| Background:As one of the most common malignancies, lung cancer ranks the first of morbidity and mortality among other diseases in China. Its main therapies include surgery, chemotherapy and radiotherapy, accompanied by endocrine or herb monomer treatment sometimes. Due to the severe complications and side effects of these treatments, the immune cell-oriented therapy is obtaining increasing attention in recent years.NK cells (Natural Killer cells) are characterized as CD3-CD56+lymphocytes in human body, accounting for approximately 10-15% of peripheral blood lymphocytes. Working as the bridge that connects the innate immunity and adaptive immune system, NK cells play a critical role in the control of infective diseases as well as malignancies. Clinical studies show that transplantation of human NK cells had a good graft-versus-tumor effect (GVT) upon patients, remarkably improving their life quality and survival rate while the graft-versus-host disease (GVHD) was rarely seen. By virtue of its advantages, relevant NK cell-based immunotherapy researches and clinical trials are in expansion worldwide. But, despite the promising effects upon malignancy treatment, its limited number in whole blood and low survival rate after transplantation have been restricting the broad clinical applications of NK cell-based immunotherapy. With continuous development of Chinese medicine and internationalization, many studies have been shown that herb medicine can directly inhibit the growth and proliferation of tumor cell, induced apoptosis of tumor cell, and influence the key proteins of the periodic signal pathway, meanwhile playing an important role in regulating in function of immune cells at same time. It could promise a more effective method against lung cancer than current treatments. And they are less harmful to normal tissues.Some researches reveal that a range of natural components in herbs can enhance the functions of immune cells in terms of directly inhibiting the growth and proliferation of tumor cells, inducing apoptosis and regulating the key proteins in the cell-cycle signaling pathways. With relative minor side effects and affordable price compared to conventional therapies, they have been regarding as potential drugs for malignancy treatment.Objectives:Based on the above-mentioned difficulties in clinical application of NK cells, our objectives were to 1) establish a better in vitro NK cell proliferation system through construction of a novel artificial antigen presentation cells (aAPC) by modifying the surface ligands of parental K562 antigen presenting cells; 2) find out an active component in herbs that can enhance the activity of NK cells so that facilitate its anticancer effects.Materials and Methods:1. In vitro study:First we made the CD137L/p SBSO sleeping beauty expression plasmid by PCR and established the mbIL-21-CD137L-K562 stably-expressed cell line by transfection of the construct in K562 cells; and then we tested the purity and expression level of surface receptors on NK cells. Next, we counted the cell number to calculate the NK cell proliferation rate and used Calcein AM release assay to analyze the cytotoxic activity of NK cells in vitro. Thereafter, we detected the STAT-1-6 phosphorylation sites by Western blot. To screen the effective herb monomers, we treated cells with various herb components and used flow cytometry to detect if there’s elevated expression level of NK cell activating signal-specific ligands. We also detected the influence of active Chinese herb medicine to NK cell activity by CCK8 assay. Meanwhile, we recorded the NK cell activity by both calcein release assay and killing experiments; using Elisa we detected the IFN-y expression level; by flow cytometry, we analyzed the expression level of specific receptors on the surface of CD 107a or NK cell.2. In vivo study:We established the A549 tumor-bearing mouse model by subcutaneous injection of A549 tumor cells. We measured the changes in tumor size after intravenous injection of NK cells and intraperitoneal injection of active herb components.Results:By flow cytometry, we explored m-IL-21 and CD137 to filter mbIL-21-CD137L-K562. We detected the receptor ligands of CD3/CD16/CD56 on the surface of NK cell co-incubated with PBMC and determined the establishment of amplification of NK cells in vitro based on artificial antigen-presenting cells. The amplification of NK cell has a high purity by mbIL-21-CD137L-K562. The expression of most NK cell-activating and inhibitory receptors was up-regulated. By cell counting, we calculate a higher efficiency of cell expansion of NK cells. We compared the cytotoxic activity of NK cells and the expansion of NK cell showed persistent in vitro by Calcein AM release assay. IL-21 activated but JSI-124 acting as the specific inhibitor inactivated STAT-3. STAT-3 inhibition impaired NK cell proliferation and cytotoxicity.In vitro study, using a variety of monomer on tumor cells, by flow cytometry, we explored the expression of MIC A//B、HLA-ABC and speculated that artemisinin would regulate the killing function of NK cells. It was proved that artemisinin can inhibit the proliferation and growth of tumor cell detected by MTT cell viability test, and promote the surveillance of NK cells by CCK test. Low concentrations of artemisinin improved the capability of NK cells to identify and kill the cells in lung cancer by calcein release experiments and direct killing assay. It also increased the production of granzyme and IFN-γ. Up-regulation of MICA/B expression of NKG2D, and down-regulation of the expression of HLA-ABC. NK cells combined with artemisinin can inhibit the neoplasm growth on A549 tumor-bearing mice.Conclusions:We found that 1) STAT-3 phosphorylation is required for proliferation and cytotoxicity of mbIL-21-CD137L-K562-based human NK cells and 2) artemisinin can strengthen the capability of NK cells to identify and kill the cells derived from lung cancer, such as A549/H1299/H1975, by regulating MICA/B, HLA-ABC expression levels on the surface of target cell and promoting granzyme and perforin-mediated release.3) Combined treatments of NK cell-based immunotherapy and traditional Chinese herb intervention do have effect in the treatment of malignancy. This research will provide new insights for the further study and development of anticancer drugs. |
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