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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

Activation of PKCδ was involved in lanatoside C-induced apoptosis.(A) Hep3B cells were incubated with different PKC inhibitors (Ro318220: pan-PKC inhibitor, Gö 6983: classical PKC inhibitor, and Rottlerin: PKCδ inhibitor) for 18 h and subsequently analyzed by FACScan flow cytometry with PI staining to determine their sub-G1 proportion. Data are expressed as means ± SEM of three independent determinations. *P < 0.01 and **P < 0.05, untreated cell versus lanatoside C-treated cells. (B) Hep3B and HA22T cells were transfected with PKCδ or control siRNA, followed by treatment with lanatoside C (0.6 μM) for 18 h. Cell viability was determined by MTT assay. (C) Hep3B cells were treated with lanatoside C (0.6 μM) or co-incubated with rottlerin (5 μM) for the indicated time and detected of PKCδ Thr505 in membrane fraction and PKCδ from total lysates by Western blot analysis. Caveolin was a membrane marker protein and α-tubulin used as internal control. (D) Hep3B cells were treated with lanatoside C (0.6 μM), rottlerin (left panel) or PKCδ siRNA (right panel), or combination treatment for 18 h and then incubated with rhodamine123 (5 μM) for 30 min. Data acquisition and analysis were performed on a FACScan flow cytometry. (E) Hep3B Cells were incubated with Lanatoside C (0.6 μM), rottlerin (5 μM) or PKCδ siRNA, or combination treatment for 18 h. Cells were harvested from nuclear fraction and total lysates for detection of the indicated protein expressions by using Western blot analysis. C23 was a nuclear marker protein used as internal control. Results are representative of three independent experiments.
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f4: Activation of PKCδ was involved in lanatoside C-induced apoptosis.(A) Hep3B cells were incubated with different PKC inhibitors (Ro318220: pan-PKC inhibitor, Gö 6983: classical PKC inhibitor, and Rottlerin: PKCδ inhibitor) for 18 h and subsequently analyzed by FACScan flow cytometry with PI staining to determine their sub-G1 proportion. Data are expressed as means ± SEM of three independent determinations. *P < 0.01 and **P < 0.05, untreated cell versus lanatoside C-treated cells. (B) Hep3B and HA22T cells were transfected with PKCδ or control siRNA, followed by treatment with lanatoside C (0.6 μM) for 18 h. Cell viability was determined by MTT assay. (C) Hep3B cells were treated with lanatoside C (0.6 μM) or co-incubated with rottlerin (5 μM) for the indicated time and detected of PKCδ Thr505 in membrane fraction and PKCδ from total lysates by Western blot analysis. Caveolin was a membrane marker protein and α-tubulin used as internal control. (D) Hep3B cells were treated with lanatoside C (0.6 μM), rottlerin (left panel) or PKCδ siRNA (right panel), or combination treatment for 18 h and then incubated with rhodamine123 (5 μM) for 30 min. Data acquisition and analysis were performed on a FACScan flow cytometry. (E) Hep3B Cells were incubated with Lanatoside C (0.6 μM), rottlerin (5 μM) or PKCδ siRNA, or combination treatment for 18 h. Cells were harvested from nuclear fraction and total lysates for detection of the indicated protein expressions by using Western blot analysis. C23 was a nuclear marker protein used as internal control. Results are representative of three independent experiments.

Mentions: In a previous study, bufalin, a cardiac glycoside, was shown to simultaneously induce cell differentiation and apoptosis through cPKC and PKCδ in human monocytic leukemia THP-1 cells19. These PKC isozymes are involved in diverse functions, including induction of cell apoptosis, reflecting differences in their tissue distribution, subcellular localization and substrate specificity20. We co-treated Hep3B cells with lanatoside C and different PKC inhibitors, including the pan-PKC inhibitor Ro318220, the cPKC inhibitor Gö6983 or the PKCδ-specific inhibitor rottlerin, and assessed proliferation using MTT assay. These experiments showed that only rottlerin significantly reduced lanatoside C-induced inhibition of Hep3B cell proliferation and did so in a concentration-dependent manner (Fig. 4A). Consistent with this, small interfering RNA (siRNA) targeting PKCδ (siPKCδ) also markedly reversed lanatoside C-induced inhibition of Hep3B and HA22T cell proliferation (Fig. 4B).


Lanatoside C, a cardiac glycoside, acts through protein kinase C δ to cause apoptosis of human hepatocellular carcinoma cells
Activation of PKCδ was involved in lanatoside C-induced apoptosis.(A) Hep3B cells were incubated with different PKC inhibitors (Ro318220: pan-PKC inhibitor, Gö 6983: classical PKC inhibitor, and Rottlerin: PKCδ inhibitor) for 18 h and subsequently analyzed by FACScan flow cytometry with PI staining to determine their sub-G1 proportion. Data are expressed as means ± SEM of three independent determinations. *P < 0.01 and **P < 0.05, untreated cell versus lanatoside C-treated cells. (B) Hep3B and HA22T cells were transfected with PKCδ or control siRNA, followed by treatment with lanatoside C (0.6 μM) for 18 h. Cell viability was determined by MTT assay. (C) Hep3B cells were treated with lanatoside C (0.6 μM) or co-incubated with rottlerin (5 μM) for the indicated time and detected of PKCδ Thr505 in membrane fraction and PKCδ from total lysates by Western blot analysis. Caveolin was a membrane marker protein and α-tubulin used as internal control. (D) Hep3B cells were treated with lanatoside C (0.6 μM), rottlerin (left panel) or PKCδ siRNA (right panel), or combination treatment for 18 h and then incubated with rhodamine123 (5 μM) for 30 min. Data acquisition and analysis were performed on a FACScan flow cytometry. (E) Hep3B Cells were incubated with Lanatoside C (0.6 μM), rottlerin (5 μM) or PKCδ siRNA, or combination treatment for 18 h. Cells were harvested from nuclear fraction and total lysates for detection of the indicated protein expressions by using Western blot analysis. C23 was a nuclear marker protein used as internal control. Results are representative of three independent experiments.
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Related In: Results  -  Collection

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f4: Activation of PKCδ was involved in lanatoside C-induced apoptosis.(A) Hep3B cells were incubated with different PKC inhibitors (Ro318220: pan-PKC inhibitor, Gö 6983: classical PKC inhibitor, and Rottlerin: PKCδ inhibitor) for 18 h and subsequently analyzed by FACScan flow cytometry with PI staining to determine their sub-G1 proportion. Data are expressed as means ± SEM of three independent determinations. *P < 0.01 and **P < 0.05, untreated cell versus lanatoside C-treated cells. (B) Hep3B and HA22T cells were transfected with PKCδ or control siRNA, followed by treatment with lanatoside C (0.6 μM) for 18 h. Cell viability was determined by MTT assay. (C) Hep3B cells were treated with lanatoside C (0.6 μM) or co-incubated with rottlerin (5 μM) for the indicated time and detected of PKCδ Thr505 in membrane fraction and PKCδ from total lysates by Western blot analysis. Caveolin was a membrane marker protein and α-tubulin used as internal control. (D) Hep3B cells were treated with lanatoside C (0.6 μM), rottlerin (left panel) or PKCδ siRNA (right panel), or combination treatment for 18 h and then incubated with rhodamine123 (5 μM) for 30 min. Data acquisition and analysis were performed on a FACScan flow cytometry. (E) Hep3B Cells were incubated with Lanatoside C (0.6 μM), rottlerin (5 μM) or PKCδ siRNA, or combination treatment for 18 h. Cells were harvested from nuclear fraction and total lysates for detection of the indicated protein expressions by using Western blot analysis. C23 was a nuclear marker protein used as internal control. Results are representative of three independent experiments.
Mentions: In a previous study, bufalin, a cardiac glycoside, was shown to simultaneously induce cell differentiation and apoptosis through cPKC and PKCδ in human monocytic leukemia THP-1 cells19. These PKC isozymes are involved in diverse functions, including induction of cell apoptosis, reflecting differences in their tissue distribution, subcellular localization and substrate specificity20. We co-treated Hep3B cells with lanatoside C and different PKC inhibitors, including the pan-PKC inhibitor Ro318220, the cPKC inhibitor Gö6983 or the PKCδ-specific inhibitor rottlerin, and assessed proliferation using MTT assay. These experiments showed that only rottlerin significantly reduced lanatoside C-induced inhibition of Hep3B cell proliferation and did so in a concentration-dependent manner (Fig. 4A). Consistent with this, small interfering RNA (siRNA) targeting PKCδ (siPKCδ) also markedly reversed lanatoside C-induced inhibition of Hep3B and HA22T cell proliferation (Fig. 4B).

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