Glioma tissue specimens
All of the primary human glioma specimens included grade II (12 samples), grade III (12 samples) and grade IV (9 samples) were collected from the Department of Neurosurgery, the Affiliated Hospital of Xuzhou Medical University ranged from September 2016 to September 2019. Besides, no patients underwent antitumor therapies including radiotherapy and chemotherapy before the operation. In addition, 9 cases of normal brain tissue specimens were selected as control. These fresh samples were harvested and immediately frozen in liquid nitrogen. All the glioma samples were histologically diagnosed by three experienced clinical neuropathologists according to the updated WHO Classification of Tumors of the Nervous System in 2016. The histology and clinical data of the glioma samples was shown in Table 1. This study was written informed consent obtained from all patients and approved by the Medical Ethics and Human Clinical Trial Committee of the Affiliated Hospital of Xuzhou Medical University.
Cell lines and reagents
The glioma cell lines U87, U251 and human embryonic kidney cell line HEK293T were purchased from the Shanghai Cell Bank, Type Culture Collection Committee, Chinese Academy of Sciences. All cell lines were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both purchased from Hyclone (Logan, UT, USA)) and supplemented with penicillin (100 mg/L) and streptomycin (100 mg/L, AASIG01-100, Sigma-Aldrich Chemical Company, St Louis, MO, USA) in a condition of 37 °C, 5% CO2 and saturated humidity conditions. Dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). PolyJet™ was obtained from SignaGen (Gaithersburg, MD, USA). A potent and selective PAK1 inhibitor IPA-3 was bought from MedChem Express (Monmouth Junction, NJ). A stock solution of IPA-3 was prepared in DMSO, then further diluted with DMEM to yield a final stock concentration of 10 μM.
Establishment of PAK1 gene silencing model
According to the complementary DNA (cDNA) sequence of PAK1 gene in GenBank, PAK1 shRNAs were designed by Invitrogen RNAi Designer. Three pairs of specific shRNAs including shPAK1-1, shPAK1-2, shPAK1-3 and a control oligonucleotides were designed as followings:
shPAK1-1 Forward: 5’-GATCGCTTCAGGCACAGTGTATACTTTCAAGAGAAGTATACACTGTGCCTGAAGCTTTTTTG-3’
shPAK1-1 Reverse: 5’-AATTCAAAAAAGCTTCAGGCACAGTGTATACTTCTCTTGAAAGTATACACTGTGCCTGAAGC-3’
shPAK1-2 Forward: 5’-GATCGGGTTGTTATGGAATACTTGGTTCAAGAGACCAAGTATTCCATAACAACCCTTTTTTG-3’
shPAK1-2 Reverse: 5’-AATTCAAAAAAGGGTTGTTATGGAATACTTGGTCTCTTGAACCAAGTATTCCATAACAACCC-3’
shPAK1-3 Forward: 5’-GATCGCCTGAACACCTCATTGTATTTTCAAGAGAAATACAATGAGGTGTTCAGGCTTTTTTG-3’
shPAK1-3 Reverse: 5’-AATTCAAAAAAGCCTGAACACCTCATTGTATTTCTCTTGAAAATACAATGAGGTGTTCAGGC-3’
Control Forward: 5’-GATCTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTG-3’
Control Reverse: 5’-AATTCAAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAA-3’.
The oligonucleotide (100 µM) were annealed and diluted with sterile water 100 times for following use. Subsequently, the annealed oligomers were subcloned into the pLV-shRNA-EGFP(2A)Puro vector by means of both EcoR I and BamH I cloning sites. The recombinant plasmids were confirmed whether the sequence of shRNA was inserted into the plasmid vector by agarose gel electrophoresis and DNA sequence analysis. The lentiviruses were packaged by three-plasmid-based expression system, namely co-transfecting the core plasmid and the packaging plasmids (psPAX2 and pMD2.G plasmid) in HEK293T cells, which was carried out by PolyJet (SignaGen, Gaithersburg, MD, USA) according to the manufacturer's instructions. The lentiviruses were collected and used for glioma cell infection. 48 h after infection, the lentivirus-infected cells were cultured in the medium containing 2.5 μg/mL puromycin selection to establish stable shPAK1 cell lines (Sigma, St. Louis, MO, USA).
Cell viability assay
For the experiment of IPA-3 treatment, 8*103 cells/mL U87 and U251 cells were treated with DMSO vehicle and the different concentrations of IPA-3 ranged from 0 to 25 μM for 24 h and then subjected to CCK-8 assay to determine the optimal efficiency concentration of IPA-3 on cell proliferation. We found that U87 and U251 cells displayed more sensitive to 25 μM of the half maximal inhibitory concentrations (IC50) value treated with IPA-3 for 24 h. Thus, the IPA-3 at a concentration of 25 μM was adopted for the subsequent experiments. For the experiment of PAK1 shRNA treatment, approximately 3*103 cells/mL U87 and U251 cells after infection PAK1 shRNA were plated in 96-well plate per well, each group cells were repeated in four wells, adhered for 20 h later, then removed the supernatants, at the indicated time (0, 24, 48, 72, 96 and 120 h), medium mixed with 10% Cell Counting Kit-8 (CCK-8) reagent (VICMED Life sciences, Xuzhou, China) was added to each well and incubated at 37 °C for 2 h, the absorbance was measured at 450 nm by a SynergyMx Multi-Mode Microplate Reader (Biotek, Winooski, VT). All experiments were performed in triplicate independently.
EdU incorporation assay
This assay aimed to detect cell proliferation using Cell-Light™ EdU Cell Proliferation Detection Kit (Ruibo Biotech, Guangzhou, China) according to the manufacturer’s instructions. For the experiment treated with IPA-3, 8*103 cells/mL U87 and U251 cells were placed in 96‐well plate per well for 20 h, then treated with DMSO vehicle and IPA-3, respectively. Then treated with 50 μM 5-ethynyl-20-deoxyuridine (EdU) in 37 °C for 2 h. Afterwards, the cells were fixed by 4% paraformaldehydrate for 30 min and treated with 0.5% Triton X-100 for 10 min, separately. Thereafter, the cells were incubated with 1×Apollo® reaction solution for 30 min and stained with 1X Hoechst33342 reaction cocktail for 30 min in the dark, respectively. Finally, the cells were washed by phosphate buffered saline (PBS) and observed with Olympus IX-71 inverted microscope (Olympus, Japan). The percentage of EdU-positive cells was calculated by the ratio of EdU-positive cells to total Hoechst-stained cells. For the experiment treated with PAK1 shRNA, a total of 8 *103 cells/mL U87 and U251 cells after infection PAK1 shRNA were seeded into 96-well plate, the following disposals were similar to the above descriptions. All experiments were performed in triplicate independently.
Colony formation assay
For the experiment of IPA-3 treatment, 1*103 cells/mL U87 and U251 cells after treatment with or without IPA-3 per well were seeded in 6-well plate, each group cells were repeated in three wells, then placed in 37 °C, 5% CO2 incubator, after 14 days, the plates were washed by PBS, then fixed with 4% paraformaldehyde for 30 min, finally stained with 0.3% crystal violet diluted in methanol for 15 min, and visualized immediately on an Olympus IX-71 inverted microscope. Colonies containing more than 50 cells were counted. For the experiment of PAK1 shRNA treatment. A total of 1*103 cells/mL U87 and U251 cells after infection PAK1 shRNA were seeded in 6-well plate, each group cells were repeated in three wells, the following disposals were conducted as previously described. The data was analyzed by the Image J software (National Institutes of Health, Bethesda, MD). All experiments were performed in triplicate independently.
Cell cycle and cell apoptosis detection
For the cell cycle experiment, this assay aimed to detect cell cycle distribution using kFlour647 Click-iT EdU Flow Detection Kit (KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s instructions. Cell fixation and permeabilization using the eBioscienceTM Intracellular Fixation &Permeabilization Buffer Set (Invitrogen). 2*105 cells/mL U87 and U251 cells which were pre-treated with or without IPA-3 were seeded into 6-well plate for 20 h, once attached to the plate, treated with 10 μM EdU in 37 °C for 2 h. Afterwards, cells were harvested and resuspended in 1 ml Fixation Buffer and incubated the at room temperature for 15 minutes in the dark. Afterwards, the cells were resuspended with 1×Permeabilization Buffer and incubated for 20 min. Next, added Click-iT reaction working mixture to each tube and incubated for 30 minutes at room temperature. Removed the supernatant and resuspended in 1 mL 1×Permeabilization Buffer containing 5 μg/mL 4,6-diamino-2-phenyl indole (DAPI) (Sigma, St. Louis, MO, USA) to stain the cell nucleus in the dark for 15 min. Finally, the cell suspension was filtered by a 50-mm nylon mesh and immediately analyzed by flow cytometry (BD, Franklin Lakes, NJ, USA). Cell cycle analysis was performed by flowjo 7.6 software. For the experiment of PAK1 shRNA treatment, 2*105 cells/mL U87 and U251 cells pre-transfected PAK1 shRNA were seeded into 6-well plate, the following disposals was conducted as the above protocol.
For cell apoptosis assay, 2*105 cells/mL U87 and U251 cells pre-treated with or without IPA-3 were performed using PE Annexin V Apoptosis Detection Kit I (BD, Biosciences, USA). The cells were harvested and resuspended in cold PBS, then centrifuged and discarded the supernatants thoroughly. Subsequently, resuspended cells in 1×Binding Buffer, transfered 100 µL of the solution to a 5 ml culture tube, and added 5 µL phycoerythrin (PE) Annexin V and 5 µL 7-amino-actinomycin-D (7-AAD), respectively. Vortexed the cells and incubated for 15 min at room temperature (RT) in the dark. Eventually, added 400 µl of 1X Binding Buffer to each tube, the cell suspensions were filtered by a 50-mm nylon mesh and immediately analyzed by flow cytometry, wherein 10,000 cells were counted from each sample. For the experiment treated with PAK1 shRNA was conducted as previously described. All experiments were performed in triplicate independently.
Wound healing assay
The experiment aimed to evaluate the migration behavior of glioma cells. U87 and U251 cells pre-treated with or without IPA-3 were seeded in 6-well plates. When the cells were at 80% confluence, the scratches were performed along the straight line with a sterile pipette tip, rinsed with PBS three times to remove detached cells completely. Subsequently, changed serum-free medium and incubated in a condition of 37 °C, 5% CO2 and saturated humidity conditions, at the designated time 0 and 24 h, randomly chose six 100х magnification fields per sample around the lesion border by Olympus IX-71 inverted microscope. Finally, counted the migrating cells across the wound to quantify the relative cell migration rates. For the experiment treated with PAK1 shRNA was conducted as previously described. All experiments were performed in triplicate independently.
Transwell migration and invasion assays
The two experiments were performed using a chemotaxis chambers with polycarbonate nucleopore membrane. The filters with a diameter of 6.5 mm and pore size of 8 μm (Invitrogen). To detect cell invasion ability, firstly, the filters were pre-coated with 50 uL Matrigel (1 mg/mL) (BD, Biosciences, USA), then rehydrated with 100 μL FBS-free medium in 37 °C incubator for 2 h. Subsequently, a density of 6*103 cells/mL U87 and U251 cells pre-treated with or without IPA-3 suspended with serum-free medium were plated in the upper layer part of each chamber, 600 μL culture medium containing 10% FBS as a source of chemoattractants was added to the each lower chamber and incubated in 37 °C incubator. 36 h later, removed the non-invasive cells of the upper chamber with cotton swabs. Afterwards, invasive cells on the lower surface of the filter were fixed with 4% paraformaldehyde for 30 min and stained with 0.3% crystal violet (w/v) diluted with methanol for 15 min. Finally, five fields of adherent cells in each well were randomly photographed to quantify the cell invasion rates under an Olympus IX71 inverted microscope (Olympus, Japan). The assessment of cell migration ability was the same conduction except that filters were not pre-coated with Matrigel. For the experiment treated with PAK1 shRNA was conducted as previously described. All experiments were performed in triplicate independently.
Western Blotting analysis
The cells were lysed with RIPA lysis buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.25% Na-deoxycholate) supplemented with protease and phosphatase inhibitor cocktail, and lysates were incubated on ice for 30 min to yield a homogenate, subsequently, centrifuged and removed cell debris to obtain the final supernatants. The protein concentrations of whole cell lysates were determined using the BCA protein assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. After the protein extract was mixed with 5×SDS buffer (Beyotime, Shanghai, China), the protein was denatured by boiling for 10 min at 100 °C, then equal amount of protein lysates was subjected to 10-12% sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS–PAGE) at the voltage of 70 V to 110 V. And then electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane (Merk Millipore, Billerica, MA, USA) at 110 mV, then the membranes were blocked with 3% bovine serum albumin for 60 min at room temperature, washed with Tris-buffered saline Tween-20 (TBST) (20 mM Tris pH 7.4, 137 mM NaCl and 0.1% Tween-20), subsequently incubated in specific primary antibodies at 4 °C overnight or at room temperature for 2 h, then the membrane was washed with TBST for three times, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology (CST), Danvers, MA, USA) at room temperature for 2 h. Using enhanced chemiluminescence (ECL) (Thermo Fisher Scientific, NM, USA) and exposed with a ChemiDocTM Imaging System (Biorad, Hercules, CA, USA). β-actin was regarded as a loading control. Immunoblots were quantified by densitometry using Image J version 1.46 softwar (National Institute of Health, Bethesda, MD, USA). All experiments were performed in triplicate independently. Antibodies against Bax (50599-2-Ig), MMP2 (10373-2-AP), MMP9 (10375-2-AP) and β-actin (60008-1-Ig) were purchased from proteintech (Chicago, USA). Antibodies against PAK1(#2602), p21 (#2647), p53 (#2524), p38 (#9212), p-p38 (Thr180/Tyr182, #9211), CDK4(#12790), Cyclin D1 (#2922), Cofilin (D3F9, #5175) and Cleaved Caspase-3 (#9664) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against MDM2 (sc-965) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Most antibodies were used in 1:1000 dilutions, unless specified otherwise.
Intracranial mouse model
All mice experiments were conducted in accordance with protocols approved by the Xuzhou Medical University Animal Care and Use Committee and followed guidelines for animal welfare. For the purpose of establishing intracranial GBM xenografts, 6 to 8-week-old female BALB/c-nude mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Company and housed in Individually Ventilated Caging (IVC) systems in a 12 h light/dark cycle. A total of 3*105 U87 cells/mL expressing mCherry routinely treated by mycoplasma per well were seeded in 6-well plate. When the cell confluence reached 50%, the cells were infected with PAK1 shRNAs and control lentivirus, respectively. After 48 h of infection, the cells were trypsinized and resuspended with sterile PBS. A total of 5*105 shPAK1 and control cells (in 5 μL PBS) were collected and injected into the intracranial striatum of nude mice with a stereotactic instrument and a microsyringe. Before injection, mice were anesthetized with 3% chloral hydrate, subsequently, a hole about 5 mm in diameter was burred located in 1 cm anterior and 2 cm lateral to the bregma, then with the stereotactic guidance, the microsyringe needle penetrated 3 cm from the calvaria to the striatum and injected cells at a rate of 1 μL/min[13-16]. The animals were randomly divided into 3 groups, with 8 mice in each group, including shControl, shPAK1-2, shPAK1-3, respectively. Starting on day 28 d after tumor cell implantation, sacrificed 2 mice by cervical dislocation from each group every week, and the brain samples were fixed in 4% paraformaldehyde. Afterwards, performed frozen slices for hematoxylin and eosin (HE) staining to obtain tumor growth status from different groups.
All experiments were performed in triplicate independently. Data were represented as the mean ± SD as indicated. The Student’s t-test (two-tailed) was used for comparison of two groups, comparisons among multiple groups were analyzed with One-way analysis of variance (ANOVA). P<0.05 was considered statistically significant. All statistical analyses were performed using the SPSS 16.0 software (SPSS Inc., Chicago, IL, USA).