Total tissue lactate dehydrogenase activity in endometrial carcinoma.

Lactate dehydrogenase (LDH) is essential for continuous glycolysis necessary for accelerated tumor growth. The aim of this study was to reconsider if assay of total tissue activity of this enzyme could be useful as marker for endometrial carcinoma (EC). Activity of LDH was measured spectrophotometrically in homogenate supernatants of uterine tissue samples of 40 patients (10 normal endometria, 27 normal myometria, and 33 EC), including 30 matched pairs. Data obtained were analyzed in relation to clinical and histopathologic findings and compared with our previously published results on the tissue levels of the same enzyme in ovarian cancer and on the proteolytic activity of dipeptidyl peptidase III (DPP III) in EC (suggested biochemical indicator of this malignancy). Significantly increased (1.8-3.0 times; P < 1 x 10(-4)) LDH activity was observed in EC samples if compared with normal uterine tissues. This rise was not related to the clinicopathologic findings, however. In contrast to previous results on LDH in ovarian carcinomas, a significant rise in LDH activity was found already in grade 1 EC. Using the cutoff value of 1.06 U/mg, diagnostic sensitivity of 82%, specificity of 100%, and accuracy of 91% for total tissue LDH assay have been calculated. A correlation of tissue's LDH and DPP III activities was found, and their combined assay for EC showed increased diagnostic sensitivity (94%) and accuracy (96%).

Selective cytotoxicity of diazenecarboxamides towards human leukemic cell lines.

In this study, the cytotoxicity of 16 diazenes towards four human leukemic cell lines was tested. Regarding their structure these 16 diazenes belong to three subclasses: diazenecarboxamides (11 compounds), diazenedicarboxamides (4 compounds) and alkyl aminocarbonyldiazenecarboxylate (1 compound). The leukemic cell lines used in this study were NALM-1, JURKAT, HL-60 and K-562. Fifteen out of 16 tested diazenes were cytotoxic towards the leukemic cell lines: 11 with high efficacy (IC(50)<50 microM) at least towards two to three leukemic cell lines, and 4 with medium efficacy (IC(50)>50 microM). Ten out of these 11 diazenes have a common structure and belong to the subclass of diazenecarboxamides. Five diazenes (SB-681, LK-34, UP-39, JK-1197, UP-11) were highly cytotoxic (IC(50) values 3.3-38.9 microM) towards all four leukemic cell lines. The selectivity of the cytotoxicity towards leukemic cells was tested by using resting and Con-A-stimulated peripheral blood mononuclear cells (PBMC) isolated from healthy donors and towards normal mouse fibroblast cell line, 3T3. The diazenes cytotoxic towards leukemic cells, did not affect the viability of the resting PBMC suggesting selectivity of their action. Moreover, eight diazenes did not affect the normal dividing cells (Con-A-stimulated PBMC and fibroblasts). Thus, we present eight diazenes which are selectively cytotoxic towards leukemic cells, not affecting normal cells even when activated to proliferation. These compounds may represent new potential agents for the treatment of leukemia patients.

Lithium increases expression of p21(WAF/Cip1) and survivin in human glioblastoma cells.

Lithium is the most widely prescribed mood stabilizer, but the precise molecular mechanisms underlying its therapeutic function are not yet fully elucidated. Recent preclinical and clinical evidence indicates its neuroprotective and neurotrophic effects. As a tight coupling of function and metabolism in the central nervous system between glial cells and neurons has recently been detected, lithium's effect on glial cells may participate also in the total beneficial effects of this drug. The aim of the present study was to analyze molecular mechanisms induced in human glioblastoma A1235 cells by the treatment with lithium, especially its influence on the expression of apoptosis-related genes. Lower levels of lithium (0.5 mmol/L and 2 mmol/L) did not cause any cytotoxicity or changes in the cell cycle phase distribution following 72 h incubation. However, a higher dose (20 mmol/L) was cytostatic for glioblastoma cells, and caused accumulation of cells in G(2)/M phase of the cell cycle. The treatment with lithium did not alter the levels of Bcl-2 or procaspase-3 and did not cleave PARP, but increased the levels of p21(WAF/Cip1) and survivin. Thus, increased expression of p21(WAF/Cip1) (a protein with antiapoptotic function), and survivin (a protein that supports the growth of cells by suppression of apoptosis and promotion of cell proliferation) may be the early events in the long-term cell response to lithium that are involved in the beneficial effects of this drug.

Structurally similar diazenes exhibit significantly different biological activity.

We have previously synthesized various diazenecarboxamides (subsequently referred to as diazenes) that were cytotoxic to several tumor cell lines. To increase their biological activity, the structure has been modified appropriately. In the present study we examined the effects of N(1)-phenyl-N(2)-(2-pyridinylmethyl)diazenedicarboxamide (RL-337) obtained from the previously examined cytotoxic compound N(1)-phenyl-N(2)-(2-pyridinyl)diazenecarboxamide (JK-279), and compared them with those of diazene JK-279. Using a modified colorimetric MTT assay, the cytotoxicity of RL-337 was determined on human cervical carcinoma HeLa cells, glioblastoma A1235 cells, and prostate adenocarcinoma PC-3 cells. The possible synergistic effect of diazene RL-337 with cisplatin, doxorubicin, and vincristine, and its influence on intracellular GSH content was examined on HeLa cells. Diazene RL-337 was cytotoxic against all three human tumor cell lines, being more cytotoxic to HeLa cells than diazene JK-279. The higher efficacy of RL-337 than of JK-279 can be connected with higher basicity of the 2-picoline moiety present in the former diazene comparing with the pyridine fragment that is a part of the latter. The diazene RL-337 acted synergistically with cisplatin, doxorubicin, and vincristine (diazene JK-279 exhibited synergistic effect only with cisplatin). Glutathione (determined by Tietze's method) was not a target molecule of diazene RL-337 (but was for JK-279, as shown earlier). After just 1 h treatment with diazene RL-337, the cells started to lose membrane integrity. There was no cleavage of caspase-3 in RL-337-treated samples, and the majority of cells died 6 h after the treatment through necrosis (previously, apoptosis-like cell death was detected for diazene JK-279). Thus, although diazenes JK-279 and RL-337 are very similar in their structure, they exhibit widely different biological activity.

Diazene JK-279 induces apoptosis-like cell death in human cervical carcinoma cells.

Diazene N-phenyl-2-(2-pyridinyl)diazenecarboxamide (JK-279) is a newly synthesized compound, cytotoxic for several tumor cell lines and their drug-resistant sublines. In human cervical carcinoma cells (HeLa), this compound reduced intracellular glutathione content and increased sensitivity to cisplatin. The aim of the present study was to elucidate the molecular mechanisms involved in the cytotoxic effect of diazene JK-279 on HeLa cells. Cytotoxicity was determined by the MTT method. Flow cytometry analysis showed that diazene JK-279 induces G(2)/M phase arrest, mediated by the increase in p21 expression, and accompanied by an alteration in the expression of survivin. The highest concentration of JK-279 altered nuclear morphology in intact cells, showing "apoptosis-like" features. No cleavage of procaspase-3, procaspase-9 and PARP, or altered expression of apoptotic proteins Bcl-2 and Bax were detected. At the same time, PS externalization and internucleosomal DNA cleavage were observed. Partial necrosis was detected as well. Our results demonstrate that cytotoxicity of diazene JK-279 is mostly the consequence of caspase-independent cell death, which is in some aspects "apoptosis-like". Taking into account the multiplicity of mechanisms used by cancer cells to prevent apoptosis, the drugs (like diazene JK-279) that would activate alternative cell death pathways could provide a useful tool for new types of cancer therapy.

Quantitative biochemical analysis of lactate dehydrogenase in human ovarian tissues: correlation with tumor grade.

In an attempt to identify glycolytic capacity of normal and neoplastic human ovary, total lactate dehydrogenase (LDH) activity was measured in tissue cytosol originating from 69 patients (18 with benign ovarian tumor, 34 with ovarian carcinoma, six with nonepithelial ovarian malignant tumors, and 11 with tumor metastatic to ovary) and compared to the LDH activity of normal ovarian tissues (n = 19). Median value of total LDH-specific activity expressed as U/mg protein was 0.546 in normal tissues, 0.584 in benign tumors, 1.071 in malignancies metastatic to ovaries, 0.872 in nonepithelial primary ovarian tumors, and 0.818 in primary carcinomas. A significant rise in LDH-specific activity was found in malignant primary and secondary tumors of epithelial and nonepithelial origin, but not in benign neoplasms, compared to the activity in normal tissue. Ovarian carcinomas of serous histologic type did not differ in LDH activity from mucinous tumors. However, poorly differentiated carcinomas (grade 3) showed significantly enhanced activity of this glycolytic enzyme when compared to its grade 1 counterpart. The subgroup of grade 1 tumors did not differ in LDH activity from normal and benign ovarian tissue. Obtained results suggest that direct correlation might exist between ovarian epithelial tumor grade and lactate dehydrogenase activity.

Examination of cytotoxicity and mutagenicity of AH26 and AH Plus sealers.

AIM: To study in vitro the cytotoxic and mutagenic effects of AH26 and AH Plus. METHODOLOGY: Cytotoxic effects on Chinese hamster V79 cells were determined by counting viable cells following incubation with eluations of AH26 and AH Plus. In one set of experiments, the materials were mixed, set for 1 h and then eluted with dimethyl sulphoxide (DMSO) for 1 h, 24 h and 7 days. In the other set, AH26 and AH Plus were mixed and set for 1 h, 24 h and 7 days in physiological saline then crushed and eluted in DMSO for 24 h. The cytotoxic effects of these eluates were evaluated. Three concentrations were chosen to examine the mutagenic effects of AH26 and AH Plus: 5.57, 16.7 and 55.7 microg mL(-1). The structural chromosomal aberration analysis and micronucleus test were performed on human lymphocytes according to standard procedures. RESULTS: Dose-response curves of cell survival were obtained. Both materials were shown to be cytotoxic in doses larger than 55.7 microg mL(-1), except for AH26, after 7 days setting time. AH Plus was also shown to be toxic in concentrations of 16.7 microg mL(-1), except after 7 days setting time. Neither AH26 nor AH Plus induced a significant increase of chromosomal aberrations or micronuclei induction at any setting time or concentration. CONCLUSION: There was no mutagenicity found for AH26 and AH Plus on human lymphocytes in highly controlled conditions in vitro.

Methyl 2-(2-chloroethylaminocarbonyl)diazenecarboxylate SB-166 inhibits the growth of different tumour cell lines, including drug-resistant sublines.

Recently we synthesized new drugs, diazenecarboxamides (shortly diazenes), that were cytotoxic for several tumour cell lines. Because the solubility and biological activity of these drugs was relatively low, new compounds have been synthesized. In the present study we examined the cytotoxic effect of nine compounds: an imidazolidin-2-one (SB-282: methyl 5-benzoyl-3-(2-chloroethyl)-2-oxo-4-phenyl-2,3-dihydro-1H-imidazol-1-ylcarbamate), two diazenecarboxamides (UP-140: N-phenyl-2-(2-quinolinyl)diazenecarboxamide; JK-1090: N-(4-iodophenyl)-2-(2-pyridinyl)diazenecarboxamide), two aminocarbonyl substituted diazenecarboxylates (SB-178: methyl 2-[(cyclohexylamino)carbonyl]diazenecarboxylate; SB-166: methyl 2-[[(2-chloroethyl)amino]carbonyl]diazenecarboxylate) and four diazenedicarboxamides (SB-410: N(1)-(2-chloroethyl)-N(2)-(2-pyridinylmethyl)-1,2-diazenedicarboxamide; SB-472: N(1)-(2-chloroethyl)-N(2)-(4-isopropylphenyl)-1,2-diazenedicarboxamide; SB-503: N(1)-(4-sec-butylphenyl)-N(2)-(2-chloroethyl)-1,2-diazenedicarboxamide; SB-474: N(1)-(4-tert-butylphenyl)-N(2)-(2-chloroethyl)-1,2-diazenedicarboxamide). Using a modified colorimetric MTT assay, their cytotoxicity was determined on eight human cell lines: laryngeal carcinoma parental and two drug-resistant cell lines, glioblastoma parental and drug-resistant cell lines, cervical carcinoma parental and drug-resistant cell lines and breast adenocarcinoma cells. Results show that diazene SB-166 was very effective, reducing significantly the cell survival of all eight examined cell lines, including four drug-resistant cell lines. Compound SB-410 was cytotoxic for all examined cell lines, but mostly only in the highest concentration. Other compounds were not significantly cytotoxic to any of the treated cell lines. Our results, especially those obtained on drug-resistant cells, encourage further research on compound SB-166 as a potential anticancer drug.

Glutathione and glutathione S-transferases as early markers for ovarian carcinomas: case series.

AIM: To determine the activity of glutathione (GSH) and concentrations of glutathione S-transferases (GST), urokinase type plasminogen activator (uPA), and plasminogen activator inhibitor type 1 (PAI-1), and to evaluate their diagnostic and prognostic value and possible correlation with clinical and histopathological prognostic factors for ovarian carcinomas. METHODS: The concentrations of GSH, uPA, PAI-1, and activity of GST were analyzed in 35 tissue samples taken from 10 normal ovaries, 10 benign, 10 primary malignant, and 5 metastatic ovarian tumors. The GSH level and GST activity were determined by spectrophotometric methods, and uPA and PAI-1 concentrations by ELISA commercial kits. RESULTS: GSH concentrations were significantly higher in primary malignant (126.3+/-12.8 nmol/mg protein) and metastatic (160.5+/-24.3 nmol/mg protein) ovarian tumor specimens than in normal ovarian tissue (48.9+/-8.1 nmol/mg protein, p<0.003 for both carcinoma groups) or benign ovarian tumor samples (35.2+/-5.0 nmol/mg protein, p=0.001). The GST activity was significantly higher in primary malignant (245.8+/-22.7 nmol/min/mg protein) and metastatic (303.7+/-48.8 nmol/min/mg protein) ovarian tumor tissues than in benign tumor specimens (105.9+/-16.2 nmol/min/mg protein, p<0.004 for both carcinoma groups) or normal ovarian tissue samples (133.2+/-32.0 nmol/min/mg protein, p<0.044 for both carcinoma groups). There were no statistical differences in uPA and PAI-1 concentrations between normal, benign, and malignant tumor samples. Concentrations of GSH, uPA and PAI-1, and activity of GST were independent from histopathological and clinical prognostic factors. CONCLUSION: Increased GSH concentration and GST activity found in primary malignant and metastatic ovarian tumor samples were independent of histopathological and clinical prognostic factors, suggesting that they could be early markers for ovarian carcinomas.

Induction of heat shock protein 70 in drug-resistant cells by anticancer drugs and hyperthermia.

The altered constitutive and inducible levels of heat shock proteins 70 (Hsp70) in drug-resistant cells may influence the efficiency of combined hyperthermia and anticancer drug treatment. In the present study, the constitutive levels of Hsp70 and induction of these proteins by hyperthermia and two anticancer drugs (used for resistance development) were determined in cervical and laryngeal carcinoma cells. The levels of Hsp70 were quantified by Western blot. Constitutive levels of Hsp70 were similar in parental and drug-resistant cells suggesting that Hsp70 is not involved in drug-resistance. Hyperthermic treatment induced Hsp70 in all examined cell lines but with different kinetics between drug-resistant and parental cells. Following the treatment with anticancer drugs, Hsp70 was induced only in cisplatin-resistant laryngeal cells. Kinetics of Hsp70 induction (stress-type and cell-type specific) was different in drug-resistant cells as compared to parental cells. The observed alterations in Hsp70 induction in drug resistant and parental cells should be taken into account when combined treatments (i. e. hyperthermia and anticancer drugs) are planned.

Drug-resistant human laryngeal carcinoma cells have increased levels of cathepsin B.

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.

Plasminogen activator inhibitor type 2: potential prognostic factor for endometrial carcinomas.

The clinical determination of proteases which are involved in carcinogenesis, invasion and metastasis may contribute to the detection of the early stage of disease, and to the prognostic assessment of patients with the cancer. The aim of the present study was to determine the level of urokinase plasminogen activator (uPA), plasminogen activator inhibitor type 1 (PAI-1) and plasminogen activator inhibitor type 2 (PAI-2) in normal and malignant tissues of corpus uteri and to evaluate the possible correlation with clinical and histopathological prognostic factors. UPA, PA-I and PAI-2 were determined by the ELISA assay in tissue cytosol of matched pair samples from 27 patients with endometrial carcinoma. Results show that significantly higher levels of these proteins were found in malignant than in normal tissue samples (uPA: 1.266 versus 0.633 ng/mg protein, PAI-1:4.468 versus 1.958 ng/mg protein, and PAI-2:3.428 versus 0.483 ng/ml protein). The levels of uPA and PAI-1 did not correlate with clinical staging or pathohistological grading. However, in tumor tissues with clinical stages II and III, myometrial invasion > 50%, and lymphovascular invasion, increased levels of PAI-2 were determined. Our results indicate that components of the plasminogen activation cascade are up-regulated in endometrial cancer and suggest the role of PAI-2 in determining invasive potential of endometrial carcinomas.

The mutagenic potential of AH+ and AH26 by Salmonella/microsome assay.

The aim of this study was to examine the mutagenic potential of canal sealers AH+ and AH26 by Salmonella/microsome assay. The materials were tested immediately after mixing, 1 hr and 1 month later, respectively. The dimethyl sulfoxide extracts of sealers in amounts of 3.0, 1.5, and 0.75 microliters/plate were used. The plated bacterial strains of Salmonella were TA 98 and TA 100. The results showed that AH+ is mutagenic toward strain TA 100 1 hr after mixing. One month after mixing, mutagenic activity was expressed only in TA 98. Paste A showed strong mutagenicity toward TA 100. AH26 was more mutagenic to the TA 100 immediately after mixing, 1 hr later, and 1 month after it was polymerized. Also it was mutagenic toward TA 98 in the polymerized condition. Further examinations should be conducted to establish a definitive conclusion about mutagenic potential for these two endodontic materials.

Cytotoxic effect of four root filling materials.

The aim of this study was to evaluate the cytotoxic effect of four root canal sealers: AH26, AH Plus, Diaket and Apexit. In the experiment two cell lines, human cervical carcinoma (HeLa) cells and mouse skin fibroblasts (L929), were used. Under aseptic conditions, the sealers were prepared according to the manufacturers' directions, and 0.01 mL of each material was placed in a 24-well plate. The sealers were covered with cell suspension. The cytotoxicity was estimated by determining the number of viable cells by a light microscope, as well as the total number of cells 24 h, 48 h and 120 h after the treatment with mentioned materials. The results obtained in this study showed the high cytotoxcity of the new AH Plus root canal sealer, which was shown to be equally or more toxic to the standard AH26 and Diaket materials. Apexit was the least toxic sealer.

Diazenes as modificators of drug-resistance in tumor cells.

To overcome the drug resistance, which is the major obstacle in the successful treatment of cancer patients, various compounds have been tested. Glutathione is one of the most promising targets for modulation. In the present study, we examined the influence of five new synthesized compounds--diazenes on the reduction of the intracellular level of GSH. Further, we investigated their ability to increase the cytotoxicity of cisplatin, vincristine and doxorubicin. In experiments human parental cervical (HeLa) and laryngeal (HEp2) carcinoma cells and their drug-resistant cell sublines (HeLaCA and CK2, respectively) were used. Intracellular GSH content was examined spectrophotometrically by the procedure developed by Tietze. The cell sensitivity to drugs was determined using a modified colorimetric MTT assay. Results showed that the rate of reduction of GSH concentration was dependent on the cell type and the type of diazenes. We did not find a correlation between the reduction in GSH level and increased cytotoxicity to selected anticancer drugs. Nevertheless, we found that: a) diazenes LV-35 and VZ-19 increased the cytotoxicity of cisplatin in HEp2 cells, b) diazene MG-19 potentiated the cytotoxicity of vincristine in HEp2 cells, and c) diazene VZ-19 in HeLaCA cells. These data suggest that specific combination of diazene and anticancer drug may be useful in the treatment of certain tumor types.

Diazenes: modificators of tumor cell resistance to cisplatin.

Most studies indicate that modulation of glutathione metabolism may be one of the most promising means of reversing clinical drug resistance. Five new diazene compounds have been synthesized: JK-279, JK-835, JK-913, JK-925 and LV-57 that should, according to their structure and biochemical properties, lower the GSH concentration. In the present study, we examined the influence of diazenes on cisplatin resistance in human cervical (HeLa) and laryngeal carcinoma (HEp2) cells as well as in their cisplatin-resistant sublines (HeLaCA and CK2, respectively). Intracellular GSH content was examined spectrophotometrically by the procedure developed by Tietze. The cell sensitivity to drugs was determined using a modified colorimetric MTT assay. Results show that all examined diazenes lowered GSH concentration. This decrease was insignificant for JK-835 and JK-925 in HeLa and HeLaCA cells, and JK-925 in CK2 cells. In human cervical carcinoma HeLa and HeLaCA cells, JK-279 was mostly active in sensitizing the cells to cisplatin, especially in drug-resistant cells. JK-913, JK-835 and LV-57 reverted partially resistance to cisplatin in HEp2 cells, while none of the diazenes was active in CK2 cells. In conclusion, diazene JK-279 may be useful in the combined treatment (cisplatin + diazene) for the certain type of cancer.

Diazene JK-279: potential anticancer drug.

The aim of this study was to examine the cytotoxic effect of 10 newly synthesized diazenecarboxamides (diazenes). Using a modified colorimetric MTT assay, their cytotoxicity was determined on 10 human cell lines: cervical carcinoma parental and cisplatin-resistant cells, laryngeal carcinoma parental and cisplatin- and vincristine-resistant cells, glioblastoma parental and cisplatin-resistant cells, breast adenocarcinoma parental and doxorubicin-resistant cells, and mammary carcinoma cells. Results show that diazene JK-279 was most effective, reducing significantly the cell survival of all 10 cell lines examined, including five drug-resistant cell lines. A cytotoxic effect was observed also on nine from 10 cell lines for diazene JK-835. A small reduction in cell survival was obtained (mainly for highest drug concentrations) for diazenes LV-57 and MG-19 on two cell lines, and JK-429 and JK-913 on one cell line. Other diazenes did not demonstrate any cytotoxic activity. The results encourage further research on diazene JK-279 as a potential anticancer drug.

Cisplatin resistant glioblastoma cells may have increased concentration of urokinase plasminogen activator and plasminogen activator inhibitor type 1.

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.

Reversal of carboplatin resistance in human laryngeal carcinoma cells.

The effectiveness of carboplatin in the treatment of patients with tumors is limited by drug resistance. Because of that, there is a great interest to find a way to revert the resistance and improve the success of cancer treatment. The aim of the present study was to examine five potential modulators of carboplatin resistance with different mode of action: buthionine sulfoximine, ethacrinic acid, amphotericine B, cyclosporine A and aphidicoline. The effect of these compounds on the sensitivity of human laryngeal parental (HEp2) and carboplatin-resistant (HEp7T) cells to carboplatin was examined by MTT spectrophotometric assay. The results have shown that buthionin sulfoximine and, to a lesser extent, ethacrinic acid reduced the resistance of HEp7T cells to carboplatin. Aphidicolin increased the sensitivity of both HEp2 and HEp7T cells to carboplatin, but this effect was more expressed in parental HEp2 cells. Our data suggest that human laryngeal carcinoma cells treated with clinically relevant doses of carboplatin became resistant to this drug due to multifactorial molecular mechanisms. Accordingly, the resistance to carboplatin could be reduced by different modulators.

Drug resistant tumor cells have increased levels of tumor markers for invasion and metastasis.

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.