Prokaryotic expression, purification and characterization of a novel pro-apoptosis protein hPNAS-4.

Recently, hPNAS-4 [human PNAS-4; also known as C1orf121 (Genbank accession number NP-057160)] was reported to be a novel pro-apoptotic protein in mammalian cells. However, at present there is little information about hPNAS-4 and its molecular mechanisms. In our present studies, the hPNAS-4 gene was first cloned into the pGEX-6p-1 vector with a GST (glutathione transferase) tag to express in soluble form in Escherichia coli induced with 0.5 mM IPTG (isopropyl beta-D-thiogalactoside) at 25 degrees C, and the recombinant hPNAS-4 was purified to near homogeneity with 96% purity by affinity chromatography and anion-exchange chromatography. The purified hPNAS-4 protein was further identified by liquid chromatography-ESI (electrospray ionization)-MS analysis and used to raise polyclonal antibodies that could specifically recognize the hPNAS-4 protein. In addition, hPNAS-4 is localized to the cytoplasm and the perinuclear compartment. Furthermore, the overexpression of hPNAS-4 in human A549 lung cancer cells resulted in decrease of cell viability via apoptosis. This study represented an important step to investigate the characterization for the new pro-apoptosis protein hPNAS-4, which should aid the discovery of new drug targets for the development of effective therapeutic approaches to cancer in the future.

Genetic transfer of PNAS-4 induces apoptosis and enhances sensitivity to gemcitabine in lung cancer.

PNAS-4 has been demonstrated to induce apoptosis in U2OS cells. To evaluate its feasibility as a new strategy for cancer therapy, we analyzed its anti-tumor effect with or without gemcitabine in A549 lung cancer cells. MTT assay, Hoechst 33258 staining and flow cytometric analysis were used to determine the cytotoxicity of PNAS-4 alone or plus gemcitabine. The anti-tumor efficacy was further investigated in vivo with nude mice. PNAS-4 plasmid/liposome complexes were injected by tail vein every 4 days. Gemcitabine was given ip on a weekly schedule for 4 weeks. PNAS-4 alone and plus gemcitabine induced apoptosis in A549 cells in vitro. The xenograft lung cancer treated with PNAS-4 retarded growth compared with the empty vector. The combination of PNAS-4 with gemcitabine induced anti-tumor activity accompanied by an increase in apoptotic cells compared with PNAS-4 or gemcitabine alone. No other obvious toxicity was found. PNAS-4 therefore suppresses tumor growth in vivo and enhances sensitivity to gemcitabine. This suggests that the PNAS-4 gene could be a potential candidate for lung cancer therapy alone or in combination with gemcitabine.