Biochemical and Biophysical Research Communications 499 (2018) 927e933

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

PMN inhibits colorectal cancer cells through inducing mitotic arrest

and p53-dependent apoptosis via the inhibition of tubulin
polymerization
Xingkang Wu a, Yuemao Shen a, b, *
a
Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, No. 44 West Wenhua Road, Jinan,
Shandong 250012, PR China
b
State Key Laboratory of Microbial Technology, Shandong University, No. 27 South Shanda Road, Jinan, Shandong 250100, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Colorectal cancer (CRC) is the third most prevalent malignancy worldwide. New understandings about
Received 21 March 2018 this disease are urgently required to guide clinical therapies. In this study, we focused on the effects of
Accepted 3 April 2018 the small molecule PMN on CRC cells. PMN dose-dependently inhibited CRC cell proliferation through
Available online 9 April 2018
inducing mitotic arrest and apoptosis. PMN induced mitotic arrest via the disruption of spindle apparatus
by inhibiting microtubule polymerization. PMN-induced mitotic arrest resulted in apoptosis and p53
Keywords:
upregulation. Furthermore, p53 upregulation sensitized PMN-induced mitotic cells to apoptosis. This
PMN
study deepens the understanding of the effects of p53 on the response of CRC cells to PMN, providing the
Colorectal cancer
Mitotic arrest
basis for the potential development of PMN as a lead compound of microtubule-destabilizers for p53-
Microtubule positive CRC treatment.
Apoptosis © 2018 Elsevier Inc. All rights reserved.
p53

1. Introduction sarcoma, lymphoma, and leukemia [6]. Although effective in

various cancers, the use of MTAs in colorectal cancer therapy is
Colorectal cancer (CRC) is the third most prevalent cancer currently limited.
worldwide, resulting in 1,361,000 new cases and over 690,000 The tumor suppressor p53 is an evolutionarily conserved pro-
deaths in 2012 [1]. The median overall survival of metastatic tein that regulates many proteins involved in cell-cycle arrest,
colorectal cancer patients is about 24e30 months [2]. Total 34 apoptosis, senescence, DNA repair, and metabolic responses and
drugs have been approved by FDA to treat colorectal cancer, responds to a variety of stress signals [7,8]. It plays essential roles in
including 8 drugs for combination treatments [3]. the development and treatment outcomes of CRC. The p53
Microtubules (MTs) are highly dynamic components of the dysfunction is one of most important molecular pathways leading
cytoskeleton [4]. Microtubules have been developed as attractive to CRC development, which has been reported in 50e70% of pa-
chemotherapeutic targets for anti-cancer drugs [5]. Microtubule- tients with CRC [9]. Several clinical and experimental trials have
targeting agents (MTAs) are commonly classified into two major shown that inactivation of p53 was correlated with worse clinical
groups: microtubule-destabilizers and -stabilizers. FDA-approved outcome of chemotherapy in CRC [10,11]. Hence, investigations that
MTAs such as vinblastine, vincristine, vinorelbine, estramustine evaluate the effects of p53 on the sensitivity of CRC cells to small
and vindesine inhibit MT polymerization, whereas agents such as molecules are crucial for the development of a drug for treating
paclitaxel, docetaxel, and ixabepilone enhance MT polymerization CRC.
[6]. MTAs are clinically applied to treat a variety of cancers, PMN (Fig. 1A), namely 6-(pyrimidin-5-yl)naphthalen-2-ol, has
including breast, ovary, lung, prostate, head-and-neck, Kaposi's been found to exhibit anti-cancer properties against MDA-MB-231,
A549 and HeLa cells previously [12], but the mechanism of action
remains unknown. In this study, we explored the cytotoxic mech-
anism of PMN against CRC cells. Our study demonstrated that PMN
* Corresponding author. Key Laboratory of Chemical Biology (Ministry of Edu-
cation), School of Pharmaceutical Sciences, Shandong University, No. 44 West displayed dose-dependent inhibitory activity against three CRC cell
Wenhua Road, Jinan, Shandong 250012, PR China. lines through inducing mitotic arrest and p53-mediated apoptosis.
E-mail address: [email protected] (Y. Shen).

https://doi.org/10.1016/j.bbrc.2018.04.021
0006-291X/© 2018 Elsevier Inc. All rights reserved.
928 X. Wu, Y. Shen / Biochemical and Biophysical Research Communications 499 (2018) 927e933

Fig. 1. PMN shows potent antitumor activity against CRC cells. (A) The chemical structure of PMN. (B) PMN dose-dependently inhibited cell growth. Data were obtained by SRB
assay and presented as mean ± SD (n ¼ 3). (C) PMN inhibited colony formation of cells. Images displayed the colony formation in PMN-treated cells. The scar bar represents 7 mm.
(D) PMN showed dose-dependent inhibition of colony formation in cells. Data were presented as mean ± SD (n ¼ 3).

Mechanistically, PMN disrupted spindle apparatus by inhibiting concentrations of tested agents in PEM buffer (80 mM PIPES,
microtubule polymerization. 0.5 mM EGTA, 2 mM MgCl2, pH 6.9) containing 6.5 mM DAPI, 1 mM
GTP and 15% glycerol. Microtubule polymerization curves were
2. Materials and methods obtained by measuring the fluorescence intensity (excitation at
360 nm, emission at 450 nm) every minutes during 60 min at 37 " C
2.1. Chemicals and antibodies with a fluorimeter Microplate Readers (Synergy H1, BioTek, USA).

PMN was synthesized according to the methods of Chen et al. 2.4. Cell viability assay
[12]. Nocodazole was purchased from Selleckchem and paclitaxel
was purchased from MedChemExpress. Anti-PARP, anti-p53 (1C12) The cell viability was determined using a sulforhodamine B
and anti-phospho-Aurora kinase antibodies were purchased from (SRB) assay [13]. Briefly, cells were seeded in 96-well plate and
Cell Signaling Technology. Anti-GAPDH and anti-phospho-H3 (S10) treated for 72 h with indicated concentrations of agents. Cells were
antibodies were purchased from Abcam. Anti-b-tubulin antibody fixed for 1 h with 10% trichloroacetic acid (TCA) at 4 " C. The plate
was purchased from Proteintech. was washed five times with slow-running water and dried at room
temperature. After drying, the cells were stained for 10 min with
2.2. Cell culture 0.4% SRB solution and the unbound dye was rinsed five times with
1% (vol/vol) acetic acid. The protein-bound dye was dissolved in
HCT116, SW480, RKO and RKO-E6 cells were purchased from the 10 mM Tris solution (pH 10.5) and the OD value was measured at
Cell Bank of the Shanghai Institute for Biological Sciences, Chinese 510 nm in a microplate reader. The cell viability (%) was calculated
Academy of Science. HCT116 (p53!/!) cells were obtained from by (ODS e ODB)/(ODC ! ODB) # 100, where ODC is the OD value in
School of Life Sciences, Xiamen University (Fujian, China). HCT116, the presence of 0.1% DMSO, ODS is the OD value in the presence of
HCT116 (p53!/!), RKO and RKO-E6 cells were cultured in DMEM PMN, and ODB is the OD value in the absence of cells.
(Gibco, 12800-017). SW480 cells were cultured in RPMI 1640 me-
dium (Gibco, 31800-022). All media were supplemented with 10% 2.5. Cell transfection
fetal bovine serum (Biological Industries), 100 U/mL penicillin, and
100 mg/mL streptomycin. The cells were incubated in a 37 " C incu- The siRNA transfection was performed using Lipofectamine™
bator with 5% CO2. RNAiMAX according to the manufacturer's instructions. Control
siRNA (ACGUGACACGUUCGGAGAAdTdT) and Mad2 siRNA (GGAA-
2.3. In vitro tubulin polymerization assay CAACUGAAAGAUUGGdTdT) were purchased from GenePharma.

For in vitro tubulin polymerization assay, a fluorescence-based 2.6. Colony formation assay
tubulin polymerization assay kit (Cytoskeleton, BK011P) was
used. In brief, tubulin protein was mixed with different Cells were plated at 10000e20000 cells/well in 6-well plate
 X. Wu, Y. Shen / Biochemical and Biophysical Research Communications 499 (2018) 927e933 929

according to cell lines and incubated overnight. The following day increasing concentrations of PMN. SRB assays showed that PMN
PMN stocks in DMSO were diluted in medium then added to cells dose-dependently inhibited CRC cell proliferation (Fig. 1B). To
with a maximum final DMSO concentration on cells of 0.1%. After further investigate the long-term efficacy of PMN on CRC cells,
incubation for two weeks, cells were fixed in methanol/acetic acid colony formation assay was applied. In accordance with the results
(3:1) and then stained with Giemsa stain. The plate was imaged on of SRB assays, PMN dose-dependently inhibited the colony forma-
ChemiDoc XRS þ imaging system (Bio-Rad). The number of col- tion of CRC cells (Fig. 1C and D). Interestingly, HCT116 cells were
onies was determined by the colony area through Image-Pro Plus more sensitive than SW480 cells to high dose PMN, whereas
software. The relative colony area was calculated by (As e A0)/(Ac e SW480 cells were more sensitive to low-dose PMN (Fig. 1B and D).
A0) # 100%, where Ac is the colony area in the presence of 0.1% Besides, RKO cells were most sensitive to PMN. Together, these
DMSO, As is the colony area in the presence of PMN, and A0 is the results provide evidence that PMN shows potent antitumor activity
colony area on the first day before PMN treatment. against CRC cells.

2.7. Cell cycle assay 3.2. PMN induces mitotic arrest and apoptosis in CRC cells

Cells were treated with indicated concentrations of PMN and To understand the mechanism of growth inhibition in response
totally harvested by tyrpsinisation and centrifugation. Cells were to PMN, the effect of PMN on cell cycle distributions was examined.
then fixed in cold 75% ethanol overnight at -20 " C. After fixation, By comparison with DMSO-treated cells, cells showed a dose-
cells were stained for 30 min at 37 " C with 10 mg/mL propidium dependent accumulation of G2/M phase following 12 h exposure
iodide (PI) in PBS plus 100 mg/mL RNase A. Then cells were analyzed to increasing concentrations (10, 20 and 40 mM) of PMN (Fig. 2A). To
by a flow cytometer (ACEA NovoCyte™). address whether the PMN-treated cells are arrested at G2 or M
phases, phospho-Aurora kinases and phospho-H3 (S10), well-
2.8. Immunofluorescence known mitotic markers, were detected. PMN dose-dependently
increased the expression of phospho-Aurora kinases and
Cells were plated on glass coverslip and incubated overnight. phospho-H3 (S10) in CRC cells (Fig. 2B). Further evidence coming
After 24 h exposure to PMN, cells were fixed for 15 min with 4% from live cell imaging analysis showed that PMN prevented mitotic
paraformaldehyde at room temperature. After washing with PBS, progression in HCT116 and RKO cells (Fig. S1). These results
cells were blocked for 1 h with PBS plus 0.3% Triton X-100 and 10% demonstrate that PMN induces mitotic arrest in CRC cells.
FBS. Primary antibodies were diluted to suitable concentration with Following, the effects of PMN on apoptosis were explored by
PBS plus 10% FBS and incubated overnight with cells at 4 " C. Then western blot. PMN dose-dependently increased the levels of
cells were washed with PBS and incubated for 1 h with Alexa Fluor- cleaved-PARP and p53 in HCT116 and RKO cells but had no effect on
conjugated secondary antibodies at room temperature. Cells were those in SW480 cells (Fig. 1B). Typically, cleaved-PARP serves as a
then stained with 1 mg/mL Hoechst 33342. The coverslip was marker of cells undergoing apoptosis [14] and p53 promotes
mounted in ProLong® Gold Antifade Mountant (Lifetechnologies). apoptosis under different stress stimuli through multiple mecha-
Cells were observed and imaged on a fluorescent inverted micro- nisms [15]. In addition, PMN dose- and time-dependently induced a
scope (Zeiss, Axio Observer A1). sharp accumulation of sub-G1 cells (Fig. 1A and S2A). These results
reveal that PMN induces apoptosis in CRC cells.
2.9. Western blot
3.3. PMN exerts its cellular effects via the induction of mitotic arrest
Cells were collected by cell scraper and directly sampled in
1 # loading buffer (250 mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM To investigate whether PMN exerts its cellular effects via the
DTT, 0.01% bromophenol blue, pH ¼ 6.8). Samples were separated induction of mitotic arrest, the effects of PMN on CRC cells trans-
on a SDS-PAGE gel and transferred to PVDF membrane (Millipore) fected with Mad2 siRNA were tested. Mad2 is an essential protein
by wet transfer. The membrane was blocked for 1 h with 5% BSA in for spindle checkpoint, which is a primary cell cycle checkpoint that
TBST (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.6) at prolongs mitotic progression [16]. Immunoblot analysis showed
room temperature and incubated overnight with specific primary that siRNA transfection silenced Mad2 expression and suppressed
antibodies at 4 " C. Then, the PVDF membrane was washed with the upregulation of phospho-Aurora kinase and phospho-H3 (S10)
TBST and incubated for 1 h with horseradish peroxidase- in PMN-treated cells (Fig. 2D), indicating that Mad2 knockdown
conjugated secondary antibodies (Abcam) at room temperature. successfully reduced PMN-induced mitotic arrest. Mad2 knock-
Chemiluminescence signals were probed with ECL reagent on the down significantly increased the cell viability of PMN-treated cells
ChemiDoc XRS þ imaging system (Bio-Rad). (Fig. 2C) and suppressed PMN-induced reduction in G1 phase and
accumulation of sub-G1 cells (Fig. S2B and S2C), indicating that
2.10. Statistical analysis PMN-induced cytotoxicity was Mad2 dependent. Meanwhile, Mad2
knockdown inhibited PMN-induced upregulation of cleaved-PARP
Data analysis was carried out using Prism 7 (GraphPad). Sig- and p53 in RKO and HCT116 cells (Fig. 2D), suggesting that PMN-
nificant differences of all pairwise combinations were determined induced mitotic arrest contributed to apoptosis and p53 upregu-
by two-way ANOVA multiple comparison test. Values of P < 0.05 lation. Collectively, these findings suggest that PMN exerts its
were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, cellular effects via the induction of mitotic arrest.
****P < 0.0001, ns: not significant.
3.4. PMN disrupts microtubule organization
3. Result
Since defects in microtubule organization usually lead to mitotic
3.1. PMN shows potent antitumor activity against CRC cells arrest, the effects of PMN on cellular microtubule were investigated
by immunofluorescence. In the absence of PMN, CRC cells in the
To evaluate the effects of PMN on CRC cells, three CRC cell lines interphase exhibited the normal microtubules that were thick,
including HCT116, SW480 and RKO cells, were treated with strong and clearly visible (Fig. S3). In contrast, PMN treatment
930 X. Wu, Y. Shen / Biochemical and Biophysical Research Communications 499 (2018) 927e933

Fig. 2. PMN triggers apoptosis via the induction of mitotic arrest. (A) PMN induced G2/M phase arrest in cells. CRC cells were exposed to the indicated concentrations of PMN for
12 h and analyzed by flow cytometry. (B) PMN promoted the activation of mitotic and apoptotic markers. Cells were treated for 24 h with the indicated concentrations of PMN and
subjected to immunoblot analysis with indicated antibodies. (C) PMN-induced decrease in cell viability was Mad2 dependent. CRC cells were transfected with indicated conditions,
and treated with indicated agents. Data were obtained by SRB assay and presented as mean ± SD (n ¼ 3). (D) PMN-induced apoptosis was Mad2 dependent. Cells were treated with
indicated conditions and subjected to immunoblot analysis with indicated antibodies.
 X. Wu, Y. Shen / Biochemical and Biophysical Research Communications 499 (2018) 927e933 931

generated disarrangement of microtubules, which presented in HCT116 cells (Fig. 4C), the sub-G1 cells increased from 2.75% to
diffused and unclear staining (Fig. S3). During mitosis, microtubules 26.31% in HCT116 (p53!/!) cells (Fig. 4C), indicating that p53
in cells without PMN treatment were arranged into a bipolar array, deletion reduced sub-G1 cells in PMN-treated HCT116 cells. Similar
such that each half spindle contains uniformly oriented microtu- results were observed in RKO and RKO-E6 cells (Fig. 4C). Further-
bules (Fig. 3A). However, PMN treatment led to a specific more, immunoblot analysis showed that p53 deletion obviously
arrangement of microtubules, which were organized into short and suppressed the cleavage of PARP but not the phosphorylation of
multipolar spindles (Fig. 3A). These results indicated that PMN Aurora kinases and H3 (S10) in HCT116 and RKO cells (Fig. 4D),
disrupted cellular microtubule organization. To better understand indicating that p53 mediated PMN-induced apoptosis but not
how PMN affects microtubule organization, the effects of PMN on mitotic arrest. Collectively, these results suggest that p53 sensitizes
microtubule dynamics were assessed by using in vitro tubulin PMN-induced mitotic cells to apoptosis.
polymerization assay. The time-dependent curves of tubulin poly-
merization in the presence of PMN were similar to that of noco- 4. Discussion
dazole but not that of paclitaxel, indicating that PMN inhibited
microtubule polymerization (Fig. 3B). Thus, these findings corrob- Herein, we showed that PMN is a potential cytotoxic agent
orate that PMN disrupts spindle apparatus via the inhibition of against the human CRC cell lines: HCT116, SW480 and RKO. PMN
tubulin polymerization. dose-dependently inhibited CRC cell proliferation. PMN arrested
CRC cells at mitotic phase, leading to cell growth inhibition,
3.5. P53 sensitizes PMN-induced mitotic cells to apoptosis apoptosis and p53 upregulation. Moreover, PMN disrupted the or-
ganization of spindle apparatus via the inhibition of tubulin poly-
Given that p53 was sharply increased in PMN-treated HCT116 merization. Notably, p53 upregulation sensitized PMN-induced
and RKO cells, we wondered how p53 contributes to cytotoxicity of mitotic cells to apoptosis. Based on this, we supposed that tubulin
PMN in HCT116 and RKO cells. To achieve this, two p53-deficient and p53 are key proteins that speak to the mechanism by which
cell lines, HCT116 (p53!/!) cells in which exon 2 of p53 was dis- PMN elicits these cellular effects.
rupted by homologous recombination [17] and RKO-E6 cells in PMN clearly exerted its inhibitory effects on microtubule poly-
which E6 expression induced a dramatic decrease in p53 protein merization in vitro, and induced multipolar and fragmented spin-
levels [18], were used. SRB and colony formation assay showed that dles in CRC cells. It is well known that spindle apparatus is crucial
HCT116 (p53!/!) and RKO-E6 cells were less sensitive to PMN than for the accomplishment of mitosis [19], further supporting that the
their parent cells containing p53 (Fig. 4A and B). These results inhibition of microtubule polymerization by PMN enables cells to
suggest that p53 enhances the sensitivity of HCT116 and RKO cells develop mitotic spindle defect and be unable to complete mitosis.
to PMN. Therefore, PMN induces mitotic arrest via the disruption of spindle
To ascertain how p53 regulates the sensitivity of PMN to HCT116 apparatus through inhibiting microtubule polymerization, result-
and RKO cells, the effects of p53 deficiency on cell cycle distribution ing in the failure of cell division.
were evaluated. While the sub-G1 cells dose-dependently PMN-induced mitotic arrest resulted in p53-mediated apoptosis
increased from 4.55% to 40.40% following 24 h of PMN treatment in p53-expressing CRC cells. This is consistent with the key role of

Fig. 3. PMN perturbs microtubule organization. (A) PMN disrupted the formation of spindle apparatus in CRC cells. Cells were treated for 24 h with 20 mM PMN and processed for
immunofluorescence with anti-b-tubulin antibody. The scale bar represents 5 mm. (B) PMN inhibited tubulin polymerization in a cell-free assay. Graph showed microtubule
polymerization curves in the presence of indicated agents. The depolymerization and polymerization controls were nocodazole and paclitaxel, respectively.
932 X. Wu, Y. Shen / Biochemical and Biophysical Research Communications 499 (2018) 927e933

Fig. 4. PMN sensitizes p53-expressing cells to apoptosis. (A) The deletion of p53 decreased the cytotoxicity of PMN against RKO and HCT116 cells. Graph showed growth inhibition
rates of PMN against RKO, RKO-E6, HCT116 and HCT116 (p53!/!) cells by SRB assay. Data were presented as mean ± SD (n ¼ 3). (B) The deletion of p53 increased the colony
formation in PMN-treated cells. Graph showed relative colony area of RKO, RKO-E6, HCT116 and HCT116 (p53!/!) cells in the presence of increasing concentrations of PMN. Data
were presented as mean ± SD (n ¼ 3). (C) The deletion of p53 attenuated the PMN-induced cell death in RKO and HCT116 cells. Cells were treated for 24 h with increasing con-
centrations of PMN and subjected to cell cycle analysis. (D) The deletion of p53 reduced the expression of cleaved-PARP in PMN-treated RKO and HCT116 cells. Cells were treated for
24 h with increasing concentrations of PMN and subjected to immunoblot analysis with indicated antibodies.

p53 in mitotic stress, as revealed by previous literature. Protein p53 sensitivity of CRC cells to PMN through suppressing apoptosis, thus
functions upstream of Bax to promote apoptosis in HCT116 cells suggesting that PMN sensitizes p53-positive CRC cells to apoptosis.
treated with microtubule-destabilizers, including vinblastine and In conclusion, the present study shows that PMN potentially
2-methoxyestradiol [20,21]. In another study, p53 deletion pre- sensitizes p53-positive CRC cells to apoptosis through inducing
vents apoptosis in HCT116 cells treated with GSK923295 (CENPE mitotic arrest via the inhibition of microtubule polymerization. Our
inhibitor), MLN8054 (Aurora A inhibitor) and ZM447439 (Aurora B study reveals the antitumor mechanism of PMN against CRC cells,
inhibitor) [22]. Take together with previous findings, our results do opening opportunities for the development of PMN as a lead
favor the mitotic stress-response model that p53 acts as the inducer compound of microtubule-destabilizers for p53-positive CRC
of apoptosis during mitotic arrest. treatment.
Confusingly, by comparison with RKO and HCT116 cells,
SW480 cells with high level expression of p53 were less sensitive to
PMN. Notably, PMN had no effect on the expression of p53 and
cleaved-PARP in SW480 cells, and induced fewer sub-G1 cells in
SW480 cells than in HCT116 and RKO cells (Fig. S2A). It is reported
Acknowledgments
that the R273H mutation of p53 abolishes its function by inacti-
vating its sequence-specific DNA binding activity in SW480 cells
This work was supported by the National Natural Science
[23,24]. This lead to deficient transcriptional activity of p53 in
Foundation of China (81373304, 81530091, 31329005), Science
SW480 cells, probably contributing to that SW480 cells responded
Foundation of Two sides of Strait (U1405223) and Program for
to PMN in a manner similar to HCT116 (p53!/!) and RKO-E6 cells.
Changjiang Scholars and Innovative Research Team in University
Collectively, both deletion and inactivation of p53 weaken the
(IRT_17R68).
 X. Wu, Y. Shen / Biochemical and Biophysical Research Communications 499 (2018) 927e933 933

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