Original Article

Cancer Gene Therapy (2003) 10, 141–151 doi:10.1038/sj.cgt.7700546

Selective suppression of cathepsin L by antisense cDNA impairs human brain tumor cell invasion in vitro and promotes apoptosis

Natas carona Levic caronar1,2, Ricardo A Dewey3, Emma Daley2, Timothy E Bates4, Derek Davies5, Janko Kos6, Geoffrey J Pilkington2 and Tamara T Lah1

  1. 1Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia
  2. 2Experimental Neuro-Oncology Group, Department of Neuropathology, Institute of Psychiatry, King's College London, De Crespigny Park, Denmark Hill, London, UK
  3. 3Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany
  4. 4MRC Applied Neuroscience Group, School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre Nottingham, UK
  5. 5FACS Laboratory, London Research Institute, Lincoln's Inn Fields Laboratories, Cancer Research, 44 Lincoln's Inn Fields, London, UK
  6. 6Department of Biochemical Research and Drug Design, Krka, d.d., Cesta na Brdo 49, Ljubljana, Slovenia

Correspondence: Dr Tamara T Lah, Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Vec caronna Pot 111, Ljubljana 1000, Slovenia. E-mail: tamara.lah@uni-lj.si

Received 19 September 2002.

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Abstract

Invasion and metastasis of certain tumors are accompanied by increased mRNA protein levels and enzymatic activity of cathepsin L. Cathepsin L has also been suggested to play a role in the proteolytic cascades associated with apoptosis. To investigate the role of cathepsin L in brain tumor invasion and apoptosis, the human glioma cell line, IPTP, was stably transfected with full-length antisense and sense cDNA of cathepsin L. Down-regulation of cathepsin L by antisense cDNA significantly impaired (up to 70%) glioma cell invasion in vitro and markedly increased glioma cell apoptosis induced by staurosporine. Compared to control and parental cell lines, antisense down-regulation of cathepsin L was associated with an earlier induction of caspase-3 activity. Up-regulation of cathepsin L activity by sense cDNA was associated with reduced apoptosis and later induction of caspase-3 activity. Moreover, down-regulation of cathepsin L lowered the expression of antiapoptotic protein Bcl-2, whereas up-regulation increased the expression of Bcl-2, indicating that cathepsin L acts upstream of caspase-3. These data show that cathepsin L is an important protein mediating the malignancy of gliomas and its inhibition may diminish their invasion and lead to increased tumor cell apoptosis by reducing apoptotic threshold.

Keywords:

apoptosis, Bcl-2, brain tumor, cathepsin L, human glioma, invasion

Lysosomal cysteine peptidases comprise the largest family of lysosomal enzymes and belong to the group of papain-like peptidases. These are divided, based on their commonality of protein sequence, into two families: the cathepsin L–like family (L, H, V, K, S, W, F) and the cathepsin B–like family (B, C, O, X, and so forth), which also differ in other structural and functional biochemical characteristics.1 Lysosomal peptidases are regulated at every level of their biosynthesis, including transcription, translation, posttranslational processing, carbohydrate maturation, and trafficking to lysosomes. The activation of lysosomal enzymes is triggered by lowering pH and limited proteolysis during the process in which endosomes maturate into lysosomes. Apart from the lysosomes, cathepsin activation may occur both at the plasma membrane and extracellularly.2 One of the most interesting characteristics of cathepsins is their trafficking between various intracellular organelles, which is influenced by a variety of factors and appears to be strongly associated with tumor cell invasiveness.3

Local invasive growth is a key feature of primary malignant brain tumors. Malignant brain cells invade and spread into normal, healthy brain tissue and cause its destruction,4 although the mechanisms of invasion are not well understood. The diffusely infiltrative nature of glioblastoma multiforme is one of the major problems in its successful surgical removal and radiation therapy. Proteolytic degradation of extracellular matrix (ECM) components, such as laminin and fibronectin, is believed to facilitate invasive spread of tumor cells.5 Proteolytic enzymes, including cathepsins, mediate this process, either acting alone or in proteolytic cascades.6

In brain tumors, cathepsin B is the most investigated of lysosomal enzymes to date,7 and several studies are in agreement that cathepsin B expression is elevated in high-grade human glioma cells in vitro. In biopsies of human glioma, cathepsin B levels correlated with pathological evidence of invasion into normal brain tissue and with survival rate of the patients.8 Cathepsin L was also significantly increased in high-grade astrocytomas compared with low-grade astrocytomas and normal brain.9 Specific cathepsin L antibodies significantly lowered glioblastoma cell invasion. We have previously demonstrated that cathepsin L protein levels and activity were significantly higher in anaplastic astrocytoma and glioblastoma cell lines than in benign astrocytomas, although the relative role of cathepsin L in local invasion is not known.10

One of the most important aspects of therapeutic failure in malignant brain tumors is the high resistance of glioma cells to induction of apoptosis, which should be triggered by chemotherapy and/or radiation therapy. Malfunctions of apoptotic signaling pathways are common in many forms of human cancers,11 as cancer cells have raised apoptotic thresholds.12 In general, the activation of cell death pathways depends on both the triggering stimulus and the cell type,13 although these pathways appear to converge into a cascade of caspase activation, leading to DNA degradation and cell death. Staurosporine induces mitochondria-mediated apoptosis, which is regulated by the Bcl-2 family of proteins, certain members of which (such as Bcl-2 and Bcl-xL) are antiapoptotic and others (such as Bid and Bax) are proapoptotic.14,15 In addition to inhibiting apoptosis, the dimerization of Bcl-xL with Bcl-2 may also prevent labilization of lysosomes, secondary to interactions with pro-oxidants, although the precise mechanisms of Bcl-2 interactions with lysosomal hydrolyses are still unknown.16

Besides caspases, other endopeptidases may be involved in the proteolytic cascades of apoptosis,17 including other lysosomal cysteine proteases and their inhibitors stefins A and B.1,18,19 As proteolytic cascades involved in invasion may interfere with those involved in apoptosis,21 this present study was designed to investigate possible roles of lysosomal cysteine endopeptidase cathepsin L in both invasion and staurosporine-induced apoptosis. We demonstrated that in the human glioblastoma cell line IPTP (a) alteration in cathepsin L expression simultaneously affects both invasion and apoptosis, and (b) decreased invasiveness is associated with increased response to apoptotic stimuli.

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Methods

Cell culture

The human cell line IPTP was cultured from a biopsy of a glioblastoma multiforme (World Health Organization, grade IV) obtained from King's College Hospital, London. The biopsy was rinsed with HBSS and the tissue disaggregated into small fragments. The resulting cell suspension was transferred into tissue culture flasks containing DMEM (Gibco BRL, Paisley, UK), supplemented with 10% fetal bovine serum (Sigma-Aldrich, Gillingham, UK) and 1% antibiotics/antimycotics (Life Technologies, Paisley, UK), and incubated at 37°C and 5% CO2. The cell suspension was left undisturbed for 3–4 days until settled and proliferated from the small explants. The cells were then routinely passaged, grown to 80% confluency, and harvested by gentle scraping prior to the various cellular and biochemical assays. Cells were transfected at passage 15 and all experiments were carried out within a five-passage difference.

The human glioma cell line IPTP was chosen because it resembles closer the properties of a patient's tumor cells than high passages of commercial cell lines do.

Preparation of constructs

The 1.3-kb human Prepro-cathepsin L (PreproCatL) cDNA was excised from pGEM/PreproCatL plasmid using SalI (Promega, Southampton, UK) and subcloned into the XhoI site of the pcDNA3.1(-) mammalian expression vector (Invitrogen, Paisley, UK). PreproCatL cDNA was subcloned in either antisense (PreproCatLasn) or sense (PreproCatLsn) orientation using T4 DNA ligase (Promega). The orientation of PreproCatL cDNA in the pcDNA3.1(-) plasmid was determined by cleavage with BglII (Promega). The 730-bp green fluorescent protein (GFP) cDNA was excised from pFLX-5CG plasmid (kindly provided by Prof Garry Nolan, Stanford University, USA) by double digestion with BamHI and SalI (Promega). GFP cDNA was ligated using T4 DNA ligase (Promega) into pcDNA3.1(-) digested with BamHI and SalI. Before the transfection, plasmid DNA of all three constructs were purified using a Qiagen Plasmid Kit (Qiagen, West Sussex, UK) under sterile conditions.

Cell transfection and cell clone selection

IPTP cells (7.5times105) were transfected with 5 mug of plasmid DNA of either pcDNA3.1(-)/PreproCatLasn, pcDNA3.1(-)/PreproCatLsn, or pcDNA3.1(-)/GFP using the calcium phosphate precipitation method.21 Selection was initiated 16 hours after transfection by addition of 800 mug/mL geneticin (G418) (Calbiochem, Nottingham, UK) to the cell culture medium. Stably transfected clones were selected for 2–3 weeks in the presence of G418. Twenty independent G418-resistant clones were isolated from each transfection group.

Clone detection by flow cytometry

To determine the presence of positive antisense and sense cathepsin L clones, the intracellular content of cathepsin L was measured by flow cytometry. Cells were harvested, fixed in ice-cold 4% paraformaldehyde, permeabilized, and incubated for 12–16 hours at room temperature in 100 muL of cathepsin L monoclonal antibody (CATHLM1, dilution 1/2) (Krka, Ljubljana, Slovenia). Cells were then washed and incubated with 100 muL of sheep antimouse FITC-conjugated (dilution 1/10) secondary antibodies (Sigma). Cells treated in the same manner, except that the incubation with the primary antibody was omitted, served as negative control. Analysis of 104 cells per sample was performed on FACS Calibur (Becton Dickinson, Oxford, UK). Data analysis was performed with Cellquest software.

Clones expressing pcDNA3.1(-)/GFP were detected using the fluorescence microscope (Zeiss, Welwyn Garden City, UK). Clone IPTPvgfp, with more than 80% of the cells expressing pcDNA3.1(-)/GFP, was used as the negative control in all experiments.

Quantification of RNA

Total RNA from 1times106 cells was extracted using the StrataPrep total RNA miniprep kit (Stratagene, Amsterdam, The Netherlands) following the recommendations of the supplier. One microgram of RNA of each cell line was converted to cDNA using primer p(DT)15 for cDNA synthesis (Roche Diagnostics, Mannheim, Germany) and 50 U of Expand Reverse Transcriptase (Roche Diagnostics) in a total volume of 40 muL. Two microliters of the cDNA mixture was amplified in duplicates using 5 muM of each cathepsin L primers (forward 5'-cagtgtgggagaagaacatg-3' and reverse 5'-tgattcttcacaggagtcac-3'), 1 mM MgCl2, 5% DMSO, and the LightCycler–FastStart DNA Master SYBR Green I kit (Roche Diagnostics) in capillar tubes. The cathepsin L RNA content in each cell line was determined in real time in a Light Cycler thermal cycler (Roche Diagnostics). The cycling program was as follows: one cycle at 95°C for 10 minutes, followed by 35 cycles at 95°C for 15 seconds, 50°C for 10 seconds and 72°C. Using the same cDNA in the independent capillar tubes, and in the same run and same conditions, GAPDH primers (forward 5'-ttcaccaccatggagaaggc-3' and reverse 5'-tgatggcatggactgtggtc-3') at a concentration of 5 muM were added to the reaction mix. cDNA quantification of cathepsin L and GAPDH was analyzed by the second derivative maximum algorithm and the specificity of the products by the melting point analysis algorithm provided in the Light Cycler system. The DNA amplification values obtained from cathepsin L were normalized to the values obtained from GAPDH for each cell line.

Quantification of cathepsins L and B by ELISA

Cathepsins L and B antigens in cytoplasmic fractions were measured using a commercially available human cathepsin L and human cathepsin B ELISA kits (Krka) according to the manufacturer's instructions. Total, free, and complexed forms of cathepsins L and B are detected by this method. Cathepsins L and B content was expressed in nanograms per milligram of total protein. Protein concentration was determined by Protein assay (Bio-Rad, München, Germany), using bovine serum albumin as a standard according to the manufacturer's protocol.

Cathepsin L-type activity assay

Cathepsin L-type activity in cell lysates was measured using the previously described method22 with modifications. Briefly, an aliquot of 15 mug of total protein diluted in 100 muL of double distilled water was incubated in 870 muL of 0.34 M sodium acetate buffer, pH 5.5, containing 2 mM EDTA–Na2 (Sigma). Freshly prepared free 1.4-dithiothreitol (DTT) (Sigma) was added to a final concentration of 4 mM. To discriminate between cathepsin L-type and cathepsin B-type activities, a selective cathepsin B inhibitor, CA074 (L-trans-epoxysuccinyl-Ile-Pro-OH propylamide) (Bachem, Meyerside, UK), was added at a final concentration of 5 muM and preincubated for 15 minutes at 37°C. The negative controls were incubated with a 5-muM final concentration of 5 muM of general inhibitor of cysteine protease E64c (L-trans-epoxysuccinyl-Leu-3-methyl-butylamide) (Bachem) instead of CA074. The reaction was started by the addition of 5 muM of fluorogenic substrate Z-Phe-Arg-AMC (benzoylcarbonyl-Phe-Arg-7-amino-4-methylcoumarin; Bachem) at a final concentration of 16.7 muM and samples were incubated for further 60 minutes at 37°C. The reaction was terminated by 500 muL of 1 mM iodoacetic acid (Sigma). Fluorescence of the degradation product, 7-AMC (7-amino-4-methylcoumarin), was measured at an excitation wavelength of 370 nm and an emission wavelength of 460 nm, using a spectrometer (Perkin Elmer LS-50B, Beaconsfield, UK). One enzyme unit (EU) was defined as the quantity releasing 1 nmol of 7-AMC per minutes. Specific activity was calculated as milli enzyme units per milligram of total protein in the sample. Due to the lack of (a) absolute substrate specificity and (b) a selective cathepsin L inhibitor, the assay may also detect the activities of other cathepsin L–like types of cysteine proteinases.

Proliferation assay

This procedure is based on the reduction of the yellow tetrazolium salt MTT (1-(4,5 dimethyltiazol-2-yl)-2,5 diphenyl tetrazolium bromide) to purple formazan crystals by the mitochondrial enzyme succinate dehydrogenase. This conversion is proportional to the mitochondrial activity of viable cells.23 Each well, containing growing cells, was incubated with a final concentration of 0.5 mg/mL MTT (Sigma) for 3 hours at 37°C and the medium was centrifuged at room temperature for 5 minutes at 13,000timesg. Pelleted formazan crystals were dissolved in DMSO, the resulting solution was transferred to a microtiter plate, and absorbance was measured at 570 nm on a Dynatech MR 700 microplate reader. Aliquots of cell culture medium alone were incubated with MTT and used as controls.

Cell adhesion assay

The wells of a 96-well microtiter plate were coated with 0.5 mg/mL Matrigel matrix (Collaborative Biomedical Products, Bedford, MA), dried overnight under sterile conditions at room temperature, reconstituted with 70 muL of serum-free medium, and 3.5times104 cells were added per well. Cells were seeded in seven wells for each cell line. After 30 minutes at 37°C and 5% CO2, nonadherent cells were removed by gentle washing with HBSS. The MTT assay was then used to assess the percentage of viable adherent cells.

Cell motility assay

Cellular motility was evaluated using 24-well transwell chambers (Falcon, Oxford, UK) with 8-mum pore size polycarbonate membrane filters, separating the upper and the lower culture compartments. Cells (2times105 cells/well) were incubated on uncoated filters for 24 hours at 37°C in 5% CO2. The percentage of migrated cells was assessed by MTT colorimetric assay. The percentage of migration (%) after 24 hours was calculated as the ratio OD (lower)/(OD upper+OD lower)times100.

Cell invasion assay

Invasion of parental and transfected cells was evaluated using 24-well transwell chambers (Falcon) with polycarbonate membrane filters of 8 mum pore size, coated with 250 muL of growth factor–reduced Matrigel (0.9 mug/mm2). Matrigel matrix was dried at room temperature overnight and reconstituted with 200 muL of cold serum-free DMEM (Gibco BRL). Cells were harvested at 80% confluency and incubated (2times105 cells/well) on the reconstituted Matrigel for 24 hours at 37°C and 5% CO2. The percentage of invading cells was assessed by MTT colorimetric assay. The percentage of invasion (%) after 24 hours was calculated as the ratio OD (lower)/(OD upper+OD lower)times100.

Assessment of apoptosis

The stage of apoptosis was measured using the technique, which employs the differential uptake of the fluorescent DNA binding dyes acridine orange and ethidium bromide.24 These dyes can be used to determine whether the cell is in the early or late stages of apoptosis based on membrane integrity. Quantitative assessments were made by using fluorescence confocal microscopy (LSM5 PASCAL; Zeiss), determining the number of apoptotic cells whose nuclei were highly condensed or fragmented.

FACS analysis of number of apoptotic cells

Cells were treated with 1 muM straurosporine (STS) for periods of 0, 4, 8, 16, and 24 hours. Apoptotic cells were determined using previously described method.25 Cells were incubated with 200 muL of propidium iodide (PI) (50 mug/mL) and fluorescence was analysed by flow cytometer (Becton Dickinson). Apoptotic cells were designated as those that fell within the sub-G1 region. At least 104 cells were counted.

Caspase-3 activity assay

Caspase-3 activity was measured in cell lysates using the fluorigenic substrate Ac-DEVD-AMC (acetyl-Asp-Glu-Val-Asp-amino-methylcoumarin; Calbiochem) as described previously.26 Fifty microliters of cell lysates was diluted with 150 muL of reaction buffer (100 mM HEPES, 100 mM NaCl, 10% sucrose, 0.1% CHAPS, 1 mM EDTA, and 10 mM DTT, pH 7.4). Into control samples, 2 muL of the inhibitor, 20 mM Z-VAD-FMK (benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone; R&D Systems, Abingdon, UK), was added and preincubated for 30 minutes at 37°C. The reaction was started by adding 1 muL of 10 mM substrate. Samples were further incubated for 1 hour at 37°C. Production of 7-AMC was measured at room temperature in a fluorimeter (Perkin Elmer LS50-B) at an excitation wavelength 375 nm and an emission wavelength of 450 nm. Background fluorescence, where no sample was added to the reaction mixture, was subtracted from sample fluorescence.

Determination of intracellular content of Bcl-2

To determine the intracellular content of Bcl-2 protein, flow cytometry was used (see Clone detection by flow cytometry). One hundred microliters of human monoclonal anti–Bcl-2 antibody (NCL-Bcl-2, dilution 1/10) (Novocastra Laboratories, Newcastle upon Tyne, UK) was used as primary antibody and 100 muL of conjugated antimouse CY3 (dilution 1/10) (Sigma) was used as secondary antibody.

Statistical analysis

Data are presented as meanplusminusstandard error of the mean (SEM) of at least three independent experiments. All quantifications were performed in triplicate. Statistical comparison was made by Student's t test and data were considered to be statistically significantly different at P<.05.

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Results

Characterization of glioblastoma clones for cathepsin L expression

IPTP cells were transfected with the mammalian expression vector pcDNA3.1(-) containing PreproCatL cDNA sequence in antisense or sense orientation. Transfected clones were analyzed for their cathepsin L protein levels by flow cytometry. Among the sense-transfected clones, the clone IPTP24sn showed the highest levels of cathepsin L protein compared to untransfected parental IPTP cells (Fig 1A) and the highest cathepsin L levels of all the colonies analyzed. Among the antisense-transfected clones, the clone IPTP4asn showed the lowest levels of cathepsin L protein of all the colonies analyzed, demonstrating that antisense cathepsin L cDNA was able to efficiently reduce the production of cathepsin L protein.

Figure 1.
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A: Flow cytometric analysis of intracellular content of cathepsin L in human glioblastoma IPTP cells and IPTP-transfected clones. Cells were stably transfected with PreproCatL cDNA in antisense and sense orientation. Cathepsin L content was determined using monoclonal antibodies and flow cytometric analysis. The relative amounts of cathepsin L in the transfected IPTP cells are presented as the overlaid histogram and compared with the control parental IPTP cells (thick black line). The IPTP4asn clone (dark grey line) showed the lowest content of cathepsin L (shift to the left), whereas the IPTP24sn (light grey line) cells showed the highest cathepsin L protein levels (shift to the right) compared to the parental IPTP cells. IPTP cells where primary antibodies were omitted represented negative control (thin black line). B: Cathepsin L RNA quantification in IPTP parental and IPTP-transfected clones. Cathepsin L RNA content was measured by real-time PCR in the parental IPTP, and transfected IPTPvgfp, IPTP4asn, and IPTP24sn cell lines. Cathepsin L RNA levels were expressed relative to GADPH levels. Significantly (29%) lower cathepsin L RNA levels in IPTP4asn cells and significantly (66%) higher cathepsin L RNA levels in IPTP24sn cells were found compared to untransfecetd IPTP cells. Bars represent mean value of two independent experiments and standard error of the mean (meanplusminusSEM). ***Indicates statistically significant (P<.05) difference compared with the parental IPTP cell line. C: Cathepsin L protein concentration in cell lysates of selected clones of IPTP cells. Cathepsin L protein was measured by ELISA in the parental IPTP, and transfected IPTPvgfp, IPTP4asn, and IPTP24sn cell lines. Cathepsin L protein concentrations were expressed relative to total protein concentration in the cell lysates. Significantly (41%) lower cathepsin L concentrations in IPTP4asn cells and significantly (150% higher) cathepsin L concentrations in IPTP24sn cells were found compared to untransfecetd IPTP cells. Bars represent mean value of three independent experiments and standard error of the mean (meanplusminusSEM). ***Indicates statistically significant (P<.05) difference compared with the parental IPTP cell line.

Full figure and legend (56K)

Similar results were obtained by RNA quantification (Fig 1B) and ELISA (Fig 1C). Figure 1B shows that in the antisense-transfected cell line IPTP4asn, the cathepsin L RNA levels were significantly decreased by 29% compared to the parental IPTP levels and the IPTPvgfp (control) cell line. In the case of the sense-transfected cell line IPTP24sn, the levels of cathepsin L RNA significantly increased by 66% compared to the controls (untransfected IPTP and IPTPvgfp cell lines).

The IPTP4asn clone showed a 41% reduction in cathepsin L protein levels and IPTP24sn showed a 150% increase in cathepsin L when compared with nontransfected IPTP cells. IPTPvgfp (control) cells expressed nonsignificantly altered levels of cathepsin L, when compared with parental IPTP cells (Fig 1C).

Cathepsin L-type activity in cell lysates of IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells is presented in Figure 2. Cathepsin L-type activity was significantly reduced (by 50%) in IPTP24asn cells and significantly increased (by 143%) in IPTP24sn cells in comparison to nontransfected cells. In IPTPvgfp cells, cathepsin L activity was slightly higher compared to parental IPTP cells, but the difference was nonsignificant. Cathepsin L activity, therefore, paralleled its protein levels, suggesting that other cathepsin L-type cathepsins are present in minute amounts.

Figure 2.
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Cathepsin L-type activity in cell lysates of selected clones of IPTP cells. Cathepsin L-type activity was measured using the fluorogenic substrate Z-Phe-Arg-AMC. The mean values of specific enzymatic activities in IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells were compared. Cathepsin L-type activity was reduced by 50% in IPTP4asn and significantly elevated by 143% in IPTP24sn cells compared with the parental cells. Bars represent mean value of three independent experiments and standard error of the mean (meanplusminusSEM). ***Indicates a statistically significant (P<.05) difference compared with the parental IPTP cell line.

Full figure and legend (26K)

Expression of cathepsin B determined by ELISA assay was not affected by PreproCatL cDNA transfection (Fig 3). Parental IPTP and transfected IPTPvgfp, IPTP4asn, and IPTP24sn cells expressed similar protein levels of cathepsin B.

Figure 3.
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Cathepsin B protein concentration in cell lysates of selected clones of IPTP cells. Cathepsin B protein was measured by ELISA in the parental IPTP and transfected IPTPvgfp, IPTP4asn, and IPTP24sn cell lines. Cathepsin B protein concentrations were expressed relative to total protein concentration in the cell lysates. No significant changes in cathepsin B concentrations were observed between cell lines analyzed. Bars represent mean value of three independent experiments and standard error of the mean (meanplusminusSEM).

Full figure and legend (34K)

Effects of cathepsin L on cell proliferation, adhesion, motility, and invasion in vitro

The presence of PreproCatL cDNA in sense and antisense orientations did not influence cell proliferation rate (data not shown). Other vital characteristics, such as motility and adhesion to Matrigel, were also comparable to the control and nontransfected IPTP cells (Fig 4).

Figure 4.
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Panel A: Adhesion of parental and transfected glioblastoma cell lines in vitro. IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells were tested for adhesion on the Matrigel substratum. No significant differences in adhesion were observed between all four types of cell lines. Bars represent mean value of three independent experiments and standard error of the mean (meanplusminusSEM). Panel B: Motility of parental and transfected glioblastoma cell lines in vitro. IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells were tested for motility in transwell chambers, using 8-mum polycarbonate filters. No significant differences in motility were observed among all four types of cell lines. Bars represent mean value of three independent experiments and standard error of the mean (meanplusminusSEM). Panel C: Invasion of parental and transfected glioblastoma cell lines in vitro. IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells were tested for penetration into Matrigel in transwell chambers with 8-mum polycarbonate filters. The relative invasive potential of IPTP4asn was significantly reduced by 70%, compared with the parental IPTP cells. Bars represent mean value of three independent experiments and standard error of the mean (meanplusminusSEM). ***Indicates statistically significant (P<.05) difference compared with the parental IPTP cell line.

Full figure and legend (46K)

To test the involvement of cathepsin L in cellular invasion, an in vitro Matrigel invasion assay was used to assess the invasive potential of parental IPTP and IPTP4asn-, IPTP24sn-, and IPTPvgfp-transfected cells. In IPTP4asn cells, reduced cathepsin L expression caused a significant (70%) impairment of cellular invasion (Fig 4) compared with the parental IPTP cells. In IPTP24sn cells, where cathepsin L protein levels were increased, no differences in invasiveness were detected when compared with nontransfected IPTP cells. Control IPTPvgfp cells showed 25% lower invasiveness compared to nontransfected cells, but the difference was not statistically significant.

Effects of cathepsin L on apoptosis

Treatment of IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells with 1 muM STS for 24 hours resulted, on average, in 33%, 36%, 54%, and 13% of apoptotic cells, respectively (Fig 5). IPTP4asn cells had a significantly higher rate of apoptosis, whereas IPTP4sn cells had significantly less apoptotic cells compared with the nontransfected IPTP cells. The presence of apoptotic cells after 1 muM-STS treatment was examined by flow cytometric cell cycle analysis at different time intervals (0, 4, 8, 16, and 24 hours) (Fig 6). The amount of apoptotic cells, presented in a sub-G1 peak (left-hand side peak), increased with time in all cell lines (Fig 6). After 24 hours, IPTP and IPTPvgfp cells showed a similar sub-G1 pattern, ranging between 0.83% and 25.0% of apoptotic cells. IPTP4san cells died faster compared to nontransfected cells within a range of 2.0–45.0%, whereas only 1.0–13.7% of IPTP24sn cells were found in sub-G1 peak in the 24-hour time interval studied. Figure 5 shows typical data obtained 24 hours after STS treatment.

Figure 5.
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Apoptosis of human glioblastoma cell lines induced by STS. IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells were treated with 1 muM STS and the relative rate of apoptosis was determined after 24 hours, using acridine orange/ethidium bromide staining. IPTP4asn cells had significantly higher rate of apoptosis (54% of cells), whereas IPTP24sn cells were more resistant to apoptosis (13% of cells). Bars represent mean value of three independent experiments and standard error of the mean (meanplusminusSEM). ***Indicates statistically significant (P<.05) difference compared with the parental IPTP cell line.

Full figure and legend (28K)

Figure 6.
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Analysis of cellular DNA content in human glioblastoma cell lines. Cell cycle analysis in IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells was performed during 24-hour exposure to 1 muM STS and the amount of the apoptotic cells in sub-G1 region was determined by flow cytometry, ranging between 1% and 25%, 1% and 23%, 2% and 45%, and 1% and 14%, respectively. Relative percentages of apoptotic cells in sub-G1 peak are presented after 24 hours of exposure to STS. Histograms are from a representative experiment.

Full figure and legend (18K)

Profiles of caspase-3 activity were determined in cell lysates of IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells (Fig 7). Cells were treated with 1 muM STS and caspase-3 activity was measured using the fluorigenic substrate Ac-Asp-Glu-Val-Asp-AMC at different time points (0, 1, 2, 4, 8, 16, and 24 hours). IPTP and IPTPvgfp cells showed similar time course of activation profiles with induction of caspase-3 activity at 4 hours and peak activity at 8 hours. In highly apoptotic IPTP4asn cells, earlier induction of caspase-3 activity, starting at 2 hours and reaching peak activity at 8 hours, was observed. The caspase-3 activity profile for IPTP4sn cells showed the lowest apoptotic rate among the four cell lines. IPTP24sn cells showed a slight increase in caspase-3 activity starting at 4 hours with a delayed peak activity at 16 hours.

Figure 7.
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Time course of caspase-3 activation after STS exposure of human glioblastoma clones. Caspase-3 activity was measured in the lysates of IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells during exposure to 1 muM STS at the indicated time intervals. Data were normalized for easier comparison. For each cell line, mean value of peak activity was taken as 1. For each time point, the mean value was than calculated as a percentage of peak activity. IPTP, IPTPvgfp, and IPTP4asn cells showed a peak of caspase-3 activity at 8 hours, whereas IPTP24sn showed delayed peak at 16 hours.

Full figure and legend (44K)

The data showed that cathepsin L affects expression and the rate of caspase-3 activation after induction of apoptosis, suggesting that cathepsin L possibly interferes with an apoptotic cascade upstream of the caspase-3.

To assess whether cathepsin L affects other regulatory proteins, which in turn affect apoptosis in the mitochondrial pathway, the antiapoptotic Bcl-2 protein was measured by flow cytometry (Fig 8). IPTP and IPTPvgfp showed a similar expression of Bcl-2 protein (63% and 67% positive Bcl-2 cells, respectively). IPTP4asn cells expressed significantly less Bcl-2 protein (44% positive Bcl-2 cells) and IPTP24sn cells expressed significantly higher Bcl-2 protein levels (81% positive Bcl-2 cells). Expression of Bcl-2, reflecting the apoptotic rate of the respective cell lines, is also in line with the expression of cathepsin L, suggesting that cathepsin L regulates apoptosis upstream of Bcl-2, inducing its expression.

Figure 8.
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Flow cytometric analyses of intracellular amount of Bcl-2 in human glioblastoma cell lines. Relative amounts of Bcl-2 protein were assessed by flow cytometry using specific Bcl-2 antibodies in IPTP, IPTPvgfp, IPTP4asn, and IPTP24sn cells. The relative amounts of Bcl-2 are presented in two overlaid histograms (A,B) and compared with the control parental IPTP cells (thick black line, 63% of positive cells). A: Bcl-2 was significantly less expressed in IPTP4asn cells (grey line, 44% of positive cells). B: IPTP24sn cells expressed significantly more Bcl-2 protein (grey line, 81% of positive cells) compared to parental IPTP cells.

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Discussion

Cathepsin L is an endopeptidase with high affinity for collagen and other fibrillar proteins as substrates. It is a ubiquitous lysosomal cysteine proteinase, participating in general protein catabolism,27 although its specific functions in physiological28 and in pathological processes, including cancer progression,29 have been demonstrated.

In brain tumors, degradation of the ECM — consisting of components such as type IV collagen, laminin, and fibronectin — is thought to be crucial for neoplastic cell invasion. In a previous study, we demonstrated that penetration of glioblastoma cells into these protein substrates in an in vitro invasion assay was significantly impaired by a general cysteine proteases inhibitor E-64c and by intracellular and extracellular inhibition of cathepsin B by its selective inhibitors, Ca074Me and Ca074, respectively.10

However, the impairment of inhibition was not complete, suggesting that other endopeptidases, including another cysteine peptidases, were involved. Indeed, CLIK 148, a specific cathepsin L inhibitor, also reduced the invasion of tumor cells through Matrigel.30 To unambiguously establish the involvement of cathepsin L in the proteolytic cascade during glioblastoma cell invasion, we have stably transfected human glioblastoma IPTP cells with the constructs that contained the complete coding region of cathepsin L in antisense and sense orientation. Our results demonstrate that a successful transfection with cathepsin L antisense construct led to decreased cathepsin L enzymatic activity and protein content, compared with nontransfected parental cell line and control cells transfected with the vector/GFP construct. In the sense-transfected cells, up-regulation of cathepsin L protein levels and cathepsin L enzymatic activity was achieved. Antisense cathepsin L clone showed significantly reduced invasiveness in Matrigel, indicating that cathepsin L participates in invasion into this natural reconstituted ECM. It has been published only recently, very similar to our data, that the in vitro motility and the ability of osteosarcoma cell line MNNG/HOS to invade through the Matrigel were inhibited by 35–75%, using several cathepsin L antisense oligonucleotides.31 Furthermore, we observed that cathepsin L sense-transfected cells were not more invasive than untransfected cells. This could be due to the fact that these cells already express optimum levels of the protease needed for the effective penetration into Matrigel. This also suggests that cathepsin L is not a rate-limiting factor in Matrigel degradation, but more likely acts upstream of the proteolytic cascade, activating other proteases involved in invasion.6,32,33,34 Recently, an antisense strategy was used to down-regulate expression of another proinvasive molecule, urokinase plasminogen activator receptor (uPAR). This resulted in inhibition of invasion of a malignant glioma cell line20 and reduced growth of tumors in nude mice, as a consequence of induction of tumor cell apoptosis. Therefore, we were interested to know whether the impairment of invasion in cathepsin L antisense clones is associated with a simultaneous increase of apoptosis in our experimental system.

Indeed, we found that apoptotic rate increased in antisense-transfected cells treated with staurosporine, whereas in cathepsin L sense-transfected cells, fewer cells were apoptotic than in nontransfected and control cells. In antisense-transfected cells, caspase-3 activity started to increase earlier (2 hours) after treatment with staurosporine, compared with nontransfected cells (4 hours). Overexpression of cathepsin L seemed to indirectly suppress the caspase-3 activity and delayed its activation (16 hours). Cathepsin L also induced expression of the antiapoptotic Bcl-2 protein, which probably had a major effect in protecting the IPTP cells from staurosporine-induced apoptosis. High levels of Bcl-2 are known to promote cancer by inhibiting apoptosis, thereby prolonging cell survival. On the other hand, Bcl-2 itself has been reported to enhance malignant phenotype of glioma cells by increasing invasion and altering the expression of a set of metalloproteases and their inhibitors.35 Down-regulation of uPAR also increased apoptosis by Bcl-2 induction.20 All of these studies demonstrate the complex nature of Bcl-2 interactions with proteolytic enzymes, such as cathepsin L, and add further weight to the concept that cathepsins, in addition to caspases, play a role in apoptosis.

In support of our findings, the antiapoptotic activity of cathepsins B and L was reported by Zhu and Uckun,36 who found that the cysteine cathepsin inhibitor Z-Phe-Gly-NHO-Bz induced rapid apoptotic death in various human cancer cells, including glioblastoma. Tobin et al37 found that apoptosis levels in keratinocytes and melanocytes were higher in cathepsin L(-/-) than in cathepsin L(+/+) hair follicles.

Felbor et al38 showed brain atrophy of cathepsin B(-/-)/cathepsin L(-/-) mice due to the massive apoptosis of neurons in the cerebral cortex, cerebellar Purkinje, and granule cell layers. Similar to the findings in our experimental system, Kirschke et al27 reported that antisense RNA inhibited cathepsin L expression and also reduced tumorigenicity of various types of malignant cells, possibly indicating an increased apoptotic rate of tumor cells. An indirect support to our hypothesis comes from a clinical study of breast carcinoma patients,39 where high levels of cathepsin L in primary tumors, but none of other proteolytic factors, had significant prognostic impact for shorter survival in lymph node–positive patients, despite the fact that patients received adjuvant chemotherapy and hormone therapy. The therapy may have failed due to the high levels of cathepsin L, which may protect tumors from chemotherapeutically induced apoptosis. In analogy to cathepsin L, increased expression of cathepsin B rescued cells from apoptotic death induced by serum deprivation,40 whereas transfection with antisense oligonucleotides of cathepsin B induced apoptosis.41

In contrast, some studies have suggested that cathepsin B, calpain, granzyme B (a serine protease), and aspartic protease cathepsin D stimulated apoptosis.17,42,43,44,45 It has been proposed that lysosomal dysfunction and destabilization induced by various factors, result in the release of cathepsins, particularly cathepsin L, which may then activate caspase-3.16,46,47 However, the ability of cathepsins to activate/up-regulate caspase-3 activity may be dependant on cell type. It has been demonstrated on the human neuronal cell line NT2 that cysteine cathepsins did not activate caspase-3, although total lysosomal extract from these cells was effective in stimulating apoptosis by cleaving the proapoptotic protein Bid.18 This effect may also be due to not only cathepsins, but also other lysosomal proteolytic enzymes, such as recently discovered apoptases.19 Katunuma et al19 reported that apoptases are tightly bound to the lysosomal membranes and released into the cytoplasm — what results in proteolytic activation of procaspase-3. Furthermore, the authors suggested that proapoptases were activated by cathepsin L in normal cells, whereas in cancer cells, significantly lower levels of apoptases were observed. Presumably, overexpression of cathepsin L in tumors may lead to further degradation of apoptases, protecting them from apoptosis.

In conclusion, our findings support the hypothesis that successful migration and invasion of glioma cells require their resistance to triggering of intrinsic apoptotic cascade, once the cells become detached from the primary tumor. This transformation of the tumor cells to a more invasive and less apoptotic phenotype appears to be dependent on cathepsin L activity and expression of Bcl-2 protein. Therefore, it is likely that cathepsin L is an important factor for glioblastoma cell survival and the effective inhibition of cathepsin L activity could considerably diminish, if not completely oblate, the invasive potential of glioblastoma cells and also lead to tumor cell death by apoptosis. For clinical application, the data are both relevant for possible prediction of the response of glioma patients to chemotherapy and relevant for designing of adjuvant therapy, based on administration of cathepsin L inhibitors.

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Acknowledgements

This study was supported by the Ministry of Education, Science and Sport of Republic of Slovenia (program no. 0105-509, to TL); the PhD fellowship to N L by the Ministry of Education, Science and Sport; the British Council grant support for Partnerships in Science PSP 6; and the Samantha Dickinson Research Trust (G J P) and the European Union (G J P). We thank Astrid Fitter (Department of Pediatric Hematology and Oncology, Hannover Medical School) for her excellent technical assistance.

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