Trans‐3,5,4´‐trimethoxystilbene reduced gefitinib resistance in NSCLCs via suppressing MAPK/Akt/Bcl‐2 pathway by upregulation of miR‐345 and miR‐498

Abstract Despite initial dramatic efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR‐TKIs) in EGFR‐mutant lung cancer patients, subsequent emergence of acquired resistance is almost inevitable. Resveratrol and its derivatives have been found to exert some effects on EGFR‐TKI resistance in non‐small cell lung cancer (NSCLC), but the underlying mechanisms remain unclear. We screened several NSCLC cell lines with gefitinib resistance by MTT assay and analysed the miR‐345/miR‐498 expression levels. NSCLC cells were pre‐treated with a resveratrol derivative, trans‐3,5,4‐trimethoxystilbene (TMS) and subsequently challenged with gefitinib treatment. The changes in apoptosis and miR‐345/miR‐498 expression were analysed by flow cytometry and q‐PCR respectively. The functions of miR‐345/miR‐498 were verified by CCK‐8 assay, cell cycle analysis, dual‐luciferase reporter gene assay and immunoblotting analysis. Our results showed that the expression of miR‐345 and miR‐498 significantly decreased in gefitinib resistant NSCLC cells. TMS pre‐treatment significantly upregulated the expression of miR‐345 and miR‐498 increasing the sensitivity of NSCLC cells to gefitinib and inducing apoptosis. MiR‐345 and miR‐498 were verified to inhibit proliferation by cell cycle arrest and regulate the MAPK/c‐Fos and AKT/Bcl‐2 signalling pathways by directly targeting MAPK1 and PIK3R1 respectively. The combination of TMS and gefitinib promoted apoptosis also by miR‐345 and miR‐498 targeting the MAPK/c‐Fos and AKT/Bcl‐2 signalling pathways. Our study demonstrated that TMS reduced gefitinib resistance in NSCLCs via suppression of the MAPK/Akt/Bcl‐2 pathway by upregulation of miR‐345/498. These findings would lay the theoretical basis for the future study of TMS for the treatment of EGFR‐TKI resistance in NSCLCs.

study of targeting the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has become a hot spot in NSCLC treatment. [2][3][4][5] That is, by competitively binding extracellular ligand binding sites with ATP or other substrates, blocking EGFR tyrosine autophosphorylation and tyrosine kinase activation, leading to EGFR activation inhibition and downstream signal transduction disorder, ultimately inhibiting cell cycle progression, accelerating apoptosis, disrupting angiogenesis, invasion and metastasis. 2,3 Gefitinib is a commonly used EGFR-TKI drug for NSCLC treatment. EGFR-TKIs can prolong patients' progression free survival and overall survival in NSCLC clinical treatment, especially in relatively sensitive patients, both in one-line and multi-line treatment and the side effects are mild. 4 However, there is no significant effect of EGFR-TKs on some patients, EGFR mutations for example; or patients initially sensitive to EGFR-TKIs with good efficacy for some time (about 1-1.5 years), but subsequently prone to develop disease progression and EGFR-TKI resistance, the so-called secondary resistance. 5 Trans-3,5,4′-trimethoxystilbene (TMS), a polyphenolic compound known by the chemical name of Stilbene, is a synthetic analogues of resveratrol, a constituent of red wine, vegetables and Chinese medicines, Polygonum cuspidatum for example. 6 TMS has been reported to possess more potent anticancer and antiangiogenic activities than resveratrol. [7][8][9][10] Resveratrol and its derivatives have been found to exert some effects on NSCLC, 11 especially on EGFR-TKI resistance, but the underlying mechanisms remain unclear.
MiRNAs are a group of non-coding small RNAs about [22][23][24][25][26] nucleotides, involved in the regulation of gene expression at the posttranscriptional level. In recent years, miRNAs have become the focus of oncology research. Although only about 1% of human genes, miR-NAs regulate about 30% of the human-encoded protein genes involved in the occurrence and development of many tumours, including lung cancer. 12,13 Recent research found that miRNAs involved in a variety of tumour drug resistance, especially in NSCLC, can affect the chemosensitivity of gefitinib and other drugs involved in EGFR-TKIs resistance. 14,15 MiR-345 and miR-498 were found to be downregulated in NSCLC patients and cell lines and closely associated with the tumour progression and poor prognosis, 16,17 but there were few reports about the molecular regulation mechanism of miR-345 and miR-498 in NSCLC, especially in the EGFR-TKI resistance.
In this study, we have identified a remarkable sensitization to gefitinib and the anticancer effects of TMS by miR-345/miR-498 and their downstream targeted signalling pathways in NSCLC providing a better understanding of the biological activities and functions of TMS. Our findings provide new evidence for TMS as an effective complementary medicine for combination treatment with EGFR-TKI in NSCLC.
The gefitinib-acquired resistant cell subline PC-9/GR was established by chronic exposure of PC-9 cells to medium with increasing concentrations of gefitinib as described previously. 18 To confirm the best fit for TMS (Sigma) treatment, a certain concentration range (0, 0.5, 5, 50, 500 μmol/L) was administered for 24, 48 or 72 hours.
After treatment with TMS and/or gefitinib, cells were collected for analysis.

| Flow cytometric analysis
The apoptosis analysis was performed with a fluorescein isothio-

| Subcutaneous xenograft construction
The research protocol was approved and conducted in accordance with the institutional ethical guidelines from the Institutional Animal Care and Use Committee of Xiangya Hospital affiliated to Central South University.
Four-to six-week-old BALB/c athymic mice were injected subcutaneously into the right flank with 10 7 H1299 cells. When palpable tumours (approximately 75 mm 3 in diameter) developed, the mice were randomly divided into seven groups: (1) Control, left untreated; (2) Gefitinib, 25 mg/kg daily orally by gavage; (3)  were given every 4 days and for four times in total. Each treatment group consisted of at least five mice. Tumour volume was calculated using the formula π/6 × larger diameter × (smaller diameter) 2 as described previously. 19 About a month later after administration, all the mice were killed and the tumour samples were collected for further analysis and detection.

| Statistical analysis
Data are indicated as mean ± SD for at least three different determinations. Statistical analysis was performed with the SPSS software (Chicago, IL, USA). Differences between variants were analysed by the Student's t test or one-way ANOVA. Date were regarded as statistically significant when P value was at least less than 0.05.   All data was illustrated as mean ± SD and the asterisks show significant difference as *P < 0.05, **P < 0.01, ***P < 0.001 compared with the miR-NC group or between the two groups shown by a horizontal line ( Figure 3A) and PC-9/GR and cell growth curve and cell cycle were analysed by MTT assay and flow cytometry. Results displayed that miR-345 and miR-498 overexpression inhibited H1299 and PC-9/GR cell proliferation ( Figure 3B) and induced cell cycle arrestin G1/G0 phage ( Figure 3C) compared with blank control and miR-NC groups.
In order to further confirm whether it was the upregulation of miR-345 and miR-498 by TMS to promote cell apoptosis, miRNA inhibitors were used to antagonize miR-345/miR-498 and flow cytometry analysis was performed. As shown in Figure 3D, inhibition of miR-345 or miR-498 weakened the effect of TMS and restored cellular resistance to gefitinib by decreasing apoptosis.  Recent studies have indicated that, the PC-9/GR cell subline achieves acquired resistance (AR) through the development of EMT. So, the expression of EMT markers after TMS treatment was analysed. Data indicated that TMS treatment significantly reduced EMT by upregulating E-cadherin and downregulating vimentin and N-cadherin in PC-9/GR cells, but these effects were reversed by the overexpression of MAPK1 or PIK3R1 suggesting that the gefitinib-AR of PC-9/ GR cells was reduced. However, these effects were relatively less dramatic in H1299 cells because of the weaker EMT in H1299 cells compared to PC-9/GR cells ( Figure 5F). All these results suggested that TMS overcame gefitinib resistance in H1299 and PC-9/GR cells by inhibiting the MAPK/c-Fos and Akt/Bcl-2 signalling pathways.

|
3.6 | TMS enhanced gefitinib chemosensitivity on tumour growth inhibition by suppressing the MAPK/ Akt/Bcl-2 pathway through miR-345 and miR-498 can suppress proliferation, invasion and apoptosis by regulating NF-κB and activator protein-1 activities. 18,19 One of the resveratrol derivatives TMS was found to be more potent than resveratrol as an anticancer agent. 9,11 In this study, we evaluated the anticancer effects in vitro and in vivo. To uncover the underlying molecular mechanisms of TMS, we also studied the cell cycle and gene expres- (F) Immunoblotting analysis of EMT marker expression in H1299 and PC-9/GR cells described in E. All data are illustrated as mean ± SD and the asterisks show significant difference as *P < 0.05, **P < 0.01, ***P < 0.001 compared with the Control/GFP group ERK signalling pathways downstream of EGFR in highly sensitive NSCLC cell lines. 25,27,28 However, in resistant cell lines, EGFR inhibition by EGFR-TKIs could not lead to apoptosis of cancer cells. 25,29,30 Therefore, inhibition of PI3K/Akt and MAPK signalling pathways through other ways may be an effective strategy for improving EGFR-TKI resistance. In our study, we have verified that MAPK1 The PC-9/GR cell is a gefitinib-acquired resistant cell subline of PC-9 cells, which has an EGFR-activating mutation, which is a 15bp deletion in the EGFR exon 19 and is initially sensitive to gefitinib. 18 The gefitinib resistance mechanism is very complex, may relate to secondary mutation of EGFR, c-Met amplification, compensatory signal establishment and the change in regulatory factors expression and the tumour microenvironment transformation.
Cheng et al detected that there was a T790M mutation which may be the cause of gefitinib-AR for PC-9/GR cells, although it occurs less frequently. 18 Recent studies have also indicated that there was not a T790M mutation in the PC-9/GR cell subline; however, it achieved AR through development of EMT. The expression of Ecadherin decreased but vimentin and N-cadherin increased in PC-9/ GR cells compared to the PC-9 cells indicating that PC-9/GR cells acquired the EMT phenotype. 31 As we know, MAPK and PI3K signalling pathways are involved in inducing tumour EMT and play roles in drug resistance. 32,33 In our study, we have verified that TMS increased the sensitivity to gefitinib by reduced EMT by upregulating E-cadherin and downregulating vimentin and N-cadherin in PC-9/GR cells after suppressing MAPK and PI3K/AKT signalling pathways by upregulating miR-345/miR-498. But these effects were reversed by the overexpression of MAPK1 or PIK3R1, further suggesting that the gefitinib-AR of PC-9/GR cells was reduced by TMS. However, these effects were relatively less dramatic in H1299 cells because of the weaker EMT in H1299 cells, which were primarily resistant to gefitinib caused by TP53 deficiency and NRAS mutation. 34 In conclusion, we have demonstrated that the resveratrol derivative TMS could reduce gefitinib resistance in NSCLC cells and xenograft tumour tissues by blocking the PI3K/Akt and MAPK pathways by upregulating miR-345 and 498. Our data would lay the theoretical basis for the future study of TMS for the treatment of EGFR-TKI resistance in NSCLCs.

CONFLI CT OF INTEREST
All authors declare no conflicts of interest.