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A Natural Anticancer Agent Thaspine Targets Human Topoisomerase IB
Supervisor By…Prof. Alessandro Desideri Prafulla Katkar
Topoisomerases are the key enzymes that control the topological state of DNA through the breaking and rejoining of DNA strands.
Two kinds of Topoisomerases : Type I (IA, IB) and Type II
These enzymes are involved in many vital process such as DNA replication, transcription, translation and chromosomal segregation etc.
Most anticancer drugs have their topoisomerase as their molecular target.
Cell Death (Apoptosis)
1. Topo poisons : Stabilizes Topo-DNA cleavable complex and inhibit religation.(Camptothecin and its derivatives topothecan, Gematecan approved in clinical trials)
2. Topo Inhibitors : Inhibit cleavage of DNA
Topoisomerase IB Topo poison
An alkaloid present in the cortex of south American tree Croton lechleriBeing used as traditional medicine for several diseases.
Thaspine been known to exhibit promising anticancer properties. (Stig Linder et al 2009)
1. Has strong cytotoxic effect on multicellular spheroids and induce apoptosis in human xenograft in vivo.
2. by intercalation of the planar complexes between the DNA base pairs.
3. and/or by inhibition of enzyme activities.
Dragon’s Blood (Sangre di grado)
1. Investigation of Mechanism of effect of Thaspine on Topoisomerase IB whether it is Topo poison or inhibitor.
2. Analyzing its effect on different steps of enzyme catalysis.
Effect of Thaspine on topoisomerase IB relaxation :
Thaspine inhibits the human topoisomerase IBin a dose dependent manner
1 2 3 4 5 6 7
C 0 5 10 15 20 50 µM
Thaspine
NC
Relaxed
SC
DMSO Thaspine Thaspine+E
Pre-incubation
NC
1 2 3 4 5 6 7 8 9 10 11 12 13
C 1 7.5 15 30 1 7.5 15 30 1 7.5 15 30 Time (Min)
SC
Relaxed
Pre-incubation of drug increase the inhibitory effect for short time
Then decreases gradually
1 2 3 4 5 6 7 8 9
C 1:1 1:2 1:4 1:8 1:1 1:2 1:4 1:8DMSO Thaspine+E
Pre-incubation
NC
Relaxed
SC
The inhibitory effect of thaspine is reversible, as the effect is lost as the
dilution increases
1 2 3 4 5 6 7 8 9 10 11 12 13
C 5 15 30 5 15 30 5 15 30 5 15 30
DMSO Thaspine CPT Taspine+E
Pre-incubation
Either thaspine inhibits the cleavage or induces a faster religation,
so that the cleavable complex cannot be formed.
Religated oligo
Cleavable Complex
1 2 3 4 5 6 7 8 9 10 11 1213 14 15
C 0 0.5 1 5 10 20 30 0 0.5 1 5 10 20 30
DMSO Thaspine
----- Tasp
DMSO
The religation rate is slowed down in presence of thaspine as compared with DMSO
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
C 0.5 1 5 10 20 60 0.5 1 5 10 20 60 0.5 1 5 10 20 60
DMSO Thaspine Thaspine+E
Pre-incubation
The ability of topoisomerase to cut the DNA is reduced in presence
of Thaspine and the effect is more intense when the thaspine pre-incubated
with enzyme.
Thaspine binds to enzyme with good interaction energy, before its interaction with DNA
A. The intercalation of thaspine between the bases of DNA, evidence for inhibition of religation.
B. Binding of thaspine to the active site of topoisomerase IB
1. The Plasmid relaxation assay demonstrate that thaspine is topoisomerase IB inhibitor.
2. The Inhibition is reversible.
3. Thaspine inhibits the cleavage of DNA, this Inhibition is due to the inability of enzyme to binds with DNA.
4. The inhibition mechanism of religation is same as that of Camptothecin.
5. Thus , finally thaspine act as both inhibitor and poison.
Prof. A.DesideriProf. Mattia FalconiPaola FioraniSilvia CastelliOscar VassaloCinzia TeausaroBarbara ArnoLaura ZuccaroZeenat JahanSara Viera
Aalborg University, Denmark : Erasmus Mundus Coordinator&European Commission .