Tumor Biol. (2013) 34:2241–2247 DOI 10.1007/s13277-013-0764-5

RESEARCH ARTICLE

Scytonemin inhibits cell proliferation and arrests cell cycle through downregulating Plk1 activity in multiple myeloma cells Guojun Zhang & Zhe Zhang & Zhuogang Liu

Received: 16 February 2013 / Accepted: 22 March 2013 / Published online: 13 April 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract Multiple myeloma is the second most common hematologic malignancy. During the pursuit for novel and more selective anticancer drugs, different approaches have pointed to polo-like kinase 1 (Plk1) as a promising target. So we used a novel agent, scytonemin, to inhibit the activity of Plk1 to investigate the effect of Plk1 in multiple myeloma cells. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to examine the effect of scytonemin on the cell proliferation of three multiple myeloma cell lines with different concentration and different time. Flow cytometry was used to examine the effect of scytonemin on the cell cycle of multiple myeloma U266 cells with different concentration and different time. Moreover, the expression of Plk1 was analyzed by Western blot and real-time PCR in myeloma U266 cells with the treatment of scytonemin. Statistical analysis was used to analyze the effect of scytonemin on the cell proliferation and cell cycle with different concentration and different time and the association between Plk1 expression and activity with the treatment of scytonemin. Scytonemin was able to inhibit the proliferation of three myeloma cells in a G. Zhang Department of Hematology, Shengjing Hospital of China Medical University, Shenyang City 110022 Liaoning Province, China e-mail:[email protected]. Z. Zhang Department of Urology, The First Hospital of China Medical University, 155 Nanjing North Street, Heping District, Shenyang City 110001 Liaoning Province, China e-mail: [email protected]. Z. Liu (*) Department of Hematology, Shengjing Hospital of China Medical University, 39 Huaxiang Road, Tiexi District, Shenyang City 110022 Liaoning Province, People’s Republic of China e-mail: [email protected]

dose-dependent manner, while U266 was the most sensitive one to scytonemin. Treatment with 3 and 4 μM scytonemin gradually increased the percentage of cells in the G2-M phase in U266 cells upon 48- and 72-h treatment. Scytonemin (at 3 and 4 μM concentration) inhibited multiple myeloma cell growth associated with downregulation of the activity of Plk1 but no effect on the expression of Plk1. Scytonemin, representing a novel Plk1 inhibitor, induced the inhibition of cell growth and cell cycle arrest in multiple myeloma cells by specifically decreasing Plk1 activity. Taken together, scytonemin is a promising novel agent for the treatment of multiple myeloma. Keywords Scytonemin . Multiple myeloma . Polo-like kinase 1 . Cell proliferation . Cell cycle

Introduction Multiple myeloma (MM) is the second most common hematologic malignancy [1]. The disease is characterized by the accumulation of mature antibody-producing plasma cells in the bone marrow [2]. Despite the recent advances in the therapy of MM and the emergence of novel agents such as bortezomib, thalidomide, and lenalidomide [3–5], the disease remains incurable. Patients eventually become resistant to chemotherapy and die of disease progression or therapy complications [6]. Novel drugs are urgently needed to overcome drug resistance. During the pursuit for novel and more selective anticancer drugs, different approaches have pointed to polo-like kinase 1 (Plk1) as a promising target. Plk1 is a key cell cycle regulator promoting entry into mitosis, spindle formation, sister chromatid segregation and cytokinesis [7, 8].

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This protein is not only highly expressed in various human malignant neoplasms but is also closely related to poor prognosis, corroborating its importance as a diagnostic marker. Scytonemin, a pigment isolated from cyanobacteria [9], is the first described small molecule inhibitor of human polo-like kinase. Scytonemin inhibited Plk1 activity in a concentration-dependent manner with an IC50 of 2 mM against the recombinant enzyme [10]. So, in this study, we used the novel agent to inhibit the activity of Plk1 to investigate the effect of Plk1 in MM cells.

Materials and methods Cell line and cultures The MM cell line U266, RPMI8226 and NCI-H929 were obtained from ATCC (Rockville, MD, USA). All cell types were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) containing 10 % fetal bovine serum (Invitrogen) and incubated in 5 % CO2 at 37 °C. Treatment with scytonemin Scytonemin was given by Dr. Peter Richter (Department of Plant Ecophysiology, Friedrich-Alexander University, Erlangen-Nürnberg, Germany) as a gift and initially dissolved in dimethylsulfoxide (DMSO), stored at −20 °C, and then thawed before use. For all experiments, cells were treated at various concentrations (1, 2, 3 and 4 μM) in accordance with previous experiments [11, 12]. Corresponding control cultures received an equal volume of solvent. Growth inhibition assay The antiproliferative effect of sytonemin on U266 cells was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) dye uptake method. In brief, the cells (50,000 per well) were incubated in quintuplicate in 96-well plates. Sytonemin was added, with a final concentration of 1, 2, 3 and 4 μmol/l, and the cells were treated for 24, 48, and 72 h. Thereafter, 20 μl MTT (5 mg/ml in phosphate-buffered saline) was added to each well. After 4 h at 37 °C, the supernatant was removed, and 150 μl Table 1 The inhibition rate of U266 cell proliferation with treatment of scytonemin (%, mean ± SD) *P<0.05, significantly different compared with the former time # P < 0.05, different compared with the former concentration

DMSO was added. When the blue crystal was dissolved, the optical density (OD) was detected at a 490-nm wavelength using a 96-well multiscanner autoreader (Model 680; Bio-Rad, UK), with 630 nm as the reference wavelength. The following formula was used: cell proliferation inhibited =(1−A490 of experimental well/A490 of blank control well)× 100 %. IC50 was the concentration that caused a 50 % inhibition of cell proliferation. Cell cycle assay by flow cytometry U266 cells in logarithmic growth phase were plated in 25-cm2 flasks and incubated overnight. Then they were treated with different concentrations of scytonemin for 48 h. Briefly, the cells were collected by centrifugation, washed in PBS and fixed in precolding 70 % ethanol for 1.5 h at 4 °C. After fixation, the cells were washed in PBS again and centrifuged 5 min at 1,000 rpm. The PBS was discarded and PI (KeyGEN Biotech Co., Ltd., Nanjing, China) was added to final concentration 50 μg/ml with dark at 4 °C for 30 min. Flow-cytometric analysis was performed on the FACSCalibur flow-cytometer (Becton Dickinson, Oxford, UK). Finally, the cells were analyzed using BD FACSDiva flow cytometry (BD FACS Canto™ II, Florida, USA) with Cell Quest software. Real-time PCR analysis Total RNA was isolated from cultured cells with ice-cold TRIZOL reagent (Invitrogen), according to the manufacturer’s specifications. RNA concentration was determined by Thermo Scientific NanoDrop ND-100 (Wilmington, DE, USA). Next, reverse transcription of 2 μl of total RNA was performed using Perfect Real-Time SYBR® PrimeScript® RT-PCR Kit (Takara, Kyoto, Japan). Real-time PCR analysis of the cDNA was quantified using Thermal Cycler Dice™ Real-Time System TP800 (Takara, Kyoto, Japan). The RT reaction system was maintained at 50 °C for 2 min and heated to 95 °C for 10 min. Then the PCR reaction was done by 35 cycles, denaturing the mixture at 95 °C for 15 s, annealing at 50 °C for 30 s and extension at 72 °C for 30 s. β-Actin was chosen as the internal control. Primer sequences used for Plk1 and β-actin were as follows: Plk1 sense primer: 5′CAAGAAGAATGAATACAGTA-3′, Plk1 antisense primer:

Concentration of scytonemin

Time with scytonemin

24 h 48 h 72 h

0 μM

1 μM

2 μM

3 μM

4 μM

0±0 0±0 0±0

8.51±1.63 13.23±2.58 11.92±2.29

16.59±1.46 22.66±4.97 18.49±3.57

24.92±2.88* 48.60±5.64*# 45.69±11.36*

33.86±5.44 55.92±4.83# 55.11±5.12

Tumor Biol. (2013) 34:2241–2247 Table 2 The inhibition rate of RPMI8226 cell proliferation with treatment of scytonemin (%, mean ± SD) *P<0.05, significantly different compared with the former time

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Concentration of scytonemin

Time with scytonemin

P<0.05, significantly different compared with the former concentration

#

24 h 48 h 72 h

5′-GGATATAGCCAGAAGTAA-3′, β-actin sense primer: 5′CTCCATC CTGGCCTCGCTGT −3′, β-actin antisense primer: 5′-GCTGTCACCTTCACCGTTC-3′. Negative controls consisted of distilled H2O. The expression levels of Plk1 were normalized with β-actin using a 2−ΔΔCT method. A melting curve analysis was performed for the PCR products to evaluate primer specificity. Western blot Frozen cells were washed twice with ice-cold phosphatebuffered saline (PBS), homogenized on ice in 10 volumes (w/v) of lysis buffer containing 20 mM Tris–HCl, 1 mM EDTA, 50 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 1 % Triton-×100, 1 mM PMSF and phosphatase inhibitor using a homogenizer (Heidoph, DLA×900, Germany). The homogenate was centrifuged at 10,000 rpm for 30 min at 4°C. The supernatant was collected and stored at −70°C. Protein content was determined by the BCA assay (BCA protein assay kit-23227; Pierce Biotechnology, Rockford, IL, USA). From each sample preparation, 40 μg of total protein was separated by 8 % SDS-PAGE and then transferred to PVDF blot membranes. The total protein extracts were analyzed by immunoblot with indicated antibodies following SDSPAGE analysis. Immunoblots were performed using mouse monoclonal antibodies for Plk1 (Abcam, Massachusetts, USA) and mouse monoclonal antibody for β-actin (Abcam, Hong Kong; a housekeeping protein used as a loading control to assure equal amounts of protein in all lanes). After blocking nonspecific binding with 5 % BSA in TBS (pH 7.5) containing 0.05 % Tween-20 (TBST), primary antibodies were incubated on the membranes for Plk1 (1:2,000) and β-actin (1:2,000) overnight at 4°C in TBST. After washing three times in TBST, the membranes were incubated for 2 h at 37°C with goat polyclonal secondary antibody to mouse IgG (1:5,000; Abcam, Hong Kong) Table 3 The inhibition rate of NCI-H929 cell proliferation with treatment of scytonemin (%, mean ± SD)

0 μM

1 μM

2 μM

3 μM

4 μM

0±0 0±0 0±0

4.35±0.94 7.39±1.32 8.99±1.54

6.77±2.30 11.68±3.03 14.38±1.35

12.5±2.98 19.64±3.21 22.35±3.44*

20.16±3.83 37.94±5.95*# 40.41±4.97#

labeled with horseradish peroxidase. The proteins were detected using an ECL detection system (Pierce), as directed by the manufacturer. Specific bands for Plk1 and β-actin were identified by prestained protein molecular weight marker (MBI Fermentas, USA). The EC3 Imaging System (UVP Inc., Pennsylvania, USA) was used to catch up the specific bands, and the OD of each band was measured using Image J software. The ratio between the OD of interest proteins and β-actin of the same sample was calculated as relative content and expressed graphically. Assay of Plk1 activity U266 cells with treatment of different concentration of scytonemin were collected. Kinase reactions were initiated by adding 20 μM cold ATP and 5 μCi of [γ-32P] ATP to 30 ml of Plk1 kinase buffer (50 mM Tris–HCl, pH 7.4, 2 mM EDTA, pH 8.0, 2 mM dithiothreitol, 10 mM MgCl2, 50 mM NaF, 2 mM Na 3 VO 4 , and 10 mM glycerophosphate). Dephosphorylated casein of 6 μg was used as substrate and the reaction buffer was incubated at 30°C for 7 min. After 25-μl aliquots were spotted on Whatman p81 paper, the reaction was stopped with 5 % H3PO4 solution. After thorough washing, the radioactivity on the filter paper was counted with a Beckman scintillation counter (Beckman Coulter Inc., Florida, USA). Each experiment was repeated at least three times. Statistical analysis SPSS 17.0 was used to perform data analysis. The t-test was used to analyze the data from real-time PCR, Western blot and activity assay in the cells. Spearman’s correlation test was used to analyze the rank data and Fisher’s exact test to compare the different rates. P values of <0.05 were considered statistically significant. Concentration of scytonemin

Time with scytonemin

24 h 48 h 72 h

0 μM

1 μM

2 μM

3 μM

4 μM

0±0 0±0 0±0

3.78±0.59 5.15±1.29 7.81±2.49

5.64±1.20 11.52±2.83 13.18±3.69

8.84±1.35 14.20±2.07 19.76±1.50*

15.07±5.19 19.48±1.92 22.36±2.72

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Results Scytonemin inhibits cell proliferation of MM cells in vitro To explore whether scytonemin might be a potential therapeutic agent against MM, we investigated its antiproliferative effects on three MM cell lines: U266, RPMI8226 and NCIH929. Although the three MM cell lines exhibited different sensitivities, scytonemin was able to inhibit proliferation of all cells in a dose-dependent manner (Tables 1, 2 and 3; Fig. 1a– c). While NCI-H929 was the least sensitive cell line, U266 was the most sensitive one to scytonemin. Scytonemin induces G2-M cell cycle arrest of MM From the results described above, it became clear that scytonemin was efficient in inhibiting proliferation of myeloma cells. We then wished to examine if sytonemin induced cell cycle arrest which then led to the observed decrease in proliferation. For this, we treated U266 cells with 1, 2, 3 and 4 μM of the drug for 24, 48 or 72 h. Following the incubation, we measured the population of cells in the different stages of the cell cycle. U266 cells treated with 1, 2, 3 and 4 μM of scytonemin were collected at different time points (24, 48, or 72 h), and then analyzed with flow cytometry. Treatment with 3 and 4 μM scytonemin gradually increased the percentage of cells in the G2-M phase in U266 cells upon 48- and 72-h treatment, it had no significant effect on G2-M phase in U266 cells either with 1 and 2 μM scytonemin or with 12-h treatment (Table 4, Fig. 2). Scytonemin inhibits MM cell growth associated with downregulation of the activity of Plk1 but has no effect on the expression of Plk1 It remains unclear why scytonemin affected G2-M phase in U266 cells only by 48-h treatment; however, scytonemin is a specific inhibitor to Plk1. In order to observe whether Plk1 plays a role in the cell cycle of U266 cells, the expression of Plk1 was examined by real-time PCR and Western blot, and the activity of Plk1 was assayed by using dephosphorylated casein as a substrate. We found that with the treatment of scytonemin the expression of Plk1 did not change in an obvious manner (Fig. 3a–c). However, the activity of Plk1 decreased with the treatment of 3–4 μM of scytonemin at 48 h (Fig. 3d). The result suggested that scytonemin inhibited the activity of Plk1, which may be one of the reasons why scytonemin could lead to G2-M arrest of MM. Fig. 1 Proliferation ability of three multiple myeloma cell lines were„ inhibited by scytonemin. a MTT assay was used to examine U266 cell proliferation. b MTT assay was used to examine RPMI8226 cell proliferation. c MTT assay was used to examine NCI-H929 cell proliferation. The data are representative of three individual experiments

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Tumor Biol. (2013) 34:2241–2247 Table 4 Flow cytometry analysis of U266 cells treated with different concentrations of scytonemin (%, mean ± SD)

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Concentration of scytonemin

*P<0.05, significantly different compared with former concentration group

Discussion MM, a type of malignant plasma cell dyscrasia originating from B lymphocytes, produces abundant monoclonal immunoglobulins [13]. These neoplastic plasma cells are believed to arise from a post-germinal center B cell that migrates to the bone marrow, adheres to the marrow stroma and triggers subsequent bone resorption and a paracrine cytokine loop [14]. The natural course of the disease may progress through monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma, intramedullary myeloma and finally, extramedullary myeloma [15]. MM is still an incurable disease. Although conventional cytotoxic chemotherapy prolongs survival in symptomatic patients, the prognosis of these patients remains poor. After treatment with conventional cytotoxic agents, relapses still inevitably occur, indicating the need for continued investigation of novel agents to treat this disease. Multiple studies have suggested that scytonemin possesses potent anti-tumor activity in hematologic malignancies, although the effects and mechanisms behind this activity are still not very clear. Recent studies have suggested that Plk1 is involved in the proliferation of cell types such as breast cancer, lung cancer, prostate cancer and Fig. 2 G2-M cell cycle arrest of U266 cell was induced by scytonemin. Flow cytometry system (FCS) was used to examine U266 cell cycle. With the increasing concentration of scytonemin, the number of U266 cells at G0–G1 stage decreased and that of U266 cells at G2-M stage increased, but the number of U266 at S stage did not obviously change. The data were representative of three individual experiments

0 1 2 3 4

μM μM μM μM μM

G0/G1

S

G2/M

52.2±3.12 43.47±1.46 36.33±2.27 22.57±1.80* 12.63±3.43*

15.93±11.86 13.27±1.60 12.13±1.31 8.5±0.79* 5.6±0.62

31.87±1.52 43.27±3.05 51.53±1.00 68.93±1.12* 81.77±2.80*

esophageal cancer [16–19]. We have also shown that scytonemin, a small molecule inhibitor of Plk1, significantly reduced cell proliferation and cell invasion [11, 12, 20]. Plk1 is therefore a promising gene candidate for targeted therapy in treating not only solid tumors but hematological malignancies as well. In the present study, we demonstrated the antiproliferative effects of scytonemin, a specific Plk1 inhibitor on three myeloma cell lines U266, RPMI8226 and NCI-H929. Abrogation of capacity of forming colonies by BI2536, a synthetic ATP-competitive Plk1 inhibitor, has previously been reported for myeloid leukaemia cells [21] and in two osteosarcoma cell lines [22]. Our results showed that concentrations (3 and 4 μmol/l) of scytonemin significantly decreased cell proliferation and inducing G2-M arrest. We found that U266 was the most sensitive one to scytonemin. Our results also showed that treatment with scytonemin provoked a clear disturbance of cell cycle phase distribution, with an accumulation of cells in G2-M after 48 h. This increase in doubled-DNA cells was previously reported after Plk1 inhibition by BI2536 in osteosarcoma cell lines [22, 23]. Specially, the latter further demonstrated by cytogenetic analysis a blockage in mitosis with higher accumulation (~60 %) of dividing cells after 24 h, and increased aneuploidy in the subsequent cycles [22].

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Fig. 3 Expression of Plk1 in U266 cells with treatment of scytonemin. a Band intensities indicated expression level of Plk1 in U266 cells with different concentration of scytonemin. β-actin was used as a loading control to assure equal amounts of protein in all lanes. b The ratio between the optical densities of Plk1 and β-actin of the same group

through Western blot was calculated and expressed graphically. c The ratio between the CT value of Plk1 and β-actin of the same group by real-time PCR was calculated and expressed graphically. The data were representative of three individual experiments. *P<0.05, relative to former concentration (d)

In the present study, U266 cells with the treatment of scytonemin resulted in a striking elevation in the activity of Plk1 in a concentration- and time-dependent manner, but did not change the protein and mRNA levels of Plk1, suggesting that scytonemin inhibited the proliferation of U266 and induced the cell cycle arrest in U266 cells by downregulating the activity of Plk1. In conclusion, in the present study we have shown that scytonemin, representing a novel Plk1 inhibitor, induces the inhibition of cell growth and cell cycle arrest in MM cells by specifically decreasing the Plk1 activity. Taken together, scytonemin is a promising novel agent for the treatment of MM. Further studies will investigate the in vivo efficacy of scytonemin alone and particularly in combination regimens with some chemotherapy drugs.

Conflicts of interest None

Acknowledgements We thank Dr. Peter Richter (Department of Plant Ecophysiology, Friedrich-Alexander University Erlangen-Nürnberg, Germany) for presenting sytonemin as a gift.

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