Elsevier

Biomedicine & Pharmacotherapy

Volume 96, December 2017, Pages 378-383
Biomedicine & Pharmacotherapy

Original article
Ginsenoside Rg3 attenuates cisplatin resistance in lung cancer by downregulating PD-L1 and resuming immune

https://doi.org/10.1016/j.biopha.2017.09.129Get rights and content

Abstract

Programmed death ligand 1 (PD-L1) as one the most important immune checkpoint was verified to involve in chemotherapy resistance in non-small cell lung cancer (NSCLC). Ginsenoside Rg3 is isolated from Chinese herb-Panax ginseng which is recognized to boost immune and has anti-cancer activity against a majority of carcinomas including NSCLC. In this study, we aim to identify whether Rg3 could attenuate the PD-L1 expression induced by resistance to cisplatin and draw out the underlying mechanisms of PD-L1 in this process. Human lung cancer cell lines A549 and A549/DDP (cisplatin-resistance) were used. Cell viability was detected by MTT assay, the PD-L1, Akt and NF-κB p65 protein expression were detected using Western blot analysis, the T cells cytotoxity to tumor cells was detected by crystal violet staining living residual tumor cells after coculture of tumor cells and T cells. The results showed that Rg3 could inhibit the growth and alleviate the resistant to cisplatin of A549/DDP cells. PD-L1 was overexpression in A549/DDP cells than A549 cells. Rg3 could decrease the PD-L1 expression induced by chemoresistance and resume the T cells cytotoxity to cancer cells. NF-κB p65 and Akt were involved in the PD-L1 overexpression and restrained by Rg3. Therefore, Rg3 could be regarded as a new agent targeting PD-L1 in chemotherapy refractory NSCLC.

Introduction

Lung cancer is the most common malignancy with high morbidity and mortality around china and worldwide, and non-small cell lung cancer (NSCLC) is the predominant subtype of lung cancer, accounts for nearly 85% of all cases [1], [2]. Combination chemotherapy based on platinum is still the first choice for more than half of patients with locally advanced or metastatic NSCLC, although target therapy for EGFR and ALK has made great achievements [3], [4]. Unfortunately, most patients will be progressed 4 to 6 months after first-line chemotherapy containing cisplatin (DDP) or carboplatin [5].

Programmed death ligand 1 (PD-L1), a major ligand for programmed death 1 (PD-1), is a well-known immune checkpoint in the B7 family that is involved in the modulation of immune evasion through the inhibition of T-cell function [6]. Several drugs targeting PD-L1 and PD1 have been approved in NSCLC as first-line [7], [8] and second-line treatment [9], [10], [11]. In addition to being a target for treatment, recent studies uncovered that PD-L1/PD1 was involved in chemotherapy resistance, and upregulation of PD-L1 after chemotherapy was recognized as a signal of poor prognosis in patients with NSCLC [12], [13], [14]. Clinical study results also confirmed that antibodies targeting PD-L1 and PD-1 could improve efficacy and survival for those patients progressed after first-line chemotherapy [9], [10], [11].

Ginsenoside Rg3 is a steroidal saponin isolated from Traditional Chinese medicine (TCM) named Panax Ginseng and has been approved for NSCLC by china food and drug administration (CFDA). A prospective randomized controlled study has showed that Rg3 could improve the efficacy of chemotherapy and delay the occurrence of drug resistance for treatment of advanced NSCLC [15]. Furthermore, a recent study also confirmed that ginseng-containing Chinese herbal formulation could reverse cisplatin resistance in lung cancer [16]. Moreover, Panax Ginseng is usually prescribed to enhance immunity guided by the theory of TCM, and it has been confirmed that Rg3 could also resume the ratio of CD4 and CD8T cells in NSCLC patients [17]. However, whether the role of Rg3 on improving the resistance to chemotherapy through inhibiting PD-L1 mediated immune escape in lung cancer are elusive.

In the present study, we want to identify whether Rg3 could decrease the PD-L1 expression induced by resistance to cisplatin and draw out the underlying mechanisms of PD-L1 in this process. To investigate the effect of Rg3 on the expression of PD-L1 and chemo-resistance in NSCLC, we used A549 cisplatin-resistance cells (A549/DDP) to analyze the mechanism in vitro. Finally, we found that PD-L1 was overexpression in A549/DDP cells compared with A549 cells, Rg3 could suppress the expression of PD-L1 in A549/DDP cells and resume the T cells function. Simultaneously, we revealed Akt and NF-κB were inhibited by Rg3 and involved in the regulation of PD-L1. Our study results affirmed that Ginsenoside Rg3 could serve as a novel agent targeting PD-L1 in treating chemotherapy refractory NSCLC.

Section snippets

Cells and reagents

A549 cells (human lung adenocarcinoma cells; Catalogue number: TCHu150) and A549/DDP cells (DDP-resistant cells; Catalogue number: JL10093) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and cultured in RPMI-1640 medium (Cyclone, GE Healthcare, UT, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone) and Penicillin/Streptomycin (1:100, SigmaeAldrich, St. Louis, MO, USA) in a humid atmosphere incubator with 5% CO2 at 37 °C. Ginsenoside 20(R)-Rg3

Rg3 inhibited A549/DDP and A549 cells viability

To evaluate the effect of Rg3 on cell viability in A549/DDP and A549 cells, different concentrations of Rg3 (5, 10, 20, 40, 80 and 160 μg/ml) were added to the cells for 12 h, 24 h and 48 h. The results showed that high concentration of Rg3 (80 and 160 μg/ml) significantly inhibited the viability of A549 and A549/DDP cells after 12 h, 24 h and 48 h (cells viability  50%), while low concentration of Rg3 (5, 10, 20 and 40 μg/ml) exerted poor function (cells viability >50%). The role of Rg3 on inhibiting

Discussion

The PD-1/PD-L1 axis plays an important role in the tumor microenvironment, protecting cancer cells from immune-mediated surveillance [18]. Several monoclonal antibodies targeting PD-1/PD-L1 axis have been approved in advanced NSCLC [8], [9], [19]. Moreover, a plenty of studies identified PD-L1 as a predictive and prognosis biomarker. Recently, it was confirmed that upregulation of PD-L1 was related to chemoresistance [12], [14], [20], [21]. Although many studies have focused on the relationship

Conclusions

In summary, our results provide new insights, suggesting that Rg3 may be a novel anti-PD-L1 agent to treat chemotherapy refractory NSCLC. In addition, the attenuation of PD-L1 expression is associated with downregulation of Akt and NF-κB signaling pathway.

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the article.

Acknowledgments

This work was financially supported by National Key Clinical Specialist Construction Programs of China (No: 2013-544) and CSCO-LiZhu Chinese Medicine foundation for cancer research, China (No: Y-L2012-004)

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    These authors contributed equally to this work and should be considered co-first authors.

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