Noscapine targets EGFRp-Tyr1068 to suppress the proliferation and invasion of MG63 cells

Osteosarcoma, the most common primary malignant bone tumor, usually arises in the metaphysis of long bones. Amplification and mutation of the epidermal growth factor receptor (EGFR) gene represent signature genetic abnormalities encountered in osteosarcoma. Noscapine is a benzylisoquinoline alkaloid derived from the opium poppy Papaver somniferum. Recently several studies have suggested its anti-cancer effect in melanoma, ovarian cancer, gliomas, breast cancer, lung cancer, and colon cancer. However, the underlying molecular mechanism for its anti-cancer effect still remains unclear. In this paper, we found the mechanism of noscapine effectively suppressed proliferation and invasion of MG63 cell line by inhibiting the phosphorylation of EGFR and its downstream pathway.

. Noscapine inhibits the growth and the invasion of MG63 and U2OS (A) The structure of noscapine. (B) Protein level of EGFR in tissues was detected by western blot. (C) MRNA level of EGFR in tissues was detected by RT-PCR. *P < 0.05 vs. normal group, **P < 0.01 vs. normal group. (D) MG63cells were cultured with indicated concentrations of noscapine for indicated hours in 96-well plates, then MTT assay was performed, results represent the mean ± SD of three experiments done in triplicate. (E) MG63cells were preincubated with DMSO or noscapine for 24 h, then cells were analyzed using a FACS vantage flow cytometer with the Cell Quest acquisition and analysis software program (Becton Dickinson and Co., San Jose, CA), the experiment was repeated three times. (F) MG63cells were pre-incubated with noscapine for 24 h then cells were treated with ANNEXIN-V-FITC apoptosis detection kit and analyzed with FCAS. The experiment was repeated for three independent times. (G,H) MG63cells were pre-incubated with noscapine for 24 h; transwell assay without matrigel was performed. Cells were counted and results represent the mean ± SD of three experiments. *P < 0.05 vs. DMSO treated group, **P < 0.01 vs. DMSO treated group. (I,J) MG63cells were pre-incubated with noscapine for 24 h; transwell assay with matrigel was performed. Cells were counted and results represent the mean ± SD of three experiments. *P < 0.05 vs. DMSO treated group, **P < 0.01 vs. DMSO treated group.
Scientific RepoRts | 6:37062 | DOI: 10.1038/srep37062 Medical University. The study protocol was approved by Medical Ethics and Human Clinical Trial Committee. All the tissues were immediately frozen in liquid nitrogen after surgery for research. We confirm that all methods were performed in accordance with the relevant guidelines and regulations. We confirm that informed consent was obtained from all subjects.
Cell culture. Osteosarcoma cell lines MG63 and U2OS were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (Invitrogen, Carlsbad, CA, USA). Cells were cultured at 37 °C with humidified atmosphere of 5% CO 2 .
MTT assays. Cells were plated in 96-well plates (1 × 10 4 /well). 24 hours later, the medium was replaced with 100 ul medium containing different concentrations of noscapine and cultured for 0, 12, 24, 36 and 48 h. 20 μ l of 5 mg/ml MTT solution was added to well. After incubation for 4 hours, the medium was removed and remaining MTT formazan was dissolved in 150 μ l of DMSO. Solution was measured at 490 nm using a Microplate Reader (BIO-RAD).
Cell-cycle analysis by flow cytometry. After incubation with different concentrations of noscapine, MG63 cells were washed using cold PBS and suspended in staining buffer with 10 ug/ml propidium iodide. The cells were analyzed using BD FACS Vantage flow cytometer (Becton Dickinson) and analyzed using CellQuest software program.
Cell migration and invasion assay. Matrigel cell invasion assay was performed using a 24-well transwell chamber (8-μ m pore size) (Corning). The transwell chambers were coated with 20 μ l matrigel (1:4, BD Bioscience). Transfected cells were trypsinized and suspended in 100 μ l of DMEM medium without serum, which was transferred to the upper chamber. 600 μ l culture medium with 10% FBS was added to the lower chamber. After 18 hours incubation, the non-invaded cells on the upper membrane surface were removed using cotton swab and the cells that passed through the filter were fixed and stained with hematoxylin. The numbers of cells that pass through the membrane were counted in using microscope. This experiment was performed in triplicate.
Cell apoptosis. Apoptosis was examined using Annexin-V-PI apoptosis detection kit. MG63 cells are washed with cold PBS and suspended using binding buffer. 5 μ l of Annexin-V-FITC and 10 μ l of PI were added. Cells were incubated in the dark for 15 minutes. 400 μ l of binding buffer was added and the apoptosis rate was examined using BD FACS Vantage flow cytometer (Becton Dickinson).
Reverse transcription and quantitative real-time PCR. Quantitative real-time PCR was performed using SYBR Green PCR master mix (TAKARA) in a total volume of 20 μ l on Mx 3000 P Real-Time PCR System as follows: 95 °C for 30 seconds, 50 cycles of 95 °C for 10 seconds, 60 °C for 30 seconds. A dissociation step was performed to generate a melting curve to confirm the specificity of the amplification. β -actin was used as the reference gene. The relative levels of gene expression were calculated by the 2 −ΔΔCt method. Primer sequences were synthesized as the Table 1.
Transfection of siRNA. Cells were transfected using pRS-siEGFR (Shanghai GeneChem Company) and then selected with puromycin (1.5 μ g/mL) for 2 weeks. The cells transfected with pRS-si-NC (negative control) and cells without treatment were used as the control.
(C) EGFR kinase were pre-incubated with the indicated concentrations of noscapine for 1 hour respectively, then Kinase Glo assay were performed and inhibition rate were calculated, linear fit curve was drawn with an equation of y = 0.02505x + 0.01751 R 2 = 0.8715. (D) Flag-EGFR-KM and Flag-EGFR-WT proteins were preincubated with the indicated concentrations of noscapine for 1 hour respectively, then Kinase Glo assay were performed. Data are shown as mean ± SD. *P < 0.05 vs. DMSO treated Flag-EGFR-WT group; **P < 0.01 vs. DMSO treated Flag-EGFR-WT group; # P < 0.05, ## P < 0.01 vs. DMSO treated Flag-EGFR-KM group; $ P < 0.05, $$ P < 0.01 vs. Flag-EGFR-WT group treated Flag-EGFR-KM group; (E) MG63cells expressing Flag-vector, Flag-EGFR-KM or Flag-EGFR-WT and then treated with or without noscapine were detected with indicated antibody by western blot. (F) MG63cells transfected with si-EGFR or si-NC (negative control) and those cells were treated with or without noscapine were detected with indicated antibody by western blot.

Noscapine inhibits the growth and the invasion of MG63 and U2OS cells.
Through the detection of protein and RNA levels, we found that the EGFR in osteosarcoma tissue was higher than that in adjacent tissues (Fig. 1B,C). The inhibitory effects on growth of noscapine were detected in MG63 and U2OS cells. As shown in MTT assay, noscapine inhibited the proliferation of MG63 and U2OS cells in a concentration dependent manner (Figs 1D and 6B). To further study the mechanisms of noscapine inhibiting the growth of osteosarcoma cells, MG63 cells were exposed to the indicated concentrations of noscapine for 24 h, and then cell cycle analysis was performed. Noscapine prominently induced a dose-dependent increase in the percentage of cells in G1 phase and a decrease in S phase compared with the control (Fig. 1E), indicating that noscapine arrest MG63 cells at the G1 phase of the cell cycle. To test whether noscapine could induce apoptosis of MG63cells, we detected the apoptosis rate by Annexin-V-FITC. By Annexin-V-FITC staining, the noscapine-induced MG63 cell apoptosis was increased with the increased concentration (Fig. 1F). Inhibitory effect of noscapine on migration and invasion of MG63cell was analyzed by transwell assay (with or without matrigel). Results showed that noscapine significantly decreased invasion and migration potential of osteosarcoma cells MG63 and U2OS(Figs 1G-J and 6E,F) in a dose-dependent manner. The above results showed that noscapine can inhibit the growth and the invasion of MG63 cells in a concentration dependent manner.

Noscapine suppresses the kinase acivity of EGFR by inhibiting EGFR p-Tyr1068 in MG63 and U2OS
cells. Because of the important role of EGFR phosphorylation in the signal pathway, we wonder whether noscapine could inhibit the phosphorylation level of EGFR and suppress the activation of EGFR in MG63cells and U2OS cells. By western blot analysis, we found that treatment of noscapine could markedly inhibit the level of EGFR p-Tyr1068 but without affected the total expression of EGFR (Figs 2A and 6A). To further detect the selective inhibition profile of noscapine, we chose four types of kinase-EGFR/ErbB1/HER1, ErbB2/HER2/neu, ErbB3/ HER3 and ErbB4/HER4; all of them are belong to the epidermal growth factor receptor family, which share a certain homology, to test the inhibition effect of noscapine on their kinase activation. By Kinase-Glo ® Luminescent Kinase Assays (Promega) which is a homogeneous non-radioactive methodin vitro, we got the kinase inhibition rate of noscapine (Fig. 2B). The results showed that noscapine markedly inhibited EGFR kinase activity in a dose-dependent manner, but had relatively weak effect on the other three kinases, there were statistically significant difference between EGFR and the other three kinases at different concentration of noscapine (Fig. 2B).
Then the inhibitory effect of noscapine on EGFR kinase was also tested by the assay. Data showed that noscapine inhibited EGFR activity with an IC 50 value of 19.26 μ mol/l (Fig. 2C).
To further verify the specific targeting of noscapine on EGFR, we performed two group experiments. Firstly, MG63 cells were transfected with Flag-vector, Flag-EGFR-WT (wild type) and Flag-EGFR-KM respectively and treated with the indicated noscapine for 24 h, the kinase inhibition rate of noscapine between EGFR-WT and EGFR-KM were compared by Kinase-Glo ® Luminescent Kinase Assays. The results showed that noscapine markedly inhibited EGFR-WT kinase activity in a dose-dependent manner, but had relatively weak effect on were detected by western blot. (E) MG63 cells expressing Flag-VECTOR, Flag-EGFR-KM or Flag-EGFR-WT were incubated with 20 μ mol/l of noscapine, MTT assay was performed after indicated hours and results represent the mean ± SD of three experiments done in triplicate. **P < 0.01 vs. Flag-Akt-KM/Flag-Akt-WT. (F) MG63 cells transfected with si-EGFR or si-NC (negative control) were treated with 20 μ mol/l of noscapine, MTT assay was performed after indicated hours and results represent the mean ± SD of three experiments done in triplicate. *P < 0.05 vs. si-EGFR/si-NC (negative control). (G) MG63cells expressing Flag-vector, Flag-EGFR-KM or Flag-EGFR-WT and then treated with or without noscapine were detected with Cyclin D1, CDK4, CDK6, EGFR, EGFR p-Tyr1068 , Akt and Akt p-Ser473 antibody by western blot. (H) MG63cells expressing Flag-vector, Flag-EGFR-KM or Flag-EGFR-WT and then treated with or without noscapine were detected with Caspase3, Bax, Bcl-2, EGFR, EGFR p-Tyr1068 , Akt and Akt p-Ser473 antibody by western blot.
EGFR-KM (Fig. 2D). Then the indicated proteins level was tested by western blot analysis (Fig. 2E). As the results showed, in the DMSO treated control group, the level of EGFR p-Tyr1068 in cells expressed Flag-EGFR-KM was significantly less than that in the cells expressed Flag-EGFR-WT. In the noscapine treated group, in both cells expressed Flag-EGFR-WT and Flag-EGFR-KM, the level of EGFR p-Tyr1068 dramatically decreased compared with the DMSO treated control group, but the total EGFR level were not changed (Fig. 2E).
In another set of experiments, the MG63/si-NC (negtive control) stable cell line and MG63/si-EGFR stable cell line were treated with or without noscapine, the level of EGFR p-Tyr1068 and total EGFR were tested by western blot analysis (Fig. 2F). The data showed that the phosphorylation degree of EGFR p-Tyr1068 significantly reduced after noscapine treatment in MG63/si-NC stable cell lines but not changed significantly in MG63/si-EGFR stable cell line compared with the DMSO control group.
The above results indicated that noscapine suppressed the kinase acivity of EGFR, at least partly by inhibiting the phosphorylation of EGFR p-Tyr1068 in MG63 cells.

Noscapine inhibits the growth of MG63 and U2OS by inhibiting EGFR/Akt/CDKs and EGFR/Akt/ Bad pathway.
Since Cyclin D1, CDK4 and CDK6 are key regulators in the G 1 phase of the cell cycle, here we examined the indicated regulators expression level in noscapine-treated cells. Western blot analysis showed that exposure of MG63 and U2OS to 10/20/30 μ mol/l noscapine for 24 h dramatically decreased the expression of Akt, Akt p-Ser473 , Cyclin D1, CDK4 and CDK6 (Figs 3A and 6C), indicating noscapine arrests cells at G1 phase and then suppresses cells growth via down-regulated Akt p-Ser473 , Cyclin D1, CDK4 and CDK6. Furthermore, real time RT-PCR showed that expression of Cyclin D1, CDK4 and CDK6 in MG63 were down-regulated at mRNA level after exposure to noscapine (Fig. 3B). Furthermore the expressions of apoptosis regulators were also examined by western blot. The expression of Bcl-2 was obviously decreased and the levels of Caspase3 and Bax were increased in noscapine treated MG63 and U2OS cells (Figs 3D and 6D). Then real time RT-PCR results verified that changes of these factors were coincidence with protein levels (Fig. 3C).  To further confirm the involvement of EGFR in noscapine -induced MG63 cells growth arrest, cells respectively expressed Flag-EGFR-WT and Flag-EGFR-KM plasmids were treated with 20 μ mol/l noscapine for 24 h. Then cell proliferation was analyzed by MTT assay. Results showed that the proliferation of cells expressed EGFR-WT was dramatically inhibited by noscapine compared with cells transfected with EGFR-KM plasmid, but there was no significant difference in the inhibition rate between EGFR-KM expressed cells and cells transfected with vector cells (Fig. 3E). Then, the same experiments were performed in MG63/si-NC (negtive control) cell line and MG63/si-EGFR cell line. Results showed that, there was significantly difference in the proliferation inhibition rate between MG63/si-NC and MG63/si-EGFR cell lines (Fig. 3F). While, western blot analysis showed that under the effect of noscapine, when the Akt p-Ser473 was over-expression EGFR p-Tyr1068 , Cyclin D1, CDK4, CDK6, Bcl-2, Caspase3 and Bax changed more significantly (Fig. 3G,H). Noscapine represses the migratory and invasive potential of MG63 by inhibiting EGFR/Akt/ MMP2 pathway. To further investigate the mechanisms of noscapine migratory of osteosarcoma cells, MG63 and U2OS cells were exposed to various concentrations of noscapine for 24 h. Western blot analysis showed that levels of MMP2 dramatically decreased (Figs 4A and 6G). Real time RT-PCR showed that expression of MMP2 in MG63 was down-regulated at mRNA level after exposure to noscapine (Fig. 4B). The migration capacity was detected in EGFR-WT, EGFR-KM (kinase mutation EGFR) and MG63/si-EGFR cell lines with or without noscapine. And results suggested that EGFR is the target of noscapine (Fig. 4C,D). Western blot analysis showed that under the effect of noscapine, when the EGFR was over-expression EGFR p-Tyr1068 and MMP2 decreased more significantly (Fig. 4E).
The anti-tumorigenic effect of noscapine in vivo. The anti-tumorigenic effect of noscapine on MG63 cells was further illustrated in vivo in a nude mouse xenogfraft. On the day of sacrifice (day 16), fangchinoline treatments at the given doses resulted in about 41.23% tumor suppression (Fig. 5A,B). Then the protein levels of EGFR and its pathways were detected by western blot (Fig. 5C). The above results suggested that noscapine inhibits the migratory and invasive of MG63 cells by inhibiting EGFR pathway. These results indicated that noscapine effectively suppressed proliferation and invasion of MG63 cells by inhibiting EGFR p-Tyr1068 (Fig. 5D).

Discussion
In cancer cells, EGFR aberrations impact a variety of cell signaling pathways, notably the PI3K-AKT and JAK/ STAT pathways 18 . In osteosarcoma, in vitro data from early passage osteosarcoma cells demonstrate constitutive EGFR phosphorylation whose abrogation leads to growth inhibition 19 . Overexpression of EGFR has been shown to promote cancer cell motility and invasion. In vitro data indicate that EGFR and Akt signaling play a role in the pathogenesis of osteosarcoma 20 .
Noscapine was shown to have potent antitumor activity against murine lymphoid tumors 21 . Since then, noscapine has been shown to exhibit activity against a wide variety of tumors in vitro and in vivo [22][23][24][25] . There are findings suggest that noscapine can promote apoptosis by suppressing Bcl-2 26 . Besides antiapoptotic proteins, noscapine also downregulates the expression of proteins linked to cell proliferation, inflammation, invasion, adhesion, and angiogenesis. These observations imply that noscapine has anti-inflammatory, antiangiogenic, and antimetastatic activities 27 . At the same time in hypoxic human glioma cells, noscapine has been shown to inhibit the secretion of VEGF 28,29 .
In this study, MG63cells were used to detect the anti-cancer effect of noscapine. As shown in MTT assay and transwell assay, noscapine treatment inhibited the proliferation and migration of MG63cells in a concentration-dependent manner. We found that the phosphorylation of EGFR (Tyr1068) dramatically decreased with the increasing concentration of noscapine, which suggested noscapine suppressed the phosphorylation of EGFR and inhibited the proliferation and migration of MG63cells. We first found that noscapine did suppress the phosphprylation levels of EGFR, so it is reasonable to conclude that noscapine suppressed Cyclin D1 and CDK4/6 expression via suppression of EGFR pathway, and inhibited the transition of cells from G1 phase to S phase, and resulted in the anti-proliferative effect on MG63 cells together with the induction of apoptosis.
In addition to the effect on cell proliferation, we demonstrated the inhibition mechanism of noscapine on invasion of MG63 cells. One of the key steps in cancer invasion and metastasis is the degradation of extracellular matrix. MMP2 has been demonstrated to play important roles in the process 30 . Our results showed that noscapine significantly suppressed the invasive ability of MG63cells in parallel with down-regulation of MMP2 and inhibit EGFR pathway.
In summary, our data showed that noscapine could inhibit the malignant phenotype of MG63 cells by inhibit the phosphorylation of EGFR (Tyr1068) and further to suppress the EGFR associated signaling pathway, EGFR/ Akt pathway. Also the anti-tumorigenic effect of noscapine on MG63 cells was illustrated in vivo. Although these results are warranted further testing, the present findings do support the conception that noscapine may offer a novel therapeutic strategy for advanced metastatic osteosarcoma.