Geraniin Differentially Modulates Chromosome Stability of Colon Cancer and Noncancerous Cells by Oppositely Regulating Their Spindle Assembly Checkpoint
Abstract
Geraniin has been reported to specifically induce apoptosis in multiple human cancers, but the underlying mechanism is poorly defined. The spindle assembly checkpoint (SAC) is a surveillance system to ensure high-fidelity chromosome segregation during mitosis. Weakening of SAC to enhance chromosome instability (CIN) can be therapeutic because very high levels of CIN are lethal. In this study, we have investigated the effects of geraniin on the SAC of colorectal cancer HCT116 cells and noncancerous colon epithelial CCD841 cells. We find that treatment of HCT116 cells with geraniin leads to dose-dependent decrease of cell proliferation, colony formation and anchorage-independent growth. Geraniin is found to induce apoptosis in mitotic and post-mitotic HCT116 cells. Furthermore, geraniin weakens the SAC function of HCT116 cells by decreasing the transcriptional expression of several SAC kinases (particularly Mad2 and Bub1), which in turn leads to premature anaphase entry, mitotic aberrations and CIN in HCT116 cells. In contrast, the proliferation of CCD841 cells is slightly inhibited by geraniin. Even more interestingly, geraniin increases the transcriptional expression of several SAC kinases (e.g., Mad1 and BubR1) to strengthen SAC efficiency, which contributes to the reduction of mitotic aberrations and CIN in CCD841 cells. Altogether, our findings reveal that the SAC pathway in human colon cancer and noncancerous cell lineages responses oppositely to geraniin treatment, resulting CIN promotion and suppression, respectively. Specific abrogation of SAC to induce catastrophic CIN in HCT116 cells may account for the selective anticancer action of geraniin.
1. Introduction
Mitosis is considered to be the most fragile period of the cell-cycle, during which it is highly susceptible to mitotic infidelity—the inability to faithfully segregate equal chromosome complements to two daughter cells during mitosis—when exposed to various insults [Schvartzman et al., 2010; Chan et al., 2012]. The spindle assembly checkpoint (SAC, also known as mitotic checkpoint) safeguards against mitotic infidelity by ensuring each and every chromosome is stably and correctly attached to microtubules before anaphase is allowed to proceed. The SAC pathway comprises several checkpoint kinases including Mad1, Mad2, Bub1, BubR1 and Mps1 that are specifically recruited to kinetochores either not attached to microtubules or lacking a bi-orientation. These proteins cooperatively form the mitotic checkpoint complex (MCC) to inhibit the mitotic ubiquitin ligase termed anaphase-promoting complex/cyclosome (APC/C) by sequestering its co-activator Cdc20 until all kinetochore–microtubule attachments are stabilized and all chromosomes have achieved bipolar orientation at the spindle equator [Musacchio, 2015].Many human tumors are sensitive to mitotic stress and this sensitivity is being exploited for therapy. Antimitotic drugs are frontline treatments for breast, ovarian and lung cancer, as well as various hematological malignancies [Dominguez-Brauer et al., 2015]. The current antimitotic cancer drugs such as vinca alkaloids and taxanes interfere with microtubule dynamics and therefore prevent the mitotic spindle from attaching amphitelically to kinetochores.
This activates the SAC and induces mitotic aberrations, blocking the cells from progressing through mitosis.Following prolonged arrest, cells either undergo mitotic stress-induced death or undergo―adaptation‖, allowing cells to progress through mitosis aberrantly and produce daughter cells harboring massive chromosomal instability (CIN) and resulting in aneuploidy or polyploidy [Gascoigne and Taylor, 2008]. The second-generation of antitumor compounds, often referred to as mitotic blockers, target microtubule motor proteins or various mitotic kinases that are highly expressed in cancer [Haschka et al., 2018]. This results in SAC abrogation and subsequent mitotic aberration and CIN in cancer cells [Haschka et al., 2018]. Amounting evidence from human, mice, and cell lines points towards a model whereby cancer cells can only tolerate a permissible range of CIN and that elevating CIN beyond a tolerable range is deleterious to tumor cells [Funk et al., 2016]. Thereafter, post-mitotic stress repsonses induce cell cycle arrest, senescence, or apoptosisin CIN cells [Rieder and Maiato, 2004; Dikovskaya et al., 2015; Hinchcliffe et al., 2016].Phyllanthus emblica Linn. is a medicinal plant distributed in China, India, Thailand and Indonesia. Our previous studies have shown that the extract of P. emblica fruits could be an ideal candidate for the treatment and prevention of human colorectal cancer [Guo et al., 2013; Guo and Wang, 2016; Guo et al., 2017b].
In recent years, phytochemicals isolated from P. emblica have gained attention owing to their diverse bioactivities. Geraniin (Figure 1) is a polyphenolic compound and has been found in the fruits of P. emblica [Liu et al., 2008]. The natural occurrence of geraniin has been verified in other ethnopharmacological important plants, such as a broad species of genus Geranium (e.g., G. sibiricum and G. thunbergii), highlighting its role as an active ingredient in traditional medicines [Cheng et al., 2017]. Recently, a large number of in vitro and in vivo experiments have reported that geraniin induces apoptosis in human glioma [Ren et al., 2017], breast cancer [Zhai et al., 2016], ovarian cancer [Wang et al., 2017a], lung cancer [Li et al., 2013;Ko, 2015], melanoma [Lee et al., 2008] and osteosarcoma cells [Wang et al., 2017b], while leaving untransformed cells unaffected. This selectivity makes geraniin ideal for cancer treatment. However, the model of action by which geraniin induces selective cytotoxicity to tumor cells has not been fully resolved.Recently, we found that geraniin induces excessive CIN in human colorectal cancer Colo205 and Colo320 cells in vitro [Guo et al., 2018b], suggesting geraniin may undermine the efficiency of SAC in colorectal cancer cells. Assessment of SAC activity and mitotic aberrations has been standardized previously in our lab [Guo and Wang, 2016; Guo et al., 2017a; Guo et al., 2017c; Guo et al., 2018a].
In present study, we find that geraniin treatment causes human colorectal cancer HCT116 cells to decrease the expression of several core SAC kinases, thereby weakening the SAC activity. Furthermore, geraniin-treated HCT116 cells display mitotic stress and CIN, and undergo mitotic and post-mitotic cell death. In contrast, geraniin increases the expression of several core SAC kinases in noncancerous human colon epithelial CCD841 cells, thereby strengthing SAC efficiency, reducing mitotic aberrations and ensuring chromosome stability. Our findings reveal that the SAC pathway responses oppositely to geraniin treatment in human colon cancer and noncancerous cell lineages, resulting CIN promotion and supression, respectively.Specific abrogation of SAC to induce catastrophic CIN in HCT116 cells may account for the selective anticancer action of geraniin.
2Materials and Method
Geraniin (purity ≥ 99%) was obtained from Amresco (Cleveland, OH, USA), and cytochalasin-B and nocodazole (Noc) was obtained from Sigma-Aldrich (St.Louis, MO, USA). Stock solutions of geraniin (5 mg/ml), cytochalasin-B (600 μg/ml) and Noc (4 mg/ml) were prepared in dimethyl sulfoxide. These solutions were stored at –20 °C and diluted to the desired concentrations in medium immediately before use.In order to evaluate the effects of geraniin on SAC, we chose chromosome stable human HCT116 colon carcinoma cells, which harbor a functional SAC [Michel et al., 2001]. The cell line CCD-841-CoN (abbreviated as CCD841 thereafter) was chosed as a noncancerous cell control.Both cell lines were maintained as a monolayer in 75 cm2 flasks (Corning, NY, USA) in RPMI 1640 medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin [5000IU/mL]/ streptomycin [5mg/mL] solution (Gibco), 1% L-glutamine (2 mM) (Sigma), and kept at 37 °C in a 5% CO2 environment. In order to ensure that endogenous CIN had not occurred significantly, HCT116 and CCD841 cells at early passages (ranging from P15 to P25) were used for this study.HCT116 and CCD841 cells were seeded into 24-well plates (Corning, NY, USA) at a density of 1 × 105 cells/mL and exposed to different concentrations of geraniin (0, 10, 20, 40 μg/ml).
After 24 hours incubation, adherent and non-adherent cells were detached from plates and collected. Cells were incubated with trypan blue to exclude dead cells, and counted with a hemacytometer. This procedure was repeated three times in duplicate for each geraniin concentration. The clonogenic survival assay was used to assess cellular sensitivity to cytotoxic treatments as it tests the fundamental aspect of survival, a cell’s ability to undergo sufficient proliferation so asto form a colony [Guo et al., 2017b]. HCT116 cells were seeded at a density of 1000 cells per well (in 6-well plates) into fresh medium contain 0, 10, 20, 40 μg/ml geraniin in triplicate. After 7 days of incubation without disturbance, colonies were fixed with cold ethanol, stained with Giemsa (San’ersi, Shanghai, China), counted (only aggregates of >30 cells were scored as colonies), and their survival was calculated as a percentage relative to the untreated control (0 μg/ml geraniin).Experiments were repeated three times for statistical analysis.Soft agar colony formation assays were carried out as previously described [Borowicz et al., 2014; Horibata et al., 2015]. Briefly, 2 ml of a 1:1 mixture of 0.6% agar (Sigma, USA) and 2× RPMI1640 (Gibco, NY, USA) media was poured evenly into 6-well plates.
After cooling, 2 ml of a 1:1 mixture of 0.3% low melting agar (Sigma, USA) and 2× RPMI1640 (prepared according to the indicated concentrations of geraniin) media was used to re-suspend 1 × 10 5 HCT116 cells and then poured on the bottom solidified agar layer. After solidification of the top agar layer at 4 °C for 15 min, the samples were incubated in a humidified incubator at 37 °C with 5% CO2 for 14 days. Ten randomly selected fields of view were imaged on a Nikon Eclipse Ti inverted microscope (Nikon Instruments Inc., NY, USA). The total number of colonies was quantified for all ten fields of view and the size of each colony was quantified by measuring the length of the longest axis. Only colony sizes of 70 µm or larger was included in the data analysis.Cytokinesis-block micronucleus (CBMN) assays were carried out as previously described [Guo and Wang, 2016; Guo et al., 2017a; Guo et al., 2017c; Guo et al., 2018a]. Briefly, HCT116 and CCD841 cells were seeded into 24-well plates at a density of 1 × 105 cells/ml and cultured in RPMI1640 medium without or with geraniin (40 μg/ml) for 24 hours. The medium was discarded after treatment, and cells were washed twice with phosphate buffer saline (PBS, pH 7.2). Fresh medium with 1.5 μg/mL cytochalasin B was added to each culture to block cytokinesis and rinsed with PBS after a further 24 hours. Cells were centrifuged onto glass slides using a cyto-centrifuge for 5 min at 800 rpm (100 g).
The final cell density per slide was kept between 0.5 × 105 and 1 × 105 cells and cell density was confirmed by using a phase contrast microscope (400×). Afterdrying briefly in air, slides were fixed in 100% cold methanol at –20 °C for 15 min and stained with 10% Giemsa. The slides were washed twice in ddH2O, then allowed to air dry and mounted using a cover slip. Slides were stored at room temperature in a black box until the scoring. Stained slides were encoded to ensure a blind microscopic analysis. All biomarkers of CBMN assay were scored under 1000 × magnification with optical microscope (Olympus, Tokyo, Japan) using previously described criteria [Fenech, 2006]. 1000 binucleated cells (BNCs) were scored per treatment group to determine the frequency of MN, nucleoplasmic bridges (NPB) and/or nuclear buds (NB). In addition, 1000 mononucleated cells (MNCs) were scored per treatment group to determine the frequency of MN and/or NB. Cells with an apoptotic or necrotic appearance were exlcuded.After treatment, both rounded-up and attached cells were harvested by trypsinization. Cell suspension was centrifuged to slides, fixed and stained as mentioned above. The mitotic cells which possessed condensed chromosomes were microscopically distinguished from the interphase cells. Stages of mitosis (prophase, prometaphase, metaphase, anaphase and telophase) were identified manually. The analysis of apoptosis was performed on the same slides. As defined previously [Fenech, 2006], cells with chromatin condensation and intact cytoplasmic and nuclear boundaries as well as cells exhibiting nuclear fragmentation into smaller bodies within an intact cytoplasmic membrane were classified as apoptotic.
In each culture, a total of at least 200 cells were scored. This procedure was repeated three times in duplicate.As decribed previously [Guo and Wang, 2016; Guo et al., 2017a; Guo et al., 2017c; Guo et al., 2018a], chromosome misalignment was defined as one or a few chromosomes scattered apart from the metaphase plate in the cytoplasm, lagging chomosome as one or a few chromosomes lagging behind at the spindle equator while all other chromosomes moved toward the spindle poles during ana-telophase; chromatin bridge as one or a few DNA fibers with the thickness of an entire arm connecting both anaphase chromosome packs during ana-telophase. Multipolar segregation as chromosomes pulled towards 3 or more poles. Atypical mitotic figures were determined in ablinded fashion. A total of at least 200 metaphase cells and 50 ana-telophases were counted in each group per experiment. This procedure was repeated at least three times in duplicate.To assess the effect of geraniin on SAC activity, we performed a dose-response proliferation curve with Noc. HCT116 and CCD841 cells were treated in combination with different concentrations of geraniin (0 or 40 μg/ml) and increasing concentrations of Noc (0, 50, 100, 200, 400 and 800 ng/ml) for 24 hours. Changes in proliferation were set relative to the values in cells treated by geraniin alone.After geraniin treatment for 24 hours, total RNA was prepared with high pure RNA isolation kit (Roche diagnostics, IN, USA), which was utilized to synthesize cDNA with PrimeScript RT reagent Kit with gDNA eraser (Takara, Japan) according to the manufacturer’s protocol.
RT-qPCR was performed in triplicate using the Kapa SYBR fast qPCR kit (KAPA Biosystems, MA, USA) and Applied Biosystems StepOne Plus RT-qPCR system (ABI, CA, USA). The primer sequences for Mps1, BubR1, Plk-4, Mad2, Mad1, Bub1, Aurora-A, Aurora-B, Plk-1, CENP-E and GAPDH were previously described [Guo and Wang, 2016; Guo et al., 2017a; Guo et al., 2017c]. The samples were heated at 95 °C for 3 min followed by 40 cycles at 95 °C for 3 s and 60 °C for 30 s. Expression of these genes was normalized to the expression of GAPDH in each sample and fold change was calculated using the 2 –ΔΔCt method [Livak and Schmittgen, 2001].All statistical analyses were performed using SPSS 17.0 for windows. Before analysis, the Kolmogorov-Smirnov test was used to test the normality of all data sets, the results showed that all data sets were normally distributed (p > 0.05). The differences of observed values among the control and various geraniin-treated groups were analyzed using one-way analysis of variance (ANOVA). First, Levene’s test was performed to examine the homogeneity of variances among the control and geraniin treated groups. Post-hoc tests (Tukey’s test was used when the equality ofvariances assumption holds (p > 0.05), and the Dunnett T3 test was used otherwise (p < 0.05)) followed in case a significant effect was detected. Differences between control group and 40 μg/ml geraniin treated group were analyzed using the two-tailed student t test. We considered as being significant only differences having a p-value (two-tailed) lower than 0.05. All data were expressed as mean ± standard error of the mean (SEM). 3Results To explore the effect of geraniin on proliferation of HCT116 cells, we assessed a number of properties, including proliferative capacity, clony formation capacity and anchorage-independent growth. We found that exposure of HCT116 cells to increasing concentrations of geraniin resulted in dose-dependent cell killing. At 40 μg/ml, geraniin induced a ~ 50% reduction in cell number as compared to control (Figure 1A and 1B). Changes in cell morphology were assessed by microscopic examination. HCT116 cells treated with 10 μg/ml geraniin became rounded up, detached from the bottom of the plate, and aggregated as assessed by phase-contrast microscopy. In contrast, the noncancerious CCD841 cells were less sensitive to geraniin in the proliferation assay. CCD841 cells showed no obvious change in cell morphology (Figure 2A) and markedly less growth inhibition than HCT116 cells upon exposure to geraniin (Figure 2B).To evaluate the long-term cytotoxicity of geraniin to HCT116 cells, we performed the cologenic assay. As expected, geraniin treatment elicited anticancer effects in a dose-dependent manner, with the greatest suppression of the colony formation at the concentration of 40 μg/ml (Figure 2C). The colony forming ability of HCT116 cells was decreased by about 60%, 93% and 100% at 10, 20 and 40 μg/ml geraniin, respectively (All p < 0.001; Figure 1E).We next performed a soft agar assay to examine the ability of the HCT116 cells to grow independent of anchorage in vitro, a phenotype that correlates with tumorigenic capacity. As expected, HCT116 cells formed colonies in control culture, consitent with anchorage-independent growth being the phenotype most consistently associated with tumorigenicity. Cells treated with geraniin formed significantly less colonies compared with control (Figure 2D and 2E). Moreover, the colonies formed at 20 and 40 μg/ml geraniin were significantly smaller than those formed from control (Figure 2D).Taken together, these results show that geraniin potently and selectively inhibits the proliferation of HCT116 cells in vitro. A concentration of 40 μg/ml of geraniin was chosen for the subsequent mechanistic studies, since it displayed the strongest response to inhibiting HCT116 cells.HCT116 cells treated with 40 μg/ml geraniin for 24 h exhibited morphological alterations such as cell shrinkage and membrane blebbings that are normally associated with the occurrence of apoptotic cell death (Figure 2A). As shown by microscopic analyses, cell death of HCT116 preceded by chromatin condensation (pyknosis) and nuclear fragmentation (karyorrhexis), two morphological hallmarks of apoptosis (Figure 3A). Quantative analysis showed that geraniin induced a over 5-fold higher percentage of apoptotic cells (1.10% vs 5.59%, p < 0.001; Figure 3B).To explore whether the geraniin-induced apoptosis is cell-cycle dependent, HCT116 cells synchronized at various cell stages were treated with geraniin (Figure 3C). HCT116 cells were synchronized in G2 by 6 hr release from a 24 hr thymidine arrest. Thereafter, cells were treated with Noc alone or in conjunction with geraniin for a total of 4 h. Mitotic cells were then collected by shake off and their apoptotic rates were determined (Figure 3C). The results showed that 3.62% of cells arrested at prometaphase underwent apoptosis, while this was 9.76% in prometaphase arrested cells co-cultured with geraniin (p < 0.001; Figure 3D).We further evaluated the impacts of geraniin on post-mitotic and interphase cells. G2 synchronized HCT116 cells were arrested in prometaphase by Noc, and mitotic cells were collected by shake off. Cells remained adhered to the plate following this procedure were cells that arrested in interphase. Both mitotic and interphase cell fractions were treated with or without geraniin for 4 h (Figure 3C). The data showed that the frequency of apoptosis in control cells exited from mitosis was 3.93%, whereas this was 12.83% in geraniin treated cells (p < 0.001; Figure 3E). However, geraniin showed no significant influence on apoptosis in interphase cells (Figure 3F).Together, our data demonstrate that geraniin specifically induces apoptosis in mitotic and post-mitotic HCT116 cells. In other words, mitosis is necessary for geraniin to exert its cell-killingeffects.When analysizing the proapoptotic effects of geraniin to HCT116 cells after release from Noc arrest, we also determined the status of viable HCT116 cells. We observed that 15.74% of control cells had exited the block after 4 h, whereas 36.04% of the geraniin treated cells had exited mitosis (p < 0.001; Figure 3E). These data suggest that geraniin-treated HCT116 cells displayed a weakened SAC.To begin to define the effects of geraniin on SAC activity, we first tested for changes in cell cycle progression of geraniin treated cells. In HCT116 cells, there was a large increase in the proportion of anaphase/telophase cells in geraniin treated cells (17.51% vs 26.8%, p < 0.05; Figure 4A). However, there was a large increase in the proportion of prometaphase/metaphase cells (67.13% vs 80.14%, p < 0.05) and decrease in the proportion of anaphase/telophase cells in geraniin treated CCD841 cells (24.41% vs 8.76%, p < 0.01; Figure 4B). Of note here, geraniin did not significantly alter the percentage of mitotic HCT116 or CCD841 cells (Figure 4C and 4D). These observations were highly evocative of that the SAC pathway of HCT116 and CCD841 cells after geraniin treatment was impaired and activated, respectively. To further explore it, the status of SAC was determined by Noc-challenge assay. Noc is a spindle poison which activates SAC by precluding both attachment and tension on kinetochores. The results showed that geraniin significantly decreased the percentage of HCT116 cells arrested in mitosis by Noc (50.05% vs 40.69%, p < 0.01; Figure 4C), but significantly increased this percentage in CCD841 cells (37.48% vs 45.99%, p < 0.05; Figure 4D). Moreover, SAC disruption is always associated with cytokinesis abnormalities and multinucleatation [Guo et al., 2017c]. Consistent with this, geraniin treatment was found to significantly increase the frequency of HCT116 cells containing 2 and 3 nuclei by 2.48 and 6.10 times, respectively (both p < 0.001; Figure 4E). In contrast, geraniin showed no significant effect on the multinucleation of CCD841 cells (Figure 4F).In addition, we performed dose–response proliferation curves with Noc (Figure 4G and 4H). When HCT116 cells were cultured in the presence of 50–200 ng/ml Noc, the proliferation of control cells were modestly decreased (~40–50%), whereas the proliferation of geraniin treatedcells dropped down less (0–34%; p < 0.05; Figure 4G). These findings were consistent with reduced SAC function and accelerated progression through the cell cycle in geraniin-treated HCT116 cells. I n CCD841 cells, the proliferation of control cells were modestly decreased (~22– 44%), whereas the proliferation of geraniin treated cells dropped down more (40–54%; p < 0.05; Figure 4H). These data supported the view that geraniin makes CCD841 cells more efficient at sustaining the SAC.Together, these results indicate that, upon geraniin treatment, the SAC of HCT116 cells was impaired and overridden, whereas the SAC of CCD841 cells was strengthened.Next, we attempted to link the observed changes of SAC function with alterations in the expression level of genes involved in the control of SAC pathway. Analysis of the mRNA levels of several SAC genes, including Mad1, Mps1, CENP-E, BubR1, PLK-1, PLK-4, Aurora-B, Aurora-A, Bub1 and Mad2, showed that they were overexpressed (9/10) in HCT116 cells compared with those of CCD841 cells (Figure S1). Specifically, there was a 5.10-, 3.25-, 2.59- fold increase in the expression of BubR1, Mad2 and Mps1 in HCT116 cells, respectively, as compared with those of CCD841 cells (Figure S1). These data confirmed the fact that most mitotic kinases are highly expressed in cancer cells than their normal counterparts. We found that geraniin treatment led to a significant downregulation of the majority of core SAC genes (9/10) in HCT116 cells. Among these, the expression of Mad2 and Bub1 were decreased ~50% (both p < 0.001; Figure 5A). In contrast, 8/10 core SAC genes were significantly upregulated in CCD841 cells after geraniin treatment. Notably, geraniin induced a 2.07-fold increase in the expression of Mad1. Other SAC genes such as CENP-E and PLK-4 were not affected (Figure 5B).Together, these results demonstrate that dual roles for geraniin in systematically modulating SAC activity is accompanied with differential expressing of SAC kinases in HCT116 and CCD841 cells.Since SAC is a major cell-cycle regulatory pathway which monitors the chromosome segregation during mitosis, we next investigated the impact of geraniin on mitotic fidelity. We analyzed the effect of geraniin on mitotic fidelity of fixed HCT116 and CCD841 cells. Geraniin treatment caused a considerable increase in the proportion of cells displaying abnormal mitoses in HCT116 cells (Figure 6). Whereas the frequency of chromosome misalignment was 9.14% in control cells, this percentage increased to 19.16% in geraniin-treated cells (p < 0.001; Figure 6A). The frequency of chromosome lagging was 6.83-fold higher in cells treated with geraniin (15.44%) when compared to those of control (4.50%, p < 0.001; Figure 6B). By comparison with control cells, the frequency of chromatin bridges was increased by 1.9 times in geraniin-treated cells (7.98% vs 22.65%, p < 0.001; Figure 6C). Moreover, we investigated the effect of geraniin on spindle polarity. The frequency of multipolar segregation in geraniin-treated cells (16.32%) remained 2.37 times higher than that of control cells (6.88%; p < 0.001; Figure 6D). Conversly, the frequenciesof these abnormal mitotic figures, except that of multipolar segregation, were significantly reduced by geraniin in CCD841 cells (All p< 0.01; Figure 6).Having demonstrated that geraniin displayed dual roles in mitotic stress, we perfomed experiments to determine whether it exhibited dual roles in regulating CIN. We started with CIN analysis in BNCs, and five abnormal endpoints were examined, including the presence of MN, NPB, NB, and coincidence of MN with NPB (MN+NPB) and MN with NB (MN+NB).Representative cell images are dispalyed in Figure 7A. As expected, the frequency of BNCs with MN (Figrue 7B), NPB (Figrue 7C), NB (Figrue 7D), MN+NPB (Figrue 7E) and MN+NB (Figrue 7F) were significantly increased in HCT116 cells (All p < 0.01) but decreased in CCD841 cells (expcet MN+NB), following geraniin treatment. Overall, after geraniin treatment, CIN was increased by 3.20-fold in binucleated HCT116 cells, respectively, while decreased 1.73-fold in binucleated CCD841 cells (Figure 7G).In addition, CIN events were also examined in MNCs. Representative images of MNCs harboring MN, NB and MN+NB are dispalyed in Figure S2A. Similar to BNCs, the fractions of MNCs harboring MN (Figrue S2B), NB (Figrue S2C) and MN+NB (Figrue S2D) were significantly increased in HCT116 cells (All p < 0.01) but decreased in CCD841 cells (All p < 0.01) following geraniin treatment. Overall, after geraniin treatment, CIN was increased byIn aggregate, geraniin displays a dual role in modulating CIN between cancer cells and their normal counterparts. 4.Disscussion The SAC is a conserved surveillance mechanism that governs the fidelity of chromosome transmission during mitosis. Targeted disruption of SAC offers a novel approach to cancer treatment: driving tumor cells into cell division despite unattached/misattached chromosomes, resulting in a lethal degree of mitotic stress and CIN [Funk et al., 2016]. Comparison with the primary fibroblasts and noncancerous cell lines has supported the possibility that cancer cells may be more sensitive to the consequences of SAC disruption than their normal counterparts [Diogo et al., 2017]. This means SAC is a more favorable therapeutic target since it is more critical for the survival of a cancer cell than a normal cell. Geraniin has been found to inhibit proliferation and induce apoptosis in multiple human tumor cells [Lee et al., 2008; Li et al., 2013; Ko, 2015; Zhai et al., 2016; Ren et al., 2017; Wang et al., 2017a; Wang et al., 2017b]. However, molecular mechanism underlying the antitumor effects of geraniin has not been fully evaluated. Previously, we found that geraniin induced gross CIN in human colorectal cancer cells in vitro [Guo et al., 2018b]. Therefore, we ask the question whether geraniin treatment induces SAC defects in cancer cells. Here, we show that geraniin-treated HCT116 cells exhibit a weakened SAC due to the decreased expression of several core SAC genes. The dysfunction of SAC leads to mitotic stress and increased CIN in HCT116 cells. We further show that geraniin-treated HCT116 cells undergo mitotic and post-mitotic cell death. These results might reflect that enhanced diffficulty of cancer cells to tolerate an elevated level of CIN in context of SAC abrogation induced by geraniin. We find that geraniin decreases the transcriptional expression of microtubule motor protein CENP-E and multiple mitotic kinases such as Bub1. CENP-E is a highly elongated kinesin that transports pole-proximal chromosomes during congression in prometaphase. During metaphase, it facilitates kinetochore–microtubule end-on attachment required to achieve and maintain chromosome alignment [Wood et al., 1997]. Cells lacking CENP-E consistently fail to align their chromosomes into a metaphase plate [Schaar et al., 1997]. Mitotic kinase Bub1 is also engaged in chromosome alignment by itself as well as recruiting CENP-E and some kinases (e.g., BubR1 and Mad2) [Johnson et al., 2004]. Accordingly, ΔBUB1 cells display a significant delay in the time to obtain full chromosome alignment as compared to wild type cells [Raaijmakers et al., 2018]. We expect that the decrease of CENP-E and Bub1 expression may account for the increased chromosome misalignment observed in geraniin-treated HCT116 cells. The key finding in this study is that geraniin abrogates the integrity of SAC in HCT116 cells. Before all chromosomes are connected to the mitotic spindle, anaphase is delayed by a MCC that inhibits the ubiquitylation activity of APC/C. The MCC assembles on the unattached kinetochore and contains the Mad2, BubR1 and Bub3 mitotic kinases in a complex with Cdc20. Mad2 and BubR1 inhibit Cdc20 by binding to substrate and APC/C recognition motifs, thereby preventing Cdc20 from activating the APC/C. The strength of SAC depends on the amount of Mad2 recruited to kinetochores and on the amount of MCC formed [Collin et al., 2013]. In addition, Aurora-A kinase is essential for maintaining the Mad2 on unattached kinetochores and that inhibition of Aurora-A leads to the loss of SAC [Courtheoux et al., 2018]. Aurora-B kinase is required for SAC integrity and inhibition of which leads the misaligned metaphase cells to override the SAC [Kallio et al., 2002]. Based on these data, we can speculate that the reduced expression of Mad2, BubR1, Aurora-A and Aurora-B after geraniin treatment may compromise the SAC activity and induce SAC override in HCT116 cells . We find that geraniin induces mitotic/post-mitotic cell death and reduces colony formation in HCT116 cells. In most cases, SAC is essential for the viability of human cells since the inhibition of SAC has been found to be lethal to human cancer cells due to massive chromosome loss [Kops et al., 2004]. Consitent with this, heterozygous reduction of CENP-E [Silk et al., 2013], Mad2 [Michel et al., 2001] or BubR1[Baker et al., 2004] leads to mitotic stress and apoptotic cell death. However, in some cases, SAC is not essential for viability of human cells. In these cases, increased expression of Bub1 is essential for cell viability upon loss of SAC activity [Raaijmakers et al., 2018]. In light of this, we propose that the reduction of Bub1 may enhance the proapoptotic action of geraniin following SAC dysfunction. Furthermore, we find that geraniin induces multipolar spindle and multinucleation in HCT116 cells, suggesting that geraniin may interfere with the centrosome homeostasis and cytokinesis. The polo kinase family member Plk-4 localizes to centrioles throughout the cell cycle and is essential for centriole duplication. Plk-4+/− murine embryonic fibroblasts show a high incidence of supernumerary centrosomes and multipolar spindles [Rosario et al., 2010]. Moreover, cytokinesis failure and multinucleation, is seen as an indirect consequence of the spindle abnormalities caused by loss of Plk-4 [Holland et al., 2012]. In addition, the kinase Plk-1 has an important role in bipolar spindle formation. Depletion of Plk-1 causes fragmentation and dissociation of the pericentriolar material from centrioles at prometaphase, resulting in centrosome amplification [Oshimori et al., 2006]. Although centrosome amplification is a common characteristic of solid and hematological cancers, cancer cells tend to cluster supernumerary centrosomes into two group to enable a bipolar mitosis [Kwon et al., 2008]. Recently, it has shown that Aurora-A inhibition limits centrosome clustering and promotes mitotic multipolarity in cells with supernumerary centrosomes [Navarro-Serer et al., 2018]. With these in mind, it is therefore tempting to speculate that multipolar spindle and multinucleation induced by geraniin in HCT116 cells may be the combined consequences of centrosome overduplication and declustering resulting from reduced expression of Plk-4, Plk-1 and Aurora-A. One of the striking findings in this study is the opposite roles of geraniin in regulating the expression of SAC kinases between colorectal cancer HCT116 cells and noncancerous CCD841 cells. As discussed above, the levels of BubR1 and Mad2 determine the number of MCC and therefore the SAC strength. Moreover, Bub1 and Mad1 act in coordination to facilitate BubR1 localization and hyperphosphorylation at kinetochores [Chen, 2002]. Mps1 plays a key role in chromosome alignment by promoting rapid centromere accumulation of Aurora-B [van der Waal et al., 2012]. Together, the increased expression of BubR1, Mad2, Bub1, Mad1, Mps1 and Aurora-B could explain the enhanced SAC activity in geraniin-treated CCD841 cells. Strengthening SAC efficiency thereby helps to reduce mitotic stress and ensure chromosome stability in CCD841 cells. In addition to SAC weakening, another factor contributing to spotaneous mitotic aberrations in normal cells is merotelic kinetochore-microtubulin attachment [Cimini et al., 2003], a type of error in which single kinetochores attach to microtubules emanating from different poles. Merotelic attachment is a major limitation for accurate chromosome segregation since it does not block chromosome alignment at the metaphase plate and is poorly detected by SAC [Cimini et al., 2003]. At anaphase entry, a proportion of merotelically attached chromosomes are lagged because they are disproportionally pulled towards the spindle poles [Cimini et al., 2002]. As a result, these lagged chromosomes fail to reach the main chromosome mass and are therefore partitioned into MN or lost in the cleavage furrow. Extensive evidence supports that Aurora-B kinase plays a critical role in merotelic attachment correction by phosphorylating key substrates at the kinetochore and promoting turnover of kinetochore microtubules [Cimini, 2007]. Since geraniin upregulates the expression of Aurora-B and reduces the frequency of chromosome lagging and MN in CCD841 cells, these findings highlight the possbility that geraniin-induced upregulation of Aurora-B might involve in the correction of merotelic attachments that spontaneously arised in CCD841 cells. Coupled with our previous results [Guo et al., 2018b], we find that geraniin is non-genotoxic in human noncancerous colon epithelial NCM460 and CCD841 cells. This finding suggest that geraniin may possess better cancer-cell selectivity and therefore fewer side effects. Furthermore, we find that geraniin decreases the spotaneous frequency of CIN events (e.g., MN, NPB and NB) in noncancerous NCM460 and CCD841 cells cells. Given that increased levels of mitotic aberrations and CIN are key drivers of oncogenic tranformation [Funk et al., 2016], these findings suggest a potential role of geraniin as a cancer chemopreventive agent. Because of its pharmacological safety, geraniin may be used alone to prevent cancer and in combination with chemotherapy to treat cancer. One limitation of this study is that only the transcriptional expression of core SAC genes was determined. An obvious weakness is that gene expression data may not directly correlate with protein levels or function. Therefore, an evaluation of the expression of these genes using Western blotting and/or immunohistochemistry might be one way of strengthening our conclusions. In future, we plan to define the precise molecular mechanisms underly the opposite roles of geraniin in modulating the SAC activity of HCT116 and CCD841 cells. Furthermore, the question of whether the differential effect of geraniin observed within cell lineages is more widely applicable to in vivo models remains open and interesting. An important future direction would be extending the findings reported here to mouse models of cancers or preclinical models of human cancers. 5.Conclusions In summary, our work shows that geraniin has selective antineoplastic activity against human colorectal cancer cells. Geraniin treatment readily causes SAC impairment in HCT116 cells through downregulation of the majority of core SAC kinases. This leads to mitotic stress, which trigger apoptotic death during mitosis and post mitosis. While some of the affected cells die in mitosis and post mitosis, the ones that escape a mitotic catastrophe exhibit massive CIN (model in Figure 8A). This way, we demonstrate that SAC abrogation serves as a previously unappreciated mechanism underlying the anticancer action of geraniin. On the other hand, the present study reveals that geraniin strengthens the robustness of SAC establishment in noncancerous CCD841 cells through BAY-1816032 moderately upregulating the expression of core SAC kinases. These regulations may account for the decreased baselines of mitotic stress and CIN in CCD841 cells (model in Figure 8B).