The aim of the study was to evaluate the prognostic significance of tumour and serum concentrations of urokinase-type plasminogen activator (uPA), its type 1 inhibitor (PAI-1) and cathepsin D (Cath ...D) in patients with squamous cell carcinoma of the head and neck (SCCHN).
Determinations of uPA and PAI-1 were made using enzyme-linked immunosorbent assays in tumour and serum samples of 47 and 32/47 patients, respectively. For the determination of tumour (94 patients) and serum (34/94 patients) Cath D concentrations, an immunoradiometric assay was used.
In an univariate survival analysis, the risk of disease recurrence and SCCHN-related death was significantly higher in the patients with high uPA (P = 0.046, P = 0.010) tumours, compared to those with low uPA tumours. In addition, the high serum levels of uPA correlated positively with the rate of relapse (P = 0.007), but not with the mortality rate (P = 0.200). There was no statistically significant difference between low and high PAI-1 groups, regarding either tumour or serum concentration of the inhibitor, and between low and high Cath D tumours. Low Cath D serum levels appeared to be related to longer disease-free interval (P = 0.055), but not to disease-specific survival (P = 0.120).
The tumour levels of uPA, as well as serum levels of uPA and Cath D could potentially predict the survival probability of patients with SCCHN. However, the strength of this association remains to be investigated on a larger and more homogeneous group of patients.
The aim of this study was to determine urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) concentrations in tumour and adjacent normal tissue samples from ...58 patients, and in serum samples from 40 of 58 patients with squamous cell carcinoma of the head and neck obtained at diagnosis and after completion of therapy. uPA and PAI-1 serum concentrations were also measured in 28 healthy volunteers who served as controls. Measurements were made using enzyme-linked immunosorbent assay (ELISA) techniques. For both uPA and PAI-1, significantly elevated concentrations were measured in tumour tissue as compared with normal tissue (uPA: 8.89 versus 0.41
ng/mg total protein (mgp),
P<0.0001; PAI-1: 23.9 versus 1.47
ng/mgp,
P<0.0001). A statistically significant difference in uPA concentrations was found between normal laryngeal and non-laryngeal tissue (0.52 versus 0.3
ng/mgp,
P=0.008), and in PAI-1 concentrations between T
1+2 and T
3+4 stage of disease (17.32 versus 35.63
ng/mgp,
P=0.04). The uPA concentrations positively correlated with those of PAI-1 measured in both tumour (
R
S=0.62,
P<0.0001) and normal tissue (
R
S=0.30,
P=0.02). In serum samples, lower concentrations of PAI-1 were measured in the control group than in patients with cancer (412.0 versus 680.5
ng/ml serum (mls),
P=0.0006). The time of collection of the serum sample did not influence uPA and PAI-1 concentrations, and no association was observed between their concentrations and any clinical and histopathological prognostic factors tested. Our results indicate that both uPA and PAI-1 may play a specific role in the process of invasion and metastasis, and might also be of prognostic value in squamous cell carcinoma of the head and neck.
Cysteine
proteinases cathepsins (Cats) B and L and their endogenous inhibitors
stefins (Stefs) A and B are implicated in the processes of local and
metastatic tumor spread. They were identified as ...potential
prognosticators in various malignant diseases, particularly in breast
cancer. The aim of the present study was to determine the
concentrations of Cats B and L and Stefs A and B in the tumor and
adjacent normal tissue samples collected from 49 patients (the present
group) with squamous cell carcinoma of the head and neck (SCCHN), using
quantitative immunosorbent assays (ELISA; KRKA d.d., Novo mesto,
Slovenia). Their clinical significance was compared with that from a
previous study (the reference group, 45 patients; Budihna et
al. , Biol. Chem. Hoppe-Seyler, 377: 385–390,
1996). The follow-up of patients from the latter report was updated for
this purpose.
In the present group, significantly higher concentrations of Cat B
( P < 0.0001), Cat L ( P <
0.0001) and Stef A ( P = 0.006) were found in tumors
compared with concentrations in their normal tissue counterparts. Cat
concentrations in normal laryngeal tissue were significantly/marginally
elevated compared with nonlaryngeal tissue (Cat B,
P = 0.02; Cat L, P = 0.06). The
tumor concentration of Cat L was found to correlate with pT
classification ( P = 0.005) and
tumor-node-metastasis stage ( P = 0.05),
whereas the concentrations of Stefs A and B correlated with pN
classification ( P = 0.007 and P = 0.03, respectively) and tumor-node-metastasis stage of the
disease ( P = 0.02 and P = 0.03,
respectively). There was no statistically significant difference
between low and high Cat B or Cat L groups, regarding either
disease-free survival or disease-specific survival, using a
minimum P approach to determine cutoff concentrations.
The risk of disease recurrence and SCCHN-related death was
significantly higher in patients with low Stef A ( P = 0.0006 and P = 0.0005, respectively) and Stef B
( P = 0.0009 and P = 0.0007,
respectively) tumors, compared with those with high-Stef A and Stef B
tumors. These results remained significant even after P s
were adjusted for a possible bias in the estimated effect on survival.
The survival analysis in the reference group also confirmed these
findings (Stef A: P = 0.0009 and
P = 0.002, respectively; Stef B:
P = 0.03 and P = 0.009,
respectively). To avoid any possible bias arising from the differences
between the laboratories that performed the biochemical analysis, the
concentrations of both Stefs in the present group and in the reference
group were standardized and coupled together to form a uniform group.
In univariate survival analysis, standardized values of Stef A and Stef
B correlated inversely with the rate of relapse ( P = 0.0000) and mortality rate ( P = 0.0000).
Multivariate regression analysis showed that the standardized value of
Stef A is the strongest independent prognostic factor for both
disease-free survival and disease-specific survival. These findings
show the specific role of Cats B and L and Stefs A and B in the
invasive behavior of SCCHN. Furthermore, Stef A proved to be a reliable
prognosticator of the risk of relapse and death in patients with this
type of cancer.
Further experiments were performed to explain a difference in chromosomal aberration yield found between samples cultivated immediately after fission neutron irradiation and samples which were ...cultivated with 96 h delay after irradiation.
Human peripheral blood samples were irradiated in mixed fission neutron/gamma field (1800 s) and biological effect assessed in the mean of analysis of unstable chromosome aberrations with a time delay in culturing cells of 12, 24, 48, and 96 h. Additional measurements were performed on irradiated and blank blood samples with the aim to detect any increase in alpha and beta activity after fission neutron irradiation. No difference was found. Results were compared to theoretically calculated values of the alpha and beta activity released from natural radioactive isotopes.
As a conclusion it is shown that in our experimental conditions the secondary effects resulting from nuclear transformations of natural or induced radioactive isotopes, recoil reactions and accompanying alpha, beta, and gamma radiation are not the reason for the increase observed in chromosomal aberration yield in blood samples cultured with a time delay of at least 24 hours.
P-glycoprotein (P-gp), a membrane protein that was originally found to be involved in the efflux of cytotoxic drugs out of the tumor cells, is also present in a variety of normal human and animal ...tissues, such as the adrenal cortex. The function of P-gp in the adrenal cortex has not been defined yet. The aim of our study was to determine whether the blockade of P-gp by cyclosporine A (CsA) dissolved in Cremophor EL (Crem) inhibits cortisol secretion in rabbits. In 14 rabbits, the baseline and ACTH stimulated serum cortisol levels were measured before and after CsA treatment. Seven rabbits were treated with 2 x 30 mg/kg CsA and seven with 2 x 90 mg/kg CsA injected s.c. Serum cortisol levels were determined by radioimmunoassay adjusted for expected values. The whole blood CsA levels were determined by a commercially available fluorescence polarization immunoassay. Serum cortisol levels, both baseline and ACTH stimulated, significantly increased after both low and high dose CsA treatment. The increase was dose dependent. The mean baseline cortisol levels increased from 5.7 (SD = 6.3) to 15.0 nmol/l (SD = 7.2) in the low dose group and from 7.7 (SD = 4.9) to 44.9 nmol/l (SD = 13.8) in the high dose group. The mean cortisol levels 8 h after ACTH stimulation increased from 53.3 (SD = 34.5) to 106.0 nmol/l (SD = 33.0) in the low dose group and from 47.7 (SD = 12.2) to 153.0 nmol/l (SD = 55.1) in the high dose group.
In our previous work we showed that the drug-resistance of cervical carcinoma, laryngeal carcinoma and glioblastoma cells may be accompanied by increased levels of tumor markers for invasion and ...metastasis (i.e. urokinase-type plasminogen activator, plasminogen activator inhibitor type 1, and/or cathepsin D). In the present study we examined the concentration of cathepsins B, L and H in three drug-resistant clones isolated from human laryngeal carcinoma (HEp2). The basal levels of cathepsins B, L and H were determined by enzyme linked immunoabsorbent assay (ELISA). Our results showed that all three clones had an increased level of cathepsin B (in two clones an almost 4-fold increase was determined). The level of cathepsin L was altered (increased) only in VK2 clone, while the levels of cathepsin H were similar in parental cells and drug-resistant clones. Thus, our results suggest that drug-resistance may be accompanied by an increased level of cathepsin B, i.e. tumor associated protease, involved in invasion and metastasis.
For effective boron neutron capture therapy (BNCT) it is important that a sufficient concentration of boron (10B) is present in the tumour during irradiation. This requirement represents a specific ...problem. The aim of this study was to test whether electroporation can be used as a non-specific drug delivery system to increase the delivery of sodium borocaptate-10B (BSH) into MCF7 (breast carcinoma) and B16F1 (melanoma) tumour cells in vitro and in B16F1 tumours in vivo. For the in vitro determination of 10B uptake, the cells were incubated in medium containing BSH and exposed to electric pulses. Boron levels were determined by inductively coupled plasma atomic emission spectrometry. In vivo, tumours were exposed to electric pulses 3 min after intravenous BSH injection. At different times after exposure the 10B concentration was determined in tumours and in blood. A difference in the 10B accumulation in the two cell lines was observed after continuous incubation of cells with BSH. No accumulation of 10B was observed in MCF7 cells, whereas in B16F1 cells, 10B accumulated well and reached a plateau within 30 min. Electroporation of these cells resulted in an accumulation of 10B into MCF7 cells up to the level of 10B in B16F1 cells. In vivo, the application of electric pulses increased and prolonged the entrapment of 10B (BSH) in the B16F1 melanoma tumours. A sufficient concentration of 10B was present in the tumour exposed to electric pulses for up to 24 h. Boron was quickly washed out from the blood and the level was below the concentrations in the tumours exposed to electric pulses at 2 h. The results of this study show that electroporation may provide a tool to increase boron concentration in the cells that have impaired transport of BSH through the plasma membrane. Furthermore, prolonged entrapment of BSH in tumours in vivo may, in addition to electroporation, be caused by the modifying effect of electric pulses on blood flow.
Gliomas are the most common form of intrinsic primary brain tumors, that extensively invade the surrounding normal brain tissue. The failure of chemotherapy treatment of these tumors is chiefly ...attributed to drug-resistance. From human glioblastoma we developed two cell sublines resistant to cisplatin due to acute (AT cells) or continuous (CT cells) treatment with clinically relevant doses of cisplatin. We examined their sensitivity to different cytostatics by colorimetric MTT assay. The concentrations of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) were determined by the ELISA assay. The results reveal that both AT and CT cells became resistant to cisplatin and vincristine; AT cells became resistant also to etoposide. Both AT and CT cells did not significantly change their sensitivity to doxorubicin, 5-fluorouracil and chlorambucil. Concentrations of uPA and PAI-1 were increased in CT cells, with no change in AT cells. In the conditioned medium of both, AT and CT cells, the level of uPA were increased. No differences in concentrations of PAI-1 in the conditioned medium of these cells were found. Thus, our results show that drug-resistance of glioblastoma cells may be accompanied with the increased levels of markers for tumor invasion.
The association between drug-resistance and three markers for invasive capacity: cathepsin D (Cath D), urokinase type plasminogen activator (uPA) and inhibitor of plasminogen activator type 1 (PAI-1) ...was examined in nine cervical and laryngeal carcinoma cell lines resistant to different cytostatics. The level of Cath D was measured by solid phase two-site immunoradiometric assay, while uPA and PAI-1 concentrations were determined by use of ELISA. All drug resistant cell lines had increased concentration of cathepsin D. uPA levels were similar in parental and drug resistant cervical carcinoma cells, but significantly higher in all examined drug resistant laryngeal carcinoma cells. In cervical carcinoma cells, PAI-1 concentrations were similar in parental and cisplatin resistant, but significantly higher in doxorubicin resistant cells. In laryngeal carcinoma cells, no increase in concentrations of PAI-1 was determined in the three from five resistant cell lines. There was no uPA in conditioned medium of parental or drug resistant cells. PAI-1 was detected in conditioned medium. Its levels were significantly increased in the medium of two cervical and three laryngeal drug resistant carcinoma cells. Thus, our results suggest that drug-resistance may be accompanied by increased levels of tumor associated proteases and/or its inhibitor.
