download report - Istituto Pasteur

P a r t i c i p a n t s :
Livia Di Renzo, researcher; Giulia Matusali, PhD student;
Alessandra De Leo, Giuseppe Arena, graduate students;
Egidio Lacanna, Claudia Stecca, Valentina Scalise, undergraduate
students.
C o l l a b o r a t i o n s :
The Wistar Institute, Philadelphia, USA (Prof. Paul M. Lieberman).
Report of activity
Resveratrol (3,4’,5-trihydroxy-trans-stilbene), a
polyphenolic natural product present in grapes and
other fruits, has been shown to possess chemopreventive
properties against several cancers, heart diseases,
ischemic injuries and inflammation. Moreover,
increasing evidence in the literature indicate that
resveratrol is able to exert anti-viral activity by interfering
with several intracellular signaling and to synergistically
enhance the effects of antiviral drugs.
In this study we are investigating the effects of
resveratrol on Epstein Barr Virus (EBV) infection.
EBV, the ethiologic agent of infectious mononucleosis,
is associated with several types of malignancies
of epithelial and lymphoid origin including nasopharyngeal
carcinoma (NPC), Hodgkin’s lymphoma,
Burkitt’s lymphoma and many lymphoproliferative
diseases arising in immunocompromised individuals.
In each of these tumors, the selective expression of
six nuclear antigens (EBNA1, 2,3A, 3B, 3C and LP)
and three membrane associated proteins
(LMP1,LMP2a and 2b ) characterize different forms
of latency programs (I, II and III). The lytic phase,
ending with the production of new viral particles,
expands the pool of infected cells favoring the
spread of viral infection.
For the purposes of this project, EBV-infected cell
lines, showing different latency programs, have been
treated for 24 hours with 30 µg/ml of resveratrol, a
concentration chosen because able to affect cell pro-
Principal investigator: Elena Mattia
Professor in Microbiology
Dipartimento di Scienze di Sanità Pubblica
Tel: (+39) 06 49914608; Fax: (+39) 06 49914626
Elena.Mattia@uniroma1.it
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New antimicrobial and antiviral agents - AREA 6
Effects of resveratrol on Epstein-Barr Virus latent and lytic phases
of infection
liferation without causing significant loss of cell viability
or marked morphological alterations. Burkitt’s
lymphoma-derived Raji and Jijoye cells (latency III),
Akata (latency I) and Hodgkin’s derived RPMI6666
cells (latency II) have been incubated with resveratrol
and the effects on the cell cycle have been studied
by analyzing in flowcitometry the distribution of
the cell population in the different phases, as well as
by measuring in Western blot the expression levels
of molecules functioning as regulators of the cell
cycle. We have found that the polyphenol affects cell
cycle progression by blocking Raji and Jijoye cells in
the G1 phase and by inducing apoptosis (sub G1
events) in Akata and RPMI 6666 cells. To evaluate
the extent of apoptosis, the percentages of annexin
V-positive cells have been determined; we found that
after 24 hours incubation, resveratrol induced apoptosis
in all cell lines with percentages varying
between 30 to 60%. In agreement with the cytofluorimetric
data, the levels of the cell cycle regulators
CDK1, CDK2 and cyclin A dramatically decreased
while cyclin-dependent-kinase inhibitors p27 was
strongly up-regulated. To investigate whether the
resveratrol exerts a direct effect on EBV expression,
we measured the levels of transcription of EBV
latent genes in the cells treated with the polyphenol.
The results of RT-PCR experiments carried out on
four EBV-positive cell lines incubated for up to 24
hours with resveratrol indicated that latent EBV
genes were strongly down-regulated independently
of the latency program, while the levels of the b2microglobulin
remained unchanged. From these
results it is possible to conclude that resveratrol is
able to affect both cell cycle progression of the host
cell, as well as EBV latent gene expression.
While a very restricted subset of tumor cells produces
virions by lytic infection, EBV productive cycle
represents the main EBV infection pattern in the
plasmacells of patients with infectious mononucleosis.
In vitro, EBV lytic cycle can be induced by the
addition to the cells of TGFb, sodium butyrate,