Preliminary Study On The Effect Of KAI1/CD82Small Extracellular Loop On Tumor Metastasis

KAI1/CD82is one of the important members of the tetraspanin superfamily, which participates in a wide variety of biological events such as tumor migration and invasion. KAI1/CD82is widely expressed in a variety of normal tissues, at the same time, it appears a down regulation or loss of KAI1/CD82expression in tumor tissues. KAI1/CD82was initially identified as a specific metastasis suppressor of prostate cancer, a plethora of evidence supports that KAI1/CD82is a wide-spectrum invasion-and metastasis-suppressor after the research on a variety of solid tumors.Since KAI1/CD82was first reported its ability to inhibit tumor metastasis in1995, its mechanism is gradually being revealed, but the detailed mechanism how it works remains unclear. The molecular structural bases of KAI1/CD82should be figured out in order to clarify its functional mechanism. It has been reported that the KAI1/CD82protein consists of membrane-spanning domains, extracellular domains and intracellular domains. The extracellular domains contains a small extracellular loop(SEL) and a large extracellular loop(LEL); the transmembrane(TM) domains are four hydrophobic peptides, and the intracellular domains comprise a N-terminal tail, a C-terminal cytoplasmic domain and a intracellular loop(IL). There plenty of studies on the function of KAI1/CD82-LEL, TM domains and intracellular domains. KAI1/CD82-LEL takes part in the dimerization of KAI1/CD82and mediates the interaction with some membrane proteins. The polar residues in TM domains are predicted to interact with other tetraspanin superfamily proteins such as CD9, CD81and form polymer with them. The acylation in intracellular domains likely assists the anchorage of The N-and C-terminal to membrane and the interactions between TM domains in the Tetraspanin Web. However, the function of KAI1/CD82-SEL is rarely reported and not well understood.To elucidate the effect of KAI1/CD82-SEL on tumor metastasis, we synthesized the KAI1/CD82-LEL by chemical synthesis and observe its inhibitory activity on cell migration in vitro through scratching and chemo tactic ways. Meanwhile, we made a primary research on the molecular mechanism by which KAI1/CD82-SEL affect the cell motility through immunoblotting. What’s more, a initiatory comparison between KAI1/CD82-SEL and KAI1/CD82whole molecular was conducted. The research provides a essential theoretical significance and potential applications on the mechanism by which KAI1/CD82suppresses tumor metastasis and development of anti-metastatic drugs.Results:(1) CD82-SEL can significantly inhibit the SW620and MDA-MB-231cells migration and invasion in vitro, and it provides a positive correlation between the action concentration of KAI1/CD82-SEL and its suppression. The SW620and the MD A-MB-231cells can respectively be completely inhibited in the concentration of50μg/ml (P<0.001) and80μg/ml (P<0.001).(2)Impact of KAI1/CD82-SEL on EGFR and relative signaling pathway:in SW620cells, the phosphorylation level of EGFR-Tyr-1173and Akt-Ser-473can be remarkably inhibited (P<0.001); in MD A-MB-231cells, the phosphorylation level of EGFR-Tyr-845, EGFR-Tyr-1173and Akt-Ser-473can be remarkably inhibited (P<0.001).(3)Over-expression plasmid was transfected into highly metastatic human colon cancer SW620, and its inhibition ability was lower than KAIl/CD82-SEL(P<0.001). Through the study of both mechanisms, we found that in SW620cells, the phosphorylation level of EGFR-Tyr-1173and Akt-Ser-473can be remarkably inhibited (P<0.001) under the action of KAI1/CD82-SEL; the phosphorylation level of EGFR-Tyr-1045and ERK can be remarkably inhibited (P<0.001) under the action of KAI1/CD82whole molecular (P<0.001).Conclusions:(1)KAI1/CD82-SEL shows a intense inhibition on the motility of tumor cells derived from various tissues, including SW620and MDA-MB-231.(2)The mechanism how KAI1/CD82-SEL inhibits the motility of SW620and MDA-MB-231cells shares a cell differentiation.(3)The inhibition efficiency of KAI1/CD82-SEL peptide fragment is higher than the KAI1/CD82whole molecular, and their mechanism of action is different.