Inhibition of STAT3 signaling as critical molecular event in HUC-MSCs suppressed Glioblastoma Cells

Abstract

Objective: We investigated the effect of human umbilical cord mesenchymal stem cells (HUC-MSCs) supernatants on proliferation, migration, invasion, and apoptosis in glioblastoma (GBM) cell lines RG-2, U251, U87-MG, and LN-428, as well as their apoptosis and autophagy-mediated through IL-6/JAK2/STAT3 signaling pathway to explore the molecular mechanisms. Methods: In this study, RG-2, U251, U87-MG, and LN-428 cells were treated with 9 mg/ml HUC-MSCs supernatants. Their responses to HUC-MSCs supernatants treatment and the status of STAT3 signaling were analyzed by multiple experimental approaches to elucidate the importance of HUC-MSCs supernatants for GBM. Results: The results demonstrated that after treatment with HUC-MSCs supernatants, in vitro proliferation of RG-2, U251, U87-MG, and LN-428 cells were inhibited, and their sustained growth was also blocked. RG-2, U251, and U87-MG cells showed significant S phase accumulation, while LN-428 cells were blocked in G0/G1 phase. Their migratory invasive capacities were inhibited, and their apoptosis and autophagy ratios were increased. These effects were mediated through the IL-6/JAK2/STAT3 and its downstream signaling pathway. Conclusion: Our data showed that HUC-MSCs supernatants had anti-tumor effects on GBM cells. It inhibited the proliferation, migration, and invasion of GBM cells and promoted their apoptosis. Negative regulation of the IL-6/JAK2/STAT3 signaling pathway enhanced apoptosis and autophagy in tumor cells, thereby improving the therapeutic effect on GBM.

Keywords: IL-6/JAK2/STAT3 signaling pathway; glioblastoma; human umbilical cord mesenchymal stem cell supernatant.

Figure 1

Growth inhibition of GBM cells by HUC-MSCs supernatant. (A), (B) and (C) CCK-8 assays: (A) HUC-MSCs were incubated with H-DMEM containing 10% FBS for 24 h and replaced by H-DMEM without FBS for 24 h, 48 h, and 72 h respectively, and then HUC-MSCs supernatants were collected. U251, U87-MG, LN-428, and RG-2 cells were treated with the collected HUC-MSCs supernatants for 24 h, 48 h, and 72 h, and then analyzed by the CCK-8 assay. (B) HUC-MSCs were incubated with H-DMEM containing 10% FBS for 24 h, 48 h, and 72 h, replaced by H-DMEM without FBS for 24 h. The HUC-MSCs supernatants were collected. U251, U87-MG, LN-428, and RG-2 cells were treated with the collected HUC-MSCs supernatants for 24 h, 48 h, and 72 h, respectively, and analyzed by the CCK-8 assay. (C) HUC-MSCs were incubated with H-DMEM containing 10% FBS for 48 h, replaced by H-DMEM without FBS, and continued for 24 h, then the collected supernatant of HUC-MSCs at 9 mg/ml was used as the effective concentration. (D) H&E morphological staining (inverted phase contrast microscope, × 40) was detected in GBM cells and treated for 48 h in the absence (CON) or presence (C9) of 9 mg/ml HUC-MSCs supernatants. HUC-MSCs supernatants, Human Umbilical Cord Mesenchymal Stem Cell Supernatants. ** P < 0.01, and *** P < 0.001 vs CON group; the error bars, the mean ± standard deviation.

Figure 2

Cell cycle of U251, U87-MG, LN-428, and RG-2 cells was altered after treatment with 9 mg/ml HUC-MSCs supernatants for 48 h, and HUC-MSCs had tumor tropism. (A) Cell cycle distribution was detected with PI staining. (B) Western Blot. β-actin was used as a qualitative and quantitative control. (C) Transwell experiments confirmed in vitro that HUC-MSCs could migrate to GBM cells. CON, 0 mg/ml HUC-MSCs supernatant; C9, 9 mg/ml HUC-MSCs supernatants; *P < 0.05, **P < 0.01 and ***P < 0.001 vs CON group; the error bars, the mean ± standard deviation.

Figure 3

Apoptosis of U251, U87-MG, LN-428, and RG-2 cells after 48 h treatment with HUC-MSCs supernatants. (A) The images of the TUNEL apoptosis assay (× 20). (B) Flow cytometry analysis of Annexin V and PI in four kinds of GBM cell lines for apoptosis. (C) and (D) Expression of apoptosis-related proteins Bcl-2, Caspase 3, and Caspase 8. (C) Western Blot. β-actin was used as a qualitative and quantitative control. (D) ICC (× 20 and × 40). (E) Apoptosis of the four GBM cell lines tested by flow cytometry was statistically analyzed. CON, 0 mg/ml HUC-MSCs supernatant; C9, 9 mg/ml HUC-MSCs supernatants; TUNEL, Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling; ICC, Immunocytochemistry. *P < 0.05, **P < 0.01 and ***P < 0.001 vs CON group; the error bars, the mean ± standard deviation.

Figure 4

After 48 h treatment with 9 mg/ml HUC-MSCs supernatants, the migration and invasion ability of U251, U87-MG, LN-428, and RG-2 cells were inhibited. (A) Transwell Migration assay. (B) Transwell Invasion assay. (C) Four GBM cell lines were statistically analyzed for migration. (D) Four GBM cell lines were statistically analyzed for invasion. (E) and (F) Changes in the expression of migration-associated proteins MMP-2 and MMP-9 in GBM cells: (E) Western Blot. β-actin was used as a qualitative and quantitative control. (F) ICC. CON, 0 mg/ml HUC-MSCs supernatants; C9, 9 mg/ml HUC-MSCs supernatants; ICC, Immunocytochemistry. NS, no statistical significance (P > 0.05); * with statistical significance (*P < 0.05, ** P < 0.01, *** P < 0.001 vs CON group). The error bars, the mean ± standard deviation.

Figure 5

Changes in STAT3 signaling pathway in U251, U87-MG, LN-428, and RG-2 cells after 48 h treatment without (CON) or with (C9) HUC-MSCs supernatants. (A) ICC examination (× 20 and × 40), and (B) Western Blot analyses of IL-6, JAK2, STAT3, p-STAT3, and PIAS3. β-actin was used as a qualitative and quantitative control. CON, 0 mg/ml HUC-MSCs supernatants; C9, 9 mg/ml HUC-MSCs supernatants. *P < 0.05, **P < 0.01 and ***P < 0.001 vs CON group; the error bars, the mean ± standard deviation.

Figure 6

Examinations of MCL-1, Survivin, VEGFA, c-Myc, LC3 ӀӀ/Ӏ and Beclin-1 expression in U251, U87-MG, LN-428, and RG-2 cells without (CON) and with (C9) HUC-MSCs supernatants treatments for 48 h. (A) ICC examination (× 20 and × 40) and (B) Western blot analyses of MCL-1, Survivin, VEGFA, and c-Myc. β-actin was used as a qualitative and quantitative control. (C) IF experiments were performed by laser confocal microscopy (× 20) to examine the expression of the autophagy-related proteins LC3 ӀӀӀ/Ӏ and Beclin-1. and (D) Western Blot analyses of LC3 ӀӀ/Ӏ and Beclin-1. β-actin was used as a qualitative and quantitative control. CON, 0 mg/ml HUC-MSCs supernatansts; C9, 9 mg/ml HUC-MSCs supernatants. *P < 0.05, **P < 0.01 and ***P < 0.001 vs CON group; the error bars, the mean ± standard deviation.

Figure 7

Alterations in STAT3 signaling pathway after 48 h of STAT3 inhibitor C188-9 applied to U251, U87-MG, LN-428, and RG-2 cells. (A) CCK-8 assays showing the effect of C188-9 in U251, U87-MG, LN-428, and RG-2 cells. (B) CCK-8 assays showing the effect of HUC-MSCs supernatants or/and C188-9 in U251, U87-MG, LN-428, and RG-2 cells. (C) Transwell Migration assay. (D) Four GBM cell lines were statistically analysed for migration. (E) Western Blot analyses of STAT3/p-STAT3 and downstream related proteins Survivin, MCL-1, and c-Myc expression. β-actin was used as a qualitative and quantitative control. CON, Blank control group; C9, 9 mg/ml HUC-MSCs supernatants; C188-9, N‐(1ʹ,2‐Dihydroxy‐1,2ʹ‐binaphthalen‐4ʹ‐yl)‐4‐methoxybenzenesulfonamide; STAT3 inhibitor; *P < 0.05, **P < 0.01 and ***P < 0.001 vs CON group; the error bars, the mean ± standard deviation.

Figure 8

Schematic diagram.