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Sulfonoquinovosyl diacylglyceride selectively targets acute lymphoblastic leukemia cells and exerts potent anti-leukemic effects in vivo.

Jain CK, Pradhan BS, Banerjee S, Mondal NB, Majumder SS, Bhattacharyya M, Chakrabarti S, Roychoudhury S, Majumder HK - Sci Rep (2015)

Bottom Line: Down-regulation of topoisomerase I or p53 renders the cells less sensitive for SQDG, while ectopic expression of wild type p53 protein in p53 deficient K562 cells results in chemosensitization of the cells for SQDG.We also show that constant ratio combinations of SQDG and etoposide or SDQG and doxorubicin exert synergistic effects on MOLT-4 cell killing.This study suggests that doses of etoposide/doxorubicin can be substantially reduced by combining SQDG with these agents during ALL chemotherapy and side effects caused can be minimized.

View Article: PubMed Central - PubMed

Affiliation: 1] Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata-700032, India [2] Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata-700032, India.

ABSTRACT
DNA topoisomerase II inhibitors e.g. doxorubicin and etoposide are currently used in the chemotherapy for acute lymphoblastic leukemia (ALL). These inhibitors have serious side effects during the chemotherapy e.g. cardiotoxicity and secondary malignancies. In this study we show that sulfonoquinovosyl diacylglyceride (SQDG) isolated from Azadirachta indica exerts potent anti-ALL activity both in vitro and in vivo in nude mice and it synergizes with doxorubicin and etoposide. SQDG selectively targets ALL MOLT-4 cells by inhibiting catalytic activity of topoisomerase I enzyme and inducing p53 dependent apoptotic pathway. SQDG treatment induces recruitment of ATR at chromatin and arrests the cells in S-phase. Down-regulation of topoisomerase I or p53 renders the cells less sensitive for SQDG, while ectopic expression of wild type p53 protein in p53 deficient K562 cells results in chemosensitization of the cells for SQDG. We also show that constant ratio combinations of SQDG and etoposide or SDQG and doxorubicin exert synergistic effects on MOLT-4 cell killing. This study suggests that doses of etoposide/doxorubicin can be substantially reduced by combining SQDG with these agents during ALL chemotherapy and side effects caused can be minimized. Thus dual targeting of topoisomerase I and II enzymes is a promising strategy for improving ALL chemotherapy.

No MeSH data available.


Related in: MedlinePlus

SQDG mediated MOLT-4 cell killing is topo I dependent.SQDG treatment precludes camptothecin mediated formation of topo I-DNAcomplexes and generates DNA replication stress in MOLT-4 cells. (a)Knockdown of topo I in MOLT-4 cells. MOLT-4 cells were transfected with100 nM topo I siRNA or 100 nM control siRNA (ctrlsiRNA). After 48 hours of transfection cells were treated withindicated concentrations of SQDG for 72 hours and MTT assay wasperformed. Three independent experiments were performed and data arerepresented as mean % cellviability ± SD. Solid bars indicatecells transfected with control siRNA and hollow bars indicate cellstransfected with topo I siRNA. (b) Topo I immunoband depletion assayin MOLT-4 cells. The cells were treated with 10 μMCPT or 20 μM SQDG or 20 μMBA and harvested at indicated time points. Western blotting was performedusing anti-topo I or anti-β-actin antibodies. Complete scans ofthe different blots are presented in the Supplementary Figure S11. (c)Pretreatment immunoband depletion assay. MOLT-4 cells were first treatedwith either 20 μM SQDG or20 μM BA for 2 hours and then treatedwith 10 μM CPT for indicated time points. Westernblotting was performed using anti-topo I or anti-β-actinantibodies. Complete scans of the different blots are presented in the Supplementary Figure S12.(d) Cell cycle analysis of SQDG treated MOLT-4 cells. Cells weretreated with different concentrations of SQDG(15 μM, 20 μM and25 μM) for 24 hours. Cells were fixed in70% ethanol and flow cytometry was performed. (e) ATR recruitment atchromatin in SQDG treated MOLT-4 cells. MOLT-4 cells were treated with15 μM SQDG for indicated time points and nuclear andchromatin fractionations were performed. Levels of ATR in chromatin andnuclear fractions were detected by western blot analysis. Histone-H3 wasused as loading control. ‘N’ stands for nuclearfraction and ‘C’ stands for chromatin fraction.Complete scans of the different blots are presented in the Supplementary Figure S13. (f)Histogram showing fold changes of ATR in chromatin and nuclear fractionsafter 15 μM SQDG treatment for indicated timepoints. Protein levels were quantitated by densitometry analysis of gelbands. ATR levels were normalized with respective histone H3 levels and foldchange was calculated. Error bars show standard deviation of mean for thetwo independent experiments.
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f4: SQDG mediated MOLT-4 cell killing is topo I dependent.SQDG treatment precludes camptothecin mediated formation of topo I-DNAcomplexes and generates DNA replication stress in MOLT-4 cells. (a)Knockdown of topo I in MOLT-4 cells. MOLT-4 cells were transfected with100 nM topo I siRNA or 100 nM control siRNA (ctrlsiRNA). After 48 hours of transfection cells were treated withindicated concentrations of SQDG for 72 hours and MTT assay wasperformed. Three independent experiments were performed and data arerepresented as mean % cellviability ± SD. Solid bars indicatecells transfected with control siRNA and hollow bars indicate cellstransfected with topo I siRNA. (b) Topo I immunoband depletion assayin MOLT-4 cells. The cells were treated with 10 μMCPT or 20 μM SQDG or 20 μMBA and harvested at indicated time points. Western blotting was performedusing anti-topo I or anti-β-actin antibodies. Complete scans ofthe different blots are presented in the Supplementary Figure S11. (c)Pretreatment immunoband depletion assay. MOLT-4 cells were first treatedwith either 20 μM SQDG or20 μM BA for 2 hours and then treatedwith 10 μM CPT for indicated time points. Westernblotting was performed using anti-topo I or anti-β-actinantibodies. Complete scans of the different blots are presented in the Supplementary Figure S12.(d) Cell cycle analysis of SQDG treated MOLT-4 cells. Cells weretreated with different concentrations of SQDG(15 μM, 20 μM and25 μM) for 24 hours. Cells were fixed in70% ethanol and flow cytometry was performed. (e) ATR recruitment atchromatin in SQDG treated MOLT-4 cells. MOLT-4 cells were treated with15 μM SQDG for indicated time points and nuclear andchromatin fractionations were performed. Levels of ATR in chromatin andnuclear fractions were detected by western blot analysis. Histone-H3 wasused as loading control. ‘N’ stands for nuclearfraction and ‘C’ stands for chromatin fraction.Complete scans of the different blots are presented in the Supplementary Figure S13. (f)Histogram showing fold changes of ATR in chromatin and nuclear fractionsafter 15 μM SQDG treatment for indicated timepoints. Protein levels were quantitated by densitometry analysis of gelbands. ATR levels were normalized with respective histone H3 levels and foldchange was calculated. Error bars show standard deviation of mean for thetwo independent experiments.

Mentions: To assess the role of topo I inhibition in SQDG mediated killing of MOLT-4 cells,siRNA silencing of TOP1 gene was performed by using a pool of threedifferent siRNAs. Knockdown of TOP1 gene in MOLT-4 cells rendered thecells less sensitive for SQDG treatment (Fig. 4a and Supplementary Fig. S3a).IC50 value of SQDG for control siRNA transfected cells was foundto be14.04 ± 0.71 μMwhile IC50 value of SQDG for topo I siRNA transfected cells was foundto be29.09 ± 2.08 μM(Supplementary Table S2). Foldresistance was calculated by the ratio of IC50 value of siRNAtransfected cells to IC50 value of control siRNA transfected cells.Fold increase in resistance for topo I siRNA transfected cells was found to be2.07 folds. Knockdown of TOP1 gene was corroborated by one additionalsiRNAs pool to mitigate off-target effects (Supplementary Fig S4a. and Supplementary Table S2). siRNA silencing ofTOP1 gene was also performed in Reh cell line. Similar to MOLT-4cells in Reh cells knockdown of TOP1 gene rendered the cells lesssensitive for SQDG treatment (Supplementary Fig S4b and S4c). IC50 value of SQDG forcontrol siRNA transfected cells was found to be14.48 ± 0.28 μMwhile IC50 value of SQDG for topo I siRNA transfected cells was foundto be27.51 ± 0.41 μM(Supplementary Table S2). Foldincrease in resistance for topo I siRNA transfected cells was found to be 1.89folds. These results indicate that SQDG mediated MOLT-4 and Reh cell killingsare topo I dependent.


Sulfonoquinovosyl diacylglyceride selectively targets acute lymphoblastic leukemia cells and exerts potent anti-leukemic effects in vivo.

Jain CK, Pradhan BS, Banerjee S, Mondal NB, Majumder SS, Bhattacharyya M, Chakrabarti S, Roychoudhury S, Majumder HK - Sci Rep (2015)

SQDG mediated MOLT-4 cell killing is topo I dependent.SQDG treatment precludes camptothecin mediated formation of topo I-DNAcomplexes and generates DNA replication stress in MOLT-4 cells. (a)Knockdown of topo I in MOLT-4 cells. MOLT-4 cells were transfected with100 nM topo I siRNA or 100 nM control siRNA (ctrlsiRNA). After 48 hours of transfection cells were treated withindicated concentrations of SQDG for 72 hours and MTT assay wasperformed. Three independent experiments were performed and data arerepresented as mean % cellviability ± SD. Solid bars indicatecells transfected with control siRNA and hollow bars indicate cellstransfected with topo I siRNA. (b) Topo I immunoband depletion assayin MOLT-4 cells. The cells were treated with 10 μMCPT or 20 μM SQDG or 20 μMBA and harvested at indicated time points. Western blotting was performedusing anti-topo I or anti-β-actin antibodies. Complete scans ofthe different blots are presented in the Supplementary Figure S11. (c)Pretreatment immunoband depletion assay. MOLT-4 cells were first treatedwith either 20 μM SQDG or20 μM BA for 2 hours and then treatedwith 10 μM CPT for indicated time points. Westernblotting was performed using anti-topo I or anti-β-actinantibodies. Complete scans of the different blots are presented in the Supplementary Figure S12.(d) Cell cycle analysis of SQDG treated MOLT-4 cells. Cells weretreated with different concentrations of SQDG(15 μM, 20 μM and25 μM) for 24 hours. Cells were fixed in70% ethanol and flow cytometry was performed. (e) ATR recruitment atchromatin in SQDG treated MOLT-4 cells. MOLT-4 cells were treated with15 μM SQDG for indicated time points and nuclear andchromatin fractionations were performed. Levels of ATR in chromatin andnuclear fractions were detected by western blot analysis. Histone-H3 wasused as loading control. ‘N’ stands for nuclearfraction and ‘C’ stands for chromatin fraction.Complete scans of the different blots are presented in the Supplementary Figure S13. (f)Histogram showing fold changes of ATR in chromatin and nuclear fractionsafter 15 μM SQDG treatment for indicated timepoints. Protein levels were quantitated by densitometry analysis of gelbands. ATR levels were normalized with respective histone H3 levels and foldchange was calculated. Error bars show standard deviation of mean for thetwo independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4507174&req=5

f4: SQDG mediated MOLT-4 cell killing is topo I dependent.SQDG treatment precludes camptothecin mediated formation of topo I-DNAcomplexes and generates DNA replication stress in MOLT-4 cells. (a)Knockdown of topo I in MOLT-4 cells. MOLT-4 cells were transfected with100 nM topo I siRNA or 100 nM control siRNA (ctrlsiRNA). After 48 hours of transfection cells were treated withindicated concentrations of SQDG for 72 hours and MTT assay wasperformed. Three independent experiments were performed and data arerepresented as mean % cellviability ± SD. Solid bars indicatecells transfected with control siRNA and hollow bars indicate cellstransfected with topo I siRNA. (b) Topo I immunoband depletion assayin MOLT-4 cells. The cells were treated with 10 μMCPT or 20 μM SQDG or 20 μMBA and harvested at indicated time points. Western blotting was performedusing anti-topo I or anti-β-actin antibodies. Complete scans ofthe different blots are presented in the Supplementary Figure S11. (c)Pretreatment immunoband depletion assay. MOLT-4 cells were first treatedwith either 20 μM SQDG or20 μM BA for 2 hours and then treatedwith 10 μM CPT for indicated time points. Westernblotting was performed using anti-topo I or anti-β-actinantibodies. Complete scans of the different blots are presented in the Supplementary Figure S12.(d) Cell cycle analysis of SQDG treated MOLT-4 cells. Cells weretreated with different concentrations of SQDG(15 μM, 20 μM and25 μM) for 24 hours. Cells were fixed in70% ethanol and flow cytometry was performed. (e) ATR recruitment atchromatin in SQDG treated MOLT-4 cells. MOLT-4 cells were treated with15 μM SQDG for indicated time points and nuclear andchromatin fractionations were performed. Levels of ATR in chromatin andnuclear fractions were detected by western blot analysis. Histone-H3 wasused as loading control. ‘N’ stands for nuclearfraction and ‘C’ stands for chromatin fraction.Complete scans of the different blots are presented in the Supplementary Figure S13. (f)Histogram showing fold changes of ATR in chromatin and nuclear fractionsafter 15 μM SQDG treatment for indicated timepoints. Protein levels were quantitated by densitometry analysis of gelbands. ATR levels were normalized with respective histone H3 levels and foldchange was calculated. Error bars show standard deviation of mean for thetwo independent experiments.
Mentions: To assess the role of topo I inhibition in SQDG mediated killing of MOLT-4 cells,siRNA silencing of TOP1 gene was performed by using a pool of threedifferent siRNAs. Knockdown of TOP1 gene in MOLT-4 cells rendered thecells less sensitive for SQDG treatment (Fig. 4a and Supplementary Fig. S3a).IC50 value of SQDG for control siRNA transfected cells was foundto be14.04 ± 0.71 μMwhile IC50 value of SQDG for topo I siRNA transfected cells was foundto be29.09 ± 2.08 μM(Supplementary Table S2). Foldresistance was calculated by the ratio of IC50 value of siRNAtransfected cells to IC50 value of control siRNA transfected cells.Fold increase in resistance for topo I siRNA transfected cells was found to be2.07 folds. Knockdown of TOP1 gene was corroborated by one additionalsiRNAs pool to mitigate off-target effects (Supplementary Fig S4a. and Supplementary Table S2). siRNA silencing ofTOP1 gene was also performed in Reh cell line. Similar to MOLT-4cells in Reh cells knockdown of TOP1 gene rendered the cells lesssensitive for SQDG treatment (Supplementary Fig S4b and S4c). IC50 value of SQDG forcontrol siRNA transfected cells was found to be14.48 ± 0.28 μMwhile IC50 value of SQDG for topo I siRNA transfected cells was foundto be27.51 ± 0.41 μM(Supplementary Table S2). Foldincrease in resistance for topo I siRNA transfected cells was found to be 1.89folds. These results indicate that SQDG mediated MOLT-4 and Reh cell killingsare topo I dependent.

Bottom Line: Down-regulation of topoisomerase I or p53 renders the cells less sensitive for SQDG, while ectopic expression of wild type p53 protein in p53 deficient K562 cells results in chemosensitization of the cells for SQDG.We also show that constant ratio combinations of SQDG and etoposide or SDQG and doxorubicin exert synergistic effects on MOLT-4 cell killing.This study suggests that doses of etoposide/doxorubicin can be substantially reduced by combining SQDG with these agents during ALL chemotherapy and side effects caused can be minimized.

View Article: PubMed Central - PubMed

Affiliation: 1] Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata-700032, India [2] Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata-700032, India.

ABSTRACT
DNA topoisomerase II inhibitors e.g. doxorubicin and etoposide are currently used in the chemotherapy for acute lymphoblastic leukemia (ALL). These inhibitors have serious side effects during the chemotherapy e.g. cardiotoxicity and secondary malignancies. In this study we show that sulfonoquinovosyl diacylglyceride (SQDG) isolated from Azadirachta indica exerts potent anti-ALL activity both in vitro and in vivo in nude mice and it synergizes with doxorubicin and etoposide. SQDG selectively targets ALL MOLT-4 cells by inhibiting catalytic activity of topoisomerase I enzyme and inducing p53 dependent apoptotic pathway. SQDG treatment induces recruitment of ATR at chromatin and arrests the cells in S-phase. Down-regulation of topoisomerase I or p53 renders the cells less sensitive for SQDG, while ectopic expression of wild type p53 protein in p53 deficient K562 cells results in chemosensitization of the cells for SQDG. We also show that constant ratio combinations of SQDG and etoposide or SDQG and doxorubicin exert synergistic effects on MOLT-4 cell killing. This study suggests that doses of etoposide/doxorubicin can be substantially reduced by combining SQDG with these agents during ALL chemotherapy and side effects caused can be minimized. Thus dual targeting of topoisomerase I and II enzymes is a promising strategy for improving ALL chemotherapy.

No MeSH data available.


Related in: MedlinePlus