Grandisin induces apoptosis in leukemic K562 cells

In this study, the potential antileukemic activity of grandisin, a lignan extracted from Piper solmsianum, was evaluated against the leukemic line K562. The cytotoxicity of grandisin (0.018 to 2.365 µM) was evaluated in K562 and normal peripheral blood lymphocytes by Trypan Blue Exclusion and MTT methods after 48h exposure to the drug. In both methods, cellular viability was concentration-dependent and the IC50 values were lower than 0.85µM. Analysis of K562 cells after treatment with grandisin showed that the cell cycle was arrested in the G1 phase with a 12.31% increase, while both S and G2 phases decreased. Morphological studies conducted after the exposure of K562 to grandisin revealed changes consistent with the apoptosis process, which was confirmed by anexin V stain and caspase activation. Thus, lignan grandisin showed antileukemic activities against the K562 cell line and the cell death process occurred via apoptosis.

Uniterms:
Grandisin/antileukemic activities; Leukemic; K562; Apoptosis.

Authorship

Alane Pereira Cortez

Laboratory of Celullar Toxicology and Pharmacology - FarmaTec, Faculty of Pharmacy, Federal University of Goiás, Goiânia, GO, BrazilFederal University of GoiásBrazilGoiânia, GO, BrazilLaboratory of Celullar Toxicology and Pharmacology - FarmaTec, Faculty of Pharmacy, Federal University of Goiás, Goiânia, GO, Brazil

Elizabeth Gomes Paulino Menezes

Polyana Lopes Benfica

Alexandre Pereira dos Santos

Larissa Moreira Cleres

Higor de Oliveira Ribeiro

Eliana Martins Lima

Medicinal Pharmaceutical Chemistry Laboratory, Faculty of Pharmacy, Federal University of Goiás, UFG, Goiânia, GO, BrazilFederal University of GoiásBrazilGoiânia, GO, BrazilMedicinal Pharmaceutical Chemistry Laboratory, Faculty of Pharmacy, Federal University of Goiás, UFG, Goiânia, GO, Brazil

Massuo Jorge Kato

Chemistry Laboratory of Natural Products, Institute of Chemistry, University of São PauloUniversity of São PauloBrazilBrazilChemistry Laboratory of Natural Products, Institute of Chemistry, University of São Paulo

Marize Campos Valadares ^{^*}*Correspondence: M. C. Valadares. Faculdade de Farmácia. Universidade Federal de Goiás - UFG. Praça Universitária, n.1166, Setor Universitário, 74605.220 - Goiânia - GO, Brasil. Phone / fax: +55 62 3209-6039 ext. 201. E-mail: marizecv@ufg.br

*Correspondence: M. C. Valadares. Faculdade de Farmácia. Universidade Federal de Goiás - UFG. Praça Universitária, n.1166, Setor Universitário, 74605.220 - Goiânia - GO, Brasil. Phone / fax: +55 62 3209-6039 ext. 201. E-mail: marizecv@ufg.br

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Figures

Figures (5)

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FIGURE 1
Cytotoxicity in K562 cells and normal peripheral blood lymphocytes (1 x 10⁶ cells/mL) treated with different concentrations of grandisin (0.018 - 2.365 µM) for 48 hours. (a) Cell viability was determined by the Trypan blue exclusion method. (b) Cell proliferation was assessed by the MTT reduction test. In the absence of compound, the viability was considered as 100%. Results represent the mean ± SD of three experiments run in four replicates (p< 0.05 compared with control cells).

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FIGURE 2
(a) Analysis of the cell cycle of K562 cells (control) by Flow Cytometry. (b) Analysis of the cell cycle of K562 cells after treatment with grandisin 0.036 µM for 24 hours.

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FIGURE 3
(a) Apoptosis rate of K562 cells exposed to grandisin 0.036 µM for 24 hours. The rate of apoptosis was determined by counting 100 cells, each sample was duplicated and the number of apoptotic cells was expressed as a percentage of the total number of cells. (b) (2, 3 and 4) Morphological characteristics of apoptosis induced K562 cells treated with grandisin 0.036 µM for 24 hours, determined by hematoxylin coloration. Arrows indicate vacuoles, membrane fragmentation and condensed chromatin, respectively. (b) (1) Morphological characteristics of untreated cells.

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FIGURE 4
Analysis of K562 cells treated with grandisin(1 x 10⁶ cells/mL) for 24 hours. The cells were stained with Annexin V-FITC and propidium iodide. Dot plot of untreated (a) and treated (b, c and d). (b) K562 cells treated with 0.072 µM of grandisin. (c) K562 cells treated with 0.036 µM of grandisin. (d) K562 cells treated with 0.018 µM of grandisin.

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FIGURE 5
Caspase activities in K562 (2 x 10⁶ cells/mL) cells after 24 hours of treatment with grandisin 0.036 µM. Colorimetric assay was performed to determine caspase-6, -8 and -9 activities. The enzyme activities were calculated in pmol min^-1 (mg protein)^-1 (extinction coefficient of pNA was 10.000 M^-1 cm^-1) and expressed relative to controls. In the absence of grandisin the activity was considered to be 100%. Error bars represent standard deviation.

FIGURE 1 Cytotoxicity in K562 cells and normal peripheral blood lymphocytes (1 x 10⁶ cells/mL) treated with different concentrations of grandisin (0.018 - 2.365 µM) for 48 hours. (a) Cell viability was determined by the Trypan blue exclusion method. (b) Cell proliferation was assessed by the MTT reduction test. In the absence of compound, the viability was considered as 100%. Results represent the mean ± SD of three experiments run in four replicates (p< 0.05 compared with control cells).

FIGURE 2 (a) Analysis of the cell cycle of K562 cells (control) by Flow Cytometry. (b) Analysis of the cell cycle of K562 cells after treatment with grandisin 0.036 µM for 24 hours.

FIGURE 3 (a) Apoptosis rate of K562 cells exposed to grandisin 0.036 µM for 24 hours. The rate of apoptosis was determined by counting 100 cells, each sample was duplicated and the number of apoptotic cells was expressed as a percentage of the total number of cells. (b) (2, 3 and 4) Morphological characteristics of apoptosis induced K562 cells treated with grandisin 0.036 µM for 24 hours, determined by hematoxylin coloration. Arrows indicate vacuoles, membrane fragmentation and condensed chromatin, respectively. (b) (1) Morphological characteristics of untreated cells.

FIGURE 4 Analysis of K562 cells treated with grandisin(1 x 10⁶ cells/mL) for 24 hours. The cells were stained with Annexin V-FITC and propidium iodide. Dot plot of untreated (a) and treated (b, c and d). (b) K562 cells treated with 0.072 µM of grandisin. (c) K562 cells treated with 0.036 µM of grandisin. (d) K562 cells treated with 0.018 µM of grandisin.

FIGURE 5 Caspase activities in K562 (2 x 10⁶ cells/mL) cells after 24 hours of treatment with grandisin 0.036 µM. Colorimetric assay was performed to determine caspase-6, -8 and -9 activities. The enzyme activities were calculated in pmol min^-1 (mg protein)^-1 (extinction coefficient of pNA was 10.000 M^-1 cm^-1) and expressed relative to controls. In the absence of grandisin the activity was considered to be 100%. Error bars represent standard deviation.

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Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br

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Grandisin induces apoptosis in leukemic K562 cells

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[1] *Correspondence: M. C. Valadares. Faculdade de Farmácia. Universidade Federal de Goiás - UFG. Praça Universitária, n.1166, Setor Universitário, 74605.220 - Goiânia - GO, Brasil. Phone / fax: +55 62 3209-6039 ext. 201. E-mail: marizecv@ufg.br