Yonsei Med J.  2013 Sep;54(5):1186-1193. 10.3349/ymj.2013.54.5.1186.

Growth Inhibition of Hepatocellular Carcinoma Huh7 Cells by Lactobacillus casei Extract

• Han DJ
• Kim JB
• Park SY
• Yang MG
• Kim H

• KMID: 1793166
• DOI: http://doi.org/10.3349/ymj.2013.54.5.1186

Abstract

PURPOSE
Lactobacillus casei (L. casei) is known to exert anti-proliferation effects on many types of cancer cells. However, the effect of L. casei on liver cancer has not been reported. Accordingly, the aim of this study was to determine the anti-cancer effect of L. casei extract on Huh7 cells.
MATERIALS AND METHODS
L. casei ATCC393 extract was prepared and purified. After the treatment of L. casei extract on Huh7 cells, cell viability, cell cycle arrest and cell death were analyzed by flow cytometry. The expression levels of tumor necrosis factor-alpha receptor 1 (TNFR1) and death receptor 3 (DR3) mRNA related with extrinsic apoptosis were assessed by reverse transcription polymerase chain reaction. Additionally, P21 and P27 cell cycle proteins as well as Caspase-3, -8, -9, phospho-Bad and Bcl-2 apoptosis proteins were analyzed by western blot analysis. To determine the effect of L. casei extract on cancer stem-like cells, we analyzed changes in side population fraction through flow cytometry.
RESULTS
The cell viability of Huh7 cells treated with L. casei extract was decreased by 77%, potentially owing to increases in the rates of Huh7 cells arrested in the G2/M phase (3% increase) and that underwent apoptosis (6% increase). The expression levels of TNFR1 and DR3 mRNA, as well as P21 and P27 cell cycle proteins, were increased. Meanwhile, the expressions of caspase-8, -9, phospho-Bad and Bcl-2 proteins decreased. However, in the case of side population cells, no remarkable changes were observed.
CONCLUSION
L. casei extract exerts a potent anti-tumor effect on the viability of liver cancer cells, although not on cancer stem-like cells.

Keyword

Lactobacillus casei; liver cancer; proliferation; side population; cancer stem-like cell

MeSH Terms

Apoptosis/drug effects
Carcinoma, Hepatocellular/*pathology
Caspase 8/metabolism
Caspase 9/metabolism
Cell Cycle Checkpoints/drug effects
Cell Extracts/*pharmacology
Cell Line, Tumor
Cell Proliferation/drug effects
Cyclin-Dependent Kinase Inhibitor p21/metabolism
Cyclin-Dependent Kinase Inhibitor p27/metabolism
Cytostatic Agents/*pharmacology
Flow Cytometry
Gene Expression Regulation, Neoplastic/drug effects
Humans
Lactobacillus casei/*chemistry
Liver Neoplasms/*pathology
Proto-Oncogene Proteins c-bcl-2/metabolism
RNA, Messenger/metabolism
Receptors, Tumor Necrosis Factor, Member 25/metabolism
Receptors, Tumor Necrosis Factor, Type I/metabolism
bcl-Associated Death Protein/metabolism
bcl-Associated Death Protein
Caspase 8
Caspase 9
Cell Extracts
Cyclin-Dependent Kinase Inhibitor p21
Cyclin-Dependent Kinase Inhibitor p27
Cytostatic Agents
Proto-Oncogene Proteins c-bcl-2
RNA, Messenger
Receptors, Tumor Necrosis Factor, Member 25
Receptors, Tumor Necrosis Factor, Type I

Figure

• 

  Fig. 1 Cell growth rate following the treatment of Lactobacillus casei extract. Huh7 cells seeded at 5×105 cells per cell culture dish and treated with 0.1 mg L. casei extract for 72 hrs. The values are presented in means and standard errors for at least three independent experiments. *p value <0.01. CTL, control.

• 

  Fig. 2 Cell cycle arrest following the treatment of Lactobacillus casei extract. L. casei extract increased the rate of arrested Huh7 cells in the G2/M phase by 3%, compared to the control group. The data shown are one representative experiment from a total of three independent experiments. CTL, control.

• 

  Fig. 3 Flow cytometry of PI-Annexin-V. The cell death rate was found to increased by 6% in the 0.1 mg L. casei extract treated group, compared to the control group, after 72 hrs. The data shown are one representative experiment from a total of three independent experiments. Statistical differences between numbers of apoptotic cells were observed with p<0.05. FL1, fluorescence channel 1; FL3, fluorescence channel 3; FITC, fluorescein isothiocyanate; PI, propidium iodide.

• 

  Fig. 4 Expression level of TNFR1 and DR3 mRNA. Significant increments in the mRNA expression levels of TNFR1 and DR3 were observed in L. casei extract treated Huh7 cells. GAPDH mRNA expression was used as a control. TNFR1, tumor necrosis factor-α receptor 1; DR3, death receptor 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. CTL, control.

• 

  Fig. 5 Western blot analysis of cell cycle and apoptosis related proteins. (A) Expression levels of P21 and P27 cell cycle proteins were increased in Huh7 cells treated with L. casei extract. The increments in P21 and P27 protein expression levels were related with the induction of cell cycle arrest. (B) Expression levels of apoptosis proteins, including caspase-3, caspase-8, caspase-9, phopho-Bad and Bcl-2, were decreased when treated with L. casei extract. Actin protein was used as an internal control. CTL, control.

• 

  Fig. 6 Side population cell analysis. L. casei extract was found to increase side population fractions in the treated group, compared to the control (p<0.05). The L. casei extract was found not to possess a potent anti-tumor effect on the cancer stem-like cells. CTL, control.

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