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Cyproheptadine, an antihistaminic drug, inhibits proliferation of hepatocellular carcinoma cells by blocking cell cycle progression through the activation of P38 MAP kinase.

Feng YM, Feng CW, Chen SY, Hsieh HY, Chen YH, Hsu CD - BMC Cancer (2015)

Bottom Line: The cyproheptadine-induced G1 arrest in HepG2 cells was associated with an increased expression of HBP1 and p16, whereas the G1/S arrest in Huh-7 cells was associated with an increase in p21 and p27 expression and a dramatic decrease in the phosphorylation of the retinoblastoma protein.Our results demonstrate that a non-classical p38 MAP kinase function, regulation of cell cycle checkpoints, is one of the underlying mechanisms promoted by cyproheptadine to suppress the proliferation of HCC cells.These results provide evidence for the drug's potential as a treatment option for liver cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chia-Yi, Taiwan. fengyumin2@gmail.com.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is a major cause of cancer deaths worldwide. However, current chemotherapeutic drugs for HCC are either poorly effective or expensive, and treatment with these drugs has not led to satisfactory outcomes. In a 2012 case report, we described our breakthrough finding in two advanced HCC patients, of whom one achieved complete remission of liver tumors and the other a normalized α-fetoprotein level, along with complete remission of their lung metastases, after the concomitant use of thalidomide and cyproheptadine. We assumed the key factor in our effective therapy to be cyproheptadine. In this study, we investigated the antiproliferative effects and molecular mechanisms of cyproheptadine.

Methods: The effect of cyproheptadine on cell proliferation was examined in human HCC cell lines HepG2 and Huh-7. Cell viability was assayed with Cell Counting Kit-8; cell cycle distribution was analyzed by flow cytometry. Mechanisms underlying cyproheptadine-induced cell cycle arrest were probed by western blot analysis.

Results: Cyproheptadine had a potent inhibitory effect on the proliferation of HepG2 and Huh-7 cells but minimal toxicity in normal hepatocytes. Cyproheptadine induced cell cycle arrest in HepG2 cells in the G1 phase and in Huh-7 cells at the G1/S transition. The cyproheptadine-induced G1 arrest in HepG2 cells was associated with an increased expression of HBP1 and p16, whereas the G1/S arrest in Huh-7 cells was associated with an increase in p21 and p27 expression and a dramatic decrease in the phosphorylation of the retinoblastoma protein. Additionally, cyproheptadine elevated the percentage of Huh-7 cells in the sub-G1 population, increased annexin V staining for cell death, and raised the levels of PARP and its cleaved form, indicating induction of apoptosis. Finally, cyproheptadine-mediated cell cycle arrest was dependent upon the activation of p38 MAP kinase in HepG2 cells and the activation of both p38 MAP kinase and CHK2 in Huh-7 cells.

Conclusions: Our results demonstrate that a non-classical p38 MAP kinase function, regulation of cell cycle checkpoints, is one of the underlying mechanisms promoted by cyproheptadine to suppress the proliferation of HCC cells. These results provide evidence for the drug's potential as a treatment option for liver cancer.

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Cytotoxicity of cyproheptadine toward normal human hepatocytes (HH) and HCC cell lines HepG2 and Huh-7. Cells in 96-well plates were cultured for 24 h, starved in serum-free medium for 24 h, and then treated with various concentrations of cyproheptadine for 24 h. Viability was determined for the treated cells using Cell Counting Kit-8. Data are presented as mean ± SD (n = 6). Significant differences from the no-treatment control, determined by one-way ANOVA and Dunnett’s comparison test, are indicated by asterisks: *p < 0.05; ***p < 0.001.
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Fig1: Cytotoxicity of cyproheptadine toward normal human hepatocytes (HH) and HCC cell lines HepG2 and Huh-7. Cells in 96-well plates were cultured for 24 h, starved in serum-free medium for 24 h, and then treated with various concentrations of cyproheptadine for 24 h. Viability was determined for the treated cells using Cell Counting Kit-8. Data are presented as mean ± SD (n = 6). Significant differences from the no-treatment control, determined by one-way ANOVA and Dunnett’s comparison test, are indicated by asterisks: *p < 0.05; ***p < 0.001.

Mentions: We first performed an in vitro cell viability assay to compare the cytotoxicity of cyproheptadine in normal human hepatocytes and in HCC-derived human cancer cell lines. Analysis using Cell Counting Kit-8 revealed significant cytotoxicity of cyproheptadine to HepG2 and Huh-7 cells relative to normal hepatocytes at various concentrations and showed that cyproheptadine inhibited cell proliferation in a dose-dependent manner (Figure 1). A similar pattern was also observed in HepG2 and Huh-7 cells treated with cyproheptadine at a low-dosage range (0.5–5 μM) for 48 h (Additional file 1: Figure S1). The IC50 of cyproheptadine, determined as the concentration of the drug that inhibited cell growth by 50% after 24 h of treatment, was found to be 44.4, 44.7, and 118.1 μM in HepG2 cells, Huh-7 cells, and normal human hepatocytes, respectively. Cyproheptadine’s highly selective toxicity toward cancer cells is represented by its high selectivity index (SI) values for HepG2 and Huh-7 cells (2.7 and 2.6, respectively; Table 1).Figure 1


Cyproheptadine, an antihistaminic drug, inhibits proliferation of hepatocellular carcinoma cells by blocking cell cycle progression through the activation of P38 MAP kinase.

Feng YM, Feng CW, Chen SY, Hsieh HY, Chen YH, Hsu CD - BMC Cancer (2015)

Cytotoxicity of cyproheptadine toward normal human hepatocytes (HH) and HCC cell lines HepG2 and Huh-7. Cells in 96-well plates were cultured for 24 h, starved in serum-free medium for 24 h, and then treated with various concentrations of cyproheptadine for 24 h. Viability was determined for the treated cells using Cell Counting Kit-8. Data are presented as mean ± SD (n = 6). Significant differences from the no-treatment control, determined by one-way ANOVA and Dunnett’s comparison test, are indicated by asterisks: *p < 0.05; ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4383201&req=5

Fig1: Cytotoxicity of cyproheptadine toward normal human hepatocytes (HH) and HCC cell lines HepG2 and Huh-7. Cells in 96-well plates were cultured for 24 h, starved in serum-free medium for 24 h, and then treated with various concentrations of cyproheptadine for 24 h. Viability was determined for the treated cells using Cell Counting Kit-8. Data are presented as mean ± SD (n = 6). Significant differences from the no-treatment control, determined by one-way ANOVA and Dunnett’s comparison test, are indicated by asterisks: *p < 0.05; ***p < 0.001.
Mentions: We first performed an in vitro cell viability assay to compare the cytotoxicity of cyproheptadine in normal human hepatocytes and in HCC-derived human cancer cell lines. Analysis using Cell Counting Kit-8 revealed significant cytotoxicity of cyproheptadine to HepG2 and Huh-7 cells relative to normal hepatocytes at various concentrations and showed that cyproheptadine inhibited cell proliferation in a dose-dependent manner (Figure 1). A similar pattern was also observed in HepG2 and Huh-7 cells treated with cyproheptadine at a low-dosage range (0.5–5 μM) for 48 h (Additional file 1: Figure S1). The IC50 of cyproheptadine, determined as the concentration of the drug that inhibited cell growth by 50% after 24 h of treatment, was found to be 44.4, 44.7, and 118.1 μM in HepG2 cells, Huh-7 cells, and normal human hepatocytes, respectively. Cyproheptadine’s highly selective toxicity toward cancer cells is represented by its high selectivity index (SI) values for HepG2 and Huh-7 cells (2.7 and 2.6, respectively; Table 1).Figure 1

Bottom Line: The cyproheptadine-induced G1 arrest in HepG2 cells was associated with an increased expression of HBP1 and p16, whereas the G1/S arrest in Huh-7 cells was associated with an increase in p21 and p27 expression and a dramatic decrease in the phosphorylation of the retinoblastoma protein.Our results demonstrate that a non-classical p38 MAP kinase function, regulation of cell cycle checkpoints, is one of the underlying mechanisms promoted by cyproheptadine to suppress the proliferation of HCC cells.These results provide evidence for the drug's potential as a treatment option for liver cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chia-Yi, Taiwan. fengyumin2@gmail.com.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is a major cause of cancer deaths worldwide. However, current chemotherapeutic drugs for HCC are either poorly effective or expensive, and treatment with these drugs has not led to satisfactory outcomes. In a 2012 case report, we described our breakthrough finding in two advanced HCC patients, of whom one achieved complete remission of liver tumors and the other a normalized α-fetoprotein level, along with complete remission of their lung metastases, after the concomitant use of thalidomide and cyproheptadine. We assumed the key factor in our effective therapy to be cyproheptadine. In this study, we investigated the antiproliferative effects and molecular mechanisms of cyproheptadine.

Methods: The effect of cyproheptadine on cell proliferation was examined in human HCC cell lines HepG2 and Huh-7. Cell viability was assayed with Cell Counting Kit-8; cell cycle distribution was analyzed by flow cytometry. Mechanisms underlying cyproheptadine-induced cell cycle arrest were probed by western blot analysis.

Results: Cyproheptadine had a potent inhibitory effect on the proliferation of HepG2 and Huh-7 cells but minimal toxicity in normal hepatocytes. Cyproheptadine induced cell cycle arrest in HepG2 cells in the G1 phase and in Huh-7 cells at the G1/S transition. The cyproheptadine-induced G1 arrest in HepG2 cells was associated with an increased expression of HBP1 and p16, whereas the G1/S arrest in Huh-7 cells was associated with an increase in p21 and p27 expression and a dramatic decrease in the phosphorylation of the retinoblastoma protein. Additionally, cyproheptadine elevated the percentage of Huh-7 cells in the sub-G1 population, increased annexin V staining for cell death, and raised the levels of PARP and its cleaved form, indicating induction of apoptosis. Finally, cyproheptadine-mediated cell cycle arrest was dependent upon the activation of p38 MAP kinase in HepG2 cells and the activation of both p38 MAP kinase and CHK2 in Huh-7 cells.

Conclusions: Our results demonstrate that a non-classical p38 MAP kinase function, regulation of cell cycle checkpoints, is one of the underlying mechanisms promoted by cyproheptadine to suppress the proliferation of HCC cells. These results provide evidence for the drug's potential as a treatment option for liver cancer.

Show MeSH
Related in: MedlinePlus