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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

Silencing of p53 reduces MCD-sensitized DOX-induced cell death and down-regulation of FasR/ FasL.(A) MCF-7 and (D) Hepa1–6 cells were treated with varying concentrations of PFTα (10–80 μM) for 24 h and cell survival was evaluated by MTT assay. (B) MCF-7 and (E) Hepa1–6 cells were treated with indicated concentration of MCD, PFT-α and DOX for 24 h. Viability of cell were measured by MTT assay. (C) MCF-7 and (F) Hepa1–6 cells were treated with MCD, PFT-α and DOX as indicated and long term clonogenic assay was performed using crystal violet. (G) Representative flow cytometric histogram of intracellular uptake of DOX in MCF-7 and Hepa1–6 cells treated with MCD, PFT-α, and DOX. (H) Bar graph is representative of relative quantitation of DOX uptake in MCF-7 and Hepa1–6 cells. (I) MCF-7 and (J) Hepa1–6 cells were treated with indicated concentration of MCD, PFT-α and DOX for 24 h, the protein levels of p53, MDM2, FasR and FasL were examined by western blotting analysis. Hsp60 served as loading control. (K) MCF-7 cells were transfected with p53 siRNA as per manufacturer’s instructions. After 18 h of transfection, cells were exposed with MCD and DOX together for 24 h and whole cell lysates were prepared and immunoblotted for p53, MDM2, FasR and FasL. Hsp60 served as loading control. Bar graphs represent (Mean ± SD) experiments done in triplicate. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 5.
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f4: Silencing of p53 reduces MCD-sensitized DOX-induced cell death and down-regulation of FasR/ FasL.(A) MCF-7 and (D) Hepa1–6 cells were treated with varying concentrations of PFTα (10–80 μM) for 24 h and cell survival was evaluated by MTT assay. (B) MCF-7 and (E) Hepa1–6 cells were treated with indicated concentration of MCD, PFT-α and DOX for 24 h. Viability of cell were measured by MTT assay. (C) MCF-7 and (F) Hepa1–6 cells were treated with MCD, PFT-α and DOX as indicated and long term clonogenic assay was performed using crystal violet. (G) Representative flow cytometric histogram of intracellular uptake of DOX in MCF-7 and Hepa1–6 cells treated with MCD, PFT-α, and DOX. (H) Bar graph is representative of relative quantitation of DOX uptake in MCF-7 and Hepa1–6 cells. (I) MCF-7 and (J) Hepa1–6 cells were treated with indicated concentration of MCD, PFT-α and DOX for 24 h, the protein levels of p53, MDM2, FasR and FasL were examined by western blotting analysis. Hsp60 served as loading control. (K) MCF-7 cells were transfected with p53 siRNA as per manufacturer’s instructions. After 18 h of transfection, cells were exposed with MCD and DOX together for 24 h and whole cell lysates were prepared and immunoblotted for p53, MDM2, FasR and FasL. Hsp60 served as loading control. Bar graphs represent (Mean ± SD) experiments done in triplicate. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 5.

Mentions: We investigated whether p53 is directly involved in the augmentation of DOX induced cell death by MCD. Treatment of MCF-7 and Hepa1–6 cells with inhibitor of p53, pifithrin-α (PFT-α) at 20 μM for 24 h did not exhibit significant cytotoxicity as evaluated by MTT assay (Fig. 4A,D). When cells were pretreated with MCD followed by PFT-α and subsequent treatment of DOX, cell viability significantly increased whereas MCD together with DOX promoted cell death as assessed by MTT assay (Fig. 4B,E). By, long term clonogenic assay, more number of surviving colonies were observed in cells treated with MCD, PFT-α and DOX as compared to MCD plus DOX (Fig. 4C,F). In addition, PFT-α did not play any role in the cellular uptake of DOX as shown by graphical representation of FACS data and bar graph represents mean red fluorescence (Fig. 4G,H). Furthermore, p53 protein levels was significantly diminished and MDM2 protein was increased following co-treatment of MCD and DOX in the presence of PFT-α compared to MCD and DOX treatment. (Fig. 4I and J; p53 and MDM2 panel, highlighted by rectangular block). As FasR/FasL are downstream molecules of p53 their levels was also significantly reduced in MCD, DOX and PFT-α treated cells (Fig. 4I,J; FasR and FasL panel, highlighted by rectangular block). To ascertain the specificity of p53, in regulation of FASR/FasL, MCF-7 cells were transfected with specific p53 siRNA. As shown in Fig. 5K, in the cells transfected with p53 siRNA, the p53 and Fas/FasL protein levels were significantly reduced whereas MDM2 levels were increased in comparison to the control siRNA transfected cells (Fig. 5K; p53 and MDM2 panels, highlighted by rectangular block).


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)

Silencing of p53 reduces MCD-sensitized DOX-induced cell death and down-regulation of FasR/ FasL.(A) MCF-7 and (D) Hepa1–6 cells were treated with varying concentrations of PFTα (10–80 μM) for 24 h and cell survival was evaluated by MTT assay. (B) MCF-7 and (E) Hepa1–6 cells were treated with indicated concentration of MCD, PFT-α and DOX for 24 h. Viability of cell were measured by MTT assay. (C) MCF-7 and (F) Hepa1–6 cells were treated with MCD, PFT-α and DOX as indicated and long term clonogenic assay was performed using crystal violet. (G) Representative flow cytometric histogram of intracellular uptake of DOX in MCF-7 and Hepa1–6 cells treated with MCD, PFT-α, and DOX. (H) Bar graph is representative of relative quantitation of DOX uptake in MCF-7 and Hepa1–6 cells. (I) MCF-7 and (J) Hepa1–6 cells were treated with indicated concentration of MCD, PFT-α and DOX for 24 h, the protein levels of p53, MDM2, FasR and FasL were examined by western blotting analysis. Hsp60 served as loading control. (K) MCF-7 cells were transfected with p53 siRNA as per manufacturer’s instructions. After 18 h of transfection, cells were exposed with MCD and DOX together for 24 h and whole cell lysates were prepared and immunoblotted for p53, MDM2, FasR and FasL. Hsp60 served as loading control. Bar graphs represent (Mean ± SD) experiments done in triplicate. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f4: Silencing of p53 reduces MCD-sensitized DOX-induced cell death and down-regulation of FasR/ FasL.(A) MCF-7 and (D) Hepa1–6 cells were treated with varying concentrations of PFTα (10–80 μM) for 24 h and cell survival was evaluated by MTT assay. (B) MCF-7 and (E) Hepa1–6 cells were treated with indicated concentration of MCD, PFT-α and DOX for 24 h. Viability of cell were measured by MTT assay. (C) MCF-7 and (F) Hepa1–6 cells were treated with MCD, PFT-α and DOX as indicated and long term clonogenic assay was performed using crystal violet. (G) Representative flow cytometric histogram of intracellular uptake of DOX in MCF-7 and Hepa1–6 cells treated with MCD, PFT-α, and DOX. (H) Bar graph is representative of relative quantitation of DOX uptake in MCF-7 and Hepa1–6 cells. (I) MCF-7 and (J) Hepa1–6 cells were treated with indicated concentration of MCD, PFT-α and DOX for 24 h, the protein levels of p53, MDM2, FasR and FasL were examined by western blotting analysis. Hsp60 served as loading control. (K) MCF-7 cells were transfected with p53 siRNA as per manufacturer’s instructions. After 18 h of transfection, cells were exposed with MCD and DOX together for 24 h and whole cell lysates were prepared and immunoblotted for p53, MDM2, FasR and FasL. Hsp60 served as loading control. Bar graphs represent (Mean ± SD) experiments done in triplicate. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 5.
Mentions: We investigated whether p53 is directly involved in the augmentation of DOX induced cell death by MCD. Treatment of MCF-7 and Hepa1–6 cells with inhibitor of p53, pifithrin-α (PFT-α) at 20 μM for 24 h did not exhibit significant cytotoxicity as evaluated by MTT assay (Fig. 4A,D). When cells were pretreated with MCD followed by PFT-α and subsequent treatment of DOX, cell viability significantly increased whereas MCD together with DOX promoted cell death as assessed by MTT assay (Fig. 4B,E). By, long term clonogenic assay, more number of surviving colonies were observed in cells treated with MCD, PFT-α and DOX as compared to MCD plus DOX (Fig. 4C,F). In addition, PFT-α did not play any role in the cellular uptake of DOX as shown by graphical representation of FACS data and bar graph represents mean red fluorescence (Fig. 4G,H). Furthermore, p53 protein levels was significantly diminished and MDM2 protein was increased following co-treatment of MCD and DOX in the presence of PFT-α compared to MCD and DOX treatment. (Fig. 4I and J; p53 and MDM2 panel, highlighted by rectangular block). As FasR/FasL are downstream molecules of p53 their levels was also significantly reduced in MCD, DOX and PFT-α treated cells (Fig. 4I,J; FasR and FasL panel, highlighted by rectangular block). To ascertain the specificity of p53, in regulation of FASR/FasL, MCF-7 cells were transfected with specific p53 siRNA. As shown in Fig. 5K, in the cells transfected with p53 siRNA, the p53 and Fas/FasL protein levels were significantly reduced whereas MDM2 levels were increased in comparison to the control siRNA transfected cells (Fig. 5K; p53 and MDM2 panels, highlighted by rectangular block).

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