original article volasertib suppresses the growth of … article volasertib suppresses the growth of...

Am J Cancer Res 2016;6(11):2476-2488www.ajcr.us /ISSN:2156-6976/ajcr0037182

Original ArticleVolasertib suppresses the growth of human hepatocellular carcinoma in vitro and in vivo

Di-Wei Zheng1*, You-Qiu Xue1,2*, Yong Li3*, Jin-Ming Di4, Jian-Ge Qiu1, Wen-Ji Zhang1, Qi-Wei Jiang1, Yang Yang1, Yao Chen1, Meng-Ning Wei1, Jia-Rong Huang1, Kun Wang1, Xing Wei1, Zhi Shi1

1Department of Cell Biology & Institute of Biomedicine, College of Life Science and Technology, Jinan University, National Engineering Research Center of Genetic Medicine, Guangdong Provincial Key Laboratory of Bioen-gineering Medicine, Guangzhou 510632, Guangdong, China; 2Center for Clinic Immunology, Third Hospital at Sun Yat-sen University, Guangzhou 510630, China; 3Department of Gastrointertinal Surgery & General Surgery, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080, China; 4Department of Urology, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, China. *Equal contributors.

Received August 3, 2016; Accepted August 9, 2016; Epub November 1, 2016; Published November 15, 2016

Abstract: Hepatocellular carcinoma (HCC) is the sixth most frequent malignant tumor with poor prognosis, and its clinical therapeutic outcome is poor. Volasertib, a potent small molecular inhibitor of polo-like kinase 1 (PLK1), is currently tested for treatment of multiple cancers in the clinical trials. However, the antitumor effect of volasertib on HCC is still unknown. In this study, our data show that volasertib is able to induce cell growth inhibition, cell cycle arrest at G2/M phase and apoptosis with the spindle abnormalities in human HCC cells. Furthermore, volasertib also increases the intracellular reactive oxidative species (ROS) levels, and pretreated with ROS scavenger N-acety-L-cysteine partly reverses volasertib-induced apoptosis. Moreover, volasertib markedly inhibits the subcutaneous xenograft growth of HCC in nude mice. Overall, our study provides new therapeutic potential of volasertib on hepa-tocellular carcinoma.

Keywords: Hepatocellular carcinoma, volasertib, ROS, apoptosis

Introduction

Hepatocellular carcinoma (HCC) is the sixth most frequent malignant tumor and the third leading cause of cancer-related mortality in the world [1]. Most cases of HCC are as a result of viral infection (hepatitis B or C) and metabolic toxins (alcohol or aflatoxin), and the patients with HCC are usually diagnosed when tumor is in an advanced stage [2]. Although treatment of HCC includes the combination of surgery, chemotherapy or radiotherapy, the 5-year sur-vival rates of patients with HCC is poor [3]. Consequently, it is necessary to develop new therapeutic strategies against HCC.

The polo-like kinases (PLKs) are a family of con-served serine/threonine kinases and play the critical role in the regulation of multiple stages of cell cycle, including mitotic entry/exit, centro-some maturation, and maintenance of the bipolar spindle [4]. It has been reported that PLK1 is overexpressed in up to 80% of cancers

and associated with poorer prognosis [5-7]. In human HCC, PLK1 expression is significantly upregulated and correlated with venous inva-sion, tumor nodules, Edmondson grade and survival rates [8, 9]. Knockdown of PLK1 signifi-cantly inhibits the growth of HCC cells by induc-ing cell cycle arrest at G2/M phase and apopto-sis [10, 11]. Additionally, the HBV X protein can enhance the expression of PLK1, and inhibition of PLK1 suppresses the HBV X protein-mediat-ed oncogenic transformation in HCC [12]. Therefore, PLK1 is a promising therapeutic tar-get of HCC. Volasertib (BI 6727) is a potent small molecule inhibitor of PLK1 (IC50=0.87 nM) by competing with ATP for binding to the ATP binding pocket, and 6- and 65-fold greater activity against two closely related kinases PLK2 (IC50=5 nM) and PLK3 (IC50=56 nM) [13]. In the preclinical studies, volasertib shows broad antitumor activity in several cancer mod-els including hematological malignancies, mel-anomas, cervical cancer and sarcomas [13-17].

Volasertib suppresses HCC growth

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However, the antitumor effect of volasertib on HCC is still unknown. In this study, we demon-strated that volasertib could potently suppress the growth of human HCC in vitro and in vivo.

Material and methods

Cell culture and reagents

Human hepatoma cancer cell lines BEL7402, HepG2, SMMC7721 and SK-Hep-1 were cul-tured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and strepto-mycin (100 ng/ml) in a humidified incubator at 37°C with 5% CO2. Volasertib was purchased from ApexBio Technology. N-acetly-L-cysteine (NAC) and dihydroethidium (DHE) were pur-chased from Sigma-Aldrich. Anti-PARP (9542), anti-β-tubulin (A11031) and Anti-GAPDH (KM- 9002) antibodies were from Cell Signaling Technologies, Ruiying Biological and Sungene Biotech, respectively.

Cell viability assays

Cells were firstly seeded into a 96-well plate at a density of 5~10×103 cells per well, and incu-bated with drugs in three parallel wells for 72 h. Then 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyl-tetrazolium bromide (MTT) was added to each well at a final concentration of 0.5 mg/ml. After incubation for 4 h, formazan crystals were dis-solved in 100 μl of DMSO, and absorbance at 570 nm was measured by plate reader. The concentrations required to inhibit growth by 50% (IC50) were calculated from survival curves using the Bliss method [18, 19]. For drug com-binational experiments, cells were treated with the indicated concentrations of volasertib com-bined with cisplatin for 72 h. The data were analyzed by CompuSyn software with the results showed as combination index (CI) val-ues according to the median-effect principle, where CI <1, =1, and >1 indicate synergism, additive effect and antagonism, respectively [20, 21].

Cell cycle assays

Cells were harvested and washed twice with cold phosphate-buffered saline (PBS), then fixed with ice-cold 70% ethanol for 30 min at 4°C. After centrifugation at 200×g for 10 min, cells were washed twice with PBS and resus-pended with 0.5 ml PBS containing PI (50 μg/

ml), 0.1% Triton X-100, 0.1% sodium citrate, and DNase-free RNase (100 μg/ml), and detected by FCM after 15 min incubation at room temperature in the dark. Fluorescence was measured at an excitation wavelength of 480 nm through a FL-2 filter (585 nm). Data were analyzed using ModFit LT 3.0 software (Becton Dickinson) [22, 23].

Apoptosis assays

Cell apoptosis was evaluated with flow cytome-try (FCM) assay. Briefly, cells were harvested and washed twice with PBS, stained with Annexin V-FITC and propidium iodide (PI) in the binding buffer, and detected by FACS Calibur FCM (BD, CA, USA) after 15 min incubation at room temperature in the dark. Fluorescence was measured at an excitation wave length of 480 nm through FL-1 (530 nm) and FL-2 filters (585 nm). The early apoptotic cells (Annexin V positive only) and late apoptotic cells (Annexin V and PI positive) were quantified [24, 25].

Reactive oxygen species (ROS) assay

Cells were incubated with 10 μM of DHE for 30 min at 37°C, and observed under fluorescence microscope (Olympus, Japan) immediately after washing twice with PBS. Five fields were taken randomly for each well [26, 27].

Western blot analysis

Cells were harvested and washed twice with cold PBS, then resuspended and lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 ng/ml PMSF, 0.03% aprotinin, 1 μM sodium orthovanadate) at 4°C for 30 min. Lysates were centrifuged for 10 min at 14,000×g and supernatants were stored at -80°C as whole cell extracts. Total protein con-centrations were determined with Bradford assay. Proteins were separated on 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA and incubated with the indicated primary antibodies. Corresponding horseradish peroxidase (HRP)-conjugated sec-ondary antibodies were used against each pri-mary antibody. Proteins were detected using the chemiluminescent detection reagents and films [28, 29].

Immunofluorescence assay

BEL 7402 and Hep G2 cells grown on glass cov-erslips were washed thrice with PBS and fixed


2478 Am J Cancer Res 2016;6(11):2476-2488

in 4% paraformaldehyde. The fixed cells were washed thrice with PBS containing 0.05% Tw- een 20, and then treated with PBS containing 0.2% Triton X-100 for 10 min at room tempera-ture followed by washing, blocking and incuba-tion with anti-β-tubulin for 1 hr at 37°C. The coverslips were washed, treated with Hoechst 33342 (5 μg/ml in PBS) for 5 min, washed again. The stained cells were analyzed by using the inverted Olympus microscope IX 70 FluoView system [30, 31].

Nude mouse assays

Balb/c nude mice were obtained from the Ba- lb/c nude mice were obtained from the Guang- dong Medical Laboratory Animal Center and maintained with sterilized food and water. Six female nude mice with 5 weeks old and 20 g weight were used for each group. Each mouse was injected subcutaneously with BEL7402 and HepG2 cells (2×106 in 100 μl of medium) under the shoulder. When the subcutaneous tumors were approximately 0.3×0.3 cm2 (two perpendicular diameters) in size, mice were randomized into four groups, and were injected intraperitoneally with vehicle alone (0.5% car-boxymethyl cellulose) and volasertib alone (15 mg/kg) every two days, respectively. The body weights of mice and the two perpendicular

diameters (A and B) of tumors were recorded. The tumor volume (V) was calculated according to the formula:

v 6 2A B 3= +r ` j

The mice were anaesthetized after experiment, and tumor tissues were excised from the mice and weighted. The rate of inhibition (IR) was cal-culated according to the formula [32, 33]:

IR 1Mean tumor weight of control group

Mean tumor weight of experimental group100%= - #

Statistical analysis

All results are expressed as mean ± standard deviation (SD). Statistical analysis of the differ-ences between two groups is performed with Student’s t-test. Values of P<0.05 were consid-ered as significant differences.

Results

Volasertib inhibits the growth of HCC cells in vitro

To investigate the effect of volasertib on the growth of HCC, cell survival was detected by

Figure 1. Volasertib inhibits the growth of HCC cells in vitro. A: Chemical structure of volasertib. B: The representa-tive growth curves of cells treated with volasertib are shown. C: Summary of IC50 of volasertib in the indicated HCC cells is shown. Cells were treated with various concentrations of volasertib for 72 h and cell survival was determined by MTT assay. Datas were mean ± S.D. of three independent experiments.


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MTT assay. The four HCC cell lines BEL7402, HepG2, SMMC7721 and SK-Hep-1 were treat-ed with various concentration of volasertib for 72 hr. As shown in Figure 1, the survivals of four cell lines were decreased in a dose-depen-dent manner after volasertib treatment. BEL7402 cells were most sensitive to volasert-ib with the IC50 values of 0.006 μM. The IC50 values of volasertib in other three HCC cell lines HepG2, SMMC7721 and SK-Hep1 were 2.854 μM, 3.970 μM and 7.025 μM, respectively.

Volasertib induces cell cycle arrest at G2/M phase in HCC cells

To determine whether the growth inhibition of volasertib on HCC cells is the results of cell cycle arrest, both BEL7402 and HepG2 cells were treated with volasertib at the indicated concentrations for 48 hr. The cell cycle distribu-tion was assessed by FCM with PI staining, and analysed with ModFit LT 3.0 software. As shown in Figure 2, volasertib dose-dependently induced the accumulation in Sub G1 and G2/M

phase and reduction in G0/G1 phase in both BEL7402 and HepG2 cells.

Volasertib induces apoptosis in HCC cells

To further determine whether volasertib is able to induce apoptosis in HCC cells, both BEL7402 and HepG2 were treated with volasertib at the indicated concentrations for 48 hr, stained with Annexin V/PI and examined by FCM. As shown in Figure 3A-D, volasertib induced apoptosis in both BEL7402 and HepG2 cells in a dose-dependent manner. To investigate the molecu-lar mechanism of cell apoptosis by volasertib, the apoptosis related proteins are detected by Western blot. The marker of apoptosis, the cleaved PARP, was increased in a dose- and time-dependent manner after volasertib treat-ment (Figure 3E, 3F).

Volasertib promotes spindle abnormalities in HCC cells

PLK1 plays a critical role in the centrosome maturation and maintenance of the bipolar

Figure 2. Volasertib induces cell cycle arrest at G2/M Phase in HCC cells. BEL7402 (A) and HepG2 (B) cells were treated with volasertib at the indicated concentrations. The distribution of cell cycle was detected by FCM with PI staining. The percentages of sub G1, G1/G0, S, G2/M phase were calculated using ModFit LT 3.0 software. The rep-resentative charts, quantified results (C, D) of three independent experiments were shown. *P<0.05 and **P<0.01 vs. corresponding control.


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spindle [4]. To test whether volasertib promotes spindle abnormalities, both BEL7402 and HepG2 cells were treated with volasertib at the indicated concentrations for 24 hr and stained with Hoechst 33342 (to visualize chromosomal DNA, blue) and β-tubulin antibody (to visualize tubulin spindles, red). As shown in Figure 4, both BEL7402 and HepG2 cells after volasertib treatment exhibited profound abnormalities in

spindle formation, which lead to misalignment of chromosomes.

Volasertib enhances ROS accumulation in HCC cells

Reactive oxidative species (ROS) is important to involve in the sensitivity of cancer cells to anticancer agents [34]. To examine the role of

Figure 3. Volasertib induces apoptosis in HCC cells. BEL7402 (A) and HepG2 (B) cells were treated with volasertib at the indicated time and concentrations. The apoptosis was detected by FCM with Annexin V/PI staining. The propor-tions of Annexin V+/PI- and Annexin V+/PI+ cells indicated the early and late stage of apoptosis. The protein expres-sion was examined by Western blot, and GAPDH was used as loading control. The representative charts, quantified results (C, D) and Western blot results (E, F) of three independent experiments were shown. *P<0.05 and **P<0.01 vs. corresponding control.


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ROS in the anticancer effect of volasertib in HCC cells, the ROS fluorescent probe dihydro-ethidium (DHE) was used to stain both BEL7402

and HepG2 cells after volasertib treatment. As shown in Figure 5, volasertib dose- and time-dependently enhanced the fluorescent intensi-

Figure 4. Volasertib promotes spindle abnormalities in HCC cells. BEL7402 (A) and HepG2 (B) cells were treated with 10 nM and 100 nM of volasertib for 24 h respectively. Cells were fixed and stained with Hoechst 33342 (to stain DNA, blue) as well as with β-tubulin antibody (to visualize tubulin spindles, red). Cells were analyzed by immu-nofluorescence microscopy. Arrows indicate the spindle abnormalities of cells.


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ty of DHE in both cells, suggesting that volaser-tib can enhance ROS accumulation in HCC cells.

Inhibition of ROS partially rescues volasertib-induced apoptosis in HCC cells

To further study the role of ROS in volasertib-induced apoptosis in HCC cells, both BEL7402 and HepG2 cells were treated with volasertib

for 48 h with or without the ROS scavenger NAC at 5 mM pretreated for 1 h and stained with DHE. As shown in Figure 6A-D, the volasertib-induced DHE fluorescent signal was dramatically reversed by NAC in both cells. Additionally, the volasertib-induced apoptosis was partially inverted by NAC (Figure 6E-H), suggesting volasertib can induce both ROS dependent and independent apoptosis in HCC cells.

Figure 5. Volasertib enhances ROS accumulation in HCC cells. BEL7402 and HepG2 cells were treated with volas-ertib at the indicated times and concentrations, stained with DHE and photographed under florescent microscope. The representative micrographs (bright field - A, B and fluorescence - C, D) and quantified results (E, F) of three independent experiments were shown. *P<0.05; **P<0.01 vs. corresponding control.


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Figure 6. Inhibition of ROS partially rescues volasertib-induced apoptosis in HCC cells. BEL7402 (A) and HepG2 (B) cells were treated with volasertib at 30 nM and 10 μM respectively for 48 h in the presence or absence of 5 mM NAC pretreatment for 1 h, stained with DHE and photographed under florescent microscope. The representative micro-graphs and quantified results (C, D) of three independent experiments were shown. The apoptosis was detected by FCM with Annexin V/PI staining. The representative charts (E, F) and quantified results (G, H) of three independent experiments were shown. Vol: Volasertib. *P<0.05 and **P<0.01 versus corresponding control.

Volasertib suppresses the subcutaneous xeno-graft growth of HCC in nude mice

To further estimate the antitumor effects of volasertib in vivo, we generated the subcutaneous xenograft tumor models by transpl-

anting both BEL7402 and HepG2 cells into nude mice. As shown in Figure 7, volasertib at 15 mg/kg significantly inhibited the tumor growth by reducing the volume and weight of both BEL7402 and HepG2 tumors. The inhibi-tion rates of tumor growth in BEL7402 and


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Figure 7. Volasertib suppresses the subcutaneous xenograft growth of HCC in nude mice. Each mouse was injected subcutaneously with BEL7402 and HepG2 cells (2×106 in 100 μl of medium) under the shoulder. When the tumors developed into 0.3×0.3 cm2 (two perpendicular diameters) in size, mice were randomized into two groups, and were intraperitoneally injection with vehicle (0.5% carboxymethyl cellulose) and volasertib (15 mg/kg) every two days, respectively. The body weights of mice and the two perpendicular diameters of the tumor were recorded. The mice were anaesthetized after experiment, and tumor tissue was excised from the mice and weighed. The original tumors (A, B), tumor volume (C, D), tumor weight (E, F), body weight (G) and summary data (H) were shown. The values presented are the means ± SD for each group. *P<0.05 and **P<0.01 vs. corresponding control.


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HepG2 was 75.4% and 52.9%, respectively. Ad- ditionally, the weight of nude mice did not exhibit the statistical difference during experi-ments, which suggests volasertib at the indi-cated dose did not cause toxicities in mice (Figure 7G).

Discussion

In this study, we show that volasertib can potently induce cell growth inhibition, cell cycle arrest at G2/M phase and apoptosis with abnormal spindle formation in human HCC cells in vitro and in vivo, which are consistent with previous reports that have demonstrated that volasertib is able to induce mitotic arrest and apoptosis by targeting PLK1 in several other cancer models [13, 14]. Furthermore, volasert-ib also increases the intracellular ROS levels, and pretreated with ROS scavenger NAC par-tially reverses volasertib-induced apoptosis. It has been reported that another PLK1 inhibitor BI 2536 alone or in combination with histone deacetylase (HDAC) inhibitor vorinostat could also enhance the intracellular ROS production to trigger cell death either in imatinib mesylate-sensitive or -resistant BCR/ABL+ leukemia cells, and the ROS scavenger Mn (III) tetrakis (4-ben-zoic acid)porphyrin chloride (MnTBAP) blocks ROS generation as well as lethality in BI 2536/vorinostat-treated cells [35]. Similarly, BI 2536 alone or combined with microtubule-destabiliz-ing drug vincristine dramatically promotes the production of ROS to induce cell apoptosis in rhabdomyosarcoma cells, and the antioxidants MnTBAP, NAC as well as α-tocopherol signifi-cantly rescue from BI 2536/vincristine-induced apoptosis [36]. Therefore, ROS plays an impor-tant role in cancer treatment with PLK1 inhibi-tors including volasertib, which usually triggers cancer cell death by increasing the intracellular ROS.

Currently, the clinical data indicate that volas-ertib has the promising clinical efficacy in a wide range of malignancies. The phase I clinical trials of volasertib show a favorable pharmaco-kinetic profile and encouraging preliminary anti-tumour activity with manageable toxicities such as anaemia, neutropenia and thrombocytope-nia [37-40]. But volasertib has insufficient anti-tumor activity as a monotherapy in the phase II trial of locally advanced or metastatic urothe-lial cancer [41]. Accordingly, multiple groups

have investigated the combined anticancer effects of volasertib and other chemotherapeu-tic drugs in the preclinical and clinical studies. Combination of volasertib with microtubule-interfering drugs including vincristine, vinblas-tine, vinorelbine or eribulin shows the synergis-tic induction of apoptosis in Ewing sarcoma and other pediatric malignant cells [42, 43]. Vol- asertib alone or in combination with azaciti-dine, cytarabine, decitabine and quizartinib is highly potent in multiple preclinical models of acute myeloid leukemia (AML) [44]. Moreover, volasertib powerfully inhibits the growth of bladder and cervical cancer cells, and potenti-ates the activity of cisplatin [17, 45]. Inspiringly, a recent phase I study has demonstrated that volasertib plus cisplatin or carboplatin has manageable safety and antitumor activity without pharmacokinetics interference [46]. Another phase I study shows that volasertib combined with nintedanib also has manageable safety profile and antitumor effect without clearly pharmacokinetic disturbance [47]. But volaser-tib in combination with afatinib exhibits limited antitumor activity in a phase I study of 57 patients with advanced solid tumors [48]. In a phase II trial including 87 patients with AML, co-treatment with volasertib and cytarabine significantly prolongs median overall survival and median event-free survival but increases frequency of side effects compared with cyta-rabine alone [49]. But the results of another phase II trial in patients with advanced non-small-cell lung cancer exhibits that volasertib in combination with pemetrexed does not improve efficacy without increasing adverse events and pharmacokinetics interference compared with pemetrexed alone [50]. However, the combina-tion of volasertib with other anticancer agents for the treatment of HCC still needs to be test-ed in the future.

In summary, our study demonstrated that vola-sertib could potently induce cell growth inhibi-tion, cell cycle arrest and apoptosis in HCC cells in vitro and in vivo. The beneficial therapy of volasertib appears to be potential treatment strategy in patients with HCC.

Acknowledgements

This work was supported by funds from the Chinese National Natural Science Foundation No. 31271444 and No. 81201726 (Z. S.), and


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No. 31171304 (X. W.), the Guangdong Natural Science Funds for Distinguished Young Scholar No. 2014A030306001 (Z. S), the Guangdong Special Support Program for Young Talent No. 2015TQ01R350 (Z. S.) and the Science and Technology Program of Guangdong No. 2016A- 050502027 (Z. S.) and No. 2013B090500109 (X. W.), the Science and Technology Program of Guangzhou No. 201510010123 and No. 201300000041 (X. W.), and the Foundation for Research Cultivation and Innovation of Jinan University No. 21616119 (Z. S.).

Disclosure of conflict of interest

None.

Address correspondence to: Drs. Zhi Shi and Xing Wei, Department of Cell Biology & Institute of Bio- medicine, College of Life Science and Technology, Jinan University, National Engineering Research Center of Genetic Medicine, Guangdong Provincial Key Laboratory of Bioengineering Medicine, Room 708, The 2nd Engineer and Scientific Building, 601 Huangpu West Road, Guangzhou 510632, China. Tel: +86-20-852-245-25; Fax: +86-20-852-259-77; E-mail: [email protected] (ZS); [email protected] (XW)

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