Colchicine of Colchicum autumnale, A Traditional Anti-Inflammatory Medicine, Induces Apoptosis by Activation of Apoptotic Genes and Proteins Expression in Human Breast (MCF-7) and Mouse Breast (4T1) Cell Lines

Abstract

Objective: Breast cancer is one of the major causes of mortality among women. Due to many side effects of the existing chemotherapeutic agents, the research of anti-cancer drugs, including natural products, is still a big challenge. Here, we investigated the effects of colchicine on apoptosis of two breast cancer cell lines ( human MCF-7 and mouse 4T1).

Materials and methods: In this experimental study, we evaluated the apoptotic effects of colchicine on (MCF-7) and (4T1), as well as a human cancer-associated fibroblast cell line as a control group. Extraction and chromatographic techniques were applied to isolate colchicine from Colchicum autumnale L. To compare the isolated colchicine with pure standard colchicine, we used the H-NMR technique. The methyl thiazolyl tetrazolium (MTT) assay, quantitative reverse transcriptase-polymerase chain reaction, Western blotting and annexin V/PI staining were used to evaluate the apoptotic effects of the isolated and standard colchicine.

Results: Similar to standard colchicine, the isolated colchicine inhibited cell proliferation significantly in cancer cell lines. Colchine inhibited proliferation and induced apoptosis on a dose-dependent manner. The medicine modified the expression of genes-related to apoptosis by up-regulation of P53 ,BAX, CASPASE-3, -9 and down-regulation of BCL-2 gene, which led to an increase in the BAX/BCL-2 ratio.

Conclusion: We showed that isolated colchicine from Colchicum autumnale and pure standard colchicines modulate the expression levels of several genes and therefore exerting their anticancer effects on both human (MCF-7) and mouse (4T1) breast cancer cells. Based on these results, we suggest that colchicine can be a potential candidate for prevention and treatment of breast cancer.

Keywords: Apoptosis; Breast Cancer Cell; Colchicine; Colchicum autumnale; Toxicity.

Fig 1

Cytotoxic effects of different concentrations of standard (drug) and isolated colchicine after 24-hours of treatment. A. On MCF-7 cells, B. On 4T1 cells, and C. On fibroblast cells. Data are given as mean ± SE for each point of three separate experiments, *; P<0.05, **; P<0.001 vs. control.

Fig 2

The growth inhibitory effect of different concentrations of isolated and standard colchicine (drug) after 48 hours. A. On MCF-7 cells, B. 4T1 cells, and C. Fibroblast cells. Data are given as mean ± SE for each point of three separate experiments, *; P<0.05 and **; P<0.001 vs. control.

Fig 3

mRNA expression of pro-apoptotic and anti-apoptotic genes. A. P53, BAX, BCL-2 in MCF-7 cells, B. p53, Bax, Bcl-2 in 4T1 cells. The expression of pro-apoptotic and anti-apoptotic genes in both cell lines was determined by measuring mRNA levels using real-time polymerase chain reaction (PCR). Data are given as mean ± SE for each point of three separate experiments, *; P<0.01, **; P<0.05, ***; P<0.001, between equal concentrations of colchicine and $; P<0.01, $$; P<0.05, $$$; P<0.001, between different concentrations of colchicine. C. Colchicine effects on the expression of BAX/BCL-2 in MCF-7 and 4T1 cells. Data are given as mean ± SE for each point of three separate experiments. *; P<0.01, **; P<0.05, ***; P<0.001 vs. control and $; P<0.01, $$; P<0.05, $$$; P<0.001 vs. equal concentrations of isolated and standard colchicine (drug).

Fig 4

Expression of pro-apoptotic and anti-apoptotic proteins and densitometric analysis. A. The effects of colchicine on the expression of pro-apoptotic and anti-apoptotic proteins in MCF-7 and 4T1 cells. Cells were exposed to the isolated and standard colchicine (drug) at 0.5 and 1 µg/ml and 200 and 400 µg/ml, respectively for 24 hours. The protein expression of P53, Bax, Bcl-2, caspase-3 and -9 was determined by Western blotting against controls, B. Densitometricy analysis of P53, Bax, Bcl-2, Caspase-3 and -9 proteins in MCF-7 cell line, and C. Densitometricy analysis in 4T1 cell line. Data are given as mean ± SE for each point of three separate experiments. Different letters indicate a significant difference (P<0.05) and the same characters indicate a non-significant difference between the treatments (P>0.05). I; Isolated and D; Drug.

Fig 5

Results of Fluorescence Microscope image and early and late apoptos of MCF-7 cells. A. Untreated cells as the control group, B. Treated cells with 0.5 µg/ml of standard colchicine (drug), C. Treated cells with 1 µg/ml of standard colchicine (drug), D. Treated cells with 0.5 µg/ml of isolated colchicine, E. Treated cells with 1 µg/ml of isolated colchicine (scale bar: 50 µm), and F. The results of early and late apoptosis in MCF-7 cells determined by Annexin V-PI method. **; P<0.001 vs. control, and ^$$; P<0.001 vs. equal concentrations of isolated and standard colchicine (drug).

Fig 6

Results of Fluorescence Microscope image and early and late apoptos of 4T1 cells. A. Untreated cells as control group, B. Treated cells with 200 µg/ml of standard colchicine, C. Treated cells with 400 µg/ml of standard colchicine (drug), D. Treated cells with 200 µg/ml of isolated colchicine, E. Treated cells with 400 µg/ml of standard colchicine (drug) (scale bar: 50 µm), and F. Results of early and late apoptosis in 4T1 cells determined by Annexin V-PI method. **; P<0.001 vs. control and ^$$; P<0.001 vs. same concentration of isolated and standard colchicine (drug).