Fractionation of Potamon Persicum Crabs hemolymph and their effects on MCf-7, MdA-231, and HUVEC

Introduction and aims: Breast cancer is the most common malignancy among Iranian women. The induction of apoptosis is considered a major goal in the treatment of cancer at different stages of tumor growth. The aim of this study was to fractionate the Potamon persicum crab hemolymph serum and assess the its cell toxicity, induction of apoptosis and anti-proliferation effects on cancer cell lines (MDA-231, MCF-7) and HUVEC. Materials and Methods: Crabs were transported to the laboratory in containers containing river water and anesthetized by ether and chloroform. The hemolymph was collected in a sterile tube through a cut in the forearm of the crab. Then the serum was separated from the cellular part by ion exchange chromatography. The cell toxicity and anti-proliferative effects of the total serum and fractions were investigated by LDH (Lactate dehydrogenase) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tests and their apoptotic effect was assayed by a combination of TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling)  and propidium iodide staining. Results: Four different protein fractions in terms of electrical charge were obtained from hemolymph serum by ion exchange. The fourth group (hemocyanin) contained 80% of the total protein of hemolymph serum. The fourth fraction and total serum were able to inhibit the growth and survival of MDA-231 and MCF-7 cancer cells at lower concentrations than HUVEC (noncancerous cells). They also increased the induction of apoptosis relative to the control sample, with the fourth fraction having a stronger effect than total serum. Discussion and Conclusion: Due to the fact that the total serum and fourth fraction of Hemolymph had low toxicity and showed anti-proliferation effect on cancer cells in lower concentrations and also caused the induction of apoptosis in these cells, so the hemolymph serum of this crab and especially the fourth fraction which has the highest amount of hemolymph protein could be a good option for more quantitative studied and continuing the study in in-vivo models for breast cancer treatment.