J Biosci (2019) 44:96 Ó Indian Academy of Sciences DOI: 10.1007/s12038-019-9904-4 (0123456789().,-volV)(0123456789().,-volV) Calycosin down-regulates c-Met to suppress development of glioblastomas , XIAOHU NIE ,YUE ZHOU* ,XIAOBING LI,JIE XU,XUYAN PAN,RUI YIN and BIN LU Department of Neurosurgery, Huzhou Central Hospital, Huzhou, Zhejiang, People’s Republic of China *Corresponding author (Email, [email protected]) These authors contributed equally to this work. MS received 15 October 2018; accepted 9 June 2019; published online 7 August 2019 The antitumor effect of calycosin has been widely studied, but the targets of calycosin against glioblastomas are still unclear. In this study we focused on revealing c-Met as a potential target of calycosin suppressing glioblastomas. In this study, suppressed-cell proliferation and cell invasion together with induced-cell apoptosis appeared in calycosin-treated U251 and U87 cells. Under treatment of calycosin, the mRNA expression levels of Dtk, c-Met, Lyn and PYK2 were observed in U87 cells. Meanwhile a western blot assay showed that c-Met together with matrix metalloproteinases-9 (MMP9) and phosphorylation of the serine/threonine kinase AKT (p-AKT) was significantly down-regulated by calycosin. Furthermore, overexpressed c-Met in U87 enhanced the expression level of MMP9 and p-AKT and also improved cell invasion. Additionally, the expression levels of c-Met, MMP9 and p-AKT were inhibited by calycosin in c-Met overex- pressed cells. However, an AKT inhibitor (LY294002) only effected on MMP9 and p-AKT, not on c-Met. These data collectively indicated that calycosin possibility targeting on c-Met and exert an anti-tumor role via MMP9 and AKT. Keywords. AKT; calycosin; c-Met; glioblastoma; MMP9 1. Introduction Schlessinger 2010). A gene-chip analysis in our preliminary experiment showed that calycosin inhibited the expression Glioma is the most common tumor in human brains. There are level of RTKs (Dtk, c-Met, Lyn and PYK2). Among them, five classifications in glioma based on the degree of develop- c-Met is well known as a receptor of the hepatocyte growth ments and about 75% of them are at grade III and IV which are factor (HGF) and regulates morphogenesis in embryonal considered as the highly risky grade (Silvia et al. 2014). cells (Bladt et al. 1995). Recently c-Met was reported to be Glioblastoma is a type of malignant glioma with only 14.6 sur- related to regulating cancer development in various tissues viving months on average after diagnosis (Ellingson et al. 2012). (Sennino et al. 2012; Han et al. 2014) and a series of Furthermore, the traditional treatments are still the primary tremendous progress have been made in the development of therapy but with a poor curative effect and prognosis (Ganipineni targeted agents against c-Met treating various cancers et al. 2018). Therefore, a better treatment is in great need. (Granito et al. 2015; Park et al. 2017). Calycosin is a main bioactive compound extracted from radix In this study we investigated that whether calycosin could astragali which is a common herbal medicine commonly used in regulate the development of glioblastoma through c-Met. traditional Chinese medicine (Gao et al. 2014). Early studies Our results showed that calycosin could suppress the pro- showed that calycosin has various pharmacological properties liferation and invasion in two glioblastoma cell lines U251 like anti-inflammatory, antioxidant and neuroprotective effects and U87. Additionally, we also explored the downstream (Guo et al. 2012; Gao et al. 2014). Recently several studies molecule system of c-Met which was related to regulating indicated that calycosin had a promising antitumor effect in invasion in glioblastoma cells. Therefore we detected the various tumor cells (Qiu et al. 2014; Chen et al. 2015; Tian et al. expression levels of matrix metalloproteinases (MMP9), 2017). Although it has been proved that calycosin showed AKT and P-AKT which are strongly associated with cell anticancer effects against glioblastoma, the direct binding target invasion in various cancer cells in U251 and U87 cells after of calycosin is still unclear (Nie et al. 2016). treated with calycosin. Our results showed that calycosin The receptor tyrosine kinase (RTK) family is consists of a could suppress the expression level of c-Met and the series of cell-surface receptors and play important roles in expression levels of MMP9 and P-AKT were also down- regulating various cellular processes (Lemmon and regulated followed by the inhibition of c-Met. http://www.ias.ac.in/jbiosci 1 96 Page 2 of 8 Xiaohu Nie et al. These findings suggest that c-Met may be a potential 2.5 Western blot assay target for calycosin against glioblastomas. The cell samples cultured with calycosin or LY294002 were washed three times with cold PBS and then lysed in 2. Materials and methods Radioimmunoprecipitation assay lysis buffer. The super- natant containing extracted protein was collected, after 2.1 Cell culture 10 min centrifugation at 12,000 rpm under 4°C. A bicin- choninic acid protein assay kit (Thermo, PICPI23223) was HEK293T, U251 and U81 cells used in the this research used to determine the concentrations of these protein sam- were bought from the Institute of Biochemistry and Cell ples. About 25 lg proteins from each sample were separated Biology, Chinese Academy of Sciences (Shanghai, China). by a sodium dodecyl sulfate-polyacrylamide gel elec- All these cells were cultured in Dulbecco’s minimum Eagle’s trophoresis gel followed by transferring them onto a nitro- medium (DMEM) at 37°C and 5% CO2 were added. At the cellulose (NC) membrane (Millipore, HATF00010). 5% same time, 10% fetal calf serum (FCS) and 1% antibiotic skim milk was used to block the NC membrane with target (penicillin/streptomycin) were supplemented in DMEM. proteins and then primary antibodies (MMP9, Abcam, Ab38898; C-met, Abcam, Ab51067; AKT, CST, #4685; P-AKT, CST, #4060; GAPDH, CST, #5174) were added. 2.2 Cell counting kit-8 (CCK-8) assay The appropriate secondary horseradish peroxidase-conju- gated antibodies (HRP-labeled Goat Anti-Rabbit IgG, Bey- A CCK-8 assay was used to analyze the cell proliferation. In otime, A0208; HRP-labeled Donkey Anti-Goat IgG, brief, every 5 9 103 cells were seeded onto one well of Beyotime, A0181 and HRP-labeled Goat Anti-Mouse IgG, 96-well plate and cultured in a cell incubator (Thermo Forma Beyotime, A0216) were added to the NC membranes and 3111) with 5% CO2 at 37°C overnight. Followed by treating then washed with Tris buffer with 1% Tween (TBST) three with a bunch of different concentrations (0, 25, 50, 100, 200, times. The target bands were visualized with enhanced 400 and 800 lM) of calycosin (Sigma, B9938-10MG), the chemiluminescence reagents (Millipore, WBKLS0100) and cell proliferation rate was analyzed by a spectrophotometer then the NC membranes with target bands were imaged in (Perlong, DNM-9602) after being treated for 0, 24, 48 and the Tanon-5200 system (Tanon) and then analyzed with 72 h. Image J software. 2.3 Cell apoptosis assay 2.6 Real-time polymerase chain reaction (PCR) analysis U251 and U87 cells were cultured with calycosin at 0, 100, 200 and 400 lM for 48 h separately. After this about The total RNA from cell samples was extracted by using 5–10 9 104 cells were collected from each well. Following Trizol reagent (Invitrogen, 1596-026) according to manu- the treatment with sufficient Annexin V-FITC (Beyotime, facturer’s instruction. Equal amounts of RNA were used to C1063) and propidium iodide (Beyotime, C1063), a FACS reverse transcribed into complementary DNA (cDNA) with instrument (BD, Accuri C6) was used to analyze the cell a RevertAid First Strand cDNA Synthesis Kit (Fermentas, apoptosis. K1622). The real-time PCR was conducted with a SYBR Green PCR kit (Thermo, K0223) on a ABI-7300 instrument (ABI). GAPDH was served as an internal control. The pri- 2.4 Transwell analysis mer information is shown as follows: DTK, 50 TGTGGC TCACGGTAGAAGG 30 and 50 GCTTGAAGGTGAA- The cells treated with calycosin or LY294002 (Merck, CAS CAGTGGC 30; c-Met, 50 TACCACTCCTTCCCTG 30 and 154447-36-6) were cultured in DMEM without serum for 50 CATTTCCCTTAATTTCC 30; Lyn, 50 CCCTATGATGG- 24 h, and then seeded about 5 9 104 cells into the upper CATCCAC 30 and 50 CTTTCTGCGTCCTTCCTG 30; chamber precoated with matrigel followed by adding 0.7 mL PYK2, 50 AGATTCCCGACGAAACCC 30 and 50 GACAC RPMI 1640 Medium (Gibco, 22400089) with 10% FCS into CTTCATAGACCTCCC 30 and GAPDH, 50 AATCCCAT- the lower chamber and then the cells were cultured at 37°C CACCATCTTC 30 and 50 AGGCTGTTGTCATACTTC 30. with 5% CO2. After 24 h, the cells still on the upper surface were gently removed. The invaded cells were fixed by 4% formaldehyde solution (Sinopharm, Shanghai, China) for 2.7 C-Met overexpression in U87 cells 10 min followed by dyeing with 0.5% crystal violet (Solarbio, C8470) for another 30 min. After being washed with PBS The full-length c-Met sequence was cloned into a lentiviral three times, the cells were counted under a microscope (Cai- plasmid pLVX-puro (Clontech, USA). The constructed len- kang, Shanghai, China) at 9200 magnification. tiviral vector or control vector was co-transfected into Calycosin suppress glioblastomas via c-Met Page 3 of 8 96 HEK293T cells with helper plasmids. The supernatant U87 cells compared with the untreated groups. In addition, the containing target lentivirus was collected after 48 h and then results from a transwell assay showed that calycosin could infected U81 cells. The c-Met expression level was evalu- inhibit cell invasion but only at higher concentrations. As ated by the real-time PCR and western blot. shown in figure 2A and B, when the calycosin concentration was at 100 lM, there was no difference in U251 (P[0.05) or no extremely significant (0.01\P\0.05) in U87 cells on cell 2.8 Statistical analysis invasion compared with untreated groups.