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. 2009 Sep 28;15(36):4538-46.
doi: 10.3748/wjg.15.4538.

Induction of apoptosis and cell cycle arrest in human HCC MHCC97H cells with Chrysanthemum indicum extract

Zong-Fang Li  1 , Zhi-Dong Wang, Yuan-Yuan Ji, Shu Zhang, Chen Huang, Jun Li, Xian-Ming Xia
Affiliations

Induction of apoptosis and cell cycle arrest in human HCC MHCC97H cells with Chrysanthemum indicum extract

Zong-Fang Li et al. World J Gastroenterol. .

Abstract

Aim: To investigate the effects of Chrysanthemum indicum extract (CIE) on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma (HCC) MHCC97H cell line.

Methods: Viable rat hepatocytes and human endothelial ECV304 cells were examined by trypan blue exclusion and MTT assay, respectively, as normal controls. The proliferation of MHCC97H cells was determined by MTT assay. The cellular morphology of MHCC97H cells was observed by phase contrast microscopy. Flow cytometry was performed to analyze cell apoptosis with annexin V/propidium iodide (PI), mitochondrial membrane potential with rhodamine 123 and cell cycle with PI in MHCC97H cells. Apoptotic proteins such as cytochrome C, caspase-9, caspase-3 and cell cycle proteins, including P21 and CDK4, were measured by Western blotting.

Results: CIE inhibited proliferation of MHCC97H cells in a time- and dose-dependent manner without cytotoxicity in rat hepatocytes and human endothelial cells. CIE induced apoptosis of MHCC97H cells in a concentration-dependent manner, as determined by flow cytometry. The apoptosis was accompanied by a decrease in mitochondrial membrane potential, release of cytochrome C and activation of caspase-9 and caspase-3. CIE arrested the cell cycle in the S phase by increasing P21 and decreasing CDK4 protein expression.

Conclusion: CIE exerted a significant apoptotic effect through a mitochondrial pathway and arrested the cell cycle by regulation of cell cycle-related proteins in MHCC97H cells without an effect on normal cells. The cancer-specific selectivity shown in this study suggests that the plant extract could be a promising novel treatment for human cancer.

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Figures

Figure 1
Figure 1
Effects of Chrysanthemum indicum extract (CIE) on cell viability of normal cells. A: Rat hepatocytes; B: Human umbilical vein endothelial cell line ECV304. Various concentrations of CIE were added, and the cells were incubated for 24 h. Cell viability was measured by 0.2% trypan blue exclusion (A) and MTT assay (B) respectively. Results presented are representative of 3 independent experiments.
Figure 2
Figure 2
Dose-response curves for CIE in MHCC97H cells, following 24 h, 48 h and 72 h incubation, as assessed by MTT assay. CIE produced a concentration- and time-dependent decrease in cellular proliferation. Results presented are representative of 3 independent experiments.
Figure 3
Figure 3
Effect of CIE on the induction of MHCC97H cell apoptosis. A: The apoptosis of MHCC97H cells induced by CIE were determined by flow cytometry at 24 h; B: CIE induced a concentration-dependent increase in early and late cellular apoptosis. Results presented are representative of 3 independent experiments. aP < 0.05, bP < 0.01 vs control group.
Figure 4
Figure 4
Morphological changes in MHCC97H cells with CIE. Cells were observed by phase contrast microscopy in controls and after treatment with 400, 800, 1200 μg/mL CIE (× 200).
Figure 5
Figure 5
Effect of CIE on MHCC97H cellular mitochondrial membrane potential (MMPΔψm). A: The MMPΔψm of MHCC97H cells were determined by flow cytometry at 24 h after CIE; B: Results presented are representative of 3 independent experiments. aP < 0.05, bP < 0.01 vs control group.
Figure 6
Figure 6
Effect of CIE on apoptosis-related protein expression. A: The expressions of cytochrome C, caspase-9 and caspase-3 were assessed by Western blotting; B: Results presented are representative of 3 independent experiments. aP < 0.05, bP < 0.01 vs control group.
Figure 7
Figure 7
Effect of CIE on MHCC97H cell cycle. A: The cell cycle of MHCC97H cells in the presence of CIE was determined by flow cytometry at 48 h; B and C: Results presented are representative of 3 independent experiments. aP < 0.05, bP < 0.01 vs control group.
Figure 8
Figure 8
Effect of CIE on cell cycle-related protein expression. A: The expression of CDK4 and P21 was assessed by Western blotting; B: Results presented are representative of 3 independent experiments. bP < 0.01 vs control group.
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