Anti-tumor Effect Of Adenovirus Carrying P53 Combined With Chemotherapeutic Agents In Mesothelioma

Objective: Malignant pleural mesothelioma is one kind of rare disease which comes from pleural mesothlial cells and characterized by local invasion and high degree of malignancy. Most of malignant mesothlioma are linked with asbestos exposure, remains intractable despite recent treatment modalities. The latent period is long and no preventive procedures are currently available. The patient numbers will continuously increase in the next decades in many industrialized countries as well as newly developing countries. Extrapleural pnuemonectory is a therapeutic option only applicable to an early-staged case but recurrence is common even after the radical surgery. Mesothelioma is resistant to radiotherapy and used for a palliative purpose. Chemotherapy is therefore the primary treatment in most of the cases and a combination of cisplatin (CDDP) and pemetrexede (PEM) is currently the first-line regimen. Nevertheless, an average survival rate with the CDDP plus PEM regimen is 12.1 months and the prognosis remains poor. A novel therapeutic strategy is thereby required to improve the prognosis and quality of the patients.The majority of malignant mesothelioma possesses the wild-type p53 gene with defective INK4A/ARF locus containing the p14ARF and p16INK4A genes. We examined whether forced expression of p53 inhibited the growth of mesothelioma cells and produced anti-tumor effects by a combination of cisplatin (CDDP) or pemetrexede (PEM). Transduction of mesothelioma with adenoviruses bearing the p53 gene (Ad-p53) induced phosphorylation of p53, up-regulated p21 expression and decreased phosphorylation of pRb. The transduction generated cleavage of caspase-8 and -3and then induced the cell apoptosis. Cell cycle analysis showed increased G0/G1-phase populations and subsequently sub-G1 fractions, depending on cells and Ad-p53 doses. Transduction with Ad-p53 suppressed viability of mesothelioma cells and augmented the inhibition by CDDP or PEM in a synergistic manner. Intrapleural injection of Ad-p53 and systemic administration of CDDP produced anti-tumor effects in an orthotopic animal model. These data collectively suggest that Ad-p53 is a possible agent for mesothelioma in combination of the first-line regimen.Methods:In this research, we chose 5 kinds of human mesothelioma cells, NCI-H2452, NCI-H2052, NCI-H226, NCI-H28 and MESO-211H cells. MTT method was used to examine the suppress proliferation effect introduced by the transduction of Ad-p53. Cell cycle analysis was performed to check the cell cycle changes induced by Ad-p53. We further examined apoptosis pathway of Ad-p53-mediated effects in mesothelioma cells with Western blot analyses. MTT method was used again to identify the combinatory anti-tumor effects between Ad-p53 and CDDP or PEM. Animal test was performed to determine anti-tumor effects by Ad-p53 with CDDP in vivo.Results:1 MTT assay result: Ad-p53 but not Ad-LacZ treatment inhibited growth of all the mesothelioma cells with a dose-dependent manner except NCI-H2052 cells. The IC50 values suggested that NCI-H28 cells were the most sensitive to Ad-p53 followed by NCI-H2452 cells and both NCI-H226 and MSTO- 211H cells were similar in the susceptibility. Insensitivity of NCI-H2052 cells to Ad-p53 could be partly due to the lower expression level of the major Ad receptor.2 FACS analysis resultThe mesothelioma cells, MSTO-211H and NCI-H28, were infected by Ad-p53. Transduction of MSTO-211H cells with Ad-p53 induced G0/G1-phase arrest followed by increased sub-G1 populations. Ad-p53-infected NCI-H28 cells however showed augmented G2/M-phase populations with decreased S-phase populations after the infection and then increase sub-G1-phase populations. Sub-G1 populations increased significantly in NCI-H28 cells compared with MSTO-211H cells, showing high susceptibility to Ad-p53-mediated apoptosis.3 Weston blot assay result3.1 We further examined molecular events of Ad-p53-mediated effects in MESO-211H and NCI-H28 cells with Western blot analyses. Transduction with Ad-p53 induced the phosphorylation of p53. Expression levels of p21 increased with Ad-p53 transduction in both cells. pRb was down regulated in both cells after the Ad-p53 transduction, which corresponds to the shift from phosphorylated to unphosphorylated states. These data demonstrated that Ad-p53 infection activated p53-mediated pathways.3.2 Transduction with Ad-p53 induced cleavage of caspase-3 and caspase-8, whereas Ad-p53 transduction did not induce caspase-9 cleavage in MSTO-211H and NIC-H28. Bid expression levels were unchanged and truncated Bid was not detected in both cells. These data collectively suggested that Ad-p53 transduction activated the extrinsic receptor-mediated pathways through caspase-8 but the intrinsic mitochondrial-mediated apoptosis via caspase-9 was less likely to be involved.4 MTT analyze the combinatory anti-tumor effects between Ad-p53 and CDDP or PEM4.1 Mesothelioma cells, MSTO-211H and NIC-H28, were treated with different concentrations of CDDP and then infected with Ad-53, Transduction with Ad-p53 enhanced CDDP-induced anti-tumor effects and the CalcuSyn software analysis showed that the Ad-p53 and CDDP were synergistic in the inhibitory activity.4.2 Mesothelioma cells, MSTO-211H and NIC-H28, were treated with different concentrations of PEM and then infected with Ad-53, Transduction with Ad-p53 enhanced PEM-induced anti-tumor effects and the CalcuSyn software analysis showed that the Ad-p53 and PEM were synergistic in the inhibitory activity.5 In vivo anti-tumor effectsPleural dissemination was developed by intrapleural injection of MESO- 211H cells. Nude mice received intrapleural administration of Ad-p53 or Ad-LacZ as a control. Tumor weights on day 28 showed that Ad-p53 produced anti-tumor effected whereas Ad-LacZ administration did not influence the weight. We examined the combinatory effects with Ad-p53 and CDDP. Administration of Ad-p53 and CDDP respectively inhibited the tumor growth and the combination inhibit the tumor growth greater than Ad-p53 and CDDP treatment alone.Conclusion:1 Ad-p53 treatment inhibits the growth of most mesothelioma cells with a dose-dependent manner.2 Ad-p53 make the cell cycle changes and then induced the cell apoptosis.3 Ad-p53 activate p53-mediated molecular events and the extrinsic receptor-mediated apoptosis pathways4 Ad-p53 enhance the cytotoxicity of the anti-tumor agents.5 In vivo test, administration of Ad-p53 and CDDP produce the anti-tumor effects in synergistic manner.Concluding remark:Transduction with Ad-p53 enhance CDDP and PEM-induced anti-tumor effects in vitro and the administration of Ad-p53 and CDDP produce the anti-tumor effects in synergistic manner in vivo.