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PURPOSEThe purpose of this study was to elucidate the mechanism of action of the Helix lucorum hemocyanin (HlH), b-HlH-h, and RvH2-g hemocyanins as potential agents against bladder cancer.METHODSWe evaluated the viability of 647-V, T-24, and CAL-29 bladder cancer cell lines after treatment with the tested hemocyanins. The cell viability was measured at 72 hrs with MTT and WST-1 assays. Acridine orange/propidium iodide double staining was used to discriminate between apoptotic and necrotic cells. Gene expression profiling of the 168 genes from human inflammatory cytokines and signal transduction pathways were performed on the tumor cells before and after hemocyanins' treatment.RESULTSThe results showed decreased survival of cancer cells in the presence of HlH and two functional units: b-HlH-h and RvH2-g. Acridine orange/propidium iodide double staining revealed that the decreased viability was due to apoptosis. The gene expression data showed upregulation of genes involved in the apoptosis as well as of the immune system activation, and downregulation of the CCL2, CCL17, CCL21, CXCL1, and ABCF1 genes.CONCLUSIONSThe present study is the first to report gene expression in human cells under the influence of hemocyanins. The mechanism of antitumor activity of the HlH, b-HlH-h, and RvH2-g hemocyanins includes induction of apoptosis. In addition to the antiproliferative effect, downregulation of the genes with metastatic potential was observed. Together with the already known immunogenic effect, these findings support further studies on hemocyanins as potential therapeutic agents against bladder cancer.

Changes in the gene expression profile of the bladder cancer cell lines after treatment with Helix lucorum and Rapana venosa hemocyanin.