3EZ,20Ac-ingenol, a catalytic inhibitor of topoisomerases, downregulates p-Akt and induces DSBs and apoptosis of DT40 cells

Abstract

We have previously reported that many ingenol compounds derived from Euphorbia kansui exhibit topoisomerase (topo) II inhibitory activity. Of these compounds, 3EZ,20Ac-ingenol inhibited topo I activity. Camptothecin, which inhibits the religation activity of topo I without interfering with the binding of topo I to DNA and induces topo I-mediated DNA cleavage, was used as a positive control. In this study, we found that 3EZ,20Ac-ingenol did not hamper the binding of topo I to DNA in the same manner as camptothecin but affected the inhibition of cleavage of one DNA strand. 3EZ,20Ac-ingenol inhibited cell proliferation by blocking cell cycle progression in the G2/M phase. To define the mechanism of inhibition of DT40 cell proliferation, the change in Akt activity was observed because Akt activity is regulated in response to DNA damage. Western blot analysis revealed that 3EZ,20Ac-ingenol downregulated the expression of p-Akt, and apoptosis was detected by the presence of DNA double-strand breaks and caspase 3 activation.

Mechanism of the inhibition of leukemia cell growth and induction of apoptosis through the activation of ATR and PTEN by the topoisomerase inhibitor 3EZ, 20Ac-ingenol

“…The topoisomerase I catalytic inhibitors, betulinic acid and ␤-lapachone, have also been studied extensively as cancer drugs, and although several studies reported that they induce PI3K/Akt inactivation [32,33], their ensuing apoptotic mechanisms remain unclear. We also reported previously that the catalytic topoisomerase I inhibitor 3EZ, 20Ac-ingenol induced DNA damage leading to apoptosis via Akt inactivation in B cell line, DT 40 [24]. PTEN deficient cells have higher genomic instability suggesting that PTEN modifies chromatin structure and thereby influences the repair of DNA damage [34].…”

“…Although PTEN has no direct role in ATM/ATR activation, it may affect the functions of downstream repair proteins [3]. Because catalytic inhibitors of topoisomerases I and II inhibit the relaxation of helical super coiling during the replication process and induce DNA repair responses [17,19,24,35], PTEN may be induced by 3EZ, 20Ac-ingenol. Accordingly, PTEN was upregulated after 3 h of treatment with 3EZ, 20Ac-ingenol in BALL-1 cells, and inhibition of the PI3K/Akt signaling pathway was almost concomitant.…”

“…BALL-1, HEV0011, and MCF-7 cells were incubated as described previously [24,25]. The diterpene compound 3EZ, 20Ac-ingenol (3-O-(2 E,4 Z-decadienoyl)-20-O-acetylingenol) was dissolved in dimethyl sulfoxide.…”

“…Flow cytometric analysis was performed as described previously (24). BALL-1 cells were treated with 0.5 M 3EZ, 20Ac-ingenol for 0, 6, 12, 24, or 48 h. DNA content was determined by the flow cytometry (Beckman Coulter).…”

“…ICRF-193 has been extensively analyzed as a class II catalytic inhibitor of topoisomerase II, but several reports suggest that, although the extent of DNA damage induced by ICRF-193 and merbarone is limited, this compound induces H2AX phosphorylation by acting as a topoisomerase II poison [15,[20][21][22][23]. In addition, the topoisomerase I catalytic inhibitor (class II) 3EZ, 20Ac-ingenol isolated from Euphorbia kansui induces ␥H2AX and apoptosis by downregulating p-Akt [24]. In the present study, we investigated whether 3EZ, 20Ac-ingenol alters the chromosome structure and/or induces DNA damage leading to the PTEN and ATR pathway activation and p53 stabilization in leukemia BALL-1 cells.…”