Licorice Extracts Inhibits growth of Non-Small Cell Lung Cancer by Down-Regulating CDK4-Cyclin D1 Complex and Increasing CD8+ T Cell Infiltration


 Background: Targeting tumor microenvironment (TME) may provide therapeutic activity and selectivity in treating cancers. Therefore, an improved understanding of the mechanism by which drug targeting TME would enable more informed and effective treatment measures. Glycyrrhiza uralensis Fisch (GUF, licorice), a widely used herb medicine, which has shown promising immunomodulatory activity and anti-tumor activity. However, the molecular mechanism of this biological activity has not been fully elaborated.Methods: Here, potential active compounds and specific targets of licorice that trigger the antitumor immunity were predicted with a systems pharmacology strategy. Flow cytometry technique was used to detect cell cycle profile and CD8+ T cell infiltration of licorice treatment. And anti-tumor activity of licorice was evaluated in C57BL/6 mice.Results: We reported the G0/G1 growth phase cycle arrest of tumor cells induced by licorice that is related with the down-regulation of CDK4-Cyclin D1 complex, which subsequently led to increased protein abundance of PD-L1. Further, in vivo studies demonstrated that mitigation the outgrowth of NSCLC tumor induced by licorice was reliant on increased antigen presentation and improved CD8+ T cell infiltration.Conclusions: Briefly, our findings improved understanding of the anti-tumor effects of licorice with the systems pharmacology strategy, thereby promoting the development of natural products in the prevention or treatment of cancers.Trial registration: Not applicable


Introduction
Lung cancer is the most prevalent diagnosed cancer worldwide and a major contributor of cancer mortality. And non-small cell lung cancer (NSCLC) accounts for approximately 85% of the diagnosed lung cancers [1][2][3]. In the recent years, immunotherapy targeting T cells has increasingly shown its potentiality in treatment of a wide variety of solid tumors, such as non-small cell lung cancer (NSCLC) [4][5][6]. Although encouraging, it is the fact that still only a small number of patients obtain long-term bene t, which is likely correlated with the complex network of the tumor microenvironment (TME) [7]. The tumor microenvironment (TME), a complex physical and biochemical system, playing a pivotal role in tumor initiation, progression, metastasis, and drug resistance [8]. It contains cells of the immune system, tumor cells, tumor vasculature and extracellular matrices (ECM) [9]. Among them, tumor cells could express inhibitory ligands that suppress the T-cell activity to evade immune destruction. Immune cells could produce some cytokines, growth factors, enzymes, and angiogenic mediators to promote growth of tumor [10]. And ECM consists of biological barriers around the tumor tissue to hamper lymphocyte penetration. Therefore, better understanding of the interactions in the TME that would increase the ratio of patients bene ting from these therapies are essential.
Traditional herb medicines and herbal derived components are playing increasingly critical roles in prevention and treatment of cancers [11,12]. Compared with conventional chemotherapy, they are of low toxicity and pleiotropic actions, targeting the complex network of TME by modulating multiple cellsignaling pathways involved in immune. Thereby, natural products could be a great repository for development of novel therapeutic approaches in cancer treatment. As a well-known herbal medicine used worldwide for centuries, to date, several reports have been published complicating the immunomodulatory activity of licorice on multiple cancers, including colon cancer, breast cancer, acute myeloid leukemia, gastric cancer, melanoma, and prostate cancer [13][14][15][16]. However, the molecular underpinnings of this biological activity that licorice exert its immunomodulatory potential have not been fully elaborated.
To address this question, we used a systems pharmacology strategy to elaborate that how licorice exert anti-tumor effects by regulating multiple immune-related signaling pathways and targets, in uencing cell cycle progression, and mitigate the growth of NSCLC cancer. First, by screening the poly-pharmacology molecules of licorice, predicting the targets of active compounds, constructing the networks, and linking the targets to the immune phenotype in lung cancer patients, we observed that active ingredients of licorice targeted a great variety of tumor-related signaling pathways, including cell cycle, in ammation, and migration. Then, we used in vitro and in vivo experiments to reveal anti-tumor effects of licorice. On the one hand, we found that licorice down-regulates CDK4-Cyclin D1 complex, resulting in G0/G1 phase arrest and increased PD-L1 levels in lung cancer cells. On the other hand, we also found that licorice increased antigen presentation and in ltration of CD8 + T cell, signi cantly decreased tumor volume of mouse models of NSCLC in vivo. Taken together, our studies indicate that systems pharmacology strategy greatly uncovered the action mechanism of poly-pharmacology molecules of licorice, contributing the use of natural products for further anti-cancer drug development.

Results
1, Systems pharmacology uncovers that licorice targets cell cycle progression and immune process As a comprehensive system, the systems pharmacology approach was used to investigate the complex molecular mechanisms of licorice as a treatment for NSCLC in this study (as shown in Fig. 1).
Altogether, 89 ingredients were identi ed in licorice with the searching literatures and using Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), and a total of 23 active ingredients (shown in Table 1) were screened out by in silico ADME (absorption, distribution, metabolism, and excretion) system, with the criteria of oral bioavailability (OB) ≥ 50% and drug-likeness (DL) ≥ 0.40. Then, predicted by the weighted ensemble similarity method (WES) [17] and systematic drug targeting tool (SysDT)[18], we found that these 23 ingredients in licorice were investigated interacted with 109 targets (shown in Table 2 and table S1). And we constructed the compound-target (C-T) network graph to greatly illustrate the relationships between compounds and targets. In terms of the targets interacted with licorice, we observed that most of which were related to cell cycle, immune, in ammation, cancer and neoplasm metastasis with higher scores. Speci cally, including CDK2, ESR1, PPARG, ESRRA, PRKACA, CXCL8, PLAA, RXRB, MAPK14 and so on (shown in Fig. 2a).
To detect the potential role of these targets, we performed Gene Ontology (GO) biological processes enrichment analysis, and found that most of biological processes were involved in immune progress. Including "regulation of myeloid cell differentiation", "neutrophil mediated immunity", and "regulation of cytokine production involved in in ammatory response" (shown in Fig. 2b and 2c). Then, to further understand the relationship between licorice and diseases, using the Database for Annotation Visualization, and Integrated Discovery (DAVID), we performed the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. And the results showed that the most targets of licorice mainly enriched in signaling pathways related to the cancer process. Including "non-small cell lung cancer", "small cell lung cancer" "pathways in cancer", "prostate cancer", "T cell receptor signaling pathway" and so on (Fig. 2d).
Therefore, the systems pharmacology analysis uncovers that licorice mainly targets cell cycle and immune progress to exert its anti-cancer effect, and paves the way for in-depth understanding of the multi-target molecular mechanism of licorice treating for NSCLC. Table 1. Chemical information and pharmacokinetics parameters of 23 active compounds of licorice. To further study the anti-cancer effect of licorice on NSCLC, we rst tested the effects of licorice on the growth of tumor cells. According to the CCK8 assay results shown in Fig. 3a, we could recognize that licorice induced a concentration-dependent inhibition of H1975 cell proliferation. Treating licorice two days with concentrations of 3200, 5600 and 7200µg/mL, we found that compared with DMSO treatment, the H1975 cell growth decreased by 25, 48 and 87%, respectively. Moreover, the IC50 value on it were 5400µg/mL.
Next, given the analysis of systems pharmacology for licorice, and a number of studies have shown that the negative effects of licorice or its relatives on cell cycle progression [15,16,19,20], we reasoned that licorice might in uenced cell cycle to exert the anti-tumor effect on NSCLC to some extent. To test the hypothesis, we treated H1975 cells with different concentrations of licorice followed by ow cytometry analysis of cell cycle pro le. Strikingly, H1975 cells subjected to licorice led to a signi cant increase in the number of cells arrested at G0/G1 growth phase, in a dose-dependent manner, compared with vehicle control containing media (shown in Fig. 3b and Fig. 3c). At the same time, the number of cells at both S growth phase and G2/M growth phase slightly decreased (Fig. 3c). This nding consistent with previous study that licorice induced G1 cell cycle arrest in MCF-7 human breast cancer cells [16].
It has been known that cyclin-dependent kinase (CDK)/cyclin complexes, such as CDK2/Cyclin E, CDK4, CDK6/CyclinD1, and P21 play crucial roles in cell cycle progression [21]. Therefore, to elucidate the underlying molecular mechanism with which licorice induced cell cycle arrest at G0/G1 growth phase, immunoblot analysis were performed to evaluate cell cycle-related protein abundance in vitro experiment. Notably, we found that the levels of CDK4, cyclin D1 were reduced, and the effect was concentration dependent, while the expression of CyclinB1 and CyclinA2 was relatively maintained at the level of the control group following licorice treatment ( Fig. 3d and Fig. 3e). Interestingly, the expression of p21, a CDK inhibitor, was slightly decreased in response to licorice exposure vs control group (shown in Fig. 2d).
In addition, previous work uncovered that cyclin D1 degradation occurs mainly at the G1/S phase boundary [21,22]. Collectively, these results indicated that licorice is likely to induce tumor cells arrested at G0/G1 growth phase by down-regulating CDK4-Cyclin D1 complex.

3, Licorice positively regulates PD-L1 protein abundance
It has been shown that PD-L1 expression can be modulated at both transcriptional and post-translational levels, however, it is not yet clear whether PD-L1 expression is regulated under physiological conditions for example during cell cycle progression [23][24][25][26]. In this setting, to further understand the connection between PD-L1 and cell cycle, we used cell synchronization by nocodazole arrest and immunoblot analysis to explore variation of PD-L1 during cell cycle. As shown in Fig. 4a and Fig. 4b, we found that PD-L1 protein expression increased in M/early G1 phases, followed by a great decrease in late G1/S phases.
As our results showed that licorice down-regulated Cyclin D1-CDK4 expression to arrest cell cycle progression, we probed whether licorice participated in variation of PD-L1. To do this, we treated H1975 cells with different concentration of licorice, followed by immunoblotting analysis. Strikingly, licorice administration results in a signi cant increase in the expression of PD-L1 protein ( Fig. 4c and Fig. 4d), in a dose-dependent manner. Furthermore, recent nding had shown that Cyclin D-CDK4 kinase destabilized PD-L1, inhibition of CDK4/6 in vivo increased PD-L1 protein levels [27]. Together, these ndings indicating that increased levels of PD-L1 expression by licorice correlated with down-regulation of Cyclin D1-CDK4 expression.

Licorice induce tumor regression by affecting Cyclin D1-CDK4-PD-L1 axis
Based on previous studies that various natural compounds in licorice possess effective antitumor activity [14,16,28,29], we wanted to know whether licorice can function in vivo to suppress tumor progression for NSCLC. To do so, we utilized C57/BL6 female mice bearing LLC tumor to assess the antitumor impact of licorice. And size-matched tumor-bearing mice (TBMs) were divided into 4 groups randomly and received the administrations (as depicted in Fig. 5a).
By day 20 of treatment, as expected, all control mice encountered humane endpoints. Then mouse from each group were killed and dissected tumor, mouse serum was taken out and stored for subsequent experiment.
It is critical to note that licorice treatment result in a 64.9% tumor volume regression, and we found that there was slightly inhibitory effect on tumor volume of mice treated with anti-PD-L1 antibody alone vs control mice. Interestingly, we also observed a 54.7% tumor volume reduction in licorice + anti-PD-L1 mice compared with control mice over time. (Fig. 5a).
In keeping with our nding of tumor volume, treatment of licorice led to a signi cant induction of tumor weight, this also occurred in licorice + anti-PD-L1 group compared with untreated group. However, slight reduction of tumor weight was observed in anti-PD-L1 alone group (Fig. 5b).
Having pinpointed the critical role for licorice in affecting Cyclin D1-CDK4 expression in vitro, we next examined whether licorice had similar in uence in vivo. Therefore, we assayed cell cycle-related protein for tumor tissue using the immunoblot method. Consistent with earlier observations in vitro (Fig. 3d), licorice treatment markedly reduced the abundance of CDK4 Cyclin D1, and importantly led to a dramatic PD-L1 accumulation compared with control group (Fig. 5c and 5d).
Therefore, these results coherently indicated that licorice might mainly function through down-regulating CDK4-Cyclin D1 to stabilize PD-L1 and subsequently suppress tumor progression.

5, Licorice increased antigen presentation and in ltration of CD8 + T cell
Furthermore, the results of systems pharmacology analysis indicated that kinds of compounds of licorice correlated with CD8 + T-cell (Fig. 6a, gure S2a, gure S2b), then intratumoral CD8 + T-cell in ltration in tumor tissue lysates were measured by ow cytometry analysis. Importantly, CD8 + T cell in ltration of licorice-treated mice we detected increased by 6% of that in untreated mice (Fig. 6c, Fig. 6d and gure S2c). In further support of a physiological role for licorice in promoting CD8 + T cell in ltration, we used the mice serum to perform ELISA-based assays and found a remarkable increase of IFN-γin licorice-treated mice (Fig. 6e). These results were in line with a previous study that CDK4/6 inhibitors induce breast cancer cell cytostasis and enhance their capacity to present antigen and stimulate cytotoxic T cells [30].
Next, to gain insights into the physiological role of licorice in modulating tumor regression at gene level, RT-qPCR analysis was performed. Speci cally, we sought to determine relative mRNA levels of antigen presentation genes by RT-qPCR analysis, and observed that transporter-MHC interactions (Tap-bp) had at least a 15x fold increase in licorice-treated tumor tissues compared to control tumor samples, and peptide transporters (Tap1 and Tap2) were also markedly up-regulated in licorice-treated tumors, although directing peptide cleavage (Erap1) hardly change to some extent. (Fig. 6b).
Altogether, these studies indicated that licorice increased expression of antigen presentation genes and promoted CD8 + T cell in ltration for tumor tissue.

Discussion
Natural products were shown broadly to interfere growth signals by multi-speci c actions [31], which may open an opportunity to treat NSCLC effectively. In a panel of human cancers, licorice has been uncovered to provide growth-limiting activities [16,28,32]. Although changes in the cell-cycle have been noted under licorice treatment settings [19,20], dissecting mechanism of the biological activity of licorice remains a challenge. Here, the critical ndings of our study, summarized in Fig. 5c and Fig. 2d, include the discovery that licorice limits lung cancer growth mainly related with down-regulating CDK4-Cyclin D1 complex and enhancing intra-tumoral CD8 + T cell in ltration. Our detailed investigation shows that licorice induce G1 cell-cycle arrest in lung cancer cells by inhibiting CDK4-Cyclin D1 complex, which in turn increase PD-L1 levels and antigen presentation and results in intra-tumoral CD8 + T cell in ltration. These ndings convincingly argue for a potential treatment option of licorice in the prevention and treatment of NSCLC.
Beginning with systems pharmacology analysis, ow cytometry analysis of cell cycle pro le and immunoblotting, we observed that licorice treatment led to G1 cell-cycle arrest and inhibit the expression of CDK4-Cyclin D1 complex in H1975 cells. This biological activity was further validated in licorice-treated tumor. It is well known that CDK4-Cyclin D1 complex were required for progression of cells cycle through the G0/G1 phase [33][34][35], which would suggest that G1 cell-cycle arrest is largely associated with decreased levels of CDK4-Cyclin D1 after licorice treatment. One of the strongest links between CDK4-Cyclin D1 complex down-regulation and tumor regression has come from inhibitor studies. As a kind of CDK4/6 inhibitors, abemaciclib caused regression of bulky tumors in mouse models of mammary carcinoma [30]. Furthermore, many human cancers harbor genomic or transcriptional aberrations that could activate CDK4/6[36-38]. Therefore, our nding that licorice inhibit the expression of CDK4-Cyclin D1 complex would be critically important for prevention and treatment of cancers.
Moreover, Cyclin D/CDK4 was found negatively regulates PD-L1 protein stability in several tumor cell lines [27,39]. And previous studies revealed that response to PD-1/PD-L1 blockade might correlate with PD-L1 expression levels in tumor cells [40][41][42]. Notably, we discovered that licorice treatment induced increased expression of PD-L1 levels both in vitro and in vivo. These studies, together with our ndings, shed light on a viable option for the management of NSCLC, with or without other treatments in conjunction, to enhance the e ciency of cancer immunotherapies.
The functional impairment of T cell-mediated immunity in the TME is a de ning feature sharing by many cancers, and CD8 + T cells became the central focus of new cancer therapeutics [43,44]. Due to the data that shows licorice increased expression of antigen presentation genes and promoted CD8 + T cell in ltration for tumor tissue, we reasoned that CD8 + T cell in ltration contribute to growth inhibition for tumor.
In summary, this study evidenced that licorice induced G0/G1 phase cell cycle arrest by down-regulating CDK4-Cyclin D1 complex on tumor cells, in addition, licorice increased expression of antigen presentation genes and in ltration of CD8 + T cell in tumor microenvironment. Therefore, this study illuminated a novel mechanism of anti-tumor effect of licorice in NSCLC treatment, and provide functional evidence for development of natural products in anti-tumor immunity.

Methods
Pharmacokinetic evaluation the ingredients of licorice were identi ed based on searching literatures and using Traditional Chinese Medicine Systems Pharmacology Database (TCMSP, http://tcmspw.com/) [45] and active ingredients (shown in Table 1) were further screened out by in silico ADME system, with the criteria of oral bioavailability (OB) ≥ 50% and drug-likeness (DL) ≥ 0.40.

Target shing and validation
We identi ed direct targets of licorice on the basis of a WES method and a SysDT tool, then obtained targets were uploaded to Uniprot (http://www.uniprot.org)[46] to normalize their name and organisms.
And the targets of Homo sapiens were chosen for further investigation. We used Cytoscape 3.7.0 software to construct and analyze compound-target network.
GO enrichment analysis and KEGG analysis for targets.
(GO) enrichment analysis and KEGG analysis was performed through mapping targets to DAVID (http://david.abcc.ncifcrf.gov) for classi cation. We chose the terms with P value less than 0.05.
Cell proliferation assay   Figure 1 Work ow of systems pharmacology analysis to undercover mechanism of licorice.

Figure 2
Systems pharmacology analysis of targets of licorice a Construction of compound-target network, the triangle represents compounds, the octagon represents targets, the edge represents connection between compounds and targets. b GO enrichment analysis of potential targets of licorice, the y-axis represents the enriched GO term, and the GeneRatio represents the number of targets located in this GO/the total number of targets located in the GO. c GO term associated with immune process were shown, and the size of the circle represents the count. d KEGG analysis of targets of licorice, the color represents the enrichment signi cance, the y-axis represents pathway, and the GeneRatio represents the number of targets located in this KEGG pathway/the total number of targets located in the KEGG pathways. . c The protein expression in H1975 cells pretreated with 400 600 800 µg/ml GUF or vehicle control, was measured by immunoblots, versus GAPDH as a loading control d Relative protein abundance of PD-L1 of (c). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) Figure 5 Licorice inhibit the growth of tumor volume depending on the Cyclin D1-CDK4-PD-L1 axis a C57 BL/6 mice were injected with 5*105 LLC cells. Beginning 24 hours later, 200 mg/kg GUF or vehicle or anti-PD-L1 (n = 5 per group) were administered once daily. The tumor growth curve is shown, with tumor sizes presented as mean ± SEM. *P < 0.05. b Primary tumor mass of mice is shown, presented as mean ± SEM. c Protein expression in tumors from GUF group and control group was measured by immunoblots, versus β-Actin as a loading control. d Relative protein abundance of PD-L1, CDK4, Cyclin D1, Cyclin B1 of (c), (*p < 0.05, **p < 0.01, ***p < 0.001.)

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Extendeddata.docx