Cucurbitacin B inhibits proliferation, induces G2/M cycle arrest and autophagy without affecting apoptosis but enhances MTT reduction in PC12 cells
DOI:
https://doi.org/10.3329/bjp.v11i1.23791Keywords:
Apoptosis, Autophagy, Cucurbitacin B, G2/M cycle, MTT, PC12 cellAbstract
In the present study, the effect of cucurbitacin B (a natural product with anti-cancer effect) was studied on PC12 cells. It significantly reduced the cell number, changed cell morphology and inhibited colony formation while MTT results showed increased cell viability. Cucurbitacin B treatment increased activity of succinode hydrogenase. No alteration in the integrity of mem-brane, the release of lactic dehydrogenase, the mitochondrial membrane potential, and the expression of apoptotic proteins suggested that cucurbitacin B did not induce apoptosis. The cell cycle was remarkably arrested at G2/M phase. Furthermore, cucurbitacin B induced autophagy as evidence by accumulation of autophagic vacuoles and the increase of LC3II. In addition, cucurbitacin B up-regulated the expression of p-beclin-1, p-ULK1, p-Wee1, p21 and down-regulated p-mTOR, p-p70S6K, CDC25C, CDK1, Cyclin B1. In conclusion, cucurbitacin B inhibited PC12 proliferation but caused MTT pitfall. Cucurbitacin B induced G2/M cell cycle arrest, autophagy, but not the apoptosis in PC12 cells.
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- Figure 1: Effect of cucurbitacin B on the morphology, cell number and colony formation in PC12 cell line.
- Figure 2: Effect of cucurbitacin B on PC12 cell viability using MTT assay (A); cellular ATP content (B); and activity of succinode hydrogenase (C).
- Figure 3: Effect of cucurbitacin D and E on cell viability.
- Figure 4: Effect of cucurbitacin B on membrane integrity. Trypan
- Figure 5: Effect of cucurbitacin B on JC-1 staining, Hoechst 33342 staining and the expression of apoptotic related proteins.
- Figure 6A: Effect of Cucurbitacin B on G2/M cell cycle arrest. Expression of p-Wee1, cyclin B1, CDK1, CDC25C and p21. Ctrl, Control
- Figure 6B: Flow cytometer to detect G2/M cell cycle arrest
- Figure 7: Autophagic inducible effect of cucurbitacin B on PC12. (A) MDC staining. (B) Expression of p-mTOR (Ser 2448), p-ULK1 (Ser 757), p-p70S6K (Thr421/Ser424), p-Beclin-1 (Ser 93/96), LC3B. (C) Expression of p-mTOR (Ser 2448) and LC3B
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