Synergic Action of Oxoglaucine from the Tumor and Mitosis

Oxoglaucine arrested tumor cells at G2/M phase. We observed gradual accumulation of rounded cells, which resemble the appearance of mitotic cells, after addition of oxoglaucine. We therefore analyzed the effects of oxoglaucine on cell cycle progression. Oxoglaucine induced a dose-dependent G2/M arrest after 24 hours of drug exposure. 68% of the cell population was blocked in G2/M phase when they were exposed to 30µM oxoglaucine for 24 hours (Fig. 2A). To understand the mechanism of oxoglaucine in inducing cell cycle arrest, we analyzed the effects of oxoglaucine on the microtubule polymerization and the mitotic spindle formation during mitosis. Our data showed that oxoglaucine disrupted the mitotic spindle organization, but did not inhibit polymerization of microtubule (Fig. 2B). Normal mitotic cells in metaphase displayed a bipolar spindle.

Condensed chromosomes lined up between the spindle poles to form the metaphase plate. Cells exposed to oxoglaucine for 16 hours displayed disrupted appearance of mitotic spindles (Fig. 3). These data suggested that oxoglaucine is a mitosis-targeted agent. Oxoglaucine and anti-microtubule agents acted synergistically to induce G2/M arrest and tumor cells death The fact that oxoglaucine inhibited mitosis, but did not affected microtubule polymerization suggested that oxoglaucine did not act the same as anti-microtubule agents such as vincristine and taxol. We then analyzed the effects of oxoglaucine in combination with anti-cancer agents on tumor cell growth and death. We found that combination of oxoglaucine and vincristine or taxol increased the percentage of G2/M arrested cells (Fig. 4) and decreased the relative tumor cells viability (Tab. 1). The Combination index (CI) value indicated that the combination of oxoglaucine and anti-microtubule agents acted synergistically to inhibit tumor cells growth. But there were no synergistical effects of oxoglaucine with other non-mitosis inhibitor anti-cancer drugs such as cisplatin, or doxorubicin (data not shown).

Oxoglaucine and vincristine acted synergistically to inhibited tumor growth in vivo Following the in vitro validation of oxoglaucine as an effective agent for blocking cell growth, we evaluated the anti-tumor effects of oxoglaucine in vivo in combination with vincristine in a HGC-27 bearing mice model. Drug treatment began 7 days after the HGC-27 tumor cells inoculation and continued for 14 days. All animals were sacrificed 24h after the last injection of agents and all tumors were excised and weighed. Animals treated with oxoglaucine had significantly reduced tumor volume and tumor weight in a dose of 50mg/kg compared to control animals (Fig. 5). The inhibition rate of oxoglaucine at the dose of 50mg/kg was 37.27%. Oxoglaucine (50mg/kg) in combination with vincristine (0.5mg/kg) showed a synergistic effect. Tumor volume and tumor weight was significantly lower in animals treated with oxoglaucine and vincristine than those treated with vincritine alone. The inhibition rate had significant difference in the combination treatment group and vincristine treatment group (79.42% vs 50.09%, p<0.001). These data indicated that oxoglaucine alone or in synergy with vincristine inhibited tumor growth in vivo. Discussion Oxoglaucine, an aporphine alkaloid, has been identified and isolated from several plants, including Xylopia aethiopica (Annonaceae), Glaucium flavum Crantz (Papaveraceae) and Liriodendron tulipifera L. (Magnoliaceae)[20,22,25].

In present study, oxoglaucine was isolated from YHS and was found to have anti-tumor activity. It has been reported that the ethanol extract of YHS inhibited metastasis of breast cancer cells and induced MCF-7 cell cycle arrest at G2/M phase [7,8]. We presented data to show that oxoglaucine inhibited proliferation and induced apoptosis of different types of tumor cells. It blocked tumor cells at mitosis and induced apoptotic cell death. The exact mechanism by which oxoglaucine blocked cancer cells mitosis is not completely understood. The in vitro analysis of the effect of oxoglaucine on microtubule polymerization suggests that oxoglaucine may not directly interact with tubulin and is different from anti-microtubule agents, such as taxol and vincristine. Visualization of mitotic spindle with fluorescent microscopy indicated that oxoglaucine disrupted mitotic spindle formation in cancer cells. During mitosis, many microtubule associated proteins (MAPs), such as Stathmin and Kinesins, control microtubule dynamics to achieve proper spindle assembly and chromosome segregation[26-28]. Microtubule regulatory proteins have become potential biomarkers and molecular targets for cancer therapy. Oxoglaucine may influence the expression or activity of these proteins and disrupt the spindle formation. Both anti-microtubule agents (taxol and vincristine) and oxoglaucine arrest cancer cells at G2/M phase. The target protein of oxoglaucine appeared to be not the same as anti-microtubule agents. Our results indicated that vincristine (or taxol) and oxoglaucine acted synergistically to induce G2/M arrest and inhibit tumor cells growth in vitro and in vivo. These data suggest that the combination of two anti-mitosis agents with different targets could be a useful strategy for cancer therapy.

Conclusion

The present study showed the anti-cancer effect of oxoglaucine, its mechanism of action, and the synergistic action with anti-microtubule agents. The data indicated oxoglaucine acted synergistically to inhibit the growth of tumor cells by inducing G2/M arrest. It suggests the potential therapeutic uses of oxoglaucine. Taken together, our work demonstrates that oxoglaucine is a new

type of mitosis inhibitor that sensitizes with anti-microtubule agents to inhibit cancer cells growth.