BackgroundTumor irradiation combined with adjuvant treatments, either vascular targeted or immunomodulatory, is under intense investigation. Gene electrotransfer of therapeutic genes is one of these approaches. The aim of this study was to determine, whether gene electrotransfer of plasmid encoding shRNA for silencing endoglin, with vascular targeted effectiveness, can radiosensitize melanoma B16F10 tumors.Materials and methodsThe murine melanoma B16F10 tumors, growing on the back of C57Bl/6 mice, were treated by triple gene electrotransfer and irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice. Furthermore, histological analysis of tumors (necrosis, apoptosis, proliferation, vascularization, presence of hypoxia and infiltration of immune cells,) was used to evaluate the therapeutic mechanisms.ResultsGene electrotransfer of plasmid silencing endoglin predominantly indicated vascular targeted effects of the therapy, since significant tumor growth delay and 44% of tumor free mice were obtained. In addition, irradiation had minor effects on radioresistant melanoma, with 11% of mice tumor free. The combined treatment resulted in excellent effectiveness with 88% of mice tumor free, with more than half resistant to secondary tumor challenge, which was observed also with the plasmid devoid of the therapeutic gene. Histological analysis of tumors in the combined treatment group, demonstrated similar mode of action of the gene electrotransfer of plasmid encoding shRNA for silencing endoglin and devoid of it, both through the induction of an immune response.ConclusionsThe results of this study indicate that irradiation can in radioresistant melanoma tumors, by release of tumor associated antigens, serve as activator of the immune response, besides directly affecting tumor cells and vasculature. The primed antitumor immune response can be further boosted by gene electrotransfer of plasmid, regardless of presence of the therapeutic gene, which was confirmed by the high radiosensitization, resulting in prolonged tumor growth delay and 89% of tumor free mice that were up to 63% resistant to secondary challenge of tumor. In addition, gene electrotransfer of therapeutic plasmid for silencing endoglin has also a direct effect on tumor vasculature and tumors cells; however in combination with radiotherapy this effect was masked by pronounced immune response.

Background: Treatment options for recurrent head and neck tumours in the previously irradiated area are limited, including re-irradiation due to radioresistance of the recurrent tumour and previous dose received by surrounding normal tissues. As an in vitro model to study radioresistance mechanisms, isogenic cells with different radiosensitivity can be used. However, they are not readily available. Therefore, our objective was to establish and characterize radioresistant isogenic human pharyngeal squamous carcinoma cells and to evaluate early radiation response in isogenic parental, radioresistant and radiosensitive cells. Methods: Radioresistant cells were derived from parental FaDu cells by repeated exposure to ionizing radiation. Radiosensitivity of the established isogenic radioresistant FaDu-RR cells was evaluated by clonogenic assay and compared to isogenic parental FaDu and radiosensitive 2A3 cells. Additional phenotypic characterization of these isogenic cells with different radiosensitivity included evaluation of chemosensitivity, cell proliferation, cell cycle, radiation-induced apoptosis, resolution of DNA double-strand breaks, and DNA damage and repair signalling gene expression before and after irradiation. Results: In the newly established radioresistant cells in response to 5 Gy irradiation, we observed no alteration in cell cycle regulation, but delayed induction and enhanced resolution of DNA double-strand breaks, lower induction of apoptosis, and pronounced over-expression of DNA damage signalling genes in comparison to parental cells. On the other hand, radiosensitive 2A3 cells were arrested in G 2 /M-phase in response to 5 Gy irradiation, had a prominent accumulation of and slower resolution of DNA double-strand breaks, and no change in DNA damage signalling genes expression. Conclusions: We concluded that the emergence of the radioresistance in the established radioresistant isogenic cells can be at least partially attributed to the enhanced DNA double-strand break repair, altered expression of DNA damage signalling and repair genes. On the other hand, in radiosensitive isogenic cells the reduced ability to repair a high number of induced DNA double-strand breaks and no transcriptional response in DNA damage signalling genes indicate on a lack of adaptive response to irradiation. Altogether, our results confirmed that these isogenic cells with different radiosensitivity are an appropriate model to study the mechanisms of radioresistance.

Electrochemotherapy is an established local ablative method used for the treatment of different tumor types, including tumors of the head and neck area. Clinical studies have demonstrated a lower response rate of tumors that recur in pre-irradiated area. The aim of the present study was to explore the response of experimentally induced radioresistant cells and tumors to electrochemotherapy with cisplatin or bleomycin. The radioresistant cells (FaDu-RR) were established by fractionated irradiation of parental human squamous cell carcinoma cell line, FaDu. We compared the 2 cell lines in response to chemotherapy and electrochemotherapy with cisplatin or bleomycin in vitro and in vivo. Using specific mass spectrometry-based analytical methods we determined the difference in the uptake of chemotherapeutics in tumors after electrochemotherapy. Additionally, we compared the capacity of the cells to repair DNA double-strand breaks (DSB) after exposure to the drugs used in electrochemotherapy with the γH2AX foci resolution determined by immunofluorescence microscopy. Our results indicate radio- and cisplatin cross-resistance, confirmed with the lower response rate of radioresistant tumors after electrochemotherapy with cisplatin. On the other hand, the sensitivity to electrochemotherapy with bleomycin was similar in both cell lines and tumors. While the uptake of chemotherapeutics after electrochemotherapy was comparable in both tumor models, there was a difference between the cell lines in capacity to repair DNA DSB-the radioresistant cells had a lower level of DSB and faster DNA repair rate after exposure to both, cisplatin or bleomycin. Due to the higher complete response rate after electrochemotherapy with bleomycin than with cisplatin, we conclude that the results favor bleomycin-over cisplatin-based electrochemotherapy for treatment of radioresistant tumors and/or tumors that regrow after radiotherapy.

BackgroundManagement of locoregionally recurrent head and neck squamous cell carcinomas (HNSCC) is challenging due to potential radioresistance. Pulsed low-dose rate (PLDR) irradiation exploits phenomena of increased radiosensitivity, low-dose hyperradiosensitivity (LDHRS), and inverse dose-rate effect. The purpose of this study was to evaluate LDHRS and the effect of PLDR irradiation in isogenic HNSCC cells with different radiosensitivity.Materials and methodsCell survival after different irradiation regimens in isogenic parental FaDu and radioresistant FaDu-RR cells was determined by clonogenic assay; post irradiation cell cycle distribution was studied by flow cytometry; the expression of DNA damage signalling genes was assesed by reverse transcription-quantitative PCR.ResultsRadioresistant Fadu-RR cells displayed LDHRS and were more sensitive to PLDR irradiation than parental FaDu cells. In both cell lines, cell cycle was arrested in G2/M phase 5 hours after irradiation. It was restored 24 hours after irradiation in parental, but not in the radioresistant cells, which were arrested in G1-phase. DNA damage signalling genes were under-expressed in radioresistant compared to parental cells. Irradiation increased DNA damage signalling gene expression in radioresistant cells, while in parental cells only few genes were under-expressed.ConclusionsWe demonstrated LDHRS in isogenic radioresistant cells, but not in the parental cells. Survival of LDHRS-positive radioresistant cells after PLDR was significantly reduced. This reduction in cell survival is associated with variations in DNA damage signalling gene expression observed in response to PLDR most likely through different regulation of cell cycle checkpoints.

The alkylpyridinium polymer APS8, a potent antagonist of α7 nicotinic acetylcholine receptors (nAChRs), selectively induces apoptosis in non-small cell lung cancer cells but not in normal lung fibroblasts. To explore the potential therapeutic value of APS8 for at least certain types of lung cancer, we determined its systemic and organ-specific toxicity in mice, evaluated its antitumor activity against adenocarcinoma xenograft models, and examined the in-vitro mechanisms of APS8 in terms of apoptosis, cytotoxicity, and viability. We also measured Ca2+ influx into cells, and evaluated the effects of APS8 on Ca2+ uptake while siRNA silencing of the gene for α7 nAChRs, CHRNA7. APS8 was not toxic to mice up to 5 mg/kg i.v., and no significant histological changes were observed in mice that survived APS8 treatment. Repetitive intratumoral injections of APS8 (4 mg/kg) significantly delayed growth of A549 cell tumors, and generally prevented regrowth of tumors, but were less effective in reducing growth of HT29 cell tumors. APS8 impaired the viability of A549 cells in a dose-dependent manner and induced apoptosis at micro molar concentrations. Nano molar APS8 caused minor cytotoxic effects, while cell lysis occurred at APS8 >3 µM. Furthermore, Ca2+ uptake was significantly reduced in APS8-treated A549 cells. Observed differences in response to APS8 can be attributed to the number of α7 nAChRs expressed in these cells, with those with more AChRs (i.e., A549 cells) being more sensitive to nAChR antagonists like APS8. We conclude that α7 nAChR antagonists like APS8 have potential to be used as therapeutics for tumors expressing large numbers of α7 nAChRs.