Masahiko Nishizaki

Overexpression of the wild-type p53 gene inhibits NF-κB activity and synergizes with aspirin to induce apoptosis in human colon cancer cells

The tumor suppressor gene p53 is a potent transcriptional regulator of genes which are involved in many cellular activities including cell cycle arrest, apoptosis, and angiogenesis. Recent studies have demonstrated that the activation of the transcriptional factor nuclear factor κB (NF-κB) plays an essential role in preventing apoptotic cell death. In this study, to better understand the mechanism reponsible for the p53-mediated apoptosis, the effect of wild-type p53 (wt-p53) gene transfer on nuclear expression of NF-κB was determined in human colon cancer cell lines. A Western blot analysis of nuclear extracts demonstrated that NF-κB protein levels in the nuclei were suppressed by the transient expression of the wt-p53 in a dose-dependent manner. Transduced wt-p53 expression increased the cytoplasmic expression of IκBα as well as its binding ability to NF-κB, thus markedly reducing the amount of NF-κB that translocated to the nucleus. The decrease in nuclear NF-κB protein correlated with the decreased NF-κB constitutive activity measured by electrophoretic mobility shift assay. Furthermore, parental cells transfected with NF-κB were better protected from cell death induced by the wt-p53 gene transfer. We also found that the wt-p53 gene transfer was synergistic with aspirin (acetylsalicylic acid) in inhibiting NF-κB constitutive activity, resulting in enhanced apoptotic cell death. These results suggest that the inhibition of NF-κB activity is a plausible mechanism for apoptosis induced by the wt-p53 gene transfer in human colon cancer cells and that anti-NF-κB reagent aspirin could make these cells more susceptible to apoptosis.

Myristoylation of the Fus1 Protein Is Required for Tumor Suppression in Human Lung Cancer Cells

FUS1 is a novel tumor suppressor gene identified in the human chromosome 3p21.3 region that is deleted in many cancers. Using surface-enhanced laser desorption/ionization mass spectrometric analysis on an anti-Fus1-antibody-capture ProteinChip array, we identified wild-type Fus1 as an N-myristoylated protein. N-myristoylation is a protein modification process in which a 14-carbon myristoyl group is cotranslationally and covalently added to the NH2-terminal glycine residue of the nascent polypeptide. Loss of expression or a defect of myristoylation of the Fus1 protein was observed in human primary lung cancer and cancer cell lines. A myristoylation-deficient mutant of the Fus1 protein abrogated its ability to inhibit tumor cell-induced clonogenicity in vitro, to induce apoptosis in lung tumor cells, and to suppress the growth of tumor xenografts and lung metastases in vivo and rendered it susceptible to rapid proteasome-dependent degradation. Our results show that myristoylation is required for Fus1-mediated tumor-suppressing activity and suggest a novel mechanism for the inactivation of tumor suppressors in lung cancer and a role for deficient posttranslational modification in tumor suppressor-gene-mediated carcinogenesis.

Synergistic Tumor Suppression by Coexpression of FHIT and p53 Coincides with FHIT-Mediated MDM2 Inactivation and p53 Stabilization in Human Non-Small Cell Lung Cancer Cells

Aberrations of the tumor suppressor genes FHIT and p53 are frequently associated with a wide range of human cancers, including lung cancer. We studied the combined effects of FHIT and p53 proteins on tumor cell proliferation and apoptosis in human non-small cell lung carcinoma (NSCLC) cells in vitro and on tumor growth in animal models by adenoviral vector-mediated cotransfer of wild-type FHIT and p53 genes. We found that the coexpression of FHIT and p53 synergistically inhibited tumor cell proliferation in NSCLC cells in vitro and suppressed the growth of human tumor xenografts in nude mice. Furthermore, we found that this synergistic inhibition of tumor cell growth corresponded with the FHIT-mediated inactivation of MDM2, which thereby blocked the association of MDM2 with p53, thus stabilizing the p53 protein. Our results therefore reveal a novel molecular mechanism consisting of FHIT-mediated tumor suppression and the interaction of FHIT with other cellular components in the pathways regulating p53 activity. These findings show that combination treatment with synergistic tumor-suppressing gene therapy such as Ad-FHIT and Ad-p53 may be an effective therapeutic strategy for NSCLC and other cancers.

CACNA2D2-mediated apoptosis in NSCLC cells is associated with alterations of the intracellular calcium signaling and disruption of mitochondria membrane integrity

The CACNA2D2 gene, a new subunit of the Ca2+-channel complex, was identified in the homozygous deletion region of chromosome 3p21.3 in human lung and breast cancers. Expression deficiency of the CACNA2D2 in cancer cells suggests a possible link of it to Ca2+ signaling in the pathogenesis of lung cancer and other cancers. We investigated the effects of overexpression of CACNA2D2 on intracellular Ca2+ contents, mitochondria homeostasis, cell proliferation, and apoptosis by adenoviral vector-mediated wild-type CACNA2D2 gene transfer in 3p21.3-deficient nonsmall cell lung cancer cell lines. Exogenous expression of CACNA2D2 significantly inhibited tumor cell growth compared with the controls. Overexpression of CACNA2D2 induced apoptosis in H1299 (12.5%), H358 (13.7%), H460 (22.3%), and A549 (50.1%) cell lines. Levels of intracellular free Ca2+ were elevated in AdCACNA2D2-transduced cells compared with the controls. Mitochondria membrane depolarization was observed prior to apoptosis in Ad-CACNA2D2 and Adp53-transduced H460 and A549 cells. Release of cyt c into the cytosol, caspase 3 activation, and PARP cleavage were also detected in these cells. Together, these results suggest that one of the pathways in CACNA2D2-induced apoptosis is mediated through disruption of mitochondria membrane integrity, the release of cyt c, and the activation of caspases, a process that is associated with regulation of cytosolic free Ca2+ contents.

Combination of TRAIL gene therapy and chemotherapy enhances antitumor and antimetastasis effects in chemosensitive and chemoresistant breast cancers

We recently found that breast cancer cell lines that are resistant to chemotherapy or to the recombinant TRAIL protein are susceptible to TRAIL gene therapy. However, it is unclear whether a combination of TRAIL gene therapy and chemotherapy will have enhanced antitumor activity or can be used for the treatment of metastasis. In this study, we investigated the combined effect of TRAIL gene therapy and chemotherapeutic agents, including doxorubicin, paclitaxel, vinorelbine, gemcitabine, irinotecan, and floxuridine, in different breast cancer cell lines. In all the cell lines tested, including a breast cancer cell line that is resistant to chemotherapy, the combination of TRAIL gene therapy and cytotoxic agents had either a synergistic or an additive effect. An in vivo study showed that aerosolized administration of an adenovector expressing the GFP-TRAIL fusion protein from the human telomerase reverse transcriptase promoter (designated Ad/gTRAIL) also decreased the number of lung metastases from both doxorubicin-sensitive and doxorubicin-resistant breast cancer cell lines. The combination of TRAIL gene therapy and chemotherapy resulted in a further reduction of lung metastatic nodules with minimal toxicity. These results suggest that a combination of TRAIL gene therapy and chemotherapy is effective in the treatment of metastatic diseases.