INDUCTION OF INDUCIBLE NO-SYNTHASE IS AN ESSENTIAL PART OF TNFα-INDUCED APOPTOSIS IN McF-7 CELLS

Abstract

The TNFa-induced cytotoxic signal is mediated by the intracellular 'death domain' of the 55kD-TNF-receptor. In various cell types the death signal has been demonstrated to be coupled with induction of the inducible NO-Synthase (iNOS). However, it is still widely unknown, whether iNOSinduction is essential for TNF-cytotoxicity, especially in cells derived from sold t u r n .NO-producbjon and iNOS expression in relation to TNFa-induced cytotoxicity were therefore investigated in the human breast cancer cell line MCF-7 and a TNFa-resistent variant. Nogeneration was evaluated by photometric detection of the stable end product NO2 in the supernatant Differential RT-PCR was used for semiquantitative measurement of iNOS-mRNA. Incubation with TNFa (max 3600 IUlml) for 24 hrs led to induction of iNOS-mRNA after 2-4 hrs as well as generation of N O (min 0.46 f 0.18, max 3.23 f 0.24 nmoU1O' cells124hrs) and was followed by cell death (max 34%) in a concentrationdependent way. Dying celb showed the characteristic features of apoptosis and wem quantitated by flow cytometry with propidium iodide. NO-produdion and cell death were further enhanced (5.39 f 0.57 nmoU10' calls124hn. 65%) by addition of cycloheximide (CX, 10 pg/ ml), while CX alone had no such effect. Addition of the competitive iNOSinhibitor L-NAME (1 00 pM) greatly diminished Nogeneration and apoptosis. In MCF-7 variants iNOS-mRNA was still induced by TNFa, however. NO-production was much lowar than in sensitive cells. TNFa-sensitivity and NO-production were restored by CX. No difference was seen in basal expression of bcl-2-mRNA in sensitive and resistent cell lines as measured by differential RT-PCR. Taken together, iNOS-induction plays an essential role in TNFa-triggered apoptotic cell death of MCF-7cells. Prolongation of iNOS-mRNA half l i by CX may partly account for its additive effect on NOproduction and cytotoxicity. The mechanisms leading to decreased NO-generation in resistent variantse. g. reduced iNOS-activity and/ or upregulation of scavenger moleculesstill need further clarification. I n v i t r o s t u d i e s c a r r i e d out i n r a t l i v e r homogenate and c e l l u l a r membranes c l e a r l y i n d i c a t e t h a t at o c o p h e r o l b i n d i n g t o p r o t e i n i s c l o s e l y r e l a t e d t o i t s a n t i o x i d a n t e f f e c t . Ascorbate/FeSo4/Heparin b l o c k s o r p r e v e n t s p r o t e i n b i n d i n g t o a-tocopherol and thereby reduced a n t i o x i d a n t e f f e c t and increased l i p i d p e r o x i d a t i o n . I r r a d i a t i o n causes damage t o proteins, thus unable t o recognize and b i n d t o a-tocopherol r e s u l t i n g i n reduced a n t i o x i d a n t e f f e c t and increased l i p i d p e r o x i d a t i o n . Among t h e c e l l u l a r membranes studied, mitochondria possess s i g n i f i c a n t l y l a r g e amount o f p r o t e i n s f o r @tocopherol binding. Therefore, a small amount o f added crtocopherol C10 pml could completely p r o t e c t mitochondria from p e r o x i d a t i o...

Regulation and measurement of oxidative stress in apoptosis

“…In this study, binding of TNFα to TNFR1 induced the expression of iNOS. Inhibition of iNOS activity abolished the cytotoxicity of TNFα in MCF7 and other TNFα-susceptible cells (Binder et al, 1999). In a separate experiment, HeLa cells transfected with eNOS were found to be resistant to TNFα-mediated apoptosis.…”

HER2/neu reduces the apoptotic effects of N-(4-hydroxyphenyl)retinamide (4-HPR) in breast cancer cells by decreasing nitric oxide production

“…It remains to be determined whether HER2/neu-induced suppression of NO in breast cancer is a mechanism specific to retinoids or whether it has a broader effect on chemotherapy and hormone resistance. This is particularly important, as NO production has been linked to apoptosis induction by chemotherapeutic and biological agents, including doxorubicin (Kalivendi et al, 2001) and tumor necrosis factor-a (Binder et al, 1999). Future studies will be conducted to determine the mechanisms involved in the HER2/neu-mediated suppression of 4-HPR-induced NOSII expression and NO production in breast cancer cells.…”

TNF-α in Cancer Treatment: Molecular Insights, Antitumor Effects, and Clinical Utility

“…Nitric oxide (NO) is involved in survival of TNF-α-treated cells through NF-κB-induced expression of inducible NO synthase (iNOS) [91,92]. We have previously shown that inhibition of NOS by the addition of L-NAME (NG-nitro-L-arginine methyl ester) during TNF-α-based ILP resulted in an increased tumor response in rats bearing STSs [93].…”