Valproic acid radiosensitizes anaplastic thyroid cells through a decrease of the DNA damage repair capacity.

BACKGROUND: Anaplastic thyroid cancer (ATC) represents a rare lethal human malignancy with poor prognosis. Multimodality treatment, including radiotherapy, is recommended to improve local control and survival. Valproic acid (VA) is a clinically available histone deacetylase inhibitor with a well-documented side effect profile. In this study, we aim to investigate the combined effect of VA with photon irradiation in vitro. METHODS: Anaplastic thyroid cancer cells (8505c) were used to investigate the radiosensitizing effect of VA. RESULTS: VA sensitized cells to photon irradiation. VA increased radiation-induced apoptosis and radiation-induced DNA damage measured by γH2AX foci induction. Furthermore, VA prolonged γH2AX foci disappearance over time in irradiated cells and decreased the radiation-induced levels of mRNA of key DNA damage repair proteins of the homologous recombination (HR) and the nonhomologous end joining (NHEJ) pathways. CONCLUSIONS: VA at a clinically safe dose enhance the radiosensitivity of 8505c cells through an increase in radiation-induced apoptosis and a disruption in the molecular mechanism of HR and NHEJ DNA damage repair pathways.

Interferon-ß lipofection I. Increased efficacy of chemotherapeutic drugs on human tumor cells derived monolayers and spheroids.

We evaluated the effect of hIFNß gene transfer alone or in combination with different antineoplastic drugs commonly used in cancer treatment. Five human tumor-derived cell lines were cultured as monolayers and spheroids. Four cell lines (Ewing sarcomas EW7 and COH, melanoma M8 and mammary carcinoma MCF-7) were sensitive to hIFNß gene lipofection. Although this effect appeared in both culture configurations, spheroids showed a relative multicellular resistance (insensitive colon carcinoma HT-29 excluded). EW7 and M8 hIFNß-expressing cells were exposed to different concentrations of bleomycin, bortezomib, carboplatin, doxorubicin, etoposide, methotrexate, paclitaxel and vincristine in both configuration models. In chemotherapy-sensitive EW7 monolayers, the combination of hIFNß gene and antineoplastic drugs displayed only additive or counteractive (methotrexate) effects, suggesting that cytotoxic mechanisms triggered by hIFNß gene lipofection could be saturating the signaling pathways. Conversely, in chemotherapy-resistant EW7 spheroids or M8 cells, the combination of hIFNß with drugs that mainly operate at the genotoxic level (doxorubicin, methotrexate and paclitaxel) presented only additive effects. However, drugs that also increase pro-oxidant species can complement the antitumor efficacy of the hIFNß gene and clearly caused potentiated effects (bleomycin, bortezomib, carboplatin, etoposide and vincristine). The great bystander effect induced by hIFNß gene lipofection could be among the main causes of its effectiveness, because only 1 or 2% of EW7 or M8 hIFNß-expressing cells killed more than 60 or 80% of cell population, respectively.

Interferon-ß lipofection II. Mechanisms involved in cell death and bystander effect induced by cationic lipid-mediated interferon-ß gene transfer to human tumor cells.

We evaluated the cytotoxic effects (apoptosis, necrosis and early senescence) of human interferon-ß (hIFNß) gene lipofection. The cytotoxicity of hIFNß gene lipofection resulted equivalent to that of the corresponding addition of the recombinant protein (rhIFNß) on human tumor cell lines derived from Ewing's sarcoma (EW7 and COH) and colon (HT-29) carcinomas. However, it was stronger than rhIFNß on melanoma (M8) and breast adenocarcinoma (MCF7). To reveal the mechanisms involved in these differences, we compared the effects of hIFNß gene and rhIFNß protein on EW7 and M8 (sensitive and resistant to rhIFNß protein, respectively). Lipofection with hIFNß gene caused a mitochondrial potential decrease simultaneous with an increase of oxidative stress in both cell lines. However, rhIFNß protein displayed the same pattern of response only in EW7-sensitive cell line. The great bystander effect of the hIFNß gene lipofection, involving the production of reactive oxygen species, would be among the main causes of its success. In EW7, this effect killed >60% of EW7 cell population, even though only 1% of cells were expressing the transgene. As hIFNß gene was effective even in the rhIFNß protein-resistant M8 cell line and in a way not limited by low lipofection efficiency, these results strongly support the clinical potential of this approach.

Suicide gene therapy on spontaneous canine melanoma: correlations between in vivo tumors and their derived multicell spheroids in vitro.

To validate the use of multicellular spheroids to predict the efficacy of herpes simplex thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene therapy in the respective in vivo tumors, we established and characterized 15 melanoma-derived cell lines from surgically excised melanoma tumors. Three HSVtk-lipofected cell lines were not sensitive to GCV in any culture configuration, other five displayed similar sensitivity as monolayers or spheroids, and only one resulted more sensitive when grown as spheroids. Other six cell lines manifested a relative multicellular resistance (MCR) phenotype growing as spheroids, compared with the same cells growing as monolayers. The reverse correlation between the MCR and the monolayers survival to HSVtk/GCV suggests that one of the main causes of MCR would be the rapid cell repopulation after suicide gene treatment. The high correlation of MCR with the spheroids radial growth and with the mitotic index of the respective originary tumors supported this re-growth involvement. A remarkable finding was the high correlation in HSVtk/GCV sensitivity between in vivo tumor and the corresponding derived cell lines growing as spheroids (R(2) = 0.85). This strongly encourages the implementation of spheroids as highly realistic experimental model for optimizing and predicting the in vivo response of the respective tumors to therapeutic strategies.