The Intrabody Targeting CDK4 Inhibits Growth And Metastases Of Melanomas

Melanoma is a type of malignant tumor originating from melanocytes,and its incidence is gradually increasing worldwide.For the early-stage melanoma,it can be surgically removed,but it is easy to recur after surgery,while for the advanced and metastatic melanoma,there is still a need for effective treatment methods.Thus exploring new melanoma treatments has important scientific value and significance for human health.Dysregulation of the cell cycle leads to malignant proliferation of cells,which in turn leads to tumors.CDK4 is an important molecule in cell cycle regulation.It interacts with Cyclin D1 to form a Cyclin D1-CDK4 complex,which in turn catalyzes the phosphorylation of Rb protein,releases E2 F transcription factors,and enables the cell cycle to enter from G1 phase to S phase.Overexpression of CDK4 has been found in various tumors and cancers,and CDK4 has become an important target for tumor therapy.Intrabody is a new type of immunotherapy strategy.Its gene fragments can be delivered into cells through a carrier,and then expressed in cells,targeting and binding to intracellular antigens and inhibiting the activity of target proteins.In the previous study,our group has successfully prepared a single-chain antibody AK that can specifically bind to CDK4 protein,and fused its gene fragment with the nuclear localization signal sequence NLS to successfully prepare an intracellular antibody NLS-AK targeting intracellular CDK4.The previous experimental studies have proved that NLS-AK can inhibit the growth and proliferation of cervical cancer cells,liver cancer cells and breast cancer cells,but the therapeutic effect and mechanism of NLS-AK on melanoma are still unclear.Here,the inhibitory effects of targeting CDK4 intrabody NLS-AK on the proliferation and migration of three melanoma cells（B16-F0,B16-F10,A375）were analyzed in vitro,and the in vivo therapeutic effect was evaluated by the animal model bearing the B16-F10 cells,and the following results were obtained:co-immunoprecipitation and Western blot results showed that NLS-AK was effectively expressed in melanoma cells and specifically interacted with CDK4protein;indirect immunofluorescence results showed that NLS-AK and CDK4co-localized in the nucleus;MTT and flow cytometry results showed that NLS-AK inhibited the growth and proliferation of melanoma cells and promoted the apoptosis of melanoma cells;the results of scratch wound healing and transwell experiments showed that NLS-AK inhibited the migration and invasion of melanoma cells;the results from NLS-AK treatment for mice bearing melanoma further showed that NLS-AK inhibited the growth and metastasis of melanoma in vivo;moreover by using Q-PCR and Western blot,we explored the effects of NLS-AK on m RNA and protein related to tumor proliferation and invasion in melanoma cells.In conclusion,the anti-CDK4 intrabody NLS-AK can effectively inhibit the growth and migration of melanoma cells in vitro and in vivo.This study provides an experimental basis for the treatment of melanomas with specific intrabody against CDK4,and also offers a new idea for the melanoma therapy targeting CDK4.