1223-1230

INTERNATIONAL JOURNAL OF ONCOLOGY 30: 1223-1230, 2007 Ribozyme-targeting procathepsin D and its effect
on invasion and growth of breast cancer cells:
An implication in breast cancer therapy
ARUNA VASHISHTA1, SUJATA SARASWAT OHRI1, MARY PROCTOR2, 1Department of Pathology, University of Louisville, Louisville, KY; 2Research Resource Center, University of Louisville and VA Medical Center, Louisville, KY, USA; 3Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Received November 20, 2006; Accepted January 17, 2007 Abstract. Procathepsin D (pCD), a zymogen of lysosomal
and secreted in a disease-specific manner provide possible aspartic peptidase cathepsin D, overexpression is correlated targets for therapeutics, early detection and monitoring of with highly invasive malignancies, including breast cancer.
Recently, different studies have shown the role of secreted Procathepsin D (pCD) is a proform of lysosomal aspartic pCD as mitogen acting both in an autocrine and a paracrine peptidase cathepsin D (CD) that is overexpressed (2- to 50- manner. The aim of the present study is to examine the anti- fold) and secreted by breast cancer tissues and cell lines (1-3).
tumor effects elicited by a decrease in the protein level of In normal cells, CD is translated as preprocathepsin D in pCD by ribozyme and to explore the therapeutic potential rough endoplasmic reticulum and is translocated to golgi of this specific targeting. Using the mFold program, we apparatus as pCD (52 kDa) after removal of signal peptide.
designed seven anti-pCD ribozymes and checked the acces- The pCD is then sorted to lysosomes, where it is activated sibility to target pCD mRNA by RNase H cleavage experiment at low pH to a single-chain proteolytically active form by in a cell-free system. The sequences of the 4 most effective removal of activation peptide (AP) and further cleaved into a ribozymes were cloned and stably transfected in a highly two-chain mature form of 33 and 14 kDa respectively (4).
metastatic human breast cancer cell line, MDA-MB-231, to The mature CD participates in intracellular protein catabolism, knock down the expression of pCD. Downregulation of pCD hormone and antigen processing, and apoptotic pathway (5,6).
due to ribozyme expression was observed by Western blotting However, the proform, pCD, is correlated with enhanced and real-time RT-PCR. Stably transfected cells with anti- proliferation and neoplastic transformation (7,8). Clinical pCD ribozymes exhibited a significant lowering of in vitro studies using immunoassays have reported that pCD/CD is a invasion (p<0.001) and reduction in lung colonization potential marker of poor prognosis for breast cancer and is associated in nude mice when compared to control ribozyme transfected with a high risk of metastasis (9-13). Various studies performed cells. We also found that downregulation of pCD by ribo- on the cell culture as well as on a mouse model have shown zyme promotes apoptosis of MDA-MB-231 cells on serum that the transfection of pCD in cancer cell lines influences the deprivation. These results suggest that we have generated a growth of cancer cells and that the rate of growth was related biologically functional ribozyme against pCD with possible to the level of pCD expression (14,15). Therefore, targeting therapeutic implications in breast cancer cells.
pCD by genetic means could help to better understand its rolein tumor growth. For this purpose, we used ribozyme directed Introduction
Hammerhead ribozymes are small catalytic RNAs with Breast cancer is one of the most lethal malignancies among the potential to ablate gene expression in a highly sequence- women in the Western world. Proteins that are overexpressed specific manner and are therefore useful in analyzing genefunction and as a therapeutic approach against various human _________________________________________ diseases (16,17). Ribozymes possess the unique characteristicof cleaving RNA without the additional help of an enzyme Correspondence to: Dr Vaclav Vetvicka, Department of (16). Ribozyme has the antisense effect as well as catalytic Pathology, University of Louisville, 511 S. Floyd Street, MDR activity, both of which contribute to the activity of ribozyme (18). Multiple turnovers, i.e. binding and cleaving of the substrate, dissociation and binding to a new target molecule,provide an additional advantage to ribozyme. We used a Key words: breast cancer, invasion, procathepsin D, ribozyme, hammerhead ribozyme approach to target and suppress pCD gene expression in vitro and in vivo and further determinepCD's role in cancer invasion and metastasis.
VASHISHTA et al: EFFECTS OF RIBOZYME-MEDIATED pCD KNOCKDOWN ON BREAST CANCER In the present study, we first tested all the designed ribo- were PCR amplified using the overlapping oligos which have zymes under physiological conditions in a cell-free system a SalI site at the 5' end and a HindIII site at the 3' end. The PCR followed by cloning of the effective ribozymes in expression products were digested with SalI and HindIII and cloned in vector. The constructs were stably transfected in breast SalI and HindIII digested pHßApr-1-neo vector to produce cancer cell line MDA-MB-231 and downregulation of pCD a plasmid where the expression of ribozyme was driven by expression was monitored both at mRNA and protein levels.
ß-actin promoter. The direction and sequence were verified Attenuation of pCD expression by ribozyme resulted in a using plasmid-specific primers: 5'-CCTCCGACCAGTGT great reduction of cell invasion in vitro and lung colonization TTGC-3' and 5'-GTCTGGATCCCTCGAAGC-3'.
Transfection. The MDA-MB-231 human breast cancer cell Materials and methods
line was cultured in normal growth medium overnight toobtain 70-80% confluency. The following day, the cells were Cell lines and cell culture. Breast cancer cell line MDA- transfected with 1.5 μg of plasmid DNA mixed with 2.5 μl MB-231 was obtained from Dr R. Ceriani of the John Muir of Lipofectamine-2000 reagent (Invitrogen) in Optimem-I Cancer and Aging Research Institute, Walnut Creek, CA.
media (Invitrogen). After 5 h, the transfection medium was The cell line was cultured in RPMI-1640 (Sigma Chemical replaced with normal medium and the cells were grown for Co., St. Louis, MO) with 10% fetal calf serum (FCS; an additional 24 h. For generation of stable clones, the cells Hyclone Lab., Logan, UT), 100 U/ml penicillin (Sigma) and were incubated in the presence of Geneticin (G418-sulfate; 100 μg/ml streptomycin (Sigma) at 37˚C in 5% CO .
Invitrogen) over a period of 3 weeks. Finally, clonal expansionwas performed in a 96-well plate and the positive stable clones Design of anti-pCD ribozymes and analysis of accessible site using RNase H mapping. The procedure has been previouslyreported (15). Briefly, the secondary structure of human Western blot analysis. Control and stably transfected MDA- pCD was generated using RNA mFOLD program version MB-231 cells (2x105 cells/ml) were seeded in normal growth 3.1 (19). The suitable GUC sites within the pCD mRNA were medium followed by replacement with medium containing identified. The sense and antisense oligodeoxyribonucleotide 0.1% FCS and allowed to grow for another 48 h. Super- (ODN) corresponding to ribozyme target sequences identified natants were collected, concentrated (10X) using centricon by mFOLD program were designed. MDA-MB-231 cells (Pall Life Sciences, Ann Arbor, MI) and 10 μg protein (3x107) were pelleted and washed twice in phosphate-buffered (estimated by BCA protein Assay Kit; Pierce, Rockford, IL) saline (PBS). The pellet was resuspended in hypotonic buffer was resuspended in Laemmli buffer (BioRad, Hercules, (7 mM Tris-HCl, pH 7.5, 7 mM KCl, 1 mM MgCl , 1 mM CA). For lysates, the control and transfected cells were ß-mercaptoethanol), incubated for 10 min on ice, sonicated trypsinized, and washed twice with cold PBS. The cell pellet with pulse of 10 sec followed by addition of 1/10 of the final was then dissolved in cold lysis buffer (10 mM Tris, 5 mM volume of neutralizing buffer (21 mM Tris-HCl, pH 7.5, EDTA, 150 mM NaCl, 1% Triton-X 100, pH 7.4 containing 116 mM KCl, 3.6 mM MgCl , 6 mM ß-mercaptoethanol).
protease inhibitors cocktail; Sigma). Lysis was performed The homogenate was centrifuged at 13,000 rpm for 15 min at on ice for 30 min. Protein lysate was then cleared by centri- 4˚C. The RNaseH-mediated cleavage experiment was carried fugation and 20 μg of each protein sample was resuspended out in a total volume of 30 μl, containing 20 μl of cell in Laemmli buffer. Electrophoresis and immunoblotting was extract, 1 mM DTT, 40 U of RNase inhibitor (Ambion Inc., performed as described (20). The monoclonal antibody anti-AP Austin, TX) and 50 nM of the various antisense ODNs and (21), anti-CD antibody (Calbiochem, San Diego, CA) and the sense ODN (control). The mixture was incubated for 15 min polyclonal anti-ß-actin antibody (Ambion Inc.) were used.
at 37˚C followed by digestion with 10 U of DNase I (AmbionInc.) for 45 min at 37˚C and then subjected to phenol RT-PCR analysis. Total cellular RNA was extracted from the extraction and ethanol precipitation. Reverse transcription control and transfected MDA-MB-231 cells using TRIzol (RT) was performed using two-step RT-PCR kit (Invitrogen, reagent (Invitrogen) according to the manufacturer's instr- Carlsbad, CA) in 20 μl using 50 ng of random hexamer uctions and extensively treated with RNase-free DNase. Total primer and 10 U of Moloney murine leukemia virus reverse cellular RNA (1 μg) of control and each transfectant were transcriptase. An aliquot of 18 μl of each RT reaction was reverse transcribed in a volume of 20 μl using the Superscript™ PCR-amplified using pCD primers: (5'-CCAGTACTACG first-strand synthesis system (Invitrogen). An aliquot of 2 μl of RT product was PCR amplified using the plasmid-specific CGA-3') and 2 μl using the ß-actin primers (5'-TGCTATC primers (described above) to check for ribozyme expression and also with pCD primers and ß-actin primers (internal CTGAGTC-3') for 25 cycles. PCR was carried out in 50 μl control) to check the pCD expression and the equal amount with 1.5 U of Taq DNA polymerase at 94˚C for 30 sec, 62˚C of RNA respectively. PCR amplification for pCD and actin for 45 sec, 72˚C for 90 sec with final extension at 72˚C for was performed for 25 cycles as described above and for RZ 7 min. The PCR products were resolved on 1.5% agarose gel expression initial denaturation was performed at 94˚C for and visualized after staining with ethidium bromide.
2 min followed by 30 cycles of 94˚C for 30 sec, 55˚C for30 sec and 72˚C for 30 sec and final extension at 72˚C for Construction of ribozyme expression vector. The ribozymes 7 min. The PCR products were separated on 1.5% agarose designated as control RZ, RZ385, RZ644, RZ763 and RZ1254 gel and visualized by ethidium bromide staining.
INTERNATIONAL JOURNAL OF ONCOLOGY 30: 1223-1230, 2007 Real-time RT-PCR. Messenger RNA was isolated from Table I. Sequences of antisense and control sense ODNs transfected MDA-MB-231 cells and control cells using the corresponding to ribozyme target sequences identified by TRIzol reagent (Invitrogen). The cDNA was synthesized using 250 ng of total RNA and TaqMan reverse transcription ––––––––––––––––––––––––––––––––––––––––––––––––– reagents (Applied Biosystems, Foster City, CA) in 25 μl RT reaction. Real-time PCR reaction was then carried out in a total of 20 μl of reaction mixture using 2 μl cDNA per reaction, 5 μM of each cat D primer (forward: 5'-GGACATCGCTTG CTGGAT-3' and reverse: 5'-CTTGGCTGCGATGAAGGT-3') or ß-actin primer (forward: 5'-CACTGGCATCGTGATGGA-3' and 5'-GGCCATCTCTTGCTCGAA-3') and 10 μl of 2X SYBR-Green PCR Core Reagents on ABI PRISM SequenceDetection System 7300 (Applied Biosystems). The PCR ––––––––––––––––––––––––––––––––––––––––––––––––– program was initiated by 10 min at 95˚C before 40 thermalcycles, each for 15 sec at 95˚C and 1 min at 60˚C. At the endof the PCR cycle, a dissociation curve was generated to ensurethe amplification of a single product and the threshold cycle The medium was then aspirated to ease the formation of time (Ct values) for each gene was determined. Relative mRNA the formazan product, which was then solubilized with the levels were calculated based on the Ct values normalized to addition of 100 μl of acidic isopropanol. The optical density was measured at 570 nm with reference wavelength of630 nm using a SLT ELISA reader (Tecan, Research Triangle In vitro invasion analysis and proliferation assay. A Park, NC). Similar experiments were repeated in triplicates.
commercial kit by Chemicon International (Temecula, CA)was used to evaluate the invasion across Matrigel layers Cell death detection ELISA. Apoptosis was studied with according to the manufacturer's instructions. Briefly, 3x105 the detection and quantification of cytoplasmic histone- cells were added to each well and after 24 h, the cells that associated DNA fragments (mono- and oligo-nucleasomes) had migrated through the matrix and adhered to other side by photometric enzyme immunoassay (Cell Death Detection of the insert were fixed and stained with 0.5% (w/v) crystal ELISA, Roche Diagnostics, Mannheim, Germany). The control violet. Cells that had invaded and stained with crystal violet cells and stably transfected cells were seeded in 12-well were extracted with 10% (v/v) acetic acid. An aliquot of stained plates and after 24 h the cells were deprived of serum for cells was transferred to a 96-well plate and absorbance was 48 h. The cells were lysed and centrifuged followed by obtained at 560 nm using a multiplate reader.
analysis of cytoplasmic histone-associated DNA fragments For cell growth assay, control and stably transfected cells according to the manufacturer's instructions. Results were were plated into a 96-well plate at 5x104 cells/ml (150 μl/ from three different experiments performed in triplicate.
well). Cells were evaluated for proliferation on the day ofplating and day 1, 2, 3 and 4 after plating using a Biotrak Cell Statistical analysis. All data were expressed as mean ± proliferation ELISA system (Amersham Pharmacia Biotech, standard deviation (SD). Statistical comparisons between Piscataway, NJ) according to the manufacturer's instructions.
two groups or among groups were determined using t-test The growth of cells is shown here as absorbance.
or one-way ANOVA. A p-value of <0.05 was consideredstatistically significant.
In vivo studies in nude mice. Four- to six-week-old athymicnude mice (Jackson lab, Bar Harbor, ME) were injected i.v.
with 0.2 ml of a cell suspension containing 4x106 MDA-MB-231 cells or cells stably transfected with RZ1254 or with Designing of ribozymes and demonstration of accessibility of control RZ construct. Six weeks after cell injection, mice ribozyme to target pCD mRNA in cell-free system. Using were euthanized by inhalation of CO . A complete necropsy mFOLD program version 3.1, seven hammerhead ribozymes was performed on all mice and the heart, lungs, kidney, liver, against pCD mRNA ‘GUC motifs’ were designed (15). Ribo- spleen and parts of the intestinal tract were removed and zymes were numbered according to the position of the cleavage fixed in 10% buffered formalin. Fixed tissues were trimmed site in pCD mRNA sequence. To evaluate the accessibility and paraffin-embedded for processing. The blocks were cut of different sites in intracellular environment, we designed into 4-μm sections and stained with hematoxylin and eosin antisense and control sense ODNs corresponding to ribozyme for histopathology examination. A minimum of six mice per target sequences as shown in Table I. The antisense ODNs (asODN) were allowed to bind to native pCD mRNA in cellextracts followed by cleavage with endogenous RNase H MTT assay. Control and stably transfected MDA-MB-231 and quantification by RT-PCR of the remaining uncleaved cells (7x103) were plated onto each well (100 μl/well) of mRNA. The asODNs corresponding to RZ385, RZ644, RZ763 96-well culture plates in 2% FCS for the specified time.
and RZ1254 were most effective in cell extracts, reducing MTT (Sigma) solution (10 μl from 5 mg/ml stock) was then the pCD mRNA levels to almost negligible (Fig. 1). As an added to each well, and the plates were further incubated efficiency control, sense ODN was used. These results indicate at 37˚C for 4 h in a humidified incubator with 5% CO .
that the ‘GUC motif’ at positions 385, 644, 763 and 1254 VASHISHTA et al: EFFECTS OF RIBOZYME-MEDIATED pCD KNOCKDOWN ON BREAST CANCER Figure 1. RNase H-mediated cleavage of endogenous pCD mRNA in cellextracts using anti-pCD ODNs. MDA-MB-231 cell extract was treated withthe sense ODN as well as various antisense ODNs (50 nM) correspondingto ribozyme target sites. The cleaved RNA was reverse transcribed usinghexamer primer. Different aliquots of RT products were amplified usingpCD primers and ß-actin primers as internal control. A band of ~1.2 kbpCD mRNA was obtained with sense ODN and reduction of pCD mRNAwith various antisense ODNs was compared to control sense ODN in theethidium bromide gel.
in the pCD mRNA is accessible to ribozyme in a cell-freesystem.
Intracellular model system for studying the specificity andefficacy of pCD ribozyme. The catalytic activity of the four Figure 2. Ribozyme-mediated downregulation of pCD at both protein (A) promising ribozymes designated as RZ385, RZ644, RZ763 and mRNA (B and C) level in MDA-MB-231 cells. (A) MDA-MB-231 cellsas well as stably transfected with pHßApr-1-neo, vector with control RZ and RZ1254 was then tested in cells. These hammerhead sequence and four effective ribozymes were checked for the secreted pCD ribozymes were cloned into the pHßApr-1-neo expression and intracellular cathepsin D level. Equal amounts of total protein were vector where a ribozyme expression is under the control of loaded on SDS-PAGE and subsequently analyzed by Western blotting.
ß-actin promoter. Catalytically inactive mutant ribozyme Expression of pCD protein in cell supernatant was detected using anti-APantibody (upper panel), CD expression was detected in cell lysates using was designed by changing the G-A in the catalytic domain anti-CD antibody, where purified CD was used as control in the last lane of ribozyme that resulted in a loss of catalytic activity. The (middle panel) and equal protein was confirmed by immunoblotting with human breast cancer cell line MDA-MB-231 was transfected anti-ß-actin antibody (lower panel). (B) To check the expression of pCD- with empty vector, vector with control, and effective ribozymes.
mRNA and transfected ribozymes, total RNA was extracted from non-transfected as well as transfected MDA-MB-231 cells. RT-PCR analysis Stable transfectants were selected for each and analyzed was performed using pCD primers (upper panel, 25 cycles), plasmid- by Western blotting and RT-PCR. As shown in Fig. 2A, specific primers (middle panel, 30 cycles) or ß-actin primers (lower panel, expression of pCD in MDA-MB-231 cells transfected with 25 cycles). (C) The level of reduction of pCD-mRNA was assessed by empty vector and control RZ was not altered while the real-time RT-PCR, and the results are expressed as relative pCD mRNAlevel compared to the control cells based on the Ct values normalized to the RZ385, RZ644, RZ763 and RZ1254 led to a drastic reduction of pCD protein amount in conditioned media (Fig. 2A,upper panel). The level of CD protein in cell lysates wasalso affected by transfection of these effective ribozymes(Fig. 2A, middle panel). Equal loading of protein is shown mRNA (50-60%) was also confirmed by real-time RT-PCR by detection of actin on the identical blot (Fig. 2A, lower (Fig. 2C). Expression of ribozymes was seen in RT-PCR demonstrating that the transfection of ribozyme vectors To ensure that downregulation of pCD was due to ribo- resulted in ribozyme expression. These results demonstrate zyme activation, RT-PCR analysis was performed using pCD the efficacy, efficiency, and specificity of pCD ribozymes in primers (Fig. 2B, upper panel), RZ primers (Fig. 2B, middle panel) and actin primers ( Fig. 2B, lower panel) as control.
Further, the expression of active ribozymes from pHßApr- As shown in Fig. 2B, the anti-pCD ribozymes were capable 1-neo vector was detected in MDA-MB-231 cells for multiple of downregulating pCD mRNA expression significantly in passages following transfection. The expression of ribozymes MDA-MB-231 cells, while no change was observed in control in cells was seen further than passage 20 (data not shown).
RZ and empty vector transfected cells. The reduction of pCD This demonstrates that the construct is stable in the cells.
INTERNATIONAL JOURNAL OF ONCOLOGY 30: 1223-1230, 2007 Table II. Inhibition of experimental metastasis by knockdownof pCD.
–––––––––––––––––––––––––––––––––––––––––––––––––Cell line –––––––––––––––––––––––––––––––––––––––––––––––––MDA-MB-231 Figure 3. Effect of anti-pCD ribozymes on cell invasion. In vitro invasiveness through Matrigel membrane of MDA-MB-231 cells, stably transfected control ribozyme and active ribozymes cells was measured. The cells (3x105) were ––––––––––––––––––––––––––––––––––––––––––––––––– incubated on the top of Matrigel-coated filters and the extent of cell invasion Nude mice were injected with a specified number of control or anti- was measured after staining the invasive cells by colorimetric reading at pCD ribozyme transfected MDA-MB-231 cells via the tail vein. Mice . The experiment was repeated thrice and the data represent the were euthanized after 6 weeks and metastatic foci were checked on mean ± SD of three independent experiments. *p<0.05; **p<0.001 vs. mean the lung surface macroscopically. Number of mice with metastasis/ number of mice injected.
–––––––––––––––––––––––––––––––––––––––––––––––––––––– The cell growth of control RZ and RZ1254 transfected MDA-MB-231 cells was then accessed. In proliferation assay,we observed only 5% reduction between the control RZ andRZ1254 transfected cells over a period of 96 h in the presenceof 10% FCS (Fig. 4).
Anti-tumor activity of pCD-ribozyme in breast cancer cells.
For in vivo analysis, 4x106 MDA-MB-231 cells or thosestably transfected with control RZ or RZ1254 were injectedintravenously through the tail veins of athymic nude mice.
After 6 weeks, the mice were euthanized and lung tissue wasexamined. All the mice injected with wild-type or transfectedwith control RZ cells showed macroscopic lung colonization Figure 4. Effect on proliferation of MDA-MB-231 cells by downregulation while only 2 of the 10 recipients of RZ1254 transfected of pCD expression. MDA-MB-231 cells as well as cells stably transfected cells developed macroscopic lung metastases (Table II).
with control RZ and RZ1254 were seeded in 96-well plates at a density of Additionally, microscopic examinations of tissue sections 5x104 cells/ml (150 μl/well) in the presence of 10% FCS and incubated fortime intervals of 0, 19, 43, 67 and 93 h followed by evaluation of proliferation revealed infiltration of tumor cells and almost complete using a Biotrak ELISA system. The figure shows a representative cell growth replacement of normal lung parenchyma by tumor cells in analysis of three similar independent experiments.
the case of MDA-MB-231 cells and cells transfected withcontrol RZ while the cells transfected with RZ1254 showedno infiltration of tumor cells. Instead, they were nested Downregulation of pCD in cell lines expressing and secreting adjacent to bronchioles and capillaries (Fig. 5). Further, pCD affects the in vitro invasiveness of breast cancer cells. The inflammation and necrosis associated with the periphery correlation between the pCD secretion and invasive potential of tumor masses was observed in the case of MDA-MB-231 of different cancer cell lines has been established (15,20,22).
cells and cells transfected with control RZ whereas no Preliminary study using one of the designed ribozymes has necrosis and only minimal inflammation was observed shown reduction in invasion through Matrigel in the ZR-75-1 with cells transfected with RZ1254. In a separate set of breast cancer cell line (15). Therefore, the effect of down- experiments, 1x107 cells were injected into mice through the regulation of pCD expression by anti-pCD ribozymes on the tail vein and checked for survival after six weeks. None of invasive potential of MDA-MB-231 cells was evaluated using the recipients of the MDA-MB-231 cells and cells transfected the Matrigel invasion assay. All the active ribozyme transfected with control RZ survived while 80% of the RZ1254 trans- cells showed reduction in invasion through Matrigel membrane and the most significant effect was seen in cells transfectedwith RZ1254 (Fig. 3). In contrast, the empty vector and control Induction of apoptosis in anti-pCD ribozyme transfected RZ transfected cells showed similar invasion as wild-type MDA-MB-231 cells on serum deprivation. Interestingly, the growth inhibitory effects of ribozyme expression appeared to VASHISHTA et al: EFFECTS OF RIBOZYME-MEDIATED pCD KNOCKDOWN ON BREAST CANCER Figure 6. Inhibitory effect on cell viability in low serum condition and apoptosis Figure 5. Effect of anti-pCD ribozyme on lung colonization of MDA-MB-231 induction after serum deprivation by downregulation of pCD in MDA-MB-231 cells in vivo. Histological analysis of hematoxylin and eosin-stained sections cells. (A) Cell viability of the control cell and cells transfected with control of lung tissue from nude mice injected with MDA-MB-231 cells, or cells RZ and RZ1254 was estimated by MTT assay. All values of OD are relative stably transfected with control RZ and RZ1254. One representative slide of to the OD at 0 h. (B) Control and stably transfected cells were deprived of each is shown. As seen in the slide of wild-type cells and cells transfected serum for 48 h and apoptosis was studied by cell death ELISA kit. RZ1254 with control RZ, the lung parenchyma were totally replaced by tumor cells showed a loss of cell viability and increase in apoptosis after 48 h in contrast and arrows mark the region of inflammation and tissue necrosis. In the case to control cells and cells transfected with control RZ. The data are expressed as of cells transfected with RZ1254 tumor cells nested adjacent to bronchiolesand capillaries (arrow mark) and no necrosis was apparent. Original mean ± SD from three independent experiments. *p<0.05.
have shown that pCD affects proliferation of cancer cells be more profound in vivo than in vitro. To determine whether in vitro and in vivo and it plays an essential role in tumor attenuation of pCD could promote death of tumor cells on angiogenesis and apoptosis (23). In fact, a positive correlation stress, MTT assay and cell death detection ELISA were has been established between the proportion of pCD secreted performed upon serum starvation. The control cells, control by human breast cancer cell lines and their invasive potential RZ and RZ1254 transfected cells were seeded in 2% FCS (22). Moreover, studies performed in our laboratory have and checked for cell viability by MTT assay every 24 h. As shown that pCD acts as a mitogen on both parental and shown in Fig. 6A, the RZ1254 transfected cells showed loss neighborhood cancer cells through the interaction of its AP of cell viability after 48 h in contrast to control cells and with an unidentified cell surface receptor (24,25). Recently, control RZ. To clarify that the reduction in cell proliferation we showed that the AP-treatment of ZR-75-1 cells induced was a result of induced apoptosis, control cells, control RZ the expression of genes involved in signal transduction, cell and RZ1254 cells were deprived of serum for 48 h and cycle regulation, tumor invasion, and metastasis (26). Another analyzed by cell death detection ELISA. The data showed study has shown a paracrine communication between cancer that MDA-MB-231 cells transfected with RZ1254 underwent and stromal cells where the pCD secreted by cancer cells 36% apoptosis after serum starvation compared to control stimulates fibroblast proliferation, survival, motility and invasion as well as activates the ras-MAP kinase pathway(27). Taken together, these studies suggest an important role Discussion
of the pCD molecule in the biology of cancer and make it anattractive candidate as a therapeutic target.
Elevated levels of pCD have been seen in many types of In the present study, we used the ribozyme approach to malignancies as reviewed by Leto et al (7). Different studies cleave the pCD mRNA and to further study the effect of INTERNATIONAL JOURNAL OF ONCOLOGY 30: 1223-1230, 2007 this depletion on invasion and growth of cancer cells. The of pCD might lead to altered signal transduction pathways ribozymes were designed to cleave distinct GUC-recognition sites of the pCD-mRNA using the mFOLD program. Although In conclusion, the efficacy of the anti-pCD hammerhead computer programs could predict the accessible sites on the ribozyme as a potential gene therapy agent in human breast RNA substrate, the most effective cleavage sites of the target cancer has been studied. The specific inhibition and secretion are found by experimental approaches (28). As in living of pCD in breast cancer cells by anti-pCD ribozyme not only cells, RNA structure and thus accessibility depend on the inhibits invasion and growth but also induces apoptosis of intracellular environment. Common methods to determine MDA-MB-231 cells under stress. Our findings provide a new effective cleavage sites are the RNase H treatment of the insight into targeting the pCD by hammerhead ribozyme to substrate after binding to antisense ODN predicted by the intervene aggressiveness of breast cancer and an experimental computer program (29) and in vitro cleavage assays (18). As basis for future therapeutic implications.
expected, the seven anti-pCD hammerhead ribozymes showedstrikingly different accessibility in the RNase H experiment.
Acknowledgements
The RNase H reaction with asRZ385, asRZ644, asRZ763and asRZ1254 showed that these sites are most accessible.
This study was supported by research grant from National The ribozyme transgene constructs were made by using ß-actin Institute of Health (NIH RO1CA82159-01A2).
promoter vector and succeeded in having a highly active,non-regulated activation of ribozymes. The effect of ribozymes References
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