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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 and DOX combination inhibits growth of Hepa1–6 isografted tumors in C57BL/6J mice.Hepa1–6 cell-derived tumors were developed in C57BL/6J mice. Tumor bearing mice were administered normal saline, 64 mg/kg of MCD (i. p./alternative day), 1 mg/kg of DOX (i. p. every alternative day) and a combination of MCD and DOX. (A) Tumor initiation and progression (B) Changes in body weight (C) Median overall survival of mice (D) Representative panels of H&E staining of major vital organs such as heart, liver, kidney and lungs of mice (Magnification 10x). (E) Western blot analysis of indicated proteins (p53, MDM2, FasR, FasL, Bcl-2, Bax and Hsp60) from tumor lysates of mice administered MCD, DOX and combination of both. (F) Representative immunohistochemical analysis of CD31, Ki67 and apoptosis in tumor sections was examined by TUNEL staining. Cell nuclei stained blue with 4’,6-diamidino-2-phenylindole (DAPI) (Magnification 60x). TUNEL assay shows induced apoptotic nucleus (green) in tumors of MCD and DOX together administered mice. (G) Bar graph (Mean ± SD) showing the quantitation of average number of CD31, Ki67 and TUNEL positive cells selected from different fields by Image J software. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 6.
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f5: MCD and DOX combination inhibits growth of Hepa1–6 isografted tumors in C57BL/6J mice.Hepa1–6 cell-derived tumors were developed in C57BL/6J mice. Tumor bearing mice were administered normal saline, 64 mg/kg of MCD (i. p./alternative day), 1 mg/kg of DOX (i. p. every alternative day) and a combination of MCD and DOX. (A) Tumor initiation and progression (B) Changes in body weight (C) Median overall survival of mice (D) Representative panels of H&E staining of major vital organs such as heart, liver, kidney and lungs of mice (Magnification 10x). (E) Western blot analysis of indicated proteins (p53, MDM2, FasR, FasL, Bcl-2, Bax and Hsp60) from tumor lysates of mice administered MCD, DOX and combination of both. (F) Representative immunohistochemical analysis of CD31, Ki67 and apoptosis in tumor sections was examined by TUNEL staining. Cell nuclei stained blue with 4’,6-diamidino-2-phenylindole (DAPI) (Magnification 60x). TUNEL assay shows induced apoptotic nucleus (green) in tumors of MCD and DOX together administered mice. (G) Bar graph (Mean ± SD) showing the quantitation of average number of CD31, Ki67 and TUNEL positive cells selected from different fields by Image J software. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 6.

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)

MCD and DOX combination inhibits growth of Hepa1–6 isografted tumors in C57BL/6J mice.Hepa1–6 cell-derived tumors were developed in C57BL/6J mice. Tumor bearing mice were administered normal saline, 64 mg/kg of MCD (i. p./alternative day), 1 mg/kg of DOX (i. p. every alternative day) and a combination of MCD and DOX. (A) Tumor initiation and progression (B) Changes in body weight (C) Median overall survival of mice (D) Representative panels of H&E staining of major vital organs such as heart, liver, kidney and lungs of mice (Magnification 10x). (E) Western blot analysis of indicated proteins (p53, MDM2, FasR, FasL, Bcl-2, Bax and Hsp60) from tumor lysates of mice administered MCD, DOX and combination of both. (F) Representative immunohistochemical analysis of CD31, Ki67 and apoptosis in tumor sections was examined by TUNEL staining. Cell nuclei stained blue with 4’,6-diamidino-2-phenylindole (DAPI) (Magnification 60x). TUNEL assay shows induced apoptotic nucleus (green) in tumors of MCD and DOX together administered mice. (G) Bar graph (Mean ± SD) showing the quantitation of average number of CD31, Ki67 and TUNEL positive cells selected from different fields by Image J software. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 6.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: MCD and DOX combination inhibits growth of Hepa1–6 isografted tumors in C57BL/6J mice.Hepa1–6 cell-derived tumors were developed in C57BL/6J mice. Tumor bearing mice were administered normal saline, 64 mg/kg of MCD (i. p./alternative day), 1 mg/kg of DOX (i. p. every alternative day) and a combination of MCD and DOX. (A) Tumor initiation and progression (B) Changes in body weight (C) Median overall survival of mice (D) Representative panels of H&E staining of major vital organs such as heart, liver, kidney and lungs of mice (Magnification 10x). (E) Western blot analysis of indicated proteins (p53, MDM2, FasR, FasL, Bcl-2, Bax and Hsp60) from tumor lysates of mice administered MCD, DOX and combination of both. (F) Representative immunohistochemical analysis of CD31, Ki67 and apoptosis in tumor sections was examined by TUNEL staining. Cell nuclei stained blue with 4’,6-diamidino-2-phenylindole (DAPI) (Magnification 60x). TUNEL assay shows induced apoptotic nucleus (green) in tumors of MCD and DOX together administered mice. (G) Bar graph (Mean ± SD) showing the quantitation of average number of CD31, Ki67 and TUNEL positive cells selected from different fields by Image J software. Cropped blots are used in the main figure and full length blots are included in Supplementary figure 6.
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