The construction of the eukaryotic expression plasmid pcDNA3.1/azurin and the increased apoptosis of U2OS cells transfected with it

Abstract

Abbreviations used: FCM -flow cytometry; GFP -green fluorescent protein; HA -haemagglutinin 9 peptide HA epitope; hCMV -human cytomegalovirus; IAP -inhibitor apoptosis protein; PCR -polymerase chain reaction; RT -reverse transcription; UV -ultraviolet Abstract: In our previous study, we demonstrated that azurin could selectively trigger apoptosis in human osteosarcoma cell line U2OS cells. However, the rate of apoptosis (35.8 ± 3.2%) is not very high, and azurin is too expensive to obtain readily. To solve these problems, we constructed a eukaryotic expression plasmid containing the azurin gene with an influenza virus haemagglutinin 9 peptide HA epitope tag, and transfected the recombinant plasmid pcDNA3.1(+)/azurin into U2OS cells. RT-PCR and Western blot analysis validated the successful transfection and the expression of the azurin-HA protein. Conspicuous apoptosis of the transfected cells was detected by flow cytometry (FCM) and the DNA ladder test. The apoptosis rate reached 64.3 ± 13.1%. The transcriptional levels of the Bax and p53 genes increased significantly in U2OS cells transfected with pcDNA3.1(+)/azurin, but the Bcl-2 mRNA level decreased. There was no difference in the levels of Bcl-xl mRNA and Survivin mRNA. We propose that the transfection of the recombinant plasmid pcDNA3.1(+)/azurin can significantly induce apoptosis in U2OS cells. This is closely associated with the up-regulation of the transcriptional level of the Bax and p53 genes, and the down-regulation of that of the Bcl-2 gene.

Recent Patents on Bacterial Proteins as Potential Anticancer Agents

“…Enhanced Bax levels then triggered apoptosis in the cancer cells [32,33]. Similar induction of apoptosis in human osteosarcoma cell line U20S cells has also been reported [34,35], and a patent application filed ( Table 1, part C). One unique feature of azurin is that unlike targeted anticancer drugs that inhibit a key step in one of the multiple pathways through which cancer cells replicate, azurin intervenes in multiple pathways of cancer growth and thus constitutes a potentially promiscuous drug [36].…”

“…The azurin-mediated induction of apoptosis in cancer cells [32][33][34][35] is interesting since azurin enters into cancer cells much more preferentially than into normal cells [38] which allows preferential induction of apoptosis in cancer cells. This might explain the lack of toxicity of azurin to live mice [33].…”

Noncationic Peptides Obtained From Azurin Preferentially Enter Cancer Cells

“…Azurin inhibits the growth of several human cancer cell lines in vitro (14,(24)(25)(26) and in vivo (13). Figure 6A and B illustrate the effect of p18 and p28 relative to azurin and dacarbazine (DTIC) on UISO-Mel-2 cells as determined by MTT and cell count.…”

Modulation of Angiogenesis and Immune Response in Canine Osteosarcoma by BMP-2 and Mesenchymal Stem Cells

“…Our results demonstrated significant caspase-3 activation with cytochrome c release into the cytoplasm with decreased anti-apoptotic protein Bcl-2 and increased pro-apoptotic BAX expression levels in all groups under the given experimental conditions using MSC/rhBMP-2. The Bcl-2 is known to be expressed at high levels in osteosarcomas and plays a role in the modulation and decrease of BAX gene expression through the induction of apoptosis and the suppression of the growth of tumor cells including osteosarcoma [71][72]. Therefore, an increased Bcl-2 expression can contribute to the intrinsic osteosarcoma chemoresistance, and may provide a promising target for gene therapy [73].…”