Antisense oligodeoxynucleotide targeting HER2 mRNA sensitized docetaxel in breast cancer treatment

Abstract

Context: Human epidermal growth factor receptor 2 (HER2) is one of the oncogenes closely associated with the development and prognosis of breast carcinoma. Down-regulation of HER2 mRNA by antisense oligodeoxynucleotide (ASO) HER2 has been suggested to be a feasible treatment for patients with breast carcinoma.

Objective: The antitumor effects of ASO HA6722 were investigated in vitro and in vivo. Materials and methods: In this study, SK-BR-3, a HER2-overexpressing breast carcinoma cell line, was used as the model for in vitro experiments. Inhibitory effects of the ASO HA6722 were detected by methyl-thiazoldiphenyl tetrazolium (MTT) assay. Meanwhile, HER2 mRNA levels were monitored by reverse transcription polymerase chain reaction (RT-PCR). The in vivo antitumor effects were evaluated in nude mice xenograft model.

Results: Our results showed that HA6722 alone could inhibit the growth of SK-BR-3 cells in a dose-dependent manner with the IC₅₀ value of 41.8 ± 8.1 nM. In addition, the antitumor effect of docetaxel (TXT) could be sensitized by low dose of HA6722 both in vitro and in vivo, suggesting that ASO HA6722 could inhibit the growth of breast cancer cells and enhance the cytotoxic effects of TXT.

Discussion and conclusion: The combination treatment of TXT and HA6722 could be a more effective approach for breast cancer treatment. The future study should focus on the antitumor effect in other models.

Keywords::

• Chemosensitizer
• breast neoplasms
• knock down

Introduction

Breast cancer has become the leading cause of death in Chinese women in recent years. Standard treatments of breast tumor include surgery, radiotherapy, chemotherapy, and endocrinotherapy. Even though great progress has been made both in adjuvant or rescue setting (Gralow, 2006; Moulder & Hortobagyi, 2008), the outcome of these treatment is very poor for patients with advanced or metastatic disease (Orlando et al., 2007). New molecular targeting drugs, such as antisense agents, monoclonal antibodies, and antiangiogenic agents, are still behind the therapeutic development for treating solid tumors (Sleijfer et al., 2006). It is predicted that advances in new molecular targeting drugs will significantly improve the survival of patients with advanced and recurrent breast carcinoma (Hatake et al., 2007; Fujii et al., 2008).

Previously, secondary structures of HER2/neu mRNA have been simulated by our laboratory via application of computer software or online analysis tools; the conservation of the secondary structure has been analyzed and parameters involved in the interaction between antisense and sense mRNAs have been proposed (Song et al., 2000). A series of antisense phosphorothioate oligodeoxynucleotides (AS-ODNs) were then designed, synthesized, and screened for inhibitory effects in breast cancer cell lines. Preliminary studies showed that one of the sequences, HA6722, possessed satisfactory effects in inhibiting the growth of cell lines of breast cancer (Sun et al., 2005). The aim of this study was to systemically examine the inhibitory effects of HA6722 on breast cancer when administered alone and in combination with the docetaxel (TXT), a highly effective drug for breast cancer and a cytotoxic agent, both in vitro and in vivo.

Materials and methods

Chemicals, drugs, and supplies

Chemicals and supplies were purchased from the following suppliers: RPMI 1640 and fetal bovine serum (FBS) were from GIBCO BRL (Grand Island, NY). The 20-mer oligodeoxynucleotides HA6722 and its control sequence Scramble6722 were synthesized by SBS Genetech Co. Ltd. (Beijing, China). Lipofectamine^™ 2000 for transfection of antisense oligodeoxynucleotide (ASO) and Invitrogen SuperScript^™ for reverse transcription polymerase chain reaction (RT-PCR) were from Invitrogen Corporation (Beijing, China). TXT from Aventis Pharma (Beijing, China) was dissolved in dimethyl sulfoxide (DMSO) before use.

Cell line

Human breast carcinoma cell line, SK-BR-3, was purchased from the Cell Culture Center of the Institute of Chinese Basic Medical Science. This cell line overexpresses HER2 and was used as the model cell line in this study. Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS plus 1% penicillin/streptomycin and 1% glutamine. Cultures were maintained in a 5% CO₂-humidified incubator at 37°C, and experiments were performed with cells at the exponential growth phase.

Methyl-thiazoldiphenyl tetrazolium assay

The methyl-thiazoldiphenyl tetrazolium (MTT) assay was performed as previously described (Mohammad et al., 2005). In brief, SK-BR-3 cells (2 × 10⁴ cells/well) were added to a 96-well culture plate (Costar, Cambridge, MA). When cells reached exponential growth phase, they were either treated with the anticancer drug TXT or transfected with AS oligodeoxynucleotide HA6722/Scramble6722 (with Lipofectamine^™ 2000). Final concentrations of TXT were 1, 4, 16, 64, 250, and 1000 nM. Finally, concentrations of Scramble6722 and HA6722 were 12.8, 32, 80, 200, and 1250 nM. After 24 h of transfection, the tetrazolium agent was then added to each well followed by a 4-h incubation. Subsequently, the culture supernatant was removed and cells were dissolved in DMSO. After thorough formazan solubilization, the absorbance of each well was measured with a microculture plate reader at the wavelength of 540 nm. Growth inhibition was expressed as the ratio of the mean absorbance of treated cells to that of control cells. Each experiment was carried out three times independently, and each assay was performed in six replicate wells. The growth inhibition rate was calculated to determine the 50% inhibitory concentration (IC₅₀) value.

Reverse transcription polymerase chain reaction

Primers for human-specific HER2 were designed using the sequence from GenBank (Accession No.) and synthesized by SBS Genetech Co. Ltd. (Beijing, China). The sequences of primers for HER2 and glyceraldehyde-3-phosphate (G3PDH) are as follows:

For RT-PCR, total RNA was extracted with the Invitrogen SuperScript^™ RNA extraction kit (Carlsbad, CA). Five microliters of the total RNA was then transcribed into cDNA using 0.2 mM primers of HER2, 0.2 mM dNTP, RNasin 40 U, 1× buffer, and 2 U AMV in a final volume of 27 μL following the kit protocol from Promega (Madison, WI). The cDNA was then submitted for PCR assay, whose reaction mixture consisted of 1× PCR buffer, 200 mM dNTP, 20 pmol of each primer and 2 U of Golden Taq DNA polymerase (Invitrogen Corporation, China). The mixture was pre-incubated for 5 min at 94°C, followed by 30 cycles of amplification at 94°C for 50 sec, 56°C for 50 sec, and 72°C for 60 sec. A final extension was performed at 72°C for 7 min.

Table

HER2	Forward	5′-CAATGGAGACCCGCTGAAC-3′
	Reverse	5′-CAGTGCGCGTCAGGCTCT-3′
G3PDH	Forward	5′-ACCACAGTCCATGCCATCAC-3′
	Reverse	5′-TCCACCACCCTGTTGCTGTA-3′

Transfection with AS HER2 oligodeoxynucleotides

The sequences of AS HER2 mRNA phosphorothioate oligonucleotide (HA6722) and its control Scramble6722 are as follows:

For transfection, HA6722 and control ODNs were added to SK-BR-3 cells in the form of complexes with cationic Lipofectamine^™ 2000 according to the manufacturer’s manual (Roh et al., 1999). In brief, the growth medium was removed, and serum-free RPMI was added to the cells. The ODN–Lipofectamine complex was prepared and then added drop-wise to the cells. After 12 h of incubation, cells were washed with serum-free RPMI before RPMI medium containing 10% fetal bovine serum was added. Cells were then treated with AS HER2 ODN for another 24–48 h. Thereafter, cells were incubated with or without (control) TXT for 24 h, and cell viability was assessed by MTT assay.

Table

HA6722	5′-CAGCAGCCGAGCCAGCCCGA-3′
Scramble6722	5′-ACAGCAGCCGAGCCAGCCCG-3′

In vivo experiments using the nude mouse model

All animal experiments were performed using female BALB/c nude mice purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Science. The experimental protocols were approved by the Chinese Laboratory Animal Center. All animal experiments were carried out according to the standards of animal care as outlined in the Guide for the Care and Use of Experimental Animals of Union Medical University of Beijing. In brief, the animals were maintained and handled under aseptic conditions with 12 h dark/light conditions and were allowed free access to food and water throughout the study.

The in vivo antitumor effect was evaluated with the xenograft transplanted into BALB/c nude mice following previously described protocols (Alpaugh et al., 1999; Ueno et al., 2002). In brief, SK-BR-3 breast carcinoma cells were transplanted into the flank of nude mice. When the tumor size reached ~100 mm³, the mice were divided arbitrarily into seven treatment groups (n = 7 per group), that is, no treatment, treatments with Scramble6722, TXT, and HA6722 alone, and combination treatments of Scramble plus TXT and HA6722 plus TXT (with two different HA6722 dosages, 2 and 5 mg/kg/day). HA6722 and Scramble6722 were administered intravenously at a dose of 2–5 mg/kg for 12 consecutive days per treatment, whereas TXT was administered intravenously twice a week. Tumors from those mice surviving the whole treatment period were excised and the tumor volume was calculated using the following equation (Tanaka et al., 1996):

1

Tumor inhibition rate was calculated using the following equation:

2

where a stands for the average weight of the control group and b stands for the average weight of the treatment group.

Statistical analysis

Statistical significance was determined with the SPSS software (version 10.0, SPSS Corporation, Chicago, IL) using either Student’s t-test or analysis of variance (one-way ANOVA). A P-value of <0.05 was considered statistically significant.

Results

Inhibitory effects of HA6722 on SK-BR-3 cell lines

As shown in Figures 1A and 1B, MTT assay revealed that both TXT and HA6722 could inhibit the growth of SK-BR-3 cells in a dose-dependent manner. The IC₅₀ of TXT was 14.7 ± 5.3 nM, while it was 41.8 ± 8.1 nM for HA6722. Scramble6722, however, nearly had no inhibitory effect on the growth of this cell line with an IC₅₀ of 489.4 ± 12.1 nM.

Figure 1.  HA6722 enhanced the inhibitory effects of Docetaxel (TXT) on growth of SK-BR-3 cell lines. Cells were either transfected with HA6722/scramble6722 alone (b), treated with various concentrations of TXT alone (a), or had the combination treatment (c), for 24 h before the MTT assay was performed. Each experiment was carried out three times independently, and each assay was performed in six replicate wells. Inhibition rate was calculated as the ratio of the mean absorbance of treated cells to that of control cells.

Enhanced antitumor effect of TXT to SK-BR-3 when administered with HA6722 in vitro

To clarify whether there is a synergistic antiproliferation effect between HA6722 and TXT, a fixed concentration of HA6722 (12.8 nM) was combined with serial concentrations of TXT to treat SK-BR-3 cells. At 12.8 nM, HA6722 had only a very weak inhibitory effect (close to nontoxic) on SK-BR-3 cell (Figure 1B). However, MTT results of the combination treatment revealed that IC₅₀ of TXT decreased from 14.7 ± 5.3 nM to 9.1 ± 3.6 nM due to the addition of HA6722 (P = 0.039, n = 3), indicating a chemosensitive effect of HA6722 to TXT (Figure 1C).

Down-regulation of HER2 by HA6722

To determine whether the antiproliferation effects of HA6722 on SK-BR-3 cells were related to the down-regulation of its target mRNA, we detected the expression of HER2 mRNA in SK-BR-3 cells before and after they were treated with HA6722 by means of RT-PCR. As shown in Figure 2A, the inhibition of HER2 mRNA by HA6722 was in a dose-dependent manner. At the concentration of 200 nM for 9 h, HA6722 dramatically down-regulated the expression of HER2 mRNA in SK-BR-3 cells (Figure 2B). Data indicated that HA6722 enhanced the inhibitory effect of TXT by the mechanism of specifically inhibiting the expression of HER2 mRNA. The down-regulation of HER2 by HA6722 was also evaluated by immunohistochemistry stain in vivo. As shown in Figure 3, HER2 expression is almost negative in HA6722-treated groups.

Figure 2.  Reverse transcription polymerase chain reaction (RT-PCR) demonstrating the down-regulation of HER2 mRNA by HA6722. (A) SK-BR-3 cells were transfected with various amounts of HA6722 for 24 h before the mRNA expression was evaluated. (B) SK-BR-3 cells were either transfected with Scramble6722, a random oligonucleotide (random), 200 nM HA6722 for 9 h or not transfected (baseline) before the mRNA evaluation via RT-PCR. The house-keeping gene GAPDH was used as an internal control.

Figure 3.  Down-regulation of HER2 by HA6722 in vivo. Each treatment group was consisted of seven mice. Tumors from those mice surviving the entire treatment period were excised and photographed. (A) Blank group; (B) T (M) group; (C) TScr (M) group; (D) HA (M) group; (E) THA (M) group.

Enhancement of antitumor effect of TXT by AS HER2 ODNs in vivo

To study the in vivo effect of HA6722 on TXT in the inhibition of breast cancer, nude mice models were utilized. As shown in Figure 4A, multiple intravenous injections of HA6722 (q.d. × 12) could significantly inhibit the growth of breast cancer in nude mice. Compared with the control group, 30 days after the first administration of HA6722 (5 mg/kg/day) alone, the tumor volume almost did not increase from pretreatment. In addition, HA6722 enhanced the antitumor effects of TXT (Figure 4A). The inhibitory rate of HA6722 on the tumor growth reached a level of 76.3%. Moreover, HA6722 improved the sensitivity to the routine chemotherapeutic agent of the tumors and resulted in the elevation of the tumor growth inhibitory rate of docetaxol. At the dosage of 5 mg/kg/day, HA6722 significantly increased the inhibitory effect of low-dose docetaxol (7.5 mg/kg/day) on tumor growth (from <50% to 88.3%), which was comparable with the inhibitory rate (88.7%) of high-dose docetaxol alone (15 mg/kg/day). On the other hand, at the same dosage of 5 mg/kg/day, Scramble6722 did not alter the anticancer activity of TXT (see Figure 4B).

Figure 4.  Effects of HA6722 on tumor volume and tumor weight in the nude mouse model. Each treatment group consisted of seven mice. (A) HA6722 alone (M, medium dosage, 5 mg/kg/day) could inhibit tumor growth (P < 0.05 compared with blank treatment). THA (L), docetaxel (TXT) 7.5 mg/kg/day + HA6722 2 mg/kg/day; (B) HA6722 sensitized TXT on tumor growth. Inhibitory effect of TXT (medium dosage, 7.5 mg/kg/day) plus HA6722 (5 mg/kg/day) was comparable with that of high TXT dosage treatment [T (H), 15 mg/kg/day]. [P < 0.05 compared with blank treatment, P > 0.05 between T (H) and THA (M) groups]; (C) final tumor weights of different treatment groups compared with blank treatment *P < 0.01; **P < 0.01; ***P < 0.05; ^#P < 0.05.

Discussion

Great progress has been made in the diagnosis and treatment of breast cancer in recent years. However, prognosis of those patients with advanced or metastatic disease after treatment still remains rather poor. Searching for new remedies for these patients has become one of the most important topics of this field.

It is generally accepted that multiple gene mutations and/or overexpressions are involved in the development of breast carcinoma. Of all the abnormal genes, HER2/neu (also called c-erbB-2) is one of the oncogenes most closely associated with the occurrence, development, and prognosis of breast carcinoma. Overexpression of the HER2 is detected in ~30% of human breast carcinoma (Miles, 2001) and its overexpression is an important negative prognostic factor that may associate with increased resistance to routine chemotherapies (Del Mastro et al., 2005), radiation therapies, and hormone therapies (Lipton et al., 2002). Drugs that specifically target and inhibit the expression of individual gene are the ideal therapy for cancer and other diseases. Herceptin, a specific humanized monoclonal antibody targeting HER2 receptor, has been approved in clinic for treatment of breast cancers and demonstrated encouraging results (Yeon & Pegram, 2005). With the advancement of technology, other molecular drugs and mechanisms have been reported to down-regulate HER2 expression in the protein level. AS-ODNs came to existence and have been developed rapidly from laboratory to clinics after over two decades of arduous research. It has found application in the treatment of neoplasm, cardiovascular disease, viral and inflammatory diseases. Recently, several groups demonstrated that intervention of gene expression with small molecular oligonucleotide could down-regulate the expression of certain types of oncogenes, therefore reducing drug resistance in breast cancer cells (Gao et al., 2007), inhibiting neuroepithelioma cell growth (Hau et al., 2007), and retarding melanoma growth in mice (Schlingensiepen et al., 2008). In addition, several oligonecleotides specifically targeting oncogenes have showed promising outcome in animal models and clinical trials (Pirollo et al., 2003). These studies suggested that the genetic approach could be utilized to develop specific oncogene inhibitors. Antisense oligonucleotides represent a genre of effective small molecule inhibitors (Redell & Tweardy, 2005; Canales et al., 2006). Antisense oligonucleotide drugs have previously been shown to be safe and more effective than other types of anticancer agents in animal models and in patients with cancer. With the advantage of low molecular weight, an antisense oligonucleotide is easily amenable for knocking down oncogene expression that leads to poor prognosis in the development and progression of malignant tumors.

Breast cancer has become the primary cause of cancer death in woman worldwide. TXT has been recognized as one of the most effective drugs in the treatment of breast cancer. However, the dose-limiting side effects severely limited its application and breast cancer is far from cure. Previously, our laboratory designed an antisense oligonucleotide HA6722, a 20-mer HER2 mRNA-specific AS-ODN, and demonstrated that it could inhibit the growth of MDA-MB-453 breast carcinoma cells in vitro in a dose-dependent manner (Sun et al., 2005). In this study, we extended our findings to show whether HA6722 could inhibit the growth of another HER2 overexpressing breast carcinoma cell line, SK-BR-3, and to investigate the inhibitory effects of HA6722 on SK-BR-3 cells both in vitro and in vivo, although it was administered either alone and in combination with a cytotoxic agent, TXT.

Our results showed that: (a) at a concentration of 5 mg/kg/day, antisense oligonucleotide HA6722 could inhibit the proliferation of SK-BR-3 cells and down-regulate the HER2 mRNA expression in a dose-dependent manner, and (b) combinations of HA6722 and TXT had synergistic/additive effects on cellular retention and could inhibit the growth of breast cancer in xenograft models. These findings coincided with data from others (Waterhouse et al., 2003). In contrast, synergism was not observed with the control sequence Scramble6722. Therefore, a conclusion could be made that HA6722 worked in a sequence-specific manner. The current study provides evidence for the chemosensitizing effect of HA6722 in breast carcinoma and opens new perspectives in improving the therapeutic efficacy of cytotoxic agents against malignancies. The fact that HA6722 could markedly enhance the cytotoxicity of TXT suggests that HA6722 alone or in combination with chemotherapy might become an effective therapeutic approach for patients with other HER2-overexpressing solid tumors. In addition, our finding that HA6722 sensitized tumor cells to TXT by down-regulation of HER2 mRNA provides insight into potential mechanisms by which antisense oligonucleotide work.

In summary, we have collected evidence that HA6722 was effective for HER2-overexpressing breast carcinoma both in vitro and in vivo, and it could be used as a chemosensitizer to the microtubule-affecting antimitotic agent, TXT. In addition, this synergistic effect of HA6722 and TXT correlates with the down-regulation of HER2 mRNA. Therefore, it is safe to say that the combined administration of HA6722 and TXT makes it possible to achieve a relatively higher efficacy at a relative lower dose of the cytotoxic agent, hence avoiding the severe side effects when high-dose chemotherapeutic agents was administered. For the management of patients with breast cancer, the combination treatment might be an ideal strategy.

Conclusions

ASO HA6722, when administered alone or in combination with cytotoxic agent TXT, could inhibit the growth of HER2 overexpression breast cancer both in vitro and in vivo. The combination treatment of TXT and HA6722 could potentially be a more effective approach for breast cancer treatment.

Acknowledgements

The authors thank Prof. Zefei JIAND for his helpful advice and discussions throughout this study.

Declaration of interest

This study was sponsored by National Science Foundation of China (No. 30970592) and Wuzuze Foundation for Sci-Tech Development, Beijing. The authors claim no competing financial interests with any individual or institutions.