A specific dispiropiperazine derivative that arrests cell cycle, induces apoptosis, necrosis and DNA damage

Abstract

Dispiropiperazine compounds are a class of molecules known to confer biological activity, but those that have been studied as cell cycle regulators are few in number. Here, we report the characterization and synthesis of two dispiropiperazine derivatives: the previously synthesized spiro[2',3]-bis(acenaphthene-1'-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (SPOPP-3, 1), and its previously undescribed isomer, spiro[2',5']-bis(acenaphthene-1'-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (SPOPP-5, 2). SPOPP-3 (1), but not SPOPP-5 (2), was shown to have anti-proliferative activity against a panel of 18 human cancer cell lines with IC₅₀ values ranging from 0.63 to 13 µM. Flow cytometry analysis revealed that SPOPP-3 (1) was able to arrest cell cycle at the G2/M phase in SW480 human cancer cells. Western blot analysis further confirmed the cell cycle arrest is in the M phase. In addition, SPOPP-3 (1) was shown to induce apoptosis, necrosis, and DNA damage as well as disrupt mitotic spindle positioning in SW480 cells. These results warrant further investigation of SPOPP-3 (1) as a novel anti-cancer agent, particularly for its potential ability to sensitize cancer cells for radiation-induced cell death, enhance cancer immunotherapy, overcome apoptosis-related drug resistance and for possible use in synthetic lethality cancer treatments.

Figure 1

Synthesis of dispiropiperazine derivatives SPOPP-3 (1) and SPOPP-5 (2).

Figure 2

SPOPP-3 (1) inhibited human colon cancer cell viability. Cell viability in SW480, HT29 and HCT116 human colon cancer cells was assessed using the MTT assay. Cells were treated with different concentrations of SPOPP-3 (1) or SPOPP-5 (2) for 48 h. Results shown are representative from three separate experiments. Error bars are SEM.

Figure 3

SPOPP-3 (1) arrested cell cycle at G2/M phase in SW480 cells. (a) SW480 cells were treated with 2% DMSO, 20 µM SPOPP-3 (1), or 20 µM SPOPP-5 (2) for 24 h after which cells were harvested and subjected to cell cycle analysis using flow cytometry. (b) The results from (a) and two another additional biological replicates (n = 3) were combined and expressed as shown. The cell cycle percentages were calculated based on the Watson Pragmatic model. Two-way ANOVA was performed: *p < 0.0001.

Figure 4

SPOPP-3 (1) induced phospho-histone H3 in SW480 cells. (a) Immunoblots showing phospho-histone H3 and GAPDH expression upon treatment with 2% DMSO, 20 µM SPOPP-3 (1) or 20 µM SPOPP-5 (2) for 24 h. (b) The results from (A) and another two biological replicate (n = 3) were averaged and expressed as shown. t-test was used: *p < 0.05.

Figure 5

Defective cyclin B1 induction in SPOPP-3 treated cells as shown by immunofluorescence experiments. SW480 cells were treated with 40 µM SPOPP-3 or 2% DMSO for 24 h before fixation and immunostaining with cyclin B1 antibody and DAPI. (a) Representative image of cells treated with SPOPP-3 (lower images) showing a large population of tetraploid cells (indicated by arrows) without cyclin B1 staining. In contrast, DMSO-treated cells have a relatively small population of tetraploid cells all of which expressed cyclin B1. (b) Tetraploid cells (with doubled DAPI signal) (left) and the percentage of tetraploid cells positive for cyclin B1 (right) were quantified in each group and presented as average ± SD. One-way ANOVA was used: *p < 0.05. Scale bar 20 mm.

Figure 6

SPOPP-3 (1) induced apoptosis and necrosis in SW480 cells. (a) SW480 cells were treated with 2% DMSO, 20 µM SPOPP-3 (1), or 20 µM SPOPP-5 (2) for 24 h, after which cell lysates were isolated and subjected to cell death analysis using flow cytometry. (b) The results from (a) and two other additional biological replicates (n = 3) were combined and expressed as shown. Two-way ANOVA was used: *p < 0.005, **p < 0.0001.

Figure 7

SPOPP-3 (1) does not cause microtubule disruption. SW480 cells were treated with 50 nM vinblastine, 1 µM colchicine or 40 µM SPOPP-3 (1) for 24 h before fixation and immunostaining with α-tubulin antibody and DAPI. Mitotic spindles were clearly observed in SPOPP-3 (1) treated cells while colchicine and vinblastine induced microtubule disruption via different mechanisms. Diffuse α-tubulin staining in the cytoplasm and para-crystal formation (arrows) can be observed with colchicine and vinblastine treatment respectively. Scale bar 5 mm.

Figure 8

SPOPP-3 (1) induced DNA damage. DNA lesion quantification using LORD-Q in SW480 cells treated with 40 µM SPOPP-3 (1) for the indicated time periods. Short and long amplicons amplified from the mtDNA gene was used in the quantification of lesions in mitochondrial DNA. Data presented are averaged from three biological replicates ± SEM (n = 3).