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Abstract

The aim of the present study was to investigate the possible use of mouse double-minute 2 (MDM2) molecular imaging to predict chemotherapeutic sensitivity in breast cancer xenografts (BCXs). MCF-7 cells were transfected with MDM2 antisense oligonucleotides (ASONs), and MDM2 expression levels were determined by Western blotting. Cell viability was assessed by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in MCF-7 cells transfected with ASONs and treated with paclitaxel. BCXs were established in nude mice by injection of ASONs, and tumor volumes were measured after paclitaxel treatment. MDM2 ASONs were labeled with ^99mTc to generate an MDM2 molecular probe, and MDM2 expression levels were evaluated by imaging and Western blotting. MDM2 ASONs downregulated MDM2 expression in a dose-dependent manner and increased the rate of paclitaxel-induced cell growth inhibition. Imaging of tumors revealed significant differences in the tumor to skeletal muscle (T/M) ratio between groups. Tumor MDM2 protein expression was correlated with T/M ratios at 4 hours (R = .880) and 10 hours (R = .886). The effect of paclitaxel varied among nude mice bearing BCXs with different concentrations of ASONs, as shown by differences in tumor growth. MDM2 molecular imaging could be a promising method for predicting the sensitivity of BCXs to chemotherapy.

DESPITE THE WIDESPREAD USE of chemotherapy for the treatment of breast cancer and advances in chemotherapy regimens, some breast cancers are associated with poor clinical outcomes. Important prognostic factors in breast cancer include the number of lymph nodes involved, tumor size, histologic grade, and hormone receptor status; the first two are the basis for the American Joint Committee on Cancer (AJCC) staging system. The sixth edition of the AJCC staging system was designed to allow better prediction of prognosis by tumor stage.¹ However, tumor behavior is not fully determined by these characteristics, and the disease prognosis can vary. Advances in molecular biology techniques have enabled the analysis of several prognostic and predictive factors to explain this phenomenon at the molecular level. Studies have identified relevant prognostic factors, such as HER2/neu gene amplification and protein expression, the expression of other members of the epithelial growth factor receptor family, p53 gene mutations, and mouse double-minute 2 (MDM2) gene overexpression, among others.²

The MDM2 oncoprotein is an important regulator of p53 turnover that acts by promoting p53 ubiquitination and proteasomal degradation. The activation of p53 target genes induces apoptosis, cell-cycle arrest, and senescence, which are important tumor suppressor mechanisms.³ In normal cells, p53 and MDM2 are involved in an autoregulatory negative feedback loop by which activation of p53 after DNA damage triggers the transcription of MDM2. The upregulated MDM2 protein binds to the transactivation domain of p53, inhibiting its transcriptional activity, and targets the p53 protein for degradation by the ubiquitin-proteasome system, resulting in the irreversible deactivation of p53. The MDM2-p53 pathway is an important regulator of homeostasis.⁴ However, the overexpression of MDM2 by gene amplification or other pathways in certain cancers can lead to the dysregulation of the feedback system, resulting in alterations in cell growth and tumor cell characteristics.⁵

MDM2 is overexpressed in many human cancers⁶,⁷ and is associated with advanced cancer disease states, such as invasive^8,9 and high-grade or late-stage tumors,¹⁰ recurrence,^11,12 metastasis,^13,14 resistance to chemotherapy,^15–17 and radiotherapy.^18,19 Therefore, MDM2 is considered not only an important therapeutic target^20–22 but also an important predictor of response to treatment.^23–25 The timely and accurate assessment of MDM2 expression is of clinical significance for cancer therapy. In a previous study, we successfully synthesized an MDM2 molecular probe and performed MDM2 molecular imaging.²⁶ In the present study, MDM2 expression levels were determined by molecular imaging and correlated with the response to chemotherapy in a breast cancer xenograft (BCX) model to investigate the possibility of predicting chemotherapeutic sensitivity in BCXs.

Materials and Methods

General

The antisense oligonucleotide (ASON) (5′-GATCACTC CCACCTTCAAGG-3′) was purchased from Sangon Biologic Engineering Technology and Services Co., Ltd (Shanghai, China). All internucleotide linkages were phosphorothioate bonds. Each DNA molecule contained a primary amine on the 5′ terminus attached through a 6- carbon alkyl linker. Hydrazinonicotinamide (HYNIC) was purchased from Gil Biochemical Co. Ltd (Shanghai, China). Radioactive pertechnetate (^99mTcO₄⁻) was obtained from a ⁹⁹Mo-^99mTc radionuclide generator made by the China Institute of Atomic Energy. N,N- Dimethylformamide (DMF), dimethyl sulfoxide tricine (DMSO), and Sncl₂-2H₂O were purchased from Sigma-Aldrich (St. Louis, MO). Antihuman MDM2 (SMP-14) monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Lipofectamine 2000 and Trizol were purchased from Invitrogen (Carlsbad, CA). Sep-Pak C18 reverse-phase columns and Sephadex G25 columns were obtained from Waters Co, USA. Paclitaxel was provided by Lukang Chenxin Pharmaceutical Industrial (Shandong, China). BALB/c mice were purchased from Vital River Laboratory (Beijing, China).

Western Blot Analysis of MDM2 Gene Silencing in MCF-7 Cells

The MCF-7 cell line was a gift from the Tumor Hospital of Harbin Medical University. MCF-7 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 mg/mL of penicillin-streptomycin. MCF-7 cells were detached by trypsin, counted, plated in six-well flat-bottomed culture plates in DMEM with 10% FBS (2 mL/well, 2.5 × 10⁶ cells/well), and incubated at 37°C and 50 mL/L CO₂ for 24 hours. Aliquots containing 10 μg ASONs diluted in 500 μL DMEM and 25 μL Lipofectamine 2000 diluted in 475 μL DMEM were combined and incubated for 20 minutes at room temperature, yielding a final ASON concentration of 0.01 μg/μL. MCF-7 cells were incubated with a control mixture consisting of 10% FBS and liposome-coated ASONs (100 or 500 nmol/L) in six-well plates for 6 hours at 37°C (five wells/group). The medium was then removed and changed to DMEM with 10% FBS. Cells were collected after a further 24 hours of incubation at 37°C.

The protein level of MDM2 was analyzed by Western blotting. Equal amounts of lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Bio-Rad Trans-Blot nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were incubated in blocking buffer (Tris-buffered saline containing 0.1% polysorbate 20 and 5% nonfat milk) for 1 hour at room temperature followed by incubation with the appropriate primary antibody (anti-MDM2, 1:1,000 dilution) overnight at 4°C with gentle shaking. Membranes were washed three times (15 minutes each) with washing buffer (Tris-buffered saline containing 0.1% polysorbate 20) and incubated with goat antimouse secondary antibody (1:3,000 dilution) for 1 hour at room temperature. After washing as described above, the protein of interest was detected using enhanced chemiluminescence reagents from Amresco (Solon, OH). Protein bands were detected and analyzed with a gel imaging system. Relative levels of each protein are expressed as a percentage of the control and normalized to the corresponding β- actin level.

Evaluation of Paclitaxel-Induced Cell Growth Inhibition after MDM2 Gene Silencing in MCF-7 Cells

MCF-7 cells transfected with 10% FBS and liposome-coated ASONs (100 and 500 nmol/L) were plated in 96-well flat-bottomed culture plates in DMEM with 10% FBS (100 μL/well, 5 × 10³ cells/well, 10 wells/group). Paclitaxel (1 μmol/L) was added to the culture medium (five wells/group). An equal volume of 0.9% physiologic saline was added to the culture medium (five wells/group) as a control. After 20 hours, 20 μL of 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL) was added to each well, and the plates were returned to the incubator for 4 hours. After this final incubation, the medium and MTT reagents were discarded, 150 μL DMSO was added to each well, and the plates were shaken for 10 minutes to ensure full dissolution of the crystals. The absorbance (A) of the wells was measured at a wavelength of 490 nm by the Freedom EVOLyzer (Tecan, Maennedorf, Switzerland) enzyme-linked immunosorbent assay analyzer. The experiment was repeated five times, and mean values were determined. The cell growth inhibition rate, defined as (1 – experimental group optical density value/control group optical density) × 100%, was recorded.

Animal Tumor Model

Thirty-five BALB/c mice (female; mean weight [± SD]: 20 ± 2 g; age: 3-4 weeks) were used in the study. The mice were inoculated with 1 × 10⁷ MCF-7 cells (100 μL) in the right hind limbs, and the tumors were allowed to grow to a diameter of 1 to 1.5 cm. The mice were maintained using a standard diet, bedding, and environment, with free access to food and drinking water. All animal procedures were approved by the Harbin Medical University Animal Care and Use Committee.

Thirty-five BALB/c mice with BCXs were randomly divided into four groups (A, B, C, D) of 10 mice (A, B, C) and 5 mice (D). Groups A, B, and C were further divided into two subgroups of five mice each (A1/A2, B1/B2, C1/C2, respectively). Mice in groups A and D were treated with 150 μL phosphate-buffered saline administered intraperitoneally once daily (5 days per week) for 15 days. Mice in groups B and C were treated with MDM2 ASONs at 25 mg/kg (B) and 50 mg/kg (C) intraperitoneally once daily (5 days per week) for 15 days in 150 μL phosphate-buffered saline.

MDM2 Molecular Probe Preparation

The synthesis of the MDM2 molecular probe and the assay of the labeling efficiency and specific activity were performed as described previously.²⁶ The radiochemical purity was calculated by paper chromatography on Xinhua #1 filter paper with acetone and normal saline as the mobile phase.

MDM2 Molecular Imaging in Mice Bearing BCXs

The MDM2 molecular probe (7.4 MBq) was injected into the mouse via the tail vein on day 16 after 15 days of ASON treatment. At 4 and 10 hours after injection, imaging was performed using a single-photon emission computed tomographic (SPECT) scanner (Millennium VG, Hawkeye, GE Healthcare, Wauwatosa, WI) equipped with a low-energy, high-resolution, parallel-hole collimator. Static images (100,000 counts), obtained with a zoom factor of 2.0, were digitally stored in a 256 × 256 matrix. The ratio of tumor to skeletal muscle (T/M) was calculated over the region of interest.

Western Blot Evaluation of MDM2 Expression in Mice Bearing BCXs

Animals in the three subgroups (A1, B1, C1) were sacrificed by cervical dislocation, and tumor tissues were dissected. Portions of tumor tissues (100 mg) were frozen in liquid nitrogen, mechanically ground with mortar and pestle, homogenized using lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.5 mmol/L ethylenediaminetetraacetic acid [EDTA], 1 mmol/L dithiothreitol, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/L phenylmethanesulfonyl fluoride), sonicated, and centrifuged at 12,000g for 20 minutes at 4°C. Samples containing equal amounts of proteins were analyzed by Western blotting according to the above method.

In Vivo Chemotherapy

Paclitaxel treatment began on day 16 after 15 days of ASON administration in three subgroups (A2, B2, C2). Paclitaxel (10 mg/kg/day) was administered intraperitoneally on days 16, 19, 23, 26, and 30, and mice in group D were given the same volume of physiologic saline. Tumor growth was monitored by measuring two perpendicular diameters using calipers on days 0, 3, 6, 9, 12, 15, and 18. Tumor size (weight in grams) was calculated by the following formula: 0.5 a × b², where a is the long diameter (cm) and b is the short diameter (cm). A tumor growth curve was generated based on tumor size, and differences in tumor size at different times were analyzed in the four groups.

Statistical Analysis

All statistical analyses were performed using SPSS software version 17.0 (SPSS, Inc., Chicago, IL). Data are expressed as mean ± standard deviation (SD). Spearman correlation was used to calculate correlations between the T/M ratios obtained by molecular imaging and MDM2 expression level in the tumor, and other statistical comparisons of mean values were performed using one-way analysis of variance (ANOVA). A p value < .05 was considered significant.

Results

Expression of the MDM2 Protein in MCF-7 Cells

MDM2 protein expression levels decreased gradually with increasing ASON doses. Significant differences in MDM2 protein expression were detected between cells treated with 100 or 500 nmol/L ASONs and the control group (p < .01, n = 5) (Figure 1).

Figure 1.

Effects of antisense oligonucleotides (ASONs) on MDM2 protein levels in MCF-7 cells. The inhibitory effects of ASONs on MDM2 protein expression are shown in a dose-dependent manner. Relative levels of each protein are expressed as a percentage of the control and normalized to the corresponding β-actin level. MDM2/β-actin was analyzed by one-way ANOVA. *p < .01 compared to the control group.

MCF-7 Cell Growth Inhibition Rate

MCF-7 cells with different MDM2 expression levels showed different reactions to paclitaxel, with an increase in the rate of cell inhibition associated with decreasing MDM2 expression levels. The cell growth inhibition rates in the 500 nmol/L ASON, 100 nmol/L ASON, 10% FBS, and control groups were 14.45% ± 1.44%, 6.91% ± 1.15%, 1.88% ± 0.48%, and 0.006% ± 0.48% (n = 5), respectively. Significant differences in cell growth inhibition rates were observed between cells treated with 100 or 500 nmol/L ASONs and the 10% FBS group (p < .01, n = 5). The cell growth inhibition rate of the 0.9% physiologic saline control group was significantly lower than that of the paclitaxel treatment groups (p < .01, n = 5) (Figure 2).

Figure 2.

Comparison of paclitaxel-induced cell growth inhibition rates in MCF-7 cells. Cell growth inhibition rates were compared by one-way ANOVA. *p < .01 compared to the 10% phosphate-buffered saline (PBS) + paclitaxel groups. ^#p < .01 compared to the 0.9% physiologic saline control groups. ASON – antisense oligonucleotide.

Oligonucleotide Conjugation and Radiolabeling

The labeling efficiency of the MDM2 molecular probe was 55.98% ± 4.46% (n = 5), the specific activity was 1,406.2 ± 52.6 kBq/μg (n = 5), and the radiochemical purity was greater than 95% after purification.

Molecular Imaging

In nude mice bearing BCXs, the tumors were clearly imaged at 4 and 10 hours after injection of the MDM2 molecular probe. Because the molecular probe is cleared primarily through the urinary and digestive systems, the liver and bladder were also imaged at 4 and 10 hours.

A gradual decrease in the T/M ratio was observed in animal models treated with increasing MDM2 ASON doses. Significant differences in the T/M ratio were detected between groups B1/B2 and C1/C2 and group A1/A2 at 4 and 10 hours, respectively (p < .01, n = 5) (Figure 3).

Figure 3.

Molecular imaging of MDM2 expression in A (a), B (b), and C (c) group mice at 4 and 10 hours. The tumors (arrows) were clearly visualized after injection of the MDM2 molecular probe. At all time points, the tumor to skeletal muscle (T/M) ratios of two subgroups (A1, B1, C1 and A2, B2, C2) were analyzed by one-way ANOVA (d). *p < .01, compared to the A1 subgroups. ^#p < .01 compared to the A2 subgroups.

MDM2 Protein Expression in Mice Bearing BCXs

Consistent with the MDM2 molecular imaging results, MDM2 protein expression levels in MCF-7 xenograft-bearing mice decreased gradually with increasing ASON doses. MDM2 protein expression in tumors correlated well with the T/M ratios at 4 hours (R = .880, p < .01) and 10 hours (R = .886, p < .01) obtained from the MDM2 molecular imaging analysis (Figure 4).

Figure 4.

Correlation of MDM2/β-actin with tumor to skeletal muscle (T/M) ratio in nude mice. The MDM2/β-actin level was significantly correlated with the T/M ratios obtained from the MDM2 molecular imaging at 4 (a) and 10 (b) hours.

Response to Paclitaxel in Tumors with Different MDM2 Expression Levels

Nude mice bearing different MDM2 ASON–expressing BCXs (A2, B2, and C2 groups) showed different responses to paclitaxel treatment. Molecular imaging experiments showed that the level of MDM2 expression had a significant effect on tumor growth in response to paclitaxel in the three subgroups, but especially in the C2 group. No significant differences in tumor volume were observed between the three subgroups on day 3 (p > .05, n = 5). However, tumor volume differed significantly between groups B2 and C2 and group A2 on days 6, 9, 12, 15, and 18 (p < .05, n = 5). Xenograft tumors grew at a faster rate in group D than in the three subgroups (A2, B2, C2), with significant differences in tumor volume observed after day 3 (p < .05, n = 5) (Figure 5).

Figure 5.

Growth curve of tumors in the paclitaxel treatment groups (A2, B2, C2) and the physiologic saline control group. Beginning on day 6, significant differences were observed in tumor volume between the A2 group and the B2, C2 groups. The tumor volume of untreated mice (D group) was significantly different from the tumor volume of mice treated with paclitaxel after day 3 (A2, B2, C2 groups).

Discussion

Personalized medicine is often heralded as one of the major leaps forward for twenty-first century medical practice. It aims to develop new therapies and optimize prescribing by steering patients to the right drug at the right dose at the right time.²⁷ The success of personalized medicine depends on the availability of accurate diagnostic tests capable of identifying patients who can benefit from targeted therapies. At the intersection of molecular biology, bioinformatics, and in vivo imaging, molecular imaging allows the detection of specific molecular targets, from those pertaining to gene expression to protein products and their specific functions. Molecular imaging has become an important diagnostic test in personalized medicine for cancer.^28–31

The oncogene MDM2 is closely related to the occurrence, development, and biological behaviors of tumors. MDM2 has been considered a target for gene therapy, and several drugs targeting MDM2 have been tested in preclinical studies.^32–34 High expression of the MDM2 oncogene has been associated with resistance to conventional radiotherapy and chemotherapy, resulting in poor clinical outcomes. The MDM2 gene has thus become a therapeutic target to improve tumor sensitivity to radiotherapy and chemotherapy. However, MDM2 gene expression levels vary in different tumors and at different times in the same tumor, and a consensus has not been reached regarding the optimal conditions for gene therapy. Therefore, the accurate evaluation of MDM2 expression levels in different tumors and throughout tumor development is particularly important.

MDM2 expression is mainly evaluated in vitro by reverse transcriptase–polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry. We successfully synthesized an MDM2 molecular probe and showed its specific detection in tumor-bearing mice by a series of in vitro and in vivo methods in a previous study. In the present study, we evaluated MDM2 expression levels in MCF-7 cells and xenograft breast cancer tumors with differential expression of MDM2. We then analyzed the effects of conventional chemotherapy treatment in correlation with MDM2 expression levels to investigate the possibility of using MDM2 molecular imaging for the prediction of chemotherapeutic sensitivity.

Paclitaxel, one of the most widely used anticancer drugs, is effective for the treatment of many types of tumors, especially breast cancer. Although paclitaxel is a common clinical breast cancer chemotherapy drug,^35–38 certain breast cancer patients remain insensitive to paclitaxel, resulting in poor clinical outcomes and undesirable side effects. Therefore, the development of effective and less toxic treatments would be of value, and accurate evaluation of tumor characteristics is necessary for the design and selection of specific therapies. Because overexpression of the MDM2 gene is one of the most common causes of tumor insensitivity to chemotherapy,^15–17 assessment of MDM2 expression levels enables prediction of the response to paclitaxel and is therefore of clinical significance to predict the outcomes of treatment.

In the present study, we used ASON-mediated inhibition of gene expression, which relies on the base-pairing principle, to silence the expression of MDM2. ASONs bind specifically to the messenger ribonucleic acid (mRNA) of a target gene, effectively inhibiting gene expression.³⁹ We transfected cells with different concentrations of ASONs and evaluated the response to paclitaxel treatment by measuring cell growth inhibition rates. The results showed significant differences in cell growth among cells with different MDM2 expression levels, with a negative correlation between MDM2 expression and the response to paclitaxel. These in vitro results confirmed the role of MDM2 expression in the response to paclitaxel and laid the foundation for in vivo animal experiments.

BCX tumors with differential MDM2 expression were generated in mice by transfection with ASONs using the method described by Zhang and colleagues, who showed that MDM2 ASONs downregulated the expression of MDM2 in nude mice bearing breast cancer tumors.⁴⁰ The expression of MDM2 in BCXs was assessed by molecular imaging at 4 and 10 hours after injection of a specific MDM2 molecular probe based on previous experiments showing that tumors can be more clearly imaged with less background noise at 4 and 10 hours than at 1 hour.²⁶ Analysis of tumor tissues showed that MDM2 protein expression levels in the tumor correlated well with the T/M ratios obtained by MDM2 molecular imaging, confirming that molecular imaging can accurately evaluate the expression of MDM2. Mice were then treated with paclitaxel at five time points, and tumor volume was assessed at seven time points based on a study by Wang and colleagues, who reported that transfection with MDM2 ASONs can increase the sensitivity to paclitaxel in nude mice bearing breast cancer tumors.⁴¹ Our results showed a significant positive correlation between MDM2 expression and the rate of tumor growth starting at 6 days after paclitaxel treatment, which was consistent with the molecular imaging results showing that MDM2 expression level was negatively correlated with the efficacy of paclitaxel treatment.

In previous studies, mutations in the p53 gene were associated with the response to chemotherapy in breast cancer patients.^42,43 However, a recent study showed that mutations in TP53 and an MDM2 polymorphism resulting in MDM2 overexpression were not associated with the response to paclitaxel in breast cancer patients, whereas they conferred poor disease-specific survival in patients treated with paclitaxel.⁴⁴ These results are in disagreement with those of the present study and suggest that further experiments are needed to determine whether our findings can be generalized in the clinical setting.

Summary

Silencing of MDM2 by ASONs in cells and xenograft tumors provided a model of differential MDM2 expression to test the efficacy of MDM2 molecular imaging for the prediction of response to paclitaxel treatment in breast cancer in vitro and in vivo. Our results indicate that assessment of MDM2 expression levels by molecular imaging is a valuable noninvasive tool to predict the response to paclitaxel in breast cancer and warrants further investigation for its potential clinical application as a prognostic marker for the selection of patients who would benefit from chemotherapy.

Footnotes

Acknowledgment

Financial disclosure of authors: This study was supported by the National Natural Science Foundation of China (NSFC 81171362), the Natural Science Foundation of Heilongjiang Province (D201060), and the Harbin Technological Innovation Talent Special Funds Project (2011RFQYS100).

Financial disclosure of reviewers: None reported.

SUPPLEMENTARY MATERIAL

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MDM2 Molecular Imaging for the Prediction of Chemotherapeutic Sensitivity in Human Breast Cancer Xenograft

Abstract

Materials and Methods

General

Western Blot Analysis of MDM2 Gene Silencing in MCF-7 Cells

Evaluation of Paclitaxel-Induced Cell Growth Inhibition after MDM2 Gene Silencing in MCF-7 Cells

Animal Tumor Model

MDM2 Molecular Probe Preparation

MDM2 Molecular Imaging in Mice Bearing BCXs

Western Blot Evaluation of MDM2 Expression in Mice Bearing BCXs

In Vivo Chemotherapy

Statistical Analysis

Results

Expression of the MDM2 Protein in MCF-7 Cells

MCF-7 Cell Growth Inhibition Rate

Oligonucleotide Conjugation and Radiolabeling

Molecular Imaging

MDM2 Protein Expression in Mice Bearing BCXs

Response to Paclitaxel in Tumors with Different MDM2 Expression Levels

Discussion

Summary

Footnotes

Acknowledgment

SUPPLEMENTARY MATERIAL

References