Dual targeting of microtubule and topoisomerase II by α-carboline derivative YCH337 for tumor proliferation and growth inhibition

Abstract

Both microtubule and topoisomerase II (Top2) are important anticancer targets and their respective inhibitors are widely used in combination for cancer therapy. However, some combinations could be mutually antagonistic and drug resistance further limits their therapeutic efficacy. Here we report YCH337, a novel α-carboline derivative that targets both microtubule and Top2, eliciting tumor proliferation and growth inhibition and overcoming drug resistance. YCH337 inhibited microtubule polymerization by binding to the colchicine site and subsequently led to mitotic arrest. It also suppressed Top2 and caused DNA double-strand breaks. It disrupted microtubule more potently than Top2. YCH337 induced reversible mitotic arrest at low concentrations but persistent DNA damage. YCH337 caused intrinsic and extrinsic apoptosis and decreased MCL-1, cIAP1 and XIAP proteins. In this aspect, YCH337 behaved differently from the combination of vincristine and etoposide. YCH337 inhibited proliferation of tumor cells with an averaged IC50 of 0.3 μM. It significantly suppressed the growth of HT-29 xenografts in nude mice too. Importantly, YCH337 nearly equally killed different-mechanism-mediated resistant tumor cells and corresponding parent cells. Together with the novelty of its chemical structure, YCH337 could serve as a promising lead for drug development and a prototype for a dual microtubule/Top2 targeting strategy for cancer therapy.

Keywords: YCH337; antitumor activity; microtubule; topoisomerase II; α-carboline derivative.

Figure 1. The chemical structure and synthesis of YCH337

Reagents and conditions used in the preparation of YCH337: (a) NaOAc, AcOAc, 90°C, 85%; (b) Fe, AcOH, reflux 2 h, 55%.

Figure 2. YCH337 inhibits microtubule polymerization by binding to the colchicine site and disrupts spindle assembly

(A) HeLa cells were treated with YCH337, vincristine or paclitaxel at 0.5 μM for 1 h and then interphase microtubule was imaged by immunofluorescence microscopy. Scale bar: 10 μm. (B) YCH337 inhibited in vitro polymerization of microtubulin in a cell-free system. Colchicine and paclitaxel were used as references. (C) SK-OV-3 cells were treated with different agents at 0.2 μM for 24 h. Then, the polymerized fraction and the free fraction of tubulin were separated by ultracentrifugation and processed for Western blotting. (D) the binding site of YCH337 on tubulin was determined by the competitive binding assay. Vincristine and CA4 were used as references. (E) HeLa cells were treated with 1 μM YCH337 for 1 h, and then the mitotic spindle assembly was shown by immunofluorescence microscopy. Scale bar: 10 μm.

Figure 3. YCH337 inhibits Top2, which is weaker than it suppresses microtubule in cells

(A–B) YCH337 inhibited DNA decatenation catalyzed by Top2 (A) rather than by Top1 (B). The electrophoresis assay was described in Materials and methods. Each reaction contained the same amount of Top2 (A) or Top1 (B) and DMSO except the control (pBR322 DNA). The Top2 inhibitor etoposide (VP-16) and the Top1 inhibitor SN38 were used as positive controls. RLX: the relaxed form of pBR322 DNA. SC: the supercoiled form of pBR322 DNA. (C–D) HeLa cells were treated with YCH337 at indicated concentrations for 48 h (C) or at 1 μM for the indicated time (D). Then the cells were lyzed and immunoblotted for γH2AX. (E) HeLa cells were treated with YCH337. Tubulin and γH2AX foci were then imaged by the immunofluorescence-based laser confocal microscopy. The magnified images of microtubule in the cells pointed to by the arrows (a, b and c) were presented at the bottom of the figure. Scale bar: 10 μm.

Figure 4. YCH337 induces reversible mitotic arrest but irreversible DNA damage

(A–C) HeLa cells were treated with 1 μM YCH337 for the indicated time. Cell cycle was analyzed by flow cytometry (A). The statistical data of three independent experiments were shown in (B). The cells were treated with 1 μM YCH337 for the indicated time, and then the cells were lyzed and immunoblotted for mitotic molecular markers MPM2 and p-H3 (Ser10-phosphorylated histone 3) (C). (D) HeLa cells were treated with YCH337 at 0.5 μM for the indicated time or at indicated concentrations for 48 h. Then, the cells were lyzed and immunoblotted for CyclinB1 and phosphorylated CDK1 at Thr14/Tyr15 (p-T14/Y15-CDK1). (E–F) HeLa cells were treated with 0.2 μM YCH337 for 12 h. Then the medium was changed to remove YCH337 and the cells continued to be cultured in the drug-free medium for indicated time for flow cytometry analysis (E). The statistical data of three independent experiments were shown in (F). (G–H) HeLa cells were treated as in (E) and collected for Western blotting (G) or flow cytometry analysis (H).

Figure 5. YCH337 induces apoptosis, different from that elicited by the combination of the tubulin inhibitor VCR and the Top2 inhibitor VP-16

(A–B) loss of mitochondrial membrane potential (MMP) in HeLa cells treated with 0.5 μM YCH337 for the indicated time was analyzed by flow cytometry. Representative images were presented in (A) and the data from three independent experiments were expressed as mean ± SD in (B). (C–F) HeLa cells were treated with different agents at the indicated concentrations for 48 h or with 0.5 μM YCH337 for the indicated time. VCR and VP-16 were used at 0.2 μM and 10 μM, respectively. Then cells were lyzed and immunoblotted for PARP and caspases (C) or anti-apoptotic proteins (D). Apoptosis was analyzed by the Annexin V-PI co-staining-based flow cytometry. Representative histograms were presented (E). The data from three independent experiments were expressed as mean ± SD (F).

Figure 6. YCH337 inhibits proliferation and growth of human tumor cells in vitro and in vivo

(A) YCH337 exerted potent antiproliferative activity on a panel of cancer cell lines. SRB assays (for solid tumor cells) or luminescent cell viability assays (for leukemia cells) were used to determine the 72-h IC₅₀ values. Data from three independent experiments were expressed as mean ± SD. (B) YCH337 affected the growth of human colon cancer HT-29 xenografts in nude mice (upper) and the body weight of the tested animals (lower). Vehicle, 0.5% carboxymethylcellulose sodium; MMC, mitomycin C; RTV, relative tumor volume. There is statistically significant difference of RTV between the vehicle and YCH337 treatment groups beginning on day 7, P < 0.005. (C) a schematic presentation of possible mechanisms of action of YCH337.