Chemotherapeutic Drugs Sentisize Human Lung Adenocarcinoma Cells To TRAIL Induced Apoptosis

Background:Primary carcinoma of lung is one of the most common maliganant tumors that do harm to human being's health. Epidemic disease investigation showed that there was obviously high rate of incidence of lung cancer in most countries. Primary carcinoma of lung is the first lethal factor of all tumors in city. Lung cancer will have become the first or second most common malignant tumor in most advanced countries and other ones by the end of this century. One of every four or five patients who will be dead of cancer will be suffered from lung cancer. Therefore the research of treating lung cancer is urgent. Now, most of chemotherapeutic drugs do harm to the normal cells which proliferate rapidly in bodies as well as tumor cells, especially to bone marrow cells and gastrointestinal tract mucosa epithelial cells. Tumor necrosis factor related apoptosis inducing ligand(TRAIL) is a recently identified member of the TNF family and is capable of inducing apoptosis in various transformed cells. TRAIL has shown a relative sparing of normal cells. TRAIL is a promising agent for development as a cancer therapeutic agent. Objective:To study the effects and mechanisms of TNF related apoptosis inducing ligand(TRAIL) alone or in combination with chemotherapeutic drugs on apoptosis in A549 cells.Methods:1. Cytotoxicity Determination Assays: Human lung adenocarcinoma cell strain A549 cells were cultured in vitro. The cell growth curve of A549 cells was delineated in order to analyze the cell proliferating dynamic values. 4,5-dimethyl thiazole(MTT) was applied to assay the growth inhibitory rates(GIR) of various concentrations of TRAIL, cisplatin(DDP), etoposide(VP16), docetaxel(TXT) alone or combination of TRAIL and subtoxic dose of each chemotherapeutic drug.At the same time, growth inhibitory curves(GIC) were delineated and half inhibitory concentrations(IC50) were analyzed.2. Analysis of Apoptosis: Morphological changes of A549 cells were investigated by using microscope before and after the treatment of TRAIL alone, a fixed dose of each chemotherapeutic drug(Cisplatin,Etoposide,Docetaxel) alone or the combination of TRAIL and each drug. AnnexinV assay for apoptosis was conducted with AnnexinV kit. Cells were stained with Annexin V -FITC and Propidium iodide(PI),and were analyzed by flow cytometry(FCM) to determin the apoptosis rates after the treatment of TRAIL alone,a fixed subtoxic dose of each chemotherapeutic drug(Cisplatin, Etoposide, Docetaxel) alone or the combination of TRAIL and each drug.3. RT-PCR assay: Reverse transcription-polymerase chain reaction(RT-PCR) was applied to semi-quantitatively assay mRNA expression of death receptors DR4 and DR5 in human lung adenocarcinoma cells A549 before and after the treatment of sub-toxic doses of VP16, DDP and TXT for different hours.Results:l.Cell double time of A549 cells was 32.lh. A549 cells were at the logarithmic growth phase in the density between 4.1xlO4/ml and 22.5><104/ml. There was significant positive correlation between A549 cell numbers and A values(r =0.93), which showed that A549 cells were suitable for MTT assay.Cytoxity Determination Assays showed that the growth inhibitory rates(GIR) of TRAIL at the concentration of lOOng/mk 1600ng/ml and in an acting time of 24 hours to cell strain A549 were 6.84+ 1.14%, 26.10+4.00%. The growth inhibitory rates(GIR) of TRAIL became larger with increasing concentrations of TRAIL. But the value of IC50 of TRAIL alone was more than 1600ng/ml. Chemotherapeutic drugs DDP , VP16 or TXT could significantly inhibit the proliferation of A549 cells in a dose-dependent fashion. The values of IC50 of DDP, VP16 and TXT were respectively 44.0ug/ml, 55.1ng/ml, 62.1ng/ml. The growth inhibitory rates(GIR) of TRAIL lOOng/ml in the presence of sub-toxic levels of DDP 5ug/ml, VP16 7.5ug/ml and TXT 4ng/ml were 21.40+ 4.48%, 26.10+4.02%, 19.98±4.15%. There was siganificant difference in the growth inhibitory rates(GIR) of TRAIL before and after the combination of DDP, VP16 or TXT (P< 0.05).2. After 24 hours treatment of A549 cells by TRAIL(100ng/ml), DDP(5ug/fnl), VP16(7.5ug/ml) or TXT(4ng/ml), little more apoptosis was observed under phase-contrast microscopy and 4.44%, 5.51%, 7.54%, 12.26% apoptotic rates were detected by FCM. Combination of TRAIL(100ng/ml) with low concentrations of DDP(5ug /ml), VP16(7.5ng/ml) or TXT(4ng/ml) could effectively induce apoptosis in A549 cells. The apoptotic rates were as follows: 22.84%, 24.32%, 16.84%.3. Sub-toxic dose of VP16 could up-regulate the expression of DR4 and DR5mRNA in A549 cells. The ratios of DR4/p-actin and DR5/p-actin in A549 cells treated by VP16(7.5ug/ml) for 8h were 1.13±0.13and 1.06+0.25. And the ratios treated for 12h were 1.18+0.20 and 1.02+0.02. There was significantly statistical difference between the above two groups and normal control group(P <0.05).Sub-toxic doses of DDP or TXT had no significant effect on the expression of DR4 and DR5 mRNA in A549 cells. The expression of DR4 and DR5 mRNA in A549 cells treated by DDP(5p.g/ml) or TXT(4ng/ml) for 4h and 8h was not significantly higher than that of normal control groups(P>0.05). Conclusions:1. The combination of TRAIL and sub-toxic doses of chemotherapeutic drugs DDP, VP16 or TXT can sufficiently inhibit the proliferation of human lung adenocarcinoma cell strain A549 cells in synergistic manner. 2. The combination ofTRAIL and sub-toxic doses of DDP, VP16 or TXT can greatly enhance their pro-apoptotic effect on A549 cells. 3. Sub-toxic dose of VP16 can up-regulate the expression of DR4 and DR5 mRNA in A549 cells, which may be the main mechanism of this synergy. 4. Sub-toxic doses of DDP or TXT has no significant effect on the expression of DR4 and DR5 mRNA in A549 cells, which may not be related to the synergy.