The Effects And Mechanisms Of KAI1 On The Biological Functions Of Multiple Myeloma Cells

Multiple Myeloma (MM), a neoplastic hematologic disorder with malignant plasmacytoid lymphocyte proliferans, has high prevalence and poor prognosis. The feature of MM is the secretion of monoclonal immunoglobulin companied with the decrease of immunoglobulin and extensive osteolytic lesions and/or osteoporosis. Except for hematopoietic stem cell transplantation, the standard chemotherapy regimen which alleviates only the symptoms often results in failure because of the drug resistance and palindromia during the treatment. The elucidation of the molecular and cellular mechanism of MM's occurrence and development is thus of great significance for the drug target searching and finally the conquest of this disease.The activation of oncogene or the silence of anti-oncogene in tumor cells is the primary factor in determining the occurrence and development of carcinoma. KAI1/CD82 is an important tumor suppressor gene, generally distributing in high abundance in prostate, lung, liver, bladder, marrow and placenta. As a member of transmembrane 4 super family (TM4SF), KAI1 has 4 transmembrane domains. KAI1 has been proved to play important roles in the regulation of cell cluster, adhesion, migration and proliferation. Our previous research showed that KAI1 suppresses the proliferation and migration, induces apoptosis of pancreatic carcinoma cells. The present study focused on the influence of CD82 on the biological functions of myeloma cell and the potential mechanisms underlying, unsealing the effective drug target for the treatment of myeloma.Firstly, we identified the recombinant adenovirus Ad5-KAI1 constructed by our laboratory on the genome level. The results showed that target gene KAI1 but not replication competent adenovirus could be detected by PCR, indicating that Ad5-KAI1 was replication-defective vector carrying KAI1 gene. After amplification and purification, the virus particle titer and purity was determined by the measurement of OD260 and OD280. The particle titer of the purified virus was 2.97×1012 vp/ml with a purity of 1.37. And the infection titer determined by fast CPE and TCID50 (extreme dilution method) was 1×1011 pfu/ml and 6.3×1010 IU/ml, the ratio of which to the particle titer was 47:1,These results demonstrated that recombinant adenovirus Ad5-KAI1 with high purity and titer was obtained, meeting the requirement of function experiments.Secondly, the KAI1 molecular in myeloma cell lines and MM patients'primary CD 138+ cells were detected by flow cytometry. The results showed that KAI1 was hardly expressed on the cell surface of myeloma cell lines, such as XG-7, SKO-007 and U-266, while the percentage of KAI1 positive cells in CD138+ cells isolated from two MM patients by magnetic bead were 17.8% and 16.7% respectively. These results demonstrated that the expression of KAI1 was significantly suppressed both in myeloma cell lines and in primary cells. To analyze the influence of KAI1 on the biological functions of myeloma cells, the myeloma cell line SKO-007 was infected by Ad5-GFP and Ad5-KAI1, after which the adenovirus infection efficiency and the KAI1 expression was detected by fluorescence microscopy and flow cytometry. The results showed that at 24 h after infection, the GFP+ cell of 50, 100 and 200 MOI Ad5-GFP infected SKO-007 was nearly 99% and no significant difference was observed between cells infected for 48 h and those for 24 h; the expression efficiency of KAI1 on 100 MOI Ad5-KAI1 infected SKO-007 was up to 90% at 24 h. These results indicated that the optimum infection intensity for subsequent experiments was 100 MOI.Thirdly, we assessed the influence of KAI1 on the cell biological functions, including proliferation, migration and apoptosis using Ad5-KAI1-infected SKO-007 cells as modle. CCK-8 and CFSE were employed to determine the effects of KAI1 on proliferation of SKO-007. Compared to control, Ad5-KAI1 significantly inhibited the proliferation of SKO-007. The cell migration experiment showed that compared to control, KAI1 effectively suppressed the migration of SKO-007 and the migration stimulating effect induced by HGF. The apoptosis was detected using Annexin V/PI double labeling method and the result showed that compared to the control, KAI1 effectively promoted SKO-007 apoptosis. The above results proved that KAI1 suppressed the proliferation and migration of SKO-007 and the migration stimulation effect induced by HGF while promoting apoptosis.Finally, we investigated the underlying mechanism by which KAI influence the biological functions of myeloma cells. The expression of Caspase-3 and PARP in SKO-007 was significantly upregulated by KAI1, whereas the Capase-3 suppressor blocked KAI1-induced apoptosis. It is indicated that KAI1 promotes apoptosis through a Capase-3 dependent pathway. We also demonstrated that KAI1 activated ERK signal pathway evidenced by the upregulation of ERK phosphorylation in myeloma cells infected by adenovirus carrying KAI1 gene. This was consistent with other reports showing the excessive activation of ERK pathway induced apoptosis per contra. PD98059, a specific inhibitor of ERK pathway was used to elucidate whether the induction of apoptosis by KAI1 was ERK dependent. The result that the induction of apoptosis by KAI1 was inhibited after the block of ERK pathway, indicated that the KAI1-induced apoptosis was regulated by ERK. As a growth factor, HGF can stimulate the expression and the phosphorylation of ERK. We conducted experiments to identify the inhibition of HGF-induced ERK phosphorylation by KAI1. The results showed that ERK phosphorylation in the cells without KAI1 infection was upregulated by HGF while that in the cells with KAI1 infection showed no significant differences with that of without HGF. We also examined the changes of AKT and STAT3 signal pathway induced by KAI1 and found that the phosphorylation of AKT was inhibited but STAT3 was no change. The results indicate that multiple pathways were involved in the function of myeloma cells by KAI1.In conclusion, KAI1 plays a role in the regulation of the biological functions of myeloma cells, such as suppressing proliferation and migration and inducing apoptosis. The regulation of apoptosis by KAI1 was Caspase-3-dependent and related to ERK pathway. The present work sets up foundations for the further elucidation of the mechanism by which KAI1 regulates myeloma cell and provides perspectives to develop new drug target for MM.