Digitoxin is a naturally occurring cardiac glycoside and anti-cancer agent extracted mianly from Digitalis lanata[1]. Digitoxin actively inhibits herpes simplex virus type 1 (HSV-1) replication with a EC50 value of 0.05M and exhibited the highest cytotoxicity against HeLa cervical cancer cells with an IC50 value of 28nM at 48h[2][3].
In vitro, human cervical cancer cell line HeLa cells were treated overnight with various concentrations of Digitoxin (0, 4, 20, or 100nM) for 24h or with 20nM Digitoxin for 12, 24, or 36h. Digitoxin caused DNA damage to block the cell cycle at the G2/M phase by triggering the activation of the ATM/ATR-CHK1/CHK2-Cdc25C signaling pathway[3]. Prostat cancer cells PC3 cells and HeLa cells were treated with digitoxin(10 and 25nM) for 24h. Fresh medium and drug were added every day. Cells were continuously treated in this manner for up to 3 days. Digitoxin inhibited NFκB-driven TGFBR2 expression, as well as Vimentin, while elevating E-cadherin expression. Digitoxin also significantly reduces HSPB1 mRNA and the HSPB1/RBFOX2 mRNA ratio in PC3 cells[4]. HeLa cervical cancer cells were treated with Digitoxin (25nM to 1μM; 24 h). Digitoxin caused dose-dependent cytotoxicity and activated intrinsic apoptosis via cleavage of caspase-9, caspase-3 and PARP. Carcinotoxic activity of Digitoxin includes suppression of NFAT-driven c-MYC expression[5].
In vivo, male and female Long Evans rats were injected i.p. once daily for 4 consecutive days with pregnenolone-16a-carbonitrile (PCN) (50mg/kg) then injected i.p. with an ethanolic solution of Digitoxin (1-10mg (1.3-13gmol)/kg) twenty-four hours after the last injection. PCN protected rats from Digitoxin toxicity, by elevating both liver microsomal cytochrome P-450p and UDP-GT-dt₁ activities[6]. Digitoxin (0, 3, 10 and 30μg per 100g of body weight) was administered to 3 cotton rats in one dose intraperitoneally 6.25h before intranasal infection with 107TCID50/100g of cotton rat with influenza strain A/Wuhan/H3N2/359/95 for 4 days. Digitoxin significantly and differentially suppressed levels of the cytokines TNFα, oncogene/keratinocyte chemoattractant (GRO/KC), MIP2, monocyte chemoattractant protein 1 (MCP1) and IFNγ, in the cotton rat lung[7].
References:
[1] Haux J. Digitoxin is a potential anticancer agent for several types of cancer. Med Hypotheses. 1999 Dec;53(6):543-8.
[2] Su C T, Hsu J T A, Hsieh H P, et al. Anti-HSV activity of digitoxin and its possible mechanisms. Antiviral Res. 2008 Jul;79(1):62-70.
[3] Gan H G, Qi M, Chan C P. et al. Digitoxin inhibits HeLa cell growth through the induction of G2/M cell cycle arrest and apoptosis in vitro and in vivo. Int J Oncol. 2020 Aug;57(2):562-573.
[4] Pollard B S, Suckow M A, Wolter W R, et al. Digitoxin Inhibits Epithelial-to-Mesenchymal-Transition in Hereditary Castration Resistant Prostate Cancer. Front Oncol. 2019 Aug 2:9:630.
[5] Yang Q F, Dalgard C L, Eidelman O, et al. Digitoxin induces apoptosis in cancer cells by inhibiting nuclear factor of activated T-cells-driven c-MYC expression. J Carcinog. 2013 May 20:12:8.
[6] Arlotto M P, Sonderfan A J, McKinney M M, Parkinson A. Digitoxin metabolism by liver microsomal cytochrome P-450 and UDP-glucuronosyltransferase and its role in the protection of rats from digitoxin toxicity by pregnenolone-16 alpha-carbonitrile. Arch Biochem Biophys. 1986 Nov 15;251(1):188-97.
[7] Pollard B S, Blancoi J C, Pollard J R. Classical Drug Digitoxin Inhibits Influenza Cytokine Storm, With Implications for Covid-19 Therapy. In Vivo. 2020 Nov-Dec;34(6):3723-3730.