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1‑calcium phosphate‑uracil, a synthesized pyrimidine derivative agent, has anti‑proliferative, pro‑apoptotic and anti‑invasion effects on multiple tumor cell lines.

Peng J, Chen X, Hu Q, Yang M, Liu H, Wei W, Liu S, Wang H - Mol Med Rep (2014)

Bottom Line: Meanwhile, the increased expression of the pro‑apoptotic protein B-cell lymphoma-2 (Bcl-2)-associated X and a marked reduction of Bcl‑2 levels were associated with increased 1‑CP‑U concentrations.Additionally, anti‑migration and anti‑invasion effects of 1‑CP‑U were evidently associated with the downregulation of matrix metalloproteinase proteins.These results indicated that 1‑CP‑U appeared to be a more effective inhibitor of cell migration and invasion, rather than of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, West China Institute of Women and Children's Health, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China.

ABSTRACT
1‑calcium phosphate‑uracil (1‑CP‑U), a synthetic pyrimidine derivative, has been documented to demonstrate a variety of different biological activities. However, the potency and mechanisms of this agent's anti-cancer activity have not been elucidated to date. In the present study, the anti‑cancer effects of 1‑CP‑U were examined in a range of in vitro assays. Different cell lines were treated with 1‑CP‑U at varied concentrations (0.7, 1.0, 1.4 µmol/l) for indicated durations. The cell proliferation was then examined by MTT assay. The cellular apoptotic effects were detected by Hoechst 33342 and Annexin V/propidium iodide staining, while the capacity of 1‑CP‑U on invasion and migration were examined by cell invasion and wound healing assays. The expression of matrix metalloproteinase proteins, as well as pro‑ and antiapoptotic proteins was detected by western blotting analysis. The results identified that 1‑CP‑U was able to inhibit the viability of SKOV3, HeLa, SMMC‑7721 and A549 cell lines in a dose‑ and time‑dependent manner, while it exerted only marginal toxic effects on non‑cancerous cells. The IC50 concentration of 1‑CP‑U for tumor cell lines was ~1.0 µmol/l. The growth inhibition induced by 1‑CP‑U was accompanied by a broad spectrum of pro‑apoptotic activities, in which different cell lines varied in their sensitivity to 1‑CP‑U. Meanwhile, the increased expression of the pro‑apoptotic protein B-cell lymphoma-2 (Bcl-2)-associated X and a marked reduction of Bcl‑2 levels were associated with increased 1‑CP‑U concentrations. Additionally, anti‑migration and anti‑invasion effects of 1‑CP‑U were evidently associated with the downregulation of matrix metalloproteinase proteins. Of note, it was observed that 1‑CP‑U significantly inhibited both the migration and invasion at a lower concentration, as compared with the dose required to achieve significant inhibition of apoptosis. These results indicated that 1‑CP‑U appeared to be a more effective inhibitor of cell migration and invasion, rather than of apoptosis. In conclusion, the present study was the first, to the best of our knowledge, to demonstrate the function of 1‑CP‑U in tumor proliferation, apoptosis and invasion with specific effects against cancer cells in vitro, suggesting 1‑CP‑U as a potential novel anticancer agent.

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Effects of 1-CP-U on apoptosis. (A) Tumor cells were treated with various concentrations of 1-CP-U (0.7, 1.0 and 1.4 μmol/l) for 48 h, and then stained with Hoechst 33342 dye. The strong blue signals indicate apoptotic cells of SMMC-7721 with nuclear chromatin condensation and the formation of nuclear fragments and apoptotic bodies (arrows; magnification, ×40). (B) Tumor cells were stained with Annexin V/PI dye at the indicated time. Analyses were conducted on 10,000 cells in each trail. The cells in the early stages of apoptosis were only Annexin V-FITC positive. The cells in the late stage of apoptosis and those moving toward secondary necrosis stained positive with both Annexin V-FITC and PI (Q3, live cells; Q4, early stages of apoptosis; Q2, late stage of apoptosis and secondary necrosis); (C) The percentage of Annexin V positive cells representing apoptosis in (B) was quantified and statistically analyzed as the mean ± standard deviation. *P<0.05; **P<0.001 vs. controls. 1-CP-U, 1-calcium phosphate-uracil; PI, propidium iodide; FITC, fluorescein isothiocyanate.
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f3-mmr-10-05-2271: Effects of 1-CP-U on apoptosis. (A) Tumor cells were treated with various concentrations of 1-CP-U (0.7, 1.0 and 1.4 μmol/l) for 48 h, and then stained with Hoechst 33342 dye. The strong blue signals indicate apoptotic cells of SMMC-7721 with nuclear chromatin condensation and the formation of nuclear fragments and apoptotic bodies (arrows; magnification, ×40). (B) Tumor cells were stained with Annexin V/PI dye at the indicated time. Analyses were conducted on 10,000 cells in each trail. The cells in the early stages of apoptosis were only Annexin V-FITC positive. The cells in the late stage of apoptosis and those moving toward secondary necrosis stained positive with both Annexin V-FITC and PI (Q3, live cells; Q4, early stages of apoptosis; Q2, late stage of apoptosis and secondary necrosis); (C) The percentage of Annexin V positive cells representing apoptosis in (B) was quantified and statistically analyzed as the mean ± standard deviation. *P<0.05; **P<0.001 vs. controls. 1-CP-U, 1-calcium phosphate-uracil; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Mentions: Following observing the decline in cell viability caused by 1-CP-U, particularly at higher concentrations, the induction of apoptosis by 1-CP-U was assessed. Firstly, tumor cells were cultured for 48 h with or without 1-CP-U, and apoptosis-specific morphology was visualized by Hoechst 33342 staining. Condensed chromatin and apoptotic bodies were identified in the 1-CP-U-treated cells, as determiend by fluorescence microscopy (Fig. 3A). Secondly, flow cytometric analysis was performed following staining of the cells with Annexin-V-FITC plus PI (Fig. 3B). The apoptotic rates in the 1.0 μmol/l 1-CP-U group were 25.50±4.33, 31.00±12.04, 22.87±8.57 and 6.03±1.76 for SKOV3, HeLa, SMMC-7721 and A549 cell lines, respectively. The apoptotic rates in the 1.4 μmol/l 1-CP-U groups were 37.17±5.13, 69.57±12.63, 46.07±11.01 and 7.97±1.61%, respectively. The apoptotic rates in the two treated groups were significantly higher than those in the control group (15.93±5.18, 7.60±4.31, 5.43±1.40% and 3.23±0.47%, respectively; P<0.05), enhanced with increasing doses of 1-CP-U (Fig. 3C). Among the four cell lines exposed to 1-CP-U, the number of apoptotic cells in the SKOV3, HeLa and SMMC-7721 populations increased markedly, particularly in the HeLa cells (9.15-fold at 1.4 μmol/l 1-CP-U treatment as compared with the corresponding control group), while a comparably modest increase was detected in the A549 cells (2.47-fold at 1.4 μmol/l 1-CP-U treatment as compared with the corresponding control group), suggesting that different cell lines varied in their sensitivity to 1-CP-U.


1‑calcium phosphate‑uracil, a synthesized pyrimidine derivative agent, has anti‑proliferative, pro‑apoptotic and anti‑invasion effects on multiple tumor cell lines.

Peng J, Chen X, Hu Q, Yang M, Liu H, Wei W, Liu S, Wang H - Mol Med Rep (2014)

Effects of 1-CP-U on apoptosis. (A) Tumor cells were treated with various concentrations of 1-CP-U (0.7, 1.0 and 1.4 μmol/l) for 48 h, and then stained with Hoechst 33342 dye. The strong blue signals indicate apoptotic cells of SMMC-7721 with nuclear chromatin condensation and the formation of nuclear fragments and apoptotic bodies (arrows; magnification, ×40). (B) Tumor cells were stained with Annexin V/PI dye at the indicated time. Analyses were conducted on 10,000 cells in each trail. The cells in the early stages of apoptosis were only Annexin V-FITC positive. The cells in the late stage of apoptosis and those moving toward secondary necrosis stained positive with both Annexin V-FITC and PI (Q3, live cells; Q4, early stages of apoptosis; Q2, late stage of apoptosis and secondary necrosis); (C) The percentage of Annexin V positive cells representing apoptosis in (B) was quantified and statistically analyzed as the mean ± standard deviation. *P<0.05; **P<0.001 vs. controls. 1-CP-U, 1-calcium phosphate-uracil; PI, propidium iodide; FITC, fluorescein isothiocyanate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214340&req=5

f3-mmr-10-05-2271: Effects of 1-CP-U on apoptosis. (A) Tumor cells were treated with various concentrations of 1-CP-U (0.7, 1.0 and 1.4 μmol/l) for 48 h, and then stained with Hoechst 33342 dye. The strong blue signals indicate apoptotic cells of SMMC-7721 with nuclear chromatin condensation and the formation of nuclear fragments and apoptotic bodies (arrows; magnification, ×40). (B) Tumor cells were stained with Annexin V/PI dye at the indicated time. Analyses were conducted on 10,000 cells in each trail. The cells in the early stages of apoptosis were only Annexin V-FITC positive. The cells in the late stage of apoptosis and those moving toward secondary necrosis stained positive with both Annexin V-FITC and PI (Q3, live cells; Q4, early stages of apoptosis; Q2, late stage of apoptosis and secondary necrosis); (C) The percentage of Annexin V positive cells representing apoptosis in (B) was quantified and statistically analyzed as the mean ± standard deviation. *P<0.05; **P<0.001 vs. controls. 1-CP-U, 1-calcium phosphate-uracil; PI, propidium iodide; FITC, fluorescein isothiocyanate.
Mentions: Following observing the decline in cell viability caused by 1-CP-U, particularly at higher concentrations, the induction of apoptosis by 1-CP-U was assessed. Firstly, tumor cells were cultured for 48 h with or without 1-CP-U, and apoptosis-specific morphology was visualized by Hoechst 33342 staining. Condensed chromatin and apoptotic bodies were identified in the 1-CP-U-treated cells, as determiend by fluorescence microscopy (Fig. 3A). Secondly, flow cytometric analysis was performed following staining of the cells with Annexin-V-FITC plus PI (Fig. 3B). The apoptotic rates in the 1.0 μmol/l 1-CP-U group were 25.50±4.33, 31.00±12.04, 22.87±8.57 and 6.03±1.76 for SKOV3, HeLa, SMMC-7721 and A549 cell lines, respectively. The apoptotic rates in the 1.4 μmol/l 1-CP-U groups were 37.17±5.13, 69.57±12.63, 46.07±11.01 and 7.97±1.61%, respectively. The apoptotic rates in the two treated groups were significantly higher than those in the control group (15.93±5.18, 7.60±4.31, 5.43±1.40% and 3.23±0.47%, respectively; P<0.05), enhanced with increasing doses of 1-CP-U (Fig. 3C). Among the four cell lines exposed to 1-CP-U, the number of apoptotic cells in the SKOV3, HeLa and SMMC-7721 populations increased markedly, particularly in the HeLa cells (9.15-fold at 1.4 μmol/l 1-CP-U treatment as compared with the corresponding control group), while a comparably modest increase was detected in the A549 cells (2.47-fold at 1.4 μmol/l 1-CP-U treatment as compared with the corresponding control group), suggesting that different cell lines varied in their sensitivity to 1-CP-U.

Bottom Line: Meanwhile, the increased expression of the pro‑apoptotic protein B-cell lymphoma-2 (Bcl-2)-associated X and a marked reduction of Bcl‑2 levels were associated with increased 1‑CP‑U concentrations.Additionally, anti‑migration and anti‑invasion effects of 1‑CP‑U were evidently associated with the downregulation of matrix metalloproteinase proteins.These results indicated that 1‑CP‑U appeared to be a more effective inhibitor of cell migration and invasion, rather than of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, West China Institute of Women and Children's Health, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China.

ABSTRACT
1‑calcium phosphate‑uracil (1‑CP‑U), a synthetic pyrimidine derivative, has been documented to demonstrate a variety of different biological activities. However, the potency and mechanisms of this agent's anti-cancer activity have not been elucidated to date. In the present study, the anti‑cancer effects of 1‑CP‑U were examined in a range of in vitro assays. Different cell lines were treated with 1‑CP‑U at varied concentrations (0.7, 1.0, 1.4 µmol/l) for indicated durations. The cell proliferation was then examined by MTT assay. The cellular apoptotic effects were detected by Hoechst 33342 and Annexin V/propidium iodide staining, while the capacity of 1‑CP‑U on invasion and migration were examined by cell invasion and wound healing assays. The expression of matrix metalloproteinase proteins, as well as pro‑ and antiapoptotic proteins was detected by western blotting analysis. The results identified that 1‑CP‑U was able to inhibit the viability of SKOV3, HeLa, SMMC‑7721 and A549 cell lines in a dose‑ and time‑dependent manner, while it exerted only marginal toxic effects on non‑cancerous cells. The IC50 concentration of 1‑CP‑U for tumor cell lines was ~1.0 µmol/l. The growth inhibition induced by 1‑CP‑U was accompanied by a broad spectrum of pro‑apoptotic activities, in which different cell lines varied in their sensitivity to 1‑CP‑U. Meanwhile, the increased expression of the pro‑apoptotic protein B-cell lymphoma-2 (Bcl-2)-associated X and a marked reduction of Bcl‑2 levels were associated with increased 1‑CP‑U concentrations. Additionally, anti‑migration and anti‑invasion effects of 1‑CP‑U were evidently associated with the downregulation of matrix metalloproteinase proteins. Of note, it was observed that 1‑CP‑U significantly inhibited both the migration and invasion at a lower concentration, as compared with the dose required to achieve significant inhibition of apoptosis. These results indicated that 1‑CP‑U appeared to be a more effective inhibitor of cell migration and invasion, rather than of apoptosis. In conclusion, the present study was the first, to the best of our knowledge, to demonstrate the function of 1‑CP‑U in tumor proliferation, apoptosis and invasion with specific effects against cancer cells in vitro, suggesting 1‑CP‑U as a potential novel anticancer agent.

Show MeSH
Related in: MedlinePlus