Bax gene therapy for human osteosarcoma using cationic liposomes in vivo.

The purpose of this study was to evaluate the anti-tumor effect of human osteosarcoma (HOSM-1) tumor xenografts in nude mice via transfer of the Bax gene using cationic liposomes. The HOSM-1 tumors transplanted into nude mice grew to 5-6 mm in diameter. Following growth of the tumor to this size, liposomes with the Bax plasmid were applied locally to the peripheral tumor (day 0) and were applied 3 times per week for 2 weeks (6 times in total). The tumor growth inhibitory effect was evaluated by measuring the tumor volume up to day 40. The expression of Bax was observed by immunohistochemical analysis and apoptosis was detected using the TUNEL assay. Tumor growth increased only slightly during the administration period, and tumor volume on day 50 was 43% of that in the saline control group. In the tumor margin 48 h after the completion of administration, Bax immunoreactivity was detected and apoptotic cells were clearly increased. Since these results suggested that Bax gene therapy using cationic liposome induced apoptosis in HOSM-1 tumor in vivo, we anticipate that this therapy will be useful for the treatment of osteosarcoma.

G3139 induces cell death by caspase-dependent and -independent apoptosis on human melanoma cell lines.

G3139 is an 18-mer phosphorothioate oligodeoxynucleotide (ODN) which has been targeted on the initiation codon region of the bcl-2 gene. Currently, clinical trials on G3139 for diverse tumors are underway in phase II and phase III. However, basic investigations of bcl-2 antisense ODN (G3139) and reverse ODN (G3622) have not been fully examined. In this report, we investigate cell death caused by G3139 and G3622 and the impact of antisense ODN in melanoma cell lines. We confirmed that G3139 reduced the level of bcl-2 protein and both G3139 and G3622 inhibited cell proliferation and induced apoptosis. G3139 was noted to produce a more intense effect than G3622. Although the general caspase inhibitor, Z-VAD-fmk, prevented apoptosis incompletely, the inhibition ratio of both ODNs was approximately equivalent. Our results suggested that inhibition of cell proliferation by ODNs is produced by apoptosis, but that the apoptotic pathway is not fully induced by the caspase-dependent pathway. Upon examination of the intracellular apoptotic protein dynamics, AIF localized within the mitochondria was translocated to the cytosol within 24 h, and subsequently to the nuclei after 48 h of treatment with G3139. Our results imply the following: the transfection of ODNs can induce apoptosis, the anti-tumor effect of G3139 is better than G3622, and the difference in the anti-tumor effect is specifically based upon the reduction of expression of the target DNA in malignant tumors. We consider that antisense ODNs may be an important tool for anti-tumor chemotherapy and the targeting of specific DNA is important in enhancing the anti-proliferative effect against tumors.

mHFE7A, a newly identified monoclonal antibody to Fas, induces apoptosis in human melanoma cells in vitro and delays the growth of melanoma xenotransplants.

The Fas receptor is a potentially valuable therapeutic target in cancer treatment. However, the clinical application of antibodies directed to this target is hindered by their serious side effects in vivo, including liver toxicity. One murine monoclonal antibody, mHFE7A, binds both to human Fas and murine Fas, without inducing any obvious side effects. However, the potential therapeutic effects of mHFE7A are unclear in human cancer cells or tumors. Here, we determined whether mHFE7A could induce apoptosis in vitro, and assessed its effects on major apoptotic pathways in a human melanoma cell line, MMN9. We also investigated its anti-cancer effects on transplanted melanoma MMN9 tumors in BALB/c nude mice. Treatment of mHFE7A cross-linking with Ig induced cell death similar to CH-11 treatment. Apoptosis induced by mHFE7A was defined by Hoechst 33342 DNA staining and DNA fragmentation assay. Furthermore, mHFE7A-mediated apoptosis was dependent on activation of a caspase signaling cascade involving caspases-8 and -3. Administration of mHFE7A also delayed the growth of melanoma xenografts. Thus, we provide the first evidence that the murine anti-Fas monoclonal antibody, mHFE7A, induces apoptosis of human malignant melanoma cells in vitro and is anti-tumorigenic in vivo.

The influence of oral tumor cell proliferation activity and membrane potential on the transfection efficiency of a cationic liposome.

The transfection efficiency of cationic liposomes varies according to cell type, but the specific cellular characteristics that affect transfection efficiency have not yet been defined. We investigated whether the transfection efficiency of cationic liposomes correlates with cell proliferation activity or cell membrane potential in oral malignant melanoma (HMG) and oral osteosarcoma cell lines (HOSM-1 and HOSM-2). The cell membrane potential was assessed by uptake of a cationic probe. Three oral tumor cell lines were exposed to a cationic liposome complexed with a beta-galactosidase expression plasmid, and beta-galactosidase expression was compared. Cell proliferation was about 2-fold higher in HOSM-1 cells than in HMG cells. The cell membrane potential in HMG and HOSM-1 cells was comparable, while the membrane potential in HOSM-2 cells was 1.6-fold higher. beta-galactosidase expression was measured by X-Gal staining in 7.0% of HMG, 17.0% of HOSM-1 and 11.5% of HOSM-2 cells. The present study demonstrates that gene therapy with cationic liposomes may be a promising new strategy for treatment of oral malignant melanoma and osteosarcoma. In addition, the transfection efficiency of cationic liposomes appears to be influenced by cell proliferation activity, but not cell membrane potential.

Experimental therapy using interferon-gamma and anti-Fas antibody against oral malignant melanoma cells.

The Fas/FasL signalling system plays an important role in chemotherapy-induced apoptosis in several different cell types. After interferon-gamma (IFN-gamma) treatment, we have previously reported a significant increase in Fas expression in oral malignant melanoma cell lines (MMN9, PMP, MAA, HMG) in vitro, and combination therapy using IFN-gamma and anti-Fas antibody (CH-11) has shown a synergistic anti-proliferative effect in MMN9 cells. There have been several in-vitro studies using CH-11, but there are few reports of its anti-tumour effect in vivo. In this study, we investigated experimental therapy using anti-Fas antibody against MMN9 in vivo in a mouse model, and histologically examined tumour tissue removed from BALB/c nude mice. Animals that received both IFN-gamma and CH-11 showed a 53.8% increase in anti-tumour effect (P=0.0018) 20 days after the first administration. In the histological study, the combined administration group tested positive in terminal deoxynucleotidyl transferase-mediated nick end labelling staining, and showed significantly increased levels of Fas expression on immunostaining compared with the vehicle group. These results show the efficacy of anticancer therapy using IFN-gamma and anti-Fas antibody via the modulation of Fas-mediated apoptosis. Moreover, inhibition of IFN-gamma/CH-11-induced apoptosis with a general caspase inhibitor (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) reduced cell death significantly in vitro. Bcl-2 cleavage did not occur under these conditions, suggesting a relationship between caspase activation and Bc1-2 cleavage in MMN9 cells.

p53 gene therapy of human osteosarcoma using a transferrin-modified cationic liposome.

Gene delivery via transferrin receptors, which are highly expressed by cancer cells, can be used to enhance the effectiveness of gene therapy for cancer. In this study, we examined the efficacy of p53 gene therapy in human osteosarcoma (HOSM-1) cells derived from the oral cavity using a cationic liposome supplemented with transferrin. HOSM-1 cells were exposed to transferrin-liposome-p53 in vitro, and the growth inhibition rate, expression of p53 and bax, and induction of apoptosis were measured 48 hours later. Treatment of HOSM-1 cells with transferrin-liposome-p53 resulted in 60.7% growth inhibition. Wild-type p53 expression and an increase in bax expression were observed following transfection with transferrin-liposome-p53, and 20.5% of the treated HOSM-1 cells were apoptotic. In vivo, the HOSM-1 tumor transplanted into nude mice grew to 5 to 6 mm in diameter. Following growth of the tumor to this size, transferrin-liposome-p53 was locally applied to the peripheral tumor (day 0) and then applied once every 5 days for a total of six times. During the administration period, tumor growth did not occur, and the mean tumor volume on the last day of administration (day 25) was 10.0% of that in the saline control group. These results suggest that p53 gene therapy via cationic liposome modification with transferrin is an effective strategy for treatment of osteosarcoma.

Interferon-gamma and anti-Fas antibody-induced apoptosis in human melanoma cell lines and its relationship to bcl-2 cleavage and bak expression.

Interferon-gamma (IFNgamma) has been shown to induce apoptosis through the induction of the Fas antigen in certain cell lines. In this study, we used four melanoma cell lines (MMN9, PMP, MAA and HMG) to study the antiproliferative effect of exogenous IFNgamma treatment, the expression of IFNgamma-induced Fas antigen, and the combined effect of IFNgamma and anti-Fas antibody (CH-11). We also investigated the relationship between Fas-mediated apoptosis and the expression of the bcl-2 family, measured using Western blotting. IFNgamma increased the mean fluorescence intensity of Fas in MMN9 and PMP cells as measured by flow cytometry. Combined therapy had a significant antiproliferative effect on MMN9 and PMP cells, as measured by the MTT assay. After exposure to IFNgamma and anti-Fas antibody, cleavage of bcl-2 occurred in apoptotic cells, and the signal intensity of both bcl-2 and bak decreased in surviving MMN9 cells, as shown by Western blotting analysis. Our results indicate that IFNgamma induces overexpression of Fas and consequently enhances the sensitivity of melanoma cells to Fas-mediated apoptosis. Furthermore, it is possible that cleavage of bcl-2 correlates with the induction of apoptosis induced by IFNgamma and anti-Fas antibody in melanoma cells. We conclude that combined therapy with IFNgamma and anti-Fas antibody may provide an alternative and more efficient chemotherapeutic approach against melanoma cells by inducing the overexpression of Fas after exposure to IFNgamma.