Heterocyclic Organobismuth(III) Compound Targets Tubulin to Induce G2/M Arrest in HeLa Cells

Abstract

Abstract. Our previous study showed that organobismuth compounds induce apoptosis in human promyelocytic leukemia cells, although solid tumor cell lines were relatively resistant. Herein, we investigated the primary cellular target of these compounds in HeLa cells. One organobismuth compound, bi-chlorodibenzo[c,f][1,5]thiabismocine (compound 3), arrested the cell cycle at G 2 /M as assessed by flow cytometry and by upregulating the expression of cyclin B1. At a low concentration (0.5 μM), compound 3 caused cell cycle arrest at the mitotic phase and induced apoptosis. At a higher concentration (>1.0 μM), it induced an arrest in the G 2 /M phase, leading to apoptosis. In many cells blocked at the M phase, the organization of microtubules was affected, indicating depolymerization of the microtubule network. Western blotting demonstrated that compound 3 depolymerized microtubules similar to colchicine and nocodazole. Experiments in vitro also showed that compound 3 inhibited the assembly of purified tubulin in a concentrationdependent manner by interacting with the colchicine-binding site of tubulin through its SH groups. Heterocyclic organobismuth compounds are novel tubulin ligands.

Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

“…Most antimicrotubule agents that target the cellular microtubule network result in an aberrant formation of the mitotic spindle, subsequent blockage of the cell cycle in G 2 /M phase and causing apoptotic cell death [20,21]. Since α-santalol caused G 2 /M phase arrest and apoptosis, we examined whether it affects the organization of microtubules in UACC-62 cells, which are relatively big cells and their microtubules are easily visualized compared to A431 cells.…”

New 2-Aryl-1,4-naphthoquinone-1-oxime Methyl Ether Compound Induces Microtubule Depolymerization and Subsequent Apoptosis

“…Microtubule polymerization dynamics was monitored by measuring the change in absorbance at 340 nm every 1 or 5 min for 60 min at 37°C using a SmartSpec TM 3000 (Bio-Rad Laboratories, Hercules, CA, USA). As positive controls of the microtubule sensitizer, TAX and VNR were prepared, of which concentrations were set according to previous reports (31,32).…”

A novel organobismuth compound, 1-[(2-di-p-tolylbismuthanophenyl)diazenyl]pyrrolidine, induces apoptosis in the human acute promyelocytic leukemia cell line NB4 via reactive oxygen species

“…However, our preliminary experiments with heterocyclic organobismuth(III) using an in vitro assay system with GSH as substrate did not show any evidence of the direct inhibition of glutathione peroxidase activity. In addition, we have revealed that one specific biological target of heterocyclic organobismuth(III) is tubulin [24]. However, as shown in Fig.…”

“…The assembly of porcine tubulin was monitored using porcine brain tubulin purified as described earlier [24]. Tubulin was resuspended on ice in ice-cold tubulin-polymerization buffer (80 mM piperazine-1, 4-bis (2-ethane sulfonic acid (PIPES) pH 6.9, 2 mM MgSO 4 , 1 mM ethylene glycol tetra acetic acid (EGTA), 1 mM Mg-GTP, and 1.5% (v/v) glycerol) and 100 μl (250 μg) was pipetted into the designated wells of a half area 96-well plate pre-warmed to 25°C in 0.20 mM of each compound.…”

“…We have previously shown that heterocyclic organobismuth(III) targets tubulin resulting in depolymerization in vitro and destroys the microtubular network of HeLa cells [24]. Microtubules function in a number of cellular processes and numerous anti-tumor drugs have been known to target microtubules [40].…”