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MMP-9 signal pathway
Biomedicine & Pharmacotherapy 74 (2015) 77–82
Available online at
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Original Article
Schisandrin B suppresses glioma cell metastasis mediated by inhibition of mTOR/MMP-9 signal pathway Yan Jiang, Qiuli Zhang, Jinsuo Bao, Chenghua Du *, Jian Wang, Qiang Tong, Chang Liu Department of Neurosurgery, The Affiliated Hospital of Inner Mongolia National University, Tongliao, Inner Mongolia 028007, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 June 2015 Accepted 9 July 2015
Background: Malignant glioma is the aggressive tumor in the brain and is characterized by high morbidity, high mortality. The main purpose of the present study was to investigate the therapeutic effects of Schisandrin B on glioma cells and preliminary explore the possible mechanism underlying antimetastasis of Schisandrin B. Methods: Two glioma cell lines, U251 and U87, were used in present study. The ability of metastasis of glioma cells was evaluated using transwell migration assay and invasion assay. Expression of Akt, mTOR, MMP-2 and MMP-9 was determined using Western blotting. Antagonist or agonist was used to activated or inactivated signal molecules. Results: Schisandrin B suppressed cell migration and invasion in manner of dose dependent as well as inhibited expression of p-Akt, p-mTOR and MMP-9. Activation of PI3K by 740Y-P treatment leaded to upregulation of p-Akt, mTOR and MMP-9; inactivation of mTOR by Rapamycin treatment inhibited expression MMP-9 while activation of mTOR by L-Leucine treatment enhanced MMP-9 expression in Schisandrin B incubated cells. Anti-migration and invasion action of Schisandrin B was also reversed by mTOR activation. Conclusion: Our findings demonstrate that Schisandrin B can suppress migration and invasion of glioma cell via PI3K/Akt-mTOR-MMP-9 signaling pathway. ß 2015 Published by Elsevier Masson SAS.
Keywords: Akt mTOR Cell invasion Cell migration PI3K
1. Introduction Glioma is one of the most common malignant central nervous system tumors in human and is characterized of morbidity, disability, mortality and high recurrence rate [1]. Tumor recurrence usually recurs within 6–9 months, resulting in a median patient survival of just 13–16 months following diagnosis [16]. Clinical treatment for glioma focuses on surgery with a combination of radiotherapy and high fat soluble chemotherapy drugs. However, surgery is unable to excise all malignant glioma cells because of its infiltration to adjacent brain parenchyma and also easily cause complications [6,19]. The residual tumors can behave in a very aggressive and malignant fashion. Hence, a new optimal pharmacotherapy drugs that target multiple mechanisms of tumor growth and metastasis are urgently needed to improve outcomes for patients with malignant gliomas [18]. Schisandrin B is the most abundant dibenzocyclooctadiene derivative in extracted from the fruit of Schisandra chinensis Baill
* Corresponding author. E-mail address: [email protected] (C. Du). http://dx.doi.org/10.1016/j.biopha.2015.07.006 0753-3322/ß 2015 Published by Elsevier Masson SAS.
and has been used in traditional Chinese medicine to treat physiological functions disorders, especially improve cardiac [18] and injured liver [10] as well as protects neural cell from oxidative stress [8]. In addition, Schisandrin B has been reported to possess antitumor activity in various human cancers. For example, Schisandrin B induces apoptosis and cell cycle arrest of gallbladder cancer cells [20]. Schisandrin B attenuates cancer invasion and metastasis via inhibiting epithelial–mesenchymal transition [12]. Schisandrin B has been also showed to suppressed glioma proliferation [11]. However, to our present knowledge, it’s not clear whether Schisandrin B affects glioma cells migration and invasion. Human tumor development is a very complex process and the pathological process is high regulated by signal transduction [7]. PI3K/Akt/mTOR signaling, one of the three major signaling pathway that have been identified as important in cancer, is highly active in glioma and plays a pivotal role in cell survival, proliferation, and angiogenesis, and is frequently deregulated in human cancer [14]. Notably, various antitumor substances, such as PCI-24781 and progesterone, have been proved to against glioma via inactivating PI3K/Akt/mTOR signal [3,23].
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In this study, we were to investigate the anti-invasion action of Schisandrin B in glioma cells (including U251 and U87 cell lines) as well as attempt explore the underlying mechanism. Here, we show that Schisandrin B inactivated the PI3K/Akt-mTOR signaling pathway that suppresses the expression of the matrix metalloproteinase-9 (MMP-9) which involved in the degradation and breakdown of the environmental extracellular matrix (ECM) and the basement membrane, promoting cancer metastasis. 2. Material and methods 2.1. Reagents and antibodies Schisandrin B and Rapamycin, an inhibitor for mTOR were purchased from Sigma–Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO, Sigma–Aldrich, St. Louis, MO, USA). The concentrations of stock solution of Schisandrin B were 40 M, 80 M and 160 M and that of Rapamycin is 20 mM, which were then diluted in the culture medium to obtain the desired dose. An equal volume of DMSO in complete culture medium was used as the vehicle control. To eliminate the cytotoxicity of DMSO, the final concentration of DMSO for all experiments was maintained at less than 0.1%. PI3K activator peptide 740Y-P (500 mg/mL; Tocris Bioscience, Ellsville, MO, USA) and mTOR activator L-Leucine (5 mM; Calbiochem, CA, USA) were dissolved in phosphatebuffered saline (PBS, pH 7.4). An equal volume of PBS in complete culture medium was used as the vehicle control. Antibodies for Western blotting is as followed: antibodies against phospho-mTOR (ser2448), phospho-Akt (ser473), mTOR and Akt were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against MMP-9 and MMP-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and antibody against a-tubulin was purchased form Abcam (Cambridge, MA, USA).
2.4. Cell migration assays Analysis of cell migration was performed using 24-well transwell plates (8.0-mm pore membranes; Costar, UK). About 1 104 cells which have been treated with Schisandrin B and/or L-Leucine were loaded into the upper chambers of transwell. Accordingly, the lower chambers were filled with medium (plus 1% FBS). The transwell plates were then incubated at condition of 37 8C, 5% CO2 for 48 h. The cells were harvest in the upper side of polycarbonate membrane and washed by PBS buffer. Cells in polycarbonate membrane were received hematoxylin–eosin staining and was cut, placed on a microscope slide with covered by cover slide. The samples were examined under the microscope and photographed. The total migrated cell number and percentage were then counted. 2.5. Matrigel invasion assay Analysis of Matrigel cell invasion was performed using the BD Biocoat Matrigel Invasion Chamber (pore size: 8 mm, 24-well; BD Biosciences, San Jose, CA, USA). Cells (5 104) were plated in the upper chamber of transwell in serum-free medium containing 1 mg/mL Matrigel. The bottom chamber contained medium with 10% FBS. The transwell plates were then incubated at condition of 37 8C, 5% CO2 for 48 h. The bottom of the chamber insert was stained with Calcein AM (Invitrogen, Carlsbad, CA, USA). The cells that had invaded through the membrane to the lower surface were evaluated in a fluorescence plate reader at excitation/emission. 2.6. Statistical analysis The results are expressed as mean SD. A Student’s t-test was used to compare individual data with control value or vehicle control using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). A probability of P < 0.05 was considered to be significantly different from control data.
2.2. Cell lines and culture 3. Results The human glioma cell lines, U251 and U87, were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (CAS, Shanghai, China) and were incubated in high-glucose Dulbecco’s Modified Eagle Medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA), and 100 U/mL penicillin (HyClone, USA). The cells were cultured in condition of atmosphere containing 5% CO2 at 37 8C. 2.3. Western blotting Western blot analysis was performed to determine protein expression. Cells were collected after various treatments and lysed in lysis buffer containing protease inhibitors from Calbiochem (CA, USA). Soluble proteins were obtained by centrifugation at 15,000 rpm for 5 min at 4 8C and protein concentration was determined using BCA kit following instruction. Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were then electrophoretic transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocked by 5% T skim milk powder (diluted in TBS), the target protein were blotted via membrane incubated with primary antibodies (the dilution rate are as follows: p-mTOR, p-Akt (ser473), mTOR and Akt-1:1000; MMP-9 and MMP-2,1:500; a-tubulin, 1:1000). The blotted proteins were labeled by peroxidase-conjugated secondary antibodies (1:2000, Sigma Co., USA). The target proteins were visualized using SuperSignal West Pico chemiluminescence substrate system (Pierce). The band intensity was analyzed using Scion image software (Frederick, MD).
3.1. Schisandrin B suppresses glioma cell invasion To evaluate the effect of Schisandrin B on cell invasion, U251 and U78 were incubated with doses of Schisandrin B (40, 80, and 160 mmol/L) with DMSO as control. As shown in Fig. 1A, Schisandrin B suppressed cell migration in a manner of dose dependent and significantly effective inhibited doses are 80 and 160 mmol/L. In result of invasion assay, we observed that Schisandrin B also significantly resulted in reduction of number of invasion cells (Fig. 1B). 3.2. Profiles of signal molecules To determine the effect of Schisandrin B on expressions of signal molecules, U251 and U78 cells were incubated with 40, 80 and 160 mmol/L respectively followed by Western blotting assay. As shown in Fig. 2, Schisandrin B treatment attenuated expression of p-Akt, p-mTOR and MMP-9 in dose manner while has no effect on levels of total Akt, total mTOR and MMP-2. 3.3. Activation of PI3K reverses action of Schisandrin B To investigate the mechanism underlying Schisandrin B induced inhibition of cell invasion, we firstly examined whether PI3K/Akt/mTOR is involved in Schisandrin B incubated U251 and U87 cell. PI3K/signal was activated by cells treated with cellpermeable phospho-peptide (740Y-P) capable of binding to the SH2 domain of regulatory subunit of PI3K to stimulate enzymeactivity with PBS as control. This activated peptide treatment
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Fig. 1. Effect of Schisandrin B on migration and invasion of glioma cell. Glioma cells of U251 and U78 lines were incubated with 40, 80 and 160 mmol/L Schisandrin B for 24 h with DMSO as control. Ability of (A) cell migration and (B) cell invasion was determined. Data were presented as mean SD. *P < 0.05 compares with DMSO.
reversed effect of Schisandrin B and leaded to upregulation of pmTOR and MMP-9 (Fig. 3A). While activation of PI3K/Akt induced increase of MMP-9 expression was abrogated by mTOR inhibition by Rapamycin treatment (Fig. 3B).
for 24 h. The cells were harvest for expression analysis of MMP-9. As shown Fig. 4, activation of mTOR promoted MMP-9 expression compared with negative control group of treatment with Schisandrin B and PBS.
3.4. Activation of mTOR abrogated Schisandrin B induced downregulation of MMP-9
3.5. Activation of mTOR enhanced anti-glioma of Schisandrin B
Next, we evaluated the action of mTOR in Schisandrin B incubated U251 and U87 cells. Glioma cells were treated with L-Leucine, an agonist for mTOR, plus Schisandrin B was treated
To test the role of mTOR in anti-glioma of Schisandrin B, mTOR was activated by L-Leucine treatment before U251 and U87 cells incubated with Schisandrin B followed by cell invasion analysis. As shown in Fig. 5A, the number of migrated cell in Schisandrin
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Fig. 4. Activation of mTOR enhanced MMP-9 expression in Schisandrin B incubated U251 and U87 cells. Cells were pre-incubated with 5 mmol/L L-Leucine, an agonist for mTOR, for 2 h with PBS as control before 80 mmol/L Schisandrin B treatment. MMP-9 expression was presented by using Western blotting.
Fig. 2. Expression of signal molecules in Schisandrin B incubated glioma cells. Cells were treated with 40, 80 and 160 mmol/L Schisandrin B for 24 h and expression of Akt, mTOR, MMP-2 and MMP-9 was determined using Western blotting. p-Akt, phosphorylated Akt; T-Akt, total Akt; p-mTOR, phosphorylated mTOR; T-mTOR, Total mTOR.
B + L-Leucine group is greater than that in Schisandrin B group (Fig. 5A). There is the same trend of variability in result of cell invasion analysis in which activation of mTOR significantly reversed anti-cell invasion action of Schisandrin B (Fig. 5B).
4. Discussion As relapse in glioma patients largely caused by cancer cell metastasis, to reduce metastasis is a major strategy to combat cancer. A far-reaching proposal pointed out that ‘given the fact that
Fig. 3. Activation of PI3K enhanced expression of p-mTOR and MMP-9 in Schisandrin B incubated U251 and U87 cells. (A) Cells were pretreated with 500 mg/mL 740Y-P, an agonist for PI3K, for 2 h and then incubated with 80 mmol/L Schisandrin B for 24 h; levels of p-mTOR and MMP-9 were examined. (B) Cells were pretreated with 20 nM Rapamycin, an inhibitor for mTOR, for 2 h with DMSO as control and incubated with 740Y-P for 2 h; MMP-9 expression was determined.
primary tumors can often be controlled using conventional therapies, could agents that act specifically on the process of metastasis be more likely to increase long term patient survival’ [11]. Cancer cell metastasis is related primarily to cell migratory ability and invasiveness. In the present study, we demonstrated that Schisandrin B significantly suppressed invasion and migration of U251 and U87 glioma cell. Moreover, we found the potential mechanisms might be associated with inactivation of PI3K/Akt/ mTOR signaling and expressive suppression of MMP-9. These findings suggested that Schisandrin B could be an effective and therapeutic drug against progression of malignant glioma. In vitro U251 and U87 glioma cell culture, Schisandrin B was effective in inducing cell migration and invasion in a trend of dose dependent. This finding provides an important piece of information regarding one of the possible biological activities of Schisandrin B that suppressed glioma metastasis. Here we propose that the involvement of the glioma activity related signal pathway, PI3K/Akt/mTOR, could be an important mechanism in mediating the effects of Schisandrin B in view of genetic comparison of Schisandrin B incubated glioma cells and control cells, in which we observed that Schisandrin B treatment inactivated Akt and mTOR as well as induced expressive inhibition of MMP-9. Common alterations in primary glioma include phosphatase and phosphatidylinositol-3 kinases (PI3K), and seen in approximately 30% of GBMs each [15]. In cellular signaling transmission, PI3K phosphorylates phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) in order to further recruit Akt to the membrane where Akt can be phosphorylated [21]. One of the major downstream signaling targets of AKT is mTOR, a critical effector of cell signaling pathways generally deregulated in a variety of cancers including glioma [2,17]. Our present data showed that invoked PI3K by cellpermeable phospho-peptide (740Y-P) leaded to increase of mTOR activation in Schisandrin B incubated U251 and U87 cells. Also, mTOR by L-Leucine reversed Schisandrin B’s anti-cell metastasis action, and significantly increased number of cell migrated and invasive cell. Our finding suggested the involvement of PI3K/Akt/ mTOR cascade was in Schisandrin B’s anti-metastasis and were consistent with previous researches that pointed out that resultant deregulation of the Akt/mammalian target of mTOR signaling pathway from these alterations has shown to be an important determinant of gliomagenesis [17,22]. The change in adhesive capability between tumor cells and extracellular matrix is one key factor in cancer cell metastasis. Thus, the degradation of the extracellular matrix mediated by proteases, such as matrix metallo proteinases (MMPs) allows glioma migration [13]. Matrix metalloproteinase-9 (MMP-9) is highly expressed in human GBM tissue [4,24] and was reported to be associated with slightly shorter overall survival in glioblastoma patients [5]. While cellular level of MMP-9 is highly regulated by mTOR [9]. The directive evidence for PI3K/Akt/mTOR regulating
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Fig. 5. Activation of mTOR enhanced cell migration and invasion in Schisandrin B incubated U251 and U87 cell. Cells were pre-incubated with 5 mmol/L L-Leucine before Schisandrin B treatment. Ability of (A) cell migration and (B) cell invasion was examined. Data were presented as mean SD. *P < 0.05 compared with corresponding control.
MMP-9 is activation or inactivation of mTOR can alter MMP-9 expression in Schisandrin B incubated glioma cells in present study. Overall, our findings demonstrate a possible role of PI3K/ Akt/mTOR signaling associated downregulation of MMP-9 in antitumor effects of in Schisandrin B incubated glioma cell. In summary, our present study for the first time demonstrated that Schisandrin B effectively inhibited migration and invasion of human glioma cell via inactivating a PI3K/Akt/mTOR cascade pathway, which leads to expressive inhibition of MMP-9. This
observation may provide a potential candidate for development of preventive agents against glioma metastasis and invasion in the future.
Competing interests The authors have no actual or potential conflicts of interest to declare.
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Available online at
ScienceDirect www.sciencedirect.com
Original Article
Schisandrin B suppresses glioma cell metastasis mediated by inhibition of mTOR/MMP-9 signal pathway Yan Jiang, Qiuli Zhang, Jinsuo Bao, Chenghua Du *, Jian Wang, Qiang Tong, Chang Liu Department of Neurosurgery, The Affiliated Hospital of Inner Mongolia National University, Tongliao, Inner Mongolia 028007, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 June 2015 Accepted 9 July 2015
Background: Malignant glioma is the aggressive tumor in the brain and is characterized by high morbidity, high mortality. The main purpose of the present study was to investigate the therapeutic effects of Schisandrin B on glioma cells and preliminary explore the possible mechanism underlying antimetastasis of Schisandrin B. Methods: Two glioma cell lines, U251 and U87, were used in present study. The ability of metastasis of glioma cells was evaluated using transwell migration assay and invasion assay. Expression of Akt, mTOR, MMP-2 and MMP-9 was determined using Western blotting. Antagonist or agonist was used to activated or inactivated signal molecules. Results: Schisandrin B suppressed cell migration and invasion in manner of dose dependent as well as inhibited expression of p-Akt, p-mTOR and MMP-9. Activation of PI3K by 740Y-P treatment leaded to upregulation of p-Akt, mTOR and MMP-9; inactivation of mTOR by Rapamycin treatment inhibited expression MMP-9 while activation of mTOR by L-Leucine treatment enhanced MMP-9 expression in Schisandrin B incubated cells. Anti-migration and invasion action of Schisandrin B was also reversed by mTOR activation. Conclusion: Our findings demonstrate that Schisandrin B can suppress migration and invasion of glioma cell via PI3K/Akt-mTOR-MMP-9 signaling pathway. ß 2015 Published by Elsevier Masson SAS.
Keywords: Akt mTOR Cell invasion Cell migration PI3K
1. Introduction Glioma is one of the most common malignant central nervous system tumors in human and is characterized of morbidity, disability, mortality and high recurrence rate [1]. Tumor recurrence usually recurs within 6–9 months, resulting in a median patient survival of just 13–16 months following diagnosis [16]. Clinical treatment for glioma focuses on surgery with a combination of radiotherapy and high fat soluble chemotherapy drugs. However, surgery is unable to excise all malignant glioma cells because of its infiltration to adjacent brain parenchyma and also easily cause complications [6,19]. The residual tumors can behave in a very aggressive and malignant fashion. Hence, a new optimal pharmacotherapy drugs that target multiple mechanisms of tumor growth and metastasis are urgently needed to improve outcomes for patients with malignant gliomas [18]. Schisandrin B is the most abundant dibenzocyclooctadiene derivative in extracted from the fruit of Schisandra chinensis Baill
* Corresponding author. E-mail address: [email protected] (C. Du). http://dx.doi.org/10.1016/j.biopha.2015.07.006 0753-3322/ß 2015 Published by Elsevier Masson SAS.
and has been used in traditional Chinese medicine to treat physiological functions disorders, especially improve cardiac [18] and injured liver [10] as well as protects neural cell from oxidative stress [8]. In addition, Schisandrin B has been reported to possess antitumor activity in various human cancers. For example, Schisandrin B induces apoptosis and cell cycle arrest of gallbladder cancer cells [20]. Schisandrin B attenuates cancer invasion and metastasis via inhibiting epithelial–mesenchymal transition [12]. Schisandrin B has been also showed to suppressed glioma proliferation [11]. However, to our present knowledge, it’s not clear whether Schisandrin B affects glioma cells migration and invasion. Human tumor development is a very complex process and the pathological process is high regulated by signal transduction [7]. PI3K/Akt/mTOR signaling, one of the three major signaling pathway that have been identified as important in cancer, is highly active in glioma and plays a pivotal role in cell survival, proliferation, and angiogenesis, and is frequently deregulated in human cancer [14]. Notably, various antitumor substances, such as PCI-24781 and progesterone, have been proved to against glioma via inactivating PI3K/Akt/mTOR signal [3,23].
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Y. Jiang et al. / Biomedicine & Pharmacotherapy 74 (2015) 77–82
In this study, we were to investigate the anti-invasion action of Schisandrin B in glioma cells (including U251 and U87 cell lines) as well as attempt explore the underlying mechanism. Here, we show that Schisandrin B inactivated the PI3K/Akt-mTOR signaling pathway that suppresses the expression of the matrix metalloproteinase-9 (MMP-9) which involved in the degradation and breakdown of the environmental extracellular matrix (ECM) and the basement membrane, promoting cancer metastasis. 2. Material and methods 2.1. Reagents and antibodies Schisandrin B and Rapamycin, an inhibitor for mTOR were purchased from Sigma–Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO, Sigma–Aldrich, St. Louis, MO, USA). The concentrations of stock solution of Schisandrin B were 40 M, 80 M and 160 M and that of Rapamycin is 20 mM, which were then diluted in the culture medium to obtain the desired dose. An equal volume of DMSO in complete culture medium was used as the vehicle control. To eliminate the cytotoxicity of DMSO, the final concentration of DMSO for all experiments was maintained at less than 0.1%. PI3K activator peptide 740Y-P (500 mg/mL; Tocris Bioscience, Ellsville, MO, USA) and mTOR activator L-Leucine (5 mM; Calbiochem, CA, USA) were dissolved in phosphatebuffered saline (PBS, pH 7.4). An equal volume of PBS in complete culture medium was used as the vehicle control. Antibodies for Western blotting is as followed: antibodies against phospho-mTOR (ser2448), phospho-Akt (ser473), mTOR and Akt were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against MMP-9 and MMP-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and antibody against a-tubulin was purchased form Abcam (Cambridge, MA, USA).
2.4. Cell migration assays Analysis of cell migration was performed using 24-well transwell plates (8.0-mm pore membranes; Costar, UK). About 1 104 cells which have been treated with Schisandrin B and/or L-Leucine were loaded into the upper chambers of transwell. Accordingly, the lower chambers were filled with medium (plus 1% FBS). The transwell plates were then incubated at condition of 37 8C, 5% CO2 for 48 h. The cells were harvest in the upper side of polycarbonate membrane and washed by PBS buffer. Cells in polycarbonate membrane were received hematoxylin–eosin staining and was cut, placed on a microscope slide with covered by cover slide. The samples were examined under the microscope and photographed. The total migrated cell number and percentage were then counted. 2.5. Matrigel invasion assay Analysis of Matrigel cell invasion was performed using the BD Biocoat Matrigel Invasion Chamber (pore size: 8 mm, 24-well; BD Biosciences, San Jose, CA, USA). Cells (5 104) were plated in the upper chamber of transwell in serum-free medium containing 1 mg/mL Matrigel. The bottom chamber contained medium with 10% FBS. The transwell plates were then incubated at condition of 37 8C, 5% CO2 for 48 h. The bottom of the chamber insert was stained with Calcein AM (Invitrogen, Carlsbad, CA, USA). The cells that had invaded through the membrane to the lower surface were evaluated in a fluorescence plate reader at excitation/emission. 2.6. Statistical analysis The results are expressed as mean SD. A Student’s t-test was used to compare individual data with control value or vehicle control using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). A probability of P < 0.05 was considered to be significantly different from control data.
2.2. Cell lines and culture 3. Results The human glioma cell lines, U251 and U87, were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (CAS, Shanghai, China) and were incubated in high-glucose Dulbecco’s Modified Eagle Medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA), and 100 U/mL penicillin (HyClone, USA). The cells were cultured in condition of atmosphere containing 5% CO2 at 37 8C. 2.3. Western blotting Western blot analysis was performed to determine protein expression. Cells were collected after various treatments and lysed in lysis buffer containing protease inhibitors from Calbiochem (CA, USA). Soluble proteins were obtained by centrifugation at 15,000 rpm for 5 min at 4 8C and protein concentration was determined using BCA kit following instruction. Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were then electrophoretic transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocked by 5% T skim milk powder (diluted in TBS), the target protein were blotted via membrane incubated with primary antibodies (the dilution rate are as follows: p-mTOR, p-Akt (ser473), mTOR and Akt-1:1000; MMP-9 and MMP-2,1:500; a-tubulin, 1:1000). The blotted proteins were labeled by peroxidase-conjugated secondary antibodies (1:2000, Sigma Co., USA). The target proteins were visualized using SuperSignal West Pico chemiluminescence substrate system (Pierce). The band intensity was analyzed using Scion image software (Frederick, MD).
3.1. Schisandrin B suppresses glioma cell invasion To evaluate the effect of Schisandrin B on cell invasion, U251 and U78 were incubated with doses of Schisandrin B (40, 80, and 160 mmol/L) with DMSO as control. As shown in Fig. 1A, Schisandrin B suppressed cell migration in a manner of dose dependent and significantly effective inhibited doses are 80 and 160 mmol/L. In result of invasion assay, we observed that Schisandrin B also significantly resulted in reduction of number of invasion cells (Fig. 1B). 3.2. Profiles of signal molecules To determine the effect of Schisandrin B on expressions of signal molecules, U251 and U78 cells were incubated with 40, 80 and 160 mmol/L respectively followed by Western blotting assay. As shown in Fig. 2, Schisandrin B treatment attenuated expression of p-Akt, p-mTOR and MMP-9 in dose manner while has no effect on levels of total Akt, total mTOR and MMP-2. 3.3. Activation of PI3K reverses action of Schisandrin B To investigate the mechanism underlying Schisandrin B induced inhibition of cell invasion, we firstly examined whether PI3K/Akt/mTOR is involved in Schisandrin B incubated U251 and U87 cell. PI3K/signal was activated by cells treated with cellpermeable phospho-peptide (740Y-P) capable of binding to the SH2 domain of regulatory subunit of PI3K to stimulate enzymeactivity with PBS as control. This activated peptide treatment
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Fig. 1. Effect of Schisandrin B on migration and invasion of glioma cell. Glioma cells of U251 and U78 lines were incubated with 40, 80 and 160 mmol/L Schisandrin B for 24 h with DMSO as control. Ability of (A) cell migration and (B) cell invasion was determined. Data were presented as mean SD. *P < 0.05 compares with DMSO.
reversed effect of Schisandrin B and leaded to upregulation of pmTOR and MMP-9 (Fig. 3A). While activation of PI3K/Akt induced increase of MMP-9 expression was abrogated by mTOR inhibition by Rapamycin treatment (Fig. 3B).
for 24 h. The cells were harvest for expression analysis of MMP-9. As shown Fig. 4, activation of mTOR promoted MMP-9 expression compared with negative control group of treatment with Schisandrin B and PBS.
3.4. Activation of mTOR abrogated Schisandrin B induced downregulation of MMP-9
3.5. Activation of mTOR enhanced anti-glioma of Schisandrin B
Next, we evaluated the action of mTOR in Schisandrin B incubated U251 and U87 cells. Glioma cells were treated with L-Leucine, an agonist for mTOR, plus Schisandrin B was treated
To test the role of mTOR in anti-glioma of Schisandrin B, mTOR was activated by L-Leucine treatment before U251 and U87 cells incubated with Schisandrin B followed by cell invasion analysis. As shown in Fig. 5A, the number of migrated cell in Schisandrin
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Fig. 4. Activation of mTOR enhanced MMP-9 expression in Schisandrin B incubated U251 and U87 cells. Cells were pre-incubated with 5 mmol/L L-Leucine, an agonist for mTOR, for 2 h with PBS as control before 80 mmol/L Schisandrin B treatment. MMP-9 expression was presented by using Western blotting.
Fig. 2. Expression of signal molecules in Schisandrin B incubated glioma cells. Cells were treated with 40, 80 and 160 mmol/L Schisandrin B for 24 h and expression of Akt, mTOR, MMP-2 and MMP-9 was determined using Western blotting. p-Akt, phosphorylated Akt; T-Akt, total Akt; p-mTOR, phosphorylated mTOR; T-mTOR, Total mTOR.
B + L-Leucine group is greater than that in Schisandrin B group (Fig. 5A). There is the same trend of variability in result of cell invasion analysis in which activation of mTOR significantly reversed anti-cell invasion action of Schisandrin B (Fig. 5B).
4. Discussion As relapse in glioma patients largely caused by cancer cell metastasis, to reduce metastasis is a major strategy to combat cancer. A far-reaching proposal pointed out that ‘given the fact that
Fig. 3. Activation of PI3K enhanced expression of p-mTOR and MMP-9 in Schisandrin B incubated U251 and U87 cells. (A) Cells were pretreated with 500 mg/mL 740Y-P, an agonist for PI3K, for 2 h and then incubated with 80 mmol/L Schisandrin B for 24 h; levels of p-mTOR and MMP-9 were examined. (B) Cells were pretreated with 20 nM Rapamycin, an inhibitor for mTOR, for 2 h with DMSO as control and incubated with 740Y-P for 2 h; MMP-9 expression was determined.
primary tumors can often be controlled using conventional therapies, could agents that act specifically on the process of metastasis be more likely to increase long term patient survival’ [11]. Cancer cell metastasis is related primarily to cell migratory ability and invasiveness. In the present study, we demonstrated that Schisandrin B significantly suppressed invasion and migration of U251 and U87 glioma cell. Moreover, we found the potential mechanisms might be associated with inactivation of PI3K/Akt/ mTOR signaling and expressive suppression of MMP-9. These findings suggested that Schisandrin B could be an effective and therapeutic drug against progression of malignant glioma. In vitro U251 and U87 glioma cell culture, Schisandrin B was effective in inducing cell migration and invasion in a trend of dose dependent. This finding provides an important piece of information regarding one of the possible biological activities of Schisandrin B that suppressed glioma metastasis. Here we propose that the involvement of the glioma activity related signal pathway, PI3K/Akt/mTOR, could be an important mechanism in mediating the effects of Schisandrin B in view of genetic comparison of Schisandrin B incubated glioma cells and control cells, in which we observed that Schisandrin B treatment inactivated Akt and mTOR as well as induced expressive inhibition of MMP-9. Common alterations in primary glioma include phosphatase and phosphatidylinositol-3 kinases (PI3K), and seen in approximately 30% of GBMs each [15]. In cellular signaling transmission, PI3K phosphorylates phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) in order to further recruit Akt to the membrane where Akt can be phosphorylated [21]. One of the major downstream signaling targets of AKT is mTOR, a critical effector of cell signaling pathways generally deregulated in a variety of cancers including glioma [2,17]. Our present data showed that invoked PI3K by cellpermeable phospho-peptide (740Y-P) leaded to increase of mTOR activation in Schisandrin B incubated U251 and U87 cells. Also, mTOR by L-Leucine reversed Schisandrin B’s anti-cell metastasis action, and significantly increased number of cell migrated and invasive cell. Our finding suggested the involvement of PI3K/Akt/ mTOR cascade was in Schisandrin B’s anti-metastasis and were consistent with previous researches that pointed out that resultant deregulation of the Akt/mammalian target of mTOR signaling pathway from these alterations has shown to be an important determinant of gliomagenesis [17,22]. The change in adhesive capability between tumor cells and extracellular matrix is one key factor in cancer cell metastasis. Thus, the degradation of the extracellular matrix mediated by proteases, such as matrix metallo proteinases (MMPs) allows glioma migration [13]. Matrix metalloproteinase-9 (MMP-9) is highly expressed in human GBM tissue [4,24] and was reported to be associated with slightly shorter overall survival in glioblastoma patients [5]. While cellular level of MMP-9 is highly regulated by mTOR [9]. The directive evidence for PI3K/Akt/mTOR regulating
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Fig. 5. Activation of mTOR enhanced cell migration and invasion in Schisandrin B incubated U251 and U87 cell. Cells were pre-incubated with 5 mmol/L L-Leucine before Schisandrin B treatment. Ability of (A) cell migration and (B) cell invasion was examined. Data were presented as mean SD. *P < 0.05 compared with corresponding control.
MMP-9 is activation or inactivation of mTOR can alter MMP-9 expression in Schisandrin B incubated glioma cells in present study. Overall, our findings demonstrate a possible role of PI3K/ Akt/mTOR signaling associated downregulation of MMP-9 in antitumor effects of in Schisandrin B incubated glioma cell. In summary, our present study for the first time demonstrated that Schisandrin B effectively inhibited migration and invasion of human glioma cell via inactivating a PI3K/Akt/mTOR cascade pathway, which leads to expressive inhibition of MMP-9. This
observation may provide a potential candidate for development of preventive agents against glioma metastasis and invasion in the future.
Competing interests The authors have no actual or potential conflicts of interest to declare.
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