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Anti-Proliferative Effect of Rosmarinus officinalis L. Extract on Human Melanoma A375 Cells.

Cattaneo L, Cicconi R, Mignogna G, Giorgi A, Mattei M, Graziani G, Ferracane R, Grosso A, Aducci P, Schininà ME, Marra M - PLoS ONE (2015)

Bottom Line: Although there is a growing body of experimental work, information about rosemary's anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action.Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays.Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of "Tor Vergata", Rome, Italy.

ABSTRACT
Rosemary (Rosmarinus officinalis L.) has been used since ancient times in traditional medicine, while nowadays various rosemary formulations are increasingly exploited by alternative medicine to cure or prevent a wide range of health disorders. Rosemary's bioproperties have prompted scientific investigation, which allowed us to ascertain antioxidant, anti-inflammatory, cytostatic, and cytotoxic activities of crude extracts or of pure components. Although there is a growing body of experimental work, information about rosemary's anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action. Melanoma is a skin tumor whose diffusion is rapidly increasing in the world and whose malignancy is reinforced by its high resistance to cytotoxic agents; hence the availability of new cytotoxic drugs would be very helpful to improve melanoma prognosis. Here we report on the effect of a rosemary hydroalcoholic extract on the viability of the human melanoma A375 cell line. Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays. The effect on cell cycle was investigated by using flow cytometry, and the alteration of the cellular redox state was evaluated by intracellular ROS levels and protein carbonylation analysis. Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed.

No MeSH data available.


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Intracellular ROS levels (panel A) and carbonylation of total proteins (panel B) of A375 melanoma cells treated with Rosmarinus officinalis extract.(A) Cells were incubated with 1:120 and 1:240 dilutions of the rosemary extract for 24 h. ROS production was evaluated as CM-H2DCFDA fluorescence. Values are expressed as relative fluorescence of treated samples as compared to control ones and are the mean ± SD from three independent experiments. *P≤0.05 versus vehicle treated control cells. (B) Carbonylation was evaluated by derivatization of extracted proteins with 2,4-dinitrophenylhydrazine, SDS-PAGE separation and immunoblotting with anti 2,4-dinitrophenylhydrazone antibodies. Values are expressed as relative optical density of treated samples as compared to control ones and are the mean ± SD from three independent experiments. *P≤0.05 versus vehicle treated control cells.
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pone.0132439.g005: Intracellular ROS levels (panel A) and carbonylation of total proteins (panel B) of A375 melanoma cells treated with Rosmarinus officinalis extract.(A) Cells were incubated with 1:120 and 1:240 dilutions of the rosemary extract for 24 h. ROS production was evaluated as CM-H2DCFDA fluorescence. Values are expressed as relative fluorescence of treated samples as compared to control ones and are the mean ± SD from three independent experiments. *P≤0.05 versus vehicle treated control cells. (B) Carbonylation was evaluated by derivatization of extracted proteins with 2,4-dinitrophenylhydrazine, SDS-PAGE separation and immunoblotting with anti 2,4-dinitrophenylhydrazone antibodies. Values are expressed as relative optical density of treated samples as compared to control ones and are the mean ± SD from three independent experiments. *P≤0.05 versus vehicle treated control cells.

Mentions: Since the anti-proliferative effects of different phytochemicals on various cancer cell lines has been attributed to their pro-oxidant, rather than anti-oxidant properties [50], the intracellular ROS concentration of melanoma cells treated with rosemary crude extract, compared to that of control cells, was estimated by FACS, using CM-H2DCFDA as fluorescent probe. Results from FACS analysis, reported in Fig 5 (panel A) showed that treating melanoma cells with 1: 120 and 1:240 dilutions of the extract for 24 h, brought about a significant reduction of intracellular ROS levels, thereby indicating that cytotoxicity was not triggered by cellular oxidative damage. This result was confirmed also by protein carbonylation analysis. Carbonylation is a common protein modification induced by cellular oxidative imbalance and can be easily detected by protein derivatization with DNHP and recognition with anti protein-hydrazone antibodies. The overall carbonylation level of proteins from control and treated cells was quantified by estimating the total optical density of extracted proteins after SDS PAGE, Western Blotting and immunodecoration, using the Quantity One software from Bio-rad. Results reported in Fig 5 (Panel B) demonstrated that treatments with 1:120 and 1:240 extract dilutions after 24 h incubation, determined a reduction of cell protein carbonylation, thus confirming the anti-oxidant action of the rosemary extract under our experimental conditions.


Anti-Proliferative Effect of Rosmarinus officinalis L. Extract on Human Melanoma A375 Cells.

Cattaneo L, Cicconi R, Mignogna G, Giorgi A, Mattei M, Graziani G, Ferracane R, Grosso A, Aducci P, Schininà ME, Marra M - PLoS ONE (2015)

Intracellular ROS levels (panel A) and carbonylation of total proteins (panel B) of A375 melanoma cells treated with Rosmarinus officinalis extract.(A) Cells were incubated with 1:120 and 1:240 dilutions of the rosemary extract for 24 h. ROS production was evaluated as CM-H2DCFDA fluorescence. Values are expressed as relative fluorescence of treated samples as compared to control ones and are the mean ± SD from three independent experiments. *P≤0.05 versus vehicle treated control cells. (B) Carbonylation was evaluated by derivatization of extracted proteins with 2,4-dinitrophenylhydrazine, SDS-PAGE separation and immunoblotting with anti 2,4-dinitrophenylhydrazone antibodies. Values are expressed as relative optical density of treated samples as compared to control ones and are the mean ± SD from three independent experiments. *P≤0.05 versus vehicle treated control cells.
© Copyright Policy
Related In: Results  -  Collection

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pone.0132439.g005: Intracellular ROS levels (panel A) and carbonylation of total proteins (panel B) of A375 melanoma cells treated with Rosmarinus officinalis extract.(A) Cells were incubated with 1:120 and 1:240 dilutions of the rosemary extract for 24 h. ROS production was evaluated as CM-H2DCFDA fluorescence. Values are expressed as relative fluorescence of treated samples as compared to control ones and are the mean ± SD from three independent experiments. *P≤0.05 versus vehicle treated control cells. (B) Carbonylation was evaluated by derivatization of extracted proteins with 2,4-dinitrophenylhydrazine, SDS-PAGE separation and immunoblotting with anti 2,4-dinitrophenylhydrazone antibodies. Values are expressed as relative optical density of treated samples as compared to control ones and are the mean ± SD from three independent experiments. *P≤0.05 versus vehicle treated control cells.
Mentions: Since the anti-proliferative effects of different phytochemicals on various cancer cell lines has been attributed to their pro-oxidant, rather than anti-oxidant properties [50], the intracellular ROS concentration of melanoma cells treated with rosemary crude extract, compared to that of control cells, was estimated by FACS, using CM-H2DCFDA as fluorescent probe. Results from FACS analysis, reported in Fig 5 (panel A) showed that treating melanoma cells with 1: 120 and 1:240 dilutions of the extract for 24 h, brought about a significant reduction of intracellular ROS levels, thereby indicating that cytotoxicity was not triggered by cellular oxidative damage. This result was confirmed also by protein carbonylation analysis. Carbonylation is a common protein modification induced by cellular oxidative imbalance and can be easily detected by protein derivatization with DNHP and recognition with anti protein-hydrazone antibodies. The overall carbonylation level of proteins from control and treated cells was quantified by estimating the total optical density of extracted proteins after SDS PAGE, Western Blotting and immunodecoration, using the Quantity One software from Bio-rad. Results reported in Fig 5 (Panel B) demonstrated that treatments with 1:120 and 1:240 extract dilutions after 24 h incubation, determined a reduction of cell protein carbonylation, thus confirming the anti-oxidant action of the rosemary extract under our experimental conditions.

Bottom Line: Although there is a growing body of experimental work, information about rosemary's anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action.Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays.Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of "Tor Vergata", Rome, Italy.

ABSTRACT
Rosemary (Rosmarinus officinalis L.) has been used since ancient times in traditional medicine, while nowadays various rosemary formulations are increasingly exploited by alternative medicine to cure or prevent a wide range of health disorders. Rosemary's bioproperties have prompted scientific investigation, which allowed us to ascertain antioxidant, anti-inflammatory, cytostatic, and cytotoxic activities of crude extracts or of pure components. Although there is a growing body of experimental work, information about rosemary's anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action. Melanoma is a skin tumor whose diffusion is rapidly increasing in the world and whose malignancy is reinforced by its high resistance to cytotoxic agents; hence the availability of new cytotoxic drugs would be very helpful to improve melanoma prognosis. Here we report on the effect of a rosemary hydroalcoholic extract on the viability of the human melanoma A375 cell line. Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays. The effect on cell cycle was investigated by using flow cytometry, and the alteration of the cellular redox state was evaluated by intracellular ROS levels and protein carbonylation analysis. Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed.

No MeSH data available.


Related in: MedlinePlus