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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.


Lanatoside C induced apoptosis in HCC cells.(A) Hep3B cells were treated with the indicated concentrations of lanatoside C (0.1–0.6 μM) for 18 h and detected of procaspase-2, -3, -6, -7, -8, -9 and PARP protein expressions by using Western blot analysis. (B) Hep3B cells were treated with lanatoside C (0.6 μM) for the indicated time (6–24 h), and cells were harvested from total lysates for detection of PARP protein expressions by using Western blot analysis. Data are representative of three independent experiments. (C and D) Hep3B cells were incubated in 0.6 μM lanatoside C with or without 100 μM z-VAD-fmk for 24 h. (C) The cell viability was determined by using MTT assay as described in methods. Data are repeated at least three independent determinations. *P < 0.05. (D) The apoptotic cells were stained with PI and analyzed by flow cytometry as described in methods. Data are repeated at least three independent determinations. **P < 0.01. (E) HA22T cells were treated with lanatoside C for 18 h to detect the expressions of caspases and mitochondrial proteins.
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f3: Lanatoside C induced apoptosis in HCC cells.(A) Hep3B cells were treated with the indicated concentrations of lanatoside C (0.1–0.6 μM) for 18 h and detected of procaspase-2, -3, -6, -7, -8, -9 and PARP protein expressions by using Western blot analysis. (B) Hep3B cells were treated with lanatoside C (0.6 μM) for the indicated time (6–24 h), and cells were harvested from total lysates for detection of PARP protein expressions by using Western blot analysis. Data are representative of three independent experiments. (C and D) Hep3B cells were incubated in 0.6 μM lanatoside C with or without 100 μM z-VAD-fmk for 24 h. (C) The cell viability was determined by using MTT assay as described in methods. Data are repeated at least three independent determinations. *P < 0.05. (D) The apoptotic cells were stained with PI and analyzed by flow cytometry as described in methods. Data are repeated at least three independent determinations. **P < 0.01. (E) HA22T cells were treated with lanatoside C for 18 h to detect the expressions of caspases and mitochondrial proteins.

Mentions: Mitochondrial outer membrane permeabilization allows soluble proteins to diffuse into the cytosol from the intermembrane space of mitochondria. One such soluble protein is apoptosis-inducing factor (AIF), which translocates into the nucleus to trigger caspase-independent apoptosis18. Our results indicate that lanatoside C markedly increased the amount of AIF in the nuclear fraction in a time-dependent manner (Fig. 2D). Similar results were obtained in HA22T cells, suggesting that lanatoside C causes caspase-independent apoptosis in human HCC cells (Fig. 3E and Supplementary Fig. S2A).


Lanatoside C, a cardiac glycoside, acts through protein kinase C δ to cause apoptosis of human hepatocellular carcinoma cells
Lanatoside C induced apoptosis in HCC cells.(A) Hep3B cells were treated with the indicated concentrations of lanatoside C (0.1–0.6 μM) for 18 h and detected of procaspase-2, -3, -6, -7, -8, -9 and PARP protein expressions by using Western blot analysis. (B) Hep3B cells were treated with lanatoside C (0.6 μM) for the indicated time (6–24 h), and cells were harvested from total lysates for detection of PARP protein expressions by using Western blot analysis. Data are representative of three independent experiments. (C and D) Hep3B cells were incubated in 0.6 μM lanatoside C with or without 100 μM z-VAD-fmk for 24 h. (C) The cell viability was determined by using MTT assay as described in methods. Data are repeated at least three independent determinations. *P < 0.05. (D) The apoptotic cells were stained with PI and analyzed by flow cytometry as described in methods. Data are repeated at least three independent determinations. **P < 0.01. (E) HA22T cells were treated with lanatoside C for 18 h to detect the expressions of caspases and mitochondrial proteins.
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f3: Lanatoside C induced apoptosis in HCC cells.(A) Hep3B cells were treated with the indicated concentrations of lanatoside C (0.1–0.6 μM) for 18 h and detected of procaspase-2, -3, -6, -7, -8, -9 and PARP protein expressions by using Western blot analysis. (B) Hep3B cells were treated with lanatoside C (0.6 μM) for the indicated time (6–24 h), and cells were harvested from total lysates for detection of PARP protein expressions by using Western blot analysis. Data are representative of three independent experiments. (C and D) Hep3B cells were incubated in 0.6 μM lanatoside C with or without 100 μM z-VAD-fmk for 24 h. (C) The cell viability was determined by using MTT assay as described in methods. Data are repeated at least three independent determinations. *P < 0.05. (D) The apoptotic cells were stained with PI and analyzed by flow cytometry as described in methods. Data are repeated at least three independent determinations. **P < 0.01. (E) HA22T cells were treated with lanatoside C for 18 h to detect the expressions of caspases and mitochondrial proteins.
Mentions: Mitochondrial outer membrane permeabilization allows soluble proteins to diffuse into the cytosol from the intermembrane space of mitochondria. One such soluble protein is apoptosis-inducing factor (AIF), which translocates into the nucleus to trigger caspase-independent apoptosis18. Our results indicate that lanatoside C markedly increased the amount of AIF in the nuclear fraction in a time-dependent manner (Fig. 2D). Similar results were obtained in HA22T cells, suggesting that lanatoside C causes caspase-independent apoptosis in human HCC cells (Fig. 3E and Supplementary Fig. S2A).

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 (PKC&delta;) via Thr505 phosphorylation and subsequent membrane translocation. Inhibition of PKC&delta; reversed lanatoside C-induced MMP loss and apoptosis, confirming that lanatoside C caused apoptosis through PKC&delta; activation. We also found that the AKT/mTOR pathway was negatively regulated by lanatoside C through PKC&delta; activation. In conclusion, we provide the first demonstration that the anticancer effects of lanatoside C are mainly attributable to PKC&delta; activation.

No MeSH data available.