Journal of Surgical Oncology 2011;103:704–715

Downregulation of Stathmin Is Involved in Malignant Phenotype Reversion and

Cell Apoptosis in Esophageal Squamous Cell Carcinoma

FENG WANG, MD,1 LIU-XING WANG, MD,1 SHENG-LEI LI, MD,2 KE LI, MD,1 WEI HE, MD,1

HONG-TAO LIU, MD,3* AND QING-XIA FAN, MD1**

1Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, P.R. China2Department of Pathology, The First Affiliated Hospital of Zhengzhou University, Henan Key Laboratory of Tumor Pathology, Zhengzhou,

Henan, P.R. China3Laboratory for Cell Biology, Department of Bioengineering of Zhengzhou University, Zhengzhou, Henan, P.R. China

Background and Objectives: Stathmin plays a critical role in the regulation of mitosis and mediates the development of malignant tumors. Here, we

investigated the potential role of stathmin in cell cycle and apoptosis in esophageal squamous cell carcinoma (ESCC).

Methods: A stathmin short hairpin RNA (shRNA) plasmid was employed to downregulate stathmin expression in the ESCC cell line EC9706 cells.

Cell proliferation was measured by cell counting, MTT, and colony formation assay. Cell migration was measured by Boyden chamber. Western blot

was used to analyze the expressions of stathmin, survivin, and apoptosis-related proteins in transfected cells. Cell cycle and apoptosis were

determined by flow cytometry and DNA ladder. Oncogenicity assay in nude mice was utilized to analyze phenotypic changes of transfected cells in

vivo.

Results: After transfection with stathmin shRNA plasmid, stathmin expression markedly decreased in EC9706 cells. Stathmin downregulation

significantly inhibited cell proliferation, cell migration in vitro, and tumorigenicity in vivo, meanwhile arrested cell cycle in the G2/M phase and

induced cell apoptosis. Further, stathmin downregulation resulted in downregulation of Bcl-2 and survivin proteins, activation of Caspase-3.

Conclusions: These findings demonstrate that stathmin may play an essential role in carcinogenesis of ESCC, which will lay a foundation for target

therapy of ESCC.

J. Surg. Oncol. 2011;103:704–715. � 2011 Wiley-Liss, Inc.

KEY WORDS: esophageal squamous cell carcinoma; stathmin; cell cycle; apoptosis; RNA interference

INTRODUCTION

The dynamics of microtubule polymerization/depolymerization

during the different phases of the cell cycle are regulated by two major

classes of proteins, microtubule-stabilizing and microtubule-destabiliz-

ing factors [1]. Stathmin family phosphoproteins (stathmin, SCG10,

SCLIP, RB3/RB30/RB300) are important signal transduction molecules

and regulators of microtubule dynamics [2]. Stathmin (Oncoprotein 18)

is a confirmed member of the family of microtubule-destabilizing

proteins that play a critical role in the regulation of mitosis [3]. It is

well documented that stathmin may have a direct role in the regulation of

mitosis came from genetic studies that stathmin expression interferes

with the progression of cells during mitosis [4,5]. Stathmin is overex-

pressed across a broad range of human malignancies (leukemia, lym-

phoma, neuroblastoma; ovarian, prostatic, breast, lung cancers;

mesothelioma; Wilms tumor; adenoid cystic carcinoma of the salivary

glands). Most importantly, those cancers with overexpression of stath-

min would lead to poor prognosis [6,7], suggesting stathmin plays a

crucial role in maintenance of malignant phenotypes in various human

cancers [8,9].

In addition, inhibition of stathmin expression results in growth

suppression, cell cycle arrest in the G2/M phase and malignant tumor

phenotype reversion [10,11]. A previous study from our laboratory

showed that stathmin is also expressed at high level in esophageal

squamous cell carcinoma (ESCC). Thus, it is believed that stathmin

may provide an attractive molecular target for disrupting the mitotic

apparatus and arresting the proliferation of malignant cells. RNA inter-

ference (RNAi) has been verified to be an extremely useful experimental

tool for the study of genes functions [12]. In the present study, we

employed short hairpin RNA (shRNA)-triggered RNAi targeting stath-

min to explore the potential of new therapeutic targets in the treatment of

human esophageal carcinoma. The experiments described in this report

support that anti-stathmin could reverse the malignant phenotype of

esophageal carcinoma.

MATERIALS AND METHODS

Construction of Plasmid Expressing shRNA for Stathmin

The DNA oligonucleotides coding for the short hairpin (sh) stathmin

were designed and synthesized as follows:

50-GATCCCCctggagaagcgtgcctcagTTCAAGAGActgaggcacgcttctc-

cagTTTTTGGAAA-30;50-GATCCCCgaaagacgcaagtcccatgTTCAAGAGAcatgggacttgcgtctttc-

TTTTTGGAAA-30;50-GATCCCCacgagagcacgagaaagaaTTCAAGAGAttctttctcgtgctctcgt-

TTTTTGGAAA-30;

Grant sponsor: Natural Science Foundation of Henan Province; Grant num-ber: 072102310054; Grant sponsor: Natural Science Foundation of Edu-cation Department of Henan Province; Grant number: 2010A310008.

*Correspondence to: Hong-Tao Liu, MD, Laboratory for Cell Biology,Department of Bioengineering of Zhengzhou University, Zhengzhou, Henan450001, P.R. China. E-mail: liuht1230@126.com

**Correspondence to: Qing-Xia Fan, MD, Department of Oncology, TheFirst Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, P.R.China. Fax: 86-371-66295953 E-mail: fqx2243@yahoo.com.cn

Received 13 June 2010; Accepted 3 January 2011

DOI 10.1002/jso.21870

Published online 24 February 2011 in Wiley Online Library(wileyonlinelibrary.com).

� 2011 Wiley-Liss, Inc.

50-GATCCCCcgtttgcgagagaaggataTTCAAGAGAtatccttctctcg-

caaacgTTTTTGGAAA-30.

All these sequences were inserted between BglII and HindIII restric-

tion sites of pSUPER-EGFP plasmid (pSUPER RNAi SystemTM,

OligoEngine). pSUPER-EGFP plasmid named as pSE was control

plasmid. The recombinant plasmids were named as pSE-sh1, pSE-

sh2, pSE-sh3, and pSE-sh4, respectively. The recombinant vectors were

confirmed by the digestion analysis of restriction endonuclease and

DNA sequencing by TaKaRa Biotech Company using ABI PRISM

SigDyeTM Terminator Cycle Sequencing Ready Reaction Kit with

AmpliTaq DNA Polymerase FS (Perkin-Elmer, Waltham, MA).

Stable Transfection of Plasmids and Selection

ESCC cell line EC9706 cells, as a gift from Tumor Research Insti-

tute, Chinese Academy of Medical Sciences, was used in this study.

EC9706 cells were seeded in a six-well plate at 5 � 105 cells/well, and

cultured overnight about 90% confluence prior to transfection. Sub-

sequently, transfection was performed using Lipofectamine 2000 (Invi-

trogen, Inc. Carlsbad, CA) according to the manufacturer’s instructions.

Forty-eight hours after pSE-sh1–4 transfections, stable cell lines were

selected with G418 (600 mg/ml). Five stably transfected EC9706 cells

(transfected with pSE-sh1, pSE-sh2, pSE-sh3, pSE-sh4, or pSE plas-

mid) were established.

Antibodies

Stathmin PcAb (CST-3352) was purchased from Cell Signaling

Technology, Inc. (Danvers, MA); Caspase-3 PcAb (sc-1225), bcl-2

McAb (sc-7382), survivin McAb (sc-8807), P53 PcAb (sc-100) and

human Actin PcAb (sc-1616), horseradish peroxidase (HRP) conju-

gated goat anti-mouse (sc-2060), and goat anti-rabbit IgG antibodies

(sc-2004) were all purchased from Santa Cruz Biotechnology (Santa

Cruz, CA).

RT-PCR Analysis of Stathmin in Transfected Cells

Total cellular RNA was isolated using Trizol reagent (Invitrogen)

according to the manufacturer’s instructions. Briefly, 5 mg of total RNA

was reverse-transcribed to the cDNA using AMV reverse transcriptase

(Takara Bio Inc. Shiga, Japan). Human GAPDH RNA was used as an

internal control. Stathmin (F: 50-atggcttcttctgatatccagg-30; R: 50-ttagt-

cagcttcagtctcgtca-30, product size is 450 bp) and Human GAPDH (F: 50-aaggtcggagtcaacggatttg-30; R: 50-cttgacaaagtggtcgttgagg-30, product

size is 915 bp) primers were designed to amplify the specific band

using the procedures described in the following: initial denaturation at

948C for 3 min, followed by of 30 sec at 948C, 30 sec at 558C, and

60 sec at 728C for a total of 35 cycles of stathmin and 25 cycles of

GAPDH; and a final extension at 728C for 10 min. After amplification,

10 ml of PCR products were resolved on 1.5% agarose gel. DNA bands

were visualized by UV light and documented with a Gene Tools (Model

P67UA).

Western Blot Analysis of Stathmin in Transfected Cells

All the group of EC9706 cells were collected by centrifugation for

protein extraction using RIPA lyses buffer and 1 tablet/10 ml of pro-

tease inhibiter cocktail tablets (Roche Inc company, Nutley, NJ),

respectively. Briefly, 60 mg of total cellular proteins were separated

on 12% SDS–PAGE, then transferred to polyvinylidene difluoride

(PVDF) membranes (Promega Corporation, Madison, WI). Non-

specific binding sites were blocked by incubating the membranes in

TBS–0.1% Tween-20 with 5% skimmed milk for 1 hr at room tempera-

ture (RT), the membranes were incubated with primary antibodies anti-

stathmin (1:200 dilution for stathmin; 1:1,000 dilution for human b-

actin) for 3 hr at RT, and then washed three times with TBS-T. The

membranes were then incubated with HRP-conjugated goat anti-mouse

(or goat anti-rabbit) IgG (1:3,000 dilution) for 1 hr at RT, and washed

three times with TBS-T. The blots were developed according

to enhanced chemiluminescence (ECL) detection kit (Promega

Corporation, Madison, WI), and protein relative expressions were

analyzed using Scion Image Software. Human b-actin was used as

an internal control.

Cell Proliferation Was Measured by Cell Counting Assay

EC9706 cells transfected steadily with pSE-sh2 or pSE were seeded

in a 24-well plate at a concentration of 4 � 104 cells/well. The cell

cultures were measured for cell proliferation levels at different time

points (24, 48, 72, and 96 hr) using ADAM-MC CELL COUNTESS

(Digital Bio, Beijing, China). All the experiments were at least from

three independent repeats. Cell proliferation was counted according to

Patterson formula, Doubling time (Td) ¼ T � lg2/lg (N/N0).

Cell Proliferation Was Measured by MTT Assay

Three groups of stably transfected cells were grown in 96-well plates

(4 � 103 cells/well). At 24, 48, 72, and 96 hr following innoculation,

20 ml of MTT was added to each well to a final concentration of 0.5%.

After a 4 hr incubation at 378C in the dark, 150 ml DMSO was added to

each well for 10 min to dissolve the formazan crystals. The absorbance

was measured using an ELISA reader (EXL800) (Bio-Tek Instruments,

Winooski, VT) at 490 nm. The viability of the transfected cells was

expressed as a percentage of population growth plus the standard error

of the mean relative to that of untransfected control cells.

Flat Plate Colony Formation Assay

EC9706 cells transfected steadily with pSE-sh2 or pSE were har-

vested and inoculated (1 � 103 cells) into culture capsules. Four weeks

later, the cells were fixed with 95% ice-cold (48C) ethanol for 15 min

and dyed with Giemsa for 20 min. Colonies more than 50 cells were

counted under microscope and calculated cloning efficiency. Colony

formation efficiency ¼ (number of average colony/number of inocu-

lated cells) � 100%. Each group was at least from three independent

repeat experiments.

Cell Migration Assay

A modified Boyden chamber (Costar Transwell inserts; Corning,

Lowell, MA; with a pore size of 8.0 mm) covered with 120 mg/ml

matrigel was used. The bottom chamber of the transwell chamber

was filled with 600 ml DMEM containing 10% FBS. Cells were then

suspended at a density of 1 � 106 cells/ml in 200 ml of DMEM supple-

mented with 0.5% FBS and placed in the upper chamber. The cells were

incubated for 6 hr at 378C in 5% CO2. After the upper side of the filter

had been scraped with a cotton tip to eliminate EC9706 cells that had not

migrated through it, the filter was removed and fixed in 10% trichloro-

acetic acid before staining with 0.1% crystal violet for 20 min. The cell

number in five randomly chosen fields was determined using a light

microscope [13,14]. Experiments were performed in triplicate and

repeated three times.

Flow Cytometry Analysis of Cell Cycle and Apoptosis

EC9706 cells transfected steadily with pSE-sh2 or pSE

(4 � 104 cells) were harvested by trypsinization and fixed in 70%

ice-cold (48C) ethanol for 2 hr. Cell pellets were resuspended in

1 mg/ml RNase solution for 30 min at 378C, and then in 0.1 mg/ml

PI solution (DNA-PrepTM Reagents Kit, Beckman Coulter, Fullerton.

CA) at 48C for 1 hr in the dark. Cell cycle analysis was performed on a

Downregulation of Stathmin in ESCC 705

Journal of Surgical Oncology

flow cytometer. DNA composition and cell cycle distribution was

analyzed with CELL Quest software. Apoptosis of stable transfectants

was also measured with an annexin V-fluorescein isothiocyanate

apoptosis detection kit (Zymed; Invitrogen) that was used to detect

the cell apoptosis of stable transfectants. Set up three repeat wells in

each group.

Apoptotic DNA Ladder Detection

EC9706 cells transfected steadily with pSE-sh2 or pSE were har-

vested. Total DNA was extracted from each sample by the apoptotic

DNA ladder kit (Roche Inc company, Nutley, NJ) according to man-

ufacturer’s instructions, respectively; the extracted DNA was separated

by 2% (w/v) agarose gel electrophoresis in order to analyze the inter-

nucleosomal DNA cleavage.

Western Blot Analysis of Stathmin, Caspase-3, Bcl-2,

Survivin, and P53 in Transfected Cells

Three groups of EC9706 cells (including blank, pSE-sh2, and pSE)

were collected by centrifugation for protein extraction using RIPA lyses

buffer and 1 tablet/10 ml of protease inhibiter cocktail tablets (Roche),

respectively. Briefly, 40 mg of total cellular proteins (60 mg of protein

for stathmin) were separated on 12% SDS–PAGE, then transferred to

PVDF membranes (Promega Corporation, Madison, WI). Non-specific

binding sites were blocked by incubating the membranes in TBS–0.1%

Tween-20 with 5% skimmed milk for 1 hr at RT. The membranes were

incubated with primary antibodies (1:200 dilution for stathmin; 1:400

dilution for Caspase-3, bcl-2, survivin, and P53; 1:1,000 dilution for

human b-actin) for 3 hr at RT, and then washed three times with TBS-T.

The membranes were then incubated with HRP-conjugated goat anti-

mouse (or goat anti-rabbit) IgG (1:3,000 dilution) for 1 hr at RT, and

washed three times with TBS-T. The blots were developed according to

ECL detection kit (Promega), and protein relative expressions were

analyzed using Scion Image Software. Human b-actin was used as an

internal control.

Oncogenicity Assay in Nude Mice

Oncogenicity studies in vivo were performed according to institu-

tional guidelines and a protocol improved by the animal research

committee. Athymic Nude mice (male, 4–5 weeks of age) were pur-

chased from Chinese Acadamy of Science, Shanghai Experimental

Animal Centre (China), and given subcutaneous injections of 0.1 ml

untransfected EC9706 cells, stably transfected EC9706 cells with pSE-

sh2 or pSE suspension at a concentration of 2 � 107 cells/ml in the

RPMI-1640 medium without serum. The inoculations were performed

in eight mice for one group, which were maintained under pathogen-free

conditions. Tumor growth from days 7 to 35 after inoculation was

monitored, and tumor diameters were measured with a caliper. Tumor

volumes (mm3) were calculated by the following formula: V ¼ 1/

2 � L2 � W (L, tumor length; W, tumor width). After a 35-day fol-

low-up period, all mice were killed, and subcutaneous tumors were

resected and weighed to evaluate the tumor growth. At the same time,

tumors of different groups were protected in liquid nitrogen for further

assays.

Statistical Analysis

All experiments were performed at least in triplicate and all quan-

titative data are presented as means � SD. All statistical analyses

were performed with SPSS 13.0. Comparisons among all groups were

performed with the One-way analysis of variance (ANOVA) and

Student Newman Keuls method. P < 0.05 was considered statistically

significant.

RESULTS

Transfection of Recombinant Plasmids Into

EC9706 Cells

According to protocol of LipofectamineTM 2000 Kit, four recombi-

nant plasmids and control plasmid pSE were transfected into EC9706

cells. After incubation for 48 hr, green fluorescence could be seen under

the invert fluorescence microscope. GFP expressed mainly in nucleus.

Cell colonies transfected steadily were obtained by screening with G418

(Fig. 1).

Downregulation of Stathmin Expression by

Stathmin-Specific shRNA in EC9706 Cells

To inhibit stathmin gene expression with shRNA, we constructed

four plasmids expressing shRNA for stathmin under the control of the

human H1 promoter using pSUPER-EGFP plasmid. RT-PCR analysis

was performed to examine the effects of stathmin shRNA on stathmin

expression at transcription in EC9706 cells after transfection. The

results showed that stathmin mRNA levels in EC9706 cells after

transfection with pSE-sh1, pSE-sh2, pSE-sh3, and pSE-sh4 were

48.1 � 6.7%, 15.4 � 1.5%, 40.8 � 5.1%, and 20.5 � 1.7%, respect-

ively, of that with blank control. The inhibition effects of pSE-sh2 and

pSE-sh4 on stathmin mRNA expression was obvious stronger than that

of pSE-sh1 and pSE-sh3 (Fig. 2A). To confirm whether the stathmin-

specific shRNA expressing plasmid influence stathmin protein expres-

sion, we determined stathmin protein levels in EC9706 cells after

transfection with shRNA expressing plasmids using Western blot with

stathmin PcAb. The protein level of stathmin in EC9706 cells after

transfection with pSE-sh2, pSE-sh4 was about 13.9 � 3.8% and

17.7 � 4.7% of that with blank control, respectively (Fig. 2B). These

findings suggested that the levels of stathmin mRNA and protein

Fig. 1. Transfection of recombinant plasmids into EC9706 cells (A: 200�; B: 400�; C: 20�) EC9706 cells were seeded in a 6-well plate at5 � 105 cells/well, and cultured overnight. Forty-eight hours after transfections with recombinant plasmids, GFP expressed mainly in nucleus. Theratio of transfected cells was about more than 40%. After selected with G418, stably transfected cells colonies were established. A: UntransfectedEC9706 cells. B: EC9706 cells transfected with plasmids having the indicated EGFP constructs. C: Colonies of EC9706 cells transfected withplasmids. Data were representative figures from pSE-sh2 transfected cells and colonies. [Color figure can be viewed in the online issue, available atwileyonlinelibrary.com.]

706 Wang et al.

Journal of Surgical Oncology

expression were significantly decreased in EC9706 cells stably express-

ing pSE-sh2 and pSE-sh4.

Effects of Stathmin-shRNA on Cell Proliferation and Cell

Colony Formation In Vitro

Stably transfected EC9706 cells were seeded into 24-well plates

(4 � 104 cells/well) and counted cells number with cell countess to

test the cells proliferation for 96 hr in vitro. Untransfected EC9706 cells

and pSE transfected cells were exponential multiplication at 24–96 hr,

and the doubling time was 21–22 hr. There was no difference between

the two groups (P > 0.05); the doubling time of pSE-sh2 transfected

cells were 26–28 hr. The proliferation of pSE-sh2 transfected cells

decreased at 24 hr. Cells growth became even slower at 48, 72, and

96 hr, compared with the control groups. The cell proliferation curve

showed that the stable transfectants expressing stathmin shRNA had

incomplete inhibition but moderate proliferation retardation (P < 0.01)

(Fig. 3A).

Fig. 2. Stathmin mRNA and protein expression in ESCC EC9706 cells after transfected with stathmin shRNA expressing plasmids EC9706 cellswere transfected with stathmin shRNA expressing plasmids or control plasmid. Total RNA and protein was isolated respectively after steadytransfection. RT-PCR and Western blot were performed as described in the Materials and Methods Section. A: RT-PCR assay. Data wererepresentative figure from three independent experiments. B: Quantification of stathmin mRNA determined by RT-PCR analysis of stablytransfected cells at 1 week posterior to the colony formation, the inhibition ratios in EC9706 cells transfected with pSE-sh2, pSE-sh4 were morethan 80%. Data are shown mean � SD from three independent experiments, �P < 0.01. C: Western blot assay. Data were representative figure fromthree independent experiments. D: Quantification of stathmin protein determined by Western blot analysis, which validated the inhibition ratios ofstathmin protein in EC9706 cells after transfection with pSE-sh2, pSE-sh4 were also more than 80%. Data are shown mean � SD from threeindependent experiments, �P < 0.01.

Downregulation of Stathmin in ESCC 707

Journal of Surgical Oncology

To detect the effect of stathmin downregulation on the proliferation

of cells, stably transfected EC9706 cells and untransfected cells were

seeded into 96-well plates (4 � 103 cells/well). Cultures were collected

at different time points for analysis of cell proliferation level using MTT

assay. The results indicated that the proliferation level of pSE-sh2

transfected EC9706 cells were no different at 24 hr (90.1 � 8.2% of

untransfected cells), but obviously decreased at 48 hr and were main-

tained for 96 hr. The viable cell percentages at 48, 72, and 96 hr were

76.1 � 7.7%, 60.7 � 6.4%, and 53.2 � 5.8% of the PBS negative

control, respectively (P < 0.01) (Fig. 3B).

To examine the effect of stathmin downregulation on the colony

formation of cells, stably transfected EC9706 cells and untransfected

cells were inoculated into culture capsules (1 � 103 cells). Colony

formation efficiency of three groups of EC9706 cell (blank, pSE, and

pSE-sh2) were 65.41 � 8.30%, 63.75 � 9.62%, and 33.24 � 5.65%

respectively. The colonies formed in the pSE-sh2 transfected EC9706

cells group were much less than both blank group and pSE group

(P < 0.05). There was no difference of the colonies formed in the blank

group and pSE group (P > 0.05) (Fig. 3C and 3D).

As shown above, all these results showed that RNAi-mediated

stathmin downregulation resulted in marked inhibition of ESCC

EC9706 cells proliferation in vitro.

Effects of Stathmin-shRNA on Cell MorphologicChange In Vitro

Stably transfected cells with pSE or pSE-sh2 were obtained by

screening with G418. After colonies formed for 1 week, many of

pSE-sh2 transfected cells appeared swelled, multi-nucleus, microtubule

could not rupture, cell mitotic arrest, and mitotic slippage occurred. At 2

weeks, lots of transfectants appeared cytoplasm running off, karyopyk-

nosis, and apoptosis. But these phenomena did not occur in pSE trans-

fected cells (Fig. 4). The result suggested that stathmin downregulation

might inhibit the growth of EC9706 cells through changing the balance

of microtubule dynamics (polymerization/depolymerization) to prevent

cell division.

Effects of Stathmin Downregulation on Cell

Migration In Vitro

To investigate the role of stathmin in the migration of EC9706 cells, a

modified Boyden chamber method was adopted. Three groups (blank,

pSE, and pSE-sh2) of EC9706 cells migrated through a porous mem-

brane. The results of this experiment are shown in Figure 5, the number

of migrated cells per filter in three groups of EC9706 cells were

Fig. 3. Stathmin downregulation inhibits cell growth and colony formation ability in EC9706 cells. EC9706 cells were transfected with stathmin-specific shRNA expressing plasmids pSE-sh2 or negative control shRNA expressing plasmids pSE. Cells were collected at different time point foranalysis of proliferation level using Cell counting assay and MTTassay, respectively. A: Cell counting assay showed the growth curves of cells stablytransfected EC9706 cells. Data are shown mean � SD from three independent experiments, �P < 0.01. B: MTTassay. The relative SDH activity ofthe blank cells was set as 1. The proliferation of EC9706 cells transfected with pSE-sh2 was inhibited obviously. Data are shown mean � SD fromthree independent experiments, �P < 0.01. C: Test the effect of stathmin downregulation on EC9706 cell colony formation ability by cell colonyformation assay. Data were representative figure from three independent experiments. D: Quantification of cell colonies determined by cell coloniescounting analysis of stably transfected cells. Each column represents a mean value of triplicate experiments in each group. Data are mean � SD,�P < 0.05.

708 Wang et al.

Journal of Surgical Oncology

Fig. 4. Stathmin downregulation inhibits cell division in EC9706 cells (200�). A: Morphologic observation of transfected EC9706 cells with theindicated EGFP constructs. Green fluorescence could be seen under the invert fluorescence microscope. B: Morphologic observation of transfectedEC9706 cells after stained with Giemsa. Data were representative figures from three independent experiments. Pink arrow indicated the microtubulecould not rupture, cell mitotic arrest and mitotic slippage occurred, cell division did not accomplished. Red arrow indicated the swelled, multi-nucleus cells which are several times than control cells in volume. Green arrow indicated the cell appeared cytoplasm running off, karyopyknosis,and apoptosis. [Color figure can be viewed in the online issue, available at wileyonlinelibrary.com.]

Fig. 5. Effects of stathmin downregulation on migration of EC9706 cells. A: Cell migration assay. EC9706 cells transfected with pSE or pSE-sh2(2 � 105) were added in the upper chamber, the bottom chamber was filled with DMEM containing 10% FBS. The migratory activity of the cells wasestimated based on the number of cells migrating to the lower chamber. Data were representative figures from three independent experiments. B:Quantification of migration cells determined by cells counting analysis of stably transfected cells. Data are shown mean � SD from threeindependent experiments, �P < 0.01. [Color figure can be viewed in the online issue, available at wileyonlinelibrary.com.]

Downregulation of Stathmin in ESCC 709

Journal of Surgical Oncology

128.52 � 7.82, 121.78 � 6.50, and 30.24 � 2.57, respectively. Stath-

min downregulation by stathmin shRNA impeded EC9706 cell

migration by 76% (P < 0.01). There was no significant difference in

the ability of migration between pSE transfected cells and untransfected

cells (P > 0.05).

Effects of Stathmin-shRNA on Cell Cycle and Apoptosis

In Vitro

ESCC EC9706 cells proliferation inhibition by downregulation of

stathmin expression was caused by disrupting the cell cycle and affect-

ing microtubule assembly shown in other types of mammalian cells. To

reveal the mechanisms underlying RNAi-mediated proliferation inhi-

bition, we used flow cytometric analysis to detect changes in the cell

cycle rates in ESCC EC9706 cells. The cell cycle analysis results

showed: percentage of cell cycle in G2/M phase was obviously

increased after transfection with pSE-sh2. Compared with untransfected

cells, the proportion of cells in G0/G1 phase decreased significantly

from 66.8 � 6.1% to 47.7 � 6.9% (P < 0.05), and the cells in G2/M

phase increased obviously from 5.7 � 0.9% to 20.8 � 3.4%

(P < 0.01), but the proportion of cells in S phase were not different

from the other groups. The populations of each phase in pSE group and

blank control group have no change. (P > 0.05) (Table I, Fig. 6A).

Next, an annexin V-fluorescein isothiocyanate apoptosis detection

kit (Zymed) was used to detect cell apoptosis of EC9706 cells stable

transfectants. Cell apoptosis analysis by flow cytometry showed that

compared with untransfected EC9706 cells (2.0 � 0.4%), the apoptosis

rate of pSE-sh2 transfected EC9706 cells significantly increased by

18.2 � 2.5% (P < 0.05), whereas there was no obvious change in pSE

transfected EC9706 cells (2.4 � 0.5%, P > 0.05) (Fig. 6B).

To test whether downregulation of stathmin causes apoptosis of

esophageal carcinoma cells, EC9706 cells were stably transfected with

pSE-sh2 plasmid or pSE control plasmid for 1 week and then treated by

3% ethanol for 6 hr. Total DNAwas extracted for apoptotic DNA ladder

detection using DNA fragmentation assay. As shown in

Figure 6C, downregulation of stathmin obviously enhanced apoptotic

response to 3% ethanol of EC9706 cells.

All above results suggested that RNAi-mediated downregulation of

stathmin expression in EC9706 cells could induce cell accumulation in

the G2/M phase and initiate cell division arrest, DNA fragmentation and

final apoptosis.

Effects of RNA Interference Targeting Stathmin on CellApoptosis-Related Proteins

Total proteins were extracted from untransfected EC9706 cells and

stably transfected EC9706 cells expressing pSE-sh2 or pSE plasmids,

respectively, and immunoblot analyses were carried out using anti-

stathmin, anti-Bcl-2, anti-Caspase-3, anti-survivin, anti-P53, and

anti-b-actin as described in the Materials and Methods Section. The

results showed that Bcl-2 and survivin were markedly downregulated,

and cleaved Caspase-3 was significantly activated in stable transfectants

with pSE-sh2 compared with that of blank and pSE (P < 0.05). Other-

wise, P53 had no change in stable transfectants with pSE-sh2

(P > 0.05) (Fig. 7).

Inhibition of Tumor Growth by Stathmin

Downregulation In Vivo

With the above findings of the inhibitive effects of stathmin-shRNA

on EC9706 cells. Subsequently, we explore whether stathmin plays a

critical role in tumor formation in vivo, and whether it may be extended

to clinical gene therapy. We subcutaneously injected aliquots of

2.0 � 106 untransfected EC9706 cells and stably transfected EC9706

cells with pSE or pSE-sh2 into three groups of mice and monitored

tumor growth. After inoculating 8–10 days, subcutaneous neoplasma

nodule was visible. Tumor growth rates were equal in three groups of

nude mice. As shown in Figure 8A, the growth of tumors formed from

the pSE-sh2 transfected xenografts was significantly inhibited com-

pared with tumors formed from untransfected xenografts or pSE trans-

fected xenografts. At 35 days after inoculation, the average tumor

volume of the mice was decreased by 55.1% in pSE-sh2 xenografts

compared with untransfected control xenografts (P < 0.01). After 5

weeks, the weight of tumors from the mice were measured, the average

weight of neoplasma body were 863.8 � 251.4 mg in pSE-sh2 trans-

fected xenografts, much smaller than that in pSE transfected xenografts

(1,705.25 � 556.1 mg) and untransfected control xenografts

(1,886.5 � 594.2 mg, P < 0.01, Fig. 8B,C). Furthermore, according

to the results of HE staining (Fig. 8D), necrosis occurred in most of the

stathmin downregulation group xenografts in the process of tumor

formation. The oncogenicity of pSE-sh2 transfected xenografts weak-

ened compared with pSE transfected xenografts and untransfected

control xenografts. These results indicated that RNAi-mediated stath-

min downregulation exerted a strong growth-suppressive effect on

ESCC EC9706 cells in vivo.

DISCUSSION

Stathmin (Op18), a cytosolic phosphoprotein, is a member of a

family of microtubule-destabilizing proteins that regulate the dynamics

of microtubule polymerization and depolymerization [15]. Blocking up

stathmin phosphorylation will halt cell division at G2/M phases of the

cell cycle [16]. Stathmin is an important regulatory factor in the process

of microtubule-associated protein phosphorylation in cells and impact

directly on cell division and proliferation [17]. Antisense inhibition of

stathmin expression results in abrogation of the transformed phenotype

of leukemic cells in vitro and inhibition of tumorigenicity of leukemic

cells in vivo [18]. These observations in erythroleukemic cells are

similar to that in osteosarcoma SSOP-9607 cells and cervical cancer

Hela cells [19]. These findings suggest that high levels of stathmin

expression are necessary for the transformation of tumor cells. However,

if the rate of proliferation of the transformed cells is profoundly reduced

by inhibiting stathmin expression, they may lose the ability to behave in

a malignant fashion, as reflected by their failure to cause tumors in mice

[18]. Stathmin is a p27-binding partner, low p27 and high stathmin were

found to correlate with the metastatic behavior of sarcoma cells in vivo

[20]. Stathmin is validated to influence sarcoma cell shape, motility, and

metastatic potential [21].

Stathmin is overexpressed in various types of human cancers, includ-

ing esophageal carcinoma, and its high expression levels could affect the

distribution of cell cycle [22]. A previous study from our laboratory

showed that stathmin also expressed at high level in esophageal carci-

noma (data not shown), suggesting that stathmin was closely associated

with occurrence and development of ESCC. In addition, the high level of

stathmin expression was recently shown to correlate with established

TABLE I. Cell Cycle of Stable Transfectants Detected by Flow Cytometry

Group

Cell cycle phase (%)

G0/G1 S G2/M

Blank 66.8 � 6.1 27.5 � 3.8 5.7 � 0.9

pSE 63.1 � 5.8 30.4 � 3.7 6.5 � 0.8

pSE-sh2 47.7 � 6.9� 31.5 � 4.2 20.8 � 3.4��

Data are shown mean � SD from three independent experiments.�

P < 0.05.��

P < 0.01.

710 Wang et al.

Journal of Surgical Oncology

prognostic factors in breast carcinoma, lung adenocarcinomas, and oral

squamous-cell carcinoma [7,23,24].

The ability to perturb gene expression selectively in a target cell

provides a powerful tool for probing the function of a protein of interest.

RNAi is characterized by high efficiency, high specificity, and low

toxicity [25,26]. This novel technology is becoming a conventional

application for in vivo cancer therapy [27–29]. In this study, to explore

the possibility of stathmin as an effective therapeutic target, we

employed an RNAi technique to silence endogenous stathmin expres-

sion in EC9706 cells and analyzed phenotypic changes of stably trans-

fected EC9706 cells. In this study, we achieved almost complete

downregulation of stathmin expression by using a shRNA treatment

strategy in EC9706 cells. Experimental data showed that stathmin

downregulation led to significant inhibition of proliferation, migration,

Fig. 6. Stathmin downregulation induces G2/M-phase cell cycle arrest and apoptosis in EC9706 cells. A: Quantification of cell cycle distributiondetermined by flow cytometry analysis of stably transfected cells at 1 week posterior to the colony formation. Data were representative graphs fromthree independent experiments. B: Cell apoptosis of stably transfected EC9706 cells detected by flow cytometry at 1 week posterior to the colonyformation. Data are shown mean � SD from three independent experiments, �P < 0.05. C: Cell apoptosis of stably transfected EC9706 cellsdetected by DNA ladder at 1 week posterior to the colony formation. Data were representative ladders from three independent experiments. [Colorfigure can be viewed in the online issue, available at wileyonlinelibrary.com.]

Downregulation of Stathmin in ESCC 711

Journal of Surgical Oncology

Fig. 7. Effect of stathmin downregulation on the expression of P53, Bcl-2, Caspase-3, and survivin in EC9706 cells. A: Western blot analysis ofthree groups of EC9706 cells (untransfected cells and stably transfected cells expressing pSE-sh2 or pSE plasmids) with anti-stathmin, anti-Bcl-2,anti-Caspase-3, anti-survivin, and anti-P53. Data were representative figures from three independent experiments. B: Quantification of P53 proteinexpression determined by Western blot analysis of stably transfected cells at 1 week posterior to the colony formation. P53 expression had no changein stable transfectants with pSE-sh2. #P > 0.05. C: Quantification of survivin protein expression determined by Western blot analysis of stablytransfected cells. Survivin expression were markedly downregulated in stable transfectants with pSE-sh2. D: Quantification of Caspase-3 proteinexpression determined byWestern blot analysis of stably transfected cells. Cleaved Caspase-3 was significantly activated in stable transfectants withpSE-sh2. E: Quantification of stathmin protein expression determined by Western blot analysis of stably transfected cells. Stathmin expression weremarkedly downregulated in stable transfectants with pSE-sh2. F: Quantification of Bcl-2 protein expression determined byWestern blot analysis ofstably transfected cells. Bcl-2 expression were markedly downregulated in stable transfectants with pSE-sh2. b-actin was used as an internal controlfor protein equal loading control. Data are shown mean � SD from three independent experiments, �P > 0.05.

712 Wang et al.

Journal of Surgical Oncology

and colony formation in vitro, and resulted in accumulation of G2/M

phase and final apoptosis of ESCC EC9706 cells.

Stathmin downregulation is determined to inhibit EC9706 cell

growth in vitro. Thus, it is of great interest to investigate whether it has

similar effect in vivo. Through tumor formation assay, stathmin down-

regulation is demonstrated to suppress the growth of EC9706 xeno-

grafts. Moreover, necrosis was found in stathmin downregulation

EC9706 xenografts by HE staining. Since stathmin downregulation

is found to induce cell cycle arrest and cell apoptosis in EC9706 cells,

it can be deduced that the necrosis in EC9706 cell xenografts may be a

result of cell apoptosis induced by stathmin downregulation. All these

results suggest that stathmin is a potential target for suppressing pro-

liferation and triggering apoptosis, which can be explained by its

key roles in mitosis. Thus, we have reasons to believe that stathmin

may provide an excellent molecular target for esophageal carcinoma

therapy.

Apoptosis is a complex, multistage, and many genes involved

process. Now, it has been understood to be triggered by two distinct

signaling pathways [30,31]. One is the death receptor pathway, regarded

as the extrinsic pathway; and the other is the mitochondrial pathway,

regarded as the intrinsic pathway. For extrinsic pathway, the apoptotic

cell death can be triggered from the outside of cells by activating death

receptors. Then, through their ligands, the initiator Caspase-8 and -10

are cleaved and activated, which lead to the motivation of their down-

stream effector Caspases to kill the cell. For intrinsic pathway, apoptosis

is mediated by the release of cytochrome c from the mitochondria,

which promotes the activation of Procaspase 9 into its activated form

Caspase-9, and activates the downstream effector Caspases to trigger

Fig. 8. Effects of stathmin downregulation on xenografts growth in vivo. The tumors growth in mice developed from untransfected EC9706 cellsand stably transfected EC9706 cells (transfected with pSE-sh2 or pSE control plasmid). The inoculation was performed in three groups (n ¼ 8). A:The tumor volume curves. Data were the means � SD (n ¼ 8 tumors), �P > 0.01. B: The final tumor weight at necropsy 35 days after seeding. Datawere the means � SD (n ¼ 8 tumors), �P > 0.01. C: The representative EC9706 cell xenografts of each group. D: Paraffin embedded sections ofrepresentative EC9706 xenografts were analyzed by HE staining. Lots of swelled multi-nucleus cells could be seen. Arrows indicate necrosis intumor tissues. [Color figure can be viewed in the online issue, available at wileyonlinelibrary.com.]

Downregulation of Stathmin in ESCC 713

Journal of Surgical Oncology

cell death. In the intrinsic apoptotic pathway, P53 is proved to promote

cell apoptosis; while Bcl-2 has an anti-apoptotic effect. Survivin is

known experimentally to protect normal and transformed cells from

apoptosis [32–34]. It has been proposed that survivin localizes to the

mitochondria and is released into the cytoplasm in response to a cell-

death signal, which in turn inhibiting Caspase-9 activity and preventing

apoptosis [35]. Following microtubule inhibitor treatment, when mitotic

arrest and mitotic slippage occur, survivin is downregulated [36] ena-

bling apoptosis to occur in G1. Inhibition of survivin by mitotic inhibi-

tors increase paclitaxel-induced apoptosis and cell death in colonic

carcinoma cells [37].

To date, molecular regulation mechanism of stathmin downregula-

tion mediated cell apoptosis remains elusive, therefore, in this study, cell

apoptosis related gene expressions were detected by Western blot. The

results showed that stathmin downregulation in EC9706 cells could

decrease the Bcl-2 and survivin protein levels, but there was no change

of P53 expression. Furthermore, with the activation of the downstream

effector, Caspase-3, apoptotic pathway is determined to be activated by

stathmin downregulation in EC9706 cells. Therefore, stathmin down-

regulation was found to trigger apoptosis in EC9706 cells, and stathmin

is regarded to take a crucial part in the regulation of EC9706 cell growth

through its roles in cell division and apoptosis regulation. However,

microtubule inhibitor induce apoptosis in cancer cells through multiple

signaling pathways, which are not yet fully elucidated and remain an

area of much interest and debate [38–40]. The exact mechanisms of

apoptotic signaling pathways triggered by stathmin downregulation

remain unclear and need to be further investigated.

In summary, RNAi-mediated stathmin downregulation effectively

inhibited cell proliferation in vitro and tumorigenicity in vivo, induced

cell accumulation in the G2/M phase, and led to apoptotic cell death in

human esophageal carcinoma cells. All these findings suggest that

stathmin may be a pivotal determinant for tumorigenesis, thus it is

expected to be a potential therapeutic target for the treatment of esoph-

ageal carcinoma.

ACKNOWLEDGMENTS

We are grateful to the Tumor Center of Zhengzhou University for

their technical assistance. This work was supported by a grant from

Natural Science Foundation of Henan Province (072102310054) and

Natural Science Foundation of Education Department of Henan Prov-

ince (2010A310008).

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