Sulforaphane counteracts aggressiveness of pancreatic cancer driven by dysregulated Cx43-mediated gap junctional intercellular communication.

The extreme aggressiveness of pancreatic ductal adenocarcinoma (PDA) has been associated with blocked gap junctional intercellular communication (GJIC) and the presence of cancer stem cells (CSCs). We examined whether disturbed GJIC is responsible for a CSC phenotype in established and primary cancer cells and patient tissue of PDA using interdisciplinary methods based in physiology, cell and molecular biology, histology and epigenetics. Flux of fluorescent dyes and gemcitabine through gap junctions (GJs) was intact in less aggressive cells but not in highly malignant cells with morphological dysfunctional GJs. Among several connexins, only Cx43 was expressed on the cell surface of less aggressive and GJIC-competent cells, whereas Cx43 surface expression was absent in highly malignant, E-cadherin-negative and GJIC-incompetent cells. The levels of total Cx43 protein and Cx43 phosphorylated at Ser368 and Ser279/282 were high in normal tissue but low to absent in malignant tissue. si-RNA-mediated inhibition of Cx43 expression in GJIC-competent cells prevented GJIC and induced colony formation and the expression of stem cell-related factors. The bioactive substance sulforaphane enhanced Cx43 and E-cadherin levels, inhibited the CSC markers c-Met and CD133, improved the functional morphology of GJs and enhanced GJIC. Sulforaphane altered the phosphorylation of several kinases and their substrates and inhibition of GSK3, JNK and PKC prevented sulforaphane-induced CX43 expression. The sulforaphane-mediated expression of Cx43 was not correlated with enhanced Cx43 RNA expression, acetylated histone binding and Cx43 promoter de-methylation, suggesting that posttranslational phosphorylation is the dominant regulatory mechanism. Together, the absence of Cx43 prevents GJIC and enhances aggressiveness, whereas sulforaphane counteracts this process, and our findings highlight dietary co-treatment as a viable treatment option for PDA.

Sulforaphane increases drug-mediated cytotoxicity toward cancer stem-like cells of pancreas and prostate.

Despite intense efforts to develop treatments against pancreatic cancer, agents that cure this highly resistant and metastasizing disease are not available. Considerable attention has focused on broccoli compound sulforaphane (SF), which is suggested as combination therapy for targeting of pancreatic cancer stem cells (CSCs). However, there are concerns that antioxidative properties of SF may interfere with cytotoxic drugs-as suggested, e.g., for vitamins. Therefore we investigated a combination therapy using established pancreatic CSCs. Although cisplatin (CIS), gemcitabine (GEM), doxorubicin, 5-flurouracil, or SF effectively induced apoptosis and prevented viability, combination of a drug with SF increased toxicity. Similarly, SF potentiated the drug effect in established prostate CSCs revealing that SF enhances drug cytotoxicity also in other tumor entities. Most importantly, combined treatment intensified inhibition of clonogenicity and spheroid formation and aldehyde dehydrogenase 1 (ALDH1) activity along with Notch-1 and c-Rel expression indicating that CSC characteristics are targeted. In vivo, combination treatment was most effective and totally abolished growth of CSC xenografts and tumor-initiating potential. No pronounced side effects were observed in normal cells or mice. Our data suggest that SF increases the effectiveness of various cytotoxic drugs against CSCs without inducing additional toxicity in mice.