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Keywords:

  • survivin;
  • endometrial cancer;
  • RNA interference;
  • proliferation;
  • apoptosis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

Endometrial cancer is a common gynecologic malignancy among women. The molecular mechanisms involved in the progression of endometrial cancer are unclear, which has hampered the development of an effective treatment. Survivin, a newly identified member of the inhibitor of apoptosis (IAP) family, regulates 2 critical processes in neoplastic transformation: cell proliferation and apoptosis.

METHODS

Survivin mRNA and protein expression levels were analyzed in human normal cycling endometrium, atypical endometrium, and endometrial adenocarcinoma by immunohistochemical, reverse-transcriptase polymerase chain reaction (RT-PCR), and Western blot analyses. To study the biological function of survivin in endometrial cancer, RNA interference was applied to knock down survivin expression in the Ishikawa endometrial cancer cell line by recombinant plasmids producing survivin small hairpin RNA. Furthermore, the signal pathway that regulates survivin expression was investigated.

RESULTS

Higher levels of survivin mRNA and protein expression were observed in endometrial adenocarcinomas than in atypical or normal endometrium. Immunohistochemical staining revealed that 83.3% (50 of 60) of endometrial adenocarcinoma samples, 55.0% (11 of 20) of atypical endometrium samples, and 25.0% (5 of 20) of normal endometrium samples were positive for survivin protein. Inhibition of survivin by RNA interference reduced cell proliferation and induced apoptosis in Ishikawa cells by down-regulating cyclin D1 and phosphorylated RB and activating caspase-3 and caspase-8. The authors also found that the MAPK pathway was a signal transduction pathway upstream of survivin. Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) up-regulated survivin protein expression by activating the MAPK pathway in endometrial cancer cells.

CONCLUSIONS

Survivin is an attractive target for endometrial cancer treatment. Growth factors could regulate survivin expression by activating the MAPK pathway. Cancer 2006. © 2006 American Cancer Society.

Endometrial cancer is the most common gynecologic malignancy in many developed countries and affects about 2% to 3% of women.1 Unopposed estrogen stimulation of the endometrium is the classic etiologic factor associated with the development of endometrial cancer.2 However, a large proportion of endometrial cancer cases cannot be explained by hormonal risk factors. Recent advances in the molecular genetics of endometrial cancer have shown that molecular changes play an important role in the development of this cancer.3

Survivin, a newly identified member of the inhibitor of apoptosis protein (IAP) family, is capable of regulating both cell proliferation and apoptosis. Survivin is regulated in a highly cell cycle-dependent manner; its expression is markedly increase in the G2/M-phase and is associated with mitotic-spindle microtubules, centromeres, and intracellular mid-bodies.4 It has been shown that survivin inhibits cell death induced by a variety of apoptotic stimuli, including Fas/FasL, caspases, and anticancer drugs.5 The overexpression of survivin confers cytoprotection against a variety of apoptotic stimuli, whereas loss of survivin expression or function causes spontaneous apoptosis or sensitizes cancer cells to apoptotic stimuli.6

Survivin has been shown to be abundantly expressed in embryonic and fetal organs, but it has not been reported in differentiated normal tissues.7 However, survivin is overexpressed in 60 cancer cell lines and most human tumor types, including lung, breast, stomach, liver, bladder, and ovarian cancers.8 Overexpression of this protein has also been associated with increased cancer aggressiveness and decreased patient survival.9 Despite this knowledge of survivin, its role in endometrial cancer is largely unknown. Discovery of the function and signal pathway of survivin in endometrial cancer may provide clues to endometrial carcinogenesis and offer therapeutic alternatives for endometrial cancer treatment.

To understand the function and regulation of survivin in endometrial cancer, we investigated the expression of survivin in human endometrial adenocarcinomas. We used RNAi to knock down survivin expression in endometrial cancer cells and studied the signal transduction pathway of survivin. Our results confirmed that survivin plays a critical role in endometrial cancer.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Tissue Selection

Tissue samples from 17 patients with endometrial adenocarcinoma and 4 patients with atypical endometrium were collected consecutively for reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis from the Obstetrics and Gynecology Hospital, Fudan University. Samples of normal cycling endometrium were selected from hysterectomy specimens of 14 consecutive patients from the same hospital who had no history of endometrial pathology. None of the patients was undergoing hormone replacement therapy at the time she was selected for inclusion in the study. The carcinomas consisted of 12 Grade 1 tumors, 2 Grade 2 tumors, and 3 Grade 3 tumors; 13 cases were categorized as Stage I, 2 cases as Stage II, and 2 cases as Stage III. The average age of the patients with carcinoma was 55.0 years (range, 31–77 years).

In addition, 100 samples of paraffin-embedded tissues (60 endometrial adenocarcinoma, 20 atypical endometrium, and 20 normal endometrium) from the same hospital were examined immunohistochemically. The carcinomas consisted of 40 Grade 1 tumors, 13 Grade 2 tumors, and 7 Grade 3 tumors; 47 cases were categorized as Stage I, 9 cases as Stage II, 3 cases as Stage III, and 1 case as Stage IV. The average age of the patients with carcinoma was 47.3 years (range: 24–74 years). Histologic diagnoses and tumor grading were based on the International Federation of Gynecology and Obstetrics (FIGO) classification system.10

The Ethics Committee of the Medical Faculty of Fudan University approved this study. Informed consent was obtained from each subject.

Cell Cultures

The human endometrial adenocarcinoma cell line Ishikawa was kindly provided by Dr. Wei (Second Hospital, Peking University, China) and was cultured in DMEM/F12 (Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum. The cells were maintained in 75-cm2 tissue culture flasks (Corning Glass Works, Corning, NY) under sterile conditions at 37°C in water-saturated air containing 5% carbon dioxide.

Design and Preparation of Constructs

Three 19-nucleotide (nt) DNA sequences targeting survivin were designed by Kangchen (Shanghai, China). The 3 sequences (5′-GCATCTCTACATTCAAGAA-3′, 5′-GAAGCAGTTTGAAGAATTA-3′, and 5′-AGGAAACCAACAATAAGAA-3′) corresponded to nt 174–192, 391–409, 465–483, respectively, of the human survivin mRNA (GenBank accession NM-001168). The 19-nt oligonucleotide 5′-TTCTCCGAACGTGTCACGT-3′, which had no significant homology to any known human mRNA in the databases, was used as a negative control. Synthetic sense and antisense oligonucleotides (Shanghai Sangon Biological, Shanghai, China) were used to create the template for generating RNA composed of 2 identical 19-nt sequence motifs in an inverted orientation separated by a 9-base pair (bp) spacer to form a double-stranded hairpin of small interfering RNA. Two micrograms of each oligonucleotide was annealed for 10 minutes at 95°C and for 1 hour at 37°C and then ligated into 2 μg of pRNAT-U6.1/Neo plasmid (containing the ampicillin resistance gene and the U6 promoter; Genscript, Piscataway, NJ) and linearized with BamHI and HindIII. The four constructs were named pRNAT-suv1, pRNAT-suv2, pRNAT-suv3, and pRNAT-neg. These constructs were cloned in TOP10 chemically competent Escherichia coli cells according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). The sequence of each insert was confirmed by automated sequencing.

In Vitro Transfection

Transfection of plasmids was performed with Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Briefly, the day before transfection 5 × 105 Ishikawa cells per well were plated onto 6-well plates and grown for 1 day, when the cells had reached 80% to 85% confluency. The plasmids and Lipofectamine 2000 were each diluted in 250 μL of serum-free Opti-MEM (Gibco BRL) and incubated for 5 min at room temperature. Then the diluted plasmid and the diluted Lipofectamine 2000 were combined at a 1:2 ratio (4 μg of plasmid with 8 μL of Lipofectamine 2000). This combination was mixed gently and incubated for 20 minutes at room temperature. A total of 500 μL of the combination was added to each well in a final volume of 2 mL per well. The cells were incubated for another 72 hours before the experiments were conducted.

RNA Extraction and RT-PCR

Trizol (Invitrogen) was used to extract total RNA from the fresh tissue samples. The RNA was reverse-transcribed into cDNA with a reverse transcription kit (Promega, Madison, WI). Primers for survivin were 5′-GACCACCGCATCTCTACATTC-3′ (sense) and 5′-TGCTTTTTATGTTCCTCTATGGG-3′ (antisense).11 The PCR product was 194 bp long. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Primers for GAPDH were 5′-ACCACAGTCCATGCCATCAC-3′ (sense) and 5′-CCACCACCCTGTTGCTGTAG-3′ (antisense), and the PCR product was 450 bp long. PCR conditions were as follows: 95°C for 5 minutes, 28 cycles of 94°C for 30 seconds, 53°C for 30 seconds, 72°C for 30 seconds, and 72°C for 7 minutes. PCR products were detected by 2% agarose electrophoresis. The intensity of survivin and GAPDH were evaluated. The relative levels of survivin were as the ratio of survivin/GAPDH.

Western Blot Analysis

Cells were harvested and lysed with RIPA buffer (0.15 M NaCl, 1% NP40, 0.01 M deoxycholate, 0.1% sodium dodecyl sufate [SDS], 0.05 M Tris-HCl [pH 8.0], 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 μg/ml each of aprotinin, pepstatin, and leupeptin). Fifty micrograms of each soluble protein sample was separated by 12% or 15% SDS-polyacrylamide gel electrophoresis (PAGE), blocked in 2.5% skim milk/TPBS (1× phosphate-buffered saline [PBS] containing 0.1% Tween 20), and probed with each primary antibody overnight at 4°C. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ). The intensity of survivin and GAPDH were evaluated. The relative levels of survivin were as the ratio of survivin/GAPDH. Antibodies against survivin, Bcl-2, phosphorylated ERK (p-ERK), ERK, and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-caspase-3 and anti-caspase-8 antibodies were purchased from Cell Signaling Technology (Beverly, MA); anti-cyclin D1 and anti-cyclin E antibodies from Neomarker (Fremont, CA); and anti-p-RB (Ser780) antibody from Stressgen (Victoria, British Columbia, Canada).

Immunohistochemical Analysis

Expression of survivin protein in paraffin-embedded clinical tissues was examined using the avidin-biotin complex immunoperoxidase method. Deparaffinized sections were treated with 3% hydrogen peroxide to block endogenous peroxidase activity. After sections were blocked with 5% normal serum for 20 minutes, rabbit anti-survivin antibody (1:100 dilution; Santa Cruz Biotechnology) was applied and the sections were incubated overnight at 4°C. After rinsing by PBS the biotinylated secondary antibody and avidin-biotin complex/horseradish peroxidase (Dakopatts, Glostrup, Denmark) were applied. Peroxidase activity was visualized by applying the diaminobenzidine chromogen. The sections were counterstained with hematoxylin. Rabbit immunoglobulin G alone (without primary antibody) was used as a negative control for immunostaining. Two independent investigators evaluated the immunohistochemical staining without knowledge of the clinical pathologic parameters. Survivin staining was cytoplasmic. For semiquantitative assessment of the immunohistochemical results, the mean percentage of positive tumor cells was determined in at least 10 random fields at ×400 magnification in each section. It was graded as focal (≤10%), regional (11%–50%), or diffuse (>50%). The intensity of the survivin immunoreaction was graded weak, moderate, or intense. The mean percentage of positive tumor cells and the staining intensity were then combined to produce a survivin-IHC result. Cases that graded “intense diffuse, intense regional, moderate diffuse, and moderate regional” were considered positive for survivin; others were considered negative.

Cell Proliferation and DNA Synthesis Analyses

Cell proliferation and DNA synthesis were evaluated using 5-bromo-2′-deoxyuridine (BrdU) incorporation. Cells plated on coverslips were transfected with Lipofectamine 2000 and after 72 hours 20 μM BrdU (Sigma Chemical Company, St. Louis, MO) was added and the cells incubated at 37°C for another 1 hour. Cells were fixed in 3.5% paraformaldehyde for 30 minutes at 4°C, washed in 0.1 M PBS with 1% Triton X-100, and treated with 2 N HCl for 1 hour at 37°C. Immediately after the acid wash, 0.1 M borate buffer was added to the cells for 30 minutes at room temperature. The cells were then treated with 0.1% Triton X-100 for 2 minutes at room temperature and incubated in 10% normal goat serum for 1 hour before incubation overnight at 4°C with anti-BrdU antibody (Sigma Chemical Company). Finally, the cells were treated with biotinylated secondary antibody (Santa Cruz Biotechnology), which was followed by color development in 3,3′-diaminobenzidine. PBS alone (without primary antibody) was used as a negative control for immunostaining. The mean percentage of positive cells was determined in at least 10 random fields at ×400 magnification in each sample.

Cell Cycle and Apoptosis Analyses

Cell cycle distribution was determined by measuring the cellular DNA content with the use of flow cytometry. Cells were trypsinized, washed in PBS, fixed in 70% ice-cold ethanol at 4°C overnight, washed with PBS again, and stained with 100 μL of 50 mg/L propidium iodide at 37°C for 30 minutes. Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit I (BD Pharmingen, San Diego, CA) was used to identify apoptotic and viable cells following the manufacturer's instructions. The percentage of early apoptotic (FITC-positive and propidium iodide-negative) cells was calculated from the data originating from flow cytometry.

Assay of the Epidermal Growth Factor Receptor/MAPK Signal Transduction Pathway

Ishikawa cells were grown as described in “Cell Cultures,” serum-starved for 18 to 20 hours, washed twice with PBS, and incubated with 10 ng/mL EGF or TGF-α (Peprotech, Rocky Hill, NJ) for up to 120 minutes (for the ERK phosphorylation assay) or for up to 24 hours (for the survivin protein assay). To evaluate the effect of the MEK1/2 inhibitor U0126 (Sigma Chemical Company) on the epidermal growth factor receptor (EGFR)/MAPK pathway, we pretreated the cells with 30 μM U0126 for 30 minutes and then with 10 ng/mL EGF or TGF-α for 30 minutes (for the ERK phosphorylation assay) or 24 hours (for the survivin protein assay). The cells were then harvested and the proteins extracted for Western blot analysis. Anti-p-ERK1/2 and anti-survivin were purchased from Santa Cruz Biotechnology.

Statistical Analysis

Results for all the experiments were analyzed using Student t test or α2 analysis (α2 analysis for positive rate comparison and Student t test for the others). Differences were considered significant at P < .05. SPSS 11.0 software (SPSS Inc., Chicago, IL) was used to perform statistical analysis.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Survivin was highly expressed in endometrial carcinoma. We investigated the expression of survivin in endometrial adenocarcinoma, atypical endometrium, and normal cycling endometrium. By RT-PCR analysis, we observed that survivin mRNA levels were significantly higher in endometrial adenocarcinoma than in atypical or normal endometrium (P < .05) (Fig. 1A). Similarly, survivin protein levels were significantly higher in endometrial adenocarcinoma than in atypical and normal endometrium as determined by Western blot analysis (P < .05) (Fig. 1B).

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Figure 1. The expression of survivin in normal cycling endometrium (N), atypical endometrium (A), and endometrial adenocarcinoma (T). (A) Representative samples of reverse-transcriptase polymerase chain reaction (RT-PCR). The level of survivin mRNA was standardized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as a loading control. *P < .05 versus adenocarcinoma. (B) Representative samples of Western blot analysis. The average signal intensity of survivin protein was standardized to GAPDH. *P < .05 and **P < .01 versus adenocarcinoma.

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We also studied the expression level of survivin in clinical samples. Immunohistochemical staining showed that 83.3% (50 of 60) of the endometrial adenocarcinoma samples, 55.0% (11 of 20) of the atypical endometrium samples, and 25.0% (5 of 20) of the normal endometrium samples were positive for survivin protein. Compared with atypical endometrium and normal endometrium, endometrial adenocarcinoma had much more positive cases (P<.05) (Table 1). The staining intensity was also stronger in tumor cells than that in atypical and normal cells (Fig. 2). No statistical significant differences were found between type 1 and type 2 endometrial cancers (Table 1). The fact that survivin expression in the adenocarcinoma tissues was not correlated with clinical pathologic parameters, including patient age, tumor grade, and tumor stage (Table 1), suggests that survivin overexpression is an early event in endometrial carcinogenesis.

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Figure 2. Immunohistochemical staining of survivin protein in normal endometrium, atypical endometrium, and endometrial adenocarcinoma. (A) Survivin-negative case of normal endometrium in the proliferative phase. (B) Survivin-negative case of normal endometrium in the secretory phase. (C, D) Survivin-positive cases of atypical endometrium. (E, F) Survivin-positive cases of endometrial adenocarcinoma. GAPDH indicates glyceraldehyde-3-phosphate dehydrogenase (3,3′-diaminobenzidine [DAB], original magnification ×200).

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Table 1. Immunohistochemical Staining of Survivin Protein in Normal, Atypical, and Malignant Endometrial Tissues
CharacteristicTotal no.No. survivin-negativeNo. survivin-positivePositive rate (%)P
  • *

    Compared with normal endometrium.

Tissue type
Normal  endometrium2015525.0 
Atypical  endometrium2091155.0.02*
Endometrial  adenocarcinoma60105083.3<.005*
Patient age, y    .75
 <502432187.5 
 ≥503672980.6 
Tumor grade    .64
 14063485.0 
 2–32041680.0 
Tumor stage    .90
 I4783983.0 
 II-IV1321184.6 
Tumor type    .61
 I4473784.1 
 II1631381.3 

Survivin RNAi specifically reduced survivin expression in endometrial cancer cells. To reveal the role of survivin in the development of endometrial carcinoma, we inhibited survivin expression in Ishikawa endometrial cancer cells by RNAi and studied the resulting proliferation and apoptosis rates of these cells. Three recombinant plasmids of survivin RNAi (pRNAT-suv1, pRNAT-suv2, and pRNAT-suv3) and 1 negative control plasmid (pRNAT-neg) were constructed successfully and tested for their ability to knock down survivin expression in the Ishikawa cells. Transfection with pRNAT-suv2 and pRNAT-suv3 resulted in a reproducible decrease of 70% to 80% in the expression level of both survivin mRNA (as judged by RT-PCR) (Fig. 3A) and survivin protein (as judged by Western blot analysis) (Fig. 3B), whereas transfection with pRNAT-neg (Fig. 3A,B) or pRNAT-suv1 (data not shown) failed to reduce the level of survivin mRNA or protein expression. The effects of pRNAT-suv2 and pRNAT-suv3 were specific in that they failed to knock down expression of the unrelated protein GAPDH (Fig. 3).

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Figure 3. Selective silencing of survivin expression in Ishikawa endometrial cancer cells by RNAi. Survivin mRNA and protein expression was tested by (A) reverse-transcriptase polymerase chain reaction (RT-PCR) and (B) Western blot analysis, respectively. Transfections were performed with reagent only (Control), a negative control plasmid (pRNAT-neg), or recombinant plasmids of survivin RNAi (pRNAT-suv2 and pRNAT-suv3). The transfections with reagent only and with PRNAT-neg were used as negative controls. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

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Inhibition of survivin induced G1 cell cycle arrest, inhibited cell proliferation, and down-regulated cyclin D1 and p-RB expression in endometrial cancer cells. We next assessed the effect of inhibiting survivin on cell cycle progression in Ishikawa cells by using flow cytometry. We found that transfection with pRNAT-suv2 or pRNAT-suv3 caused cells to accumulate in the G1 and sub-G1 phases and the proportion of cells in the S+G2/M-phase to decrease, whereas transfection with pRNAT-neg resulted in little change in the cell cycle distribution (Fig. 4) (Table 2). To verify the effect on cell cycle progression, DNA synthesis and cell proliferation were assessed using BrdU incorporation. Consistent with the results of cell cycle analysis, transfection with pRNAT-suv2 or pRNAT-suv3 significantly reduced DNA synthesis and cell proliferation compared with transfection with reagent only (control) or pRNAT-neg (33% ± 2.65%, 34% ± 2.0%, 52.42% ± 3.17%, and 49.67% ± 2.52%, respectively) (P < .05 vs. control for both pRNAT-suv2 and pRNAT-suv3) (Fig. 5A).

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Figure 4. Induction of G1 cell cycle arrest in Ishikawa endometrial cancer cells by survivin RNAi. After transfection for 72 hours, cells were stained with propidium iodide and subjected to fluorescence-activated cell sorting analysis to determine the cell cycle distribution based on DNA content. The experiments were conducted 3 times with similar results.

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Figure 5. Inhibition of Ishikawa endometrial cancer cell proliferation by survivin RNAi and the effect of survivin RNAi on cell cycle regulation. (A) 5-Bromo-2′-deoxyuridine (BrdU) incorporation in transfected cells. Percentages of BrdU-positive cells were calculated based on counts of at least 10 random fields at ×400 magnification per treatment. Data are the means ± standard deviations of 3 independent experiments. *P < .01 versus control. (B) Western blot analysis for cell cycle regulators cyclin D1, cyclin E, and p-RB in transfected cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

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Table 2. Cell Cycle Distribution in Survivin RNAi-Transfected Endometrial Cancer Cells
Transfection groupCell cycle phase
Sub-G1 (%)G1 (%)S (%)G2 (%)
Control0.6553.2038.207.95
PRNAT-neg4.1553.2339.832.80
PRNAT-suv214.3158.5822.654.46
PRNAT-suv310.0360.0028.990.98

To explore the probable molecular mechanism of G1 cell cycle arrest and inhibition of cell proliferation by survivin RNAi, we used Western blot analysis to examine the levels of 3 cell cycle regulators, cyclin D1, cyclin E, and p-RB. Transfection with pRNAT-suv2 or pRNAT-suv3 markedly decreased cyclin D1 and p-RB protein expression levels compared with transfection with reagent only (control) or pRNAT-neg, whereas the cyclin E protein expression level was unaltered (Fig. 5B).

Inhibition of survivin induced apoptosis and increased cleavage of caspase-3 and caspase-8 in endometrial cancer cells. To explore the effect of survivin RNAi on apoptosis, Annexin V-FITC staining was used to detect apoptotic Ishikawa cells. Transfection with pRNAT-suv2 or pRNAT-suv3 increased the proportion of Annexin V-positive cells more than 10-fold compared with the transfection with reagent only or pRNAT-neg (Fig. 6).

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Figure 6. Induction of apoptosis in Ishikawa endometrial cancer cells by survivin RNAi. After transfection for 72 hours cells were stained with Annexin V-FITC and analyzed by flow cytometry. The experiments were conducted 3 times with similar results.

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Consistent with this observation, Western blot analysis showed increased cleavage of caspase-3 and caspase-8 in cells transfected with pRNAT-suv2 or pRNAT-suv3 compared with cells transfected with reagent only or pRNAT-neg (Fig. 7). The appearance of both caspase-3 catalytic activity and apoptosis might be associated with proteolytic cleavage of upstream caspase-8. Bcl-2 protein expression was not altered by transfection. These data indicate that survivin regulates an antiapoptotic program in Ishikawa cells and that inhibition of survivin leads to a rapid induction of apoptosis by activating caspase-3 and caspase-8.

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Figure 7. Western blot analysis for caspase-3, caspase-8, and Bcl-2 in Ishikawa endometrial cancer cells after transfection for 72 hours. GAPDH indicates glyceraldehyde-3-phosphate dehydrogenase.

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EGF and TGF-α up-regulated survivin protein expression by activating the EGFR/MAPK pathway in endometrial cells. To assess the activation of the EGFR/MAPK pathway upstream of survivin in Ishikawa cells, we investigated the phosphorylation of ERK1 and ERK2, the major molecules of this pathway. With the use of anti-p-ERK1/2 antibody, we conducted time-course analysis of ERK1/2 during treatment with 10 ng/mL EGF and TGF-α. Both growth factors rapidly activated ERK1/2 and the phosphorylation of ERK1/2 reached a maximal level 30 minutes after the cells were exposed (Fig. 8A). However, the activation of ERK1/2 was transient and decreased gradually within 2 hours. We then pretreated Ishikawa cells for 30 minutes with 30 μM U0126, a potent MEK1/2 inhibitor, and incubated them with EGF and TGF-α. U0126 completely blocked the phosphorylation of ERK1/2 activated by EGF and TGF-α (Fig. 8B).

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Figure 8. Up-regulation of survivin expression in Ishikawa endometrial cancer cells by growth factors through activation of the epidermal growth factor receptor (EGFR)/MAPK pathway. (A) Time course of ERK1/2 phosphorylation induced by 10 ng/mL of epidermal growth factor (EGF) or transforming growth factor (TGF)-α. Growth factor was added to the medium for up to 120 minutes and phosphorylated and total ERK1/2 expression levels were assessed. (B) Effects of the kinase inhibitor U0126 on induced ERK1/2 activation by EGF or TGF-α. Cells were pretreated with 30 μM U0126 for 30 minutes before stimulation by growth factor for another 30 minutes and phosphorylated and total ERK1/2 expression levels were assessed. (C) Time course of survivin protein expression regulated by 10 ng/mL of EGF or TGF-α. Growth factor was added to the medium for up to 24 hours and survivin protein expression level was assessed. (D) U0126 inhibition of survivin up-regulation induced by EGF or TGF-α. Cells were pretreated with 30 μM U0126 for 30 minutes before incubation with EGF or TGF-α for 24 hours, and survivin protein expression level was assessed. C indicates control (no reagents); E, EGF; T, TGF-α; U, U0126; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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We next investigated the regulation of survivin protein expression by EGF and TGF-α. Both growth factors up-regulated survivin expression over time (Fig. 8C). The maximal level was reached 24 hours after the cells were exposed. To verify that the EGFR/MAPK pathway was the signal transduction pathway by which EGF and TGF-α up-regulated survivin protein expression, we pretreated Ishikawa cells with U0126 for 30 minutes before EGF or TGF-α was added. Survivin protein expression markedly declined when U0126 was applied (Fig. 8D). Taken together, these data indicate that the EGFR/MAPK pathway is a signal transduction pathway upstream of survivin. EGF and TGF-α could up-regulate survivin expression by activating this pathway in endometrial cells.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Survivin, a member of the IAP family, has been shown to be overexpressed in all human cancer types studied, including endometrial carcinoma. In this study we detected survivin mRNA in almost all samples of endometrial adenocarcinoma, atypical endometrium, and normal endometrium. This result differs from those of previous studies that reported that survivin was only expressed during fetal development and in cancer.12, 13 We also demonstrated that the levels of both survivin mRNA and protein were significantly higher in endometrial adenocarcinoma than in atypical and normal endometrium. These findings indicate that survivin plays an important role in the carcinogenesis of endometrial cancer.

RNAi uses short RNA duplexes of defined sequences to silence a targeted gene14 and is an important technique in knocking down gene expression and studying gene function. For our study, we designed and successfully constructed 3 survivin gene expression vectors of double-stranded RNAi and verified that 2 of them (pRNAT-suv2 and pRNAT-suv3) were effective in that they knocked down survivin gene expression by 70% to 80% in Ishikawa cells. In contrast, the negative sequence (pRNAT-neg) was not effective. From these results, we conclude that the first 2 RNAi constructs caused selective degradation of human survivin mRNA and thereby decreased survivin protein expression levels in Ishikawa cells.

Survivin is a regulator of spindle microtubule function at mitosis and an inhibitor of apoptosis.15 The down-regulation of survivin expression by antisense oligonucleotides has been shown to significantly inhibit cell growth and induce apoptosis in melanoma16 and lung cancer.17 Carvalho et al.18 first used RNAi to specifically repress survivin in HeLa cells. They showed that survivin was no longer detectable in cultures 60 hours after transfection with survivin-specific small interfering RNA. In addition, survivin-depleted cells were delayed in mitosis and accumulated in prometaphase with misaligned chromosomes.18 We observed decreased cell proliferation and DNA synthesis when survivin expression was inhibited. Although survivin is highly expressed in the G2/M-phase of the cell cycle and overexpression of survivin may overcome the G2/M-phase checkpoint to enforce progression of cells through mitosis,19 we observed that knockdown of survivin expression increased the proportion of cells in the G1 phase and induced G1 cell cycle arrest. Thus, survivin may regulate the G1/S-phase checkpoint in endometrial cancer. Furthermore, we examined the expression of 3 proteins that play important roles in the G1/S transition: cyclin D1 and p-RB protein expression after survivin knockdown decreased markedly, whereas cyclin E protein expression was unaltered. Survivin has been shown to interact with cdk4 and enhance RB phosphorylation by releasing p16 and p21 from cdk4/p16 and cdk4/p21 complexes in hepatoma cells.20 Our results imply that survivin promotes cell proliferation and cell cycle progression by activating the cdk4/cyclinD1 complex and enhancing RB phosphorylation to release E2F transcription factor. This signal pathway is independent of the cyclin E-CDK-2 pathway. The cell cycle may be altered by various treatment through different pathways. For an example, Baicalein treatment in prostate cancer cells inhibits only the expression of survivin and cyclin D1, but not cyclin E.21

Survivin is believed to protect cells from apoptosis by suppressing caspases and procaspases (primarily caspase-3 and caspase-7) directly or indirectly.22 In our study we measured the level of apoptosis to analyze the biologic effect associated with RNAi inhibition of survivin mRNA expression. In addition to spontaneous apoptosis, transfection with pRNAT-suv2 or pRNAT-suv3 increased the apoptosis rate of Ishikawa cells. This result indicates that survivin exerts an antiapoptotic effect in endometrial cancer. We also found that cleavage of caspase-3 and caspase-8 was increased in survivin knockdown cells. Because it cannot directly inhibit caspase-3, survivin protein most likely interacts with the upstream caspase-8, thereby suppressing caspase-3 and inhibiting apoptosis.

EGFR is a transmembrane glycoprotein that belongs to the erb B family of tyrosine kinase receptors and is overexpressed in a variety of cancers, including endometrial cancer.23 EGF and TGF-α are believed to be the most important ligands for EGFR. The EGFR/MAPK pathway regulates multiple biologic processes, such as gene expression, cell proliferation, inhibition of apoptosis, cell motility, and cell adhesion, all of which contribute to the development of malignancy. In the current study, we found that EGF and TGF-α activated ERK1/2 and up-regulated survivin protein expression in Ishikawa cells. When U0126 was applied to block phosphorylation of ERK1/2, survivin protein expression decreased markedly but did not disappear completely. From these results we conclude that the EGFR/MAPK pathway is a signal pathway upstream of survivin and that EGF and TGF-α can up-regulate survivin expression by activating this pathway in endometrial cells.

In conclusion, survivin is highly expressed in endometrial cancer, knocking down survivin expression by RNAi reducing endometrial cell proliferation and inducing apoptosis by down-regulating cyclin D1 and p-RB protein expression and activating caspase-3 and caspase-8. The EGFR/MAPK pathway is a signal transduction pathway upstream of survivin, and growth factors could regulate survivin expression by activating this pathway. Survivin is a candidate as a new target for endometrial cancer treatment.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES