The Effects Of Anti-tumor Growth And Anti-tumor Angiogenesis Of RhIL-24 And EGCG On Lung Cancer In Vitro And In Vivo

Objective: To construct new hIL-24 (human interleukin-24) prokaryotic expression vector pET-21a(+)-IL-24 and make it express highly and stablely in E.coli. To assess the bioactive function of the recombinant human IL-24 (rhIL-24) after purified and annealed through examining its effect on the cytokine secretion profile of PBMC.To observe the suppression of lung tumor growth, the promotion of apoptosis and tumor angiogenesis in response to rhIL-24 and EGCG in vitro and in vivo. To establish the experimental basis for the further study of recombinant drug in prokaryotic gene expression of rhIL-24. To compare the antitumor activities between IL-24 and epigallocatechin gallate (EGCG), a major constituent of tea polyphenols, which has the same antitumor characteristics as IL-24 in some areas. Methods: Full-length hIL-24 insert flanking with BamH I and Hind III restriction sites was amplified by PCR using pBV220-hIL-24 as temeplate. Both the insert and vector, pET-21a(+),were digested with above restriction enzymes for directional subcloning. The resulting insert and vector products were purified by electrophoresis on an agarose gel, and then extracted by kit supplied via company. The above fragments were ligated overnight, transformed into DH5α, then verified by PCR amplification and futher identified by DNA sequencing. An appropriate amount of recombinant, pET-21a(+)-IL-24, was transformed into BL21. A single colony of transformed bacteria was propagated. Overnight with shaking, The culture was added into LA liquid medium and growed with shaking subsiquently. IPTG was added into the cultures when the OD600 reach 0.4~0.6, then it was incubated for additional 2hr, 3hr, 4hr, 5hr. At each time point the sample of the culture was transfered into container, collected into separate labelled tube. The appropriate culures were centrifuged, sonicated and collected. Insoluble inclusion bodies were washed, solubilized, renaturated and then dialyzed. Finally desired rhIL-24 protein was monitored by Western Blotting. The peripheral blood mononuclear cells (PBMC) were obtained by lymphocyte isolation. The mononuclear cells of two groups (rhIL-24 and PBS)were in parallel cultured for 48hr,72hr, respectively. The separate supernatant from corresponding culture medium was analyzed by detection of cytokines, including IL-6,IFN-γand TNF-αwith ELISA kit and immunostimulation function of rhIL-24 in vitro was evaluated. The inhibitory effects of rhIL-24 and EGCG on lung cancer cells, A549, were analyzed by MTT assays. The cell growth curves of inhibition rate vs time and inhibition rate vs dose were generated, and the half-maximal inhibition concentration (IC50 )meamured. The cell cycle and the apoptotic percentage of A549 cells were detected by FCM. Next we determined if administration of rhIL-24 and EGCG could inhibit angiogenesis compared with PBS(negative control) or pingyangmycin (positive control) by using chick embryo chorioallantoic membrane vascellum model(CAM). We observed the inhibitory effects of rhIL-24 and EGCG on model of lung carcinoma in nude mice finally. The immunohistochemic technique was used to investigated the expression of CD34 and NF-κB in tumor tissues. Result: The hIL-24 prokaryotic expression vector pET-21a(+)-IL-24 which was constructed was identified correctly by double enzyme cutting and PCR. There was correspondence between the protein ladder of approximate 18.5KD after the engineering bacteria sonicated and the theory's value predicted. The recombinant protein was detected by Western-Blotting with anti-IL-24 antibodies. After purified and annealed, rhIL-24 protein was expressed in E. coli high efficiently by IPTG inducing. After rhIL-24 protein stimulated PMBC for 48h, the secretion of cytokines increased gradually, reached a peak at 72h, which was significantly different from that of control group. Treatment of A549 cells with rhIL-24, EGCG resulted in significant dose-dependent and time-dependent inhibition. The estimated values IC50 were 203.36μg /ml and 46.54μg /ml, respectively. Results of FCM indicated that treatment with rhIL-24 caused G2/M arrest in A549 cells. Administration of EGCG resulted in an increase in cells in the S phase of the cell cycle and a typical apoptosis peak before the G1 phase with 19.6% of percent apoptosis. In CAM test, the result showed rhIL-24 protein and EGCG could notably inhibit angiogenesis together with pingyangmycin, especially for the second and third grade capillaries, which had great significance compared with that of negative group.The animal model was set up successfully. There were no significant difference in tumor volume in both rhIL-24 group and EGCG group compared with control grpoup(PBS) after one-week treatment, but had significant difference after two-week treatment. The tumor inhibition rate in rhIL-24 group and EGCG group was 36.4%, 40.9%, respectively. The expression of NF-кB(p50) in tumor tissues was significantly decreased in rhIL-24 group compared with that of control group, but there was no significant difference between EGCG group and control group. According to tumor MVD marked by CD34, both rhIL-24 protein and EGCG could inhibit tumor angiogenesis obviously, compared with PBS. Conclusion: 1. IL-24 prokaryotic expression vector pET-21a(+)-IL-24 system was generated successfully, expressed efficiently. 2. Approximately purified rhIL-24 protein obtained. 3. RhIL-24 protein revealed the function of immunostimulation. 4. Treatment of A549 cells with rhIL-24, EGCG resulted in significant dose-dependent and time-dependent inhibition. The IC50 was 203.36μg /ml and 46.54μg /ml, respectively. 5. Treatment with rhIL-24 caused G2/M arrest in A549 cells. Administration of EGCG resulted in an increase in cells in the S phase and a typical apoptosis peak before the G1 phase with 19.6% of percent apoptosis. 6. RhIL-24 protein and EGCG could notably inhibit angiogenesis in CAM test. 7. There were obvious suppression of lung tumor growth and tumor angiogenesis in response to rhIL-24 and EGCG in vitro and in vivo. 8. The expression of NF-кB(p50) in tumor tissues was significantly decreased in rhIL-24 group compared with control group.