Molecular Cancer Therapeutics Molecular Diagnostics in Cancer Therapeutic Development: Fulfilling the Promise of Personalized Medicine Bridging the Lab and the Clinic in Cancer Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jhaveri, M. S.
Right arrow Articles by Chang, E. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jhaveri, M. S.
Right arrow Articles by Chang, E. H.
Mol Cancer Ther. 2004;3:1505-1512
© 2004 American Association for Cancer Research

Antisense oligonucleotides targeted to the human {alpha} folate receptor inhibit breast cancer cell growth and sensitize the cells to doxorubicin treatment

Mona S. Jhaveri1, Antonina S. Rait1, Koong-Nah Chung2, Jane B. Trepel3 and Esther H. Chang1

1 Department of Oncology, Lombardi Cancer Center, Georgetown University Medical Center, Washington, District of Columbia; 2 Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri; and 3 Medical Oncology Clinical Research Unit, National Cancer Institute, NIH, Bethesda, Maryland

Requests for reprints: Esther H. Chang, Department of Oncology, Lombardi Cancer Center, Georgetown University Medical Center, Research Building, E420, 3970 Reservoir Road Northwest, Washington, DC 20007-2197. Phone: 202-687-8418; Fax: 202-687-8434. E-mail: change{at}georgetown.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Folates are essential for cell survival and are required for numerous biochemical processes. The human {alpha} isoform folate receptor ({alpha}hFR) has a very high affinity for folic acid and is considered an essential component in the cellular accumulation of folates and folate analogues used in chemotherapy. The expression of {alpha}hFR is not detected inmost normal tissues. In contrast, high levels of the expression of {alpha}hFR have been reported in a variety of cancer cells. The significance of {alpha}hFR overexpression in malignant tissues has not been elucidated, but it is possible that it promotes cell proliferation not only by mediating folate uptake but also by generating other regulatory signals. The purpose of the present study was to evaluate {alpha}hFR as a potential target for the treatment of breast cancer. Initial studies were done in nasopharyngeal carcinoma (KB) cells, which express high levels of {alpha}hFR. In KB cells, antisense oligodeoxyribonucleotides (ODN) complementary to the {alpha}hFR gene sequences were found to reduce newly synthesized {alpha}hFR protein up to 60%. To examine the effect of {alpha}hFR antisense ODNs in a panel of cultured human breast cancer cell lines, we used a tumor cell–targeted, transferrin-liposome–mediated delivery system. The data show that {alpha}hFR antisense ODNs induced a dose-dependent decrease in cell survival. Finally, we determined that {alpha}hFR antisense ODNs sensitized MDA-MB-435 breast cancer cells by 5-fold to treatment with doxorubicin. The data support the application of {alpha}hFR antisense ODNs as a potential anticancer agent in combination with doxorubicin.

Key Words: doxorubicin • 07-06-02 antisense oligonucleotides


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The human {alpha} class folate receptor ({alpha}hFR) is one member of a multigene family of surface glycoproteins with a very high affinity for folic acid and reduced folates (Kd = 0.1–20 nmol/L; refs. 1–4). Folates are essential for cell survival. They are required for numerous biochemical processes, including DNA and RNA synthesis and transmethylation reactions (1–4). Internalization of folates by {alpha}hFR involves receptor-mediated endocytosis (5). Consequently, {alpha}hFR is considered an essential component in the cellular accumulation of folates and folate analogues used in chemotherapy.

{alpha}hFR is expressed in some (6) but cannot be detected in most normal tissues. In contrast, high levels of {alpha}hFR expression have been reported in cancer cells. These include ovarian, breast, brain, lung, and colorectal cancers (7, 8). The relevance of {alpha}hFR overexpression in malignant tissues remains unclear. It is possible that elevated levels of {alpha}hFR induce cell proliferation not only by mediating folate uptake but also by generating other regulatory signals. {alpha}hFRs represent a class of cell surface proteins that are inserted into the membrane through a tail called the glycosylphosphatidylinositol anchor (9–11). Glycosylphosphatidylinositol-linked proteins are enriched in membrane clusters termed lipid rafts, in which various components of the cell signaling machinery are concentrated (12, 13). In the ovarian carcinoma cell line IGROV1, Miotti et al. (14) showed that {alpha}hFR distributes in low-density membrane microdomains and that the receptor coprecipitates with the Src family tyrosine kinase lyn and the G{alpha}i-3 heterotrimeric G-protein subunit. An invitro kinase assay revealed that both of these signaling molecules become phosphorylated in {alpha}hFR immunoprecipitates (14). Furthermore, transfection and expression of {alpha}hFR in murine NIH/3T3 fibroblast cells, which do not endogenously express {alpha}hFR, increased cell growth invitroand in vivo (15). The data suggest that {alpha}hFR participates in a macromolecular complex that generates intracellular signals potentially involved in modulating cell survival/proliferation processes.

The data presented above suggested the feasibility of targeting the {alpha}hFR for the treatment of cancers with elevated {alpha}hFR. To explore this idea, we designed {alpha}hFR antisense oligodeoxyribonucleotides (ODN). We have showed previously that ligands such as folate and transferrin can beeffective to target liposomal delivery systems to cancer cells in vitro and in vivo (16, 17). To avoid the complication of activating the {alpha}hFR, we used the transferrin-liposome–mediated gene delivery system to examine the effect of {alpha}hFR antisense ODNs in cultured human breast cancer cell lines. We found that {alpha}hFR antisense ODNs reduced {alpha}hFR levels, and this reduction correlated with decreased cell survival. Furthermore, we show that treatment of MDA-MD-435 breast cancer cells with {alpha}hFR antisense ODNs sensitized them to doxorubicin. The data support the potential use of {alpha}hFR antisense ODNs as an anticancer agent in combination with doxorubicin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines and Cell Culture
Human nasopharyngeal epidermoid carcinoma (KB cells) as well as MCF7, ZR-75-1, SK-BR-3, MDA-MB-231, MDA-MB-453, T47D, and MDA-MB-468 breast cancer cell lines were obtained from American Type Culture Collection (Rockville, MD). Human breast carcinoma cell line MDA-MB-435, normal mammary immortalized cell lines MCF10 and HS578T-BST, and normal mammary primary cells HMEC were provided by the Georgetown University Medical Center Tissue Culture Core Facility. Breast cancer cell lines were maintained in DMEM with folic acid and 10% fetal bovine serum. KB cells were maintained in DMEM without folic acid and 10% fetal bovine serum. The final folic acid concentration was ~1 to 10 nmol/L (18). Cell culture medium and medium ingredients were purchased from Biofluids, Inc. (Rockville, MD).

Oligonucleotides
Unmodified ODNs (Table 1) were synthesized by using an automated synthesizer (Applied Biosystems, FosterCity, CA). Purity of 21-mer sense and antisense ODNs was assessed by measurement of 260/280-nm absorbance ratios and by electrophoretic analysis on a 10% polyacrylamide gel. End-modified phosphorothioated ODNs were purchased from Ampligene Biotechnologies (Rockville, MD).


View this table:
[in this window]
[in a new window]
  		Table 1. {alpha}hFR antisense and sense ODN sequences and sequence location within the {alpha}hFR open reading frame

 
Immunoprecipitation
Immunoprecipitations were done as described previously (19). KB cells (106 cells per 35-mm well) were plated in 3mL DMEM without folic acid and with 10% fetal bovine serum. Cells were washed once with 2 mL DMEM (Select Amine, Life Technologies, Inc., Rockville, MD) containing no methionine or cysteine and then incubated for 1 hour in 2 mL of the same medium. Newly synthesized proteins were radiolabeled by the addition of 50 µCi [35S]methionine and 50µCi[35S]cysteine in 1 mL of growth medium containing no methionine or cysteine. Cells were incubated in medium containing radiolabeled amino acids for 16 hours in the presence of unmodified {alpha}hFR sense and antisense ODNs at the indicated concentrations. After labeling, cells were scraped and lysed by repeated freezing and thawing. Membrane proteins were solubilized in 1 mL PBS containing 1% Triton X-100 (v/v). The protein concentrations were determined by a protein assay reagent (Pierce Chemical Co., Rockford, IL). Protein samples (10 µg) were immunoprecipitated using 50 µL of polyclonal rabbit anti-{alpha}hFR antiserum and protein A-Sepharose in 750 µL of immunoprecipitation buffer [10 mmol/L Tris, 150 mmol/L NaCl, 2mmol/L EDTA (pH 7.4), 1% Triton X-100, and 0.1% SDS] for 3 hours at 4°C (20). Immunoprecipitates were boiled for5 minutes in the same buffered solution and pelleted at2,500 x g for 5 minutes. Supernatants were fractionated by12% SDS-PAGE and visualized and quantitated by PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

RNase Protection Assay
The relative abundance of {alpha}hFR transcripts in normal human tissue was determined with RNase protection assay as described previously (21). RNA was purchased from Clontech (Palo Alto, CA) and the integrity and quantity of RNA was verified by Northern analysis. The 5' EcoRI-HincII restriction fragment from the KB4 cDNA clone was radiolabeled using the Promega (Madison, WI) in vitro transcription kit and used as the {alpha}hFR riboprobe. Total RNA (25 µg), internal control RNA (2 µg), and {alpha}hFR riboprobe (100,000 counts/min) in 30 µL of hybridization buffer [40 mmol/L PIPES (pH 6.4), 80% (v/v) formamide, 0.4 mol/L NaCl, and 1 mmol/L EDTA] were denatured at 85°C for 8 minutes and then reannealed at 45°C for 18hours. The ssRNA was digested by the addition of 350µL of buffer [10 mmol/L Tris-HCl (pH 7.5), 200 mmol/L NaCl, 100 mmol/L LiCl, and 2 mmol/L EDTA] containing RNase A (14 µg) and RNase T1 (0.7 µg) followed by incubation at 25°C for 15 minutes. The digestion was terminated by the addition of 10% SDS (w/v) and proteinase K (100 µg) and incubation at 37°C for 30 minutes. After extraction in an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v), RNA was precipitated with ethanol and fractionated on a 6% sequencing wedge gel. The dried bands were visualized using a PhosphorImager (Molecular Dynamics).

Western Blot Analysis
Cellular lysates for Western analyses were prepared as described previously (22). Breast cancer and normal mammary cells were washed twice in room temperature PBS and harvested by scraping in cold lysis buffer containing PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1mg/mL phenylmethylsulfonylfluoride, 30 µg/mL aprotinin, and 1 mmol/L sodium orthovanadate. After 20minutes in an ice bath, lysates were passed through a 21-gauge needle, incubated on ice an additional 30 minutes, and then centrifuged at 15,000 x g for 20 minutes at 4°C. Protein concentrations were determined using the Pierce Micro bicinchoninic acid protein assay reagent and the proteins were stored at –80°C.

Primary anti-human {alpha}hFR polyclonal rabbit antibody was generated as described previously (20). To visualize the transferrin receptor in breast cancer cells, we used mouse anti-human transferrin receptor (Zymed Laboratories, Inc., San Francisco, CA). The anti-human glyceraldehyde-3-phosphate dehydrogenase rabbit polyclonal antibody was purchased from Trevigen (Gaithersburg, MD). The anti-actin (C-11) goat polyclonal antibody and all secondary horseradish peroxidase–conjugated antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA).

Samples containing 20 µg of total cellular protein were electrophoresed on a 4% to 20% SDS-PAGE gradient gel and electroblotted onto a 0.2-µm nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). To block nonspecific binding, the membrane was incubated at room temperature for 1 hour with 5% nonfat dry milk in 10 mmol/L Tris-HCl buffer (pH 8.0) containing 150 mmol/L NaCl and 0.05% Tween 20 (TBST). The blot was probed for 1 hour with primary antibody and washed thrice for 15 minutes each with TBST. The specific protein was detected using secondary horseradish peroxidase–conjugated immunoglobulin G. The membrane was probed with secondary antibody for 45 minutes and washed thrice for 15 minutes each with TBST. Proteins were visualized using the enhanced chemiluminescence Western blotting detection reagent and Hyperfilm enhanced chemiluminescence (Amersham, Piscataway, NJ).

Transfection and Cell Survival Assay
Transfection of {alpha}hFR S6 and AS6 end-modified phosphorothioated ODNs into breast cancer cells was accomplished using a transferrin-mediated liposomal Be delivery system as described previously (16, 17). Briefly, 2 x 104 cells were plated per well in a 96-well plate and washed twice with serum-free DMEM. Transferrin-liposome-ODN complex was prepared by mixing 50 nmol liposome B with 625 µg transferrin and then 5 nmol ODNs in 800 µL of serum-free DMEM at a final ODN concentration of 5 µmol/L and then added to cells at indicated concentrations for 5 hours. Transfections were terminated by theaddition of an equal volume of DMEM containing 10% fetal bovine serum. Cell survival was determined after 48hours by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay according to the manufacturer's instructions (Boehringer Mannheim,Indianapolis, IN). In the presence of an electron coupling reagent, XTT is converted into orange formazan by dehydrogenase in the mitochondria of living cells. The formazan absorbance, which correlates to the number of living cells, was measured at 450 nm using a microplate reader (Molecular Devices, Menlo Park, CA). The IC50 was interpolated from the graph of the log of drug concentration versus the fraction of surviving cells.

Chemosensitization
For chemosensitization studies, MDA-MB-435 cells were plated and transfected with 500 nmol/L of {alpha}hFR AS6 or S6 ODN as described above. Twenty-four hours after transfection, the medium was replaced with DMEM containing 10% fetal bovine serum and doxorubicin (Bedford Labs, Bedford, OH) at the indicated concentrations. Cell survival was determined by XTT assay, as described above, after 72hours.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Treatment with {alpha}hFR Antisense ODNs Decreases Protein Synthesis and Cell Survival in KB Cells
Eight antisense ODNs were designed to target the {alpha}hFR open reading frame (Fig. 1A) to determine whether decreased levels of {alpha}hFR impact cell survival. For preliminary experiments, we used cultured human nasopharyngeal cells (KB cells), which express high levels of {alpha}hFR. KB cells were incubated with {alpha}hFR antisense ODNs (AS1, AS2, and AS6) at the indicated concentrations for 6 hours. Figure 1B shows a dose-dependent decrease in the levels of {alpha}hFR protein up to 60% 2 days after the treatment as determined by immunoprecipitation analysis. In contrast, {alpha}hFR sense ODNs (S1, S2, and S6) had little effect on the levels of {alpha}hFR in KB cells (Fig. 1C). The data correspond to survival analysis of KB cells, which decreased after 2 days of {alpha}hFR antisense exposure (data not shown).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 1. A, schematic representation of eight antisense ODNs targeted to various regions of the {alpha}hFR open reading frame. Immunoprecipitation analysis of {alpha}hFR protein levels in KB cells treated with {alpha}hFR antisense ODNs (B) or sense ODNs (C). KB cells (106 cells per 35-mm well) were incubated with unmodified ODNs for 6 hours in serum-free medium. Relative amounts of {alpha}hFR protein were determined 36 hours after transfection using a polyclonal rabbit {alpha}hFR antibody. Immunoprecipitates were fractionated by 12% SDS-PAGE and visualized and quantitated by a PhosphorImager (Molecular Dynamics).

 
Expression of {alpha}hFR in Normal Tissue and Cultured Human Breast Cancer Cells
The levels of {alpha}hFR are reportedly higher in cancer compared with normal tissue (7, 8). We determined the levels of{alpha}hFR transcripts in a panel of normal tissue using a RNase protection assay. Figure 2 shows that {alpha}hFR transcripts are present in some tissues (i.e., the lung, kidney, breast, salivary gland, and placenta) but are not detected orare present at very low levels in most other normal tissue; in contrast, much higher levels are present inKB cells. The data confirm and extend previous observations (6).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 2. RNase protection assay of {alpha}hFR transcript levels in normal tissue. Normal human tissue RNA was purchased from Clontech. Total tissue RNAs (25 µg) were used for RNase protection assay exactly as described previously (20). The RNA species was precipitated and fractionated on a 6% (w/v) sequencing wedge gel. The dried gel was visualized using a PhosphorImager (Molecular Dynamics).

 
We were interested in evaluating {alpha}hFR antisense ODNs as a potential anticancer therapeutic agent for the treatment of breast cancer. Previous studies have reported expression of {alpha}hFR in MDA-MB-231 (22) and T47D (3) but not in ZR-75-1 (23) and MCF7 (19) cultured, human breast cancer cell lines. In Fig. 3, we did Western blot analysis of {alpha}hFR levels in a panel of eight cultured breast cancer cell lines (MCF7, ZR-75-1, SK-BR-3, MDA-MB-435, MDA-MB-231, MDA-MB-453, T47D, and MDA-MB-468), two normal mammary immortalized cell lines (MCF10A and HS578T-BST), and normal mammary primary cells (HMEC). We have detected {alpha}hFR at variable levels in all tested samples. Corrected for protein loading (based on actin levels), it seems that most of the breast cancer cell lines express higher levels of {alpha}hFR compared with that in the normal cells.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 3. Western blot analyses of {alpha}hFR and transferrin receptor (TfR) in human breast cancer cells and immortalized and primary mammary cells. Samples containing 20 µg of total cellular protein were electrophoresed on a 4% to 20% SDS-PAGE gradient gel and electroblotted onto a nitrocellulose membrane. Detection of {alpha}hFR and transferrin receptor was accomplished using polyclonal rabbit anti-{alpha}hFR and polyclonal mouse anti–transferrin receptor, respectively, horseradish peroxidase–conjugated secondary immunoglobulin G, and enhanced chemiluminescence Western blotting reagents.

 
Transferrin-Liposome–Mediated Transfection of {alpha}hFR Antisense and Sense ODNs in Human Breast Cancer Cells
Transferrin-liposome Be–mediated gene delivery has been shown both in vitro and in vivo (24, 25). Transferrin is a plasma glycoprotein used by cells to acquire iron via a process involving receptor endocytosis. There is evidence for overexpression of the receptor for transferrin on tumor cells, including oral (26, 27), prostate (28), and breast (29) cancer cells. We assessed the levels of transferrin receptors by Western blot analysis in cultured breast cancer and normal epithelial mammary cell lines. As shown in Fig. 3, breast cancer cell lines express much higher levels of transferrin receptor compared with immortalized and primary normal cell lines. We have showed previously that both transferrin and folate can be an effective ligand totarget cancer cells in vitro and invivo (16, 17). Thus, in this study, to avoid the complication of activating the {alpha}hFR, we have used the transferrin-liposome Be–mediated gene delivery system to examine the effect of {alpha}hFR antisense ODNs in cultured human breast cancer cell lines.

Impact of {alpha}hFR Antisense ODNs on Survival of Cultured Human Breast Cancer Cells
To determine the effect of {alpha}hFR antisense ODNs on the survival of cultured breast cancer cells, we transfected end-modified phosphorothioated AS6 ODNs and S6 ODNs (Table 1) at various concentrations via the transferrin-liposome Be–mediated gene delivery system. As shown inFig. 4A, increased amounts of transfected AS6 ODNs correlated with decreased cell survival as determined by XTT assay. The IC50 values (µmol/L) for the treated breast cancer cell lines were 0.633 (T47D), 0.600 (MDA-MB-231), and 0.416 (MDA-MB-435). In contrast to these data, cell lines treated with S6 ODNs had significantly less effect onthe survival of model breast cancer cell lines (Fig. 4B).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 4. Effect of various concentrations of {alpha}hFR AS6 ODNs (A) and S6 ODNs (B) on survival of cultured human breast cancer cells. End-modified phosphorothioated ODNs were transfected into 2 x 104 cells at the indicated concentrations for 6 hours using a transferrin-liposome Be–mediated delivery complex. Cell number was assessed 48 hours after transfection by XTT assay.

 
Effect of {alpha}hFR ODNs on the Levels of {alpha}hFR Protein inMDA-MB-435 Breast Cancer Cells
Antisense ODNs are believed to abrogate protein function by direct hybridization of the ODNs to exposed regions of the targeted mRNA. The resulting RNA-DNA is thought to preclude the translation of the mature, functional protein, thereby diminishing the overall cellular target protein levels (30, 31). To examine if the {alpha}hFR ODN effect on cell survival was correlated to decreased levels of {alpha}hFR protein, we transfected MDA-MB-435 breast cancer cells with end-modified phosphorothioated AS6 and S6 ODN, as shown in Fig. 5, and did Western analyses. We observed a significant decrease in the levels of {alpha}hFR after treatment with 0.25 and 0.50 µmol/L AS6 ODNs for 24 or 48 hours. However, the {alpha}hFR levels of the S6 ODN–treated samples remained relatively indistinguishable from the untreated cells or cells treated with liposome alone. These results strongly suggest that the cellular levels of {alpha}hFR protein are an important component of cell survival.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 5. Effect of {alpha}hFR AS6 and S6 ODNs on levels of {alpha}hFR protein in MDA-MB-435 breast cancer cells. MDA-MB-435 breast cancer cells were transfected, as discussed in Fig. 4, with end-modified phosphorothioated {alpha}hFR AS6 or S6 ODNs at 0.25 or 0.50 µmol/L concentrations using transferrin-liposome Be. Protein levels of {alpha}hFR were assessed by Western blot analysis using polyclonal rabbit anti-{alpha}hFR and the anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Trevigen).

 
Sensitization of MDA-MB-435 Breast Cancer Cells to Doxorubicin after Treatment with {alpha}hFR Antisense ODN
To determine whether {alpha}hFR antisense ODN sensitizes breast cancer cells to chemotherapy, we transfected, using transferrin-liposome Be, 500 nmol/L of end-modified phosphorothioated AS6 ODN or S6 ODN into MDA-MB-435 cells and treated them with varying concentrations of doxorubicin 24 hours after transfection. Cell survival was determined after 72 hours by XTT assay and the degree of sensitization was calculated by determining the ratio of IC50 values of the combined treatment relative to that of the S6 ODN and untreated controls. In Fig. 6, chemosensitization of MDA-MD-435 cells by AS6 ODN was ~5-fold (IC50S6 / IC50AS6 = 5) relative to the sense control and ~10-fold (IC50Untreated / IC50AS6 = 10) relative to the untreated control.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 6. Chemosensitization of MDA-MB-435 breast cancer cells after combination treatment with {alpha}hFR AS6 ODNs and doxorubicin. MDA-MB-435 cells were plated at 2 x 104 cells per well and transfected with 500 nmol/L {alpha}hFR AS6 or S6 end-modified phosphorothioated ODNs using transferrin-liposome Be (Tf-Lip Be). After 24 hours, cells were incubated with medium containing doxorubicin at the indicated concentrations. Cell number was determined by XTT assay after 72 hours (22). UT, untransfected.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
{alpha}hFR is one member of a multigene family of folate binding proteins. {alpha}hFR is considered an important component in the maintenance of cellular folate homeostasis. {alpha}hFR binds folates with high affinity (Kd = 0.1–20 nmol/L) and internalizes the vitamin by a process involving receptor-mediated endocytosis (1–4).

We and others (6–8) have reported that {alpha}hFR levels are higher in cancer cells than in normal tissue (Fig. 2). For example, {alpha}hFR is elevated in >90% of human epithelial ovarian malignancies as compared with normal epithelial ovarian cells (32). Other cancers with elevated {alpha}hFR relative to their normal counterpart include breast, brain, lung, and colorectal cancers (7, 8). Consequently, several strategies have been developed for the targeted delivery of drugs to {alpha}hFR-positive tumor cells (33). Covalent attachment of therapeutic agents to {alpha}hFR-targeted monoclonal antibodies has been evaluated for imaging and immunotherapy and have shown significant targeting efficacy in patients with ovarian cancer (34). Alternatively, folate-derivatized anticancer treatments have been successfully applied in vitro for {alpha}hFR-specific delivery (35). Of interest, low molecular weight radiopharmaceuticals based on folate conjugates have shown favorable pharmacokinetic properties and tumor selectivity in {alpha}hFR-positive animal tumor models (36, 37). Unlike previous reports that have used {alpha}hFR as a drug delivery vehicle, the present study isthe first account to target {alpha}hFR using gene-specific antisense ODN.

The purpose of elevated {alpha}hFR in cancer is unclear. It has been proposed that {alpha}hFR has a role in promoting cell proliferation independent of folate internalization. Bottero et al. (15) showed that transfection of {alpha}hFR into nonexpressing NIH/3T3 cells increased growth in vitro and invivo. The data presented in this report agree with these observations. We show that reducing the levels of {alpha}hFR, by {alpha}hFR-targeted antisense ODNs (Fig. 1A), inhibited cell survival in cultured human breast cancer and KB cells after36 hours (Fig. 1B). Decreased cell number is not likely to be attributable to folate starvation, a process that occurs over the order of weeks. The literature suggests that {alpha}hFR may be involved in cell processes that are unrelated to folate internalization. For example, {alpha}hFR, similar to other glycosylphosphatidylinositol-linked proteins (12, 13), may have a role in cell signaling processes leading to cell growth and proliferation (14). Further studies are required to delineate the mechanism(s) by which {alpha}hFR ODN mediates death of cancer cells.

In the present study, we were interested in testing {alpha}hFR antisense ODNs as a potential anticancer therapeutic agent for the treatment for breast cancer. We evaluated the levels of {alpha}hFR in breast cancer and normal mammary cells. As shown in Fig. 3, {alpha}hFR was present in all tested cells; however, unlike other disease models, the levels of {alpha}hFR were not significantly elevated in the cultured breast cancer cells compared with normal mammary cells. In an attempt to achieve breast cancer cell–specific targeting, we used a transferrin receptor–mediated delivery system. In Fig. 3, the transferrin receptor is more highly expressed in breast cancer cell lines than in the immortalized and primary normal mammary cells. The data show the feasibility of delivering {alpha}hFR antisense ODNs via a transferrin targeting liposomal Be delivery system even where {alpha}hFR is not overexpressed.

The mechanism by which {alpha}hFR antisense ODNs selectively decreased {alpha}hFR protein in MDA-MB-435 breast cancer cells (Fig. 5) is not entirely clear. In general, antisense ODNs are believed to abrogate protein function by direct hybridization of the ODNs to exposed regions of the targeted mRNA. The resulting RNA-DNA is thought to preclude the translation of the mature, functional protein, thereby diminishing the overall cellular target protein levels. The antisense-mediated decrease of protein production is hypothesized to occur by (a) RNase H digestion of the RNA-DNA duplex or (b) physical disruption of the translation machinery (30, 31). Nonantisense mechanisms may also be involved in selective protein targeting by ODN. For example, aptamers are a class of small nucleic acid ligand agonists that exhibit exquisite specificity for proteins (38, 39). Additional studies are required to understand the mechanism of {alpha}hFR antisense ODN–mediated selective decrease of {alpha}hFR protein in cancer cells.

The current study suggests an application of {alpha}hFR antisense ODNs for the treatment of {alpha}hFR-positive cancers, particularly ovarian cancer. {alpha}hFR has been characterized asa marker for ovarian carcinoma because it is expressed in90% of epithelial ovarian carcinomas and is absent in normal ovarian epithelial cells (40). Furthermore, {alpha}hFR appearss very early in the transformation process and even increases with tumor progression (32). The pathology of ovarian carcinogenesis has been well documented. In the early stages, the spread of the disease typically involves theopposite ovary, the uterus, and the fallopian tubes (41). In the more advanced stages, metastases occur at the peritoneal surfaces of the bladder, the rectosigmoid, or thepelvic peritoneum (42). The confinement of ovarian metastatic spread to the i.p. cavity has allowed for treatment strategies involving i.p. therapy (43). Wide expression of {alpha}hFR in ovarian carcinoma and its association with malignant transformation and cell survival processes as well as local disease metastasis support i.p. administration of {alpha}hFR-targeted antisense molecules for the treatmentof ovarian cancer.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2/11/03; revised 9/23/04; accepted 10/ 7/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Henderson GB. Folate-binding proteins. Annu Rev Nutr 1990;10:319–35.[CrossRef][Medline]
  2. Antony AC. Folate receptors. Annu Rev Nutr 1996;16:501–21.[CrossRef][Medline]
  3. Kane MA, Elwood PC, Portillo RM, et al. Influence on immunoreactive folate-binding proteins of extracellular folate concentration in cultured human cells. J Clin Invest 1988;81:1398–406.
  4. Wagner C. Folate-binding proteins. Nutr Rev 1985;43:293–9.[Medline]
  5. Rijnboutt S, Jansen G, Posthuma G, Hynes JB, Schornagel JH, Strous GJ. Endocytosis of GPI-linked membrane folate receptor-{alpha}. J Cell Biol 1996;132:35–47.[Abstract/Free Full Text]
  6. Weitman SD, Lark RH, Coney LR, et al. Distribution of the folate receptor GP38 in normal and malignant cell lines and tissues. Cancer Res 1992;52:3396–401.[Abstract/Free Full Text]
  7. Garin-Chesa P, Campbell I, Saigo PE, Lewis JL, Old LJ, Rettig WJ. Trophoblast and ovarian cancer antigen LK26. Sensitivity and specificity in immunopathology and molecular identification as a folate-binding protein. Am J Pathol 1993;142:557–67.[Abstract]
  8. Ross JF, Chaudhuri PK, Ratnam M. Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines. Physiologic and clinical implications. Cancer 1994;73:2432–43.[CrossRef][Medline]
  9. Tomassetti A, Bottero F, Mazzi M, Miotti S, Colnaghi MI, Canevari S. Molecular requirements for attachment of the glycosylphosphatidylinositol anchor to the human {alpha} folate receptor. J Cell Biochem 1999;72:111–8.[Medline]
  10. Rothberg KG, Ying YS, Kolhouse JF, Kamen BA, Anderson RG. Theglycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway. J Cell Biol 1990;110:637–49.[Abstract/Free Full Text]
  11. Chung KN, Roberts S, Kim CH, Kirassova M, Trepel J, Elwood PC. Rapid turnover and impaired cell-surface expression of the human folate receptor in mouse L(tk–) fibroblasts, a cell line defective in glycosylphosphatidylinositol tail synthesis. Arch Biochem Biophys 1995;322:228–34.[CrossRef][Medline]
  12. Ilangumaran S, He HT, Hoessli DC. Microdomains in lymphocyte signalling: beyond GPI-anchored proteins. Immunol Today 2000;21:2–7.[CrossRef][Medline]
  13. Horejsi V, Drbal K, Cebecauer M, et al. GPI-microdomains: a role in signalling via immunoreceptors. Immunol Today 1999;20:356–61.[CrossRef][Medline]
  14. Miotti S, Bagnoli M, Tomassetti A, Colnaghi MI, Canevari S. Interaction of folate receptor with signaling molecules lyn and G({alpha})(i-3) in detergent-resistant complexes from the ovary carcinoma cell line IGROV1. J Cell Sci 2000;113:349–57.[Abstract]
  15. Bottero F, Tomassetti A, Canevari S, Miotti S, Menard S, Colnaghi MI. Gene transfection and expression of the ovarian carcinoma marker folate binding protein on NIH/3T3 cells increases cell growth in vitro and in vivo. Cancer Res 1993;53:5791–6.[Abstract/Free Full Text]
  16. Xu L, Pirollo KF, Tang WH, Rait A, Chang EH. Transferrin-liposome-mediated systemic p53 gene therapy in combination with radiation results in regression of human head and neck cancer xenografts. Hum Gene Ther 1999;10:2941–52.[CrossRef][Medline]
  17. Xu L, Pirollo KF, Tang WH, Rait A, Chang EH. Transferrin-liposome-mediated systemic p53 gene therapy in combination with radiation results in human tumor regression. Tumor Target 1999;4:92–114.
  18. McHugh M, Cheng YC. Demonstration of a high affinity folate binder in human cell membranes and its characterization in cultured human KB cells. J Biol Chem 1979;254:11312–8.[Free Full Text]
  19. Chung KN, Saikawa Y, Paik TH, et al. Stable transfectants of human MCF-7 breast cancer cells with increased levels of the human folate receptor exhibit an increased sensitivity to antifolates. J Clin Invest 1993;91:1289–94.
  20. Saikawa Y, Knight CB, Saikawa T, Page ST, Chabner BA, Elwood PC. Decreased expression of the human folate receptor mediates transport-defective methotrexate resistance in KB cells. J Biol Chem 1993;268:5293–301.[Abstract/Free Full Text]
  21. Roberts SJ, Chung KN, Nachmanoff K, Elwood PC. Tissue-specific promoters of the {alpha} human folate receptor gene yield transcripts with divergent 5' leader sequences and different translational efficiencies. Biochem J 1997;326:439–47.
  22. Rait AS, Pirollo KF, Rait V, Krygier JE, Xiang L, Chang EH. Inhibitory effects of the combination of HER-2 antisense oligonucleotide and chemotherapeutic agents used for the treatment of human breast cancer. Cancer Gene Ther 2001;8:728–39.[CrossRef][Medline]
  23. Pinard MF, Jolivet J, Ratnam M, et al. Functional aspects of membrane folate receptors in human breast cancer cells with transport-related resistance to methotrexate. Cancer Chemother Pharmacol 1996;38:281–8.[CrossRef][Medline]
  24. Cristiano RJ, Curiel DT. Strategies to accomplish gene delivery via thereceptor-mediated endocytosis pathway. Cancer Gene Ther 1996;3:49–57.[Medline]
  25. Cheng PW. Receptor ligand-facilitated gene transfer: enhancement of liposome-mediated gene transfer and expression by transferrin. Hum Gene Ther 1996;7:275–82.[Medline]
  26. Thorstensen K, Romslo I. The transferrin receptor: its diagnostic value and its potential as therapeutic target. Scand J Clin Lab Invest Suppl 1993;215:113–20.[Medline]
  27. Miyamoto T, Tanaka N, Eishi Y, Amagasa T. Transferrin receptor in oral tumors. Int J Oral Maxillofac Surg 1994;23:430–3.[Medline]
  28. Keer HN, Kozlowski JM, Tsai YC, Lee C, McEwan RN, Grayhack JT. Elevated transferrin receptor content in human prostate cancer cell lines assessed in vitro and in vivo. J Urol 1990;143:381–5.[Medline]
  29. Inoue T, Cavanaugh PG, Steck PA, Brunner N, Nicolson GL. Differences in transferrin response and numbers of transferrin receptors in ratand human mammary carcinoma lines of different metastatic potentials. J Cell Physiol 1993;156:212–7.[CrossRef][Medline]
  30. Crooke ST. Molecular mechanisms of action of antisense drugs. Biochim Biophys Acta 1999;1489:31–44.[Medline]
  31. Walder RY, Walder JA. Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc Natl Acad Sci U S A 1988;85:5011–5.[Abstract/Free Full Text]
  32. Wu M, Gunning W, Ratnam M. Expression of folate receptor type {alpha} in relation to cell type, malignancy, and differentiation in ovary, uterus, and cervix. Cancer Epidemiol Biomarkers Prev 1999;8:775–82.[Abstract/Free Full Text]
  33. Sudimack J, Lee RJ. Targeted drug delivery via the folate receptor. Adv Drug Deliv Rev 2000;41:147–62.[CrossRef][Medline]
  34. Crippa F, Buraggi GL, Di Re E, et al. Radioimmunoscintigraphy of ovarian cancer with the MOv18 monoclonal antibody. Eur J Cancer 1991;27:724–9.
  35. Wang S, Lee RJ, Mathias CJ, Green MA, Low PS. Synthesis, purification, and tumor cell uptake of 67Ga-deferoxamine-folate, a potential radiopharmaceutical for tumor imaging. Bioconjug Chem 1996;7:56–62.[CrossRef][Medline]
  36. Mathias CJ, Wang S, Lee RJ, Waters DJ, Low PS, Green MA. Tumor-selective radiopharmaceutical targeting via receptor-mediated endocytosis of gallium-67-deferoxamine-folate. J Nucl Med 1996;37:1003–8.[Abstract/Free Full Text]
  37. Mathias CJ, Wang S, Low PS, Waters DJ, Green MA. Receptor-mediated targeting of 67Ga-deferoxamine-folate to folate-receptor-positive human KB tumor xenografts. Nucl Med Biol 1999;26:23–5.[CrossRef][Medline]
  38. Burgstaller P, Jenne A, Blind M. Aptamers and aptazymes: accelerating small molecule drug discovery. Curr Opin Drug Discov Devel 2002;5:690–700.[Medline]
  39. Joshi PJ, Fisher TS, Prasad VR. Anti-HIV inhibitors based on nucleic acids: emergence of aptamers as potent antivirals. Curr Drug Targets Infect Disord 2003;3:383–400.[CrossRef][Medline]
  40. Campbell IG, Jones TA, Foulkes WD, Trowsdale J. Folate-binding protein is a marker for ovarian cancer. Cancer Res 1991;51:5329–38.[Abstract/Free Full Text]
  41. Plentl AA, Friedman EA. Lymphatic system of the female genitalia. The morphologic basis of oncologic diagnosis and therapy. Major Probl Obstet Gynecol 1971;2:1–223.[Medline]
  42. Fuks Z. Patterns of spread of ovarian carcinoma: relation to therapeutic strategies. Adv Biosci 1980;26:39–51.
  43. Gibbs DD, Gore ME. Pursuit of optimum outcomes in ovarian cancer: methodological approaches to therapy. Drugs 2001;61:1103–20.[Medline]



This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
T. R. Daniels, P. P. Ng, T. Delgado, M. R. Lynch, G. Schiller, G. Helguera, and M. L. Penichet
Conjugation of an anti transferrin receptor IgG3-avidin fusion protein with biotinylated saporin results in significant enhancement of its cytotoxicity against malignant hematopoietic cells
Mol. Cancer Ther., November 1, 2007; 6(11): 2995 - 3008.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
K. L. Knutson, C. J. Krco, C. L. Erskine, K. Goodman, L. E. Kelemen, P. J. Wettstein, P. S. Low, L. C. Hartmann, and K. R. Kalli
T-Cell Immunity to the Folate Receptor Alpha Is Prevalent in Women With Breast or Ovarian Cancer
J. Clin. Oncol., September 10, 2006; 24(26): 4254 - 4261.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
V. Askoxylakis, S. Zitzmann, W. Mier, K. Graham, S. Kramer, F. von Wegner, R. H.A. Fink, M. Schwab, M. Eisenhut, and U. Haberkorn
Preclinical Evaluation of the Breast Cancer Cell-Binding Peptide, p160
Clin. Cancer Res., September 15, 2005; 11(18): 6705 - 6712.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jhaveri, M. S.
Right arrow Articles by Chang, E. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jhaveri, M. S.
Right arrow Articles by Chang, E. H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation