OBJECTIVE: Evaluate the cytotoxic effects of the nanocrystals (NCs) of Simonkolleite, ZnO, as well as their nanocomposite (NCT) in mammary cancer cells in vitro.
Study of the effect of nanocrystals of Simonkolleite and Zinc Oxide in treatment cytotoxicity in breast cancer
lines in vitro.
Annelise A. Rocha1, Anielle C. A. Silva2, Maria P. C. Costa3, Noelio O. Dantas2, Marcelo J.B. Silva4
1-Masters Program in Health Sciences, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil.
2-PhD in Physics Course, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil.
3-Graduate Program in Biotecnology, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil.
4- PhD in Imunology, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil.
OBJECTIVE: Evaluate the cytotoxic effects of the nanocrystals (NCs) of Simonkolleite, ZnO, as well as their
nanocomposite (NCT) in mammary cancer cells in vitro. METHODOLOGY: To study the effects of Ncs, was used
three cell lines of human breast, of epithelial types, MDA-MB-231 (ATCC® HTB-26 ™), which is an invasive tumor
line and MCF-7 (ATCC® HTB-22 ™), which is less invasive and MCF-10A, a non- tumorigenic breast epithelial cells,
of mammary gland tissue. The NCs and NCT were synthesized and characterized in the Laboratory of New Insulating
Materials and Semiconductors (LNMIS), Institute of Physics, Federal University of Uberlândia, 38408-100 Uberlândia,
MG, Brazil; at a concentration of 1.2 mg / ml. Three treatments were synthesized: 1) NCs Simonkolleite with 24nm in
size, 2) NCT nanoparticle, shows most of NCs Simonkolleite and small part of zinc oxide nanocrystals and 3) NCs ZnO,
only presents zinc oxide nanocrystals larger than 55nm in size, all used to treat cells in breast cancer. The final
concentrations of these NCs and (RPMI 1640), for breast cancer cell lines or Dulbecco’s Modified Eagle Medium:
Nutrient Mixture F-12 (DMEM F12) in non-tumorigenic breast cell lines, with 2.8% fetal bovine serum for the
experiments. Preparation of the solution, 5 mg of the MTT ([3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl tetrazolium
bromide] solution (SIGMA) will be diluted in 1 ml phosphate buffered saline (PBS). On the first day, the cells were
treated in 96-well plates at a density of 1×10^4 / well and incubated for 24 hours in 200 μl of culture medium plus 2.8%
fetal bovine serum. After incubation, the control (without addition of nanocrystals) was made and the treatment as
described in the above topic ab, with 20 μL of treatment in 180 μL of culture medium plus 2.8% fetal bovine serum,
leaving on one plate for 72 hours, maintained at relative humidity at 37ºC with 5% carbon dioxide (CO). Each
concentration of nanocrystals were in triplicate. After 72 hours the control and treatment with the NCs were removed
and 90 μl of medium containing 10% fetal bovine serum and 10 μl MTT (5 mg / ml) was placed in the dryer with
aluminum foil for 4 hours hours at 37 ° C. Subsequently, this medium was withdrawn with MTT and 100 μL of sodium
dodecyl sulfate (SDS). It was analyzed in the GloMax apparatus, in enzyme-linked immunosorbent assay (ELISA) plate
reader, with wavelength of 560nm. The analysis was performed using the GraphPad Prism 5 program (GraphPad
Software, La Jolla, CA, USA). To correlate the different compounds (Simonkolleite, NCT an ZnO) in differents
concentrations the ANOVA was used. The 50% growth inhibition concentration (IC 50) was calculated from a doseresponse
curve plotted graphically by the GraphPad Prisma program. RESULT: In our study it was observed the
reduction of percentages in cell viability in treatment with Simonkolleite and its concentrations were statistically
significant, meanly in higher concentration 120 μg / ml when compared to the control, consequently concentrations have
a high cytotoxicity proportional to the increase of concentration, it was note that MCF-7 was more sensitive to the drug
Simonkolleite than in MDA-MB 231, because at a low concentration already enough to be cytotoxicity in MCF-7, while
in MDA-MB 231 lines need a higher concentration than MCF-7 to be cytotoxicity. To intent to determine the
cytotoxicity, experiments in MCF-7, MDA-MB 231 and MCF-10A, were carried out at 72 hours with serial dilutions of
Simonkolleite and nanocomposites 120 μg / ml, 60 μg / ml 30 μg / ml and 15 μg / ml. In the case of MCF-7, note that
treatment with NCs Simonkolleite in 15μg / ml, there are greater statistical difference in cell viability was obtained
between the treatments. In the one-way ANOVA analysis on nanoparticle cytotoxicity revealed a significant main effect
of exposure to Simonkolleite (p <0.0001) compared to the control group. In treatments of NCT in 120 μg / ml, 60 μg /
ml and 30 μg / ml, there was a significant difference (p = 0.0005) compared to the control group. To determine the
cytotoxicity of MDA-MB 231, that the treatment with NCs Simonkolleite in 30μg / ml, a greater statistical difference in
cell viability was obtained between treatments. In the one-way ANOVA analysis NCs cytotoxicity revealed a significant
main effect of exposure to Simonkolleite (p <0.05) compared to the control group, and other treatments there aren’t
statistical difference. In the case of MCF-10A, that NCs Simonkolleite and NCT the concentration 120μg / ml, it was the
best difference in cell viability was obtained between ZnO. In the one-way ANOVA analysis on NCs cytotoxicity
revealed a significant major effect of exposure to Simonkolleite (p <0.0001) compared to the control group and between
treatments. The administration of different doses of NCs Simonkolleite (120 μg / ml, 60 μg / ml, 30 μg / ml and 15 μg /
ml) had the best significant effect in all lines when compared to the control group or in treatments of NCT or NCs ZnO,
that only NCT was different statistics in MCF-7 and MCF-10A, and Ncs ZnO wasn’t different statistics in no one lines.
CONCLUSION: In this present study, note that there is a high cytotoxicity in the MCF-7, being considered more
sensitive to the treatment of Simonkolleite. However in the MDA-MB 231 cancer lines, a cytotoxicity was observed, that
the treatment concentration is higher in MDA-MB- 231 than in MCF-7, to be more effective to reduce viability
percentage. We concluded that the expected result of cytotoxicity was obtained, but in the line less sensitive to
Simonkolleite didn’t achieve the expectations of having a higher cytotoxicity than the other treatment variables.
Key words: Simonkolleite, Breast cancer, cytotoxicity.