Growth Inhibitory Effects of Dipotassium Glycyrrhizinate in Glioblastoma Cell Lines by Targeting MicroRNAs Through the NF-κB Signaling Pathway

Abstract

It has been shown that nuclear factor kappa-B (NF-κB) is constitutively activated in glioblastoma (GBM), suggesting that the pathway could be a therapeutic target. Glycyrrhetic acid (GA), a compound isolated from licorice (Glycyrrhiza glabra), has been shown to decrease cell viability and increases apoptosis in human cancer cell lines by NF-κB signaling pathway suppression. Dipotassium glycyrrhizinate (DPG), a dipotassium salt of GA, has anti-inflammatory properties without toxicity. The current study examined the effectiveness of DPG as an anti-tumor in U87MG and T98G GBM cell lines. Additionally, we assessed DPG as a candidate for combinational therapy in GBM with temozolomide (TMZ). Our results demonstrated that the viability of U87MG and T98G cells significantly decreased in a time- and dose-dependent manner after DPG treatment, and the apoptotic ratio of DPG-treated groups was significantly higher than that of control groups. In addition, DPG in combination with TMZ revealed synergistic effects. Furthermore, the expression of NF-κB-luciferase-reporter in transfected GBM cell lines was remarkably reduced after DPG exposure by up-regulating miR16 and miR146a, which down-regulate its target genes, IRAK2 and TRAF6. A reduced neuro-sphere formation was also observed after DPG in both GBM cells. In conclusion, DPG presented anti-tumoral effect on GBM cell lines through a decrease on proliferation and an increase on apoptosis. In addition, our data also suggest that DPG anti-tumoral effect is related to NF-κB suppression, where IRAK2- and TRAF6-mediating miR16 and miR146a, respectively, might be a potential therapeutic target of DPG.

Keywords: dipotassium glycyrrhizinate; glioblastoma; miR146a; miR16; nuclear factor kappa-B.

FIGURE 1

Dipotassium glycyrrhizinate (DPG) reduces cell viability and change morphology in glioblastoma cell lines. (A) DPG inhibits cell viability of U87MG cells treated with 12, 15, 18, 20, 24, and 28 mM DPG for 24, 48, and 72 h by MTT assay and the IC₅₀ was determined (18 mM for 48 and 72 h). (B) DPG inhibits cell viability of T98G cells treated with 20, 24, 28, and 32 mM DPG for 24, 48, and 72 h by MTT assay and the IC₅₀ was determined (24 mM for 48 and 72 h). (C) U87MG cell and nuclear morphological change were observed 48 and 72 h after DPG exposure. (D) T98G cell and nuclear morphological change were observed especially after 72 h of DPG exposure.

FIGURE 2

Dipotassium glycyrrhizinate (DPG) increases apoptosis and inhibits proliferation in glioblastoma cell lines. U87MG (A) and T98G (B) cells were incubated with 18 mM and 24 mM of DPG, respectively, for 48 and 72 h. T98G was also treated using 24 mM DPG for 96 h. The genomic DNA was isolated and analyzed on 1.5% agarose gel with ethidium bromide staining. M: DNA marker 100 base pairs; C: untreated control cells. Results are representative of one of three similar experiments performed. U87MG (C) and T98G (D) cells were treated with DPG (18 mM for 72 h and 24 mM for 96 h of DPG, respectively). After DPG incubation TUNEL assay was done using in situ cell death detection kit as per the manufacturer’s protocol and the quantitative estimation of TUNEL cells after DPG exposure was measured using ImageJ. Each experiment was repeated three times. The results show a significant increase in the number of apoptotic U87MG cells (P = 0.03) after DPG treatments as compared to the untreated control cells. T98G (P = 0.19) cell also presents an increase in the apoptotic cell number. (E) Cleaved caspase-3 was measured by Western blotting analysis after treatment with 18 mM (U87MG) and 24 mM (T98G). The induction of the cleaved form of caspase-3 (11 kDa) protein increased 48 h after treatment. DPG increased cleaved caspase-3, indicating that apoptosis required caspase activation. (F) Also, DPG significantly increases PARP-1 expression in U87MG (P = 0.05) and T98G (P = 0.01) cells when compared with untreated cells. DPG inhibits the proliferating rate of U87MG (G) and T98G (H) cell lines in a time-dependent trend, in comparison with untreated control cells. Data represent means and standard deviations of a representative experiment performed in triplicate. Statistics were performed in a two-tailed t-test with P < 0.01.

FIGURE 3

DPG regulates NF-κB activity in glioblastoma cell lines by miR16- and miR146a-mediating IRAK2 and TRAF6 down-expression. U87MG (A) and T98G (B) cell lines were transfected with the NF-κB-luciferase reporter and incubated with dipotassium glycyrrhizinate (DPG) for 40 h. Equal amounts of cell extract were assayed for dual-luciferase activity. The reporter activity was observed as down-regulated by DPG when comparing with untreated transiently transfected cells. Transiently transfected cells were also treated with dehydroxymethylepoxyquinomicin (DHMEQ) or lipopolysaccharide (LPS). As expected, DHMEQ and LPS down-regulates and induces NF-κB reporter activity, respectively, in both cell lines. DPG significantly increases miR16 and miR146a expression in U87MG (P = 0.004 and P = 0.003, respectively) (C) and T98G (P = 0.01 and P = 0.006, respectively) cell lines. (D) Instead, DPG decreases IRAK2 and TRAF6 mRNA levels in U87MG (P = 0.26 and P = 0.02, respectively) and (E) T98G (P = 0.01 and P = 0.005, respectively) cells. (F) Data represent means and standard deviations of a representative experiment performed in triplicate. Statistics were performed in a two-tailed t-test with P ≤ 0.05.

FIGURE 4

Inhibitory effect of dipotassium glycyrrhizinate (DPG) in the NF-κB signaling pathway. pcDNA3.3-empty vector and pcDNA3.3-miR146a cloning vector were transiently transfected into U87MG and T98G cell lines and treated with DPG (U87MG: 18 mM; T98G: 24 mM). (A) The promoter activity of NF-κB in U87MG-pcDNA3.3-miR146a (P = 0.005) presented significantly higher compared to pcDNA3.3-empty vector cells. In contrast, the NF-κB activity presented significantly reduced when U87MG-pcDNA3.3-miR146a (P = 0.01) cells were treated with DPG. (B) The promoter activity of NF-κB in T98G-pcDNA3.3-miR146a (P = 0.11) is higher compared to T98G-pcDNA3.3-empty vector cells. Instead, the NF-κB promoter activity presented significantly reduced when T98G-pcDNA3.3-miR146a (P = 0.03) cells were treated with DPG. MiR146a expression was evaluated by qPCR. MiR146a is higher in either (C) U87MG- and (D) T98G-pcDNA3.3-miR146a untreated cells (P = 0.01 and P = 0.01, respectively) and U87MG- and T98G-pcDNA3.3-miR146a treated with DPG (P = 0.02 and P = 0.001, respectively). DPG decreases TRAF6 mRNA level in either (E) U87MG- or (F) T98G-pcDNA3.3-miR146a (P = 0.002 and P = 0.05, respectively) cells. Three independent transfections were performed in triplicate, and data are shown as the mean ± standard deviation. Statistics were performed in a two-tailed t-test with P ≤ 0.05.

FIGURE 5

Dipotassium glycyrrhizinate (DPG) decreases wound closure and inhibits cell migration in glioblastoma cell lines. (A) U87MG cell line treated with DPG fills the wound area (the area between the two dotted lines) more slowly than those untreated at 24, 48, and 72 h. (B) T98G cell line treated with DPG fills the wound area (the area between the two dotted lines) more slowly than those untreated at 24, 48, and 72 h. The wound-healing assay was expressed as relative wound width (24, 48, or 72 h average wound width divided by 0 h wound represented by the graphics). Graphics also shows average and standard deviation of three independent experiments. Statistics were performed in a two-tailed t-test with P ≤ 0.05.

FIGURE 6

Glioblastoma stem cells (GSCs) are sensitized to dipotassium glycyrrhizinate (DPG). Sphere-forming ability of GSCs in U87MG (A) and T98G (B) cell lines after 24 and 48 h after DPG treatment. Non-treated cell lines were used as controls. Cells were plated in 2% poly (2-hydroxyethyl methacrylate)-coated cell culture flasks in serum-free medium. (C) The graphic shows the average and standard deviation of three independent experiments. Statistics were performed in a two-tailed t-test with P ≤ 0.05. Differences between treated and non-treated cells were significantly observed in U87MG (P = 0.04) and T98G (P = 1E^-04) cell lines.