Expression and activity of cathepsin L2 and its effect on the degradation of melanosomes in skin lesions of seborrheic keratosis / 中华皮肤科杂志

Objective To determine the expression of cathepsin L2 (CTSL2)and evaluate its activity in skin lesions of seborrheic keratosis (SK),to observe the ultrastructural changes of melanosomes in the skin lesions of SK,and to estimate the effect of CTSL2 on the degradation of melanosomes.Methods Twenty patients with SK were enrolled from the Department of Dermatology,Renmin Hospital of Wuhan University.The lesional tissue and the perilesional normal skin were biopsied from each patient.Among 15 of the 20 patients,hematoxylin and eosin (HE)staining and Fontana-Masson silver staining were performed to observe the distribution of melanin granules,transmission electron microscopy (TEM)was conducted to observe the ultrastructural changes of melanosomes,and immunohistochemical staining was performed to estimate the cellular proliferative activity.RT-PCR and fluorogenic substrate cleavage assay were performed in the other 5 patients to determine the mRNA expression of CTSL2 and evaluate its activity,respectively.Sucrose density gradient ultracentrifugation was performed to isolate and purify melanosomes from the retinal pigment epithelium (RPE) harvested from a discarded eyeball of a 35-year old male patient with informed consent.The purified melanosomes were incubated with epidermal lysates of SK lesions,and TEM was used to observe the changes in the membrane structure of melanosomes.Statistical analysis was carried out by paired t test,and a P value ＜ 0.05 was considered statistically significant.Results A large number of melanin granules were deposited in SK lesions,while the linear deposition of melanin granules was only seen in the basal layer of the normal skin.TEM showed that the percentage of damaged melanosomes was much higher in the normal skin (49.00％ ± 4.00％) than in the SK lesions (24.33％ ± 3.06％)(t =8.49,P ＜ 0.05).RT-PCR revealed that the mRNA expression and activity of CTSL2 were both significantly lower in the SK lesions than in the normal skin (mRNA:0.35 ± 0.09 vs.0.43 ± 0.08,t =3.17,P ＜ 0.05;activity:17.46 ± 0.45 vs.28.78 ± 0.58,t =34.29,P ＜ 0.05).Moreover,TEM also showed that the percentage of damaged melanosome was lower in the SK lesion lysate-treated group (32.33％ ± 4.93％) than in the normal skin lysate-treated group (43.00％ ± 2.65％,t =3.30,P ＜ 0.05).Conclusion Decreased expression of CTSL2 in the SK lesions can affect the degradation of melanosomes by keratinocytes.However,whether CTSL2 directly takes part in the pathogenesis of SK or not is still needed to be further confirmed.

Effect of cathepsin L activation on trichloroethylene-induced skin allergy in mice / 中国职业医学

OBJECTIVE: To observe the expression of cathepsin L( CTSL) on the skin of trichloroethylene( TCE)-sensitized mice,and explore the mechanism of CTSL in TCE-induced immunological skin damage. METHODS: The specific pathogen free female BALB/c mice were randomly divided into blank control group( n = 5),solvent control group( n = 5),TCE group( n = 15) and inhibitor group( n = 15). Skin sensitization experiments were performed according to the maximum guinea pig test method. The TCE group and inhibitor group were divided into sensitized subgroups and non-sensitized subgroups according to skin sensitization results. The skin tissues were taken 72 hours after the last TCE challenge.Hematoxylin-eosin staining was used to observe the pathological structure of skin tissues and measured the thickness of epidermis. The level of Ctsl mRNA was examined by real-time quantitative polymerase chain reaction,and the expression of CTSL, interleukin( IL)-6 and IL-17 were studied by immunohistochemical staining technique. RESULTS: The sensitization rate of TCE group and inhibitor group were 40. 0%(6/15) and 33. 3%(5/15) respectively. There was no statistical difference in the sensitization rate between the two groups( P > 0. 05). The thickness of epidermis and relative expression of Ctsl mRNA,CTSL,IL-6 and IL-17 in TCE sensitized subgroup and inhibitor sensitized subgroup were higher than that in blank control group,solvent control group,TCE non-sensitized subgroup and inhibitor non-sensitized subgroup(P < 0. 05). The above-mentioned indexes were higher in TCE sensitized subgroup than that in inhibitor sensitized subgroup( P < 0. 05). The relative expression of Ctsl mRNA,CTSL,IL-6 and IL-17 in skin of TCE sensitized subgroup were positively correlated between any two indexes( P < 0. 05). CONCLUSION: CTSL activation may play an important role in immunological skin damage of TCE-sensitized mice,which may be related to the promotion of IL-6 and IL-17 release.

Correlation between plasma cathepsin L and collateral circulation in acute ischemic stroke patients with cerebral artery stenosis / 国际脑血管病杂志

Objective To investigate the correlation between plasma cathepsin L (CatL) levels and establishment of cerebral collateral circulation in acute ischemic stroke patients with cerebral artery stenosis.Methods The patients with acute ischemic stroke with at least one cerebral large artery (including internal carotid artery,middle cerebral artery,vertebral artery,and basilar artery) stenosis ＞ 70％ diagnosed by whole cerebral angiography were enrolled.ASITN/SIR blood flow classification system was used to systematically evaluate the establishment of cerebral collateral circulation.Grade 0-2 was defined as poor collateral branch and 3-4 was defined as good collateral branch,Enzyme linked immunosorbent assay was used to detect the plasma CatL level.Results A total of 79 acute ischemic stroke patients with cerebral artery stenosis were enrolled,including 63 male and 16 female.Their mean age was 58.76 ± 12.24 years old.There were 51 patients (64.56％) in the poor collateral branch group and 28 (35.44％) in the good collateral branch group.There was no significant difference in plasma CatL levels between the good collateral circulation group and the poor collateral circulation group (7.09± 2.27 mg/L vs.8.79±3.53 mg/L;t =2.751,P =0.069).Multivariable logistic regression analysis showed that only the high National Institutes of Health Stroke Scale score was the independent risk factor for poor collateral circulation (odds ratio 0.935,95％ confidence interval 0.823-0.963;P=0.046),and there was no significant independent correlation between plasma CatL levels and collateral circulation (odds ratio 0.910,95％ confidence interval 0.766-1.081;P =0.285).Conclusion There was no significant correlation between plasma CatL levels and cerebral collateral development in acute ischemic stroke patients with cerebral artery stenosis.

Expression of Cathepsin L in tumor cells and tumor-associated macrophages in patients with Ewing sarcoma family of tumors: A pilot study.

Background: Cysteine protease Cathepsin L is involved in bone remodeling and expressed in activated macrophages. It is highly expressed in metastatic tumor tissue, especially with bone metastases. Aims: We evaluated immunohistochemical expression of Cathepsin L in tumor cells and tumorassociated macrophages (TAMs) in chemo-naive Ewing sarcoma. Settings and Design: Retrospective evaluation of archived specimens of Ewing sarcoma. Materials and Methods: Immunohistochemical staining was performed on archived blocks of chemo-naive patients with Ewing sarcoma treated with uniform chemotherapy at our institute between January 2009 and November 2011. Statistical Analysis: Immunohistochemical expression was co-related with baseline demographics and survival. Results: During the study period, we had evaluable baseline samples from 62 patients with median age 15 years (range: 2-40); 26 (42%) had metastases. Cathepsin L expression in tumor cells was observed in 8/62 (13%) specimens. None of the baseline clinical characteristics correlated with Cathepsin L expression. Cathepsin L positivity was associated with poor response to neoadjuvant chemotherapy (NACT) (P = 0.05), but did not infl uence either event-free-survival (EFS) or overall survival. Cathepsin L was expressed in TAMs in all specimens. Grade 3 TAMs (>10 TAMs/high power fi eld) was associated with better response to NACT (P = 0.05). On univariate analysis Grade 3 TAMs predicted superior EFS (median EFS 28.5 months in those with Grade 3 TAMs versus 14.8 months in those with grade ½ TAMs [P = 0.04]). Conclusions: Cathepsin L expression by immunohistochemistry was low in our patient cohort, and it did not affect the outcome. In addition, Grade 3 TAMs with Cathepsin L expression was associated with improved EFS.

Expression of cathepsin B, cathepsin L and uPA mRNA levels in non-Hodgkin lymphoma and their significance / 白血病·淋巴瘤

Objective To investigate mRNA expression levels of cathepsin B,cathepsin L and uPA in non-Hodgkin lymphoma (NHL) samples,and analyze the relationship between three genes and clinicopathological factors.Methods Real-time fluorescence quantitative RT-PCR was used to quantify the relative expression levels of cathepsin B,cathepsin L and uPA mRNA in 39 NHL patients without chemotherapy or radiotherapy and 15 patients with lymph node reactive hyperplasia.Relationship between their expression and clinico-pathological factors was analyzed.Results mRNA expression levels of cathepsin B,cathepsin L and uPA in NHL group were significantly higher than those in the control group (0.403 vs 0.128,0.209 vs 0.057,0.459 vs 0.031,all P ＜ 0.05).The mRNA expression levels of cathepsin B,cathepsin L and uPA were correlated with each other in NHL.The mRNA expression levels of cathepsin B,cathepsin L and uPA in advanced staged NHL were markedly higher than those in early staged NHL (0.453 vs 0.341,0.204 vs 0.085,0.372 vs 0.196,all P ＜ 0.05).mRNA levels of cathepsin B,cathepsin L and uPA were higher in NHL patients sensitive to chemotherapy than in those resistant to chemotherapy(0.401 vs 0.556,0.085 vs 0.205,0.316 vs 0.499,all P ＜ 0.05).Conclusions The high expression of cathepsin B,cathepsin L and uPA could be detected in NHL,and their expression has a close relationship with the biological behavior characters of NHL.The dynamic examination of cathepsin B,cathepsin L and uPA may be used as clinical auxiliary diagnosis and prognosis of NHL.

Cisteínoproteasas Catepsinas L de Taenia solium: Rol biológico en la infección y potencial uso para el inmunodiagnóstico de la neurocisticercosis / Cathepsin L Cysteine Protease from Taenia solium: Its biological role in the infection and potential use for the immunodiagnosis of neurocysticercosis

Taenia solium es un helminto aplanado responsable de la teniosis y de la cisticercosis humana, siendo esta última producida por el consumo de huevos infectivos. Los cisticercos pueden desarrollarse en diferentes tejidos del hombre, frecuentemente en el sistema nervioso central causando la neurocisticercosis (NCC). Para el diagnóstico de la NCC se requiere de una adecuada interpretación de datos clínicos, resultados de neuroimagen y pruebas serológicas. Sin embargo, las pruebas serológicas podrían mejorarse con el desarrollo de antígenos candidatos capaces de incrementar su sensibilidad y especificidad. En los últimos años se han descrito una serie de proteínas de superficie y de secreción de T. solium esenciales para la interacción parásito-hospedero. Una de estas familias son las cisteínoproteasas catepsinas L, las cuales cumplen un rol preponderante para el desarrollo y supervivencia del parásito, participando en la invasión tisular, la evasión de la respuesta inmune, el desenquistamiento y enquistamiento del cisticerco. Son consideradas como antígenos potenciales para el inmunodiagnóstico de la neurocisticercosis.

Taenia solium is a plane helminth responsible for taeniasis and human cysticercosis, the latter being the result of the consumption of infective eggs. Cysticerci can develop in different human tissues, often in the central nervous system, causing neurocysticercosis (NCC). For the diagnosis of NCC, an adequate interpretation of clinical data, neuroimaging results and serological tests are required. However, serological tests could be improved by developing candidate antigens able to increase their sensibility and specificity. In the last years, a series of surface and secretory proteins of T. solium essential for the parasite-host interaction have been described. One of these families is cathepsin L cysteine proteases, which have a predominant role in the development and survival of the parasite. They take part in the tissue invasion, immune response evasion, excystation and encystment of cysticercus. They are considered potential antigens for the immunodiagnosis of neurocysticercosis.

The clinical value of serum matrix metalloproteinase-9, heparanase and cathepsin L detection in determining the degree of ovarian cancer invasion and metastasis / 中华检验医学杂志

Objective To explore the clinical value of serum cathepsin L (CL),matrix metalloproteinase-9 (MMP-9) and heparanase (Hpa) detection in determining the degree of ovarian cancer invasion and metastasis.Methods Enzyme-linked inmunosorbent assay (ELISA) and electrochemiluminescencl immunoassay (ECLIA) were used to detect the serum content of MMP-9,Hpa,CL in 217 cases with untreated ovarian cancer before surgery( in FIGO Ⅰ - Ⅱ stage 83 cases,Ⅲ-Ⅳstage 134 cases),100 cases with benign ovarian tumors and 101 healthy women control.All of the patients from Guangxi Medical University Affiliated Tumor Hospital,from September 2003 to October 2009.The relationship between the clinical pathological factors of ovarian cancer and serum content of MMP-9,Hpa,CL was analyzed.On the basis of clinical pathological diagnosis as “gold standard”,the ROC curves was drawed to evaluate the clinical value of serum CL,MMP-9,Hpa combined detection in determining the degree of ovarian cancer invasion and metastasis before surgery.Results The serum content of CL,MMP-9 and Hpat in patients with ovarian cancer were (21.23 ± 8.17),( 193.95 ± 42.49),(7.68 ± 2.32) μg/L respectively,which was higher than that in patients with benign ovarian tumors [ ( 10.97 ± 3.84),( 143.66 ± 28.47),( 4.86 ± 1.37) μg/L respectively ] and normal control [ (5.59 ± 1.75),( 57.99 ± 1 1.42),( 2.77 ± 0.80) μg/L respectively ],there was difference statistically significant ( t value CL was - 13.242,- 13.498 respectively; MMP-9 was - 14.521 and - 21.290 respectively; Hpa was - 10.896 and - 18.280 respectively,P ＜ 0.001).The serum content of CL [ ( 21.59 ± 8.24) μg/L ] in patients with epithelial ovarian cancer ( EOC) was significantly higher than that [ ( 19.57 ± 7.69) μg/L ] in non-epithelial carcinoma ( F =1 1.209,P =0.048).The serum CL,MMP-9 and Hpa content in FIGO Ⅰ -Ⅱ stage patients was (19.66 ± 7.83),(182.63 ±42.30),(7.21 ±2.05) μg/L,which was lower than that (22.64 ±8.31),(202.81 ±39.74),(8.51 ± 1.92) μg/L in FIGO Ⅲ-Ⅳ stage patients ( F value was 12.452,70.565 and 195.122respectively,P value was 0.030,0.002 and 0.000 respectively).In patients with EOC,the serum CL,MMP-9 and Hpa content in eases with poorly differentiated was ( 23.04 ± 7.67),( 200.12 ± 40.82),(8.22 ± 1.92) μg/L respectively,which was also higher than that in cases with high-moderate differentiated [ ( 18.54 ± 7.30),( 173.43 ± 39.37),(7.20 ± 2.51) iμg/L respectively;F value was 24.545,60.286 and 9.077 respectively; P was 0.004,0.035 and 0.001 respectively ].The serum content of CL and MMP-9(22.96 ± 8.41),(200.44 ±43.82) μg/L respectively in patients with invasion and metastasis in the abdominal cavity was higher than that without invasion and metastasis in the abdominal cavity [ ( 19.07 ±7.36),( 181.04 ± 36.10) μg/L,F value was 12.210,18.084 ; P value was 0.030,0.010 ] ; There was statistically significant relatioship between serum levels of Hpa and patients with distant metastasis ( F =9.430,P =0.042).On base of pathological diagnosis as gold standard,ROC curve showed the sensitivity was 60.9％ (70/115),69.6％ ( 80/115) and 72.2％ ( 83/115) and specificity was 57.4％ ( 26/62),67.2％(20/62) and 68.9％ (19/62),as serum levels of CL,Hpa,MMP-9 preoperative were detected as tumor markers to determine whether there was cancer invasion and metastasis outside the pelvis.Conclusions There is related with CL,MMP-9 and Hpa levels increase and tumor occurrence and progression in ovarian cancer.The serum content of MMP-9,Hpa,CL detection would be certain clinical reference value to determine extent of invasion and metastasis of ovarian cancer before surgery.

Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.

Relationship between cathepsin L and invasion and metastasis of ovarian carcinoma cells / 中华妇产科杂志

Objective To clone cathepsin L (CTSL) gene and construct the eukaryotic expression plasmid pcDNA3. 1-CTSL and study the relationship between CTSL and invasion and metastasis in ovarian cancer cells in vitro. Methods The total RNA was extracted from the ovarian cancer tissue and the intact cDNA of CTSL was applied by reverse transcription (RT)-PCR. The product of RT-PCR was cloned to pMD18-T vector, and subcloned to pcDNA3. 1 vector. It was tested by the enzymation and DNA sequencing.The eukaryotic expression plasmid of CTSL was introduced into HO8910 cells by liposome transfection reagent. RT-PCR was used to confirm the recombinant plasmid DNA integrated with the genomic DNA of HO8910 cells. Western blot was used to confirm the CTSL protein expression in positive clones cells. The cell growth curves, clonogenicity efficiency were observed. The cell cycles were measured by flow cytometer.The ability of invasion, metastasis and adhesion of ovarian cancer cells were detected by the matrigel invasion assay, transwell migration assay and adhesion assay, respectively. Results The results from restrictive enzyme analysis and sequencing showed that the CTSL gene was successfully inserted into pcDNA3. 1.Result from RT-PCR and western blot showed that the ovarian cancer cells which transfected by recombinant plasmid could express CTSL gene and protein. There was no difference between HO8910-CTSL and HO8910-pcDNA3. 1 cells in proliferation and adhesion ability (0.16±0.04 versus 0. 19±0. 04) of the cells (P>0.05). There was difference between HO8910-CTSL and HO8910-peDNA3.1 cells in matrigel invasion ability (0.34±0.18 versus 0.17±0.04) and metastasis ability (1.252±0.114 versus 0.486±0.027) of cancer(all P<0.05). Conclusion CTSL maybe increase the ability of invasion and metastasis of ovarian cancer cells in vitro, which may be a molecular target of blocking invasion and metastasis of ovarian cancer.

Effect of RNA interference on the bionomics of Cathepsin L of hepatoma carcinoma cells / 中华肝胆外科杂志

Objective To explore the effect of RNA interference on the bionomics of Cathepsin L of hepatoma carcinoma cells. Methods In this study, the experimental group was the Cathepsin L RNA interference group. Control groups were the normal blank hepatoma carcinoma cell group (the blank group) and the Cathepsin LRNA interference blank group (the fluorescence control group). Observing times were ld,3d and 6d after RNA interference. Transfection efficiency in each group was observed. Expression of Cathepsin L of hepatoma cells was detected by immunofluorescence, RT-PCR and WB. Cell vigor was detected by MTT assay. Changes in the cell cycle and apoptosis were observed by flow cytometry. Transwell cabin was used to detect the changes of cell invasive power.Results In the experimental group, Cathepsin L mRNA level and protein level significantly decreased, the proliferation index significantly decreased and apoptosis index significantly increased. The invasive power also decreased. Conclusion RNAi interference can inhibit Cathepsin L expression, cell proliferation and cell invasive power efficiently.

Expression of schistosomal cathepsin L1 in Escherichia coli and evaluation of its protective capacity in an animal challenge

Schistosomes use proteinases to accomplish some tasks such as tissue penetration, tissue digestion for nutrition and evasion of host immune responses. The Cathepsin L is a cysteine proteinase of the papain superfamily detected in the gut lumen indicating that this enzyme contributes to the proteolysis of ingested hemoglobin. Due to these roles they play in the schistosome biology, proteolytic enzymes are considered potential targets to develop and direct anti-schistosomal therapies. In this work, the cDNA coding Cathepsin L1 of Schistosoma mansoni was cloned into the pAE vector that provides high-level expression of heterologous proteins in Escherichia coli. The recombinant protein was expressed as inclusion bodies, purified under denaturing conditions through nickel charged chromatography and used for experimental animal vaccination. ELISA was performed with the pooled sera. Although this protein showed to be immunogenic, mice immunized with three doses of recombinant protein plus aluminum hydroxide as adjuvant did not protect against S. mansoni infection.

Isolation and characterization of a cDNA encoding a mammalian cathepsin L-like cysteine proteinase from Acanthamoeba healyi

We have cloned a cDNA encoding a cysteine proteinase of the Acanthamoeba healyi OC-3A strain isolated from the brain of a granulomatous amoebic encephalitis patient. A DNA probe for an A. healyi cDNA library screening was amplified by PCR using degenerate oligonucleotide primers designed on the basis of conserved amino acids franking the active sites of cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteinases. Cysteine proteinase gene of A. healyi (AhCP1) was composed of 330 amino acids with signal sequence, a proposed pro-domain and a predicted active site made up of the catalytic residues, Cys(25), His(159), and Asn(175). Deduced amino acid sequence analysis indicated that AhCP1 belongs to ERFNIN subfamily of C1 peptidases. By Northern blot analysis, no direct correlation was observed between AhCP1 mRNA expression and virulence of Acanthamoeba, but the gene was expressed at higher level in amoebae isolated from soil than those from clinical samples. These findings raise the possibility that Ahcp1 protein may play a role in protein metabolism and digestion of phagocytosed bacteria or host tissue debris rather than in invasion of amoebae into host tissue.

Cloning and Sequencing of Cathepsin L1(FheCL1) Gene cDNA of Fasciola hepatica / 中国寄生虫学与寄生虫病杂志

Objective To search for a candidate DNA vaccine of Fasciola hepatica. Methods Using RT-PCR and digestion with Hind III and BamHI, Fasciola hepatica secreted cathepsin L1(FheCL1) cDNA was cloned into the expression vector pcDNA3.1. Results The cloning was successful, the cDNA sequence and its deduced amino acid sequence were analyzed. There was much difference between the cloned FheCL1 and the published one. But the first 20 residues of their amino acid sequences were the same. Conclusion The recombinant plasmid pcDNA3.1-FheCL1 may be a new type of candidate DNA vaccine candidate for Fasciola hepatica. It is possible that Fasciola hepatica presents different sub-species but their amino acid residues (1 to 20) encoded by FheCL1 might build up membrane spanning helix.

Enhanced expression of cathepsin L in metastatic bone tumors

Cathepsin L is a kind of cystein proteases which are known to facilitate the invasion and metastasis of tumor cells by degrading the components of basement membrane and extracellular matrix. This study was undertaken to investigate the expression of cathepsin L by Northern blot analysis with radiolabeled cDNA specific for cathepsin L in six normal tissues, two osteosarcoma cell lines, MG-63 and Saos-2, six primary bone tumors and six metastatic bone tumors. In six normal tissues, the highest level of cathepsin L was expressed in liver with the descending order of liver > lung > thymus > ovary > kidney > esophagus. One of the two osteosarcoma cell lines established from the primary sites expressed a high level of cathepsin L mRNA. Out of six primary bone tumors, three (50%) expressed cathepsin L mRNA, while all (100%) of six metastatic bone tumors expressed the mRNA. These results demonstrating the higher frequency of expression of cathepsin L in metastatic bone tumors suggest that cathepsin L may participate in tumor invasion and metastasis.