Biology Reference
In-Depth Information
were carried out at 24 hr of infection time when the effects on the cell viability due to
the exposure to the drug or to the viral proliferation are minimal. Similar results were
obtained after 42 hr of infection but after such a prolonged time the signifi cant cell
mortality induced by RV and by the progression of the viral infection could overlap
and/or mask the actual effects attributable to the drug (infection data non shown). Fur-
thermore infection experiments performed in the presence of RV during the absorption
phase gave essentially the same results obtained in infections experiments where drug
was added after the viral penetration (not shown). This strongly suggests that virus
entry is not the main target of RV whose action is therefore exerted during the phase
of viral DNA synthesis. Furthermore, the presence of the drug for the whole duration
of the infection is necessary to abrogate completely the viral DNA production. As a
mater of fact exposure to the drug for shorter time has no effect on the overall yield
of viral DNA. Incidentally, this data also shows that the intracellular “life time” of the
viral DNA is fairly long, since removal of the drug after 12 hr exposure seems to have
little effect on the amount of the progeny DNA. These data recall a similar observation
made in our laboratory with a different natural substance [9].
At the moment the mechanism of action of RV remains to be elucidated; however
in the case of infl uenza A virus, the translocation of viral ribo-nucleoprotein complex-
es to the endoplasmic reticulum is hindered and the expression of late viral proteins is
reduced. These two phenomena could be related to the inhibition of protein kinase C
activity and its dependent pathways [22]. Also, Py utilizes proteinprotein complexes
associated to the endoplasmic reticulum and involving the viral capsid proteins VP2
and VP3 [32]. Therefore it could be speculated that the viral transfer to the nucleus, in
the case of Py, follows an analogous process.
RESULTS
Evaluation of the Cytotoxicity of Resveratrol and of the Cell Death
Consequent to Py Infection
In preliminary experiments we assessed the concentration at which RV may exert a
putative cytotoxic activity. It should pointed out, as a matter of fact, that natural sub-
stances endowed of cytoprotective and antioxidant properties, may present a threshold
effect above which they can paradoxically show cytotoxic properties. The phenom-
enon has been documented for RV and its analogues as well as for curcumin another
potent antioxidant drug with cytoprotective features [28-31].
The cytotoxicity of RV on 3T6 cells has been evaluated by the Mossman assay [27]
after treatment for 24 and 48 hr (Figure 1A and 1B, respectively), but in this latter case
the treatment with 2 μM RV was omitted since this at this concentration the drug does
not have a signifi cant effect on cell mortality. The drug is dissolved in 0.02% DMSO
(fi nal concentration) in PBS but, at this low concentration, the organic solvent has
no effects on cell survival, as shown by the second bar from the left. However cells
exposed to RV at the concentrations of 20 and 40 μM show some sensitivity to the
drug, since only about 60% of the cell population survives the treatment for 48 hr at
the higher concentration. The vital cell count observed after trypan blue staining is in
good agreement with the one obtained by the Mossman assay (Table 1, only the data
for 20 μM RV at after 24 hr of treatment are shown).
