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. 2006 May 5;6:119.
doi: 10.1186/1471-2407-6-119.

A New Extract of the Plant Calendula Officinalis Produces a Dual in Vitro Effect: Cytotoxic Anti-Tumor Activity and Lymphocyte Activation

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Free PMC article

A New Extract of the Plant Calendula Officinalis Produces a Dual in Vitro Effect: Cytotoxic Anti-Tumor Activity and Lymphocyte Activation

Eva Jiménez-Medina et al. BMC Cancer. .
Free PMC article

Abstract

Background: Phytopharmacological studies of different Calendula extracts have shown anti-inflammatory, anti-viral and anti-genotoxic properties of therapeutic interest. In this study, we evaluated the in vitro cytotoxic anti-tumor and immunomodulatory activities and in vivo anti-tumor effect of Laser Activated Calendula Extract (LACE), a novel extract of the plant Calendula Officinalis (Asteraceae).

Methods: An aqueous extract of Calendula Officinalis was obtained by a novel extraction method in order to measure its anti-tumor and immunomodulatory activities in vitro. Tumor cell lines derived from leukemias, melanomas, fibrosarcomas and cancers of breast, prostate, cervix, lung, pancreas and colorectal were used and tumor cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of LACE on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in LACE-treated cells. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously human Ando-2 melanoma cells.

Results: The LACE extract showed a potent in vitro inhibition of tumor cell proliferation when tested on a wide variety of human and murine tumor cell lines. The inhibition ranged from 70 to 100%. Mechanisms of inhibition were identified as cell cycle arrest in G0/G1 phase and Caspase-3-induced apoptosis. Interestingly, the same extract showed an opposite effect when tested on PBLs and NKL cell line, in which in vitro induction of proliferation and activation of these cells was observed. The intraperitoneal injection or oral administration of LACE extract in nude mice inhibits in vivo tumor growth of Ando-2 melanoma cells and prolongs the survival day of the mice.

Conclusion: These results indicate that LACE aqueous extract has two complementary activities in vitro with potential anti-tumor therapeutic effect: cytotoxic tumor cell activity and lymphocyte activation. The LACE extract presented in vivo anti-tumoral activity in nude mice against tumor growth of Ando-2 melanoma cells.

Figures

Figure 1
Figure 1
Dose-response effect of LACE extract in PBLs and B9 fibrosarcoma murine cell line. a) PBLs were cultured in medium alone or with various concentrations of LACE extract from 2 mg/ml (conc. 8) to 15.62 μg/ml (conc. 1) for 72 h. b) For B9 tumor cells were cultured with culture medium alone or various concentrations of LACE or CE for 72 h. Each column represents the mean of five independent experiments ± SD.
Figure 2
Figure 2
Effect of LACE in proliferation of different cancer cell lines. The MDA MB231, B16, ANDO-2, JURKAT, B9 and NKL tumor cell lines were treated or not with 250 μg/ml of LACE for 72–96 h. MDA MB231 and ANDO-2 were also treated with Taxol. Each column represents the mean of five independent experiments ± SD.
Figure 3
Figure 3
Cell cycle analysis of cells treated with LACE. PBLs were treated with LACE for 96 h, and cells were analyzed by flow cytometry. Similarly, tumor cell lines were treated with LACE for 96 h. Data indicate the percentage of cells in each phase of cell cycle. Results are representative of three independent experiments.
Figure 4
Figure 4
Effect of LACE on cyclins and CDKs. The cell lines AGS and JURKAT were cultured in alone medium or treated with LACE. a) Flow cytometric analysis was performed for the expression of cyclins D1 and E. The LACE extract produced a strong inhibition of expression of both cyclins. b) The western blot analyses showed that LACE produced down-regulation of cyclins D3 y A and CDK1/Cdc2, CDK2, CDK4, CDK6. The cyclin B expression was not modified.
Figure 5
Figure 5
Apoptosis induced by LACE. B9 cell line was untreated or treated with 250 μg/ml or 1 mg/ml of LACE for 96 h. Cells were double-stained with annexin V and 7AAD and analyzed by flow cytometry. PBLs were also analyzed and LACE extract did not produce apoptosis. All experiments were performed at least three times and gave similar results.
Figure 6
Figure 6
Flow cytometric analysis of apoptotic populations using anti-active caspase 3 antibody. AGS and HELA tumor cell lines were treated with 250 μg/ml or 1 mg/ml of LACE. Data indicate the percentage of cells positive for presence of active-caspase-3. Results are representative of three experiments
Figure 7
Figure 7
Subpopulation analysis in PBLs treated with LACE. PBLs treated or not with LACE were analyzed and results indicate an increase in CD4, CD19, and CD3-CD16-CD56 positive cells. This experiment was repeated at least three times and gave similar results.
Figure 8
Figure 8
Effect of LACE on the growth of ANDO-2 in nude mice. a) There was not differences between the different control groups, while LACE induced tumor growth inhibition reached to about 60%. The Y-axis represents long tumor diameter and the X-axis represents time (day) after ANDO-2 cell injection. Each data point represents the mean ± SD of three independent experiments. b) Effect of LACE in survival time. The percentage survival was calculated as: % survival = number of survival mice in the test group/number of mice in the group. Data are representative of three independent experiments.

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