Nitidine chloride suppresses epithelial-mesenchymal transition and stem cell-like properties in glioblastoma by regulating JAK2/STAT3 signaling

Abstract

Glioblastoma is the most aggressive and common intracranial malignant tumor, and the prognosis is still poor after various treatments. Based on the poor prognosis of glioma, new drugs that suppress the rapid progression and aggressive growth of glioma are urgently needed. It has been reported that nitidine chloride (NC) can inhibit tumor growth and epithelial-mesenchymal transition (EMT), and EMT is associated with cancer stem cell properties. The present study aimed to investigate the inhibitory effect of NC on the EMT process and stem cell-like properties in glioma cells. The results showed that the migration and invasion abilities in U87 and LN18 glioma cells were significantly increased after the induction of EMT and these effects were inhibited by NC in a concentration-dependent manner. NC treatment decreased the expression of EMT markers in glioma cells and self-renewal capacity of glioma stem-like cells. We demonstrated that these effects of NC were achieved via JAK2/STAT3 signaling. Taken together, these results indicate that NC inhibits the EMT process and glioma stem-like properties via JAK2/STAT3 signaling pathway, suggesting that NC may be a potential anti-glioma drug.

Keywords: JAK2/STAT3; apoptosis; epithelial-mesenchymal transition; glioblastoma; gliosphere; nitidine chloride.

FIGURE 1

(A, B) NC inhibited the proliferation of glioma cells treated with NC for 24 h and 48 h. The viability of glioma U87 and LN18 cells was detected by the CCK‐8 method. C NC inhibited the proliferation of NHA cells treated with NC for 24 h and 48 h. The viability of NHA cells was detected by the CCK‐8 method. Independent experiments were repeated three times, and the results were compared with those for the control group

FIGURE 2

(A, B) Glioma U87 and LN18 cells were treated with TGF‐β1 for 48 h to induce EMT. Western blotting analysis was performed to detect the expression of EMT‐related protein markers as well as β‐actin as an internal reference. (C, D) Microscopic images showing the morphology of U87 (100x) and LN18 (100x) cells after treatment with TGF‐β1 (10 ng/ml) for 48 h. (E, F) Quantitative analysis of the protein levels, Data were obtained from three independent experiments. *p<0.05, **p<0.001, ***p<0.0001

FIGURE 3

(A, B) EMT was induced in glioma cells by treatment with TGF‐β1 (control cells were not treated with TGF‐β1). Then the cells were treated with different concentrations of NC (2.5, 5.0, 7.5 and 10.0 μM) for 48 h. Western blotting analysis was performed to detect the expression of EMT‐related protein markers as well as β‐actin as an internal reference. (C, D) Quantitative analysis of the protein levels, Data were obtained from three independent experiments. *p<0.05, **p<0.001, ***p<0.0001

FIGURE 4

(A, B) U87 and LN18 glioma cells were treated with TGF‐β1 to induce EMT, and then the migration the cells was analyzed by a scratch assay after treatment with NC (2.5, 5.0, 7.5 and 10.0 μM). Microscopic images (200x) were taken after 24 and 48 h of NC treatment. (C, D) Quantitative results for glioma cell migration after induction of EMT and treatment with NC. Each independent experiment was repeated three times, and the results were compared with the those for the control group. *p<0.05, **p<0.001, ***p<0.0001

FIGURE 5

(A, B) U87 and LN18 glioma cells were treated with TGF‐β1 to induce EMT, and then the invasive ability of the cells was analyzed by Transwell assay after treatment with NC (2.5, 5.0, 7.5 and 10.0 μM). Microscopic images (200x) were taken after 24 and 48 h of NC treatment. (C, D) Quantitative results for glioma cell invasion after induction of EMT and treatment with NC. Each independent experiment was repeated three times, and the results were compared with the those for the control group. *p<0.05, **p<0.001, ***p<0.0001

FIGURE 6

(A, B) U87 and LN18 glioma cells were treated with NC (2.5, 5.0, 7.5 and 10.0 μM) for 48 h, and then apoptotic cells were detected by flow cytometry. (C, D) Quantitative data results from flow cytometry. Each independent experiment was repeated three times, and the results were compared with those for the control group. *p<0.05, **p<0.001, ***p<0.0001. (E, F) U87 and LN18 glioma cells were treated with NC (2.5, 5.0, 7.5 and 10.0 μM) for 48 h, and then the expression levels of apoptosis‐related proteins were detected by western blotting. (G, H) Quantitative analysis of the protein levels, Data were obtained from three independent experiments. *p<0.05, **p<0.001, ***p<0.0001

FIGURE 7

(A, B) U87 and LN18 glioma cells were treated first with TGF‐β1 to induce EMT and then with different concentrations of NC (2.5, 5.0, 7.5 and 10.0 μM) for 48 h. The expression levels of JAK2, p‐JAK2, STAT3 and p‐STAT3 were then detected by western blotting. (C, D) Quantitative analysis of the protein levels, Data were obtained from three independent experiments. *p<0.05, **p<0.001, ***p<0.0001

FIGURE 8

(A, B) U87 and LN18 glioma cells were treated with TGF‐β1 to induce EMT with and without 8 μM of the JAK2/STAT3 pathway inhibitor WP1066 for 48 h. Then the expression of EMT‐related marker proteins and transcription factors was detected by western blotting and compared with that in control cells. (C, D) Quantitative analysis of the protein levels, Data were obtained from three independent experiments. *p<0.05, **p<0.001, ***p<0.0001

FIGURE 9

(A, B) Gliospheres were treated first with TGF‐β1 to induce EMT and then with different concentrations of NC (2.5, 5.0, 7.5 and 10.0 μM) for 48 h. Microscopic images (100x) of the gliospheres were taken, and the numbers of second‐generation gliospheres were counted. (C, D) Quantitative results for secondgeneration gliosphere formation after EMT induction and with and without NC treatment. Each independent experiment was repeated three times, and the results were compared with those of the control group. *p<0.05, **p<0.001, ***p<0.0001

FIGURE 10

(A,B) Self‐renewal capacity of U87 and LN18 glioma stem‐like cells was detected by limited dilution assay after induction of EMT and after treatment with different concentrations of NC. The regression curve is shown, and the x‐intercept represents the number of cells required to form one gliosphere

FIGURE 11

(A, B) After treatment of gliospheres with TGF‐β1 to induce EMT and then with various concentrations of NC, western blotting was performed to detect the expression levels of tumor stem cell markers Oct4, Bmi1, and Sox2. (C, D) After the same treatments of gliospheres, western blotting was also performed to detect the expression levels of JAK2, p‐JAK2, STAT3, and p‐STAT3. (E, F, G, H) Quantitative analysis of the protein levels, Data were obtained from three independent experiments. *p<0.05, **p<0.001, ***p<0.0001

FIGURE 12

(A, B) U87 and LN18 glioma cells were treated with TGF‐β1 to induce EMT with and without 8 μM of the JAK2/STAT3 pathway inhibitor WP1066 for 48 h. Then the expression of stem cell marker proteins was detected by western blotting and compared with that in control cells. (C, D) Quantitative analysis of the protein levels, Data were obtained from three independent experiments. *p<0.05, **p<0.001, ***p<0.0001