Cytotoxic and apoptogenic properties of the hemocyanin derived from the marine mollusk Rapana thomasiana in vitro

Marine invertebrates provide a series of natural products with different biological activities. Mollusk hemocyanins have been studied intensively for many years with respect to its structure and function and are well known as biologically active substances. This prompted us to look for the cytotoxic effect of native blue copper oxygenated respiratory protein RtH, and its structural isoform RtH2 found in the hemolymph of the marine mollusk Rapana thomasiana, who lives in shallow waters of the Black sea coast. Six cell lines were used in our experiments – five human cancer cell lines (SiHa- cervical squamous cell carcinoma, CaOV- ovarian adenocarcinoma, MIA PaCa – pancreatic carcinoma, RD 64 -rhabdomyosarcoma, EJ- urinary bladder carcinoma) and Lep – nontumor human lung cell line. Following RtH and RtH2 treatment cell viability was evaluated at 24h and 48h by two methods. Cytotoxic and cytostatic effect of RtH was compared to the effect of Tamoxifen (commercially available anticancer drug). Significant cell growth inhibition (P<0.05) was observed in three of the five cell lines tested at both time treatment intervals. These were SiHa, CaOV and MIA PaCa. The cervical cell line – SiHa exhibited a mean growth inhibition and cytopatic effect (range 37 to 59%) at 48h, whereas the ovarian cell – CaOV had a range of 4 to 44% at these same concentrations. The tested substances inhibited the colony-forming ability of tumor cells in a dose-dependent manner. Native hemocyanin inhibited cell proliferation in human cel lines with different origin, and induced early apoptotic events, such as DNA degradation and caspase-3 activation. In conclusion we might suggest the native RtH could have a potential anti-tumor activity.