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. 2015 Oct 28:17:12.
doi: 10.1186/s12575-015-0026-6. eCollection 2015.

A High-Throughput Assay for Screening of Natural Products that Enhanced Tumoricidal Activity of NK Cells

Chenyuan Gong #  1 , Zhongya Ni #  1 , Chao Yao  1 , Xiaowen Zhu  1 , Lulu Ni  1 , Lixin Wang  1 , Shiguo Zhu  1
Affiliations

A High-Throughput Assay for Screening of Natural Products that Enhanced Tumoricidal Activity of NK Cells

Chenyuan Gong et al. Biol Proced Online. .

Abstract

Background: Recently, immunotherapy has shown a lot of promise in cancer treatment and different immune cell types are involved in this endeavor. Among different immune cell populations, NK cells are also an important component in unleashing the therapeutic activity of immune cells. Therefore, in order to enhance the tumoricidal activity of NK cells, identification of new small-molecule natural products is important. Despite the availability of different screening methods for identification of natural products, a simple, economic and high-throughput method is lacking. Hence, in this study, we have developed a high-throughput assay for screening and indentifying natural products that can enhance NK cell-mediated killing of cancer cells.

Results: We expanded human NK cell population from human peripheral blood mononuclear cells (PBMCs) by culturing these PBMCs with membrane-bound IL-21 and CD137L engineered K562 cells. Next, expanded NK cells were co-cultured with non-small cell lung cancer (NSCLC) cells with or without natural products and after 24 h of co-culturing, harvested supernatants were analyzed for IFN-γ secretions by ELISA method. We screened 502 natural products and identified that 28 candidates has the potential to induce IFN-γ secretion by NK cells to varying degrees. Among the 28 natural product candidates, we further confirmed and analyzed the potential of one molecule, andrographolide. It actually increased IFN-γ secretion by NK cells and enhanced NK cell-mediated killing of NSCLC cells.

Conclusions: Our results demonstrated that this IFN-γ based high-throughput assay for screening of natural products for NK cell tumoricidal activity is a simple, economic and reliable method.

Keywords: Andrographolide; High-throughput screening; IFN-γ; NK cells; Natural products.

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Figures

Fig. 1
Fig. 1
Expansion of human NK cells. NK cells were expanded as described in Methods section, and were stained with CD56, CD16 and CD3 antibodies for flow cytometry analysis. The data set presented here is a representative of three independent experiments
Fig. 2
Fig. 2
The schematic presentation of IFN-γ based high-throughput assay for screening of natural products. To identify potent natural products, human NK cells were first expanded, and then co-incubated with A549 cells in 96-well plates with or without natural products. After 24 h of co-culturing, supernatants were harvested and IFN-γ levels were measured by ELISA assay
Fig. 3
Fig. 3
Optimization of NK and target (A549) cells culturing conditions. NK cells were co-cultured with A549 cells at different E:T ratios in 96-well plates for 24 h, and then IFN-γ secretion in supernatant was detected by ELISA assay. The data shown here is a representative of three independent experiments. ***, P<0.001
Fig. 4
Fig. 4
Screening of natural products. Expanded NK cells were mixed with A549 cells in 96-well plates and natural products were added to a final concentration of 10 μM. Panel a, represents percentage increase of IFN-γ after normalization to DMSO control. Panel b, represents IFN-γ percentage ratio after normalization to IL-2 treatment
Fig. 5
Fig. 5
Analysis of andrographolide effects on cell viability of NK and NSCLC cells. Expanded NK cells and NSCLC cells were treated with different concentrations of andrographolide (Andro) for 24, 48 and 72 h, respectively. Panel a, represents NK cell viability as determined by CCK8 assay. Panel b, represents NSCLC cell viability as assessed by MTT assay. Three independent experiments were performed. Data are expressed as means ± S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001
Fig. 6
Fig. 6
Andrographolide increased IFN-γ secretion by NK cells. Expanded NK cells and A549 cells were co-cultured with various concentrations (1 μM to 50 μM) of andrographolide. After 24 h, IFN-γ release in supernatant was measured by ELISA method
Fig. 7
Fig. 7
Andrographolide enhanced NK cell tumoricidal activity. NSCLC cells or NK cells were pretreated for 24 h with andrographolide, 5 and 10 μM concentrations, respectively. Later NK cells cytotoxicity was evaluated by calcein release assay with a 5:1 ratio of effectors to target cells. Panel a, A549 cells; Panel b, H1299 cells. The data shown here are representative of three independent experiments. Data are expressed as means ± S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001
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