Interleukin-24-mediated antitumor effects against human glioblastoma via upregulation of P38 MAPK and endogenous TRAIL-induced apoptosis and LC3-II activation-dependent autophagy

Abstract

Background: Melanoma differentiation-associated gene 7 (Mda-7) encodes IL-24, which can induce apoptosis in cancer cells. A novel gene therapy approach to treat deadly brain tumors, recombinant mda-7 adenovirus (Ad/mda-7) efficiently kills glioma cells. In this study, we investigated the factors affecting cell survival and apoptosis and autophagy mechanisms that destroy glioma cells by Ad/IL-24.

Methods: Human glioblastoma U87 cell line was exposed to a multiplicity of infections of Ad/IL-24. Antitumor activities of Ad/IL-24 were assessed by cell proliferation (MTT) and lactate dehydrogenase (LDH) release analysis. Using flow cytometry, cell cycle arrest and apoptosis were investigated. Using the ELISA method, the tumor necrosis factor (TNF-α) level was determined as an apoptosis-promoting factor and Survivin level as an anti-apoptotic factor. The expression levels of TNF-related apoptosis inducing ligand(TRAIL) and P38 MAPK genes were assessed by the Reverse transcription-quantitative polymerase chain reaction(RT‑qPCR) method. The expression levels of caspase-3 and protein light chain 3-II (LC3-II) proteins were analyzed by flow cytometry as intervening factors in the processes of apoptosis and autophagy in the cell death signaling pathway, respectively.

Results: The present findings demonstrated that transduction of IL-24 inhibited cell proliferation and induced cell cycle arrest and cell apoptosis in glioblastoma. Compared with cells of the control groups, Ad/IL24-infected U87 cells exhibited significantly increased elevated caspase-3, and TNF-α levels, while the survivin expression was decreased. TRAIL was shown to be upregulated in tumor cells after Ad/IL-24 infection and studies of the apoptotic cascade regulators indicate that Ad/IL-24 could further enhance the activation of apoptosis through the TNF family of death receptors. In the current study, we demonstrate that P38 MAPK is significantly activated by IL-24 expression. In addition, the overexpression of mda-7/IL-24 in GBM cells induced autophagy, which was triggered by the upregulation of LC3-II.

Conclusions: Our study demonstrates the antitumor effect of IL-24 on glioblastoma and may be a promising therapeutic approach for GBM cancer gene therapy.

Keywords: Antitumor; Autophagy; Cell apoptosis; Glioblastoma; IL-24; Recombinant adenovirus.

Fig. 1

The expression of interleukin IL-24 in U87 transfected with Ad/GFP/IL-24. A U87cells were transfected with Ad/GFP/IL-24 and checked for the microscopy visible light. B Green fluorescent protein (GFP) expression by fluorescence microscopy

Fig. 2

Ad/IL-24 cytotoxicity in U87 cells assessed. The MTT assay results indicated that infection with different MOI can significantly reduce the viability of U87 cells vs control groups (Control and Ad/GFP) ****(P < 0.0001), **(P < 0.001), indicates a statistically significant difference between MOIs 3,5 and 10 compared with the control group by one-way ANOVA

Fig. 3

Cytotoxicity of Ad/IL-24 on U87 cells evaluated by LDH assay kit. The LDH assay results indicated that infection with various MOIs can significantly release LDH (all results normalized by uninfected internal control and Ad/GFP). *** P < 0.0001, **** P < 0.0001. All experiments are done in triplicate and repeated three times

Fig. 4

Annexin V/PI staining of U87 cells treated with Ad/IL-24. A-E U87 cells were treated with various MOI of Ad/IL-24 (3,5 and 10) for 48h, after that expose to annexin V/PI staining and analyzed via flow cytometry. Untreated and Ad/GFP cells were considered as control groups. F The total percentage of apoptotic cells was stained with annexin V/PI. *(P< 0.0316, **(P< 0.008), ****(P < 0.0001)

Fig. 5

Ad/IL-24 inhibited glioblastoma cells growth in vitro by inducing cell cycle arrest. A-F: by flow cytometry, U87 cells treated with Ad/IL-24 or Ad/GFP were harvested after 48 h and stained with PI. F: Cell cycle distribution was analyzed by flow cytometry and the percentage of cell-cycle phases was analyzed by Flow Jo Software. Each bar represents the mean ±SD of five independent experiments. *(P<0.364), **(P<0.0027), ***(P<0.001), ****(P <0.0001)

Fig. 6

Expression of IL‑24/mda‑7, Trail, and P38 MAPK in U87 cells. A: Relative expression levels of P38 MAPK B: Trail and C: IL-24/mda-7 in U87 as assessed by quantitative reverse transcriptase-PCR. The ratios of expression levels of IL-24/mda-7to β-actin are shown as relative IL-24/mda-7expression levels. Data are presented as mean ± SD.*(P<0.02), **(P<0.002), ***(P<0.0003),****(P<0.0001)

Fig. 7

Caspase-3 staining of U87 cells treated with Ad/IL-24. A-E U87 cells were treated with concentrations of Ad/IL-24 (MOI: 3, 5, and 10) for 48 h, after that expose to secondary antibody staining and analyzed by flow cytometry. Un-treated cells and Ad/GFP were considered control groups. F The total percentage of apoptotic cells was stained with Caspase-3. ** (P < 0.01), ***(P < 0.001), ****(P<0.0001)

Fig. 8

The effect of the Ad/IL-24 on protein levels of TNF-α and survivin. A Evaluation of TNF-α and (B) Survivin in U87 cell line after transfection with different concentrations of Ad/IL-24 (MOI: 3, 5, and 10) at protein level with ELISA. Un-treated cells consider and Ad/GFP as control groups. TNF-α and Survivin proteins level compared with the control groups **** (p < 0.0001), *** (P<0.0008), ** (P<0023)

Fig. 9

LC3-II staining of U87 cells treated with Ad/IL-24. A-E U87 cells were treated with different MOIs of Ad/IL-24 (MOI: 3, 5, and 10) for 48 h, after that expose to secondary antibody staining and analyzed by flow cytometry. Un-treated cells consider as control test. F. Results of compared with the control groups (Un-treated cells consider and Ad/GFP as control groups. ** (P<0.0015), *** (P < 0.003), **** (P<0001)