6,7-di-O-acetylsinococuline (FK-3000) induces G2/M phase arrest in breast carcinomas through p38 MAPK phosphorylation and CDC25B dephosphorylation

We evaluated the cytostatic effect of 6,7-di-O-acetylsinococuline (FK-3000) isolated from Stephania delavayi Diels. against breast carcinoma cell lines MDA-MB-231 and MCF-7. FK-3000 suppressed CDC25B phosphorylation directly and indirectly via p38 MAPK phosphorylation. CDC25B dephosphorylation decreased levels of cyclin B and phospho-CDC-2, and ultimately induced cell cycle arrest at the G2/M phase. The p38 MAPK inhibitor, SB 239063 blocked FK-3000-induced p38 MAPK phosphorylation and nuclear accumulation, but did not completely rescue cell death. Conclusively FK-3000 exerts its antiproliferative effect through two pathways: i) G2/M cell cycle arrest via downregulation of cyclin B and phospho-CDC2 by p38 MAPK phosphorylation and CDC25B dephosphorylation, and ii) p38 MAPK-independent induction of apoptosis.


Introduction
In 2014, the American Cancer Society reported that cancer was the second leading cause of death in the USA, estimating that >1,665,540 new cancer cases would be diagnosed that year and 585,720 cancer deaths would occur in the USA. To develop effective treatments, scientists have isolated anticancer agents from natural materials and identified a number of promising drug candidates (1).
In the search for potential therapies, we screened 509 natural products and found 14 compounds that demonstrate anticancer properties. One of these natural products was 6,7-di-O-acetylsinococuline (FK-3000), which was isolated from Stephania delavayi Diels. A literature search of the pharmaceutical properties of FK-3000 revealed a compound isolated from S. cepharantha that inhibits nuclear factor κB activity (2) and exhibits antiviral effects against herpes simplex virus type-1 (3), by inhibiting DNA synthesis (4), and against human immunodeficiency virus type-1 (5).
Mammalian cell division is controlled by cyclins and cyclin-dependent kinases (CDKs), which form various cyclin-CDK heterodimeric complexes (protein kinase holoenzymes) that regulate different phases of the cell cycle. Positive regulators of CDK function are upregulated in most cancer cells, whereas the expression of negative regulators are downregulated. Accordingly, cyclin D1, CDK4, cyclin E, cyclin A, and Wee1 are upregulated in the Long-Evans Cinnamon rat model of hepatocellular carcinoma (6); CDK4 plays a pivotal role in the progression from preneoplastic to neoplastic status in diethylnitrosamine-induced hepatocellular carcinoma in rats (7); increased expression of cell cycle regulatory proteins and kinase activities of cyclin D1, CDK4, cyclin E, cyclin A, and Wee1 was revealed by epidemiological studies of patients with liver disease (8); and inhibitors of cell division cycle 25 (CDC25) phosphatases have shown promise as anticancer agents (9). Targeting CDKs or cell cycle protein kinases is an important strategy in the discovery of novel anticancer drugs, and several preclinical and clinical trials are assessing these proteins as targets (10).
In humans, there are three homologues of CDC25: CDC25A, CDC25B, and CDC265C. In an earlier CDC25 regulation model, CDC25A controls the G 1 /S cell cycle transition, and CDC25B and CDC25C control mitosis (11) but in recent studies it was found that all three homologues have function to control both G 1 /S and G 2 /M phase transitions and mitosis (12).
In the present study, we investigated the antiproliferative mechanisms of FK-3000 by examining its effect on cell cycle regulatory proteins. Our results show that FK-3000 decreased levels of phosphorylated CDC25B (phospho-CDC25B) but neither CDC25A nor CDC25C, and induced G 2 /M phase arrest in human breast carcinoma cell lines MDA-MB231 and MCF-7.

FK-3000 isolated from S. delavayi Diels inhibits proliferation of human carcinoma cell-lines MDA-MB-231 and MCF-7.
We screened 509 natural products for anticancer activity and identified 14 candidates. The compound 6,7-di-O-acetylsinococuline (FK-3000) was isolated from S. delavayi Diels. (Fig. 1; molecular weight, 417.45), and its chemical structure was confirmed by 1 h, 13 C, and 2 D NMR. The chemical structure of FK-3000 isolated from S. delavayi Diels. was in good agreement with the compound previously isolated from S. cepharantha (22).

FK-3000 arrests MDA-MB-231 and MCF-7 cells at G 2 /M phase.
Carcinogenesis is caused by cell cycle deregulation, typically an increase in positive regulators such as CDKs and/or decrease in negative regulators such as cyclin D1, CDK4, cyclin E, cyclin A, and Wee1. Because the cell cycle is no longer controlled, cell proliferation is excessive (6). We therefore measured the effect of FK-3000 on cell cycle regulation in MDA-MB-231 and MCF-7 cells. Doses were based on the 48 h IC 50 of FK-3000 for each cell line, corresponding to 1X IC 50 to 10X IC 50 for each cell line (MDA-MB-231, 0.5-5.0 µg/ml; MCF-7, 0.7-7.0 µg/ml). As shown in Fig. 2A and B, FK-3000 treatment resulted in G 2 /M phase arrest in a time-and dose-dependent manner. In MDA-MB-231 cells treated with 1X IC 50 FK-3000 for 24 h, the percentage of G 2 /M phase arrested cells was 23.50%, increasing to 38.95% after 48-h treatment with 10X IC 50 FK-3000. In MCF-7 cells treated with 1X IC 50 FK-3000 for 24 h, the percentage of G 2 /M phase arrested cells was 28.93%, increasing to 40.13% after 48-h treatment with 10X IC 50 FK-3000.
FK-3000 induces dephosphorylation of CDC25 through p38 MAPK signaling. In cancer cells, levels of phosphorylated p38 MAPK proteins are low whereas phosphorylated CDC25B protein levels are high. CDC25B plays a key role in G 2 /M phase transition and CDC2 activation (23); phosphorylation of CDC25B is an important step leading to proliferation and metastasis of neoplastic cells. P38 MAPK induces G 2 /M arrest by inhibiting CDC25B phosphorylation  points. These results suggest that FK-3000 inhibits CDC25B through p38 MAPK activation.
We next determined the effect of FK-3000 on the G 2 /M phase regulatory factors and related proteins CDC-2, cyclin A, cyclin B, and RB. With the exception of cyclin B and phospho-CDC-2, we did not observe changes in these proteins (data not shown). Cyclin B levels were not altered by 24 h FK-3000 treatment in either MDA-MB-231 or MCF-7 cells, except in cells treated with 10X IC 50 FK-3000; however, this increase was attenuated at 48 h, and cyclin B was barely detectable after 48-h treatment with 10X IC 50 FK-3000 in both cell lines (Fig. 3B). FK-3000 decreased phosphorylation of CDC2 in a dose-and time-dependent manner, and 48-h treatment with 10X IC 50 FK-3000 in MDA-MB-231 cells and 5X IC 50 or 10X IC 50 FK-3000 in MCF-7 cell completely abolished phosphorylation of CDC2 (Fig. 3B).
p38 MAPK inhibition attenuated the antiproliferative action of FK-3000 but did not completely block FK-3000-induced apoptosis. CDC25B phosphorylation is regulated by p38 MAPK, which also blocks participation of the CDC2/cyclin B complex in G 2 /M transition (20,21). Phosphorylation of p38 MAPK plays a role in cell death, cell differentiation, and cell cycle progression. Following DNA damage, phospho-p38 MAPK  translocates from the cytoplasm into the nucleus (24), where accumulation of phospho-p38 MAPK triggers G 2 /M phase arrest and DNA repair.
We assumed that FK-3000 induced p38 MAPK phosphorylation and then suppressed CDC25B phosphorylation. Our results showed that a 90-min FK-3000 treatment stimulated p38 MAPK phosphorylation and nuclear translocation in MDA-MB-231 and MCF-7 cells (Fig. 4A and B), and this effect was suppressed by SB 239063, a potent and selective inhibitor of p38 MAPK (25). We compared phospho-p38 MAPK and phospho-CDC25B levels in FK-3000 treated cells with that of untreated cells at 90 min (Fig. 4C). Phosphorylation of CDC25B was abolished in cells treated with FK-3000 in the presence or absence of SB 239063. Together, these findings indicate that FK-3000 inhibits CDC25B phosphorylation directly as well as indirectly through p38 MAPK phosphorylation.
To evaluate the mechanism of cell cycle arrest by FK-3000, we analyzed the cell cycle distribution of treated cells. Although the distribution of cells treated with SB 239063 was similar to that of the vehicle control, SB 239063 could not completely reverse FK-3000-induced G 2 /M phase arrest (Fig. 4D).
To confirm that FK-3000 inhibited cell proliferation through p38 MAPK activation, we evaluated whether the p38 MAPK inhibitor SB 239063 could rescue the antiproliferative effect of FK-3000. Our results showed that SB 239063 attenuated but could not completely block the antiproliferative action of FK-3000. SB 239063 increased viability from 52.93 to 62.52% in FK-3000-treated MDA-MB-231 cells and increased viability from 50.59 to 60.63% in FK-3000-treated MCF-7 cells (Fig. 4E). As shown in Fig. 4E, the viability of cells treated with both SB 239063 and FK-3000 (77.69% in MDA-MB-231, 60.63% in MCF-7) did not fully recover to the level of control cells, suggesting that FK-3000 inhibits cell proliferation by an additional mechanism besides G 2 /M phase arrest through p38 MAPK phosphorylation and CDC25B dephosphorylation. We therefore analyzed the effect of SB 239063 on the rate of apoptosis in cells treated with FK-3000 (Fig. 4F). Apoptosis in cells treated with FK-3000 (SB 239063 + FK-3000 cotreatment or FK-3000 only) was significantly higher than that of cells treated with the vehicle control or SB 239063 only. Thus, FK-3000 appears to induce apoptosis by a pathway independent of the p38 MAPK-CDC25B pathway.
Taken together, these findings indicate that FK-3000 is a promising anticancer drug candidate that exerts its antiproliferative activity through two pathways: induction of G 2 /M phase arrest by p38 MAPK-CDC25B-CDC2-cyclin B modulation and stimulation of apoptosis independent of the p38 MAPK-CDC25B pathway.

Discussion
Cell cycle regulatory factors and related proteins (e.g., cyclin A, cyclin B, CDC2, CDC25A, CDC25B, CDC25C and p38 MAPK) are associated with G 2 /M transition; in particular, the CDC2-cyclin B heterodimeric complex regulates entry into mitosis (26). We found that FK-3000 induced G 2 /M phase arrest in the human breast carcinoma cell lines MDA-MB-231 and MCF-7 in a dose-and time-dependent manner. Further, phospho-CDC2 levels were significantly decreased after 24 h and cyclin B levels were decreased after 48 h, and phospho-p38 MAPK was upregulated, whereas phospho-CDC25B was downregulated in a dose-and time-dependent manner. Taken together, our findings suggest that FK-3000 induces G 2 /M arrest by inhibiting CDC2 activation via p38 MAPK phosphorylation and CDC25B dephosphorylation. To confirm these results, we evaluated the ability of the selective p38 MAPK inhibitor SB 239063 to block the antiproliferative action of FK-3000. SB 239063 increased viability from 52.93 to 62.52% in FK-3000-treated MDA-MB-231 cells and from 50.59 to 60.63% in FK-3000-treated MCF-7 cells. Moreover, SB 239063 inhibited FK-3000-induced p38 MAPK phosphorylation and nuclear accumulation (Fig. 4A-C).
however, SB 239063 did not completely rescue the effects of FK-3000, suggesting the involvement of another pathway in the antiproliferative action of FK-3000. Although SB 239063 suppressed FK-3000-induced p38 MAPK phosphorylation, it did not inhibit apoptosis (Fig. 4F). We therefore propose that FK-3000 exerts its cytostatic effect through p38 MAPK activation and its cytotoxic effect through apoptosis.
CDC25B has been proposed as a target for the development of anticancer agents (14,23). EK-6136 is a synthetic CDC25B inhibitor that inhibits cell proliferation in MCF-7  (17). FK-3000 suppresses activation of CDC25B but not CDC25C. Compared with the previously described CDC25 inhibitors, FK-3000 is a more potent inhibitor of proliferation in various cell lines and appears to be safe as assessed by animal studies at doses <10 mg/kg of body weight, administered intraperitoneally once a day for 5 days (data not shown).
We demonstrated that FK-3000 exerts an antiproliferative effect through two pathways: i) G 2 /M phase arrest via downregulation of cyclin B and phospho-CDC2 by dephosphorylation of CDC25B and phosphorylation of p38 MAPK; and ii) p38 MAPK-independent induction of apoptosis (Fig. 5). Although further studies are needed to evaluate FK-3000 in other cancer cell types and elucidate the antiproliferative mechanisms, therapeutic index, and margin of safety, our findings indicate that FK-3000 is a promising anticancer agent.