UM-164: A c-Src/p38 Kinase Inhibitor inhibits proliferationand induces apoptosis in human glioblastoma cells

doi:10.21203/rs.3.rs-1632314/v1

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UM-164: A c-Src/p38 Kinase Inhibitor inhibits proliferationand induces apoptosis in human glioblastoma cells

https://doi.org/10.21203/rs.3.rs-1632314/v1

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Glioblastoma is the most common and aggressive primary brain tumor in adults, which has high mortality and morbidity rates, and short survival time. here we tested a novel c-Src/p38 kinase inhibitor UM-164 in human glioblastoma cells in U87 and U251 cells. CCK-8 assay, EDU staining, colony formation, wound healing, cell cycle distribution analysis, migration and transwell invasion assays showed that UM-164 suppressed the proliferation and dose‑ and time‑dependent cytotoxicity in and exhibited a marked suppression after treating the U87 and U251 cells with UM-164. Flow cytometry, TUNNEL assays, immunofluorescent staining and western blotting test which tested the expression levels of BCL-2, BCL-XL, BAX, Cleaved-caspas-3, Cyclin D1, Cyclin E, MMP-2, MMP-9, P38, P-P38, EGFR, P-EGFR(Tyr1068), AKT, P-AKT, P42 and P-P42 were changed after um-164 treatment have changed and exhibited a strong inhibition for the proliferation and induces apoptosis. taken together, our findings indicate that um-164 exerts an antitumor effect by inhibiting proliferation and inducing apoptosis in human glioma cells. as for the present study provides evidence supportive of further development of multitarget kinase inhibitor UM-164 for adjuvant therapy and give a hope in GBM patients.

UM-164

glioblastoma

apoptosis

proliferation

invasion

Glioblastoma is the most common and aggressive primary brain tumor in adults and character with high rates of tumor cell proliferation, resistance to apoptosis, increase abnormal neoangiogenesis, and significant tumor cell migration, which has high mortality, morbidity rates and short survival time, namely < 15 months after the first diagnosis. meanwhile, there are no curative treatment options for the treat of GBM. despite the application of standard therapy for GBM, which includes surgery, radiation therapy, chemotherapy and TTF, only with ≤ 5% of patients living > 5 years after the first time of diagnosis[1, 2], all of them provides only short-term survival benefits after the prognosis for patients diagnosed with glioblastoma. the poor prognosis of GBM caused a heavy burden for the economy and the social development to the developing country such as here in china[3, 4], while little progress has been made toward the treat of GBM in the last few decades, so improved therapeutic strategies or drugs are required. world health organization (WHO) classificated GBM as grade IV, which is the highest grade in the brain tumors due to the defining histopathologic features of GBM are necrosis and endothelial proliferation[1]. point mutation of IDH, RTK, RAS, PI3K, p53,the methylation of MGMT and the loss of PETEN associated for the diagnose and the outbreak of the GBM patients[3]. besides, Next-generation sequencing of GBM has renewed our understanding of clonal evolution for tumor heterogeneity, changed the model that all GBM can be treated with therapies targeting a single pathway[5].

Disorder of signaling pathways plays an important role in many of cancers including the gliomas, while preclinical data showing that Src family of non-receptor protein kinases and p38 signal pathway mediate intracellular signaling pathways controlling key biologic and oncogenic processes in proliferation, angiogenesis, differentiation, migration, adhesion and apoptosis. p38 pathway is considered as a major pathway to media the inflammation and stress responses[6, 7], so Src family of protein tyrosine kinases plays key roles in regulating signal transduction. meanwhile, in human GBM, p38 MAPK is upregulated, the samples in glioblastoma cell lines and primary glioblastoma patient found that SRC and p38APK activity in GBM elevated to mediate intracellular signaling pathways controlling key biologic and oncogenic processes in cell proliferation, angiogenesis, differentiation, migration, adhesion and apoptosis, nonreceptor tyrosine kinases are cytoplasmic and include SRC and SRC-family kinases (SFKs)which plays key role in many GBM features [8–11]. in gliomas, increase expression of the epidermal growth factor (EGF), the platelet-derived growth factor (PDGF) receptors and integrin receptors mediate tumorigenesis[12]. the MAPK pathway has a close relationship with PTEN for Src can phosphorylate PTEN and decrease its activity to influence cell growth[13]and has been reported to be activated in over 88% of gliomas, contribute to proliferation, invasion, tumorigenesis, angiogenesis and apoptosis[14, 15]. as we all know, temozolomide (TMZ), a first-line chemotherapeutic drug for glioma. however, the effective rate of TMZ is only 46%, which is even lower for patients with relapsed glioma who gain resistance[16]. the application of p38 MAPK inhibitors in chemotherapy has attracted great attention for its highly expressed and activated in a variety of cancers including glioblastoma[17]. so, find a new molecule of p38 MAPK inhibitor for the treat of GBM is needed.

Previous study has showed that C-Src inhibitor (UM-164) both binds the target kinases of DFG-out inactive conformation and also inhibit the p38 kinase families, significantly suppress the tumor growth with limited in vivo toxicity [5, 18]. though previous multi-target kinase inhibitor like dasatinib which is a Src kinase inhibitor acting as an oral medication has showed failure in recurrent patients[11, 19], the advantage of UM-164 may give the world a new hope for this strategy for this new molecule binding the inactive kinase conformation combined with a better pharmacologic actions[5, 20],what’s more, the addition of fluorinated benzene ring to uM-164 make it have a great future compared with dasatinib for improved efficacy and safety[20]. potential molecular mechanism of the new c-Src/p38 kinase inhibitor UM-164 in glioma remains unknown. here, we investigated UM-164 in glioma cell line U87 cells which have no mutant PTEN and U251 cells which is the most common type of GBM cell line exhibiting mutant PTEN with UM-164.

2.1 Cell culture

U87, U251 of the cell lines for human GBM were gotten in the cell bank type culture collection of the chinese academy of sciences. identified technology of our cell lines in the laboratory was repeat profiling of short tandem, provided by Porcello Life Science &Technology Co Ltd. Dulbecco's modified Eagle's medium (DMEM), including 100µg/ml streptomycin, 10% fetal bovine serum (FBS) as well as 100µg/ml penicillin, was applied to incubate the cells at 37℃ under 5% CO2 atmosphere.

2.2 Chemicals and antibodies

UM-164 was purchased from Bio Vision and dimethyl sulfoxide was applied to dissolved it, which was purchased from Merck KGaA. and the antibodies were all from Cell Signaling Technology, Inc., except BCL-2 (GTX100064), which was from Gene Tex, Inc. and cleaved caspase-3 (ab32042), which was purchased from Abcam, Inc. including these, Anti-AKT (#4691), phosphorylated-AKT (#4060), EGFR (#1416), P-EGFR (#3777), P38 (#41666), phosphorylated- P38 (#4511), P42 (#9108), P-P42 (#4370), BCL-XL (#2764), Bax (#5023), cyclin D1 (#2978), cyclin E (#20808), MMP-9 (#13667), MMP-2 (#40994), and GAPDH (#5174).

2.3 Cell viability

The Cell Counting Kit-8 (CCK-8), purchased from dojindo molecular technologies, Inc. was employed to assess the viability of cell and confirm the inhibitory effect of UM-164 to U87 and U251. in a 96-well plate, every well includes 5000 cells, which were planted into a solution included 100µl DMEM containing 10% FBS, as well as UM-164, which was divided in multiple concentrations (0, 1, 5, 10, 50, and 100µM) for 2 days and given a fixed concentration with different times (24, 48, 72 and 96h). then, 10µl CCK-8 was applied to incubate cells for 60 minutes at 37℃. Every group was gauged the absorbance value in each well with a spectrophotometer at 450 nm for 3-time.

2.4 Colony formation assay

Adding U87 and U251 (~ 1,000) cells into 2ml DMEM with 10% FBS and put in a 6-well plate. next, various concentrations of UM-164 (0, 1, 5 and 10µM) were used to treat the cells for 14-day at 37℃ under CO2 of 5%. in 25℃, paraformaldehyde, 2ml 5%, was added in the cells for a quarter. the cells in every well were then stained by 0.5% crystal violet and taken photos by a camera.

2.5 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay

In a 96-well plate, every well includes 5000 cells, planted into a solution included 100µl DMEM, containing 10% FBS, and UM-164, which was divided in some concentrations (0, 1, 5, 10, and 20µM) about 3 days. then, cultivating the cells in 50µM Edu under 5% CO2 at 37℃, as well as fixing them by 4% paraformaldehyde about half an hour at 25℃. we treated the cells in triton X-100, 0.5%, for 20- minute as well as used PBS to wash them 3 times in 5 minutes. the cells were added 100µl 1X Apollo® reaction cocktail to incubate them for 30 minutes. hoechst 33342, 5µg/ml, was employed to stain the cell nuclei 30-minute. getting fluorescence

pictures by using fluorescence microscope in ×200 magnification.

2.6 Cell cycle distribution analysis

Distributions of cell cycle were gotten through flow cytometry, which employed a cell cycle kit of PI staining (Becton, Dickinson and Company). seeding U87 and U251 into 6-well plate and treating them with multiple concentrations (0, 1, 5, 10and 20µM) for 72h. afterward, a centrifuge, at 1000rpm, was employed in 25℃ for 5 minutes to collect cells. PBS and cold 70% ethanol was mixed into the cells to wash them for 1day at 4℃. treating the cells at 37℃ by 50 µl 100 µg/ml RNase, washing two times with PBS, centrifuging about 5 minutes at 1,000 rpm and staining in 5µl PI with stock solution, 50mg/ml. evaluating the outcomes was done by BD FACSAria. And getting assessments of the statistics from ModFit LT 4.0.

2.7 Wound-healing assay

Inoculating U87 as well as U251, 70%~80% concentration, in a plate to become a monolayer. after that, get straight cut lines in every well, we used a pipette tip to scrape respectively. Next, removing the suspended cells with PBS, adding UM-164 with several concentrations (0, 5 or 10µM) and 2ml DMEM. acquiring pictures with a microscope (Olympus BX51; Olympus Corporation) at ×10 magnification, and assessing the wound distance with imageJ software.

2.8 Invasion assays, ant stain with Phalloidin as well as Cell Migration

Transwell chambers were applied with a polycarbonate film with an 8.0µm pore diameter. mixing U87 as well as U251 with DMEM lack of 10% FBS and planting them in the top chambers. then, we used Matrigel to coated the cells, or not, in migration and invasion assay. Later, putting the chambers in a 24-well plate to incubate about 24 hours. After that, we stained the cells with the crystal violet that crossed the membranes, and carefully wiped off the cells having not penetrated membranes. quantifying the cells by using a microscope (magnification, x10; Olympus BX51; Olympus Corporation). planting the cells into 6-well plates, having glass slice at their bottom, and handling them with UM-164 in IC50 concentration. subsequently, incubating the cells with Phalloidin, which was diluted 100 times by BSA, for 1 hour and staining them for 5 minutes by DAPI. then, rinsing the cells twice for 5 minutes. finally, visualizing fluorescence images with a fluorescence microscope (Olympus BX51; Olympus Corporation), ×200 magnification.

2.9 Analyzing apoptosis with flow cytometry

Apoptosis Annexin V-PE/7-AAD kit was used to inspected to cells dealt with different UM-164 concentration (0, 1, 5, 10 and 20µM). firstly, we used centrifugation for 5 minutes (1,000rpm at 25℃) to collect cells and then rinsed them twice with PBS. After that, 1X binding buffer (0.1 mM HEPES/NaOH, 1.4 M NaCl and 25 mM CaCl2, pH 7.4)100µl was used to let the cells float in it. and add PE-Annexin V and 7-ADD both with 5µl, keep in the darkness at 25℃ for 15 minutes to stain the cells. subsequently, sucking 400µl 1X binding buffer into every tube. BD FACSAria (BD Biosciences) was used to analyze the achievements. FlowJo software (FlowJo LLC) was adopted to quantify statistics, and the total apoptosis rates were calculated and evaluated by the totality of the upper right and low right quadrants. in line with manufacturer’s protocol (Roche Molecular Diagnostics), TUNEL assay could be used to get the detection of apoptotic cells’ DNA fragments. Olympus BX51 microscope (Olympus Corporation) was used to get photos.

2.10 Measuring potential (ΔΨm) of mitochondrial membrane

One of the iconic events of early apoptosis is the disappearance of mitochondrial membrane potential (ΔΨm). firstly, planting the cells into 6-well plates, having glass slice at their bottom, and handling them with UM-164. accordance to the instructions of manufacturer. the indication of ΔΨm’s disappearance emerged as a decline of the ratio of red/green fluorescence intensity, which can be found out by flow cytometry and microscopy.

2.11 ROS level of cells treated with UM-164

The ROS level was tested by DCFH-DA. first, planting the cells into 6-well plates with glass slice at their bottom and handling them with UM-164. Then, the cells were conducted with the ROS stain, in line with the explanations of manufacturer. the flow cytometry was employed to detect the raising overall ROS level in cell. capturing images under the usage of Olympus FV1200 confocal microscope or Olympus BX51 microscope.

2.12 Western blot analysis

First, UM-164 (0, 5, 10 or 20µM) was adopted to treat the cells for 48 hours. next, lysing the cells in RIPA buffer on ice for 20 minutes. then, loading the protein onto SDS-PAGE and electro-transferring them into PVDF membranes about sixty or ninety minutes. after that, 5% skim milk was employed to block the membranes at 4°C, membrane and primary antibody were incubated at 1:1000 after one night. alexa fluor 680/790-labelled secondary antibodies, at 1:1000, were put in the membranes to incubate for one hour. the LICOR odyssey infrared imaging system can let the bands visible. and normalizing the data with the usage of GAPDH.

2.13 Immuno-fluorescence

First, fixing the cells in 4% paraformaldehyde for half an hour. Then, permeabilizing cells with Triton X-100 0.1% about 10-minute. Next, 1% bovine serum albumin was applied to block them about one hour. indicated primary antibodies were mixed in them to incubate overnight then adopting fluor-labelled secondary antibodies, purchased from Antgene, Wuhan, China. DAPI was used to stained the nuclei. Olympus FV1200 confocal microscope microscope was applied to get images.

2.14 Statistical analysis

SPSS 19.0 as well as GraphPad Prism 6.0 are used to analyze statistics. indicating all data as mean ± SD. one-way analysis of variance and t-test were used to evaluate the discrepancy between groups. every experiment repeated in triplicate. considering P < 0.05 as having statistically significant.

3.1 Inhibition of UM-164 on proliferation of U87 and U251 cells in vitro

We conducted the Cell Counting Kit-8 trail to test the proliferation effects of UM-164 (Fig. 1A) after treating U87 as well as U251 by various concentrations for 0, 5, 10, 20µM and time-points to 24, 48, 72 and 96 hours. UM-164 significant inhibited cells viability. the IC50 value of U87 is 4.38 (95% CI, 3.12–5.94), meanwhile U251 is 7.96 (95% CI, 6.1–9.43), which indicating U251 is less sensitive than U87. the IC50 value of UM-164 showed different points in time for further investment (Fig. 1B). we found that UM-164 has a significant inhibitory effect on the proliferation of U87 and U251. the colony formation assay exhibited the same results as CCK-8 under different concentrations of UM-164 (Fig. 1C, D). wound healing under concentrations (0, 5, 10 and 20µM) of UM-164 on glioma metastasis were studied. Compared with the UM-164 treatment group, the separation distance of the control group during the 48h growth period increased faster (Fig. 1E). UM-164 inhibited the wound closure of U87 as well as U251 cells and treatment group was less than that of the control group in 48 hours (Fig. 1F, P༜0.05).

3.2 Cell cycle arrestation of UM-164 on glioma cells

The flow cytometer is used to analyze the distribution of the cell cycle. after being treated with different concentrations of UM-164 for 48 hours, the cell cycle of U87 and U251 cells was significantly arrested in the G1 phase (Fig. 2A). the proportion of cells in the G1 phase in the dimethyl sulfoxide group increased significantly (P < 0.05). the proportion of glioblastoma cells in the G1, S, and G2 stages decreased in a dose-dependent manner (Fig. 2B).

3.3 The inhibitory effect of UM-164 on the migration and invasion of U251 and

U87 cells

EDU incorporation test was used to evaluate the inhibitory effect of UM-164 on U87 and U251 glioblastoma cells. results showed a great inhibitory effect which was related to the dose of UM-164 increases (Fig. 3A and B). the proliferation rate is U87 and U251 cells stained in the UM-164 group is lower than that in the DMSO group (Fig. 3C and D and B, P༜0.05). compared with the control group, the cell invasion and migration ability after UM-164 treatment was significantly reduced (Fig. 3E-H, P༜0.05). The phalloidin stain showed that under the treatment of UM-164 actin filament has been disrupted greatly (in Fig. 4).

3.4 UM-164 induces U87 and U251 cell apoptosis

Results indicating that with the concentration of UM-164 increased, the number of apoptotic cells increased (Fig. 5A, B, C and D, P༜0.05). the decrease in ΔΨm can be detected by the decrease in the red/green fluorescence intensity ratio (Fig. 5E and F). analysis by flow cytometry showed that the UM-164 can increased the ΔΨm rate obviously (Fig. 5G and H). in addition, UM-164 can induce apoptosis of U87 and U251, which can be detected by TUNEL assay (Fig. 5I and J).

3.5 UM-164 induces the increase of ROS in U87 and U251 cells

UM-164 increased the total ROS level of U87 and U251 cells. ROS level reflect the Oxidative stress level to cell exposure to different environments for 1 day (Fig. 6A, B). ROS level was increased after treatment with UM-164 on glioblastoma cells (Fig. 6C, P < 0.05), and the increase of ROS release in cells response to intra and inter mitochondrial redox-environment changes has a close relationship to mitochondria homeostasis cell death.

3.6 The expression of signal pathway was changed after treated with UM-164

Western blotting has done to show the expression of all of BCL-XL, Cyclin D1, Cyclin E, P38, P-P38, BAX, EGFR, P-EGFR(Tyr1068), AKT, BCL-2, P-AKT, P42, Cleavered-caspase3, and P-P42 changes of U87 and U251after treated with UM-164 just as showed (Fig. 7). we found that the expression amounts of lysed Caspase3, P-P38, P-P42/44, and P-AKT in U87 and U251 glioma cells were greatly higher after the treatment of UM-164 comparing with the control group (Fig. 8A, B, C, D).

GBMs are characterized by genetic alterations result in the change of cell growth, apoptosis, angiogenesis, Epigenetic alterations and invasion[21],while, epigenetic variations, viral infections and environmental factors have been identified as potential risk factors[22, 23],such as infection of HCMV, ionizing radiation, toxic agents (N-nitroso compounds, pesticides), air pollution, and radiofrequency electromagnetic waves. SRC is maintained in an inactive state by C- terminal SRC kinase (CSK)-mediated phosphorylation of a negative- regulatory tyrosine residue (Y530) and composited by eight homologous members (SRC,YES, FYN, LYN, LCK, HCK, FGR and BLK. when phosphorylated, Y530 binds to the SH2 domain, turn SRC into an inactive configuration[15]. two of most SRC family members have been shown to be activated in glioblastoma, including the malignant phenotype- promoting members LYN and FYN [24],which give SRC kinase and SFKs are promising targets for anticancer therapy like c-Src/p38 Kinase Inhibitor UM-164[12, 15, 25–29].

In the present study, glioblastoma cell line U87 and U251 cells were treated with UM-164. results showed a great suppression affection of UM-164 to U87 and U251 cells in a dose- and time-dependent manner, with IC50 values of 4.38 and 7.96 respectively and also promoted Gl arrest and strong cell apoptosis in a dose-dependent manner. the cell migration and invasion has been suppressed at the same time. Wound-healing ensured that the migration and invasion of human glioma cell lines were reduced by UM-164. the expression of BCL-2, BCL-XL, BAX, Cleavered-caspase3,Cyclin D1,Cyclin E,P38,P-P38,EGFR,P-EGFR(Tyr1068),AKTP-AKTP42and P-P42 determined by western blotting have also changed to the increasing concentration. there are various signaling pathways involved in the expose to UM-164 for GBM cells including theP38/MAPK and PI3K/AKTP-EGFRP-P42, which can affect cell survival, proliferation and apoptosis in both have and no mutant PTEN GBM cell lines. this study supports a further study of UM-164 in GBM for the improvement of structure of molecule.

Although there were obvious results of UM-164 suppressing the proliferation and inducing apoptosis effects to glioblastoma cells line of U87 and U251 in this study many limitations still exists for animal experiments which thus have not yet been done to see its pharmacokinetic parameters. nevertheless, as a prospective drug for CNS, the ability to crosses the blood-brain barrier (BBB) should be highlighted, so related test should be done in the future. although it is still necessary to further verify whether UM-164 has greater value to the glioma patients despite its limitations, this study does indicate that UM-164 is a promising drug candidate for glioma therapy.

Author Contributions

Yang kun and Chen Qianxue conceived and designed the study. Yang kun conducted the experiments. Yang kun, Xiao Meixian, Tang Xiangjun and Shan Qiang performed the statistical analysis. Yang kun wrote the manuscript. Yang kun, Xiao Meixian, Tang Xiangjun and Shan Qiang reviewed and edited the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Notes

The authors declare no competing financial interest.

The data used to support the findings of this study are available from the corresponding author upon request

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No competing interests reported.

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UM-164: A c-Src/p38 Kinase Inhibitor inhibits proliferationand induces apoptosis in human glioblastoma cells

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Cell culture

2.2 Chemicals and antibodies

2.3 Cell viability

2.4 Colony formation assay

2.5 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay

2.6 Cell cycle distribution analysis

2.7 Wound-healing assay

2.8 Invasion assays, ant stain with Phalloidin as well as Cell Migration

2.9 Analyzing apoptosis with flow cytometry

2.10 Measuring potential (ΔΨm) of mitochondrial membrane

2.11 ROS level of cells treated with UM-164

2.12 Western blot analysis

2.13 Immuno-fluorescence

2.14 Statistical analysis

3. Results

3.1 Inhibition of UM-164 on proliferation of U87 and U251 cells in vitro

3.2 Cell cycle arrestation of UM-164 on glioma cells

3.3 The inhibitory effect of UM-164 on the migration and invasion of U251 and

U87 cells

3.4 UM-164 induces U87 and U251 cell apoptosis

3.5 UM-164 induces the increase of ROS in U87 and U251 cells

3.6 The expression of signal pathway was changed after treated with UM-164

4. Discussion

Declarations

References

Additional Declarations

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