Original Contribution
Induction of caspase-independent apoptotic-like cell death of mouse mammary tumor TA3Ha cells in vitro and reduction of their lethality in vivo by the novel chemotherapeutic agent GIT-27NO

https://doi.org/10.1016/j.freeradbiomed.2010.01.026Get rights and content

Abstract

The new chemical entity GIT-27NO was created by the covalent linkage of a NO moiety to the anti-inflammatory isoxazoline VGX-1027. The compound has been shown to possess powerful anticancer effects both in vitro and in vivo. However, its effects on nonsolid and metastatic forms of tumors have not yet been investigated. We have studied the effects of GIT-27NO on the highly invasive mouse mammary TA3Ha cell line in vitro and in vivo. In contrast to the conventional exogenous NO donor sodium nitroprusside, GIT-27NO successfully enhanced intracellular NO concentration in TA3Ha cells. Intracellular accumulation of NO was followed by marked decrease in TA3Ha cell viability accompanied by typical apoptotic features. Interestingly, inverted membrane phosphatidylserine residues, reduced volume of nucleus, condensed chromatin, and terminal fragmentation of DNA were associated with inhibited caspase-3 activity and transcription of the genes encoding caspase-3, -8, and -9. In parallel, GIT-27NO rapidly but transiently prevented the loss of p53 through phosphorylation on Ser 20 and provided the necessary signals for the execution of downstream processes without p53 de novo synthesis. The caspase-independent apoptotic-like death process triggered by GIT-27NO could be mediated by markedly down-regulated expression of the antiapoptotic Bcl-2 molecule observed in TA3Ha cells exposed to GIT-27NO. In agreement with these in vitro data, GIT-27NO efficiently suppressed the growth of the ascites form and associated lethality of tumor induced by TA3Ha cells in mice.

Section snippets

Animals

Female strain A inbred mice, 5–6 weeks of age, were obtained from Charles River (Lecco, Italy) and kept under standard (non-SPF) laboratory conditions with free access to food and water. The handling of animals and the study protocol were in accordance with international guidelines and approved by the local Institutional Animal Care and Use Committee.

Reagents and cells

Fetal calf serum (FCS), RPMI 1640, phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO),

GIT-27NO suppresses TA3Ha cell growth in vitro; participation of the NO liberated from the drug

TA3Ha cells were treated with various concentrations of either GIT-27NO or its parental compound VGX-1027 for 24 h. The results of the MTT assay, presented in Fig. 1A, clearly indicate that GIT-27NO, in contrast to VGX-1027, dramatically decreased the number of viable cells in the cultures. The observed effects coincided with significant cellular uptake of drug-generated NO as early as 2 h after the start of incubation (Fig. 1B). A dose-dependent increase in nitrite accumulation in the cell

Discussion

We have demonstrated that the ascitic tumor induced by the TA3Ha cell line is sensitive to GIT-27NO treatment and we provide novel information regarding the mode of action of GIT-27NO in this setting. High expression of hyaluron receptors and mucins such as carbohydrate glycoprotein–epiglycanan on the membranes of TA3Ha cells confers on them an extremely invasive phenotype capable of escaping specific immune responses [18]. Despite these complex biochemical characteristics of TA3Ha cells,

Acknowledgments

This work was supported by the Serbian Ministry of Science (Grant 143029). We thank Dr. Gianni Garotta (Ganial Immunotherapeutics, Inc., Wilmington, DE, USA) for providing GIT-27NO and Igor Golic (Faculty of Biology, University of Belgrade, Belgrade, Serbia) for performing electron microscopy.

References (40)

Cited by (18)

  • Gasotransmitters and the immune system: Mode of action and novel therapeutic targets

    2018, European Journal of Pharmacology
    Citation Excerpt :

    This led to generation of the NO-hybridized compounds, Saquinavir, Lopinavir and Ritonavir (Donia et al., 2012; Fagone et al., 2015b; Maksimovic-Ivanic et al., 2015; Mijatovic et al., 2011; Mojic et al., 2012; Momčilović et al., 2014; Petković et al., 2013b; Rothweiler et al., 2010). We have also hybridized the anti-inflammatory VGX1027 compound with NO, obtaining a novel NO-donating compound with substantial antitumoral properties, as observed both in vitro and in vivo (Donia et al., 2009; Maksimovic-Ivanic et al., 2008; Mijatovic et al., 2010). The best characterized compound is Saq-NO (D. Maksimovic-Ivanic et al., 2017a, 2017b).

  • Effects of git-27no, a no-donating compound, on hepatic ischemia/reperfusion injury

    2019, International Journal of Immunopathology and Pharmacology
View all citing articles on Scopus
View full text