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Combined xanthorrhizol-curcumin exhibits synergistic growth inhibitory activity via apoptosis induction in human breast cancer cells MDA-MB-231.

Cheah YH, Nordin FJ, Sarip R, Tee TT, Azimahtol HL, Sirat HM, Rashid BA, Abdullah NR, Ismail Z - Cancer Cell Int. (2009)

Bottom Line: The effective dose that produced 50% growth inhibition (GI50) was calculated from the log dose-response curve of fixed-combinations of xanthorrhizol and curcumin generated from the sulforhodamine B (SRB) assay.Hoechst 33258 nuclear staining assay showed the rate of apoptosis of MDA-MB-231 cells to increase in response to the treatment.Apoptotic cell death was further confirmed by DNA fragmentation assay, where internucleosomal excision of DNA was induced upon treatment with xanthorrhizol-curcumin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur, Malaysia. yhcheah@gmail.com

ABSTRACT

Background: It has been suggested that combined effect of natural products may improve the treatment effectiveness in combating proliferation of cancer cells. The present study was undertaken to evaluate the possibility that the combination of xanthorrhizol and curcumin might show synergistic growth inhibitory effect towards MDA-MB-231 human breast cancer cells via apoptosis induction. The effective dose that produced 50% growth inhibition (GI50) was calculated from the log dose-response curve of fixed-combinations of xanthorrhizol and curcumin generated from the sulforhodamine B (SRB) assay. The experimental GI50 value was used to determine the synergistic activity of the combination treatment by isobolographic analysis and combination-index method. Further investigation of mode of cell death induced by the combination treatment was conducted in the present study.

Results: Isobole analysis revealed that substances interaction was synergistic when xanthorrhizol and curcumin were added concurrently to the cultures but merely additive when they were added sequentially. The synergistic combination treatment was then applied to the cultures to investigate the mode of cell death induced by the treatment. Immunofluorescence staining using antibody MitoCapturetrade mark revealed the possibility of altered mitochondrial transmembrane potential, which is one of the hallmark of apoptosis. Hoechst 33258 nuclear staining assay showed the rate of apoptosis of MDA-MB-231 cells to increase in response to the treatment. Apoptotic cell death was further confirmed by DNA fragmentation assay, where internucleosomal excision of DNA was induced upon treatment with xanthorrhizol-curcumin.

Conclusion: This is the first time the combined cytotoxic effect of xanthorrhizol and curcumin on MDA-MB-231 cells has been documented and our findings provide experimental support to the hypothesis that combined xanthorrhizol-curcumin showed synergistic growth inhibitory activity on MDA-MB-231 cells via apoptosis induction.

No MeSH data available.


Related in: MedlinePlus

Simultaneous treatment of xanthorrhizol and curcumin induces DNA fragmentation in MDA-MB-231 cells. At lower doses of treatment, only high molecular weight intact DNA was observed whereas small fragments of DNA were highlighted at higher doses of treatment. The fragments of DNA have interval molecular weight of ~180 bp, suggesting an apoptotic event. Results are representative of three independent experiments carried out. M – Marker. 0, 5, 10, 15, 20, 25 μg/ml – DNA samples from xanthorrhizol-curcumin (XC)-treated cells at different concentration. T – DNA sample from taxol-treated MDA-MB-231 cells (positive control). C – DNA sample from actinomycin-treated HL 60 cells (positive control).
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Figure 5: Simultaneous treatment of xanthorrhizol and curcumin induces DNA fragmentation in MDA-MB-231 cells. At lower doses of treatment, only high molecular weight intact DNA was observed whereas small fragments of DNA were highlighted at higher doses of treatment. The fragments of DNA have interval molecular weight of ~180 bp, suggesting an apoptotic event. Results are representative of three independent experiments carried out. M – Marker. 0, 5, 10, 15, 20, 25 μg/ml – DNA samples from xanthorrhizol-curcumin (XC)-treated cells at different concentration. T – DNA sample from taxol-treated MDA-MB-231 cells (positive control). C – DNA sample from actinomycin-treated HL 60 cells (positive control).

Mentions: DNA fragmentation is a biochemical hallmark of apoptotic cell death. From the agarose gel, DNA samples from the treated MDA-MB-231 cells exhibit intact genomic DNA (gDNA) and clear DNA ladders during treatment at higher doses (Figure 5). Treatments at lower doses showed only intact gDNA and do not marked any DNA laddering or even smearing effect. When the multiple bands were analyzed, they showed ~180 bp inter-nucleosomal excision, thus confirming apoptosis but not necrosis had taken place. Lane T and C consist of DNA sample from taxol-treated MDA-MB-231 cells and actinomycin-treated HL 60 cells respectively as positive control. The same pattern of DNA laddering was observed.


Combined xanthorrhizol-curcumin exhibits synergistic growth inhibitory activity via apoptosis induction in human breast cancer cells MDA-MB-231.

Cheah YH, Nordin FJ, Sarip R, Tee TT, Azimahtol HL, Sirat HM, Rashid BA, Abdullah NR, Ismail Z - Cancer Cell Int. (2009)

Simultaneous treatment of xanthorrhizol and curcumin induces DNA fragmentation in MDA-MB-231 cells. At lower doses of treatment, only high molecular weight intact DNA was observed whereas small fragments of DNA were highlighted at higher doses of treatment. The fragments of DNA have interval molecular weight of ~180 bp, suggesting an apoptotic event. Results are representative of three independent experiments carried out. M – Marker. 0, 5, 10, 15, 20, 25 μg/ml – DNA samples from xanthorrhizol-curcumin (XC)-treated cells at different concentration. T – DNA sample from taxol-treated MDA-MB-231 cells (positive control). C – DNA sample from actinomycin-treated HL 60 cells (positive control).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2630298&req=5

Figure 5: Simultaneous treatment of xanthorrhizol and curcumin induces DNA fragmentation in MDA-MB-231 cells. At lower doses of treatment, only high molecular weight intact DNA was observed whereas small fragments of DNA were highlighted at higher doses of treatment. The fragments of DNA have interval molecular weight of ~180 bp, suggesting an apoptotic event. Results are representative of three independent experiments carried out. M – Marker. 0, 5, 10, 15, 20, 25 μg/ml – DNA samples from xanthorrhizol-curcumin (XC)-treated cells at different concentration. T – DNA sample from taxol-treated MDA-MB-231 cells (positive control). C – DNA sample from actinomycin-treated HL 60 cells (positive control).
Mentions: DNA fragmentation is a biochemical hallmark of apoptotic cell death. From the agarose gel, DNA samples from the treated MDA-MB-231 cells exhibit intact genomic DNA (gDNA) and clear DNA ladders during treatment at higher doses (Figure 5). Treatments at lower doses showed only intact gDNA and do not marked any DNA laddering or even smearing effect. When the multiple bands were analyzed, they showed ~180 bp inter-nucleosomal excision, thus confirming apoptosis but not necrosis had taken place. Lane T and C consist of DNA sample from taxol-treated MDA-MB-231 cells and actinomycin-treated HL 60 cells respectively as positive control. The same pattern of DNA laddering was observed.

Bottom Line: The effective dose that produced 50% growth inhibition (GI50) was calculated from the log dose-response curve of fixed-combinations of xanthorrhizol and curcumin generated from the sulforhodamine B (SRB) assay.Hoechst 33258 nuclear staining assay showed the rate of apoptosis of MDA-MB-231 cells to increase in response to the treatment.Apoptotic cell death was further confirmed by DNA fragmentation assay, where internucleosomal excision of DNA was induced upon treatment with xanthorrhizol-curcumin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur, Malaysia. yhcheah@gmail.com

ABSTRACT

Background: It has been suggested that combined effect of natural products may improve the treatment effectiveness in combating proliferation of cancer cells. The present study was undertaken to evaluate the possibility that the combination of xanthorrhizol and curcumin might show synergistic growth inhibitory effect towards MDA-MB-231 human breast cancer cells via apoptosis induction. The effective dose that produced 50% growth inhibition (GI50) was calculated from the log dose-response curve of fixed-combinations of xanthorrhizol and curcumin generated from the sulforhodamine B (SRB) assay. The experimental GI50 value was used to determine the synergistic activity of the combination treatment by isobolographic analysis and combination-index method. Further investigation of mode of cell death induced by the combination treatment was conducted in the present study.

Results: Isobole analysis revealed that substances interaction was synergistic when xanthorrhizol and curcumin were added concurrently to the cultures but merely additive when they were added sequentially. The synergistic combination treatment was then applied to the cultures to investigate the mode of cell death induced by the treatment. Immunofluorescence staining using antibody MitoCapturetrade mark revealed the possibility of altered mitochondrial transmembrane potential, which is one of the hallmark of apoptosis. Hoechst 33258 nuclear staining assay showed the rate of apoptosis of MDA-MB-231 cells to increase in response to the treatment. Apoptotic cell death was further confirmed by DNA fragmentation assay, where internucleosomal excision of DNA was induced upon treatment with xanthorrhizol-curcumin.

Conclusion: This is the first time the combined cytotoxic effect of xanthorrhizol and curcumin on MDA-MB-231 cells has been documented and our findings provide experimental support to the hypothesis that combined xanthorrhizol-curcumin showed synergistic growth inhibitory activity on MDA-MB-231 cells via apoptosis induction.

No MeSH data available.


Related in: MedlinePlus