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A characterization of four B16 murine melanoma cell sublines molecular fingerprint and proliferation behavior.

Danciu C, Falamas A, Dehelean C, Soica C, Radeke H, Barbu-Tudoran L, Bojin F, Pînzaru SC, Munteanu MF - Cancer Cell Int. (2013)

Bottom Line: SERS bands allowed the identification inside the cells of the main bio-molecular components such as: proteins, nucleic acids, and lipids.An "on and off" SERS effect was constantly present, which may be explained in terms of the employed laser power, as well as the possible different orientations of the adsorbed species in the cells in respect to the Ag nanoparticles.MTT results showed that among the four tested cell sub-lines B16 F10 is the most proliferative and B164A5 has the lower growth capacity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Pharmacy, University of Medicine and Pharmacy "Victor Babes", EftimieMurgu Square, No. 2, 300041 Timişoara, România.

ABSTRACT

Background: One of the most popular and versatile model of murine melanoma is by inoculating B16 cells in the syngeneic C57BL6J mouse strain. A characterization of different B16 modified cell sub-lines will be of real practical interest. For this aim, modern analytical tools like surface enhanced Raman spectroscopy/scattering (SERS) and MTT were employed to characterize both chemical composition and proliferation behavior of the selected cells.

Methods: High quality SERS signal was recorded from each of the four types of B16 cell sub-lines: B164A5, B16GMCSF, B16FLT3, B16F10, in order to observe the differences between a parent cell line (B164A5) and other derived B16 cell sub-lines. Cells were incubated with silver nanoparticles of 50-100 nm diameter and the nanoparticles uptake inside the cells cytoplasm was proved by transmission electron microscopy (TEM) investigations. In order to characterize proliferation, growth curves of the four B16 cell lines, using different cell numbers and FCS concentration were obtained employing the MTT proliferation assay. For correlations doubling time were calculated.

Results: SERS bands allowed the identification inside the cells of the main bio-molecular components such as: proteins, nucleic acids, and lipids. An "on and off" SERS effect was constantly present, which may be explained in terms of the employed laser power, as well as the possible different orientations of the adsorbed species in the cells in respect to the Ag nanoparticles. MTT results showed that among the four tested cell sub-lines B16 F10 is the most proliferative and B164A5 has the lower growth capacity. Regarding B16FLT3 cells and B16GMCSF cells, they present proliferation ability in between with slight slower potency for B16GMCSF cells.

Conclusion: Molecular fingerprint and proliferation behavior of four B16 melanoma cell sub-lines were elucidated by associating SERS investigations with MTT proliferation assay.

No MeSH data available.


Related in: MedlinePlus

TEM image of silver nanoparticles inside the cytoplasm. a –B164A5 cells; b –B16F10 cells; c –B16GMCSF cells; d –B16FLT3 cells.
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Figure 1: TEM image of silver nanoparticles inside the cytoplasm. a –B164A5 cells; b –B16F10 cells; c –B16GMCSF cells; d –B16FLT3 cells.

Mentions: The colloidal nanoparticles present an absorption maximum at 420 nm and a full width at half maximum of 50 nm. TEM images of the silver colloid exhibited both spheroidal (50 to 100 nm diameter) and rod-like shapes with lengths ranging from 100 to 200 nm and about 30–40 nm rod diameter. The plasma membrane is a semi-permeable boundary between the cell and its outside environment, and the mechanisms employed for a particle to get into the cell are: diffusion, osmosis, active transport, and endocytosis. For small and non-polar molecules free diffusion is possible, instead bigger particles are incapable of crossing the plasma membrane and require uptake mechanisms such as endocytosis [23]. Due to negative charge of the cell surface, positively charged nanoparticles are preferentially taken up by living cells and the smaller particles were internalized by caveolin - independent pathway. The large nanoparticles penetrated the membrane by endocytosis, clathrin - dependent [24]. TEM observation revealed the nanoparticles uptake into the cytoplasm through both diffusion and endocytosis. Images demonstrate that the silver nanoparticles penetrated the cell membrane and were found inside the organelles and cytoplasm with perinuclear localization, free or surrounded by vesicles (Figure 1). Due to size of particles they cannot cross the nuclear pores and no particles were found in the cell nucleus. They mainly localized in the early and late lysosome, but they are also found in other organelles such as mitochondria and endoplasmic reticulum. Free aggregates appear by rapid uptake and lysosome by passing due to conjugating protein transduction domains to the surface of the nanoparticle. Localization at the level of mitochondria is realized by the presence of extra nuclear DNA [25]. These data are consistent with other experiments which underline that silver nanoparticles penetrate the cells membrane and can be used for the detection of the SERS signal [26,27]. Their aggregation is possible in the growth medium and due to their increased size endocytosis is assumed [28]. Analyzing a big number of cells from each of the four cell lines, it can be remarked that for all four B16 cell lines taken into study, incorporation was made both by diffusion and endocytosis, with no particular differences between the B16 cell lines. Pictures from each cell line can be observed in Figure 1 TEM images of silver nanoparticles inside the cytoplasm (Figure 1a –B164A5 cells; Figure 1b –B16F10 cells; Figure 1c –B16GMCSF cells; Figure 1d–B16FLT3 cells).


A characterization of four B16 murine melanoma cell sublines molecular fingerprint and proliferation behavior.

Danciu C, Falamas A, Dehelean C, Soica C, Radeke H, Barbu-Tudoran L, Bojin F, Pînzaru SC, Munteanu MF - Cancer Cell Int. (2013)

TEM image of silver nanoparticles inside the cytoplasm. a –B164A5 cells; b –B16F10 cells; c –B16GMCSF cells; d –B16FLT3 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750233&req=5

Figure 1: TEM image of silver nanoparticles inside the cytoplasm. a –B164A5 cells; b –B16F10 cells; c –B16GMCSF cells; d –B16FLT3 cells.
Mentions: The colloidal nanoparticles present an absorption maximum at 420 nm and a full width at half maximum of 50 nm. TEM images of the silver colloid exhibited both spheroidal (50 to 100 nm diameter) and rod-like shapes with lengths ranging from 100 to 200 nm and about 30–40 nm rod diameter. The plasma membrane is a semi-permeable boundary between the cell and its outside environment, and the mechanisms employed for a particle to get into the cell are: diffusion, osmosis, active transport, and endocytosis. For small and non-polar molecules free diffusion is possible, instead bigger particles are incapable of crossing the plasma membrane and require uptake mechanisms such as endocytosis [23]. Due to negative charge of the cell surface, positively charged nanoparticles are preferentially taken up by living cells and the smaller particles were internalized by caveolin - independent pathway. The large nanoparticles penetrated the membrane by endocytosis, clathrin - dependent [24]. TEM observation revealed the nanoparticles uptake into the cytoplasm through both diffusion and endocytosis. Images demonstrate that the silver nanoparticles penetrated the cell membrane and were found inside the organelles and cytoplasm with perinuclear localization, free or surrounded by vesicles (Figure 1). Due to size of particles they cannot cross the nuclear pores and no particles were found in the cell nucleus. They mainly localized in the early and late lysosome, but they are also found in other organelles such as mitochondria and endoplasmic reticulum. Free aggregates appear by rapid uptake and lysosome by passing due to conjugating protein transduction domains to the surface of the nanoparticle. Localization at the level of mitochondria is realized by the presence of extra nuclear DNA [25]. These data are consistent with other experiments which underline that silver nanoparticles penetrate the cells membrane and can be used for the detection of the SERS signal [26,27]. Their aggregation is possible in the growth medium and due to their increased size endocytosis is assumed [28]. Analyzing a big number of cells from each of the four cell lines, it can be remarked that for all four B16 cell lines taken into study, incorporation was made both by diffusion and endocytosis, with no particular differences between the B16 cell lines. Pictures from each cell line can be observed in Figure 1 TEM images of silver nanoparticles inside the cytoplasm (Figure 1a –B164A5 cells; Figure 1b –B16F10 cells; Figure 1c –B16GMCSF cells; Figure 1d–B16FLT3 cells).

Bottom Line: SERS bands allowed the identification inside the cells of the main bio-molecular components such as: proteins, nucleic acids, and lipids.An "on and off" SERS effect was constantly present, which may be explained in terms of the employed laser power, as well as the possible different orientations of the adsorbed species in the cells in respect to the Ag nanoparticles.MTT results showed that among the four tested cell sub-lines B16 F10 is the most proliferative and B164A5 has the lower growth capacity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Pharmacy, University of Medicine and Pharmacy "Victor Babes", EftimieMurgu Square, No. 2, 300041 Timişoara, România.

ABSTRACT

Background: One of the most popular and versatile model of murine melanoma is by inoculating B16 cells in the syngeneic C57BL6J mouse strain. A characterization of different B16 modified cell sub-lines will be of real practical interest. For this aim, modern analytical tools like surface enhanced Raman spectroscopy/scattering (SERS) and MTT were employed to characterize both chemical composition and proliferation behavior of the selected cells.

Methods: High quality SERS signal was recorded from each of the four types of B16 cell sub-lines: B164A5, B16GMCSF, B16FLT3, B16F10, in order to observe the differences between a parent cell line (B164A5) and other derived B16 cell sub-lines. Cells were incubated with silver nanoparticles of 50-100 nm diameter and the nanoparticles uptake inside the cells cytoplasm was proved by transmission electron microscopy (TEM) investigations. In order to characterize proliferation, growth curves of the four B16 cell lines, using different cell numbers and FCS concentration were obtained employing the MTT proliferation assay. For correlations doubling time were calculated.

Results: SERS bands allowed the identification inside the cells of the main bio-molecular components such as: proteins, nucleic acids, and lipids. An "on and off" SERS effect was constantly present, which may be explained in terms of the employed laser power, as well as the possible different orientations of the adsorbed species in the cells in respect to the Ag nanoparticles. MTT results showed that among the four tested cell sub-lines B16 F10 is the most proliferative and B164A5 has the lower growth capacity. Regarding B16FLT3 cells and B16GMCSF cells, they present proliferation ability in between with slight slower potency for B16GMCSF cells.

Conclusion: Molecular fingerprint and proliferation behavior of four B16 melanoma cell sub-lines were elucidated by associating SERS investigations with MTT proliferation assay.

No MeSH data available.


Related in: MedlinePlus