Biology Reference
In-Depth Information
MATERIALS AND METHODS
Cell Cultures
The mouse fibroblast line 3T6 and the tumor line HL60 were used throughout the
work. Cells were grown in high glucose DMEM, supplemented with newborn bovine
serum (10% final concentration), glutamine (50 mM), and penicillin-streptomycin
(10,000 U/ml). Growth temperature was 37°C in controlled humidity at 5% CO
2
. Cells
were routinely split and sub-cultured every third day.
Viral infection was performed at 4 pfu × cell
-1
, for 2 hr at 37° with occasional rock-
ing. Infection procedure and extraction (replication assays) of
de novo
synthesized
DNA were described in detail in previous works, see for instance [9, 10, 26]. Viral
DNA was visualized after agarose gel electrophoresis in the presence of ethidium bro-
mide (0.5 μg/ml, fi nal concentration). Evaluation of cell vitality: Cell viability was
assessed by the colorimetric MTT assay [27]. Absorbance was measured at 570 nm to
obtain a standard cell count. The number of cells surviving to the treatment with RV
(20 μM) was also evaluated by vital cell count in Trypan Blue in a Burker chamber.
The same approach was adopted to count the cell mortality consequent to Py infection
[18].
All experiments were repeated at least three times. The error bars indicate the
Standard Deviation of the Mean (±SEM).
DISCUSSION
In this work we report on cytotxicity versus two different cell lines: a normal mouse
firbroblast line and tumoral one. The results clearly show that RV can exert a cyto-
toxic action both against a normal stabilized fibroblast cell line and human tumor
cells. The human tumor line seems to be slightly more sensitive to the drug and this
recalls results previously obtained in our laboratory with MEX: a partially purified
natural mixture [18]. The antiviral activity of RV towards Py infection was also evalu-
ated. The exposure to the drug was carried at a concentration of RV which did not
show a significant cytotoxic effect. It is known that RV can exert anti-oxidant and
anti-inflammatory activities and, also, it regulates multiple cellular events associated
with carcinogenesis: for a relatively recent review see [28]. The cytotoxicity of RV
is only apparently paradoxical; as a matter of fact this drug induces cell cycle arrest
and stimulates the reactive oxygen species (ROS) activated mitochondrial pathway
leading to apoptosis [29, 30]. An analogous paradoxical action has been described
for another potent antioxidant: curcumin, which is able to induce apoptosis in human
cervical cancer cells [31]. Therefore, an evaluation of possible cytotoxic effects of RV
in our model system was a necessary pre-requisite. The Py productive cycle ends with
the lysis of the infected cell: hence the actual number of cells dying as a consequence
of viral proliferation had to be also assessed. The results of these experiments allowed
us to find out the best conditions where the putative antiviral activity on murine Py
could be investigated.
The results presented in this work show that like in the case of infl uenza A, HVS,
and Varicella-zoster [22-25], the viral replication is severely inhibited by RV also in
the case of murine Py. The inhibition is dose and time dependent and all experiments
