BC remains a recalcitrant cancer in women worldwide, thus novel therapeutic strategies of TCM with low side effects are urgently needed. Lycorine, the active alkaloid isolated from Lycoris radiata (family Amaryllidaceae) has a long history of application in TCM [14, 15], and has been reported to possess extensive pharmacological activities, such as anti-inflammatory, antiviral, and antitumor effects [16]. Previous studies reported that lycorine not only has inhibitory effects on tumor growth but also has extensive activity that induces apoptosis [14]. As mentioned earlier, lycorine suppressed constitutive STAT3 activation by up-regulating the expression of SHP-1 and down-regulating the expression of STAT3 target proteins, thus induced apoptosis and cell cycle arrest. However, more research is needed to fully understand potential therapeutic effect of lycorine as a drug of BC treatment and its safety profile. In the present study, we found that lycorine had inhibiting effect on BC both in vivo and in vitro for the first time.
Immunosuppression appears to be a major side effect of chemotherapy drugs, while it is not disturbing for lycorine. Instead, the lycorine suppressed tumor growth based on their altered immunostimulatory activities. Thymus and spleen are the important immune organs, which orchestrate adaptive immune responses [17]. CTX was the most effective drug evaluated against 33 kinds of cancers out of 1,000 anti-tumors compounds and medicines [18], however, it accompanied by high toxicity and severe side effects. Thus, it was worth noting that lycorine suppressed tumor growth in addition to lessening the frequency of the aforementioned adverse effects.
By contrast, stomach cancer, lung cancer and liver cancer and BC are the top four cancers with high morbidity and mortality [19–21]. Moreover, the inhibitory effects of lycorine on the growth of MCF-7, MDA-MB-231, SGC-7901, A549 and HepG-2 cells were demonstrated by MTT assay in vitro. Studied to identify the active alkaloids with anti-proliferative activities and the corresponding sensitive cell lines, lycorine had obvious inhibitory effects on MCF-7 cell proliferation in a dose-dependent manner, and the IC50 values of lycorine for MCF-7 cells was 13.98 µmol/L. Last but not least, the MCF-7 cells were considered sensitive to lycorine.
Cell cycle arrest is a process by which the normal progression of the cell cycle is disrupted. This can occur in response to various signals, such as DNA damage, replication stress, and oncogene activation [22]. It has been proposed as a therapeutic strategy for the treatment of cancer [23]. However, the mechanism that lycorine induces cell cycle arrest is still unclear. Firstly, the results demonstrated lycorine induced MCF-7 cell cycle arrest in G2/M phase arrest by flow cytometer. Furthermore, a sub of 101 genes in cell cycle were detected, among them, 20 target genes and 15 cell cycle arrest related proteins with high expression changes were discussed. More importantly, CDK1 and CyclinB1 are critical in G2 phase checkpoint. CyclinB1 progressively increases through G1 and S phase and reaches its peak in G2 phase then form complex with CDK1. It has been documented that CDK1 is activated on centrosomes, CyclinB1-CDK1 binds to the microtubules, chromosomes and to unattached kinetochores in the prometaphase [24]. Recent literature reports that evodiamine induced M-phase cell cycle arrest via the p21 signaling pathway in ESCC cells [25]. In our study, the expression of protein p21 was up-regulated by lycorine treatment. The expression of protein CDK1 was down-regulated by p21, which caused the down regulation CyclinB1 further inhibited the activity of CDK1 in MCF-7 cells. The Phosphorylation of Histone H3 (p-H3) is a critical step in the regulation of the cell cycle, playing a key role in the regulation of DNA replication and chromosome segregation [26]. During the cell cycle, p-H3 modification occurs in the early stages of mitosis and marks the chromatin for proper condensation and separation. CyclinB1 and Histone H3 phosphorylation are the markers of cells in M phase. Our experiment revealed that lycorine treatment significantly enhanced the mitotic phase marker protein p-H3 expression, indicating that lycorine triggered the M-phase cell-cycle arrest in MCF-7 cells, instead of the G2 phase. These findings suggested that lycorine induced M-phase arrest may be mediated by p21.
The phosphorylation of p-H3 is catalyzed by mitotic kinases, such as Aurora A and Aurora B, which recognize and bind to the p-H3 modified histones. Any disruptions in p-H3 phosphorylation levels may lead to a variety of chromosomal abnormalities and contribute to the development of diseases such as cancer [27]. Herein, the down-regulated expressions of protein Aurora A and Aurora B induced by lycorine in this study revealed in this study. Likewise, PLK1 was the key checkpoint to recovery from mitotic arrest mediated by the DNA damage after DNA damage repair [28]. BUBR1 is a very important protein in mitotic checkpoint, which plays a role in maintaining checkpoint activation in chromosome kinetochore and cytoplasm, and its expression peaks at G2/M phase with the progress of cell cycle. Aurora-B is involved in the enrichment and phosphorylation of BUBR1. When the lack of appropriate tension is detected at the mitotic spindle detection point, it can effectively prevent the continuation of mitosis. Meanwhile, BUBR1 can be phosphorylated by PLKl. In the present study, we demonstrated that lycorine-induced down-regulated expression in PLK1 and up-regulated expression in BUBR1. Taken together, induction of mitotic arrest in MCF-7 cells was occurred.
Apart from cell-cycle arrest, triggering microtubules dynamics is another appealing option for cancer treatment. The relationship between microtubules and cancer is complex and multifaceted. In cancer cells, alterations in microtubule dynamics can result in changes to cell division and lead to the formation of abnormal, uncontrolled growths [29]. The microtubule targeting agents (MTAs) used in cancer chemotherapy work by disrupting the normal structure and stability of microtubules, leading to cell cycle arrest and apoptosis. Here, we investigated for the first time whether lycorine hampers the mitotic effect through the inhibition of microtubule dynamics in MCF-7 cells. Oncoprotein stathmin 1 (STMN1), also known as oncoprotein 18, is involved in the regulation of the microtubule 561 dynamics system. Accumulating evidence shows that STMN1is highly expressed in multiple 562 types of human cancer, which functions as a tumor promoter contributing to malignant phenotypes [30]. Mitotic centromere-associated kinesin (MCAK/KIF2C) is a member of the kinesin-13 family, which is important for the regulation of microtubule dynamics. Moreover, disrupting the dynamic balance of microtubule depolymerization and polymerization is a potential target for the development and research of antitumor drugs [13]. In a nutshell, the results in this study proved that lycorine down-regulated expression of pro-microtubule polymerization related genes (MAP2, MAPT), and up-regulated the expression of microtubule depolymerization-related genes (STMN1, KIF2C, KATNA1) in MCF-7 cells.