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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.


Related in: MedlinePlus

Effect of lanatoside C on AKT/mTOR pathway.(A) Hep3B and HA22T cells were treated with a range of lanatoside C (0.1–0.6 μM) for 18 h. (B) Hep3B cells were treated lanatoside C (0.6 μM) for indicated time (6–24 hr) and then cells were harvested from total lysates for observation of AKT/mTOR and their downstream signaling protein expressions by using Western blot analysis. (C) Hep3B cells were incubated with Lanatoside C (0.6 μM), rottlerin (5 μM) or PKCδ siRNA, or combination treatment for 18 h and then cells were harvested from total lysates for detection of indicated protein expressions by using Western blot analysis. (D) Hep3B cells were transfected with empty vector (MOCK) or Myr-AKT for 6 h and re-serum overnight, followed by treatment with or without lanatoside C (0.6 μM) for 18 h. Cells were harvested from total lysates for detection of phospho-AKT Ser473 protein expressions by using Western blot analysis. Cell viability was measured by MTT assay. Data are expressed as means ± SEM of three independent determinations. *P < 0.01, Myr-AKT-overexpressed cells versus MOCK-transfected cells.
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f5: Effect of lanatoside C on AKT/mTOR pathway.(A) Hep3B and HA22T cells were treated with a range of lanatoside C (0.1–0.6 μM) for 18 h. (B) Hep3B cells were treated lanatoside C (0.6 μM) for indicated time (6–24 hr) and then cells were harvested from total lysates for observation of AKT/mTOR and their downstream signaling protein expressions by using Western blot analysis. (C) Hep3B cells were incubated with Lanatoside C (0.6 μM), rottlerin (5 μM) or PKCδ siRNA, or combination treatment for 18 h and then cells were harvested from total lysates for detection of indicated protein expressions by using Western blot analysis. (D) Hep3B cells were transfected with empty vector (MOCK) or Myr-AKT for 6 h and re-serum overnight, followed by treatment with or without lanatoside C (0.6 μM) for 18 h. Cells were harvested from total lysates for detection of phospho-AKT Ser473 protein expressions by using Western blot analysis. Cell viability was measured by MTT assay. Data are expressed as means ± SEM of three independent determinations. *P < 0.01, Myr-AKT-overexpressed cells versus MOCK-transfected cells.

Mentions: Phosphoinositide 3-kinases (PI3Ks) transduce signals from various growth factors and cytokines into intracellular messages by generating phospholipids, which activate the serine-threonine protein kinase Akt; moreover, dual inhibitors of PI3K/mTOR exert strong anti-proliferative activity against tumors25. Lanatoside C markedly reduced phosphorylation of Akt and its downstream effectors as well as mTOR and its downstream effectors, such as the mTOR-activated kinase p70S6K and the eukaryotic translation initiation factors 4EBP and eIF4E, in a concentration-dependent manner in both Hep3B and HA22T cells (Fig. 5A). As shown in Fig. 5B, lanatoside C inhibited these kinases in a time-dependent manner (6–24 h) in Hep3B cells. These results also inspired us to explore the relationship between the Akt/mTOR pathway and PKCδ in the context of lanatoside C treatment. Remarkably, we found that both rottlerin and siPKCδ abolished the effects of lanatoside C on the Akt/mTOR pathway (Fig. 5C and Supplementary Fig. S4B). These results strongly suggest that PKCδ regulates the Akt/mTOR pathway. Moreover, we tested the effect of enhancing AKT phosphorylation activity by transiently transfecting Hep3B or HA22T cells with Myc-tagged Akt, and found that overexpression of Akt significantly inhibited lanatoside C-induced cell death (Fig. 4D and Supplementary Fig. S4A). Collectively, these results strongly suggest that lanatoside C suppresses the Akt/mTOR signaling pathway through regulation of PKCδ, leading to enhanced apoptosis in human HCC cells.


Lanatoside C, a cardiac glycoside, acts through protein kinase C δ to cause apoptosis of human hepatocellular carcinoma cells
Effect of lanatoside C on AKT/mTOR pathway.(A) Hep3B and HA22T cells were treated with a range of lanatoside C (0.1–0.6 μM) for 18 h. (B) Hep3B cells were treated lanatoside C (0.6 μM) for indicated time (6–24 hr) and then cells were harvested from total lysates for observation of AKT/mTOR and their downstream signaling protein expressions by using Western blot analysis. (C) Hep3B cells were incubated with Lanatoside C (0.6 μM), rottlerin (5 μM) or PKCδ siRNA, or combination treatment for 18 h and then cells were harvested from total lysates for detection of indicated protein expressions by using Western blot analysis. (D) Hep3B cells were transfected with empty vector (MOCK) or Myr-AKT for 6 h and re-serum overnight, followed by treatment with or without lanatoside C (0.6 μM) for 18 h. Cells were harvested from total lysates for detection of phospho-AKT Ser473 protein expressions by using Western blot analysis. Cell viability was measured by MTT assay. Data are expressed as means ± SEM of three independent determinations. *P < 0.01, Myr-AKT-overexpressed cells versus MOCK-transfected cells.
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Related In: Results  -  Collection

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f5: Effect of lanatoside C on AKT/mTOR pathway.(A) Hep3B and HA22T cells were treated with a range of lanatoside C (0.1–0.6 μM) for 18 h. (B) Hep3B cells were treated lanatoside C (0.6 μM) for indicated time (6–24 hr) and then cells were harvested from total lysates for observation of AKT/mTOR and their downstream signaling protein expressions by using Western blot analysis. (C) Hep3B cells were incubated with Lanatoside C (0.6 μM), rottlerin (5 μM) or PKCδ siRNA, or combination treatment for 18 h and then cells were harvested from total lysates for detection of indicated protein expressions by using Western blot analysis. (D) Hep3B cells were transfected with empty vector (MOCK) or Myr-AKT for 6 h and re-serum overnight, followed by treatment with or without lanatoside C (0.6 μM) for 18 h. Cells were harvested from total lysates for detection of phospho-AKT Ser473 protein expressions by using Western blot analysis. Cell viability was measured by MTT assay. Data are expressed as means ± SEM of three independent determinations. *P < 0.01, Myr-AKT-overexpressed cells versus MOCK-transfected cells.
Mentions: Phosphoinositide 3-kinases (PI3Ks) transduce signals from various growth factors and cytokines into intracellular messages by generating phospholipids, which activate the serine-threonine protein kinase Akt; moreover, dual inhibitors of PI3K/mTOR exert strong anti-proliferative activity against tumors25. Lanatoside C markedly reduced phosphorylation of Akt and its downstream effectors as well as mTOR and its downstream effectors, such as the mTOR-activated kinase p70S6K and the eukaryotic translation initiation factors 4EBP and eIF4E, in a concentration-dependent manner in both Hep3B and HA22T cells (Fig. 5A). As shown in Fig. 5B, lanatoside C inhibited these kinases in a time-dependent manner (6–24 h) in Hep3B cells. These results also inspired us to explore the relationship between the Akt/mTOR pathway and PKCδ in the context of lanatoside C treatment. Remarkably, we found that both rottlerin and siPKCδ abolished the effects of lanatoside C on the Akt/mTOR pathway (Fig. 5C and Supplementary Fig. S4B). These results strongly suggest that PKCδ regulates the Akt/mTOR pathway. Moreover, we tested the effect of enhancing AKT phosphorylation activity by transiently transfecting Hep3B or HA22T cells with Myc-tagged Akt, and found that overexpression of Akt significantly inhibited lanatoside C-induced cell death (Fig. 4D and Supplementary Fig. S4A). Collectively, these results strongly suggest that lanatoside C suppresses the Akt/mTOR signaling pathway through regulation of PKCδ, leading to enhanced apoptosis in human HCC 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 (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.


Related in: MedlinePlus