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Synergistic growth inhibition by acyclic retinoid and phosphatidylinositol 3-kinase inhibitor in human hepatoma cells.

Baba A, Shimizu M, Ohno T, Shirakami Y, Kubota M, Kochi T, Terakura D, Tsurumi H, Moriwaki H - BMC Cancer (2013)

Bottom Line: This study examined the effects of the combination of ACR plus LY294002 on the growth of HLF human HCC cells.ACR and LY294002 cooperatively increase the expression of RARβ, while inhibiting the phosphorylation of RXRα, and that these effects are associated with the induction of apoptosis and the inhibition of cell growth in human HCC cells.This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, Gifu University Graduate School of Medicine, Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan. shimim-gif@umin.ac.jp.

ABSTRACT

Background: A malfunction of RXRα due to phosphorylation is associated with liver carcinogenesis, and acyclic retinoid (ACR), which targets RXRα, can prevent the development of hepatocellular carcinoma (HCC). Activation of PI3K/Akt signaling plays a critical role in the proliferation and survival of HCC cells. The present study examined the possible combined effects of ACR and LY294002, a PI3K inhibitor, on the growth of human HCC cells.

Methods: This study examined the effects of the combination of ACR plus LY294002 on the growth of HLF human HCC cells.

Results: ACR and LY294002 preferentially inhibited the growth of HLF cells in comparison with Hc normal hepatocytes. The combination of 1 μM ACR and 5 μM LY294002, in which the concentrations used are less than the IC₅₀ values of these agents, synergistically inhibited the growth of HLF, Hep3B, and Huh7 human HCC cells. These agents when administered in combination acted cooperatively to induce apoptosis in HLF cells. The phosphorylation of RXRα, Akt, and ERK proteins in HLF cells were markedly inhibited by treatment with ACR plus LY294002. Moreover, this combination also increased RXRE promoter activity and the cellular levels of RARβ and p21(CIP1), while decreasing the levels of cyclin D1.

Conclusion: ACR and LY294002 cooperatively increase the expression of RARβ, while inhibiting the phosphorylation of RXRα, and that these effects are associated with the induction of apoptosis and the inhibition of cell growth in human HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

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Effects of the combination of ACR and LY294002 on the phosphorylation of RXRα, ERK, and Akt proteins and the transcriptional activity of the RXRE promoter in HLF cells. (A) The cells were treated with vehicle, 1 μM ACR alone, 5 μM LY294002 alone, or a combination of 1 μM ACR and 5 μM LY294002 for 12 hours. The extracted proteins were examined by western blot analysis using the respective antibodies. Repeat western blots gave similar results. (B) A transient transfection reporter assay was performed with the RXRE luciferase reporter in the presence of vehicle, 1 μM ACR alone, 5 μM LY294002 alone, or a combination of 1 μM ACR and 5 μM LY294002. Relative luciferase activity was determined after 24 hours. Columns and lines indicate the means and SD of triplicate assays. # P < 0.01. * P < 0.05.
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Figure 5: Effects of the combination of ACR and LY294002 on the phosphorylation of RXRα, ERK, and Akt proteins and the transcriptional activity of the RXRE promoter in HLF cells. (A) The cells were treated with vehicle, 1 μM ACR alone, 5 μM LY294002 alone, or a combination of 1 μM ACR and 5 μM LY294002 for 12 hours. The extracted proteins were examined by western blot analysis using the respective antibodies. Repeat western blots gave similar results. (B) A transient transfection reporter assay was performed with the RXRE luciferase reporter in the presence of vehicle, 1 μM ACR alone, 5 μM LY294002 alone, or a combination of 1 μM ACR and 5 μM LY294002. Relative luciferase activity was determined after 24 hours. Columns and lines indicate the means and SD of triplicate assays. # P < 0.01. * P < 0.05.

Mentions: RXRα phosphorylation is involved in the development of HCC, and thus might be a promising target for HCC chemoprevention [4-9]. Therefore, the effects of the combination of ACR and LY294002 on the phosphorylation of RXRα and related signaling molecules were next investigated in HLF cells. As shown in Figure 5A, there was a significant decrease in the expression levels of p-RXRα, p-ERK, and p-Akt proteins when the cells were treated with 1 μM ACR. Treatment with 5 μM LY294002 also caused a marked decrease in the expression levels of p-RXRα and p-Akt proteins in these cells. Moreover, the decrease in the expression levels of p-RXRα, p-ERK, and p-Akt proteins was greater when the cells were treated with a combination of these agents.


Synergistic growth inhibition by acyclic retinoid and phosphatidylinositol 3-kinase inhibitor in human hepatoma cells.

Baba A, Shimizu M, Ohno T, Shirakami Y, Kubota M, Kochi T, Terakura D, Tsurumi H, Moriwaki H - BMC Cancer (2013)

Effects of the combination of ACR and LY294002 on the phosphorylation of RXRα, ERK, and Akt proteins and the transcriptional activity of the RXRE promoter in HLF cells. (A) The cells were treated with vehicle, 1 μM ACR alone, 5 μM LY294002 alone, or a combination of 1 μM ACR and 5 μM LY294002 for 12 hours. The extracted proteins were examined by western blot analysis using the respective antibodies. Repeat western blots gave similar results. (B) A transient transfection reporter assay was performed with the RXRE luciferase reporter in the presence of vehicle, 1 μM ACR alone, 5 μM LY294002 alone, or a combination of 1 μM ACR and 5 μM LY294002. Relative luciferase activity was determined after 24 hours. Columns and lines indicate the means and SD of triplicate assays. # P < 0.01. * P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852533&req=5

Figure 5: Effects of the combination of ACR and LY294002 on the phosphorylation of RXRα, ERK, and Akt proteins and the transcriptional activity of the RXRE promoter in HLF cells. (A) The cells were treated with vehicle, 1 μM ACR alone, 5 μM LY294002 alone, or a combination of 1 μM ACR and 5 μM LY294002 for 12 hours. The extracted proteins were examined by western blot analysis using the respective antibodies. Repeat western blots gave similar results. (B) A transient transfection reporter assay was performed with the RXRE luciferase reporter in the presence of vehicle, 1 μM ACR alone, 5 μM LY294002 alone, or a combination of 1 μM ACR and 5 μM LY294002. Relative luciferase activity was determined after 24 hours. Columns and lines indicate the means and SD of triplicate assays. # P < 0.01. * P < 0.05.
Mentions: RXRα phosphorylation is involved in the development of HCC, and thus might be a promising target for HCC chemoprevention [4-9]. Therefore, the effects of the combination of ACR and LY294002 on the phosphorylation of RXRα and related signaling molecules were next investigated in HLF cells. As shown in Figure 5A, there was a significant decrease in the expression levels of p-RXRα, p-ERK, and p-Akt proteins when the cells were treated with 1 μM ACR. Treatment with 5 μM LY294002 also caused a marked decrease in the expression levels of p-RXRα and p-Akt proteins in these cells. Moreover, the decrease in the expression levels of p-RXRα, p-ERK, and p-Akt proteins was greater when the cells were treated with a combination of these agents.

Bottom Line: This study examined the effects of the combination of ACR plus LY294002 on the growth of HLF human HCC cells.ACR and LY294002 cooperatively increase the expression of RARβ, while inhibiting the phosphorylation of RXRα, and that these effects are associated with the induction of apoptosis and the inhibition of cell growth in human HCC cells.This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, Gifu University Graduate School of Medicine, Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan. shimim-gif@umin.ac.jp.

ABSTRACT

Background: A malfunction of RXRα due to phosphorylation is associated with liver carcinogenesis, and acyclic retinoid (ACR), which targets RXRα, can prevent the development of hepatocellular carcinoma (HCC). Activation of PI3K/Akt signaling plays a critical role in the proliferation and survival of HCC cells. The present study examined the possible combined effects of ACR and LY294002, a PI3K inhibitor, on the growth of human HCC cells.

Methods: This study examined the effects of the combination of ACR plus LY294002 on the growth of HLF human HCC cells.

Results: ACR and LY294002 preferentially inhibited the growth of HLF cells in comparison with Hc normal hepatocytes. The combination of 1 μM ACR and 5 μM LY294002, in which the concentrations used are less than the IC₅₀ values of these agents, synergistically inhibited the growth of HLF, Hep3B, and Huh7 human HCC cells. These agents when administered in combination acted cooperatively to induce apoptosis in HLF cells. The phosphorylation of RXRα, Akt, and ERK proteins in HLF cells were markedly inhibited by treatment with ACR plus LY294002. Moreover, this combination also increased RXRE promoter activity and the cellular levels of RARβ and p21(CIP1), while decreasing the levels of cyclin D1.

Conclusion: ACR and LY294002 cooperatively increase the expression of RARβ, while inhibiting the phosphorylation of RXRα, and that these effects are associated with the induction of apoptosis and the inhibition of cell growth in human HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

Show MeSH
Related in: MedlinePlus