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Abstract

Introduction: G-protein coupled receptor 30 (GPR30) is associated with cell metastasis and drug resistance in many different cancer cells. The present study aimed to reveal the sensitivity of GPR30 to gefitinib in non-small cell lung cancer (NSCLC) cells.

Methods: Cell viability and proliferation were detected using cell counting kit 8 and 5-ethynyl-2'-deoxyuridine assays, respectively. Western blotting and quantitative real-time reverse transcription PCR were used to detect GPR30 or epithelial-mesenchyme transition (EMT)-related mRNA and protein expression.

Results: The results showed that GPR30 expression is associated with gefitinib sensitivity. G15, as a GPR30 antagonist, reduced GPR30 expression. We chose the maximum concentration of G15 with minimal cytotoxicity to detect cell viability after combined treatment with gefitinib in NSCLC cells, which indicated that G15 could increase sensitivity to gefitinib. However, the effect of G15 on gefitinib sensitivity disappeared after treatment with a small interfering RNA targeting GPR30. Further research showed that G15 or GPR30 siRNA treatment could upregulate E-cadherin and downregulate vimentin levels.

Conclusion: Taken together, these data suggested that G15 could enhance NSCLC sensitivity to gefitinib by inhibition of GPR30 and EMT.

Keywords

GPR30 gefitinib NSCLC EMT

Introduction

Non-small cell lung cancer (NSCLC) is a malignant tumor that mainly occurs in the bronchial mucosa and bronchial glands, which has become one of the main cancers worldwide.¹ Although treatment measures such as drug therapy, radiotherapy, and surgery continue to develop and progress, patients with advanced NSCLC still have poor prognosis. Gefitinib (an epidermal growth factor (EGFR) inhibitor) has been used as the front-line treatment for NSCLC.² Gefitinib could also significantly extend the median survival of patients with advanced lung cancer with drug-sensitive EGFR mutations. However, gefitinib treatment will inevitably result in the development of acquired drug resistance, leading to treatment failure.

Recently, G-protein coupled receptor 30 (GPR30) was identified as a neotype estrogen receptor, which mediates rapid and non-genomic estrogen effects, including transactivation of EGFR.³ GPR30 was proven to be highly expressed in breast cancer, ovarian cancer, cervical cancer, endometrial, NSCLC, and gastric cancer.^4–9 Its activation could promote a number of different cancers.^9–11 This receptor is also related to cell metastasis and drug resistance.^9,12 G15, as a high affinity GPR30 antagonist, could inhibit GPR30 expression.¹³ Recent studies demonstrated that inhibition of GPR30 by G15 or a GPR30 small interfering RNA (siRNA) could increase the sensitivity of gastric cancer cells and breast cancer to different drugs (cisplatin or doxorubicin).^9,14 However, the effect of GPR30 on the sensitivity of NSCLC cells to drugs has not been investigated. Therefore, the main aim of this study was to determine the involvement of GPR30 in NSCLC cell gefitinib resistance and to further explore the mechanism of GPR30 as a therapeutic target to improve the efficacy of NSCLC chemotherapy.

Materials and methods

Cell culture

Three NSCLC cell lines (HCC827 (Serial number: SCSP-538), NCI-H358 (Serial number: SCSP-583), and NCI-H1299 (Serial number: SCSP-589)) were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China) and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY, USA) containing with 10% fetal bovine serum (FBS) in humidified air containing 5% CO₂ at 37°C.

Cell viability and proliferation analysis

Cell viability was tested using cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assays. Firstly, 3 × 10³ NSCLC cells/well were seeded into 96-well plates and incubated for 3–5 h until they were close to the culture flask.^15,16 The cells were treated with drugs or transfected as follows: gefitinib (0, 5, 10, 20, or 40 μM) and G15 (10 μM) for 48 h or transfected with negative control (NC) siRNA or the GPR30 siRNA for 48 h. Next, the CCK-8 reagent was added at 10 μL/well, and culture was continued for 3 h at 37°C, after which the absorbance at X was measured. To detect cell proliferation, the NSCLC cells were treated with G15, alone or combined with gefitinib, for 48 h. 5-ethynyl-2'-deoxyuridine (EdU) staining was then carried out using a Click-iT EdU Imaging Kit (Invitrogen, Carlsbad, CA, USA). EdU (reagent A) was added to the cell culture medium and incubated for 2 h. Then, the cells were fixed using 4% paraformaldehyde at room temperature. Later, 100 μL/well of 1×Hoechst 33-342 (DAPI, 4′,6-diamidino-2-phenylindole) solution was added to the cells in a 96-well plate and kept in the dark for 30 min at room temperature. After two washes with phosphate-buffered saline (PBS), the cells were observed under a fluorescence microscope (AF488 (EDU): blue light channel excited, showing green fluorescence. Hoechst 33342 (DAPI): Ultraviolet channel excitation, showing blue fluorescence).

Cell transfection

The GPR30 siRNA (100 nM) and NC siRNA (100 nM) were designed by GenePharma (Shanghai, China).¹⁷ NSCLC cells were transfected with the siRNAs using Lipofectamine 2000 transfection reagent (Invitrogen) in a medium without serum. At 6 h after transfection, the cells were changed to a medium containing 10% FBS and incubated at 37°C for 24 h. All treatments were initiated at 24 h after transfection.

GPR30 siRNA

Forward primer 5’-GCUGUACAUUGAGCAGAAA-3’

Reverse primer 5’-CGACAUGUAACUCGUCUUU-3’;

NC siRNA:

Forward primer 5’-UUCUCCGAACGUGUCACGUTT-3’

Reverse primer 5’-ACGUGACACGUUCGGAGAATT-3’.

Quantitative real-time reverse transcription PCR (qRT-PCR)

Total RNA was extracted from cells using the Trizol reagent (Invitrogen) and reverse transcribed using SuperScript™ II reverse transcriptase (Invitrogen). The resultant cDNA was then subjected to the quantitative real-time PCR (qPCR) step of the qRT-PCR protocol using SYBR@ Premix Ex Taq™ (Takara, Dalian, China). The thermocycling conditions were as follows: 95°C for 30 s, followed by denaturation at 95°C (5 s) annealing at 60°C (30 s) for 40 cycles. ACTB mRNA (encoding β-actin) was amplified as a reference control. The results were analyzed using the 2^−ΔΔCq method.¹⁸ The relative expression of target mRNA was standardized to that of ACTB. The primers were as follows:

GPR30:

Forward primer 5’-GGGTGCCAGGACAATGAAATACTC--3’

Reverse primer 5’-ATCCGCACATGACAGGTTTATTGA--3’

ACTB:

Forward primer 5’-TGGCACCCAGCACAATGAA-3’

Reverse primer 5’-CTAAGTCATAGTCCGCCTAGAAGCA-3’

Western blotting

The NSCLC cells were lysed using lysate buffer (CST, Danvers, MA, USA) in an ice bath, and the protein concentration was determined using a bicinchoninic acid protein assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The protein extracts were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred onto polyvinylidene fluoride (PVDF) (Millipore, Billerica, MA, USA) membranes. The membranes were blocked for 1 h in blocking solution (5% nonfat dry milk (cat. no. 6342932; BD Biosciences, San Jose, CA, USA) and then incubated in Tris-buffered saline-Tween-20 buffer (TBST) for 2 h at 37°C. The remaining liquid was absorbed with filter paper, and then the membrane was placed protein side down in the primary antibody solution overnight at 4°C. The primary antibodies comprised anti-GPR30 (ab260033), anti-E-cadherin (ab40772), and anti-vimentin (ab137321) (all Abcam, Cambridge, MA, USA; 1:1000 dilution). The membranes were then decolorized and washed with TBST, before being incubated with the secondary antibody (ab205718) (Abcam; 2:1000 dilution) at 37°C for 2 h. After washing with TBST twice, the membranes were subjected to chemiluminescent detection of the immunoreactive protein bands (ECL; Bioworld Technology, Inc., Bloomington, MN, USA). The density of the protein bands was measured using Image Lab 5.0 (Bio-Rad Laboratories, Hercules, CA, USA).

Immunohistochemical staining

The NSCLC tumors and corresponding peritumoral tissue were collected from the Affiliated Lihuili Hospital, Ningbo University. Simply, paraffin sections were cut into 4 μm thick sections, dissolved in xylene, and denatured in alcohol. Then, the sections were incubated with anti-GPR30 antibodies at 4°C overnight and then with a horseradish peroxidase (HRP)-linked secondary antibody for 2 h. A 3, 3ʹ-diamino biphenyl substrate kit was used to visualize the bound antibodies.

Statistical analysis

The main data of this article are presented as means ± standard deviation (SD) and were analyzed using GraphPad Prism 8 (GraphPad Inc., La Jolla, CA, USA) using Student’s t -test and one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. The significance of the data was defined by p < .05. All experiments were repeated three times.

Results

GPR30 expression was associated with gefitinib sensitivity in NSCLC cells

To examine the connection between GPR30 expression and gefitinib sensitivity in NSCLS cells, we first performed CCK-8 assays to examine cell viability under different concentrations of gefitinib in NSCLC cells, and observed that according to the IC50 values, HCC827 cells were the most sensitive to gefitinib (Figure 1(a) and (b)). Next, we detected GPR30 expression in NSCLC cells using qRT-PCR and western blotting, which showed that the extent of GPR30 expression in the three NSCLC cells was as follows: NCI-H1299 > NCI-H358 > HCC827 (Figure 2(c) and (d)). Furthermore, immunohistochemistry showed that the expression of GPR30 was upregulated in NSCLC tumors comparted with that in corresponding peritumoral tissues (Supplementary Figure S2(a)). These data indicated that the cells with lowest expression GPR30 were the most sensitive to gefitinib.

Figure 1.

GPR30 expression was associated with gefitinib sensitivity in NSCLC cells. (a) CCK-8 assay to determine cell viability after treatment with different concentrations of gefitinib in NSCLC cells. (b) The IC50 values are indicated in a histogram. (c),(d) Confirmation of GPR30 expression using qRT-PCR and western blotting analysis. *p < .05 vs. the results in NCI-H1299 cells.

Figure 2.

The effect of G15 on NSCLC cell viability (a–c). The effect of different concentrations G15 on NSCLC cells using CCK-8 assays. *p < .05, **p < .01 vs. the 0 μM G15 group. (d–f) Western blotting determination of the level of GPR30 after treatment with or without G15 in NSCLC cells. **p < .01, ***p < .001 vs. the Control group.

The effect of G15 on NSCLC cell viability

To verify the effect of different levels of GPR30 on NSCLC cells, we chose a series of concentrations of the GPR30 inhibitor G15 (0, 2.5, 5, 10, 20, and 40 μM) for incubation with NSCLC cells. We found that 0–10 μM G15 showed little cytotoxicity in NSCLC cells; however, 20 and 40 μM G15 could reduce NSCLC cell viability (Figure 2(a)–(c)). Therefore, we chose the maximum concentration of G15 (10 μM) with little cytotoxic effect on NSCLC cells for further research. Furthermore, we examined the expression of GPR30 by western blotting following treatment with or without G15, which showed that G15 could reduce GPR30 levels in NSCLC cells (Figure 2(d)–(f)).

G15 combined with gefitinib could enhance the sensitivity of NSCLC cells to gefitinib

To reveal the synergistic effect of gefitinib combined with G15, CCK-8 assays were used to analyze cell viability following treatment with gefitinib or gefitinib combined with G15 in NSCLC cells. The results indicated that G15 plus gefitinib significantly enhanced the cell's sensitivity to gefitinib compared with gefitinib alone (NCI-H1299 CI = 0.7915, NCI-H358 CI = 0.875 HCC827 CI = 1.015) (Figure 3(a)). Similarly, EdU analysis also confirmed that G15 combined with gefitinib could reduce cell proliferation in comparison with gefitinib alone (Figure 3(b) and (c)). We also determined the expression of E-cadherin and vimentin after treatment with G15 and gefitinib, showing that compared with gefitinib group, G15 combined with gefitinib could up-regulate E-cadherin and down-regulate vimentin by Western blot and immunofluorescence analysis (Figure 3(d), Supplementary Figure S1(a)).

Figure 3.

G15 combined with gefitinib could enhance the sensitivity of NSCLC cells to gefitinib. (a) Cell viability determined in the gefitinib treatment group or the gefitinib plus G15 treatment group in NSCLC cells. (b), (c) EdU was used to examine cell proliferation between NSCLC cells treated with gefitinib and those treated with gefitinib plus G15 (magnification, ×200). *p < .05, **p < .01 vs. the gefitinib treatment group.

G15 could increase the sensitivity of NSCLC cells to gefitinib via inhibition of GPR30 and EMT

To determine the mechanisms by which G15 increased the gefitinib sensitivity of NSCLC cells, we transfected the GPR30 siRNA into NSCLC cells and examined cell viability after knockdown, with or without G15 treatment, which showed that interfering with GPR30 expression enhanced the gefitinib sensitivity of NSCLC cells (Figure 4(a)). Furthermore, we also found that the G15-mediated effect of increasing sensitivity to gefitinib disappeared following transfection with GPR30 siRNA in NSCLC cells (Figure 4(c)), suggesting that G15 affected gefitinib sensitivity by regulating GPR30. The comparison of the IC50 values in the control and GPR30 siRNA, or GRP30siRNA and G15 + GPR30 siRNA groups is presented as a bar chart in Figure 4(b) and (d). The interference effect on GPR30 was determined using qRT-PCR and western blotting (Figure 4(e), Supplementary Figure S1(a)). To verify whether G15 plays a role in sensitivity to gefitinib by regulating EMT, we transfected GPR30 siRNA into NSCLC cells and treated NSCLC cells with G15 treatment alone, which showed that treatment with either G15 alone or GPR30 siRNA alone resulted in E-cadherin upregulation and vimentin downregulation in NSCLC cells by Western blot and immunofluorescence analysis (Figure 4(f)–(g), Supplementary Figure S1(a)). The above data suggested that G15 plays an important effect in the sensitivity of NSCLC cells to gefitinib by downregulating of GPR30 and inhibiting of EMT.

Figure 4.

G15 could increase the sensitivity of NSCLC cells to gefitinib via inhibition of GPR30 and EMT (a), (c) CCK-8 assays of cell viability between the NC siRNA group and the GPR30 siRNA group or the GPR30 siRNA and G15 combined with GPR30 siRNA group following treatment with gefitinib in NSCLC cells. (b),(d) The IC50 values in corresponding groups are presented in the form of a bar chart. **p < .01 vs. the NC siRNA group. (e) Western blotting determination of the interference efficiency of the GPR30 siRNA in terms of the GPR30 protein level. *p < .05, **p < .01 vs. NC. (f)-(g) Western blotting determination of the levels of related proteins following treatment with the GPR30 siRNA, with or without with G15 in NSCLC cells. *p < .05, **p < .01 vs. the gefitinib group.

Discussion

The mortality rate of NSCLC is rising globally.¹⁹ Despite major advances in diagnosis and treatment, the 5-year survival rate for patients with lung cancer, including patients with advanced lung cancer, is still less than 15%. Gefitinib, a selective EGFR tyrosine kinase inhibitor, is a good choice to treat patients with locally advanced NSCLC who have previously received chemotherapy.²⁰ Although it could prevent angiogenesis and the continuous growth and metastasis of tumor cells to other sites, and can increase the number of apoptotic cancer cells, there will still be varying degrees of drug resistance. Thus, we need to explore the mechanism of gefitinib resistance in NSCLC to improve treatments for patients.

GPR30, as a seven transmembrane receptor, mediates non-genomic estrogen signals, including EGFR, subsequent activating mitogen activated protein kinase (MAPK) signaling pathways, and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), which are associated with cell migration, invasion, differentiation cycle progression, apoptosis, and proliferation.^21,22 The present study revealed that the expression of GPR30 was lowest in HCC827 cells and highest in NCI-H1299 cells. Interesting, the HCC827 cells had the highest sensitivity to gefitinib, while the NCI-H1299 had the lowest sensitivity to gefitinib, which revealed that the level of GPR30 was closely related to gefitinib sensitivity. Consistently, a previous study reported that NCI-H1299 cells had the highest expression of GPR30 and the lowest drug sensitivity.²³ Furthermore, immunohistochemistry for GPR30 in NSCLC tumor and corresponding peritumoral tissue supported the above data (Supplementary Figure 2(a)). G15, as a GPR30 antagonist, induces cell apoptosis and regulates drug sensitivity in several cancers.^9,13,14,24 G15 exerted its cytocidal action in vitro by co-treatment with tamoxifen and improved tamoxifen-resistant xenograft response to endocrine treatment in vivo²⁵ The inhibitory effect of G15 on proliferation could induce cell cycle arrest and apoptosis in the G2/M phase, resulting in oral cancer cell autophagy.¹³ Furthermore, our study revealed that G15 combined with gefitinib could enhance the gefitinib sensitivity of NSCLC cells. Moreover, we used HCC827GR cells to determine cell viability after treatment with Gefitinib, Gefitinib plus G15, or GPR30 siRNA, which showed that GPR30 siRNA treatment also enhances gefitinib sensitivity in HCC827GR cells (Supplementary Figure S1(c)). When we interfered with GPR30 expression in NSCLC cells, the sensitivity to gefitinib induced by G15 disappeared, suggesting that G15 could enhance sensitivity to gefitinib by inhibiting GPR30 expression in NSCLC cells. Furthermore, we also combined another chemotherapeutic drug, such as cisplatin, with G15, which also enhanced cisplatin sensitivity in NCI-H1299 and HCC827 cells (Supplementary Figure S2(b)). It has been reported that gefitinib is an EGFR tyrosine kinase inhibitor²⁶ that effectively inhibits EGFR phosphorylation and GPR30 signaling, further decreasing EGFR activation.²⁴ Consequently, we used western blotting to analyze the changes in EGFR activation after treatment with G15 or gefitinib in NSCLC. As shown in Supplementary Figure S2(c) and (d), the levels of phosphorylated (p)-EGFR (activated) were downregulated under G15 or gefitinib treatment.

EMT is a morphogenetic process, accompanied by upregulation of mesenchymal markers vimentin and downregulation of the epithelial marker E-cadherin.^27,28 EMT is closely related to chemotherapy resistance. It has been reported that GPR30 could affect the sensitivity of various tumor cells to drugs via regulation of EMT.^9,14,29 G15 could reverse doxorubicin-induced EMT and cisplatin-induced EMT in breast cancer cells with epithelial features and gastric cancer cells, respectively.^9,14 We hypothesized that G15 might regulate EMT to influence gefitinib resistance. To verify the molecular mechanism by which G15 regulates drug resistance, we treated cells with GPR30 siRNA or G15 combined with gefitinib and determined E-cadherin and vimentin levels in NSCLC cells. Compared with those in the gefitinib group, interference with GPR30 or treatment with G15 upregulated E-cadherin and downregulated vimentin levels following treatment with gefitinib. To further reveal the effect of GPR30 on the sensitivity of NSCLC cells to gefitinib, we co-transfected a Twist 1 expression plasmid and the GPR30 siRNA into NSCLC cells, and observed the changes in gefitinib sensitivity. Overexpression of Twist1, which promotes EMT, increased gefitinib resistance in NSCLC cells, whereas the GPR30 siRNA combined with Twist1 expression increased the sensitivity of the cells to gefitinib, while enhancing E-cadherin levels and reducing vimentin levels (Supplementary Figure S3(a)–(b)). These data indicate that GPR30 could enhance the sensitivity of NSCLC cells to gefitinib by regulating EMT.

The limitations of this study were as follows: (1) The signaling pathway by which GPR30 regulates EMT and whether it is regulated by transcription factors was not assessed; (2) sensitivity to gefitinib in vivo was not investigated, which should be studied in the further animal model-based research.

Conclusion

In conclusion, the above data verified that G15 could enhance the sensitivity of NSCLC cells to gefitinib via inhibition of GPR30 and prevention of EMT. This indicated that GPR30 plays a vital role in drug resistance and might be an effective target for better treatment of patients with NSCLC.

Supplemental Material

Supplemental Material - Inhibition of GPR30 sensitized gefitinib to NSCLC cells via regulation of epithelial-mesenchymal transition

Supplemental Material for Inhibition of GPR30 sensitized gefitinib to NSCLC cells via regulation of epithelial-mesenchymal transition by Hongyan Jiang, Xiaomin Yang, Jiang Ning, Shufen Zhang, Ying Cai, Liang Wang, Jinsong Yang, Guodong Xu, Wei Chen, Jianfei Wang in International Journal of Immunopathology and Pharmacology

Supplemental Material

Supplemental Material - Inhibition of GPR30 sensitized gefitinib to NSCLC cells via regulation of epithelial-mesenchymal transition

Supplemental Material for Inhibition of GPR30 sensitized gefitinib to NSCLC cells via regulation of epithelial-mesenchymal transition by Hongyan Jiang, Xiaomin Yang, Jiang Ning, Shufen Zhang, Ying Cai, Liang Wang, Jinsong Yang, Guodong Xu, Wei Chen, and Jianfei Wang in International Journal of Immunopathology and Pharmacology

Supplemental Material

Supplemental Material - Inhibition of GPR30 sensitized gefitinib to NSCLC cells via regulation of epithelial-mesenchymal transition

Supplemental Material for Inhibition of GPR30 sensitized gefitinib to NSCLC cells via regulation of epithelial-mesenchymal transition by Hongyan Jiang, Xiaomin Yang, Jiang Ning, Shufen Zhang, Ying Cai, Liang Wang, Jinsong Yang, Guodong Xu, Wei Chen, and Jianfei Wang in International Journal of Immunopathology and Pharmacology

Footnotes

Author’s note

This manuscript has not been published or submitted anywhere else.

Authors’ contributions

WC and JW designed the study and collected data. HJ, XY, SZ, and JY performed the experiments. YC and LW analyzed the data. JW wrote the manuscript. WC and JW reviewed and edited the manuscript. GX provided the pathological specimen. All authors read and approved the final manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was supported by the Zhejiang Province Traditional Medical Science Fund Project of China [grant number 2020ZB036]; the Zhejiang Province Medical and Health Science and Technology Platform Project of China [grant number 2019RC128]; and the Natural Science Foundation of Zhejiang Province [grant number LSY19H160003].

Ethical statement

ORCID iD

Wei Chen

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.³⁰

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

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