Effect of κ-opioid receptor agonist on the growth of non-small cell lung cancer (NSCLC) cells

Background: It is becoming increasingly recognised that opioids are responsible for tumour growth. However, the effects of opioids on tumour growth have been controversial. Methods: The effects of κ-opioid receptor (KOR) agonist on the growth of non-small cell lung cancer (NSCLC) cells were assessed by a cell proliferation assay. Western blotting was performed to ascertain the mechanism by which treatment with KOR agonist suppresses tumour growth. Results: Addition of the selective KOR agonist U50,488H to gefitinib-sensitive (HCC827) and gefitinib-resistant (H1975) NSCLC cells produced a concentration-dependent decrease in their growth. These effects were abolished by co-treatment with the selective KOR antagonist nor-BNI. Furthermore, the growth-inhibitory effect of gefitinib in HCC827 cells was further enhanced by co-treatment with U50,488H. With regard to the inhibition of tumour growth, the addition of U50, 488H to H1975 cells produced a concentration-dependent decrease in phosphorylated-glycogen synthase kinase 3β (p-GSK3β). Conclusion: The present results showed that stimulation of KOR reduces the growth of gefitinib-resistant NSCLC cells through the activation of GSK3β.

Opioids are small endogenously produced peptide molecules that are widely known for their analgesic and psychoactive properties (Ciccone et al, 1980;Zubieta et al, 2001;Moles et al, 2004). It has been shown that opioids can promote the growth of tumour cells (Lazarczyk et al, 2010). On the other hand, it has been controversially reported that opioids induce the apoptosis of immunocytes, cancer cells and neuroblastoma cells (Boehncke et al, 2010). Thus, it is becoming increasingly recognised that opioids have a role in tumour growth (Saurer et al, 2008).
Three major types of opioid receptors, m, d and k, have been well characterised. k-Opioid receptors (KORs) are widely expressed throughout the central nervous system (Chavkin et al, 1982;Dhawan et al, 1996). It has been reported that KOR is also expressed in the human adenocarcinoma breast cancer cell line MCF7 and small cell lung carcinoma (Kallergi et al, 2003). Furthermore, KOR agonist has been shown to inhibit the growth of H157 cell, which is a non-small cell lung cancer (NSCLC) cell (Maneckjee and Minna, 1990). However, little is known about the mechanism that underlies the inhibitory effect of KOR stimulation on the growth of NSCLC cells.
Epidermal growth factor receptor (EGFR) is a major target of molecular anti-NSCLC therapy (Wakeling et al, 2002). Non-small cell lung cancer patients with L858R or exon 19 deletion mutations in EGFR show good responses to the tyrosine kinase inhibitor gefitinib. However, patients with wild-type EGFR and acquired mutation in EGFR T790M are eventually resistant to treatment with gefitinib. In this study, we examined whether the selective KOR agonist U50,488H could inhibit the growth of gefitinibsensitive and EGFR mutant (delE746-A750, L858R) NSCLC cells (HCC827) and gefitinib-resistant and EGFR mutant (T790M) NSCLC cells (H1975), and investigated the signalling mechanism of the KOR-mediated inhibitory effect on tumour cell growth.

Cell viability assay
Cell viability was determined by a cell proliferation assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, yellow tetrazole (MTT). A 20-ml of MTT solution (5 mg ml À1 ) was added to each well of the culture medium. After incubation for an additional 2 h, the medium was removed and 100 ml of DMSO was added to resolve the formazan crystals. Optical density was measured using a microplate reader with an absorption wavelength of 600 nm. In each experiment, three replicates were prepared for each sample. The proportion of living cells was determined based on the difference in absorbance between the samples and controls.

Immunohistochemistry
The procedure for immunohistochemistry is described in the Supplementary Methods.

RNA preparation and semiquantitative analysis by reverse transcription (RT)-PCR
The RNA preparation and RT-PCR method are described in the Supplementary Methods.
Effect of KOR agonist on the growth of the EGFR exon 19 mutant NSCLC cell line HCC827 Addition of the KOR agonist U50,488H to HCC827 cells for 2 days produced a concentration-dependent decrease in tumour cell growth (Figure 2A, Po0.001 vs non-treated group). This effect was abolished by co-treatment with the selective KOR antagonist nor-BNI ( Figure 2B, ***Po0.001 vs non-treated group, ### Po0.001 vs U50,488H-treated group). In contrast, treatment of NHLF cells with U50,488H did not affect their growth ( Figure 2C). In experiments that compared the inhibition of cell growth in cells treated with gefitinib and cells treated with a combination of gefitinib and U50,488H, the growth-inhibitory effects in HCC827 cells were further enhanced in a dose-dependent manner ( Figure 2D, Po0.001 vs gefitinibtreated cells).

Effect of KOR agonist on the levels of phosphorylated Akt, GSK3b and Stat3 in H1975 cells
There were no changes in the levels of either p-Akt or p-Stat3 in H1975 cells by treatment with U50,488H for 2 days (Figures 3C and E). However, the addition of U50,488H to H1975 cells produced a significant and concentrationdependent decrease in p-GSK3b ( Figure 3D, Po0.001 vs nontreated group). Furthermore, treatment with a specific GSK-3b inhibitor BIO produced a concentration-dependent and significant decrease in tumour cell growth ( Figure 3F, Po0.001 vs non-treated group).

DISCUSSION
In the present study, we investigated the role of KOR in NSCLC cells using gefitinib-sensitive HCC827 and gefitinib-resistant H1975 cells. We found that KORs were highly expressed in both cell lines. Under these conditions, addition of the selective KOR agonist U50,488H to either HCC827 or H1975 cells produced a concentration-dependent decrease in tumour cell growth. Although some of the doses of U50,488H were relatively high, these effects were abolished by co-treatment with the selective KOR antagonist nor-BNI. These results support the idea that U50,488H can pharmacologically act on KORs to decrease tumour growth. Additionally, the inhibition of tumour growth by gefitinib in HCC827 cells was further enhanced by co-treatment with U50,488H. These findings suggest that the stimulation of KOR may provide unique opportunities for the prevention and treatment of NSCLC. GSK3b is a multifunctional serine/threonine kinase that phosphorylates and thereby regulates the functions of many metabolic, signaling, and structural proteins and transcriptional factors (Grimes and Jope, 2001). EGF can inactivate GSK3b, leading to the degradation of c-Myc and b-catenin, which are overexpressed in tumour cells. Furthermore, the tumour suppressor p53 can be inactivated because of inactive GSK3b. It has been reported that the progressive inactivation of GSK3b, which is related to the increase in phosphorylation of GSK3b, is critical for the progression of lung cancer (Tian et al, 2006). In this study, treatment of H1975 cells with U50,488H produced a significant decrease in the phosphorylation of GSK3b. It has been recognised that activated protein kinase A (PKA) leads to phosphorylation of GSK3b (Fang et al, 2000), whereas activated JNK increases GSK3b activity (Hu et al, 2009). It should be noted that the stimulation of KOR suppresses cAMP production through Gi proteins, which leads to the inactivation of PKA (Tso and Wong, 2003). Furthermore, the stimulation of KOR invokes the JNK cascade (Kam et al, 2004). Although the exact mechanism of