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Abstract ［Objectives］ This study was conducted to investigate the effect of scopoletin on the growth of breast cancer cell lines.
［Methods］ Cell culture, light microscopy and MTT assay for measuring cell viability were used to compare the inhibition of different concentrations of scopoletin to breast cancer cells, so as to provide an experimental basis for finding new therapeutic drugs for breast cancer cells.
［Results］ It was shown that a high concentration of scopoletin can inhibit the growth of breast cancer cells and promote apoptosis.
［Conclusions］ Scopoletin can be used as a potential drug for the treatment of breast cancer.
Key words Scopoletin; Breast cancer; Apoptosis
Scopoletin is an important coumarin compound with a chemical name is 7-hydroxy-6-methoxycoumarin. When plants are interfered by pathogenic bacteria, pests and other factors, a large amount of scopoletin can be synthesized to prevent interference from adverse factors. It is widely found in a variety of plants, such as Compositae, Convolvulaceae, Euphorbiaceae, Moraceae, Rubiaceae, Cruciferae, Ericaceae, Leguminosae, Meliaceae and other plants. Among them, Artemisia capillaris Thunb. leaves, Erycibe obtusifolia stems, Artemisia annua stems and leaves, and Melia azedarach seeds all show a high content. Studies have shown that scopoletin has the effects of killing pests, resisting inflammation, lowering blood fat and resisting tumors. In this study, the effects of scopoletin on cell proliferation and apoptosis in human breast cancer cells were investigated, so as to explore its possible mechanism.
Materials and Methods
Sources of materials
Human breast cancer cell line MDA-MB-435 was purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. MTT was purchased from Beijing Huarui Biotechnological Pharmaceutical Co., Ltd. DMAEM was purchased from Gibco. Scopoletin was purchased from Shanghai Yuanye Biotechnology Co., Ltd., prepared into a 100× stock solution with dimethyl sulfoxide (purchased from sigma) and stored in a refrigerator at -20 ℃.
Test methods
Cell culture and grouping
The breast cancer cell line MDA-MB-435 was cultured with a high glucose DMEM (10% fetal bovine serum) medium. When the cells were in the logarithmic growth phase, they were collected and adjusted to 5×104 cells/ml and inoculated into 96-well plates. After 24 h of culture, the experimental groups were separately added with scopoletin to a final concentration of 0, 100, 250 and 500 μg/ml, respectively. Another well was added with the culture medium containing no cells, and used as a blank control group for zero adjustment. Each group was provided with 3 duplicate wells. Detection of cell activity
After 24, 48 and 72 h of treatment with scopoletin, 20 μl of MTT (5 mg/m1) was added, and 4 h later, the supernatant was discarded. DMSO was added according to 200 μl/well, followed by shaking for 10 min. The absorbance (A value) of each well was determined with a full-wavelength microplate reader (wavelength 570 nm), and the cell growth inhibition rate was calculated according to Cell growth inhibition rate (%) = (1-Aexperimental group/Acontrol)×100%.
Detection of apoptosis rate
Breast cancer cells MDA-MB-435 in the logarithmic growth phase were added with scopoletin to a final concentration of 0, 100, 250 and 500 μg/ml, respectively, and inoculated into 6-well plates. After 24, 48 and 72 h of culture, the culture liquids were digested, centrifuged and adjusted to the cell density of 1×106 cells/ml. The cells were washed and digested with PBS twice, and then added with 5 μl of FITC Annexin and 5 μl of PI, followed by the detection and analysis of apoptosis on an FACS-Calibur flow cytometer.
Experimental data processing
The results were expressed as mean±standard deviation. The q-test was used for intra-group and inter-group comparison, in which P<0.05 was considered as having statistical significance. The calculation software was SPSS13.0.
Results and Analysis
Morphological observation
Under a 200×microscope, the breast cancer cells in the control group grew in a single layer, and were spindle shaped. After 24 h of the scopoletin treatment, the cells turned round and showed a decreased density, and the nuclei were broken into circular bodies of different sizes. Some of the cells were detached, in a suspended state, and cell debris can be seen. As the concentration of scopoletin increased, the density of breast cancer cells also decreased.
Detection of cell activity
Cell activity was measured by MTT assay, and the growth inhibition rate of each group was shown in Fig. 2.
After 24 h of treatment with different concentrations of scopoletin, the 100, 250 and 500 μg/ml treatment groups showed the inhibition rates of breast cancer cell lines of (22.62%±0.33%)*, (32.23%±0.11%)* and (41.41%±0.22%)*, respectively. After 48 h, the inhibition rates of breast cancer cell lines at 100, 250 and 500 μg/ml were (25.52%±0.28%)*, (38.43%±1.25%)* and (52.41%±1.22%)*, respectively. After 72 h, the inhibition rates of breast cancer cell lines at 100, 250 and 500 μg/ml were (32.72%±0.28%)*, (68.63%±0.36%)* and (70.53%±1.15%)*, respectively. Detection of apoptosis rate
After 24 h of the action of scopoletin, the apoptotic rates of the 0, 100, 250 and 500 μg/ml groups were 7.57%, 8.23%, 12.28% and 28.91%, respectively. The apoptosis rates after 48 h were 7.77%, 14.15%, 23.32% and 37.39%, respectively. The apoptosis rates after 72 h were 12.24%, 55.48%, 57.81% and 62.57%, respectively. As the time went, the apoptosis rate increased. The higher the concentration of scopoletin, the higher the apoptotic rate of breast cancer cells.
Discussion
Scopoletin is widely present in a variety of plants, and has a relatively high content in Arabidopsis thaliana, A. capillaris leaves, E. obtusifolia stems, A. annua stems and leaves and M. azedarach seeds. Therefore, scopoletin also has a variety of pharmacological activity including lowering blood pressure and blood lipid［1-2］, resisting inflammation, relieving pain［3-4］, protecting liver［5-7］and enhancing memory［8-9］. There are more reports about the antitumor activity of scopoletin on T lymphoma cells and prostate cancer PC3 cells［10-11］. Scopoletin is a cytotoxin which is capable of inhibiting the proliferation of PC3 cells, and its inhibition is highly dependent on time and concentration. Intensive research has shown that this drug is found to be beneficial for reducing intracellular protein content and acid phosphatase activity. In addition, in the case of cardiovascular disease, scopoletin has the effects of inducing apoptosis of human myeloid leukemia cells, activating nuclear factor KB and resisting platelet aggregation. According to related research, scopoletin is beneficial to promotion of anti-angiogenesis and blocking of autophosphorylation and downstream signaling pathway of vascular endothelial growth factor receptor-2 in tumor cells, thereby blocking tumor cell angiogenesis, inhibiting growth and metastasis of primary tumor cells, and inducing tumor cell apoptosis［12］. Studies have shown that the antitumor active sites of scopoletin are mainly the phenolic hydroxyl group of C-6 and the methoxy group of C-7. After an alkoxy group is introduced to the phenolic hydroxyl group of C-6, the inhibitory activity of the scopoletin derivative on breast cancer cells MDA-MB-231 and colon cancer HT-29 is more than 10 times higher than that of scopoletin［13］. At present, Khuda-Bukhsh et al.［14］developed the scopoletin polymer nanocapsules, which can significantly improve its anti-tumor activity of scopoletin.
Moreover, scopoletin can exert its anti-tumor effect by inhibiting tumor angiogenesis and metastasis, so it is speculated that it will be a promising natural drug for cancer treatment, which might have a significant inhibitory effect especially on fast-growing malignant tumors. This study found that scopoletin can induce apoptosis of breast cancer cells MDA-MB-435 and inhibit cell proliferation, and the apoptotic rate increased with the increase of drug concentration. When the final concentration was 500 μg/ml, the 72 h apoptosis rate reached 62.57%, indicating that the inhibition was quite obvious. The main mechanism might be achieved by up-regulating the expression of tumor suppressor genes such as p53. The specific apoptotic pathway should be further illustrated by further study on gene expression and cell cycle status associated with apoptosis. This study established a cell model for the inhibition of breast cancer cell growth by scopoletin, and will provide experimental data and theoretical basis for the clinical treatment of scopoletin in breast cancer. Through in-depth research and relevant clinical trials, it is expected to find a new class of drugs for breast cancer treatment. References
［1］MLADENKA P, MACKOV K, ZATLOUKALOVá L, et al. In vitro interact ions of counlarins with iron［J］. Biochimie, 2010, 92(9): 1108-1114.
［2］YANG JY, KOO JH, YOON HY, et al. Effect of scopoletin on lipoprotein lipase activity in 3T3-L1 adipocytes［J］. Int J Mol Med. 2007, 20(4): 527-531.
［3］MAHATTANADUL S, RIDTITID W, NIMA S, et al. Effects of Morinda citrifolia aqueous fruit extract and its biomarker scopoletin on reflux esophagitis and gastric ulcer in rats［J］. J Ethnopharmacol. 2011, 134(2): 243-250.
［4］PAN R1, GAO XH, LI Y, et al. Anti-arthritic effect of scopoletin, a coumarin compound occurring in Erycibe obtusifolia Benth stems, is associated with decreased angiogenesis in synovium［J］. International Immunopharmacology, 2010,24(4):477-490.
［5］YIN HL, LI JH, LI J, et al. Four new eoumarinolignoids from seeds of Solanumindicum［J］. Fitoterapia, 2013 ,84:360-365.
［6］NOH JR, KIM YH, GANG GT, et al. Hepatoprotective effects of chestnut (Castanea crenata) inner shell extract against chronic ethanol-induced oxidative stress in C57BL/6 mice［J］. Food Chem Toxicol, 2011, 49(7):1537-1543.
［7］YANG ZY, ZUO ZP, PAI CH, et al. Effect of scopoletin on rat liver fibrosis［J］. Pharmacology and Clinics of Chinese Materia Medica, 2017, 33(03): 51-53. (in Chinese)
［8］ANAND P, SINGH B, SINGH N. A review on coumarins as acetylcholinesterase inhibitors for Alzheimers disease［J］. Bioorg Med Chem, 2012, 20(3):1175-1180.
［9］HORNICK A, LIEB A, VO NP, et al. The coumarin scopoletin potentiates acetylcholine release from synaptosomes, amplifies hippocampal long-term potentiation and ameliorates anticholinergic- and age-impaired memory［J］. Neuroscience, 2011, 197:280-292.
［10］MANUELE MG, FERRARO G, BARREIRO ARCOS ML, et al. Comparative immunomodulatory effect of scopoletin on tumoral and normal lymphocytes［J］. Life Sci, 2006,79: 2043-2048.
［11］PAN R, GAO X, LU D, et al. Prevention of FGF-2-induced angiogenesis by scopoletin, a eoumarin compound isolated from Erycibe obtusifolia Benth, and its mechanism of action［J］. Int lmmunopharmacol, 201l, l1: 2007-2016.
［12］ZHOU JP, WANG L, WEI LJ, et al. Synthesis and antitumor activity of scopoletin derivatives［J］. Lett Drug Des Discov, 2012, 9: 397-401.
［13］LIU W, HUA J, ZHOU J, et al. Synthesis and in vitro antitumor activity of novel scopolctin derivatives［J］. Bioorg Med Chem Lett, 2012, 22: 5008-5012.
［14］KHUDA-BUKHSH AR1, BHATTACHARYYA SS, PAUL S, BOUJEDAINI N, et al. Polymeric nanoparticle encapsulation of a naturally occurring plant scopoletin and its effects on human melanoma cell A375［J］. Chin J lntegr Med, 2010, 8: 853-862.
［Methods］ Cell culture, light microscopy and MTT assay for measuring cell viability were used to compare the inhibition of different concentrations of scopoletin to breast cancer cells, so as to provide an experimental basis for finding new therapeutic drugs for breast cancer cells.
［Results］ It was shown that a high concentration of scopoletin can inhibit the growth of breast cancer cells and promote apoptosis.
［Conclusions］ Scopoletin can be used as a potential drug for the treatment of breast cancer.
Key words Scopoletin; Breast cancer; Apoptosis
Scopoletin is an important coumarin compound with a chemical name is 7-hydroxy-6-methoxycoumarin. When plants are interfered by pathogenic bacteria, pests and other factors, a large amount of scopoletin can be synthesized to prevent interference from adverse factors. It is widely found in a variety of plants, such as Compositae, Convolvulaceae, Euphorbiaceae, Moraceae, Rubiaceae, Cruciferae, Ericaceae, Leguminosae, Meliaceae and other plants. Among them, Artemisia capillaris Thunb. leaves, Erycibe obtusifolia stems, Artemisia annua stems and leaves, and Melia azedarach seeds all show a high content. Studies have shown that scopoletin has the effects of killing pests, resisting inflammation, lowering blood fat and resisting tumors. In this study, the effects of scopoletin on cell proliferation and apoptosis in human breast cancer cells were investigated, so as to explore its possible mechanism.
Materials and Methods
Sources of materials
Human breast cancer cell line MDA-MB-435 was purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. MTT was purchased from Beijing Huarui Biotechnological Pharmaceutical Co., Ltd. DMAEM was purchased from Gibco. Scopoletin was purchased from Shanghai Yuanye Biotechnology Co., Ltd., prepared into a 100× stock solution with dimethyl sulfoxide (purchased from sigma) and stored in a refrigerator at -20 ℃.
Test methods
Cell culture and grouping
The breast cancer cell line MDA-MB-435 was cultured with a high glucose DMEM (10% fetal bovine serum) medium. When the cells were in the logarithmic growth phase, they were collected and adjusted to 5×104 cells/ml and inoculated into 96-well plates. After 24 h of culture, the experimental groups were separately added with scopoletin to a final concentration of 0, 100, 250 and 500 μg/ml, respectively. Another well was added with the culture medium containing no cells, and used as a blank control group for zero adjustment. Each group was provided with 3 duplicate wells. Detection of cell activity
After 24, 48 and 72 h of treatment with scopoletin, 20 μl of MTT (5 mg/m1) was added, and 4 h later, the supernatant was discarded. DMSO was added according to 200 μl/well, followed by shaking for 10 min. The absorbance (A value) of each well was determined with a full-wavelength microplate reader (wavelength 570 nm), and the cell growth inhibition rate was calculated according to Cell growth inhibition rate (%) = (1-Aexperimental group/Acontrol)×100%.
Detection of apoptosis rate
Breast cancer cells MDA-MB-435 in the logarithmic growth phase were added with scopoletin to a final concentration of 0, 100, 250 and 500 μg/ml, respectively, and inoculated into 6-well plates. After 24, 48 and 72 h of culture, the culture liquids were digested, centrifuged and adjusted to the cell density of 1×106 cells/ml. The cells were washed and digested with PBS twice, and then added with 5 μl of FITC Annexin and 5 μl of PI, followed by the detection and analysis of apoptosis on an FACS-Calibur flow cytometer.
Experimental data processing
The results were expressed as mean±standard deviation. The q-test was used for intra-group and inter-group comparison, in which P<0.05 was considered as having statistical significance. The calculation software was SPSS13.0.
Results and Analysis
Morphological observation
Under a 200×microscope, the breast cancer cells in the control group grew in a single layer, and were spindle shaped. After 24 h of the scopoletin treatment, the cells turned round and showed a decreased density, and the nuclei were broken into circular bodies of different sizes. Some of the cells were detached, in a suspended state, and cell debris can be seen. As the concentration of scopoletin increased, the density of breast cancer cells also decreased.
Detection of cell activity
Cell activity was measured by MTT assay, and the growth inhibition rate of each group was shown in Fig. 2.
After 24 h of treatment with different concentrations of scopoletin, the 100, 250 and 500 μg/ml treatment groups showed the inhibition rates of breast cancer cell lines of (22.62%±0.33%)*, (32.23%±0.11%)* and (41.41%±0.22%)*, respectively. After 48 h, the inhibition rates of breast cancer cell lines at 100, 250 and 500 μg/ml were (25.52%±0.28%)*, (38.43%±1.25%)* and (52.41%±1.22%)*, respectively. After 72 h, the inhibition rates of breast cancer cell lines at 100, 250 and 500 μg/ml were (32.72%±0.28%)*, (68.63%±0.36%)* and (70.53%±1.15%)*, respectively. Detection of apoptosis rate
After 24 h of the action of scopoletin, the apoptotic rates of the 0, 100, 250 and 500 μg/ml groups were 7.57%, 8.23%, 12.28% and 28.91%, respectively. The apoptosis rates after 48 h were 7.77%, 14.15%, 23.32% and 37.39%, respectively. The apoptosis rates after 72 h were 12.24%, 55.48%, 57.81% and 62.57%, respectively. As the time went, the apoptosis rate increased. The higher the concentration of scopoletin, the higher the apoptotic rate of breast cancer cells.
Discussion
Scopoletin is widely present in a variety of plants, and has a relatively high content in Arabidopsis thaliana, A. capillaris leaves, E. obtusifolia stems, A. annua stems and leaves and M. azedarach seeds. Therefore, scopoletin also has a variety of pharmacological activity including lowering blood pressure and blood lipid［1-2］, resisting inflammation, relieving pain［3-4］, protecting liver［5-7］and enhancing memory［8-9］. There are more reports about the antitumor activity of scopoletin on T lymphoma cells and prostate cancer PC3 cells［10-11］. Scopoletin is a cytotoxin which is capable of inhibiting the proliferation of PC3 cells, and its inhibition is highly dependent on time and concentration. Intensive research has shown that this drug is found to be beneficial for reducing intracellular protein content and acid phosphatase activity. In addition, in the case of cardiovascular disease, scopoletin has the effects of inducing apoptosis of human myeloid leukemia cells, activating nuclear factor KB and resisting platelet aggregation. According to related research, scopoletin is beneficial to promotion of anti-angiogenesis and blocking of autophosphorylation and downstream signaling pathway of vascular endothelial growth factor receptor-2 in tumor cells, thereby blocking tumor cell angiogenesis, inhibiting growth and metastasis of primary tumor cells, and inducing tumor cell apoptosis［12］. Studies have shown that the antitumor active sites of scopoletin are mainly the phenolic hydroxyl group of C-6 and the methoxy group of C-7. After an alkoxy group is introduced to the phenolic hydroxyl group of C-6, the inhibitory activity of the scopoletin derivative on breast cancer cells MDA-MB-231 and colon cancer HT-29 is more than 10 times higher than that of scopoletin［13］. At present, Khuda-Bukhsh et al.［14］developed the scopoletin polymer nanocapsules, which can significantly improve its anti-tumor activity of scopoletin.
Moreover, scopoletin can exert its anti-tumor effect by inhibiting tumor angiogenesis and metastasis, so it is speculated that it will be a promising natural drug for cancer treatment, which might have a significant inhibitory effect especially on fast-growing malignant tumors. This study found that scopoletin can induce apoptosis of breast cancer cells MDA-MB-435 and inhibit cell proliferation, and the apoptotic rate increased with the increase of drug concentration. When the final concentration was 500 μg/ml, the 72 h apoptosis rate reached 62.57%, indicating that the inhibition was quite obvious. The main mechanism might be achieved by up-regulating the expression of tumor suppressor genes such as p53. The specific apoptotic pathway should be further illustrated by further study on gene expression and cell cycle status associated with apoptosis. This study established a cell model for the inhibition of breast cancer cell growth by scopoletin, and will provide experimental data and theoretical basis for the clinical treatment of scopoletin in breast cancer. Through in-depth research and relevant clinical trials, it is expected to find a new class of drugs for breast cancer treatment. References
［1］MLADENKA P, MACKOV K, ZATLOUKALOVá L, et al. In vitro interact ions of counlarins with iron［J］. Biochimie, 2010, 92(9): 1108-1114.
［2］YANG JY, KOO JH, YOON HY, et al. Effect of scopoletin on lipoprotein lipase activity in 3T3-L1 adipocytes［J］. Int J Mol Med. 2007, 20(4): 527-531.
［3］MAHATTANADUL S, RIDTITID W, NIMA S, et al. Effects of Morinda citrifolia aqueous fruit extract and its biomarker scopoletin on reflux esophagitis and gastric ulcer in rats［J］. J Ethnopharmacol. 2011, 134(2): 243-250.
［4］PAN R1, GAO XH, LI Y, et al. Anti-arthritic effect of scopoletin, a coumarin compound occurring in Erycibe obtusifolia Benth stems, is associated with decreased angiogenesis in synovium［J］. International Immunopharmacology, 2010,24(4):477-490.
［5］YIN HL, LI JH, LI J, et al. Four new eoumarinolignoids from seeds of Solanumindicum［J］. Fitoterapia, 2013 ,84:360-365.
［6］NOH JR, KIM YH, GANG GT, et al. Hepatoprotective effects of chestnut (Castanea crenata) inner shell extract against chronic ethanol-induced oxidative stress in C57BL/6 mice［J］. Food Chem Toxicol, 2011, 49(7):1537-1543.
［7］YANG ZY, ZUO ZP, PAI CH, et al. Effect of scopoletin on rat liver fibrosis［J］. Pharmacology and Clinics of Chinese Materia Medica, 2017, 33(03): 51-53. (in Chinese)
［8］ANAND P, SINGH B, SINGH N. A review on coumarins as acetylcholinesterase inhibitors for Alzheimers disease［J］. Bioorg Med Chem, 2012, 20(3):1175-1180.
［9］HORNICK A, LIEB A, VO NP, et al. The coumarin scopoletin potentiates acetylcholine release from synaptosomes, amplifies hippocampal long-term potentiation and ameliorates anticholinergic- and age-impaired memory［J］. Neuroscience, 2011, 197:280-292.
［10］MANUELE MG, FERRARO G, BARREIRO ARCOS ML, et al. Comparative immunomodulatory effect of scopoletin on tumoral and normal lymphocytes［J］. Life Sci, 2006,79: 2043-2048.
［11］PAN R, GAO X, LU D, et al. Prevention of FGF-2-induced angiogenesis by scopoletin, a eoumarin compound isolated from Erycibe obtusifolia Benth, and its mechanism of action［J］. Int lmmunopharmacol, 201l, l1: 2007-2016.
［12］ZHOU JP, WANG L, WEI LJ, et al. Synthesis and antitumor activity of scopoletin derivatives［J］. Lett Drug Des Discov, 2012, 9: 397-401.
［13］LIU W, HUA J, ZHOU J, et al. Synthesis and in vitro antitumor activity of novel scopolctin derivatives［J］. Bioorg Med Chem Lett, 2012, 22: 5008-5012.
［14］KHUDA-BUKHSH AR1, BHATTACHARYYA SS, PAUL S, BOUJEDAINI N, et al. Polymeric nanoparticle encapsulation of a naturally occurring plant scopoletin and its effects on human melanoma cell A375［J］. Chin J lntegr Med, 2010, 8: 853-862.