Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Apr 7:7:46134.
doi: 10.1038/srep46134.

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

Min-Wu Chao  1 , Tzu-Hsuan Chen  2 , Han-Li Huang  1 , Yu-Wei Chang  3 , Wei-Chun HuangFu  1 , Yu-Ching Lee  4   5 , Che-Ming Teng  3   6 , Shiow-Lin Pan  1   7
Affiliations

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

Min-Wu Chao et al. Sci Rep. .

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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Effect of lanatoside C on cell proliferation, cell cycle of HCC cell lines, and Hep3B xenograft model.
(A) Structure of lanatoside C. (B) Hep3B and HA22T cells were exposed to lanatoside C for 48 h, and then detected % of control cell growth by SRB assay. Lanatoside C induced Hep3B cell apoptosis in a concentration-dependent (C) and time-dependent (D) manner by FACScan flow cytometry analysis with propidium iodide (PI) staining. (E) Hep3B cells were incubated with lanatoside C in indicated concentration for 18 hr. Cells were stained with TUNEL assay (green fluorescence) and DAPI (blue fluorescence) in the same area. Magnification of TUNEL staining was 200X. (F) SCID mice were ectopically implanted with Hep3B cells. The upper curves show the effect of lanatoside C (2.5 mg/kg, ip, q3d or 2.5 mg/kg, ip, twice a week) on tumor volume and percentage of tumor growth delay (TGD), which was calculated for treatment groups relative to control group; the lower curves show the body weight of mice after indicated treatment. Data are expressed as means ± SEM of three independent determinations. *P < 0.05 and **P < 0.01, untreated cell versus lanatoside C-treated groups.
Figure 2
Figure 2. 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.
Figure 3
Figure 3. 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.
Figure 4
Figure 4. 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.
Figure 5
Figure 5. 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.
Figure 6
Figure 6. Schematic summary of lanatoside C-triggered apoptosis in human HCC cells by PKCδ activation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Siegel R. L., Miller K. D. & Jemal A. Cancer statistics, 2016. CA Cancer J Clin 66, 7–30 (2016). - PubMed
    1. Janevska D., Chaloska-Ivanova V. & Janevski V. Hepatocellular Carcinoma: Risk Factors, Diagnosis and Treatment. Open Access Macedonian Journal of Medical Sciences 3, 732–736 (2015). - PMC - PubMed
    1. Olsen S. K., Brown R. S. & Siegel A. B. Hepatocellular carcinoma: review of current treatment with a focus on targeted molecular therapies. Therapeutic Advances in Gastroenterology 3, 55–66 (2010). - PMC - PubMed
    1. Peck-Radosavljevic M. Drug Therapy for Advanced-Stage Liver Cancer. Liver Cancer 3, 125–131 (2014). - PMC - PubMed
    1. Schonfeld W. et al.. The lead structure in cardiac glycosides is 5 beta, 14 beta-androstane-3 beta 14-diol. Naunyn Schmiedebergs Arch Pharmacol 329, 414–26 (1985). - PubMed

Publication types

MeSH terms