PENTAGAMABORONON-0 (PGB-0) INCREASED CYTOTOXICITY OF AND INHIBITED METASTASIS INDUCTION BY DOXORUBICIN ON BREAST CANCER CELLS
Long term use of Doxorubicin (DOX) causes several side effects especially metastasis induction on breast cancers. Pentagamaboronon-0 (PGB-0) or 2,5-bis(4-boronic acid benzylidine) cyclopentanone is a novel curcumin analogue and performed cytotoxic effect on HER2 positive breast cancer cells. The objective of this study is to explore PGB-0 as co-chemotherapeutic agent on DOX-induced metastatic breast cancer cells, 4T1.
Potential cytotoxic and anti-metastatic activities of PGB-0 were screened by molecular docking PLANTS and revealed that PGB-0 interacted with MMP-9 and IKKβ. Cytotoxicity was conducted by MTT Assay, the 4T1 were cultured in 96-well plate. After 24h incubation, the cells were treated with series of concentration of PGB-0 and DOX. Next day, the medium was discarded and replaced with 0.5 mg/mL of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and incubated approximately 4h. The reaction was stopped with 10% SDS in 0.01 N HCl solution and incubated in dark condition for overnight to dissolve formazan salt. Cell absorbance was measured with ELISA reader at 595 nm. Cell absorbance was converted to % cell viability. PGB-0 and DOX showed cytotoxic effect on 4T1 breast cancer cells with IC50 value 294 µM and 2.4 µM, respectively and synergistically increased cytotoxicity of DOX.
Apoptosis and cell cycle investigation were conducted by flowcytometry, cells were seeded, treated with ¼ IC50 of PGB-0, ½ IC50 of PGB-0, ¼ IC50 of DOX, and combination of ¼ IC50 of PGB-0 with ¼ IC50 of DOX, for 24 h. Cells were harvested in next day. PGB-0 and its combination with DOX induced cell cycle arrest on S-phase and G2/M phase, respectively. In addition, PGB-0 and its combination with DOX induced apoptosis.
In term of anti-metastasis exploration, wound healing assay was conducted to investigate migration inhibitory. The 4T1 cells were seeded in 24 well-plate and incubated, then starvated of cells was done by replacing the medium with culture medium supplemented with 0.5% FBS in the next day for 24h. After starvation, the cells were scratched with sterile yellow tip in a straight line and treated with 2 mL of series of concentration samples (¼ and ½ IC50 of PGB-0 and ¼ IC50 of DOX, and its combination) for 24 h. The cells were documented at 0, 18, 24, and 42 h after treatment by digital camera. The results were analyzed by ImageJ software and presented as percent closure then statistically analyzed by using SPSS 17.0. Single treatment of PGB-0 inhibited cells migration, and its combination with DOX inhibited cell migration more potent than single treatment.
Gelatin zymograph was conducted to measure the expression of MMP-9. The 4T1 cells were seeded on 6 well plate, then treated by PGB-0 at concentration of ¼ and ½ IC50 and DOX at concentration of ¼ IC50 for 24 h. Each medium was collected as lysate protein then was used as sample that was subjected for electrophoresis. The 10% of SDS-PAGE supplemented with 0.1% of gelatin was used to determine the activity of MMP-9 in the culture medium. After electrophoresis, gels were washed and incubated with distilled water containing 2% of Triton-X 100 for 30 min at room temperature. The solution was removed from gels, then 100 mL of reaction buffer (40 mM Tris-HCl pH 8, 10 mM CaCl2, 0.02% NaN3) was added and incubated for 24 h at 37°C. After removal of reaction buffer, gels were stained by Coomassie Brilliant Blue R-250 solution and destained by destaining solution (20% methanol, 10% acetic acid and 70% water) until clear bands with dark blue background appear. The results were documented and analyzed by ImageJ software. The result revealed that PGB-0 and its combination with DOX inhibited MMP-9 activity. According to immunoblotting assay, PGB-0 and its combination with DOX decreased Rac1 and p120 expression.
In conclusion, PGB-0 increas
Key words: PGB-0, co-chemotherapy, cell cycle, MMP-9, metastasis
|
