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Lanatoside C, a cardiac glycoside, acts through protein kinase C δ to cause apoptosis of human hepatocellular carcinoma cells

View Article: PubMed Central - PubMed

ABSTRACT

Recent studies have revealed that cardiac glycosides, such as digitalis and digoxin, have anticancer activity and may serve as lead compounds for the development of cancer treatments. The poor prognosis of hepatocellular carcinoma (HCC) patients reflects the development of resistance to current chemotherapeutic agents, highlighting the need for discovering new small-molecule therapeutics. Here, we found that lanatoside C, an anti-arrhythmic agent extracted from Digitalis lanata, inhibited the growth of HCC cells and dramatically decreased tumor volume as well as delayed tumor growth without obvious body weight loss. Moreover, lanatoside C triggered mitochondrial membrane potential (MMP) loss, activation of caspases and translocation of apoptosis-inducing factor (AIF) into the nucleus, which suggests that lanatoside C induced apoptosis through both caspase-dependent and -independent pathways. Furthermore, we discovered that lanatoside C activated protein kinase delta (PKCδ) via Thr505 phosphorylation and subsequent membrane translocation. Inhibition of PKCδ reversed lanatoside C-induced MMP loss and apoptosis, confirming that lanatoside C caused apoptosis through PKCδ activation. We also found that the AKT/mTOR pathway was negatively regulated by lanatoside C through PKCδ activation. In conclusion, we provide the first demonstration that the anticancer effects of lanatoside C are mainly attributable to PKCδ activation.

No MeSH data available.


Effect of lanatoside C on MMP and mitochondrial related proteins.(A) Hep3B cells were incubated with DMSO (0 h) or with lanatoside C (0.6 μM) at indicated times and then cells were incubated with rhodamine123 (5 μM) for 30 minutes. Data acquisition and analysis were performed on a FACScan flow cytometry. (B) Hep3B cells were treated with a range of lanatoside C concentration (0.1–0.6 μM) for 18 hr. (C) Hep3B cells were treated with lanatoside C (0.6 μM) for indicated time (6–24 h), and then cells were harvested from total lysates for detection of BID, Bcl-xL and Mcl-1 protein expressions by using Western blot analysis. α-tubulin was used as internal control. (D) Hep3B cells were treated lanatoside C (0.6 μM) for 12 h, 18 h, and 24 h and detected of AIF in nuclear, cytosol fraction and total lysate by Western blot analysis. C23, GAPDH and α-tubulin was a nuclear, cytosol and total lysate marker protein, respectively, used as internal controls. Data are representative of three independent experiments.
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f2: Effect of lanatoside C on MMP and mitochondrial related proteins.(A) Hep3B cells were incubated with DMSO (0 h) or with lanatoside C (0.6 μM) at indicated times and then cells were incubated with rhodamine123 (5 μM) for 30 minutes. Data acquisition and analysis were performed on a FACScan flow cytometry. (B) Hep3B cells were treated with a range of lanatoside C concentration (0.1–0.6 μM) for 18 hr. (C) Hep3B cells were treated with lanatoside C (0.6 μM) for indicated time (6–24 h), and then cells were harvested from total lysates for detection of BID, Bcl-xL and Mcl-1 protein expressions by using Western blot analysis. α-tubulin was used as internal control. (D) Hep3B cells were treated lanatoside C (0.6 μM) for 12 h, 18 h, and 24 h and detected of AIF in nuclear, cytosol fraction and total lysate by Western blot analysis. C23, GAPDH and α-tubulin was a nuclear, cytosol and total lysate marker protein, respectively, used as internal controls. Data are representative of three independent experiments.

Mentions: Loss of MMP is associated with opening of the mitochondrial permeability transition pore, leakage of inner mitochondrial components into the cytosol and subsequent apoptosis16. To investigate the effect of lanatoside C on MMP, we incubated Hep3B cells with 0.6 μM lanatoside C for the indicated times and then measured rhodamine 123 fluorescence. As shown in Fig. 2A, lanatoside C induced a significant and sustained reduction in MMP after 14–18 h of treatment. These results indicate that lanatoside C induces mitochondrial dysfunction in Hep3B cells in a time-dependent manner. The Bcl-2 protein family regulate mitochondrial outer membrane permeability, which leads to the intrinsic apoptotic pathway17. We found that lanatoside C downregulated the expression of the Bcl-2 family members, BID, Bcl-2 and Mcl-1, in a concentration- and time-dependent manner (Fig. 2B and C).


Lanatoside C, a cardiac glycoside, acts through protein kinase C δ to cause apoptosis of human hepatocellular carcinoma cells
Effect of lanatoside C on MMP and mitochondrial related proteins.(A) Hep3B cells were incubated with DMSO (0 h) or with lanatoside C (0.6 μM) at indicated times and then cells were incubated with rhodamine123 (5 μM) for 30 minutes. Data acquisition and analysis were performed on a FACScan flow cytometry. (B) Hep3B cells were treated with a range of lanatoside C concentration (0.1–0.6 μM) for 18 hr. (C) Hep3B cells were treated with lanatoside C (0.6 μM) for indicated time (6–24 h), and then cells were harvested from total lysates for detection of BID, Bcl-xL and Mcl-1 protein expressions by using Western blot analysis. α-tubulin was used as internal control. (D) Hep3B cells were treated lanatoside C (0.6 μM) for 12 h, 18 h, and 24 h and detected of AIF in nuclear, cytosol fraction and total lysate by Western blot analysis. C23, GAPDH and α-tubulin was a nuclear, cytosol and total lysate marker protein, respectively, used as internal controls. Data are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: Effect of lanatoside C on MMP and mitochondrial related proteins.(A) Hep3B cells were incubated with DMSO (0 h) or with lanatoside C (0.6 μM) at indicated times and then cells were incubated with rhodamine123 (5 μM) for 30 minutes. Data acquisition and analysis were performed on a FACScan flow cytometry. (B) Hep3B cells were treated with a range of lanatoside C concentration (0.1–0.6 μM) for 18 hr. (C) Hep3B cells were treated with lanatoside C (0.6 μM) for indicated time (6–24 h), and then cells were harvested from total lysates for detection of BID, Bcl-xL and Mcl-1 protein expressions by using Western blot analysis. α-tubulin was used as internal control. (D) Hep3B cells were treated lanatoside C (0.6 μM) for 12 h, 18 h, and 24 h and detected of AIF in nuclear, cytosol fraction and total lysate by Western blot analysis. C23, GAPDH and α-tubulin was a nuclear, cytosol and total lysate marker protein, respectively, used as internal controls. Data are representative of three independent experiments.
Mentions: Loss of MMP is associated with opening of the mitochondrial permeability transition pore, leakage of inner mitochondrial components into the cytosol and subsequent apoptosis16. To investigate the effect of lanatoside C on MMP, we incubated Hep3B cells with 0.6 μM lanatoside C for the indicated times and then measured rhodamine 123 fluorescence. As shown in Fig. 2A, lanatoside C induced a significant and sustained reduction in MMP after 14–18 h of treatment. These results indicate that lanatoside C induces mitochondrial dysfunction in Hep3B cells in a time-dependent manner. The Bcl-2 protein family regulate mitochondrial outer membrane permeability, which leads to the intrinsic apoptotic pathway17. We found that lanatoside C downregulated the expression of the Bcl-2 family members, BID, Bcl-2 and Mcl-1, in a concentration- and time-dependent manner (Fig. 2B and C).

View Article: PubMed Central - PubMed

ABSTRACT

Recent studies have revealed that cardiac glycosides, such as digitalis and digoxin, have anticancer activity and may serve as lead compounds for the development of cancer treatments. The poor prognosis of hepatocellular carcinoma (HCC) patients reflects the development of resistance to current chemotherapeutic agents, highlighting the need for discovering new small-molecule therapeutics. Here, we found that lanatoside C, an anti-arrhythmic agent extracted from Digitalis lanata, inhibited the growth of HCC cells and dramatically decreased tumor volume as well as delayed tumor growth without obvious body weight loss. Moreover, lanatoside C triggered mitochondrial membrane potential (MMP) loss, activation of caspases and translocation of apoptosis-inducing factor (AIF) into the nucleus, which suggests that lanatoside C induced apoptosis through both caspase-dependent and -independent pathways. Furthermore, we discovered that lanatoside C activated protein kinase delta (PKCδ) via Thr505 phosphorylation and subsequent membrane translocation. Inhibition of PKCδ reversed lanatoside C-induced MMP loss and apoptosis, confirming that lanatoside C caused apoptosis through PKCδ activation. We also found that the AKT/mTOR pathway was negatively regulated by lanatoside C through PKCδ activation. In conclusion, we provide the first demonstration that the anticancer effects of lanatoside C are mainly attributable to PKCδ activation.

No MeSH data available.