Recently, ultrasound-targeting microbubble destruction has been employed in molecular gene therapy, and a new potent nonviral gene transfer method known as 'sonoporation' has been developed. We investigated the efficiency of sonoporation toward growth inhibition of human gingival squamous carcinoma cell line, Ca9-22, in vitro and in vivo. The cytotoxicity of bleomycin (BLM) was investigated using flow-cytometric analysis and Hoechst's staining in vitro assay systems. We found that the delivery of BLM by sonoporation induced cytotoxic effect toward Ca9-22 cells in vitro. Our in vivo results showed that tumors nearly disappeared in Ca9-22 cell-implanted nude KSN/slc mice treated with a low dose of BLM followed by sonoporation during the 4-week experimental period. Histological analysis revealed that the cytotoxic effect was mainly apoptosis. We previously reported that the cytolethal distending toxin B (cdtB) from Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, is responsible for cell cycle arrest and apoptosis in vitro. Thus, we used sonoporation to transfect a cdtB-expressing plasmid into Ca9-22 cells and examined cell viability in vitro and in vivo. We found that an administration of cdtB-expressing plasmid followed by sonoporationinduced marked growth inhibition of Ca9-22 cells and apoptotic cells were also observed in vitro and in vivo. These findings suggest that local administration of cytotoxic agents with sonoporation is a useful method for molecular cancer therapy.

No abstract

No abstract

The cytolethal distending toxin (Cdt) from Actinobacillus actinomycetemcomitans consists of three proteins, CdtA, CdtB, and CdtC, which are responsible for cell cycle arrest and apoptosis. In the present study, local delivery systems of recombinant CdtB and CdtB-expressing plasmid were established using Ca9-22, human gingival squamous cell carcinoma cell line. When CdtB was delivered to Ca9-22 cells using a BioPORTER, a 32-kDa protein was detected by Western blotting, and G2 cell cycle arrest and apoptosis occurred. In addition, the CdtB delivered upregulated the expression of phosphorylated p53 and the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in Ca9-22 cells, suggesting that these intracellular molecules might contribute to the induction of G2 cell cycle arrest and apoptosis. When the CdtB-expressing plasmid was transfected into Ca9-22 cells by lipofection or electroporation, CdtB (32 kDa) was clearly detected. Further, TdT-mediated dUTP nick end labeling positive cells were observed after transfection of the CdtB-expressing plasmid. These findings indicated that delivery of the CdtB protein and transfection of the cdtB gene induced cell cycle arrest and apoptosis in Ca9-22 cells in vitro, and we conclude that it may be possible to induce apoptosis in human gingival squamous cell carcinoma by electroporation of the cdtB gene.

Sonoporation is a drug and gene delivery system using ultrasonication that allows the intracellular delivery of foreign molecules that cannot enter cells under normal conditions. We previously reported that sonoporation with microbubbles (MBs) could achieve effective intracellular drug delivery to human gingival squamous carcinoma Ca9-22 cells. In this study, we developed anti-epidermal growth factor receptor (EGFR) antibody-conjugated MBs (EGFR-MBs) and evaluated their capacity to enhance anti-cancer drug toxicity in vitro and in vivo. We first assessed the effect of sonoporation with EGFR-MBs on Ca9-22 cells by the WST-8 assay, flow cytometry and Hoechst’s staining in vitro. Sonoporation and EGFR-MB had a strong cytotoxic effect on Ca9-22 cells with low-dose bleomycin. Furthermore, bleomycin delivery using sonoporation with EGFR-MBs remarkably increased the number of apoptotic cells. We next examined the effect of EGFR-MBs in a murine squamous cell carcinoma model. Bleomycin delivery by sonoporation with EGFR-MBs exhibited remarkable antitumor activity. Together, our results show that EGFR-MBs and ultrasound treatment increases the efficacy and specificity of intracellular drug uptake, suggesting this could be a novel drug-targeting modality for oral squamous cell carcinoma chemotherapy treatment.