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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 the effect of DOX and induces apoptosis in breast and liver cancer cells.(A) MCF-7 and (D) Hepa1–6 cells were treated with indicated concentrations of MCD (2.5–10 mM) for 4 h and the cell viability was measured by MTT assay. (B) MCF-7 and (E) Hepa1–6 cells were treated with indicated concentrations of DOX together with MCD for 24 h and subjected to MTT assay. (C) MCF-7 and (F) Hepa1–6 cells were treated with indicated concentrations of DOX together with MCD for 24 h and cells were subjected to long term clonogenic assay. (G) MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h, and apoptotic cells were analyzed by Annexin V-FITC staining using flow cytometry. (H) Quantitation of Annexin V-FITC positive cells. (I) MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h. Cells were harvested, whole cell lysates were subjected to immunoblotting and the protein levels of PARP, Bax and Bcl-2 was assessed. Hsp60 served as a loading control. (J) MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h. Cells were harvested, whole cell lysates were subjected to immunoblotting and the protein levels of caspase-8 and caspase-7 and there cleaved form were assessed. Hsp60 served as a loading control. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 1. All the bar graph represents the mean ± SD of an experiment done in triplicate (*P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001).
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f1: MCD potentiates the effect of DOX and induces apoptosis in breast and liver cancer cells.(A) MCF-7 and (D) Hepa1–6 cells were treated with indicated concentrations of MCD (2.5–10 mM) for 4 h and the cell viability was measured by MTT assay. (B) MCF-7 and (E) Hepa1–6 cells were treated with indicated concentrations of DOX together with MCD for 24 h and subjected to MTT assay. (C) MCF-7 and (F) Hepa1–6 cells were treated with indicated concentrations of DOX together with MCD for 24 h and cells were subjected to long term clonogenic assay. (G) MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h, and apoptotic cells were analyzed by Annexin V-FITC staining using flow cytometry. (H) Quantitation of Annexin V-FITC positive cells. (I) MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h. Cells were harvested, whole cell lysates were subjected to immunoblotting and the protein levels of PARP, Bax and Bcl-2 was assessed. Hsp60 served as a loading control. (J) MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h. Cells were harvested, whole cell lysates were subjected to immunoblotting and the protein levels of caspase-8 and caspase-7 and there cleaved form were assessed. Hsp60 served as a loading control. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 1. All the bar graph represents the mean ± SD of an experiment done in triplicate (*P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001).

Mentions: To investigate whether MCD has any adverse effect on MCF-7 and Hepa1–6 cells, screening experiments were performed to determine the non-toxic concentration and optimum time point of MCD suitable for use in combination treatment. Treatment of cells with various concentration of MCD (2.5 mM to 10 mM) for 4 h inhibited the cell survival in a dose-dependent manner as measured by MTT assay (Fig. 1A,D). MCD at 10 mM dose was highly toxic to cells as compared to 2.5 and 5 mM, hence, 5 mM concentration was used for further experiments. Additionally, the marginal dose of DOX for use in the combination regimen in cells treated with DOX was calculated to be 2.5 μM for both the cells (data not shown). Since DOX is used for the treatment of breast and HCC, it is necessary to define an approach to enhance the therapeutic index of DOX at lower doses. We investigated the combination effects of MCD on DOX induced effect in MCF-7 and Hepa1–6 cells. Cells were treated by MCD together with IC50 dose of DOX for 24 h. As anticipated, at IC50 dose of DOX, cell viability reduced by 50%, which was further reduced to less than 5% in the presence of MCD and these results were also verified by long term clonogenic assay (Fig. 1B,E,C,F). Next, we explored the effects of low dose (1 μM) of DOX together with MCD (5 mM) on viability of MCF-7 and Hepa1–6 cells and observed a significant reduction in cell survival as compared to MCD and DOX alone (Fig. 1B,E). Similar results were also observed in long term clonogenic assay (Fig. 1C,F). The increase in MCD potentiated DOX induced effect was due to induction of apoptosis in MCF-7 and Hepa1–6 cells as evident by graphical representation of FACS data and bar graph represents % Annexin positive cells (Fig. 1G,H). Consistent with above results combination treatment also caused PARP cleavage, decrease in the protein level of antiapoptotic protein Bcl-2, and up-regulation of proapoptotic protein Bax (Fig. 1I). Caspases play an important role in both intrinsic and extrinsic apoptotic pathways. Therefore, on examination of protein levels of caspases in MCF-7 and Hepa1–6, we found that MCD together with DOX activates caspase-8 and caspase-7 (Fig. 1J). All the above results demonstrate that MCD potentiates DOX induced cytotoxicity only in cancerous cell (MCF-7 and Hepa1–6) not in the normal hepatocytes (AML12) cells (Supplementary Figure 2A-D).


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 the effect of DOX and induces apoptosis in breast and liver cancer cells.(A) MCF-7 and (D) Hepa1–6 cells were treated with indicated concentrations of MCD (2.5–10 mM) for 4 h and the cell viability was measured by MTT assay. (B) MCF-7 and (E) Hepa1–6 cells were treated with indicated concentrations of DOX together with MCD for 24 h and subjected to MTT assay. (C) MCF-7 and (F) Hepa1–6 cells were treated with indicated concentrations of DOX together with MCD for 24 h and cells were subjected to long term clonogenic assay. (G) MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h, and apoptotic cells were analyzed by Annexin V-FITC staining using flow cytometry. (H) Quantitation of Annexin V-FITC positive cells. (I) MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h. Cells were harvested, whole cell lysates were subjected to immunoblotting and the protein levels of PARP, Bax and Bcl-2 was assessed. Hsp60 served as a loading control. (J) MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h. Cells were harvested, whole cell lysates were subjected to immunoblotting and the protein levels of caspase-8 and caspase-7 and there cleaved form were assessed. Hsp60 served as a loading control. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 1. All the bar graph represents the mean ± SD of an experiment done in triplicate (*P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: MCD potentiates the effect of DOX and induces apoptosis in breast and liver cancer cells.(A) MCF-7 and (D) Hepa1–6 cells were treated with indicated concentrations of MCD (2.5–10 mM) for 4 h and the cell viability was measured by MTT assay. (B) MCF-7 and (E) Hepa1–6 cells were treated with indicated concentrations of DOX together with MCD for 24 h and subjected to MTT assay. (C) MCF-7 and (F) Hepa1–6 cells were treated with indicated concentrations of DOX together with MCD for 24 h and cells were subjected to long term clonogenic assay. (G) MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h, and apoptotic cells were analyzed by Annexin V-FITC staining using flow cytometry. (H) Quantitation of Annexin V-FITC positive cells. (I) MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h. Cells were harvested, whole cell lysates were subjected to immunoblotting and the protein levels of PARP, Bax and Bcl-2 was assessed. Hsp60 served as a loading control. (J) MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD for 24 h. Cells were harvested, whole cell lysates were subjected to immunoblotting and the protein levels of caspase-8 and caspase-7 and there cleaved form were assessed. Hsp60 served as a loading control. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 1. All the bar graph represents the mean ± SD of an experiment done in triplicate (*P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001).
Mentions: To investigate whether MCD has any adverse effect on MCF-7 and Hepa1–6 cells, screening experiments were performed to determine the non-toxic concentration and optimum time point of MCD suitable for use in combination treatment. Treatment of cells with various concentration of MCD (2.5 mM to 10 mM) for 4 h inhibited the cell survival in a dose-dependent manner as measured by MTT assay (Fig. 1A,D). MCD at 10 mM dose was highly toxic to cells as compared to 2.5 and 5 mM, hence, 5 mM concentration was used for further experiments. Additionally, the marginal dose of DOX for use in the combination regimen in cells treated with DOX was calculated to be 2.5 μM for both the cells (data not shown). Since DOX is used for the treatment of breast and HCC, it is necessary to define an approach to enhance the therapeutic index of DOX at lower doses. We investigated the combination effects of MCD on DOX induced effect in MCF-7 and Hepa1–6 cells. Cells were treated by MCD together with IC50 dose of DOX for 24 h. As anticipated, at IC50 dose of DOX, cell viability reduced by 50%, which was further reduced to less than 5% in the presence of MCD and these results were also verified by long term clonogenic assay (Fig. 1B,E,C,F). Next, we explored the effects of low dose (1 μM) of DOX together with MCD (5 mM) on viability of MCF-7 and Hepa1–6 cells and observed a significant reduction in cell survival as compared to MCD and DOX alone (Fig. 1B,E). Similar results were also observed in long term clonogenic assay (Fig. 1C,F). The increase in MCD potentiated DOX induced effect was due to induction of apoptosis in MCF-7 and Hepa1–6 cells as evident by graphical representation of FACS data and bar graph represents % Annexin positive cells (Fig. 1G,H). Consistent with above results combination treatment also caused PARP cleavage, decrease in the protein level of antiapoptotic protein Bcl-2, and up-regulation of proapoptotic protein Bax (Fig. 1I). Caspases play an important role in both intrinsic and extrinsic apoptotic pathways. Therefore, on examination of protein levels of caspases in MCF-7 and Hepa1–6, we found that MCD together with DOX activates caspase-8 and caspase-7 (Fig. 1J). All the above results demonstrate that MCD potentiates DOX induced cytotoxicity only in cancerous cell (MCF-7 and Hepa1–6) not in the normal hepatocytes (AML12) cells (Supplementary Figure 2A-D).

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