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Strategy to enhance efficacy of doxorubicin in solid tumor cells by methyl-β-cyclodextrin: Involvement of p53 and Fas receptor ligand complex.

Mohammad N, Singh SV, Malvi P, Chaube B, Athavale D, Vanuopadath M, Nair SS, Nair B, Bhat MK - Sci Rep (2015)

Bottom Line: However, its clinical application is limited due to severe side effects and the accompanying drug resistance.MCD sensitizes MCF-7 and Hepa1-6 cells to DOX, Combination of MCD and marginal dose of DOX reduces the cell viability, and promoted apoptosis through induction of pro-apoptotic protein, Bax, activation of caspase-8 and caspase-7, down regulation of anti-apoptotic protein Bcl-2 and finally promoting PARP cleavage.Collectively, these results suggest that MCD enhances the sensitivity to DOX for which wild type p53 is an important determinant.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune- 411007, India.

ABSTRACT
Doxorubicin (DOX) is one of the preferred drugs for treating breast and liver cancers. However, its clinical application is limited due to severe side effects and the accompanying drug resistance. In this context, we investigated the effect on therapeutic efficacy of DOX by cholesterol depleting agent methyl-β-cyclodextrin (MCD), and explored the involvement of p53. MCD sensitizes MCF-7 and Hepa1-6 cells to DOX, Combination of MCD and marginal dose of DOX reduces the cell viability, and promoted apoptosis through induction of pro-apoptotic protein, Bax, activation of caspase-8 and caspase-7, down regulation of anti-apoptotic protein Bcl-2 and finally promoting PARP cleavage. Mechanistically, sensitization to DOX by MCD was due to the induction of FasR/FasL pathway through p53 activation. Furthermore, inhibition of p53 by pharmacological inhibitor pifithrin-α (PFT-α) or its specific siRNA attenuated p53 function and down-regulated FasR/FasL, thereby preventing cell death. Animal experiments were performed using C57BL/6J mouse isografted with Hepa1-6 cells. Tumor growth was retarded and survival increased in mice administered MCD together with DOX to as compared to either agent alone. Collectively, these results suggest that MCD enhances the sensitivity to DOX for which wild type p53 is an important determinant.

No MeSH data available.


Related in: MedlinePlus

MCD potentiates DOX-induced death of MCF-7 and Hepa1–6 cells in a p53-dependent manner by upregulating FasR/FasL.MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h. (A) Protein levels of p53 and MDM2 were examined by western blotting analysis. Hsp60 served as internal control was used a loading control. (B) MCF-7 and (C) Hepa1–6 cells were processed for immunostaining to detect p53 expression and its nuclear localization by immunofluorescence confocal microscopy. (D) FasR membrane staining was performed by flow cytometry. (E) Protein levels of FasR and FasL were examined by western blotting analysis and Hsp60 was used a loading control. (F) Sandwich ELISA for quantitation of secreted FasL from culture medium of MCF-7 and Hepa1–6 cells. Bar graph represents the Mean ± SD of an experiment done in triplicate. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 3.
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f3: MCD potentiates DOX-induced death of MCF-7 and Hepa1–6 cells in a p53-dependent manner by upregulating FasR/FasL.MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h. (A) Protein levels of p53 and MDM2 were examined by western blotting analysis. Hsp60 served as internal control was used a loading control. (B) MCF-7 and (C) Hepa1–6 cells were processed for immunostaining to detect p53 expression and its nuclear localization by immunofluorescence confocal microscopy. (D) FasR membrane staining was performed by flow cytometry. (E) Protein levels of FasR and FasL were examined by western blotting analysis and Hsp60 was used a loading control. (F) Sandwich ELISA for quantitation of secreted FasL from culture medium of MCF-7 and Hepa1–6 cells. Bar graph represents the Mean ± SD of an experiment done in triplicate. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 3.

Mentions: Stress induced p53 promotes cell death and it is a key molecular mechanism of antitumor agents, such as DOX31. We explored the role of p53 in potentiating DOX induced cell death by MCD in MCF-7 and Hepa1–6 cells by western blot analysis. Relatively less p53 protein level was detected in cells treated with MCD and DOX alone, whereas combination treatment of MCD and DOX significantly increased the p53 protein level and decreased the protein level MDM2, a protein involved degrading p53 (Fig. 3A). Furthermore, increased p53 expression and its localization into nucleus of cells were confirmed by immunofluorescence confocal microscopy (Fig. 3B,C). Activation of p53 upon DNA damage leads to up-regulation of several proteins. Among these, death receptor protein, FasR has been shown to be upregulated in a number of cancer cells following genotoxic stress323334. Additionally, p53 also augments the level of FasR on the plasma membrane by promoting trafficking of FasR from the Golgi35. The status of FasR was determined by flow cytometry and western blot analysis and we observed that DOX and MCD together significantly increased the expression of FasR as compared to either agent alone in MCF-7 and Hepa1–6 cells (Fig. 3D,E). Additionally, we also checked FasL protein level in cell lysates and its secreted form in the culture medium collected from the cells by western blot and by indirect ELISA respectively. The expression of FasL in whole cell lysates as well as in the culture medium was increased after treatment of DOX and MCD together (Fig. 3E,F).


Strategy to enhance efficacy of doxorubicin in solid tumor cells by methyl-β-cyclodextrin: Involvement of p53 and Fas receptor ligand complex.

Mohammad N, Singh SV, Malvi P, Chaube B, Athavale D, Vanuopadath M, Nair SS, Nair B, Bhat MK - Sci Rep (2015)

MCD potentiates DOX-induced death of MCF-7 and Hepa1–6 cells in a p53-dependent manner by upregulating FasR/FasL.MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h. (A) Protein levels of p53 and MDM2 were examined by western blotting analysis. Hsp60 served as internal control was used a loading control. (B) MCF-7 and (C) Hepa1–6 cells were processed for immunostaining to detect p53 expression and its nuclear localization by immunofluorescence confocal microscopy. (D) FasR membrane staining was performed by flow cytometry. (E) Protein levels of FasR and FasL were examined by western blotting analysis and Hsp60 was used a loading control. (F) Sandwich ELISA for quantitation of secreted FasL from culture medium of MCF-7 and Hepa1–6 cells. Bar graph represents the Mean ± SD of an experiment done in triplicate. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493576&req=5

f3: MCD potentiates DOX-induced death of MCF-7 and Hepa1–6 cells in a p53-dependent manner by upregulating FasR/FasL.MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h. (A) Protein levels of p53 and MDM2 were examined by western blotting analysis. Hsp60 served as internal control was used a loading control. (B) MCF-7 and (C) Hepa1–6 cells were processed for immunostaining to detect p53 expression and its nuclear localization by immunofluorescence confocal microscopy. (D) FasR membrane staining was performed by flow cytometry. (E) Protein levels of FasR and FasL were examined by western blotting analysis and Hsp60 was used a loading control. (F) Sandwich ELISA for quantitation of secreted FasL from culture medium of MCF-7 and Hepa1–6 cells. Bar graph represents the Mean ± SD of an experiment done in triplicate. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 3.
Mentions: Stress induced p53 promotes cell death and it is a key molecular mechanism of antitumor agents, such as DOX31. We explored the role of p53 in potentiating DOX induced cell death by MCD in MCF-7 and Hepa1–6 cells by western blot analysis. Relatively less p53 protein level was detected in cells treated with MCD and DOX alone, whereas combination treatment of MCD and DOX significantly increased the p53 protein level and decreased the protein level MDM2, a protein involved degrading p53 (Fig. 3A). Furthermore, increased p53 expression and its localization into nucleus of cells were confirmed by immunofluorescence confocal microscopy (Fig. 3B,C). Activation of p53 upon DNA damage leads to up-regulation of several proteins. Among these, death receptor protein, FasR has been shown to be upregulated in a number of cancer cells following genotoxic stress323334. Additionally, p53 also augments the level of FasR on the plasma membrane by promoting trafficking of FasR from the Golgi35. The status of FasR was determined by flow cytometry and western blot analysis and we observed that DOX and MCD together significantly increased the expression of FasR as compared to either agent alone in MCF-7 and Hepa1–6 cells (Fig. 3D,E). Additionally, we also checked FasL protein level in cell lysates and its secreted form in the culture medium collected from the cells by western blot and by indirect ELISA respectively. The expression of FasL in whole cell lysates as well as in the culture medium was increased after treatment of DOX and MCD together (Fig. 3E,F).

Bottom Line: However, its clinical application is limited due to severe side effects and the accompanying drug resistance.MCD sensitizes MCF-7 and Hepa1-6 cells to DOX, Combination of MCD and marginal dose of DOX reduces the cell viability, and promoted apoptosis through induction of pro-apoptotic protein, Bax, activation of caspase-8 and caspase-7, down regulation of anti-apoptotic protein Bcl-2 and finally promoting PARP cleavage.Collectively, these results suggest that MCD enhances the sensitivity to DOX for which wild type p53 is an important determinant.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune- 411007, India.

ABSTRACT
Doxorubicin (DOX) is one of the preferred drugs for treating breast and liver cancers. However, its clinical application is limited due to severe side effects and the accompanying drug resistance. In this context, we investigated the effect on therapeutic efficacy of DOX by cholesterol depleting agent methyl-β-cyclodextrin (MCD), and explored the involvement of p53. MCD sensitizes MCF-7 and Hepa1-6 cells to DOX, Combination of MCD and marginal dose of DOX reduces the cell viability, and promoted apoptosis through induction of pro-apoptotic protein, Bax, activation of caspase-8 and caspase-7, down regulation of anti-apoptotic protein Bcl-2 and finally promoting PARP cleavage. Mechanistically, sensitization to DOX by MCD was due to the induction of FasR/FasL pathway through p53 activation. Furthermore, inhibition of p53 by pharmacological inhibitor pifithrin-α (PFT-α) or its specific siRNA attenuated p53 function and down-regulated FasR/FasL, thereby preventing cell death. Animal experiments were performed using C57BL/6J mouse isografted with Hepa1-6 cells. Tumor growth was retarded and survival increased in mice administered MCD together with DOX to as compared to either agent alone. Collectively, these results suggest that MCD enhances the sensitivity to DOX for which wild type p53 is an important determinant.

No MeSH data available.


Related in: MedlinePlus