Limits...
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 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384006&req=5

f1: 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.

Mentions: Cardiac glycosides comprise a large family of naturally derived compounds that share a common structural motif. Their core structure consists of a steroidal framework, which is considered the pharmacophoric moiety responsible for the activity of these compounds5. Lanatoside C and digoxin are structurally closely related in that digoxin can be obtained from lanatoside C by hydrolytic removal of the acetyl and glucose moieties (Fig. 1A)6. The major pharmacological effect of cardiac glycosides, still widely used clinically, is inhibition of the plasma membrane Na+/K+ ATPase, which indirectly enhances cardiac muscle contractile force. However, recent studies suggest that several cardiac glycosides has been reported to exert anticancer activity through different mechanisms, such as the SRC/EGFR/RAS/ERK signaling pathway, p21, NF-κB, AP-1, topoisomerase and HIF-1, some of which do not involve targeting the Na+/K+ ion pump789101112. These observations suggest that cardiac glycosides could be repurposed as promising anticancer candidates.


Lanatoside C, a cardiac glycoside, acts through protein kinase C δ to cause apoptosis of human hepatocellular carcinoma cells
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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384006&req=5

f1: 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.
Mentions: Cardiac glycosides comprise a large family of naturally derived compounds that share a common structural motif. Their core structure consists of a steroidal framework, which is considered the pharmacophoric moiety responsible for the activity of these compounds5. Lanatoside C and digoxin are structurally closely related in that digoxin can be obtained from lanatoside C by hydrolytic removal of the acetyl and glucose moieties (Fig. 1A)6. The major pharmacological effect of cardiac glycosides, still widely used clinically, is inhibition of the plasma membrane Na+/K+ ATPase, which indirectly enhances cardiac muscle contractile force. However, recent studies suggest that several cardiac glycosides has been reported to exert anticancer activity through different mechanisms, such as the SRC/EGFR/RAS/ERK signaling pathway, p21, NF-κB, AP-1, topoisomerase and HIF-1, some of which do not involve targeting the Na+/K+ ion pump789101112. These observations suggest that cardiac glycosides could be repurposed as promising anticancer candidates.

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