Valproic Acid (VPA) in Combination with Knockdown of AKT3 and PI3KCA Genes Inhibits Proliferation, Induces Apoptosis and Autophagy in T98G and U87MG Glioblastoma Multiforme Cells

Research Article

Austin J Anat. 2021; 8(2): 1101.

Valproic Acid (VPA) in Combination with Knockdown of AKT3 and PI3KCA Genes Inhibits Proliferation, Induces Apoptosis and Autophagy in T98G and U87MG Glioblastoma Multiforme Cells

Paul-Samojedny M*, Liduk E, Borkowska P, Kowalczyk M, Suchanek-Raif R, Zielinska A and Kowalski J

Department of Medical Genetics, School of Pharmacy in Sosnowiec, Medical University of Silesia, Katowice, Poland

*Corresponding author: Paul-Samojedny M, Department of Medical Genetics, School of Pharmacy in Sosnowiec, Medical University of Silesia in Katowice, Jednosci 8 Street, 41-200 Sosnowiec, Poland

Received: July 13, 2021; Accepted: July 27, 2021; Published: August 03, 2021


Purpose: Glioblastoma Multiforme (GBM) is a heterogenous and highly vascularized brain tumor that avoid apoptosis due to P-glycoprotein (P-gp) mediated multi-drug resistance. Therefore, development of new therapeutic strategies that induce apoptosis, inhibit proliferation, and overcome multi-drug resistance is urgently warranted. We examined the efficacy of combination of Valproic Acid (VPA) and knockdown of AKT3 and PI3KCA genes in human glioblastoma T98G and U87MG cell lines.

Material and Methods: T98G and U87MG cells were transfected with AKT3 or PI3KCA siRNAs. Transfection efficiency was assessed using Flow Cytometry (FC) and fluorescence microscopy. The influence of AKT3 and PI3KCA siRNAs in combination with VPA on T98G and U87MG cell viability, proliferation, apoptosis and autophagy was evaluated as well. Alterations in the mRNA expression of apoptosis-related genes (CASP3 and Bid) were analyzed using QRT-PCR.

Results: The transfection of T98G and U87MG cells with AKT3 or PI3KCA siRNAs and exposition on VPA led to a significant reduction in cell viability, the accumulation of subG1-phase cells and a reduced fraction of cells in the S and G2/M phases, apoptosis or necrosis induction and induction of autophagy.

Conclusions: The siRNA-induced AKT3 and PI3KCA mRNA knockdown in combination with VPA may offer a novel therapeutic strategy to more effective control the growth of human GBM cells. Thus, knockdown of these genes in combination with valproic acid inhibits proliferation, induces apoptosis and autophagy in T98G and U87MG cells, but further studies are necessary to confirm a positive phenomenon for the treatment of GBM.

Keywords: Glioblastoma multiforme; Valproic acid; siRNA; Proliferation; Cell death


Valproic Acid (2-propylpentanoic acid; VPA) is a branched short-chain fatty acid used as an antiepileptic drug traditionally used for the treatment of certain types of seizures [1,2]. Meta-analysis performed by Yuan et al. suggests that glioblastoma patients may experience prolonged survival due to VPA administration [3]. For being identified as a potent selective histone deacetylase inhibitor, VPA has gained much attention of its potential central role in epigenetic gene regulation. Histone lysine residues acetylation and deacetylation are posttranslational modifications that influence gene expression activating and repressing gene transcription, respectively [1]. Thus, histone deacetylases play an important role in epigenetic regulation of various programmed cellular processes both in physiological and pathological conditions. VPA is often used to treat seizures in glioblastoma patients. It is known that VPA inhibits cell proliferation by causing cell-cycle arrest in the G1 and/or G2 phase and that it induces differentiation and/or apoptosis in cancer cells. There are evidences that VPA also reduced proliferation rates in glioblastoma-derived stem cells and decreased cell viability of primary human glioblastoma cells. It is very important that VPA may downregulate the expression of MGMT (O-6-methylguanine-DNA methyltransferase) and sensitize human glioma cells to temozolomide and irradiation [4]. Valproic acid has been also shown to induce cellular differentiation, growth arrest and apoptosis in gliomas [5].

Malignant gliomas are characterized by lack of response to implemented therapy leading to a quick recurrence and short lifetime, as well as chemo- and radiotherapy resistance. One of the reasons of observed clinical therapy resistance and malignance of human gliomas is overexpression of PI3K/Akt pathway molecules. PI3Ks (phosphoinositide 3-kinases) constitute a family of lipid kinases that are capable of phosphorylating the 3’OH of the inositol ring in phosphoinositides. These kinases are divided into three classes - class I consists of two subclasses-class IA and class IB, respectively. Class IA includes heterodimers that are composed of a p110 catalytic subunit and a p85 regulatory subunit. A p110 subunit has three isoforms (p110a, p110β and p110γ) that are encoded by the three different genes [6-8] and these isoforms are involved in the regulation of processes such as proliferation, cell survival, degranulation, vesicular trafficking and cell migration.

It is known that in cells in which the p110a isoform of PI3K is predominant or in which both p110a and p110β isoforms are equally important, the knockdown of PIK3CA (p110a) interferes with PI3K/ AKT signaling [9]. The PI3KCA gene has been found to be amplified and overexpressed in several types of cancers including gliomas. It has been suggested that the point mutations that activate the PI3KCA gene may represent a novel mechanism for the induction oncogenic PI3K signaling pathway [10,11]. The PI3K gene amplification may cause the increased AKT activity in tumors. The activation of AKT has been found in approximately 80% of human GBMs [12].

The AKT kinase plays an important role in the PI3K signaling pathway and AKT activity is induced following PI3K activation in various growth factor receptor-mediated signaling cascades [13]. There are three isoforms of AKT (AKT1, AKT2 and AKT3), which are encoded by three different genes. The AKT2 and AKT3 (but not AKT1) isoforms are pathologically amplified in human cancers [14]. AKT3 is primarily expressed in the brain and testis [15]. It is known that AKT2 and AKT3 are overexpressed in glioma cells and play a pivotal role in malignant gliomas [16]. Bearing in mind that cellular behavior is result of intracellular signaling network activation and depends on cross talks between different pathways, it is very interesting to verify if siRNA silencing of PI3K/Akt pathway accelerate VPA-mediated effects.

Thus, the aim of our study is concerned on proliferation, apoptosis and autophagy processes induction and changes in cell cycle AKT3 and PIK3CA genes knockdown and valproic acid exposure. Current study was also undertaken to examine the effect of the siRNAs targeting AKT3 and PIK3CA genes on T98G cells susceptibility on VPA.

Materials and Methods

Cell cultures

The T98G (American Type Culture Collection - ATCC, Manassas, VA, USA) cell line derived from a 61-year-old male [17] and U87MG (Sigma Aldrich) cell line derived from a 44-year-old male. Glioblastoma multiforme cells were cultured in modified Eagle’s Minimum Essential Medium (ATCC) supplemented with heatinactivated 10% fetal bovine serum (ATCC) and 10μg/ml gentamicin (Invitrogen). Cell lines was maintained at 37°C in a humidified atmosphere of 5% CO2 in air.

Knockdown of AKT3 and PI3KCA genes using specific siRNA

Transfection of T98G and U87MG cells with specific siRNAs targeting AKT3 or PI3KCA mRNA was performed using FlexiTube siRNA Premix (Qiagen, Italy) according to the manufacturer’s protocol, as we have described previously [18].

Valproic acid uptake

Freshly prepared stock solutions of VPA was made in serum-free medium just prior to treatment. Dose-response studies were carried out to determine the suitable doses of the drug for the inhibition of cell growth and induction of cell death and 0.5mM concentration of VPA was chosen. T98G and U87MG GBM cells transfected with AKT3 and PI3KCA specific RNAs were treated with valproic acid (0.5mM) for 96h (48h after transfection).

Cell cycle analysis

T98G an U87MG cells were seeded in 6-well plates (at a density of 1.6 x 104 cells per well), cultured overnight (24h) and after AKT3 and PI3KCA genes siRNA silencing, cells were exposure to valproic acid and cell cycle was analyzed. After incubation with specific siRNA, cells were washed with PBS buffer, trypsinized and washed once more with cold PBS buffer. Then, after suspending the cells in 300μl of cold PBS buffer, 70% cold ethanol was added dropwise in a volume of 4mL on a vortex. The cell suspension in ethyl alcohol was incubated at -20°C for 1h, and the contents of the tube were gently mixed every 10 minutes. After incubation with ethanol, the fixed cells were centrifuged and washed with PBS buffer. The cells were then resuspended in 250μl of PBS and RNaseA (10mg/ml final concentration) and propidium iodide (1mg/ml final concentration) were added. Cells were incubated for 1h under standard conditions, and the contents of the tube were gently mixed every 10 minutes. The percentage of cells at different phases of the cell cycle (subG1, G1/G0, S, G2/M, polyploidy) was assessed using a FACSAria II flow cytometer (Becton Dickinson, USA).

Apoptosis assay

T98G and U87MG cells were seeded in 6-well plates (at a density of 1.6 x 104 cells per well), cultured overnight (24h) and after AKT3 and PI3KCA genes siRNA silencing, cells were treated with valproic acid. Cells were analyzed using flow cytometry and a Vybrant® DyeCycleTM Violet/SYTOX® AADvancedTM Apoptosis Kit as described previously [18].

Total RNA extraction

Total RNA was isolated from cells cultured by TRIzol reagent (Life Technologies, Inc, Grand Island, NY, USA) according to the manufacturer’s protocol. The integrity of total RNA was checked by electrophoresis in 1% agarose gel stained with ethidium bromide. All RNA extracts were treated with DNAse I to avoid genomic DNA contamination, and assessed qualitatively and quantitatively.

Changes in mRNA copy number of apoptosis-related genes by RT-qPCR

We have performed QRT-PCR for selected genes associated with apoptosis: CASP3 and BID. RT-qPCR assays were performed using specific primers (KiCqStartTM Primers; Sigma Aldrich) and SensiFASTTM SYBR Hi-ROX One-Step kit (Bioline) according to the manufacturer’s protocol. (CFX96 Real-Time System; BIO-RAD). Real-time fluorescent RT-PCR was performed using Following conditions were taken: 45°C for 10min, 95°C for 2min, followed by 40 cycles of 5sec at 95°C, 10sec at 60°C and 5sec at 72°C. RNA for human TBP (Tata Binding Protein) was used as an endogenous control. The copy numbers for each sample were calculated by the CT-based calibrated standard curve method. Each of 12 data point for mRNA copy numbers is the average of duplicates on the same analyzed plate.

Autophagy identification using LysoTracker Red and flow cytometry

T98G and U87MG cells with or without treatment were loaded LysoTracker Red (LTR) (100nM; Invitrogen), according to the manufacturer’s instructions by incubating cells with dye for 15min at 37°C, respectively. LTR loading was shown to be optimal at 100nM after 15 minutes incubation for both cell lines. Cells were then washed in PBS buffer and resuspended in 500μl of PBS. Cells were analyzed for LTR Median Fluorescence Intensity (MFI) levels by first gating on all cell material except small debris in the origin of a FSC versus SSC dot-plot.

Statistical analysis

Data were presented as mean ± SD. One way ANOVA and post hoc Tukey’s multiple comparison test were used for comparing analyzed groups. The power of all tests was not less than 1-β=0.8. Data were analyzed with the Statistica software (StatSoft, Inc. 2008), version 10.0 ( All of the tests were two-sided and p<0.05 was considered to be statistically significant. The proliferation and apoptotic indexes were determined according to Darzynkiewicz et al. (1994) and Henry et al. (2008) [19,20]. Hierarchical clustering of the results based on the Euclidean distance was carried out using GenExEnterprise QRT-PCR analysis was performed in accordance with the mathematical rules that this program uses. In order to identify differentially expressed, autophagy- and apoptosisrelated genes, linear regression was performed. Data of QRT-PCR analysis were also clustered using SOMs.


Cell cycle changes after siRNA silencing and valproic acid treatment

In order to examine the possible mechanisms of the antiproliferative activity of AKT3 and PI3KCA siRNAs combination with Valproic Acid (VPA), cell cycle distribution using flow cytometry was performed. Proliferation index (PI), i.e. percentage of proliferating cells in S+G2/M cell cycle phases, was determined. PI was quantified in untransfected T98G and U87MG cells and after the knockdown with adequate 1nM siRNA and valproic acid exposition. It was found that AKT3 and PI3KCA siRNAs increase the percentage of the cells in the subG1 phase as compared to untransfected (control) T98G (8.4% vs. 41.1% and 52%) and U87MG cells (1.6% vs. 20.7% and 18.7%) (Figure 1A and 1B) and AKT3 and PI3KCA siRNAs in combination with 0.5mM valproic acid decreased percentage of the T98G (Figure 1A) (14% vs. 5.1% and 5% for S phase; 5.7% vs. 2.2% and 1.9% for G2/M phase) and U87MG (Figure 1B) (15.1% vs. 10.4% and 9.6% for S phase; 13.2% vs. 5.6% and 8.6% for G2/M phase) cells in the G2/M and S phases as compared to untransfected (control) after their exposition to VPA.