Nerve growth factor from Indian Russell's viper venom (RVV-NGFa) shows high affinity binding to TrkA receptor expressed in breast cancer cells: Application of fluorescence labeled RVV-NGFa in the clinical diagnosis of breast cancer.

Nerve growth factor (NGF) is a minor and neglected component of snake venom. Present study describes the purification and characterization of a NGF isoform (RVV-NGFa) from Indian Russell's viper venom (RVV). RVV-NGFa showed a protonated molecular ion [MH+] at m/z 17388.725 Da. The RVV-NGFa induced neuritogenesis in PC-12 cells but did not show cytotoxicity in mammalian cells, hemolytic activity, platelet modulation, and interference in blood coagulation system which are the characteristic pharmacological properties of RVV. By ELISA and immunofluorescence microplate reader assay, the RVV-NGFa showed appreciable binding to TrkA receptor expressed in breast cancer MDA-MB-231 and MCF-7 cells; nevertheless, pre-incubation of cells with anti-TrkA (and not TrkB or TrkC) or anti-p⁷⁵NTR antibody significantly decreased (p < 0.05) this binding. The RVV-NGFa demonstrated insignificant binding with non-cancerous cells (HEK-293, L6) lacking TrkA receptor. The binding of RVV-NGFa to TrkA receptor of breast cancer cells resulted in internalization of ligand (RVV-NGFa)-receptor (TrkA) complex to cell cytoplasm in a time-dependent manner. The spectrofluorometric study demonstrated an interaction between RVV-NGFa and cytosolic domain of the purified TrkA receptor. The fluorescence (FITC)-tagged RVV-NGFa depicted a strong fluorescence signal that was observed under a fluorescence microscope and determined by fluorescence microplate reader assay post binding to breast cancer cells; but no fluorescence signal was detected after incubating FITC-RVV-NGFa with non-cancerous L6 and HEK-293 cells. The clinical application of FITC/fluorescence nanoparticle tagged RVV-NGFa for the ex vivo and in vivo diagnosis of breast cancer is highly promising.

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