Glycyrol Alone or in Combination with Gefitinib Is Effective against Gefitinib-Resistant HCC827GR Lung Cancer Cells

Abstract

Gefitinib has been clinically demonstrated to be effective in the first-line setting for patients with advanced EGFR-mutated non-small cell lung cancer (NSCLC). However, acquired therapeutic resistance to gefitinib almost unavoidably develops, posing a major hurdle for its clinical utilization. Our previous study showed that glycyrol (GC), a representative of coumarin compounds isolated from the medicinal plant licorice, was effective against A549 lung cancer cells in both cell culture and a murine xenograft model. In this follow-up study, we evaluated the effect of glycyrol against gefitinib-resistant NSCLC and its ability to overcome the resistance using gefitinib-resistant HCC827GR cells. Results showed that glycyrol was effective against HCC827GR cells in both in vitro and in vivo. Moreover, glycyrol was able to significantly increase the sensitivity of HCC827GR cells to gefitinib, mechanistically associated with inactivating MET, which is a known important contributor to the resistance of HCC827GR cells to gefitinib. The findings of the present study suggest that glycyrol holds potential to be developed as a novel agent against gefitinib-resistant NSCLC.

1. Introduction

Carcinogenesis is a multi-step and multi-mechanism process. Numerous molecules have been identified to play a critical role in cancer initiation, progression, and metastasis. These molecular findings provide a basis for developing molecular-targeted therapy. Indeed, many molecular targeted therapies approved by the Food and Drug Administration (FDA) have demonstrated a remarkable clinical efficacy for the treatment of multiple types of cancer. However, its efficacy has often been compromised by its drug resistance in tumors, posing a major hurdle for the application of this approach [1]. Gefitinib is a small molecule that directly targets EGFR tyrosine kinase and is recommended as a standard first-line therapy for advanced EGFR-mutated non—small cell lung cancer (NSCLC) [2]. However, acquired therapeutic resistance to this drug almost unavoidably develops, leading to disease progression [3]. Novel agents that are effective against gefitinib-resistant cancer, or can overcome the resistance, are urgently needed.

Licorice is a popular Chinese herbal medicine with multiple biological functions including anti-cancer activity [4]. Numerous ingredients have been identified from licorice, including coumarin compounds [5,6]. Glycyrol (GC) is a representative coumarin compound from licorice. Our previous study showed that glycyrol was highly effective against several human NSCLC cell lines in vitro, and significantly suppressed tumor growth in an A549 xenograft mouse model [7]. Mechanistically, we demonstrated that GC can strongly bind to the TOPK protein and inhibit its kinase activity. A recent study by Xiao et al. demonstrated that the COX2/MET/TOPK signaling axis contributed to gefitinib resistance [8], which led us to hypothesize that glycyrol might be able to overcome the resistance to restore the sensitivity of gefitinib-resistant cancer cells to gefitinib. This hypothesis was tested in the present study using gefitinib-resistant HCC827GR cells.

2. Materials and Methods

2.1. Chemicals and Reagents

GC (BBP03379) was purchased from BioBioPha (Kunming, Yunnan, China). Demethylsuberosin (De, HY-N2488) and Coumestrol (Coum, HY-N2335) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Gefitinib (SML1657) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for p-MET (Tyr1234/1235) (#3077, 1:1000 dilution), total MET (#8198, 1:1000 dilution), p-TOPK (Thr9) (#4941, 1:1000 dilution), and TOPK (#4942, 1:1000 dilution) were purchased from Cell Signaling Technology (Beverly, MA, USA). β-Actin (PM053, 1:1000 dilution) and the second-antibody specific for rabbits (#458, 1:5000 dilution) or mice (#330, 1:5000 dilution) were purchased from MBL International Corporation (Woburn, MA, USA). All antibodies were used following the instructions of the respective manufacturers.

2.2. Cell Culture and Treatments

HCC827 and gefitinib-resistant HCC827 (HCC827GR) cell lines were kindly provided by Dr. Feng Zhu (Huazhong University of Science and Technology, Wuhan, China) and cultured in RPMI-1640 medium (#10491, Solarbio Life Science, Beijing, China) with 10% fetal bovine serum (#10099141, FBS; Gibco, Grand Island, NY, USA) without antibiotics. The cells at 50–60% of confluence were exposed to glycyrol, gefitinib, and/or other agents for the time indicated.

2.3. Crystal Violet Staining

The cell viability was examined by crystal violet staining. After the treatments, the cells were fixed with a 1% glutaraldehyde solution for 15 min, followed by staining with a 0.02% crystal violet solution for 30 min. After washing with PBS, 70% ethanol was added to solubilize the dye for quantitative analysis. The absorbance at 570 nm with the reference filter 405 nm was measured by a microplate reader.

2.4. Apoptosis Evaluation

Cell apoptosis was measured by flow cytometry following Annexin V staining of externalized phosphatidylserine in apoptotic cells using Annexin V/FITC Staining Kit (#4700) from MBL International (Woburn, MA, USA).

2.5. Calculation of Combination Index

HCC827GR cells were treated with gefitinib alone at concentrations of 4, 6, 8, 10 μM, or in combination with glycyrol in a fixed ratio of 1:1 for 48 h. The changes of cell viability were evaluated by crystal violet staining, and the data generated were used to calculate the combination index (CI) using the following Chou–Talalay equation [9]:

$$\mathrm{CI}=\frac{\mathrm{CA},x}{\mathrm{IC}x,\mathrm{A}}+\frac{\mathrm{CB},x}{\mathrm{IC}x,\mathrm{B}}$$

where CA, x and CB, x are the concentrations of agent A and agent B used in combination to achieve x% combinatory effect; ICx, A and ICx, B are the concentrations for single agents to achieve the same effect. CI < 1 indicates synergism, CI = 1 indicates additive effect, and CI > 1 indicates antagonism.

2.6. Western Blotting

Cells were lysed with RIPA buffer containing protease inhibitors (#539131, Sigma-Aldrich, St. Louis, MO, USA). The concentration of protein was determined by the BCA Protein Quantitation Analysis Kit (PC0020, Solarbio Life Science, Beijing, China). The proteins were separated on SDS-polyacrylamide gel electrophoresis (PAGE) gel and subsequently transferred onto a nitrocellulose membrane (#66485, Millipore, Billerica, MA, USA). After incubating with a blocking buffer (5% non-fat milk in TBST) for 1 h, the membrane was probed with specific antibodies overnight at 4 °C, followed by the corresponding secondary antibody (1 h at room temperature). The protein bands were visualized by enhanced chemiluminescence and recorded on an X-ray film.

2.7. Animals and Treatments

Male NOD-Prkdcscid Il2rgnull (NPSG) mice (6 weeks old) were purchased from Weishanglituo Company (Beijing, China). Animal Care and procedures were approved by the Institutional Animal Care and Use Committee, China Agricultural University. HCC827GR cells (5 × 10⁶) were mixed with Matrigel (50%) and injected subcutaneously into the right flank of each mouse. Tumors were measured with a caliper and tumor volumes were calculated using the following formula: length × width × width × 0.5. When the average tumor volumes reached about 100 mm³, vehicle control (l5% Tween-80) and glycyrol (20 mg/kg) was administered by intraperitoneal injection every day for 3 weeks.

2.8. Statistical Analysis

All data are presented as mean ± SD of at least three independent experiments or samples. Significant differences were determined by one-way ANOVA, followed by Tukey’s post hoc test using SPSS19.0. Graphs were drawn using GraphPad Prism (version 6.0).

3. Results

3.1. Exposure to Glycyrol Leads to Reduced Viability of HCC827 GR Cells In Vitro

To validate the resistant feature of HCC827GR cells to gefitinib, we compared the sensitivity of HCC827GR and its parental HCC827 cells to gefitinib. HCC827GR and HCC827 cells were exposed to various concentrations of gefitinib (0, 0.005, 0.01, 0.1, 1, and 10 µM) for 72 h, and cell viability was measured by crystal violet staining. As shown in Figure 1, exposure to gefitinib led to a concentration-dependent reduction of viability of these two cell lines. These cell viability data were used to calculate IC₅₀ and results showed that the IC₅₀ of HCC827GR was 5.825 µM, whereas the IC₅₀ of HCC827 cells was 0.0043 µM, indicating HCC827GR cells are highly resistant to gefitinib in comparison with HCC827 cells. We also analyzed the phosphorylation level of MET of these two cell lines, and results showed that phospho-MET and total MET in HCC827GR cells are significantly higher than that in HCC827 cells (Figure 1B,C), which is well consistent with the differential sensitivity of these two cell lines.

We next used the HCC827GR cell line as a model to determine whether glycyrol is effective against gefitinib resistant lung cancer. HCC827GR cells were treated with glycyrol at concentrations of 0, 2.5, 5, and 7.5 µM for 24 and 48 h, the changes of cell viability were assessed by crystal violet staining. As shown in Figure 1D,E, treatment with glycyrol caused a concentration-dependent and time-dependent reduction of cell viability with the lowest effective concentration at 2.5 µM (IC₅₀ is 6.65 μM). We further analyzed the influence of glycyrol on MET activation, and results showed that exposure to glycyrol resulted in a concentration-dependent inhibition of MET phosphorylation (Figure 1F,G). In addition, a decreased total MET expression by glycyrol was observed at a high concentration (5 μM). The suppression of MET activation is well consistent with its inhibitory effect on cell viability.

3.2. Glycyrol Inhibits Tumor Growth in HCC827GR Xenograft Model

Having found the inhibitory effect of glycyrol on HCC827GR cell viability and MET activation, we then questioned whether these suppressive effects could be achieved in vivo. Subcutaneous inoculation of HCC827GR cells into the right flank of male NOD-Prkdcscid Il2rgnull (NPSG) mice established the HCC827GR xenograft model. As shown in Figure 2A–C, treatment with glycyrol at a dose of 20 mg/kg significantly suppressed HCC827GR-mediated tumor growth and reduced the final tumor weight without affecting the bodyweight. In line with the in vitro finding, a significantly reduced MET phosphorylation was observed in glycyrol-treated HCC827GR tumor samples (Figure 2D,E).

3.3. Glycyrol Sensitizes HCC827GR Cells to Gefitinib

The above data demonstrated that glycyrol is able to inhibit MET activation both in vitro and in vivo. Given the critical role of elevated MET activation in the acquired resistance of cancer cells to gefitinib [8], we hypothesized that glycyrol might potentiate gefitinib-resistant HCC827GR cells to gefitinib. HCC827GR cells were exposed to gefitinib alone or in combination with glycyrol for 48 h, and the cytotoxicity was assessed by Annexin V/PI staining. As shown in Figure 3A, the cell death induction by the combination of gefitinib and glycyrol was significantly stronger than by each agent alone. To critically determine the nature (additive or synergistic) of the enhanced cytotoxic effect by the combination, we calculated the combination index using the Chou–Talalay method [9]. The cells were treated with each agent alone and in combination in a fixed ratio of 1:1 for 48 h, and the changes of cell viability were measured by crystal violet staining. As shown in Figure 3B, gefitinib or glycyrol treatment alone induced a concentration-dependent reduction of cell viability, whereas the combinations led to strongly further enhanced inhibitory effects. These data were then used to calculate the combination index using the median-effect equation of Chou–Talalay. As shown in Figure 3C, the values of the combination index were much less than 1 at all combinations tested, supporting a strongly synergistic effect induced by the combination of gefitinib and glycyrol.

3.4. Both the Benzofuranyl Group at c-3/4 and the Isopentenyl Group at c-6 Are Indispensable for Glycyrol to Exert the Sensitization Effect

To decipher the structure-activity relationship of glycyrol in terms of its sensitization effect, we assessed the combination effect of gefitinib and two analogues of glycyrol, demethylsuberosin (De) and coumestrol (Coum). The chemical structures of glycyrol, demethylsuberosin and coumestrol are shown in Figure 4A. These three compounds belong to the coumarin family, which is structurally constructed by a benzene ring fused to α-pyrone ring. In comparison with glycyrol, the benzofuranyl group at c-3/4 is absent in demethylsuberosin, while the isopentenyl group at c-6 is absent in coumestrol. HCC827GR cells were treated with gefitinib alone or in combination with demethylsuberosin or coumestrol at the same concentrations used with glycyrol for 48 h, and the changes of cell viability are shown in Figure 4B,C. The synergistically inhibitory effect of the combination of gefitinib and glycyrol was not observed by combining gefitinib with either demethylsuberosin or coumestrol. These results were consistent with the inability of these two analogues to inactivate MET (Figure 4D,E). The data indicate that the benzofuranyl group at c-3/4 and the isopentenyl group at c-6 are essential structures for the sensitization effect of glycyrol against HCC827GR cells.

4. Discussion

Drug resistance is a major factor affecting the clinical efficacy of gefitinib. A MET-based mechanism is suggested to be an important contributor to the cancer cells refractory to gefitinib [10,11]. HCC827, a human non—small-cell lung cancer (NSCLC) cell line harboring EGFR activating mutations, is initially sensitive to gefitinib. Long-term stepwise treatment with gefitinib leads to the cells’ resistance to gefitinib (HCC827GR). In the present study, we evaluated inhibitory effects of glycyrol on HCC827GR cells using both in vitro and in vivo models, and a possibility of glycyrol as a sensitizer to improve the efficacy of gefitinib against its resistant cancer. Our data demonstrated that glycyrol was able to inhibit the growth of HCC827GR cells in cell culture systems and exerted a promising in vivo efficacy in the HCC827GR xenograft mouse model. Moreover, a dramatically increased sensitivity of HCC827GR cells to gefitinib was induced in response to glycyrol. The findings of the present study suggest that glycyrol holds promising potential to be developed as a novel agent to fight against gefitinib-resistant cancer.

Proposed mechanisms that contributed to the resistance development of cancer cells to gefitinib mainly include mutation of EGFR-T790M, amplification of MET gene and its protein hyperactivation, amplification of ERBB2, transformation to small-cell lung cancer, and MET-mediated TOPK activation [8,12,13,14,15]. Our previous study showed that glycyrol was able to directly bind to and inactivate TOPK [7], which is supposed to contribute to the sensitization effect of glycyrol on HCC827GR cells to gefitinib. In the present study, we addressed the role of MET inactivation in a glycyrol-mediated sensitization effect. Western blotting analysis of phospho-MET demonstrated that MET is highly activated in HCC827GR cells, which is well in agreement with its gefitinib resistant feature, supporting a possible contribution of MET activation to HCC827GR cell resistance to gefitinib. Targeting MET is therefore a reasonable approach to fight against the gefitinib-resistant cancer cells. Treatment with glycyrol effectively suppressed MET activation in cultured HCC827GR cells, which provided a mechanistic explanation for the sensitization effect of glycyrol on HCC827GR cells to gefitinib. Accordingly, the two analogues of glycyrol that lacked the ability to inactivate MET failed to potentiate HCC827GR cells to gefitinib. Regarding the structure basis underlying the differentially suppressive effect on MET activation between glycyrol and its two analogues, our results revealed that both the benzofuranyl group at c-3/4 and the isopentenyl group at c-6 are necessary for glycyrol to exert an inhibitory effect on MET activation. These findings provided useful information for improving the anticancer activity of glycyrol through structure modification.

5. Conclusions

Glycyrol alone is effective against gefitinib resistant HCC827GR cells both in vitro and in vivo. Furthermore, glycyrol greatly sensitized HCC827GR cells to gefitinib. Mechanistically, glycyrol is able to inactivate oncogenic kinase MET and TOPK. The findings of the present study suggest that glycyrol as a novel MET inhibitory agent is worthwhile for further investigation.