Semaphoring mAb: a New Guide in RIT in Inhibiting the Proliferation of Human Skin Carcinoma

Abstract

Semaphoring is a transmembrane receptor which participates in many cytokine-mediated signal pathwaysthat are closely related to the angiogenesis, occurrence and development of carcinoma. The present study wasdesigned to access the effect of mono-antibody (mAb) guided radioimmunotherapy (RIT) on skin carcinomaand investigate the potential mechanisms. Semaphoring mAb was acquired from mice (Balb/c), purified withrProtein A column; purity, concentration and activity were tested with SDS-PAGE and indirect ELISA; specificityand expression on the cutanuem carcinoma line and tissue were tested by Western blotting; morphology changewas assessed by microscopy. MTT assay and colony inhibition tests were carried out to test the influence onthe proliferation of tumor cells; Western blotting was also carried out for expression of apoptosis-associated(caspase-3, Bax, Bcl-2) and proliferation-related (PI3K, p-Akt, Akt, p-ERK1/2, ERK1/2) proteins and analysethe change in signal pathways (PI3K/Akt and MEK/ERK). The purity of purified semaphorin mAb was 96.5%and the titer is about 1×106. Western blotting showed semaphoring mAb to have specifically binding stripeswith semaphoring b1b2 protein, B16F10, and A431 cells at 39KDa, 100KDa and 130KDa, respectively. Positiveexpression was detected both in cutanuem carcinoma line and tissue and it mostly located in cell membranes.MMT assay revealed dose-relate and time-relate inhibitory effect of semaphorin mAb on A431 and B16F10.Colony inhibition tests also showed dose-relate inhibitory effects. Western blotting demonstrated the expressionof apoptosis and proliferation-related protein and changes in signal pathway. In conclusion, we demonstratedthat semaphorin is highly expressed on the tumor cell-surfaces and RIT with semaphorin mAb has effect ini nhibiting proliferation and accelerating apoptosis of tumor cells.