phosphorylation of JNK were higher than those in control group and TAX group (P＜0.05); after SP600125 was added in, phosphorylation of JNK was inhibited (P＜0.05); they had no change with wortmannin (P＞0.05), after NAC was added in, they was lower than those in 43℃+TAX group (P＜0.05), which told us that ROS induced in thermotherapy can activate JNK signal transducting pathway.2.3 Changes of p-AktAkt pathway was inhibited in 43℃+TAX group, so phosphorylation of Akt was lower than that in control group and TAX group (P＜0.05); after wortmannin was added in, phosphorylation of Akt was inhibited completely (P＜0.05), with SP600125, phosphorylation of Akt was lower than that in 43℃+TAX group (P＜0.05), after NAC was added in, Phosphorylation of Akt was increased (P＜0.05), which showed that ROS induced in thermotherapy can inhibit activation of Akt pathway and activation of JNK signal transducting pathway would regulate activation of Akt pathway.2.4 Changes of Caspase-3 The expression of Caspase-3 in 43℃+TAX group was higher than that in control group and TAX group (P＜0.05); after SP600125 was added in, its expression was lower than that in 43℃+ TAX group (P＜0.05), after wortmannin was added in, its expression was higher than that in 43℃+ TAX group (P＜0.05), after NAC was added in, its expression was inhibited completely (P＜0.05). So apoptosis induced by thermothrapy was triggered through JNK signal transducting pathway and Akt pathway to caspase channel and ROS, JNK signal transducting pathway can activate casapase channel, Akt pathway can inhibit its activation.2.5 Changes of Apoptosis and Cell CycleThe number of cells in G₀/G₁ phase and G₂/M phase increased and those in S phase decreased in 43℃+TAX group (P＜0.05). The number of cells in S phase became less after wortmannin was added in (P＜0.05) and more after SP600125 and NAC was added in (P＜0.05), while those of G₂/M phase in SP600125 group and NAC group became less (P＜0.05). The rate of apoptosis in 43℃+ TAX group increased (P＜0.05). The rate of apoptosis went on increasing in wortmannin group and decreased in SP600125 group and NAC group (P＜0.05).3. Function of HSP70 in Therapeutic Effect of Thermotherapy with TAX on Lung Tumor CellsThe expression of HSP70 in control group and TAX group was almost identical (P＞0.05) and that in 43℃+TAX group and 43℃group was higher (P＜0.05). Its expression in 43℃+TAX group was lower than that in 43℃group (P＜0.05), which showed that HSP70 induced by thermotherapy can protect cells can be inhibited in lung tumor cells by activating or inhibiting some signal transducting pathways, but this phenomenon was not observed in normal cells.4. Function of GTSP1 in Therapeutic Effect of Thermotherapy with TAX on Lung Tumor CellsThe recombinant plasmid was successfully constructed and tramsfected into A549 cells. So transfected and non-transfected cells were obtained. The proliferation rates of transfected and non-transfected cells were consistent (P＞0.05) because GSTP1 had no effect on cell proliferation. Putting TAX in different concentrations on transfected and non-transfected cells, proliferation rates of non-transfected cells were lower than those of transfected cells (P＜0.05). With thermotherapy, proliferation rates of non-transfected cells were higher than those of transfected cells (P＜0.05). Under thermotherapy with TAX, FCM showed that the number of cells in G₀/G and G₂/M phase of transfected cells became more and those in S phase were lessConclusion and Innovetions1. BEAS-2B cells were used in the research of mechanisms of thermotherapy at the first time and it showed that thermotherapy can decrease injury of chemotherapy on normal cells. Thermotherapy can enhance therapeutic effect of themotherapy on lung tumor cells, injure cell membrane, change cells' ability of migration and induce apoptosis;2. Thermotherapy can increase expression of ROS, then activated JNK signal transducting pathway and inhibited the activation of Akt pathway, then induced apoptosis by caspase channel; thermotherapy mainly effected S phase and blocked lung tumor cells in G₂/M phase; activation of JNK signal transducting pathway in thermotherapy can influence activation of Akt pathway, which were all authenticated in vivo;3. HSP70 induced by thermotherapy can protect cells, which can be inhibited in lung tumor cells by activating or inhibiting some signal transducting pathways, but this phenomenon was not observed in normal cells;4. The recombinant plasmid, pcDNA3.0-GSTP1 and a new cell line had been successfully constructed at the first time; GSTP1 can promote metabolism of chemotherapy drugs, which increased drug-resistence of lung tumor cells, but thermotherapy can increase the sensitivity of lung tumor cells to chemotherapy drugs.