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Huachansu is a traditional Chinese medicine widely used in the clinic for cancer therapy, while the underlying mechanism is not fully clarified. This study was to investigate the targets and mechanisms of cinobufagin (CBG), an active component of Huachansu, in terms of blocking mitosis of cancer cells. Propidium iodide (PI) DNA staining was used to analyze the effect of CBG on cell cycle. The effect of CBG on mitosis of cancer cells was examined by α-tubulin and pericentrin staining after synchronization by a double thymidine block. Tubulin turbidity, tubulin polymerization and α-tubulin immunofluorescence assays were used to evaluate the effect of CBG on microtubule polymerization. CRISPR/Cas9 gene-editing technology was used to knockout microtubule-severing protein Katanin regulatory subunit B1 (KATNB1) in HCT116 cells, and the inhibitory effect of CBG on wild-type cells and knockout cells was measured by CCK-8. The engagement of CBG with KATNB1 was measured by CETSA and DARTS assays. The effect of CBG on KATNB1 protein and mRNA level was examined by Western blot and real-time PCR, respectively. Our data showed that CBG arrested HCT116 cell cycle at the G2/M phase, disrupted mitosis and induced centriole overduplication. CBG significantly inhibited tubulin polymerization in vitro and in vivo. The cytotoxicity of CBG inhibition on HCT116 was significantly attenuated upon KATNB1 depletion. Moreover, CBG bound to KATNB1 and decreased its protein level, while mutated KATNB1 weakened this effect. In conclusion, CBG inhibited microtubule polymerization via targeting KATNB1, thereby disrupting mitosis in cancer cells.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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