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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 treatment induces p53 dependent apoptotic pathway in MOLT-4 cells andectopic expression of p53 in K562 cells sensitizes the cells for SQDG.(a) FITC-annexin V-propidium iodide staining of MOLT-4 cells untreatedor treated with CPT or SQDG. Cells were treated with either CPT(2 μM) or SQDG (15 μM or20 μM) for 48 hours. (b) Analysisof p53 dependent apoptotic pathway. MOLT-4 cells were treated with15 μM SQDG for indicated time points andimmunoblotting was performed using specific antibodies for indicatedproteins involved in p53 dependent pathway. Complete scans of the differentblots are presented in the Supplementary Figure S14. (c) Fold changes of levels ofproteins involved in p53 dependent pathway upon treatment with15 μM SQDG for indicated time points. Protein levelswere quantitated by densitometry analysis of gel bands. Protein levels werenormalized with respective β-actin levels and fold change wascalculated. Error bars show standard deviation of mean for two independentexperiments. (d) Knockdown of p53 in MOLT-4 cells. MOLT-4 cells weretransfected with 100 nM p53 siRNA or 100 nM controlsiRNA. After 72 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 p53 siRNA. (e) Ectopic expression of p53 in p53deficient K562 myeloid leukemia cells. K562 cells were transfected with400 ng control vector or 400 ng p53 expressingvector pCMV-NEO-BAM. After 36 hours of transfection cells weretreated with indicated concentrations of SQDG for 72 hours andMTT assay was performed. Three independent experiments were performed anddata are represented as mean % cellviability ± SD. Solid bars indicatecells transfected with control vector and hollow bars indicate cellstransfected with p53 expressing vector.
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f5: SQDG treatment induces p53 dependent apoptotic pathway in MOLT-4 cells andectopic expression of p53 in K562 cells sensitizes the cells for SQDG.(a) FITC-annexin V-propidium iodide staining of MOLT-4 cells untreatedor treated with CPT or SQDG. Cells were treated with either CPT(2 μM) or SQDG (15 μM or20 μM) for 48 hours. (b) Analysisof p53 dependent apoptotic pathway. MOLT-4 cells were treated with15 μM SQDG for indicated time points andimmunoblotting was performed using specific antibodies for indicatedproteins involved in p53 dependent pathway. Complete scans of the differentblots are presented in the Supplementary Figure S14. (c) Fold changes of levels ofproteins involved in p53 dependent pathway upon treatment with15 μM SQDG for indicated time points. Protein levelswere quantitated by densitometry analysis of gel bands. Protein levels werenormalized with respective β-actin levels and fold change wascalculated. Error bars show standard deviation of mean for two independentexperiments. (d) Knockdown of p53 in MOLT-4 cells. MOLT-4 cells weretransfected with 100 nM p53 siRNA or 100 nM controlsiRNA. After 72 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 p53 siRNA. (e) Ectopic expression of p53 in p53deficient K562 myeloid leukemia cells. K562 cells were transfected with400 ng control vector or 400 ng p53 expressingvector pCMV-NEO-BAM. After 36 hours of transfection cells weretreated with indicated concentrations of SQDG for 72 hours andMTT assay was performed. Three independent experiments were performed anddata are represented as mean % cellviability ± SD. Solid bars indicatecells transfected with control vector and hollow bars indicate cellstransfected with p53 expressing vector.

Mentions: To study whether SQDG induces apoptosis in MOLT-4 cells, we performed annexin-Vand propidium iodide staining of SQDG treated and untreated cells followed byflow cytometric analysis. CPT (2 μM) was used as apositive control for apoptosis induction. Treatment of MOLT-4 cells with15 μM or 20 μM SQDG for48 hours markedly induced apoptosis in MOLT-4 cells (Fig. 5a). Since MOLT-4 cells express wild type p53, therefore wethought that p53 might play role in induction of apoptosis in these cells. Inorder to study p53 dependent apoptotic pathway, MOLT-4 cells were treated with15 μM SQDG for different time points and induction ofthe proteins involved in p53 dependent apoptotic pathway was checked (Fig. 5b,c). A 34 fold increase in p53 activation wasobserved after 8 hours of SQDG treatment, which reached to 60 foldsat 24 hours post-treatment. Bcl-associated X protein (Bax)activation is associated with induction of p53 dependent apoptosis. Bax is atranscriptional target of p53 and it induces cytochrome c release frommitochondrial membrane3940. Bax was activated by 2 folds after8 hours of SQDG treatment and increased to 13 folds at24 hours post-treatment. Cytochrome c together with apoptoticprotease activating factor-1 and procaspase-9 forms a complex called apoptosome,which cleaves procaspase-9 and converts it in to caspase-94142.Cleavage of procaspase-9 was increased by 9 folds after 8 hours ofSQDG treatment and reached to 50 folds at 24 hours post-treatment.Caspase-9 cleaves procaspase-3 into caspase-3, which initiates process of celldeath41. Caspase-3 is an executive caspase which activatesvarious endonucleases and proteases. During apoptosis induction PARP-1, a113 kDa protein, is cleaved into 89 and 24 kDafragments. In our study caspase-3 and PARP-1 cleavages started at12 hours of SQDG treatment and continued up to 24 hoursof the treatment. Together these findings suggest that SQDG treatment induces ap53 dependent apoptotic pathway in MOLT-4 cells. To further study p53 dependencyof SQDG mediated cell killing, siRNA gene silencing was performed to knockdownTP53 gene expression in MOLT-4 cells (Fig. 5dand Supplementary Fig. S3b). Cellviability experiments with TP53 silenced cells indicated that knockdownof p53 renders the cells less sensitive for SQDG treatment. IC50value of SQDG for control siRNA transfected cells was15.15 ± 0.13 μMwhile for p53 siRNA transfected cells it became26.85 ± 2.08 μM(Supplementary Table S5). Foldincrease in resistance for p53 siRNA transfected cells was found to be 1.77folds. Knockdown of TP53 gene was corroborated by one additional siRNAspool to mitigate off-target effects (Supplementary Fig S4d. and Supplementary Table S5). siRNA silencing of TP53 gene was alsoperformed in Reh cell line. Similar to MOLT-4 cells in Reh cells knockdown ofTP53 gene rendered the cells less sensitive for SQDG treatment (Supplementary Fig S4e and S4f).IC50 value of SQDG for control siRNA transfected cells was foundto be14.31 ± 1.04 μMwhile IC50 value of SQDG for p53 siRNA transfected cells was found tobe 22.35 ± 2.42 μM(Supplementary Table S5). Foldincrease in resistance for p53 siRNA transfected cells was found to be 1.56folds. Knockdown of TP53 gene was corroborated by one additional siRNAspool to mitigate off-target effects (Supplementary Fig S4f). On the other hand ectopic expression of p53protein in p53 deficient K562 myeloid leukemia cells amended these cellsrelatively more sensitive for SQDG treatment (Fig. 5e andSupplementary Fig. S3c).IC50 value of SQDG for K562 cells transfected with empty vectorwas found to be56.51 ± 1.14 μMwhile IC50 value of SQDG for K562 cells ectopically expressing wildtype p53 was found to be19.54 ± 2.51 μM(Supplementary Table S6). Foldincrease in sensitivity for K562 cells ectopically expressing p53 was found tobe 2.89 folds. p53 protein was also ectopically expressed in an another p53deficient cell line: HL-60. Similar to K562 cells ectopic expression of p53protein in HL-60 cells amended these cells relatively more sensitive for SQDGtreatment (Supplementary Fig S5).IC50 value of SQDG for the cells transfected with empty vectorwas found to be92.65 ± 6.35 μMwhile IC50 value of SQDG for the cells ectopically expressing wildtype p53 was found to be24.01 ± 2.0 μM (Supplementary Table S6). Foldincrease in sensitivity for HL-60 cells ectopically expressing p53 was found tobe 3.85 folds. Together these observations suggest that SQDG mediated leukemiccell killing is p53 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 treatment induces p53 dependent apoptotic pathway in MOLT-4 cells andectopic expression of p53 in K562 cells sensitizes the cells for SQDG.(a) FITC-annexin V-propidium iodide staining of MOLT-4 cells untreatedor treated with CPT or SQDG. Cells were treated with either CPT(2 μM) or SQDG (15 μM or20 μM) for 48 hours. (b) Analysisof p53 dependent apoptotic pathway. MOLT-4 cells were treated with15 μM SQDG for indicated time points andimmunoblotting was performed using specific antibodies for indicatedproteins involved in p53 dependent pathway. Complete scans of the differentblots are presented in the Supplementary Figure S14. (c) Fold changes of levels ofproteins involved in p53 dependent pathway upon treatment with15 μM SQDG for indicated time points. Protein levelswere quantitated by densitometry analysis of gel bands. Protein levels werenormalized with respective β-actin levels and fold change wascalculated. Error bars show standard deviation of mean for two independentexperiments. (d) Knockdown of p53 in MOLT-4 cells. MOLT-4 cells weretransfected with 100 nM p53 siRNA or 100 nM controlsiRNA. After 72 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 p53 siRNA. (e) Ectopic expression of p53 in p53deficient K562 myeloid leukemia cells. K562 cells were transfected with400 ng control vector or 400 ng p53 expressingvector pCMV-NEO-BAM. After 36 hours of transfection cells weretreated with indicated concentrations of SQDG for 72 hours andMTT assay was performed. Three independent experiments were performed anddata are represented as mean % cellviability ± SD. Solid bars indicatecells transfected with control vector and hollow bars indicate cellstransfected with p53 expressing vector.
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f5: SQDG treatment induces p53 dependent apoptotic pathway in MOLT-4 cells andectopic expression of p53 in K562 cells sensitizes the cells for SQDG.(a) FITC-annexin V-propidium iodide staining of MOLT-4 cells untreatedor treated with CPT or SQDG. Cells were treated with either CPT(2 μM) or SQDG (15 μM or20 μM) for 48 hours. (b) Analysisof p53 dependent apoptotic pathway. MOLT-4 cells were treated with15 μM SQDG for indicated time points andimmunoblotting was performed using specific antibodies for indicatedproteins involved in p53 dependent pathway. Complete scans of the differentblots are presented in the Supplementary Figure S14. (c) Fold changes of levels ofproteins involved in p53 dependent pathway upon treatment with15 μM SQDG for indicated time points. Protein levelswere quantitated by densitometry analysis of gel bands. Protein levels werenormalized with respective β-actin levels and fold change wascalculated. Error bars show standard deviation of mean for two independentexperiments. (d) Knockdown of p53 in MOLT-4 cells. MOLT-4 cells weretransfected with 100 nM p53 siRNA or 100 nM controlsiRNA. After 72 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 p53 siRNA. (e) Ectopic expression of p53 in p53deficient K562 myeloid leukemia cells. K562 cells were transfected with400 ng control vector or 400 ng p53 expressingvector pCMV-NEO-BAM. After 36 hours of transfection cells weretreated with indicated concentrations of SQDG for 72 hours andMTT assay was performed. Three independent experiments were performed anddata are represented as mean % cellviability ± SD. Solid bars indicatecells transfected with control vector and hollow bars indicate cellstransfected with p53 expressing vector.
Mentions: To study whether SQDG induces apoptosis in MOLT-4 cells, we performed annexin-Vand propidium iodide staining of SQDG treated and untreated cells followed byflow cytometric analysis. CPT (2 μM) was used as apositive control for apoptosis induction. Treatment of MOLT-4 cells with15 μM or 20 μM SQDG for48 hours markedly induced apoptosis in MOLT-4 cells (Fig. 5a). Since MOLT-4 cells express wild type p53, therefore wethought that p53 might play role in induction of apoptosis in these cells. Inorder to study p53 dependent apoptotic pathway, MOLT-4 cells were treated with15 μM SQDG for different time points and induction ofthe proteins involved in p53 dependent apoptotic pathway was checked (Fig. 5b,c). A 34 fold increase in p53 activation wasobserved after 8 hours of SQDG treatment, which reached to 60 foldsat 24 hours post-treatment. Bcl-associated X protein (Bax)activation is associated with induction of p53 dependent apoptosis. Bax is atranscriptional target of p53 and it induces cytochrome c release frommitochondrial membrane3940. Bax was activated by 2 folds after8 hours of SQDG treatment and increased to 13 folds at24 hours post-treatment. Cytochrome c together with apoptoticprotease activating factor-1 and procaspase-9 forms a complex called apoptosome,which cleaves procaspase-9 and converts it in to caspase-94142.Cleavage of procaspase-9 was increased by 9 folds after 8 hours ofSQDG treatment and reached to 50 folds at 24 hours post-treatment.Caspase-9 cleaves procaspase-3 into caspase-3, which initiates process of celldeath41. Caspase-3 is an executive caspase which activatesvarious endonucleases and proteases. During apoptosis induction PARP-1, a113 kDa protein, is cleaved into 89 and 24 kDafragments. In our study caspase-3 and PARP-1 cleavages started at12 hours of SQDG treatment and continued up to 24 hoursof the treatment. Together these findings suggest that SQDG treatment induces ap53 dependent apoptotic pathway in MOLT-4 cells. To further study p53 dependencyof SQDG mediated cell killing, siRNA gene silencing was performed to knockdownTP53 gene expression in MOLT-4 cells (Fig. 5dand Supplementary Fig. S3b). Cellviability experiments with TP53 silenced cells indicated that knockdownof p53 renders the cells less sensitive for SQDG treatment. IC50value of SQDG for control siRNA transfected cells was15.15 ± 0.13 μMwhile for p53 siRNA transfected cells it became26.85 ± 2.08 μM(Supplementary Table S5). Foldincrease in resistance for p53 siRNA transfected cells was found to be 1.77folds. Knockdown of TP53 gene was corroborated by one additional siRNAspool to mitigate off-target effects (Supplementary Fig S4d. and Supplementary Table S5). siRNA silencing of TP53 gene was alsoperformed in Reh cell line. Similar to MOLT-4 cells in Reh cells knockdown ofTP53 gene rendered the cells less sensitive for SQDG treatment (Supplementary Fig S4e and S4f).IC50 value of SQDG for control siRNA transfected cells was foundto be14.31 ± 1.04 μMwhile IC50 value of SQDG for p53 siRNA transfected cells was found tobe 22.35 ± 2.42 μM(Supplementary Table S5). Foldincrease in resistance for p53 siRNA transfected cells was found to be 1.56folds. Knockdown of TP53 gene was corroborated by one additional siRNAspool to mitigate off-target effects (Supplementary Fig S4f). On the other hand ectopic expression of p53protein in p53 deficient K562 myeloid leukemia cells amended these cellsrelatively more sensitive for SQDG treatment (Fig. 5e andSupplementary Fig. S3c).IC50 value of SQDG for K562 cells transfected with empty vectorwas found to be56.51 ± 1.14 μMwhile IC50 value of SQDG for K562 cells ectopically expressing wildtype p53 was found to be19.54 ± 2.51 μM(Supplementary Table S6). Foldincrease in sensitivity for K562 cells ectopically expressing p53 was found tobe 2.89 folds. p53 protein was also ectopically expressed in an another p53deficient cell line: HL-60. Similar to K562 cells ectopic expression of p53protein in HL-60 cells amended these cells relatively more sensitive for SQDGtreatment (Supplementary Fig S5).IC50 value of SQDG for the cells transfected with empty vectorwas found to be92.65 ± 6.35 μMwhile IC50 value of SQDG for the cells ectopically expressing wildtype p53 was found to be24.01 ± 2.0 μM (Supplementary Table S6). Foldincrease in sensitivity for HL-60 cells ectopically expressing p53 was found tobe 3.85 folds. Together these observations suggest that SQDG mediated leukemiccell killing is p53 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