Lipossigenasi e sopravvivenza cellulare in linee tumorali di linfoma Burkitt convertite con il virus di Epstein Barr
Author(s)
Belfiore, Maria Cristina
Date Issued
May 9, 2006
Type
Doctoral Thesis
Abstract
Numerous aggressive forms of B lymphomas are still poorly curable and among them the Burkitt Lymphoma, the oncet of which is strongly associated to Epstein-Barr virus (EBV) infection. Molecular and genetic alterations that confer apoptosis resistance are responsible for the lack of any response to a conventional chemotherapeutic treatment.
In a system of Burkitt lymphoma cell line after the EBV infection and the establishment the of the latent condition are obtained various EBV positive clones (HS1, E2b and E2R) characterized by major apoptosis resistance, over-expression of the antiapoptotic protein Bcl-2 in spite of the lack of any expression of the viral latent membrane protein LMP-1.
Moreover the different EBV positive clones show increased levels of cellular endogenous peroxides directly correlated to apoptosis resistance: interestingly a 5-lipoxygenase enzymatic activity seems to be the main source of oxidative stress and specific 5-LOX inhibitors significantly reduce the peroxide status.
Studies through RT-PCR show that in the EBV-converted E2R cell line 5-Lox gene is the mostly expressed followed by 12-Lox gene; the 15-Lox1 gene is only weakly expressed while the 15-Lox2 and cicloxygenase genes are not detected.
Moreover through quantitative PCR in Real-Time I have found an evident and direct correlation between 5-Lox gene expression and peroxides levels in the various clones.
Specific inhibitors of the 5-LOX isoform (AA861, MK886, BWA4C) induce in apoptosis the resistant cell line E2R showing that this enzyme take part in mediate survival signals.
I have seen that LOX signal transduction proceed through special LOX metabolites named hydroxyeicosatetraenoic acid or HETE.
In addition I have studied the apoptotic pathways through which the death program is realized after 5-LOX inhibition in E2R cell line.
In spite of a constitutive activation of caspases in the controls the death program is stopped by the action of the protein inhibitor of apoptosis X-IAP; so in the E2R the induction of apoptosis machinery is always accompanied by the mitochondrial release of the pro-apoptotic protein SMAC (second mitochondria-derived activator of caspases), preceding the release of cytochrome c, and by a decrement in X-IAP; moreover only the specific inhibition of caspase 3, but not the inhibition of caspases 8 and 9, saves the E2R cell line by the death (indicating that apoptotic program is caspase 3 dependent when the mitochondrial release of SMAC removes the block operates by X-IAP).
In the light of this research new chemotherapeutic approach can be hypothesized using LOX inhibitors and drugs mimicking SMAC, in combination with standard treatments, to increase lymphomas response.
Finally I have used the cellular system BL 41 vs E2R to test the apoptogenic and antioxidant action of aryltetralin lignans with a potential activity of 5-LOX inhibitors; interestingly some neo-synthetic derivatives are strongly apoptogenic on E2R resistant cell line (Saladino et al, Bioorganic & Medicinal Chemistry , 2005).
Additional information
Dottorato di ricerca in Genetica e biologia cellulare
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