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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 abrogates CPT mediated topo I-DNA complex formation in vitro andinhibits DNA binding of topo I(a) Structure of labeled 25-mer duplex oligo used for oligonucleotidecleavage assay. CL25 is the strand to be cleaved while CP25 is thecomplementary strand. CL25 is labeled withγ-32Phosphate at 5′-end, shown withasterisk (*). Arrow (↓) represents the topo I cleavage site.(b) Oligonucleotide cleavage assay. Lane 1, 1 pmol ofthe labeled 25-mer duplex oligo; lane 2, 1 pmol of the labeled25-mer duplex oligo with 20 ng topo I enzyme; lanes 3 to 10,same as lane 2 but with indicated compounds. Reactions were denatured andseparated by 7 M-urea-PAGE (20%) and visualized by autoradiography.(c) DNA cleavage assay. Lane 1, 100 fmol of pBS DNA;lane 2, 100 fmol of pBS DNA with 500 fmol of topo Ienzyme; lanes 3 to 10, same as lane 2 but with indicated compounds.Reactions were incubated at 37 °C for30 minutes and stopped with 0.5% SDS. (d) Electrophoreticmobility shift assay. Lane 1, 1 pmol of the labeled 25-merduplex oligo; lane 2, 1 pmol of the labeled 25-mer duplex oligowith 20 ng topo I enzyme; lanes 3 to 8 are same as lane 2 but inthe presence of indicated concentrations of SQDG and CPT. Complete scans ofthe autoradiogram and gel are presented in the Supplementary Figure S8 and S9.
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f2: SQDG abrogates CPT mediated topo I-DNA complex formation in vitro andinhibits DNA binding of topo I(a) Structure of labeled 25-mer duplex oligo used for oligonucleotidecleavage assay. CL25 is the strand to be cleaved while CP25 is thecomplementary strand. CL25 is labeled withγ-32Phosphate at 5′-end, shown withasterisk (*). Arrow (↓) represents the topo I cleavage site.(b) Oligonucleotide cleavage assay. Lane 1, 1 pmol ofthe labeled 25-mer duplex oligo; lane 2, 1 pmol of the labeled25-mer duplex oligo with 20 ng topo I enzyme; lanes 3 to 10,same as lane 2 but with indicated compounds. Reactions were denatured andseparated by 7 M-urea-PAGE (20%) and visualized by autoradiography.(c) DNA cleavage assay. Lane 1, 100 fmol of pBS DNA;lane 2, 100 fmol of pBS DNA with 500 fmol of topo Ienzyme; lanes 3 to 10, same as lane 2 but with indicated compounds.Reactions were incubated at 37 °C for30 minutes and stopped with 0.5% SDS. (d) Electrophoreticmobility shift assay. Lane 1, 1 pmol of the labeled 25-merduplex oligo; lane 2, 1 pmol of the labeled 25-mer duplex oligowith 20 ng topo I enzyme; lanes 3 to 8 are same as lane 2 but inthe presence of indicated concentrations of SQDG and CPT. Complete scans ofthe autoradiogram and gel are presented in the Supplementary Figure S8 and S9.

Mentions: To find whether SQDG is catalytic inhibitor or topo I poison, in vitrooligonucleotide cleavage assay was performed using synthetic oligonucleotidewith topo I binding site (Fig. 2a). Camptothecin (CPT) isa topo I poison which inhibits religation step of the enzymatic reaction cycleand stabilizes covalent topo I-DNA complexes611.Oligonucleotide cleavage assay in the presence of 10 μMand 20 μM CPT substantially increased the amount ofcleaved 12 mer oligo compared to control, as CPT stabilizes topo I-oligocovalent complexes (Fig. 2b). Betulinic acid (BA) is acatalytic inhibitor of topo I2223 and was used as anothercontrol. In presence of 10 μM and20 μM SQDG, we did not observe any increase in theamount of cleaved 12 mer oligo compared to the control reaction, indicating thatSQDG is not a topo I poison like CPT but it is a catalytic topo I inhibitor likeBA. When 20 μM BA or 20 μM SQDGwas added in the reactions and incubated for 5 minutes prior toaddition of 20 μM CPT, the amount of cleaved 12 meroligo substantially reduced in comparison to CPT alone reactions, indicatingthat SQDG/BA abrogates CPT mediated topo I-oligo covalent complex formation(Fig. 2b). Besides, DNA cleavage assay was alsoperformed to revalidate the results of oligonucleotide cleavage assay. SQDG(10 μM and 20 μM) did notstabilize the topo-DNA complexes while CPT (10 μM and20 μM) substantially increased formation of the topo-DNAcomplexes (Fig. 2c). The assay further confirmed that SQDGis a catalytic topo I inhibitor. In order to study effect of SQDG on the DNAbinding of topo I enzyme electrophoretic mobility shift assay was performedusing synthetic oligonucleotide containing topo I binding site (Fig. 2a,d). Results of EMSA indicated that in the presence of 5 and10 μM SQDG, binding of topo I to oligonucleotide wasdecreased and in the presence of 20 μM SQDG, binding oftopo I to oligonucleotide was almost completely abolished. In the presence of 5,10 and 20 μM CPT binding of topo I to oligonucleotidewas increased which may be due to the stabilization of covalent topoI-oligonucleotide complexes by CPT. Thus, EMSA results clearly suggest that SQDGinhibits DNA binding activity of topo I. In order to understand comparativeaffinities of CPT and SQDG for the enzyme, competition cleavage assays wereperformed (Supplementary Fig. S2).Upon preincubation of the enzyme with 5, 10 and 20 μMSQDG for 5 minutes, CPT mediated cleavage was decreased in 5 and10 μM SQDG preincubated conditions and the cleavage wascompletely abolished in 20 μM SQDG preincubatedcondition (Supplementary Fig. S2a).On the other hand upon preincubation of the enzyme with 5, 10 and20 μM CPT for 5 minutes, addition of20 μM SQDG did not affect the CPT mediated cleavagecomplex formation (Supplementary Fig.S2b). Besides, upon simultaneous addition of SQDG and CPT, CPTmediated cleavage was not increased in 10 and 20 μM CPTconditions and was same as 5 μM CPT condition (Supplementary Fig. S2c). Theseresults suggest that both SQDG and CPT have similar affinities for the enzyme.Together the results of cleavage assays and electrophoretic mobility shift assay(Fig. 2 and Supplementary Fig. S2) suggest that SQDG inhibits topo I upstream inthe enzymatic reaction at the DNA binding step whereas CPT inhibits topo Idownstream in the enzymatic reaction at relegation step. Thus, uponpreincubation of the enzyme is with SQDG, CPT mediated cleavage is not observedand opposite to this, upon preincubation of the enzyme with CPT, SQDG can notaffect CPT mediated cleavage complex formation. However, upon simultaneousaddition of SQDG and CPT, CPT mediated cleavage is observed but the cleavagedoes not increase with increasing concentrations of CPT, suggesting that in thepresence of SQDG the enzyme becomes unable to bind to DNA and therefore CPT cannot stabilize covalent enzyme-DNA complexes anymore.


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 abrogates CPT mediated topo I-DNA complex formation in vitro andinhibits DNA binding of topo I(a) Structure of labeled 25-mer duplex oligo used for oligonucleotidecleavage assay. CL25 is the strand to be cleaved while CP25 is thecomplementary strand. CL25 is labeled withγ-32Phosphate at 5′-end, shown withasterisk (*). Arrow (↓) represents the topo I cleavage site.(b) Oligonucleotide cleavage assay. Lane 1, 1 pmol ofthe labeled 25-mer duplex oligo; lane 2, 1 pmol of the labeled25-mer duplex oligo with 20 ng topo I enzyme; lanes 3 to 10,same as lane 2 but with indicated compounds. Reactions were denatured andseparated by 7 M-urea-PAGE (20%) and visualized by autoradiography.(c) DNA cleavage assay. Lane 1, 100 fmol of pBS DNA;lane 2, 100 fmol of pBS DNA with 500 fmol of topo Ienzyme; lanes 3 to 10, same as lane 2 but with indicated compounds.Reactions were incubated at 37 °C for30 minutes and stopped with 0.5% SDS. (d) Electrophoreticmobility shift assay. Lane 1, 1 pmol of the labeled 25-merduplex oligo; lane 2, 1 pmol of the labeled 25-mer duplex oligowith 20 ng topo I enzyme; lanes 3 to 8 are same as lane 2 but inthe presence of indicated concentrations of SQDG and CPT. Complete scans ofthe autoradiogram and gel are presented in the Supplementary Figure S8 and S9.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: SQDG abrogates CPT mediated topo I-DNA complex formation in vitro andinhibits DNA binding of topo I(a) Structure of labeled 25-mer duplex oligo used for oligonucleotidecleavage assay. CL25 is the strand to be cleaved while CP25 is thecomplementary strand. CL25 is labeled withγ-32Phosphate at 5′-end, shown withasterisk (*). Arrow (↓) represents the topo I cleavage site.(b) Oligonucleotide cleavage assay. Lane 1, 1 pmol ofthe labeled 25-mer duplex oligo; lane 2, 1 pmol of the labeled25-mer duplex oligo with 20 ng topo I enzyme; lanes 3 to 10,same as lane 2 but with indicated compounds. Reactions were denatured andseparated by 7 M-urea-PAGE (20%) and visualized by autoradiography.(c) DNA cleavage assay. Lane 1, 100 fmol of pBS DNA;lane 2, 100 fmol of pBS DNA with 500 fmol of topo Ienzyme; lanes 3 to 10, same as lane 2 but with indicated compounds.Reactions were incubated at 37 °C for30 minutes and stopped with 0.5% SDS. (d) Electrophoreticmobility shift assay. Lane 1, 1 pmol of the labeled 25-merduplex oligo; lane 2, 1 pmol of the labeled 25-mer duplex oligowith 20 ng topo I enzyme; lanes 3 to 8 are same as lane 2 but inthe presence of indicated concentrations of SQDG and CPT. Complete scans ofthe autoradiogram and gel are presented in the Supplementary Figure S8 and S9.
Mentions: To find whether SQDG is catalytic inhibitor or topo I poison, in vitrooligonucleotide cleavage assay was performed using synthetic oligonucleotidewith topo I binding site (Fig. 2a). Camptothecin (CPT) isa topo I poison which inhibits religation step of the enzymatic reaction cycleand stabilizes covalent topo I-DNA complexes611.Oligonucleotide cleavage assay in the presence of 10 μMand 20 μM CPT substantially increased the amount ofcleaved 12 mer oligo compared to control, as CPT stabilizes topo I-oligocovalent complexes (Fig. 2b). Betulinic acid (BA) is acatalytic inhibitor of topo I2223 and was used as anothercontrol. In presence of 10 μM and20 μM SQDG, we did not observe any increase in theamount of cleaved 12 mer oligo compared to the control reaction, indicating thatSQDG is not a topo I poison like CPT but it is a catalytic topo I inhibitor likeBA. When 20 μM BA or 20 μM SQDGwas added in the reactions and incubated for 5 minutes prior toaddition of 20 μM CPT, the amount of cleaved 12 meroligo substantially reduced in comparison to CPT alone reactions, indicatingthat SQDG/BA abrogates CPT mediated topo I-oligo covalent complex formation(Fig. 2b). Besides, DNA cleavage assay was alsoperformed to revalidate the results of oligonucleotide cleavage assay. SQDG(10 μM and 20 μM) did notstabilize the topo-DNA complexes while CPT (10 μM and20 μM) substantially increased formation of the topo-DNAcomplexes (Fig. 2c). The assay further confirmed that SQDGis a catalytic topo I inhibitor. In order to study effect of SQDG on the DNAbinding of topo I enzyme electrophoretic mobility shift assay was performedusing synthetic oligonucleotide containing topo I binding site (Fig. 2a,d). Results of EMSA indicated that in the presence of 5 and10 μM SQDG, binding of topo I to oligonucleotide wasdecreased and in the presence of 20 μM SQDG, binding oftopo I to oligonucleotide was almost completely abolished. In the presence of 5,10 and 20 μM CPT binding of topo I to oligonucleotidewas increased which may be due to the stabilization of covalent topoI-oligonucleotide complexes by CPT. Thus, EMSA results clearly suggest that SQDGinhibits DNA binding activity of topo I. In order to understand comparativeaffinities of CPT and SQDG for the enzyme, competition cleavage assays wereperformed (Supplementary Fig. S2).Upon preincubation of the enzyme with 5, 10 and 20 μMSQDG for 5 minutes, CPT mediated cleavage was decreased in 5 and10 μM SQDG preincubated conditions and the cleavage wascompletely abolished in 20 μM SQDG preincubatedcondition (Supplementary Fig. S2a).On the other hand upon preincubation of the enzyme with 5, 10 and20 μM CPT for 5 minutes, addition of20 μM SQDG did not affect the CPT mediated cleavagecomplex formation (Supplementary Fig.S2b). Besides, upon simultaneous addition of SQDG and CPT, CPTmediated cleavage was not increased in 10 and 20 μM CPTconditions and was same as 5 μM CPT condition (Supplementary Fig. S2c). Theseresults suggest that both SQDG and CPT have similar affinities for the enzyme.Together the results of cleavage assays and electrophoretic mobility shift assay(Fig. 2 and Supplementary Fig. S2) suggest that SQDG inhibits topo I upstream inthe enzymatic reaction at the DNA binding step whereas CPT inhibits topo Idownstream in the enzymatic reaction at relegation step. Thus, uponpreincubation of the enzyme is with SQDG, CPT mediated cleavage is not observedand opposite to this, upon preincubation of the enzyme with CPT, SQDG can notaffect CPT mediated cleavage complex formation. However, upon simultaneousaddition of SQDG and CPT, CPT mediated cleavage is observed but the cleavagedoes not increase with increasing concentrations of CPT, suggesting that in thepresence of SQDG the enzyme becomes unable to bind to DNA and therefore CPT cannot stabilize covalent enzyme-DNA complexes anymore.

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