Cloning, Expression, Purification And Antitumor Activity And Partial Mechanisms Of Human TRAIL In A549 Cell Lines

Lung cancer is the leading cause of cancer deaths in the world. Of all newly diagnosed lung cancers, 80% are non-small-cell lung cancer (NSCLC). Current protocols with present chemotherapeutic agents and radiation for therapy of NSCLC are less effective. Now, there are two obstacles in current chemotherapeutic therapy of cancer: first, chemotherapeutic agents are severely cytotoxic to normal tissue cells; secondly, cancer cells are apt to be resistant to chemotherapeutic agents. The selective treatment agents of cancer are increasingly been as an attractive anticancer target. A cloned member of the TNF family, Apo2L/TNF-related apoptosis-inducing ligand (TRAIL), may provide a new therapeutic option for cancer because they initiate apoptosis regardless of p53 phenotype and also have direct access to the caspase machinery. It specifically kills various tumor cells and transformed cells, but is nontoxic to normal cells. TRAIL induces apoptosis in a number of cancer cell lines and has broad-spectrum activity against human malignancies.In this study, human TRAIL was cloned and expressed in E.coli. We investigated the cytptoxicity and apoptosis of TRAIL based on the A549 cells in vitro and in vivo. We further evaluated the synergistic cytotoxicity of TRAIL in combination with actinomycin D (Act D), cisplatin and paclitaxel (Taxol) in A549 cell lines in vitro and in vivo. The synergisms of TRAIL in combination with chemotherapeutic agents also provide the theoretical basis for decreasing the dose of chemotherapeutic agents and cytotoxicity in the clinic.AIM: To investigate the antitumor effects of soluble human TRAIL(sTRAIL) in A549 cell lines in vitro and in vivo and develop sTRAIL as an effective and target antitumor agents, soluble human TRAIL was cloned, expressed and purified. We studied the cytotoxicity and apoptosis of sTRAIL and further evaluated the synergistic cytotoxicity of sTRAIL incombination with Act D, cisplatin and paclitaxel (Taxol) in A549 cell lines in vitro and in vivo, and whether the sensitivity was correlated with the expression level of TRAIL receptors.METHOD: The full-length human TRAIL cDNA was amplified from human placenta cDNA library by PCR. The soluble TRAIL gene (114-281 amino acids) was amplified from full-length human TRAIL cDNA template by PCR, cloned into pETlla plasmid and transformed into BL21 and expressed by lmM IPTG. Cells pastes were sonicated and centrifuged to obtain the inclusion bodies (IBs). Thus, IBs were denatured and refolded. The refolded soluble TRAIL was purified by ion exchange and filter chromatography. The purity of recombinant human sTRAIL was determined by silver staining using SDS-PAGE and HPLC. The purified sTRAIL was further identified by Western blotting and N-15 amino acides sequence analysis.We investigated the cytotoxicity of TRAIL alone and the synergistic antitumor effects in combination with chemotherapeutic drugs in A549 cells by crystal violet staining and fluorescence-activated cell sort (FACS) in vitro and the synergistic effects were evaluated by Jin's equations. The induction of apoptosis and morphological changes of sTRAIL-treated A549 cells were observed by TEM. The expression level of DR4, DR5, DcRl and DcR2 were measured in sTRAIL-treated and chemotherapeutic agents-treated A549 cells by Western blotting. The growth inhibition of tumors was evaluated in terms of incidence, volume and weight in A549-implanted nude mice model treated by sTRAIL alone and sTRAIL/cisplatin. RESULT:1. The sequence of the full length TRAIL cDNA is correct and identical to that of the report. The soluble TRAIL gene (114-281 amino acids) was subcloned into pETlla and expressed in BL21, the expression level of which is about 50%.2. TRAIL, which was expressed in BL21, exists as IBs. After IBs was denatured and refolded, the refolded soluble TRAIL was purified by ion exchange and filter chromatography. The purity of sTRAIL is about 98%, while IC50 is about 24ng/ml in sTRAIL-treated A549 cells.3. The purified recombinant sTRAIL in our laboratory exhibits the antigenicity and thecorrect sequence of N-15 amino acids identical to TRAIL protein.4. The various concentrations of sTRAIL (0.658,1.98,5.95,17.7,53.3 and 160ng/ml, 24h) demonstrated the cytotoxicity by dose-dependent relationship in A549 cells with crystal violet staining method. The dose-dependent (0.625,2.5,10,40 and 160ng/ml, 24h) and time-dependent (0.5,1,2,3 and 5h, 160ng/ml) relationships were further shown in TRAIL-treated A549 cells by FACS method.5. sTRAIL significantly induced the apoptosis in A549 cells. The apoptosis and morphological changes were observed in sTRAIL-treated A549 ceils (200ng/ml, 2h) by TEM The nucleous and nucleic membrane of the non-treated A549 cells are clear, but those of TRAIL-treated A549 cells disappeared and chromatin start to condense. In the end, the apoptotic bodies were formed and A549 cells became apoptotic.6. Chemotherapeutic agents Act D (3.09~750ng/ml), Taxol (1.11-30 u g/ml) or cisplatin (1.85~16.7 u g/ml) augmented the cytotoxicity of sTRAIL in A549 cell lines within a range of concentrations of sTRAIL (0.659~160ng/ml) in vitro (PO.01).7. The expression levels of DR4 and DR5 were not significantly different and the expression of DcR2 was slightly downregulated, but the expression of DcRl was not detected in non-treated, sTRAIL-treated , Act D (500ng/ml)-treated, Taxol (30 u g/ml)-treated and cisplatin (25 u g/ml)-treated A549 cells.8. We tested the effects of sTRAIL alone and sTRAIL/cisplatin on the establishment of new tumors. We injected nude mice with A549 cells, and 24 h later, the mice were treated with vehicle control (PBS for 10 days), sTRAIL (15mg/kg per day for 10 days, on days 2-11), cisplatin (1.5mg/kg per day for 10 days, on days 2-6 and 14-18), and the combination of sTRAIL (15mg/kg per day for 10 days, on days 2-11) and cispltin (1.5mg/kg per day for 10 days, on days 2-6 and 14-18). The tumors were established on day 13 in the control group and on day 20 in the cispltin-treated group. There are eight tumor-burdened mice out of nine mice in the control group and nine tumor-burdened mice out of nine mice in the cispltin-treated group. In contrast, the tumors were established on day 34 and there are only three tumor-burdened mice out of nine mice in sTRAIL group and the sTRAIL/cispltin-treated group. The rates of tumor inhibition following treatment sTRAIL alone and sTRAIL/cisplatin were 38.3%and 86% by tumor volume, 28.3% and 76.8% by tumor weight (PO.05 for sTRAIL alone versus control, PO.05 for TRAIL/cisplatin versus cisplatin alone and sTRAIL alone) on day 65 in vivo.9. sTRAIL demonstrated the antitumor effects on A549-established nude mice xenograft. We implanted subcutaneously the A549 tumor tissues into nude mice. After the tumors reached 260mm3 in size, CTX (60mg/kg, once on day 13) for the parallel experimental group was administered i.p. by injection. TRAIL (45mg/kg, 15mg/kg and 5mg/kg for 4 consecutive days oh days 13-16 and 3 consecutive days on days 24-26), or vehicle control (sterile normal saline) was administered i.p. by injection. The tumors that received sTRAIL injection started to decreases in size, with rates of tumor inhibition of 15.3% (5mg/kg), 29.8% (15mg/kg) and 38.7% (45mg/kg, PO.05). The body weight did not have a loss compared with the CTX-treated group.CONCLUSION:1. The expression vector pETl 1 a-sTRAIL has been constructed, which was expressed in BL21 and the expression level was about 50%.2. We have done research on the process of IB's denaturation and refolding of sTRAIL and created the manufacturing technics. The purity of purified recombinant human soluble TRAIL was about 98%.3. sTRAIL was cytotoxic to A549 cells and IC50 was about 24 ng/ml, which showed time-response and dose-response relationships in A549 cells in vitro.4. sTRAIL significantly induced apoptosis in A549 cells. The characteristic apoptosis of A549 cells induced by sTRAIL was for the first time observed under TEM.5. The combination sTRAIL with chemotherapeutic agents Act D, Taxol and cisplatin significantly augmented the cytotoxicity in A549 cells in vitro at subcytotoxic concentrations of chemotherapeutic agents.6. We found no significant changes in the expression levels of death receptors and decoy receptors of A549 cells treated by sTRAIL, Act D, Taxol and cisplatin. These findings indicate that intracellular apoptosis-inducing factors might play an important role in the modulation of the apoptotic response, rather than the expression levels of the receptors in A549 cell.