Int J Clin Exp Pathol 2014;7(9):5888-5894 www.ijcep.com /ISSN:1936-2625/IJCEP0000767

Original Article Grandinin down-regulates phosphorylation of epidermal growth factor receptor Zhuling Qu, Aiqin Song, Wei Feng, Ruyang Teng, Jie Gao, Xuanlong Yi The Affiliated Hospital of Qingdao University, Qingdao 266021, Shandong Province, China Received May 11, 2014; Accepted June 27, 2014; Epub August 15, 2014; Published September 1, 2014 Abstract: Aims: Grandinin (C46H34O30) is a compound found in Melaleuca quinquenervia leaves and in oaks. This study is to determine effects of grandinin on malignant lung cells and the related molecular mechanisms. Methods: Malignant cells were treated with grandinin with various concentrations. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide (MTT) assays and apoptosis assays were performed to determine effects of grandinin on cell viability and apoptosis. Western blotting and real time-PCR were used to determine if grandinin affects levels of phosphorylated EGFR (p-EGFR) and phosphorylated AKT (p-AKT), as well as their mRNA transcript levels. Results: It was found that grandinin treatments reduce viability of malignant lung cells and induces apoptosis. When treated with grandinin (16 µM), the apoptosis of the three lung cancer cell lines MS-1, A549, and LK-2 were increased by 8-9 folds, in comparison with the cells treated with DMSO only (the control condition). Furthermore, grandinin treatments lead to down-regulation of levels of p-EGFR and p-AKT in three malignant lung cell lines. However, grandinin does not affect mRNA levels of EGFR and AKT. Conclusions: These experimental results indicated grandinin significantly reduce malignant cell viability and effectively induces apoptosis of malignant lung cells by mediating phosphorylation down-regulation of cellular signaling proteins EGFR and AKT. It is suggested that grandinin treatments might be an effective therapeutic strategy of lung malignancies upon further studies in the future. Keywords: Grandinin, phosphorylation, epidermal growth factor receptor, down-regulates phosphorylation

Introduction Epidermal growth factor receptor (EGFR) is an important cell surface receptor, which is activated by binding of its specific ligand such as epidermal growth factor and transforming growth factor [1, 2]. Upon binding of the ligands, EGFR elicits a transition from an inactive monomeric form to its active homodimer form [2]. Dimerization of EGFR triggers its intrinsic intracellular protein-tyrosine kinase activity, resulting in autophosphorylation of several tyrosine residues in the C-terminal domain of EGFR [2]. This autophosphorylation elicits activation of downstream signaling pathways, such as the AKT-mTOR pathway [3-5]. These pathways are involved in multiple cell processes including cell proliferation, apoptosis, cell migration, and so on [3, 4]. Inhibition of EGFR activity is an important chemotherapy of various tumors. The effective chemotherapy for lung cancers includes gefi-

tinib that has significant anti-tumor activities [6-8]. Gefitinib function as EGFR tyrosinekinase inhibitors. In addition, a number of polyphenols has been found to target receptor tyrosine kinases, including EGFR [9-14]. Grandinin (C46H34O30), a compound found in Melaleuca quinquenervia leaves [15] and in oaks [16] is reported to inhibit the phosphorylation of EGFR in human colon carcinoma cells [17]. However, it is unclear if grandinin affect phosphorylation of EGFR in other types of malignancies. We have previously determined combined treatments of an Hsp90 inhibitor and TNF treatments on multiple cell resulted in synergistic killing of malignant lung cells [18]. Such effects were confirmed by the apoptosis determination using a fluorescence microscopic assay following staining of the drug-treated cells with Hoescht 33258. Moreover, the experimental results indicated that the synergistic killing due to Hsp90 inhibitor and TNF treat-

Grandinin inhibits EGFR phosphorylation Table 1. Primers used in this study Primers EGFR_F EGFR_R AKT_F AKT_R

Sequences Targets 5’ GCGTCTCTTGCCGGAATGT EGFR 5’ CTTGGCTCACCCTCCAGAAG 5’ AAGCAAATCACTGAAGGACCTGG AKT 5’ AAAGACAAGGGGCATTGGGAG

ments may be related to the reduced IKKβ levels upon treatments [18]. Phosphorylated EGFR (p-EGFR) has been identified to correlate with progression of NSCLC [19, 20]. AKT is active in most NSCLC cells [21] and high level of phosphorylated AKT (p-AKT) is often correlated with lung cancers [22]. In this paper, we investigated the effects of grandinin on malignant cells. It was found that treatment of grandinin significantly reduces cell viabilities of three malignant lung cell types in vitro. Furthermore, grandinin inhibits levels of p-EGFR and p-AKT of the three cell lines. We also found that grandinin does not affect mRNA levels of EGFR and AKT. Materials and methods Reagents and cell lines Grandinin (C46H34O30) with a purity of greater than 99% was purified from Melaleuca quinquenervia leaves and provided by the Department of Chemistry in College of Life Sciences, Ocean University of Qingdao. Two small cell lung cancer (SCLC) cell lines (SBC3 and MS-1), an adenocarcinoma cell line (A549), and a squamous-cell carcinoma cell line (LK-2) were provided by Shanghai Cell Biology Institute (Shanghai, China). The cells were maintained in RPMI-1640 medium (Sigma-Aldrich Co. Ltd, Irvine, CA) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin at 37°C with 5% CO2 and 100% humidity. Cell treatments and the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrozolium bromide (MTT) assay Briefly, cells at a density of 1 × 105 cells/well were seeded into 6-well plates in RPMI-1640 supplemented with 10% FBS and were cultured for 24 h. The cells were then treated with vehicle control (DMSO, 0.1%, v/v), grandinin (0 µM, 2 µM, 4 µM, 8 µM, 16 µM). At the end of each experiment, cells were incubated with 0.5 mg/ 5889

ml MTT for 4 h according to the protocol of manufacturer. Viability of treated cells was expressed relative to the control cells treated with DMSO. The relative viability was calculated. Apoptosis assay Cells at a density of 1 × 105 cells/well were cultured in six-well plates in RPMI-1640 supplemented with 10% FBS for 48 h, followed by addition of DMSO (0.016%, v/v), grandinin (2 µM, 4 µM, 8 µM, 16 µM). After 48 h, cells were pelleted by centrifugation, washed once with PBS, fixed by incubation in 4% paraformaldehyde for 30 min at room temperature, and then washed again with PBS to remove the fixative. The fixed cells were resuspended in PBS that contained Hoescht 33258 (5 μg/ml), followed by an incubation at room temperature for 15 min in the dark. Aliquots of cells were placed on glass slides and examined for cells with apoptotic morphology (nuclear condensation and chromatin fragmentation) via fluorescence microscopy. To quantify the apoptosis, 250 nuclei from random microscopic fields were analyzed. Data are presented as the mean percentages of apoptotic cells. Western blot assay Total proteins were harvested from cells, separated on 10% SDS/PAGE gels, and then subjected to western blot analyses. The primary antibodies against the p-EGFR (about 180 kDa), p-AKT (about 60 kDa), and β-actin were purchased from Santa Cruz, USA (anti-p-EGFR, cat # sc-81489, 1:200; anti-p-AKT, cat # sc33437, 1:200; anti-β-actin, cat # sc-130301, 1:10,000). Secondary antibodies were horseradish-peroxidase-conjugated secondary antimouse IgG (cat # 31430, 1:10,000; Pierce Biotechnology) and anti-rabbit IgG (cat # 31460, 1:5,000; Pierce biotechnology). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The experiments were repeated for at least 3 times. The mean normalized optical density (OD) of detected protein bands relative to the OD of β-actin band was calculated. Real time-PCR Quantitative RT-PCR analyses of EGFR and AKT mRNA levels in cells were performed. Briefly, total RNAs were harvested from cells using the RNeasy Kit (Qiagen, USA) according to the manInt J Clin Exp Pathol 2014;7(9):5888-5894

Grandinin inhibits EGFR phosphorylation Results Grandinin treatments reduce viability of malignant cells To investigate if grandinin affects proliferation of lung tumor cells in vitro, two SCLC cell lines (SBC3 and MS-1), an adenocarcinoma cell line (A549), and a squamous-cell carcinoma ceFigure 1. Cell treatments with DMSO (0.1%, v/v) or grandinin. Two SCLC cell lines (SBC3 and MS-1), an adenocarcinoma cell line (A549), and a squamousll line (LK-2) were treated cell carcinoma cell line (LK-2) were treated with either vehicle control (DMSO) or with either vehicle control grandinin (2 µM, 4 µM, 8 µM, 16 µM) for 72 h. (A) SBC3 cells; (B) MS-1 cells; (C) DMSO (grandinin, 0 µM) or A549 cells; (D) LK-2 cells. Cell viability was measured using 3-(4,5-dimethylthiagrandinin (2 µM, 4 µM, 8 zol-2-yl)-2,5-diphenyltetro-zolium bromide (MTT) assay immediately before (day µM, 16 µM) for 72 h. The 0) and after 1, 2, or 3 days of incubation with the compounds. Values are means ± SD for three experiments. It is considered not significant, when P > 0.05 vs. treatment with DMSO sercontrol (DMSO) cell viability of each treatment. *, it is considered as a significant ved as a drug vehicle condifference, when P < 0.05 vs. corresponding control. trol (grandinin, 0 µM). The cells were analyzed for difufacturer’s instructions. The RT-PCR experients ferences in cell killing upon various treatments were repeated at least 3 times. One microliter via number counting of living cells in the presof RNA for each condition was transcribed into ence or absence of the above compounds. cDNA using random primers in a Reverse As shown in Figure 1, the cell treatment results Transcription II system (Promega, USA) accordshowed that the treatments with the drug vehiing to the manufacturer’s instructions. Levels cle control (DMSO) did not significantly affect of EGFR and AKT mRNAs were quantified by cell viability of all of these four types of cells quantitative PCR using an ABI Prism Sequence (Figure 1). Grandinin with a concentration of 2 Detection System (Applied Biosyste-ms). PriµM or 4 µM had slight effects, if any, on cell vimers were shown in Table 1. An assay reagent ability of all of these four types of cells. Grcontaining premixed primers and a VIC labeled andinin (8 µM) decreased viabilities of the three probe (Applied Biosystems; cat. no. 4310884E) lung cancer cell lines MS-1, A549, and LK-2 to was used to quantify expression of endogenous approximately 50-60% at day 3, but no obvious GAPDH mRNA. Template-negative and RT-nedecreases for SBC3 cells, when compared to gative conditions were used as controls. The the cells treated with DMSO only (grandinin, 0 corresponding amplification plots were used to µM). When the concentration of grandinin was determine the threshold cycle value, defined as increased to 16 µM, the viabilities of the three the number of PCR cycles taken for fluorescent lung cancer cell lines MS-1, A549, and LK-2 intensity to reach a fixed threshold for each sigwere reduced to 42%, 12%, and 18%, respecnal. The relative amounts of EGFR and AKT tively, when compared with the cells treated witranscript were normalized to the amount of th DMSO only. The viabilities of SBC3 cells were GAPDH mRNA in the same condition. The levels reduced to 85% only. These results suggest (mean value) of the transcripts in the condithat such dosages (8 µM and 16 µM) of grandtions were calculated. inin effectively reduce viability of malignant cells MS-1, A549, and LK-2. Statistical analyses The experimental data are expressed as mean ± SD. Statistical software (SPSS10.0) was used for independent sample t tests, followed by oneway variance analysis. In all analyses, P < 0.05 was considered statistically significant.

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Grandinin induces apoptosis of the malignant lung cells Since grandinin effectively reduced viability of malignant cells, the effects of the compound on apoptosis in all of these 4 types of cells were

Int J Clin Exp Pathol 2014;7(9):5888-5894

Grandinin inhibits EGFR phosphorylation or grandinin (2 µM, 4 µM, 8 µM, or 16 µM) for 48 h. To quantify the apoptotic incience, we used a fluorescence microscopic as -say following staining of the drugtreated cells with Hoescht 33258. Figure 2. Detection of phenotype-dependent apoptosis induced by treatments with DMSO or grandinin. The SBC3, MS-1, A549, and LK-2 cells were treated with either vehicle control (DMSO) or grandinin (2 µM, 4 µM, 8 µM, 16 µM). Cells were harvested 48 h later. Hoechst 33258-stained cells were examined for apoptotic characteristics (nuclear margination and chromatin condensation) using a fluorescence microscope. Apoptotic incidence was calculated. Data were expressed as means ± SD for three independent experiments.

As shown in Figure 2, the treatments with the drug vehicle control (DMSO) did not significantly affect apoptosis of all of these four types of cells. Grandinin with a concentration of 2 µM or 4 µM had slight effects, if any, on apoptosis of all of these four types of cells. Grandinin (8 µM) increased viabilities of the three lung cancer cell lines MS-1, A549, and LK-2 by approximately 6-7 folds, but no obvious increases in apoptosis incidences of SBC3 cells, when compared to the cells treated with DMSO only (grandinin, 0 µM). When the concentration of grandinin was increased to 16 µM, the apoptosis of the three lung cancer cell lines MS-1, A549, and LK-2 were increased by 8-9 folds, when compared with the cells treated with DMSO only. The apoptosis incidences of SBC3 cells were not affected significantly. These results suggest that such dosages (8 µM and 16 µM) of grandinin effectively increase apoptosis incidences of malignant cells MS-1, A549, and LK-2. Grandinin treatments lead to down-regulation of levels of p-EGFR and p-AKT

Figure 3. Grandinin decreases expression of p-EGFR and p-AKT. SBC3, MS-1, A549, and LK-2 cells were treated with either vehicle control (DMSO) or grandinin (16 µM) for 48 h. Whole-cell extracts were prepared and western blot analyses were performed to analyze the expression of p-EGFR, p-AKT, and β-actin. The cellular β-actin served as a loading control. Representative blots are given. The experiments were repeated for at least three times.

further determined. The cells were treated with either vehicle control DMSO (grandinin, 0 µM)

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Since the p-EGFR and p-AKT levels are often correlated with lung cancers, we investigated the effects of grandinin on p-EGFR and p-AKT levels. The SBC3, MS-1, A549, and LK-2 cells were treated with either vehicle control (DMSO) or grandinin (16 µM) for 48 h. The total proteins were extracted and the levels of p-EGFR and p-AKT were determined by western blot analysis, with the cellular β-actin protein serving as a loading control. The mean normalized OD of p-EGFR and p-AKT bands relative to the OD of β-actin band from the same condition was all calculated and subjected to statistical analyses.

Int J Clin Exp Pathol 2014;7(9):5888-5894

Grandinin inhibits EGFR phosphorylation Discussion Grandinin was found to have cytotoxic funcions to some malignancies [17, 23]. However, it is unknown that if grandinin has effects on lung tumor cells. In this study, we have determined the effects of grandinin on several lung cancer Figure 4. Quantitative RT-PCR analysis of the mRNA expression levels of p-EGFR and p-AKT in SBC3, MS-1, A549, and LK-2 cells. One microliter of RNA for each cell lines. The cell viabicondition was transcribed into cDNA using random primers in a Reverse Transcriplity results show that tion II system (Promega, USA) according to the manufacturer’s instructions. Levels grandinin with dosages of EGFR and AKT mRNAs was quantified by quantitative PCR using an ABI Prism of 8 µM and 16 µM effeSequence Detection System (Applied Biosystems). An assay reagent containing prectively reduce viability mixed primers and a VIC labeled probe (Applied Biosystems; cat. no. 4310884E) was used to quantify expression of endogenous GAPDH mRNA. Template-negative of malignant cells MSand RT-negative conditions were used as controls. The relative amounts of EGFR 1, A549, and LK-2, but and AKT transcript were normalized to the amount of GAPDH mRNA in the same the effects on another condition. The levels (mean value) of mRNA transcripts were calculated. Error bars cell line SBC3 are relashow mean ± SD. tively smaller. It is necessary to continue stuAs shown in Figure 3, treatment with grandinin dy if the effect of grandinin on lung cancer cells did not significantly affect (or slightly affect, if is cell type-dependent. If so, this compound any) p-EGFR and p-AKT levels in SBC3 cells. may have different effects on different pathoHowever, treatments with grandinin decreased logical types of lung cancers. The smaller effelevels of p-EGFR and p-AKT levels in MS-1, cts of grandinin on SBC3 may be related to the A549, and LK-2 cells, when compared with the receptors on different types of cells. levels in the conditions treated with DMSO only. The reduced cell viabilities may result from variThese results indicated that grandinin treatous reasons, such as increased apoptosis. The ments lead to down-regulation of levels of papoptosis assays show that this compound sigEGFR and p-AKT in MS-1, A549, and LK-2 cells. nificantly increased the apoptosis rates of maGrandinin does not affect mRNA levels of lignant cells MS-1, A549, and LK-2. The effecEGFR and AKT tive dosages are 8 µM and 16 µM, which are safe to normal human cells according to previTo determine if grandinin affects mRNA levels ous reports [17]. To achieve much higher efficaof EGFR and AKT, the SBC3, MS-1, A549, and cies, the combined uses of grandinin with other LK-2 cells were treated with either vehicle condrugs such as gefitinib [6- 8] or drug candidates trol (DMSO) or grandinin (16 µM) for 48 h. The such as 17-DMAG [18] should be investigated total RNAs were isolated for all conditions and in the future. the EGFR and AKT mRNA transcript levels in the SBC3, MS-1, A549, and LK-2 cells were deterEGFR, as a major mediator of development of mined by quantitative RT-PCR. The levels of the many tumors, can induce activation of tumor mRNA transcripts in cells treated with DMSO proliferation and growth, and inhibit apoptosis only were assigned a value of 100. [24, 25]. Activation of Akt is one of the mechaAs shown in Figure 4, the mean levels of EGFR and AKT mRNA transcripts in all 4 types of cells treated with grandinin were similar to those treated with DMSO. These results suggest that grandinin does not affect mRNA levels of EGFR and AKT.

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nisms that mediate the effects of EGFR [26]. The roles of EGFR and AKT in tumor development are mediated by their phosphorylation [27, 28]. Therefore, we determined if the effects of grandinin on malignant lung cells are related to levels of their phosphorylated forms.

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Grandinin inhibits EGFR phosphorylation Our results clearly show that treatments with grandinin decreased levels of p-EGFR and p-AKT levels in MS-1, A549, and LK-2 cells, when compared with the levels in the conditions treated with DMSO only. These results indicated that grandinin treatments lead to down-regulation of levels of p-EGFR and p-AKT in MS-1, A549, and LK-2 cells. Moreover, these results are consistent to the inhibitory effects of grandinin on these cell types. Altogether, our results suggest that grandinin significantly reduce malignant cell viability and effectively induces apoptosis of malignant lung cells by down-regulating phosphorylation of cellular signaling proteins EGFR and AKT. Acknowledgements This work was supported by the following grants: Shandong province tackle key problems in science and technology program (Grant NO. Y2006C14) and Qingdao municipal science and technology commission (Grant No. 051NS-74).

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Disclosure of conflict of interest None. Address correspondence to: Dr. Zhuling Qu or Wei Feng, The Affiliated Hospital of Qingdao University, Qingdao 266021, Shandong Province, China. Tel: 86-15269236325; 86-532-81707539; Fax: 86-53281707539; E-mail: [email protected] (Zhuling Qu); [email protected] (Wei Feng)

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Int J Clin Exp Pathol 2014;7(9):5888-5894

Grandinin down-regulates phosphorylation of epidermal growth factor receptor.

Grandinin (C(46)H(34)O(30)) is a compound found in Melaleuca quinquenervia leaves and in oaks. This study is to determine effects of grandinin on mali...
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