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Ethyl acetate extract of Hypericum japonicum induces apoptosis via the mitochondria-dependent pathway in vivo and in vitro.

Zhuang Q, Li J, Chen Y, Lin J, Lai F, Chen X, Lin X, Peng J - Mol Med Rep (2015)

Bottom Line: Treatment with EAEHJ significantly reduced tumor weight, but had no effect on murine body weight.The results of the present study also showed that EAEHJ induced H22 cell apoptosis in vivo.Treatment with EAEHJ also increased the ratio of pro‑apoptotic B‑cell lymphoma 2 (Bcl‑2)‑associated X protein (Bax) to anti‑apoptotic Bcl‑2, and activated the caspase‑9 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, P.R. China.

ABSTRACT
The widely-used Chinese medicinal herb Hypericum japonicum, also known as Hypericum japonicum Thunb or Tianjihuang, displays potent anti‑carcinogenic effects against liver cancer. However, the molecular mechanism underlying the therapeutic effects of Hypericum japonicum remains to be elucidated. The present study investigated the in vivo efficacy of ethyl acetate extract of Hypericum japonicum (EAEHJ) against tumor growth in an H22 cell‑bearing liver cancer mouse model. Treatment with EAEHJ significantly reduced tumor weight, but had no effect on murine body weight. The results of the present study also showed that EAEHJ induced H22 cell apoptosis in vivo. In addition, the anti‑carcinogenic effects of EAEHJ were investigated in vitro. The results of the present study demonstrate that both phospholipid asymmetry in the plasma membrane and mitochondrial membrane potential were deregulated in HepG2 human hepatoma cells, following treatment with EAEHJ. Treatment with EAEHJ also increased the ratio of pro‑apoptotic B‑cell lymphoma 2 (Bcl‑2)‑associated X protein (Bax) to anti‑apoptotic Bcl‑2, and activated the caspase‑9 signaling pathway. These results suggest that EAEHJ is able to trigger the apoptosis of liver cancer cells via the mitochondria-dependent pathway.

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(A) Effects of Hypericum japonicum ethyl acetate extract (EAEHJ) on the expression levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) in a H22 cell-bearing liver cancer mouse model. At the end of the experiment, tumor tissue samples from the control and EAEHJ-treated groups were processed for immunohistochemical (IHC) staining in order to determine the expression levels of Bax and Bcl-2. Representative images were captured at a ×400 magnification. Quantification of the IHC assay was measured as the percentage of positively-stained cells. The data are presented as the mean ± standard deviation (n=8). *P<0.01, and **P<0.05, vs. the control. (B and C) Effects of EAEHJ on the expression levels of Bax and Bcl-2 in HepG2 human hepatoma cells. HepG2 human hepatoma cells were treated with various concentrations of EAEHJ for 24 h. (B) The mRNA expression levels of Bax and Bcl-2 in the EAEHJ-treated and untreated cells were determined by reverse transcription-polymerase chain reaction (RTq-PCR). (C) The protein expression levels of Bax and Bcl-2 were analyzed by western blotting. GAPDH and β-actin were used as internal controls for the RTq-PCR and western blotting assays, respectively. The data are representative of three independent experiments.
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f4-mmr-12-04-4851: (A) Effects of Hypericum japonicum ethyl acetate extract (EAEHJ) on the expression levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) in a H22 cell-bearing liver cancer mouse model. At the end of the experiment, tumor tissue samples from the control and EAEHJ-treated groups were processed for immunohistochemical (IHC) staining in order to determine the expression levels of Bax and Bcl-2. Representative images were captured at a ×400 magnification. Quantification of the IHC assay was measured as the percentage of positively-stained cells. The data are presented as the mean ± standard deviation (n=8). *P<0.01, and **P<0.05, vs. the control. (B and C) Effects of EAEHJ on the expression levels of Bax and Bcl-2 in HepG2 human hepatoma cells. HepG2 human hepatoma cells were treated with various concentrations of EAEHJ for 24 h. (B) The mRNA expression levels of Bax and Bcl-2 in the EAEHJ-treated and untreated cells were determined by reverse transcription-polymerase chain reaction (RTq-PCR). (C) The protein expression levels of Bax and Bcl-2 were analyzed by western blotting. GAPDH and β-actin were used as internal controls for the RTq-PCR and western blotting assays, respectively. The data are representative of three independent experiments.

Mentions: Pro-apoptotic Bax and anti-apoptotic Bcl-2 are two members of the Bcl-2 family that regulate the mitochondrial signaling pathway during apoptosis. Mitochondria-dependent apoptosis is primarily regulated by Bcl-2 family proteins. It has been suggested that mitochondrial outer membrane permeabilization (MOMP) occurs through the formation of mitochondrial pores by pro-apoptotic Bax-like proteins, which may be inhibited by anti-apoptotic Bcl-2 like members (14,15). Therefore, the ratio of Bax to Bcl-2 is crucial for determining cell fate. Higher Bcl-2 to Bax ratios, caused by the overexpression of Bcl-2 and/or downregulation of Bax, are commonly detected in various types of cancer (16), which confers a survival advantage to the cancer cells, and is associated with both chemotherapy and radiotherapy resistance. The results of the IHC assay indicated that treatment with EAEHJ reduced the expression levels of anti-apoptotic Bcl-2 in tumors, whereas the expression of pro-apoptotic Bax was increased. As shown in Fig. 4A, the percentage of Bax- and Bcl-2-positive cells in the control group was 7.8±2.5 and 38.2±6.9%, respectively, whereas those of the EAEHJ-treated mice were 16.4±3.5 (P<0.01, vs. controls) and 28±9.4% (P<0.05, vs. controls), respectively. These results indicate that treatment with EAEHJ may increase the pro-apoptotic Bax/Bcl-2 ratio. To our knowledge, this is the first time that EAEHJ has been reported to inhibit hepatoma cancer growth in vivo via the promotion of cancer cell apoptosis.


Ethyl acetate extract of Hypericum japonicum induces apoptosis via the mitochondria-dependent pathway in vivo and in vitro.

Zhuang Q, Li J, Chen Y, Lin J, Lai F, Chen X, Lin X, Peng J - Mol Med Rep (2015)

(A) Effects of Hypericum japonicum ethyl acetate extract (EAEHJ) on the expression levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) in a H22 cell-bearing liver cancer mouse model. At the end of the experiment, tumor tissue samples from the control and EAEHJ-treated groups were processed for immunohistochemical (IHC) staining in order to determine the expression levels of Bax and Bcl-2. Representative images were captured at a ×400 magnification. Quantification of the IHC assay was measured as the percentage of positively-stained cells. The data are presented as the mean ± standard deviation (n=8). *P<0.01, and **P<0.05, vs. the control. (B and C) Effects of EAEHJ on the expression levels of Bax and Bcl-2 in HepG2 human hepatoma cells. HepG2 human hepatoma cells were treated with various concentrations of EAEHJ for 24 h. (B) The mRNA expression levels of Bax and Bcl-2 in the EAEHJ-treated and untreated cells were determined by reverse transcription-polymerase chain reaction (RTq-PCR). (C) The protein expression levels of Bax and Bcl-2 were analyzed by western blotting. GAPDH and β-actin were used as internal controls for the RTq-PCR and western blotting assays, respectively. The data are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581784&req=5

f4-mmr-12-04-4851: (A) Effects of Hypericum japonicum ethyl acetate extract (EAEHJ) on the expression levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) in a H22 cell-bearing liver cancer mouse model. At the end of the experiment, tumor tissue samples from the control and EAEHJ-treated groups were processed for immunohistochemical (IHC) staining in order to determine the expression levels of Bax and Bcl-2. Representative images were captured at a ×400 magnification. Quantification of the IHC assay was measured as the percentage of positively-stained cells. The data are presented as the mean ± standard deviation (n=8). *P<0.01, and **P<0.05, vs. the control. (B and C) Effects of EAEHJ on the expression levels of Bax and Bcl-2 in HepG2 human hepatoma cells. HepG2 human hepatoma cells were treated with various concentrations of EAEHJ for 24 h. (B) The mRNA expression levels of Bax and Bcl-2 in the EAEHJ-treated and untreated cells were determined by reverse transcription-polymerase chain reaction (RTq-PCR). (C) The protein expression levels of Bax and Bcl-2 were analyzed by western blotting. GAPDH and β-actin were used as internal controls for the RTq-PCR and western blotting assays, respectively. The data are representative of three independent experiments.
Mentions: Pro-apoptotic Bax and anti-apoptotic Bcl-2 are two members of the Bcl-2 family that regulate the mitochondrial signaling pathway during apoptosis. Mitochondria-dependent apoptosis is primarily regulated by Bcl-2 family proteins. It has been suggested that mitochondrial outer membrane permeabilization (MOMP) occurs through the formation of mitochondrial pores by pro-apoptotic Bax-like proteins, which may be inhibited by anti-apoptotic Bcl-2 like members (14,15). Therefore, the ratio of Bax to Bcl-2 is crucial for determining cell fate. Higher Bcl-2 to Bax ratios, caused by the overexpression of Bcl-2 and/or downregulation of Bax, are commonly detected in various types of cancer (16), which confers a survival advantage to the cancer cells, and is associated with both chemotherapy and radiotherapy resistance. The results of the IHC assay indicated that treatment with EAEHJ reduced the expression levels of anti-apoptotic Bcl-2 in tumors, whereas the expression of pro-apoptotic Bax was increased. As shown in Fig. 4A, the percentage of Bax- and Bcl-2-positive cells in the control group was 7.8±2.5 and 38.2±6.9%, respectively, whereas those of the EAEHJ-treated mice were 16.4±3.5 (P<0.01, vs. controls) and 28±9.4% (P<0.05, vs. controls), respectively. These results indicate that treatment with EAEHJ may increase the pro-apoptotic Bax/Bcl-2 ratio. To our knowledge, this is the first time that EAEHJ has been reported to inhibit hepatoma cancer growth in vivo via the promotion of cancer cell apoptosis.

Bottom Line: Treatment with EAEHJ significantly reduced tumor weight, but had no effect on murine body weight.The results of the present study also showed that EAEHJ induced H22 cell apoptosis in vivo.Treatment with EAEHJ also increased the ratio of pro‑apoptotic B‑cell lymphoma 2 (Bcl‑2)‑associated X protein (Bax) to anti‑apoptotic Bcl‑2, and activated the caspase‑9 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, P.R. China.

ABSTRACT
The widely-used Chinese medicinal herb Hypericum japonicum, also known as Hypericum japonicum Thunb or Tianjihuang, displays potent anti‑carcinogenic effects against liver cancer. However, the molecular mechanism underlying the therapeutic effects of Hypericum japonicum remains to be elucidated. The present study investigated the in vivo efficacy of ethyl acetate extract of Hypericum japonicum (EAEHJ) against tumor growth in an H22 cell‑bearing liver cancer mouse model. Treatment with EAEHJ significantly reduced tumor weight, but had no effect on murine body weight. The results of the present study also showed that EAEHJ induced H22 cell apoptosis in vivo. In addition, the anti‑carcinogenic effects of EAEHJ were investigated in vitro. The results of the present study demonstrate that both phospholipid asymmetry in the plasma membrane and mitochondrial membrane potential were deregulated in HepG2 human hepatoma cells, following treatment with EAEHJ. Treatment with EAEHJ also increased the ratio of pro‑apoptotic B‑cell lymphoma 2 (Bcl‑2)‑associated X protein (Bax) to anti‑apoptotic Bcl‑2, and activated the caspase‑9 signaling pathway. These results suggest that EAEHJ is able to trigger the apoptosis of liver cancer cells via the mitochondria-dependent pathway.

Show MeSH
Related in: MedlinePlus