Mocetinostat as a novel selective histone deacetylase (HDAC) inhibitor in the promotion of apoptosis in glioblastoma cell line C6 and T98G

doi:10.21203/rs.3.rs-4170668/v1

Download PDF

Research Article

Mocetinostat as a novel selective histone deacetylase (HDAC) inhibitor in the promotion of apoptosis in glioblastoma cell line C6 and T98G

https://doi.org/10.21203/rs.3.rs-4170668/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Histon deacetylase (HDAC) enzyme is one of the enzymes involved in regulating gene expression and epigenetic alternation of cells by removing acetyl groups from lysine residue on a histone, allowing the histones to wrap the DNA more tightly and suppressing a tumor-suppressing gene. HDAC inhibitors play an important role in inhibiting the proliferation of tumor cells by restricting the mechanism of action of HDAC enzyme, leading to the addition of acetyl groups to lysine. Mocetinostat, also known by its chemical name (MGCD0103), is a novel isotype selective HDAC enzyme that explicitly targets HDAC isoforms inhibiting Class1(HDAC 1,2,3,8) and Class IV (HDAC11) enzymes. It was approved for treating the phase II trial of Hodgkin's lymphoma in 2010. Our study revealed that different doses of Mocetinostat inhibit the growth of glioblastoma cells, metastasis, and angiogenesis and induce the apoptosis and differentiation of glioblastoma cells C6 and T98G. Western blot has shown that MGCD0103 has many biological activities to control glioblastoma cancer cells. MGCD0103 can modulate the molecular mechanism for several pathways in cells, such as inhibition of the PI3K/AKT pathway and suppression of HDAC1 enzyme activity in charge of many biological processes in the initiation and progression of cancer. The high doses of Mocetinostat drug significantly induce apoptosis and suppress cancer cell proliferation through increased pro-apoptotic proteins (BAX) and a down level of anti-apoptotic proteins(Bid, Bcl2). Also, the mocetinostat upregulated the expression of the tumor suppressor gene and downregulated the gene expression of the E2f1 transcription factor. Additionally, MGCDO103-induced differentiation was facilitated by activating the differentiation marker GFAP and preventing the undifferentiation marker from expression (Id2, N-Myc). The MGCD0103 is a potent anticancer drug crucial in treating glioblastoma cells.

Glioblastoma

MGCD0103

Cell line T98 and C6

Apoptosis

differentiation

Invasion and Metastasis

Genetic alternation is not only involved in cancer cells, but the epigenetic modifications that cause gene expression alternation without changing the DNA sequence are responsible for carcinogenesis for many cancer cells. Recent studies indicate that epigenetic modification (DNA methylation, Histone modification, and Non-coding RNA deregulation) is the cause of initiation and cancer development (Li et al.,2010; Sharma et al.,2010). Histone modification has an essential role in regulating gene expression; it causes upregulation of oncogenes and DNA repair genes by histone acetylase and deacetylase (Perri et al.,2017). Histone deacetylase (HDAC) is a family of enzymes considered essential in the epigenetic regulation of gene expression that leads to the control of diverse cellular activities, such as growth cells, apoptosis, invasion cells, and angiogenesis. These enzymes usually remove the acylal group from the histone tail, leading to silent transcriptional gene of tumor suppressor gene (Minucci &Pelicci,2006). HDAC in humans is classified into four classes depending on how closely their sequence resembles those of yeast HDACs. Class I, II, and IV HDAC have Zn2 + dependent enzymes, while class III comprises Zn²⁺ independent (Lucio-Eterovic et al.,2008). HADC1 belongs to HDAC class I, increasing their expression in many cancer cells. Also, the study found that express this enzyme elevated in glioma cells during tumor progression, and downregulation of HDAC1 suppressed cell proliferation and invasion of glioblastoma cell line U251, T98g cells (Wang et al.,2017). Epigenetic therapy controls the regulation of epigenetic alternations that occur in tumors and convert to the normal epigenome. Some epigenetic inhibitors, including the HADC inhibitor, have been approved by the U.S. Food and Drug Administration (FDA) (Chen et al.,2013).

HDAC inhibitors have been shown to influence many pathways to inhibit growth cells and induce apoptosis and differentiation. Still, HDAC inhibitors maintain poor performance in cancer treatment because these inhibitors are almost nonselective HADC inhibitors and suppress multiple classes of HADCs, which result in toxicity and limit therapeutic efficacy (Zhang et al.,2016). One of the new classes I selective HDAC inhibitors, mocetinostat, also known as MGCD0103, specifically targets the suppression of human class I HDAC. (Fournel et al.,2008). MGCD0103 is a benzamide histone deacetylase inhibitor undergoing preclinical trial for the treatment of many cancer cells in vitro and in vivo, such as the colon (Sikandar et al.,2010), prostate (Zhang et al.,2016), Human epithelial cancer cell (Fournel et al.,2008), and Hodgkin's lymphoma (Younes et al.,2011). Also, MGCD0103 has a high ability to inhibit HADC2, HADC3, and HADC11 in vitro and increase acetylation on histone tail, which induces apoptosis and inhibits growth cells for a variety of cancer cells (Fournel et al.,2008).

Glioblastoma is the most dangerous malignant primary brain tumor that affects the adult’s central nervous system. They arise from glial cells and represent grade IV glioma (Louis et al.,2016). GBM remains one of the deadliest tumors, and its treatment methods remain limited (Stupp et al.,2005). Histone deacetylase inhibitors have a great interest in cancer treatment because they can alter the outcome of RNA sequences to induce apoptosis. Characteristics of Glioblastoma, representing increased proliferation, angiogenesis, invasion, and migration, triggered apoptosis and differentiation, are usually targeted via HDAC inhibitors. HDAC inhibitors are a promising class of therapeutic drugs being researched for treating several tumors, including GBM (Lee et al.,2017).

In the present study, we investigated the role of Mocetinostat in the treatment of human glioblastoma cell lines, and we will show inhibitory activity for Mocetinostat in suppressing cell proliferation, migration, inducing apoptosis, and differentiation of glioblastoma in two cell lines T98G and C6 in vitro. MGC0103 might be a promising anticancer drug in the treatment of glioblastoma.

1.2 Cell culture and treatment

Human glioblastoma cell lines T98G and rat glioma C6 cells were obtained from American-type culture collection (ATCC, Manassas, VA, USA). Both cells were grown in RPMI 1640 containing 1% penicillin-streptomycin and 10% fetal bovine serum (GIBCO/BRL, NY, USA). Cultures were maintained in a humidified atmosphere with 5% CO2 at 37°C. Cells were treated with different concentrations (0.0, 0.5, 1.0, 1.5, 2.0, 2.5) µM of HDAC inhibitor (MGCD0103) that was purchased from Selleckchem.com (Selleck Chemicals, Houston, TX, USA). The drug was dissolved in dimethyl sulfoxide (DMSO) to get the final concentration (5.0mM) like a sock solution. The stock solution was stored at -20°C.

2.2 A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay

The MTT assay is the best method to measure the viability of cells and the cytotoxic effects of drugs. Cellular metabolic activity of C6 and T98G was measured depending on the manufacturer’s instructions (ATCC, Manassas, VA, USA). Briefly, 0.5 x 10⁴ cells/well were seeded into 96-well plates containing growth medium RPM1 with supplements (10% PBS and 1% penicillin and streptomycin) and incubated with 5% CO2 at 37°C. After the growth cells got 70% confluence, the current medium was replaced with a new medium containing varied concentrations (0, 0.5, 1.0, 1.5, 2.0, 2.5) µM of MODC0103 for 48 h. Then, the 10µL MTT solution (ATCC, VA, USA) was put in each well and grown for 4h. Detergent reagent (ATCC, Manassas, AV, USA) was applied to each well in quantities of 100 mL to dissolve MTT formazan crystals., a wavelength absorbance was measured using a microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm, absorbance was recorded, and at 690 nm absorbance of the background was subtracted. All experiments were in triplicate.

3.2 In vitro differentiation cancer cells assay

The different concentrations of MGCD0103 were used to identify the ability of mocetinostat to induce astrocytic differentiation in glioblastoma cell lines C6 and T98G. Using 6 well plates, cells were cultivated in growth medium RPMI-1406 with 10% FBS and 1% Penicillin/Streptomycin. The cultures were placed in the incubation with 5.0% CO₂ and 37⁰C for 24h. After the culture medium was removed, the cells cultured in the medium were supplemented with different concentrations of MGCD0103. The cells were placed in the incubation conditions of 5% CO₂ at 37⁰C for 48h. After treating the cells with drugs, the culture medium was aspirated and washed twice with BPS. After that, the cells were fixed and stained using a Kwik-Diff™ kit according to the protocol of the manufacturer (Thermo-Scientific, MO, USA). The samples were examined under light microscopy to watch the differentiation features of cells.

4.2 Detection of Morphological characteristics using Apoptosis assay

The morphological feature of apoptosis cells was achieved by right staining. Both cell lines C6 and T98G at density 0.5x104 were cultured in 6 wells containing RPMI-1460 supplement with 10% FBS and 1% Penicillin /Streptomycin, then incubated in the condition of 5% CO2 at 37⁰C for 48 h. After removing the old medium, 2mL of the new medium supplement with Each well received different doses of MGCD0103 before being incubated for 48 hours. The supernatant was discarded and washed twice with PBS. Then, the cells were fixed and stained utilizing a Kwik-Diff™ staining kit following instructions provided by the manufacturer (Thermo-Scientific, St. Louis, MO, USA). The morphological features of apoptosis cells were imaged under a light microscope (Olympus, Pittsburgh, PA, USA). Image J was used to measure the percentage of apoptotic cells. The apoptosis rate was measured after counting 300 cells from each treatment.

5.2 Identification of biochemical apoptotic populations using flow cytometry and Annexin FITC/PI double staining.

a Fluorescein Isothiocyanate (FITC)-Annexin V and propidium iodide (PI) staining Apoptosis were examined to detect the Biochemical characteristics of apoptotic cells using Apoptosis Detection kit (BD Biosciences) according to the manufacturer’s instructions. Both cell lines (C6 and T98g ) were cultivated independently in 6 wells and subjected to various concentrations of MGCD0103 (0.0, 0.5, 1.0, 1.5, 2.0, 2.5) µM for 48 h. Once the cells were harvested and centrifuged at 2500x for 5 min, they were washed cold BPS twice and resuspended in 100µl 1x binding buffer (0.1 M HEPES, NaOH, PH 7.4,1.4 NaCl, and 25Mm Cacl2). The cells were transferred to a 5mL polystyrene culture tube and stained with 5µl of Annexin-V-FITC and 5µL of PI. All tubes were incubated for 15 min at darkroom temperature for the staining of cells. Then, 400µL of binding buffer was added to each tube and analyzed by flow cytometry using an Epics XL-MCL Flow Cytometry (Beckman Coulter, Fullerton, CA, USA). Three duplicates of each experiment were carried out. The cell populations were identified on the plot as a red dot, with viable cells (FITC−/PI−) appearing in the left lower quadrant (Q3), early apoptotic cells (FITC+/PI−) were displayed in the right lower quadrant (Q4), and necrotic and late apoptotic cells (FITC+/PI+) were presented in the right upper quadrant (Q2). The necrotic cells were seen in the left upper quadrant (Q1).

6.2 Scratch wounding migration assay

Determining the ability of MGCD0103 to suppress the motility of cells was evaluated by migration assay. Both 2x10⁵ T98G and C6 cells were grown in 6 well after the cells approximately reached a confluence rate of 90%. A 200-L sterile plastic pipette tip was used to gently scrape the cell monolayers in the center of the well. To eliminate the floating and debris cells, the gap was twice rinsed with a growing media. The old medium was removed from each well and a fresh medium containing various concentrations of MGCD0103 was added to each well and incubated with 5% CO₂ at 37⁰C for 24 h. Then the medium was discarded and fixed with 70% alcohol. Inverted light microscopy with a camera (BioTeck Instruments, Inc, VT, USA) was used to examine the cells' movement into the wound area. and the distance between the scratches was analyzed by Image J. To calculate the percentage of wound closure, the formula below was used: Closure of a wound is equal to [1-(wound area at Tt/wound area at T0) 100. The wound's creation time is T0, and the time since it was caused is Tt. Three times the tests were run.

7.2 Matrigel Invasion assay

Briefly, C6 and T98G cells were cultivated in a 24-transwell chamber supplied with 8.0µm pore size polycarbonate filters (Corning Life Science, Corning, NY, USA) covered in 200 µL Matrigel (Corning Life Science, Corning, NY, USA) at a final concentration 1.0 mg/mL, which was allowed to solidify at 37⁰C for 4h. 1x10⁵ cells were suspended with 100 µL of growth medium-free serum and placed into the upper chamber of the transwell. In the lower chamber of the transwell plate, 600 µL of RPMI-1640 growth medium containing 10% FBS and 1% penicillin + streptomycin supplement with various concentrations of MGCD0103 was added and then incubated for 2 days in 5% CO2 at 37⁰C for 48h. At the end of incubation, the cells had invaded the Matrigel and attached to the lower surface of the filter. Consequently, the medium from the upper chamber was discarded, and the cell migration was fixed by alcohol 70% for 1 min. Once the alcohol was removed, the cells were stained with 0.1% crystal violate for 15 min. Then, the cells were washed with double distilled water. By swabbing cotton, the non-migration cells were eliminated. Light microscopy was used to examine the cells' passage through the Matrigel and onto the lower surface of the filter. (Leica Microsystems, Inc, USA) and the percentage of cells’ migration was quantified using the Image J program (National Institutes of Health, Bethesda, MD, USA).

8.2 Invitro network angiogenesis assay

To study the effect of various concentrations of MGCD0103 on the inhibition of angiogenesis, we achieved network angiogenesis in vitro by using the Co-Culture Tube Formation Assay kit (Ibidi, Gräfelfing, Germany) as described in the company’s instructions. The Matrigel (10 µL) was placed in a 15-well chamber slide and incubated with 5% CO₂ at 37⁰C to solidify for 1h. Each of the C6 and T98G cells at a density of 2.5 x10⁴ was separately mixed with 10⁴ human mammary epithelial (HME) cells as co-culture, then they were added to each tube containing 50 µL RPMI and incubated for 6 h to network formation. After the culture medium was aspirated off the Matrigel surface, 50 µL of growth medium supplement of different concentrations was added to each well-containing network. The chamber slide was incubated for 24 h in 5% CO₂ and 37C⁰. After that, the culture medium was expelled. Cells were fixed with methanol and the image was observed by light microscopy (Leica Microsystems, Inc, USA). The tube information was counted by Image J software (National Institutes of Health, Bethesda, MD, USA).

9.2 Proteins extraction by SDS-PAGE assay

Protein extracted from both cell lines C6 and T98G were cultured in (60mm) of growth medium RPIM with 10% FBS, 1% Penicillin /Streptomycin, and incubated in 5% CO₂ at 37⁰C. After the cells cultured reached confluence, the culture medium was removed and replaced with a culture medium supplement with various concentrations of MGCD0103 for 48 h at 37⁰C. Once the treatment period was finished, the pellet from the cells was scraped and washed twice with ice-cold PBS. The cells were centrifuged at 2500rpm for 5 min and discarded in their supernatant. After that, the pellet cells were lysate with 250µL of cold RIPA buffer (25mM Tris•HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Thermofisher, IL, USA) and kept on ice for 30 minutes. The test tubes were shaken five times by vortex. After being lysate, they were centrifuged at 13,000rpm for 15 min at 4⁰C. The supernatant-containing proteins were then transferred to tubes and kept at -20⁰C for further analysis.

10.2 Western blot

The protein concertation was determined with BCA and Coomassie blue. The equal amounts of protein from each sample mixed with loading buffer and mixtures were boiled at 100⁰C for 5 min. The samples were separated by electrophoresis on 4–20% of SDS-PAGE gel for 1.5 h. After electrophoresis, the blot was transferred from the gel into the PVDF membrane (Thermofisher, IL, USA). Block with 5% nonfat dry milk in PBS-Tween 20 (0.05%, v/v) for 1 hour at room temperature. Following the membrane were incubated with the primary antibody overnight. The next day, the membrane was washed five times with PBS. Subsequently, a Suitable horseradish peroxidase-conjugated secondary antibody was incubated with the membranes. that was added to the membrane for 1h at room temperature. After washing with (PBST) five times at room temperature, the membrane was incubated with enhanced electro chemiluminescent for 5 min the results were acquired by X-ray film.

11.2 Antibody

The antibody anti-B-actin (#4967) (Cell Singling, USA) was purchased from Cell Signaling (Cell Signaling, MA, USA). The other primary antibodies used in this study were Bax(sc-4780), Bcl2(sc-7382), Bid(sc-6539) HDAC1, (sc-7872), Caspase 3(sc-7148), MMP9 (sc-13520), MMP 2(sc-1359), GAFP(sc-6170), pRB( sc-12901(), Rb(sc-15)N-Myc(sc-42), Id2 (sc-489), VEGF (sc-7269), EGFR2 (sc-271847), P-BAD( sc-133356), pAKT-1(sc-514032) were purchased from Santacruz Biotechnology (Santa Cruz, CA, USA) PI3K ( GTX50858) was purchase from Gene Tex, USA, AKT( 6744) was purchased Bio vision, USA). Horseradish peroxidase-conjugated secondary anti-rabbit was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-mouse (Santa Cruz, CA, USA)

12.2 Statically analysis

Data were represented as the mean standard error (± SEM). All experiments were repeated three times. In all experiments, results were analyzed using GraphPad Prism software (one-way ANOVA). Statically differences between groups were considered relevant at P > 0.05. All measurements were compared with the control. P*<0.05, P**<0.01,p#<0.001

1–3 Evaluation effect MGCD0103 on cell viability by MTTS assay

To investigate the ability of the anti-tumor activity of MGCD0103 on cell proliferation of glioblastoma cell lines C6 and T98G, we examined cell viability for both cell lines by a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MMT assay (Fig. 1). The glioblastoma was treated with various concentrations of MGCD0103 (0.5,1.5,2.0,2.5) µM for 48h. The MTT results showed that the viability of cells in both cell lines was significantly decreased with the increased dose of MGCD0103 compared with the control. The percentage of inhibition in growth cells was different based on the dose-dependent manner of drugs. Low concentrations of MODC0103 (0.5 µm) did not show significant effects on the growth of glioblastoma cell lines C6 and T98G, while other concentrations of MGCD0103 (1.0,1.5,2.0,25 µm) significantly reduced ((P < 0.005) growth cells in both cell lines. The higher inhibitory ratio in the proliferation of cells was with doses (2.0, 2.5) µM, which reached 35% and 41.6%, respectively in C6, while the inhibitory ratio was 54% and 53%, respectively in T98G.

2.3 Differentiation assay

The MGCD0103 potently affects the differentiation of glioblastoma cells C6 and T98G. The morphological features of differentiation cells were small and retracted cell bodies with thin, elongated, and branched processes. The changes in morphological characteristics appeared after the cells were treated with a variety of doses of MGCD0103 for 48h compared with an untreated cell (control) that kept the vast cell body with short processes (Fig. 2). The concentrations of MGCD0103(1.5) µM significantly contributed to an increase of cell length and decrease of cell width in C6. In contrast, the perfect concentration in increased length and width of cells was (2.5) µМ in T98G. The length of cells reached 481% and the width of cells 21% in C6, while the length of cells got 575%, and the width of cells was 38% in T98G. The protein expression in differentiation was achieved by the western blot after treatment. The biomarker of astrocytic differentiation in both cell lines (GFAP) was upregulated, while the proteins mediated in the dedifferentiation of cells (Id2, n-MYC) were downregulated after treatment with various diosgenin concentrations.

3.3 Wright staining

The morphological signs of apoptotic cell death for both cell lines C6 and T98G were detected by in situ wright staining assay (Fig. 3). The apoptosis percentage increased after treatment with different concentrations of MGDC0103. The morphological features for apoptosis have represented blebbing, shrinkage of cell volume, condensation of chromatin and fragmentation, nuclear fragmentation, and membrane-attached apoptotic bodies. These features significantly increased(p < 0.005) in apoptosis with the high doses of MGDC0103 (20, 25) µm. The percentage of apoptotic cells that led to apoptosis was 45% and 58% in doses (20, 25) µM, respectively, in C6 cells. In contrast, the apoptosis ratio reached 28%.36% in concentrations (20, 25) µM respectively in T98G compared with control cells. The results demonstrated that MGCD0103 plays a vital role in the induction of apoptosis.

4.3 Flow cytometry

The biochemical characteristics of apoptosis in both cell lines C6 and T98G were detected via annexin V-FIC/PI staining and analyzed by flow cytometry (Fig. 3). The early apoptotic population of Annexin V positive cells in A4 has increased after treatment with different concentrations of MGCD0103. This increase depended on the percentage of concentration added to cells. The data indicated that the inhibition rate significantly increased in high concentrations (20, 25) µM, which reached 62.6% and 68.75%, respectively, in C6, and induced apoptotic increase with increased doses. In comparison, the percentage of apoptotic cells was 43% and 56.3% in 20.25 µM, respectively in T98G.

5.3 Role protein involved in inducing activated extrinsic and intrinsic apoptosis pathway apoptosis in glioblastoma.

The morphological and biochemical features of apoptosis in glioblastoma cells after treatment with different concentrations of MGCD0103 have induced the mitochondria to activate the extrinsic and intrinsic pathways. These pathways are involved in many proteins’ expressions. Western blot assay was used to evaluate the expression of these proteins and the expression of B-actin that was used as a control (Fig. 4). The results of the western blot found that MGDC0103 activated extrinsic pathways by increasing the levels of Caspase 8 and t-bid. Also, the varied concentrations of MGCD0103 highly contributed to inducing intrinsic pathways by upregulating Bax, which represents a pro-apoptotic protein, and downregulating bcl2, which is anti-apoptotic. The increase in Bax Bcl2 usually contributes to the induction expression of caspase 3 and AIF. To demonstrate whether MGCD0103 contributes to an increased level of caspase 3. The results found that the expression of procaspase 3 has decreased in both cell lines C6 and T98G after treatment with the drug, while cleavage to caspase 3 has increased after treatment. Also, we investigated the influence of varied doses of MGCD0103 in induction cell cycle arrest in both cell lines by changing protein expression mediated in cell cycle arrest. Retinoblastoma’s suppressor gene (Rb) which represents a marker of cell cycle arrest was upregulated after treatment with different concentrations of MGCD0103 in a dose-dependent manner. In contrast, the expression of pRB and E2F involved in the S phase in cell cycle progression was low after treatment. HDAC1 enzyme is essential in epigenetic alternation, which requires cell proliferation and inhibits apoptosis. By western blot, we investigated the effect of MGCD0103 on HDAC1 protein expression in both cell lines. The level of HDAC1 protein was significantly reduced in two glioblastoma cell lines C6 and T98G after dealing with various concentrations of MGCD0103.

6.3 Wound healing

The effect of anti-cancer drugs MGCD0103 on cell migration was examined. The result of the wound healing assay showed that various concentrations of MGCD0103 had a restrained influence on the motility of both glioblastoma cell lines compared with the control that increased cell migration (Fig. 5). The high concentration of MGCD013 highly inhibited cell migration compared with a low concentration of MGCD0103. The wound closure rate had reduced to 55% and 47% when the C6 was treated with (20, 25) µM, respectively. A similar reduction in migration ability was noticed when T98G cells were treated with different concentrations of MGCD0103. The number of cell motility had decreased to 56% and 52% respectively, in concentrations (20, 25) µM of MGCD0103.

7.3 MGCD0103 inhibits the invasion cells

We investigated whether the MGCD0103 could inhibit glioblastoma cell invasion across the extracellular matrix and performed a transwell invasion assay. The results of the invasion assay showed that MGCD0103 suppressed cell invasion in both cell lines C6 and T98G compared with control in a dose-dependent manner (Fig. 5). The number of cell invasions through transwell-coated Matrigel had reduced with increasing MGCD0103 concentrations in cell lines C6 and T98G. The inhibitory percentage of cell invasion in C6 reached 20.3% and 13.3% with (20, 25) µM, while the T98G cell invasion rate was 28% and 14% with 20% and 25%, respectively, compared with untreated cell control. It has been widely recognized that the migration and invasion of glioblastoma cells always mediate the activation of the PI3k /AKT pathway. We performed a western blot to explore the role of MGCD0103 on the expression of PI3k and phosphorylation of Akt. The western blot results have shown that the expressions of two proteins expression were downregulated after treatment with MGCD0103 compared with untreated cells. Also, the expression of MMP2 and MMP9 is required for invasion by the degradation of the extracellular matrix. We assessed the effect of MGCD0103 on the expression of these two proteins by western blot. The results demonstrated that MGCD0103 could inhibit the breakdown of the extracellular matrix by downregulating protein levels of MMP2 and MMP9. The expression of MMP2 and MMP9 was significantly reduced after treatment with 2.5 µM of MGCD0103 in both cell lines c6 and T98G. These results indicate that MGCD0103 might be essential in inhibiting invasion and migration of glioblastoma cell lines.

8.3 Angiogenesis assay

To examine the inhibitory effects of MGCD0103 on in vitro angiogenesis assay, cell lines C6 and T98G were treated with various concentrations of MGCD0103. The MGCD0103 had different cytotoxicity effects depending on doses of MGCD0103 and the type of glioblastoma cells. The drug has a critical role in decreasing the number of tube formations of T98G and C6 glioblastoma cell lines compared with the control (Fig. 6). Also, the high doses significantly reduced the number of tube formations compared with low concentrations and control cells. The percentage of tube formations represented in bar graphs clearly showed that coculturing HME with C6 decreased to 54% and 45% with (20, 25) µM, respectively, while the coculturing HME with T98G was 57%, 64% with (20, 25) µM. VEGF plays a vital role in angiogenesis. EGFR attaches to the cell surface receptor and activates angiogenesis. We studied the effect of various concentrations of MGCD0103 on the activation of VEGF and EGFR expression. The results have shown the MGC0103 significantly downregulated the expression of these two proteins. Since rapamycin (mTOR) and NFkB are proteins mainly involved in the induction of VEGF mediated in angiogenesis by activating PI3K/AKT phosphorylation.

MGCD0103, one of the HDAC inhibitors, has a broad spectrum as antitumor activities against different types of cancer cells in vivo and in vitro without any cytotoxicity on animals (Founel et al., 2014). Histone deacetylase-1 (HDAC 1) is overexpressed in human glioblastoma, and they participate in varied biological processes including cell growth, invasion, and migration of cells. Knockdown of HDAC1 can inhibit the initiation and progression of cancer cells(Was et al., 2019;Wang et al.,2017). The MGCD0103 is an isotype selective HDAC inhibitor and targets HDAC1,2,3,11(Zhou et al., 2008). The present study demonstrated that MGCD0103 has an influential inhibitory role in treating two glioblastoma cell lines C6 and T98G by inducing apoptosis, differentiation, and inhibiting angiogenesis. The study has also shown a high ability of MGCD0103 in the prevention of the spread of cancer by reducing invasion and migration cells through the capability of MGCD0103 in the suppression activity of HDAC1. In addition, this study conducted the knowledge molecular mechanism of MGCD0103 in the induction of apoptosis, differentiation, and inhibition of migration and invasion cells in a dose-dependent manner.

Apoptosis is a biological process aim to eliminate proliferation cells. The western blot results revealed that Mocetinostat played a role in induction apoptosis in glioblastoma through the suppression of HDAC1 involved in regulating different proliferative pathways. Caspases induce apoptosis via cleaving specific sets of substrates, and apoptotic pathway of mitochondria is the main pathway for stimulating cell death in cancer cells through activating caspase (Liao et al.,2020). Mocetinostat were promoted the intrinsic and extrinsic pathway by increasing proapoptotic proteins (Bax) and decreasing protein expression (Bcl2 and p-Bad and bid) inducing the proliferation of cancer cells. The increase in protein expression of Bax/Bcl2 led to suppress of cells’ viability through releasing cytochrome C. As it’s known cytochrome C is considered an essential agent in the activation of caspases 3 that leads to the cleavage PARP-1 and ICAD involved in apoptosis cells (McIlroy et al.,1999: Affar et al., 2001). Also, HDACi has a significant role in regulating the activity of the Rb/E2f1 pathway, and it has vital role in initiating DNA replication and cell cycle activity (Joseph et al., 2001). Hyperphosphorylated pRB causes an increase in cell proliferation in many cancer cells through the increase of expression E2F1 and cyclin D1 activity required in cell cycle progression (Zhao et al.,2005), while hypophosphorylated RB associated with E2F1 leads to suppression of gene transcriptions responsible of cell cycle progression (Attwooll et al.,2004). HDACi largely suppresses Hyperphosphorylated pRB activity by inhibiting the expression of cyclin D and causing an increase in the hypo-phosphorylation of RB (Kenta et al.,2018). Our study appeared that the Mocetinost could also induce apoptosis by suppressing the oncogenic E2F1 transcription factor and increasing the expression of RB protein. The previous study found that Mocetinostat could induce apoptosis in liver cancer by downregulating the antiapoptotic protein E2F6 and upregulating the expression Bad in two prostate cell lines(DU-145, PC-3 )in vivo and in vitro (Zhang et al.,2016). On the other hand, HDAC1 has an influential role in the promotion of the migration, invasion, and angiogenesis of glioblastoma cells through activation of the PI3K/Akt signaling pathway in vivo and in vitro, so the knockdown of HDAC1 might lead to the suppression of the expression of p-Akt proteins (Li et al.,2018). Also, the previous study found that the suppression of the HDAC1 expression led to the upregulation of the level of cleaved Caspase 3, BAX, and E-Cadherin, and the downregulation expression of MMP9, SNAIL, and TWIST1 in T98G and U251 glioblastoma cell lines (Wang et al., 2017). Our results of the western blot demonstrated that HDACi was significantly suppressed expression of HDAC1 when glioblastoma cells were treated with MGCD0103. The mechanism of MGCD0103 for inhibition of HDAC1 occurs through the attachment of the free amino group of the benzamide of MGCD0103 with the zinc ion in the active site of the HDAC enzyme. This attachment cause inhibition of enzyme activity and induce apoptosis (Fournel et al.,2008. MGCD0103 exerts anti-cancer activity by suppressing the growth of liver cancer cells in vitro by downregulating the expression of Bcl2 and the upregulation of proapoptotic proteins Bax and Bim. At the same time, another study observed that the role of MGC0103 in the inhibition of the proliferation of cancer cells occurs by supporting HDAC1, which is involved in the increase of proliferation of tumor cells. (Liao et al.,2020).

On the other hand, the present study also found that inhibiting HDAC causes the activation of the transcription factors for genes responsible for cell differentiation (Ecker et al.., 2013). The Glial fibrillary acid protein (GFAP) represents a marker of differentiation cells in glioblastoma, and it is presented in low levels in glioblastoma cells (Lee et al., 2005). The HDAC enzyme suppresses GFAP expression in glioblastoma cells (Morita et al., 2009). The histone deacetylase inhibitors (HDACi) promote differentiation in the human glioblastoma cell line via the decrease of HDAC level and the increase of histone acetylation induced GFAP expression in primary human astrocytoma (Svechnikova et al., 2008; Kanski et al., 2014;). HDAC also increases n-Myc transcription factor expression that in turn overactivation Id2 oncogene in cancer cells (Liu et al.,2017; Yoshikawa et al.,2007). Id2 significantly play an important role in inhibition differenation and promotion growth cell in glioma cell (Yun etal.,2005). Our data found that Mocetnostate largely contributed to the upregulation of GFAP protein and downregulation of Id2, n-Myc. Khathayer et al., 2020 showed that a combination of SAHA and 4HPR increased GFAP and deceased Id2, c-Myc in the glioblastoma cell line C6 and T98G by downregulating of HDAC. Other studies indicated that other HDACi such as 4-PBA, TSA, and VPA promoted the differentiation in glioblastoma cell lines by increasing the expression of GFAP proteins that led to enhancing gap-junction communication between tumor cells and decreased expression of Id2 (Svechnikova et al, 2008; Asklund et al., 2004; Roy et al.,2011).

Cell migration is a set of multicomplex steps that start in response to migratory and chemotactic stimuli by forming protrusion in the cell membrane called invadopodia protostomes(Yamaguchi and Condeelis,2007). In the current study, Mocetinostat has been shown to have high activity in the inhibition of invasion and migration of glioblastoma cells by suppressing PI3K/AKT signaling pathway that is considered one of the most critical intracellular signaling pathways involved in cancer progressions such as invasion, migration, and angiogenesis (Crespo et al.,2016). The pathway pI3k/AKT is one of the up-streams that regulate extracellular matrix metalloprotease (MMP2, MMP9) (Zhang et al.,2015). Consequently, the results of western blot showed that mocetinostat repressed the level of MMP2, MMP9 which are considered the enzymes involved in the degradation of the extracellular matrix(ECM) in glioblastoma and highly expressed in glioblastoma (Forsyth et al.,1999). Another study observed that expression MMP2 and MMP9 are controlled by expression Class I HDAC through deacetylating of transcription factor attaching to promoter of MMP2and MMP9. Hence, treatment of cells with HDACi caused decease the level expression of MMP2,MMP9 (Mani etai.,2015)

We also examined whether mocetinostat would suppress angiogenesis in glioblastoma cells or no, which is also one of the significant biological activities in the progression of glioblastoma cancer cells (Onishi et al.,2013). The angiogenesis in glioma cells is induced by increasing expression of pI3k/Akt protein and VEGF in glioma cells (Cea et al.,2012). The result of coculture HME- glioblastoma cell line C6 and T98G has been found that mocetinostat inhibited growth angiogenesis by inhibiting EGFR singling pathway promoted VEGF gene expression via HIF independent mechanism (Zhang et al.,2008). Another study also found that mocetinostat inhibits tubule growth of cultured human endothelial cells by inducing transcription of anti-angiogenesis factor, Thrombospondin-1 human cancer cell (Liu et al.,2008).

In conclusion, our result in this study demonstrated the cytotoxic effects of different concentrations of mocetinostat (MGCD0103) on two glioblastoma cell lines C6 and T98G, and our finding in this study helped in understanding the molecular mechanism of MGCD0103 in induction the apoptosis and the differentiation in a manner dependent on dose and time. Moreover, the MGCD0103 played a vital role in suppressing migration, invasion, and angiogenesis on glioma. MGCD0103, a small molecular inhibitor, exerts anti-cancer activities against glioblastoma and is worth employing as a successful therapy to treat glioblastoma.

HDAC/ Histon deacetylase

PI3K/ Phosphatidylinositol-3-kinase

GFAP / Glial fibrillary acidic protein

Id2/ Inhibitor of DNA Binding 2

DMSO/dimethyl sulfoxide

RPMI /1640 medium/ Roswell Park Memorial Institute medium

HEPES/ 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HME/ Human Mammary Epithelial

BCA/ Bicinchoninic acid

PVDF/ Polyvinylidene fluoride

PBS /Phosphate-buffered saline

MMP-9/ Matrix metalloproteinase -9

MMP-2/ Matrix metalloproteinase -2

VEGF /Vascular endothelial growth factor

EGFR /Epidermal growth factor receptor

NF-κB / Nuclear factor kappa B

mTOR / Mammalian target of rapamycin

E2f6 / E2 transcription factor6

Bax / Bcl2-associated X protein

Bcl-2 /B-cell lymphoma 2 protein

TWIST1 /Twist-related protein 1

TSA / Trichostatin

VPA / Valproic acid.

HMVC/Human mammalian epithelia cell

PARP-1/Poly(ADP-ribose) polymerase-1

ICAD/A caspase-activated deoxyribonuclease inhibitor

ECM / Exocellular matrix

Funding

The work was supported by scholarship The Higher Committee for Education Development of Iraq and part by the R01 grants (CA-091460 and NS-057811) from the National Institutes of Health (Bethesda, MD, USA).

Acknowledgements

We thank the lab Dr. Swapan Ray lab for working in it .

Ethics approval and consent to participate.

Not applicable.

Consent for publication

Not applicable.

competing interests

The authors declare no conflict of interest.

Authors information

Department of Biology, University of Mosul School of college of sciences , Al Majmoaa Street. Postal Code : 41002. Mosul – Iraq. Dr. Firas Khathayer is Assistant Professor in Biomedical Sciences & Dr. Mohammed Hussein Mikael is Assistant Professor in Biochemistry

Corresponding author

Correspondence to Dr. Firas Khathayer

[email protected]

Li G, Margueron R, Hu G, Stokes D, Wang YH, Reinberg D (2010). Highly compacted chromatin formed in vitro reflects the dynamics of transcription activation in vivo. Mol Cell. 38(1):41-53. doi: 10.1016/j.molcel.2010.01.042. PubMed PMID: 20385088; PubMed Central PMCID: PMC3641559.
Sharma S, Kelly TK, Jones PA (2010). Epigenetics in cancer. Carcinogenesis. 31(1):27–36. doi:10.1093/carcin/bgp220.
Perri F, Longo F, Giuliano M, Sabbatino F, Favia G, Ionna F, Addeo R, Scarpati GDV, Di Lorenzo G, Pisconti S (2017). Epigenetic control of gene expression: Potential implications for cancer treatment. Crit. Rev. Oncol. Hematol 111:166–172. doi: 10.1016/j. critrevonc. 2017.01.020
Minucci S, Pelicci PG (2006). Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 6: 38–51.
Lucio-Eterovic AK, Cortez MA, Valera ET, Motta FJ, Queiroz RG, Machado HR, Carlotti CG, Jr, Neder L, Scrideli CA, Tone LG (2008). Differential expression of 12 histone deacetylase (HDAC) genes in astrocytomas and normal brain tissue: class II and IV are hypoexpressed in glioblastomas. BMC Cancer 8:243 doi: 10.1186/1471-2407-8-243.
Wang XQ, Bai HM, Li ST, Sun H, Min LZ, Tao BB, Zhong J, Li B (2017). Knockdown of HDAC1 expression suppresses invasion and induces apoptosis in glioma cells. Oncotarget 8(29):48027-48040. doi: 10.18632/oncotarget.18227. PubMed PMID: 28624794; PubMed Central PMCID: PMC5564623.
Chen QW, Zhu XY, Li YY, Meng ZQ (2014). Epigenetic regulation and cancer (review). Oncol Rep 31(2):523-32. doi: 10.3892/or.2013.2913. Epub 2013 Dec 11. Review. PubMed PMID: 24337819.
Zhang Q, Sun M, Zhou S. et al. (2016). Cell Death Discovery 2: 16036 (2016) doi:10.1038/cddiscovery.2016.36
Fournel M, Bonfils C, Hou Y, Yan PT, Trachy-Bourget MC, Kalita A, Liu J, Lu AH, Zhou NZ, Robert MF, Gillespie J, Wang JJ, Ste-Croix H, Rahil J, Lefebvre S, Moradei O, Delorme D, Macleod AR, Besterman JM, Li Z (2008). MGCD0103, a novel isotype-selective histone deacetylase inhibitor, has broad spectrum antitumor activity in vitro and in vivo. Mol Cancer Ther 7(4):759-68. doi: 10.1158/1535-7163.MCT-07-2026. PubMed PMID: 18413790.
Sikandar S, Dizon D, Shen X, Li Z, Besterman J, Lipkin SM (2010). The class I HDAC inhibitor MGCD0103 induces cell cycle arrest and apoptosis in colon cancer initiating cells by upregulating Dickkopf-1 and non-canonical Wnt signaling. Oncotarget 1(7):596-605. PubMed PMID: 21317455; PubMed Central PMCID: PMC3093052.
Younes A, Oki Y, Bociek RG, Kuruvilla J, Fanale M, Neelapu S et al. (2011). Mocetinostat for relapsed classical Hodgkin's lymphoma: an open-label, single-arm, phase 2 trial. Lancet Oncol 12(13):1222-1228 doi: 10.1016/S1470-2045(11)70265-0. Epub 2011 Oct 25.
Louis DN, Perry A, Reifenberger G, et al.. (2016). The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathol 131(6):803–820. doi:10.1007/s00401-016-1545-1
Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, et al.(2005).Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352(10):987–996. doi: 10.1056/NEJMoa043330
Lee DH, Ryu HW, Won HR, Kwon SH (2017). Advances in epigenetic glioblastoma therapy. Oncotarget 8(11):18577–18589. doi:10.18632/oncotarget.14612
Li S, Chen X, Mao L, Zahid KR, Wen J, Zhang L, Zhang M, Duan J, Duan J, Yin X, Wang Y, Zhao L, Tang X, Wang X, Xu G(20180.Histone deacetylase 1 promotes glioblastoma cell proliferation and invasion via activation of PI3K/AKT and MEK/ERK signaling pathways. Brain Res 1(1692):154-162. doi: 10.1016/j.brainres.2018.05.023. Epub 2018 May 18. PubMed PMID: 29782850.
Liao B, Sun Q, Yuan Y, Yin Y, Qiao J, Jiang P (2020). Histone deacetylase inhibitor MGCD0103 causes cell cycle arrest, apoptosis, and autophagy in liver cancer cells. J Cancer 11(7):1915-1926. doi: 10.7150/jca.34091. eCollection 2020. PubMed PMID: 32194803; PubMed Central PMCID: PMC7052879.
Mani SK, Kern CB, Kimbrough D, Addy B, Kasiganesan H, Rivers WT, Patel RK, Chou JC , Spinale FG, Mukherjee R, Menick DR(2015). Inhibition of class I histone deacetylase activity represses matrix metalloproteinase-2 and -9 expression and preserves LV function postmyocardial infarction. Am J Physiol Heart Circ Physiol. 308(11):H1391-H1401. doi:10.1152/ajpheart.00390.2014
McIlroy D, Sakahira H, Talanian RV, Nagata S (1999). Involvement of caspase 3-activated DNase in internucleosomal DNA cleavage induced by diverse apoptotic stimuli. Oncogene. 18(31):4401-8. PubMed PMID: 10442630.
Affar EB, Germain M, Winstall E, Vodenicharov M, Shah RG, Salvesen GS, Poirier GG (2001). Caspase-3-mediated processing of poly (ADP-ribose) glycohydrolase during apoptosis. J Biol Chem 276(4):2935-42. Epub 2000 Oct 25. PubMed PMID: 11053413.
Wang XQ, Bai HM, Li ST, et al. (2017). Knockdown of HDAC1 expression suppresses invasion and induces apoptosis in glioma cells. Oncotarget 8(29):48027–48040. doi: 10.18632 / onco target .18227.
Was H, Krol SK, Rotili D, et al.. (2019). Histone deacetylase inhibitors exert anti-tumor effects on human adherent and stem-like glioma cells. Clin Epigenetics 11(1):11. Published 2019 Jan 17. doi:10.1186/s13148-018-0598-5
Svechnikova I, Almqvist PM, Ekström TJ (2008). HDAC inhibitors effectively induce cell type-specific differentiation in human glioblastoma cell lines of different origin. Int J Oncol. 32(4):821-7. PubMed PMID: 18360709.
Kanski R, Sneeboer MA, van Bodegraven EJ, Sluijs JA, Kropff W, Vermunt MW, Creyghton MP, De Filippis L, Vescovi A, Aronica E, van Tijn P, van Strien ME, Hol EM(2014). Histone acetylation in astrocytes suppresses GFAP and stimulates a reorganization of the intermediate filament network. J Cell Sci 127(Pt 20):4368-80. doi: 10.1242/jcs.145912. Epub 2014 Aug 15. PubMed PMID: 25128567.
Asklund T, Appelskog IB, Ammerpohl O, Ekström TJ, Almqvist PM (2004). Histone deacetylase inhibitor 4-phenylbutyrate modulates glial fibrillary acidic protein and connexin 43 expression, and enhances gap-junction communication, in human glioblastoma cells. Eur J Cancer 40(7):1073–81. doi: 10.1016/j.ejca.2003.11.034
Lee K, Jeon K, Kim JM, Kim VN, Choi DH, Kim SU, Kim S (2005). Downregulation of GFAP, TSP-1, and p53 in human glioblastoma cell line, U373MG, by IE1 protein from human cytomegalovirus. Glia 51(1):1-12. PubMed PMID: 15779089.
Morita K, Gotohda T, Arimochi H, Lee MS, Her S (2009). Histone deacetylase inhibitors promote neurosteroid-mediated cell differentiation and enhance serotonin-stimulated brain-derived neurotrophic factor gene expression in rat C6 glioma cells. J Neurosci Res 87(11):2608-14. doi: 10.1002/jnr.22072. PubMed PMID: 19360904.
Ecker J, Witt O, Milde T (2013). Targeting of histone deacetylases in brain tumors. CNS Oncol 2(4):359–376. doi:10.2217/cns.13.24
Yamaguchi H, Condeelis J (2007). Regulation of the actin cytoskeleton in cancer cell migration and invasion. Biochim Biophys Acta. 2007;1773(5):642–652. doi: 10.1016/j.bbamcr.2006.07.001
Crespo S, Kind M, Arcaro A (2016). The role of the PI3K/AKT/mTOR pathway in brain tumor metastasis. J Cancer Metastasis Treat 2:80-89. http://dx.doi.org/10.20517/2394-4722.2015.72
Zhang FY, Hu Y, Que ZY, et al.. (2015). Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated β-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine. Int J Mol Sci 16(10):23823–23848. Published 2015 Oct 9. doi:10.3390/ijms161023823.
Forsyth P.A., Wong H., Laing T.D., Rewcastle N.B., Morris D.G., Muzik H., Leco K.J., Johnston R.N., Brasher P.M.A., Sutherland G., et al.. (1999). Gelatinase-A (MMP-2), gelatinase-B (MMP-9) and membrane type matrix metalloproteinase-1 (MT1-MMP) are involved in different aspects of the pathophysiology of malignant gliomas. Br. J. Cancer 79:1828–1835. doi: 10.1038/sj.bjc.6990291.
Liu J, Bonfils C, Dubay M, h Nguyen H, Garcia-Manero G, Yang A, Luger S , Martell R, Besterman J , Li Z(2008).Induction of an anti-angiogenesis factor, Thrombospondin 1 (TSP-1), by a novel histone deacetylase inhibitor, MGCD0103, in human cancer cells in vitro and in vivo. American Association for Cancer Research 68 (9): 739.
Cea V, Sala C, Verpelli C (2012). Antiangiogenic therapy for glioma. J Signal Transduct 2012: ID 483040 doi:10.1155/2012/483040.
Zhang Y, Zhang N, Dai B, Liu M, Sawaya R, Xie K, Huang S (2008). FoxM1B Transcriptionally Regulates Vascular Endothelial Growth Factor Expression and Promotes the Angiogenesis and Growth of Glioma Cells. Cancer Res 68(21):8733- 8742; DOI: 10.1158/0008-5472.CAN-08-1968
Zhou N, Moradei O, Raeppel S, et al.. (2008). Discovery of N‐(2‐aminophenyl)‐4‐[(4‐pyridin‐3‐ylpyrimidin‐2‐ylamino)methyl]benzamide (MGCD0103), an orally active histone deacetylase inhibitor. J Med Chem 51:4072‐4075. doi.org/10.1021/jm800251w.
Roy Choudhury, S., Karmakar, S., Banik, N.L. et al.. (2011). Valproic Acid Induced Differentiation and Potentiated Efficacy of Taxol and Nanotaxol for Controlling Growth of Human Glioblastoma LN18 and T98G Cells. Neurochem Res 36(12):2292–2305. doi:10.1007/s11064-011-0554-7
Onishi M, Kurozumi K, Ichikawa T, Date I (2013). Mechanisms of tumor development and anti-angiogenic therapy in glioblastoma multiforme. Neurol Med Chir (Tokyo) 53(11):755–763. doi:10.2176/nmc.ra2013-0200
Nevins JR (2001). The Rb/E2F pathway and cancer. Human Molecular Genetics 10(7): 699–703.doi.org/10.1093/hmg/10.7.699.
Zhao Y, Tan J, Zhuang L, Jiang X, Liu ET, Yu Q (2005). Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activation of proapoptotic protein Bim. Proc Natl Acad Sci U S A 102(44):16090–16095. doi:10.1073/pnas.0505585102.
Attwooll C, Lazzerini Denchi E, Helin K (2004). The E2F family: specific functions and overlapping interests. EMBO J. 23(24):4709–4716. doi: 10.1038/sj.emboj.7600481
Kurayoshi K, Ozono E, Iwanaga R, Bradford AP, Komori H, Araki K and Ohtani K (2018). The Key Role of E2F in Tumor Suppression through Specific Regulation of Tumor Suppressor Genes in Response to Oncogenic Changes. In book Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics, Fumiaki Uchiumi, IntechOpen. http://dx.do.org/ 10.5772/intechopen.72125.
Liu T, Tee AE, Porro A, et al. (2007). Activation of tissue transglutaminase transcription by histone deacetylase inhibition as a therapeutic approach for Myc oncogenesis. Proceedings of the National Academy of Sciences of the United States of America. 104(47):18682-18687. DOI: 10.1073/pnas.0705524104.
Yoshikawa M, Hishikawa K, Marumo T, Fujita T. Inhibition of histone deacetylase activity suppresses epithelial-to-mesenchymal transition induced by TGF-beta1 in human renal epithelial cells. J Am Soc Nephrol. 2007;18(1):58–65. doi:10.1681/ASN.2005111187

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Mocetinostat as a novel selective histone deacetylase (HDAC) inhibitor in the promotion of apoptosis in glioblastoma cell line C6 and T98G

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

1.2 Cell culture and treatment

2.2 A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay

3.2 In vitro differentiation cancer cells assay

4.2 Detection of Morphological characteristics using Apoptosis assay

6.2 Scratch wounding migration assay

7.2 Matrigel Invasion assay

8.2 Invitro network angiogenesis assay

9.2 Proteins extraction by SDS-PAGE assay

10.2 Western blot

11.2 Antibody

12.2 Statically analysis

Results

1–3 Evaluation effect MGCD0103 on cell viability by MTTS assay

2.3 Differentiation assay

3.3 Wright staining

4.3 Flow cytometry

6.3 Wound healing

7.3 MGCD0103 inhibits the invasion cells

8.3 Angiogenesis assay

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1