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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 augments intracellular uptake of DOX in MCF-7 and Hepa1–6 cells.MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD. (A) Representative flow cytometric histogram of intracellular uptake of DOX in MCF-7 and Hepa1–6 cells. (B) Bar graph is representative of relative quantitation of DOX uptake in MCF-7 and Hepa1–6 cells. (C) MCF-7 and (D) Hepa1–6 cell were subjected to immunofluorescence confocal microscopy for the detection of intracellular uptake of DOX.
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f2: MCD augments intracellular uptake of DOX in MCF-7 and Hepa1–6 cells.MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD. (A) Representative flow cytometric histogram of intracellular uptake of DOX in MCF-7 and Hepa1–6 cells. (B) Bar graph is representative of relative quantitation of DOX uptake in MCF-7 and Hepa1–6 cells. (C) MCF-7 and (D) Hepa1–6 cell were subjected to immunofluorescence confocal microscopy for the detection of intracellular uptake of DOX.

Mentions: To explore the possible mechanism of MCD potentiated DOX induced cytotoxicity, we examined the effect of MCD on intracellular uptake of DOX using flow cytometry. The amount of DOX in the cell is directly proportional to its fluorescence because DOX is an auto-fluorescent drug. The measurement of fluorescence intensity of DOX has been used to evaluate cellular uptake in the cells30. Although DOX is taken up by cells as such, interestingly, following treatment with MCD, the cellular uptake of DOX was significantly enhanced as evident by graphical representation of FACS data and bar graph represents mean red fluorescence (Fig. 2A,B). Enhancement in the accumulation of DOX by MCD was further confirmed by fluorescence microscope (Fig. 2C,D). MCD did not potentiate DOX uptake in AML12 cells (Supplementary Figure 2E-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 augments intracellular uptake of DOX in MCF-7 and Hepa1–6 cells.MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD. (A) Representative flow cytometric histogram of intracellular uptake of DOX in MCF-7 and Hepa1–6 cells. (B) Bar graph is representative of relative quantitation of DOX uptake in MCF-7 and Hepa1–6 cells. (C) MCF-7 and (D) Hepa1–6 cell were subjected to immunofluorescence confocal microscopy for the detection of intracellular uptake of DOX.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: MCD augments intracellular uptake of DOX in MCF-7 and Hepa1–6 cells.MCF-7 and Hepa1–6 cells were treated with indicated concentration of DOX together with MCD. (A) Representative flow cytometric histogram of intracellular uptake of DOX in MCF-7 and Hepa1–6 cells. (B) Bar graph is representative of relative quantitation of DOX uptake in MCF-7 and Hepa1–6 cells. (C) MCF-7 and (D) Hepa1–6 cell were subjected to immunofluorescence confocal microscopy for the detection of intracellular uptake of DOX.
Mentions: To explore the possible mechanism of MCD potentiated DOX induced cytotoxicity, we examined the effect of MCD on intracellular uptake of DOX using flow cytometry. The amount of DOX in the cell is directly proportional to its fluorescence because DOX is an auto-fluorescent drug. The measurement of fluorescence intensity of DOX has been used to evaluate cellular uptake in the cells30. Although DOX is taken up by cells as such, interestingly, following treatment with MCD, the cellular uptake of DOX was significantly enhanced as evident by graphical representation of FACS data and bar graph represents mean red fluorescence (Fig. 2A,B). Enhancement in the accumulation of DOX by MCD was further confirmed by fluorescence microscope (Fig. 2C,D). MCD did not potentiate DOX uptake in AML12 cells (Supplementary Figure 2E-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