In modulated electrohyperthermia (mEHT) the enrichment of electric field and the concomitant heat can selectively induce cell death in malignant tumors as a result of elevated glycolysis, lactate production (Warburg effect), and reduced electric impedance in cancer compared to normal tissues. Earlier, we showed in HT29 colorectal cancer xenografts that the mEHTprovoked programmed cell death was dominantly caspase independent and driven by apoptosis inducing factor activation. Using this model here, we studied the mEHT-related cell stress 0-, 1-, 4-, 8-, 14-, 24-, 48-, 72-, 120-, 168-and 216-h post-treatment by focusing on damage-associated molecular pattern (DAMP) signals.

Background: Modulated electro-hyperthermia (mEHT), a non-invasive intervention using 13.56 MHz radiofrequency, can selectively target cancers due to their elevated glycolysis (Warburg-effect), extracellular ion concentration and conductivity compared to normal tissues. We showed earlier that mEHT alone can provoke apoptosis and damage associated molecular pattern (DAMP) signals in human HT29 colorectal cancer xenografts of immunocompromised mice.Materials: Here we tested the mEHT induced stress and immune responses in C26 colorectal cancer allografts of immunocompetent (BALB/c) mice between 12-72 h post-treatment. The right side of the symmetrical tumors grown in both femoral regions of mice were treated for 30 minutes, while the left side tumors served for untreated controls.Results: Loco-regional mEHT treatment induced an ongoing and significant tumor damage with the blockade of cell cycle progression indicated by the loss of nuclear Ki67 protein. Nuclear shrinkage, apoptotic bodies and DNA fragmentation detected using TUNEL assay confirmed apoptosis. Cleaved/activated-caspase-8 and -caspase-3 upregulation along with mitochondrial translocation of bax protein and release of cytochrome-c were consistent with the activation of both the extrinsic and intrinsic caspase-dependent programmed cell death pathways. The prominent release of stress-associated Hsp70, calreticulin and HMGB1 proteins, relevant to DAMP signaling, was accompanied by the significant tumor infiltration by S100 positive antigen presenting dendritic cells and CD3 positive T-cells with only scant FoxP3 positive regulatory T-cells. In addition, mEHT combined with a chlorogenic acid rich T-cell promoting agent induced significant cell death both in the treated and the untreated contralateral tumors indicating a systemic anti-tumor effect.Conclusions: mEHT induced caspase-dependent programmed cell death and the release of stress associated DAMP proteins in colorectal cancer allografts can provoke major immune cell infiltration. Accumulating antigen presenting dendritic cells and T-cells are likely to contribute to the ongoing tumor destruction by an immunogenic cell death mechanism both locally and through systemic effect at distant tumor sites.

Modulated EHT treatment can induce programmed cell death-related tumor destruction in HT29 colorectal adenocarcinoma xenografts, which dominantly follows a caspase-independent subroutine.

Background and AimsConnexins and their cell membrane channels contribute to the control of cell proliferation and compartmental functions in breast glands and their deregulation is linked to breast carcinogenesis. Our aim was to correlate connexin expression with tumor progression and prognosis in primary breast cancers.Materials and MethodsMeta-analysis of connexin isotype expression data of 1809 and 1899 breast cancers from the Affymetrix and Illumina array platforms, respectively, was performed. Expressed connexins were also monitored at the protein level in tissue microarrays of 127 patients equally representing all tumor grades, using immunofluorescence and multilayer, multichannel digital microscopy. Prognostic correlations were plotted in Kaplan-Meier curves and tested using the log-rank test and cox-regression analysis in univariate and multivariate models.ResultsThe expression of GJA1/Cx43, GJA3/Cx46 and GJB2/Cx26 and, for the first time, GJA6/Cx30 and GJB1/Cx32 was revealed both in normal human mammary glands and breast carcinomas. Within their subfamilies these connexins can form homo- and heterocellular epithelial channels. In cancer, the array datasets cross-validated each other’s prognostic results. In line with the significant correlations found at mRNA level, elevated Cx43 protein levels were linked with significantly improved breast cancer outcome, offering Cx43 protein detection as an independent prognostic marker stronger than vascular invasion or necrosis. As a contrary, elevated Cx30 mRNA and protein levels were associated with a reduced disease outcome offering Cx30 protein detection as an independent prognostic marker outperforming mitotic index and necrosis. Elevated versus low Cx43 protein levels allowed the stratification of grade 2 tumors into good and poor relapse free survival subgroups, respectively. Also, elevated versus low Cx30 levels stratified grade 3 patients into poor and good overall survival subgroups, respectively.ConclusionDifferential expression of Cx43 and Cx30 may serve as potential positive and negative prognostic markers, respectively, for a clinically relevant stratification of breast cancers.

BackgroundPrimary systemic treatment for ovarian cancer is surgery, followed by platinum based chemotherapy. Platinum resistant cancers progress/recur in approximately 25% of cases within six months. We aimed to identify clinically useful biomarkers of platinum resistance.MethodsA database of ovarian cancer transcriptomic datasets including treatment and response information was set up by mining the GEO and TCGA repositories. Receiver operator characteristics (ROC) analysis was performed in R for each gene and these were then ranked using their achieved area under the curve (AUC) values. The most significant candidates were selected and in vitro functionally evaluated in four epithelial ovarian cancer cell lines (SKOV-3-, CAOV-3, ES-2 and OVCAR-3), using gene silencing combined with drug treatment in viability and apoptosis assays. We collected 94 tumor samples and the strongest candidate was validated by IHC and qRT-PCR in these.ResultsAll together 1,452 eligible patients were identified. Based on the ROC analysis the eight most significant genes were JRK, CNOT8, RTF1, CCT3, NFAT2CIP, MEK1, FUBP1 and CSDE1. Silencing of MEK1, CSDE1, CNOT8 and RTF1, and pharmacological inhibition of MEK1 caused significant sensitization in the cell lines. Of the eight genes, JRK (p = 3.2E-05), MEK1 (p = 0.0078), FUBP1 (p = 0.014) and CNOT8 (p = 0.00022) also correlated to progression free survival. The correlation between the best biomarker candidate MEK1 and survival was validated in two independent cohorts by qRT-PCR (n = 34, HR = 5.8, p = 0.003) and IHC (n = 59, HR = 4.3, p = 0.033).ConclusionWe identified MEK1 as a promising prognostic biomarker candidate correlated to response to platinum based chemotherapy in ovarian cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-837) contains supplementary material, which is available to authorized users.