In our previous work (Alexandrov et al., 1996) was reported that the rat sarcoma cells induced by SR-RSV express two tumor associated antigens (TAA). The one TAA has a molecular weight of 52 kD and is detected by the help of a monoclonal antibody 2C2 only on the outer side of the plasma membrane of the sarcoma cells. The other antigen, with molecular weight of 28 kD, is expressed on the outher and inner side of the membrane. The antigens were isolated as a pure fraction by polyacrylamide electrophoresis and prepared for aminoacid analysis after that. The consisting 16 bound aminoacids were in different amounts. Both antigens are rich in glycine and poor in aromatic and sulphur-containing aminoacids. The presence of glucosamine and galactosamine in the antigens proves their glycoprotein nature. The received data show that the both TAA-s differ not only in molecular weights, place of expression and functional activity, but also in the amount of the bound aminoacids which constitute their proteins.
Combined antitumor activity of CPT-11 and 5-fluorouracil (5-FU) was evaluated in a human cultured cell line derived from lung cancer. After 24 h culture with SN-38 followed by 5-FU 24 h, synergistic effect was observed in the cell line. In addition, the antitumor effect of this combination was studied in in vivo experiments using Donryu rat with Yoshida sarcoma cells. CPT-11 and 5-FU synergistically inhibited tumor growth. There was no significant increase of toxicity as assessed by the body weights. These results might support for the combination with 5-FU and CPT-11 in a chemotherapy for cancer.
We analyzed lactoferrin expression in 78 samples from patients with sporadic breast cancer and found 31/78 negative for mRNA expression. Similar results were obtained by immuno-histochemical localization of the lactoferrin protein. We did not find relationship between lactoferrin expression and clinical parameters. We investigated for the absent lactoferrin expression in some cases of breast cancer. In 68 of the samples analyzed, we found an inverse correlation between estrogen receptor expression and lactoferrin expression (P < 0,0001), thus indicating that regulation by the estrogen receptor is not the main element responsible for the expression of lactoferrin in breast cancer. Analysis of methylation of the lactoferrin genomic DNA extracted from the same patients revealed that the degree of methylation does not explain the observed absence of lactoferrin. The 937 bp lactoferrin promoter was investigated for possible mutations. By single-strand conformation polymorphism analysis one polymorphic site was found and characterized.
Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption.
Chemically induced and spontaneous liver tumors share some metabolic alterations. The decline in hemoprotein levels during hepatocarcinogenesis may result from a diminution of the intracellular heme pool. To elucidate if the onset of the pre-initiation stage alters the natural regulation mechanism of heme pathway, animals were fed with p-dimethylaminoazobenzene (DAB) and treated or not with 2-allylisopropylacetamide (AIA). The induction of 6-Aminolevulinic acid synthase (ALA-S) activity and the diminution in microsomal heme oxygenase (MHO) did not change when DAB fed animals were treated with AIA. Cytochrome P-450 (P-450) levels and glutathione S-transferase activity were increased in all the groups tested. Tryptophan pyrrolase, sulphatase and beta-glucuronidase activities were altered in DAB fed animals but AIA treatment did not produce any effect. Changes in drug metabolizing enzymes in livers of DAB fed animals could be the result of a primary deregulation of heme metabolism. These results give additional support to our hypothesis about a mechanism for the onset of hepatocarcinogenesis.
Echitamine chloride (EC), an indole alkaloid, extracted from the bark of Alstonia scholaris has got highly promising anticancer effect. The effect of this drug on the microsomal drug detoxifying system was studied in sarcoma-180 induced mice. When given sub-cutaneously at a dosage of 5 mg/kg body weight, it was able to alter the impaired drug detoxifying system which was observed in the Sarcoma-180 bearing mice. The levels of microsomal protein, Cyt-P450, Cyt-b5, NADH-Cyt-C-reductase, NADPH-Cyt-C-reductase, and glu-6 phosphatase were determined. The levels of these drug metabolizing enzymes were decreased in S-180 bearing mice. EC treatment corrected to near normal levels of these enzymes and microsomal hemeproteins. In order to understand the mechanism responsible for the decreased protein level and its normalization after treatment with EC, 3H-Phenylalanine incorporation study was carried out. From the results, it is observed that the synthesis of apoproteins is also altered in tumor-bearing animals. All these changes which were observed in tumor-bearing animals were corrected to near normal levels after treatment with EC.
Morbidity resulting from the toxicity of chemotherapeutic drugs suggests that novel approaches are worthy of investigation. Development of the use of low intensity magnetic fields as an adjuvant to current treatment regimens to prevent metastatic disease may prove to be efficacious. Using a cell culture model, we have developed a magnetic field (MF) treatment that offers the possibility of lowering the therapeutic dose of these drugs and thereby reducing morbidity. Our studies have found that a low intensity (approximately 2 gauss) MF signal and a relatively low dose (0.1 microg/ml) of Adriamycin (ADR) inhibited proliferation of human osteosarcoma cells by 82%, whereas the MF and ADR acting individually caused only 19% and 44% inhibition, respectively.
The modulating effect of newly synthesized alkylating spin labeled triazene and spin labeled nitrosourea derivatives on the DOPA-oxidase activity of mushroom tyrosinase has been investigated by Bumett's spectrophotometric method (Burnett et al., 1967). All spin labeled triazenes have exhibited activating effect on DOPA-oxidase activity of tyrosinase, whereas clinically used triazene (DTIC), which does not contain nitroxide moiety, have showed inhibiting effect. At the same experimental conditions the spin labeled aminoacid nitrosoureas have showed dual effect - activating, in the beginning of the enzyme reaction and inhibiting later on. It is deduced that the activating effect of the spin labeled compounds is due to the nitroxide moiety and the inhibiting effect of all compounds depends on their half-life time. This study might contribute to make more clear the mechanism of action of the new compounds and on the other hand would come in quite useful as a preliminary prognosis for their antimelanomic activity.
The arginase activity and ornithine level were determined in tissue obtained from patients with non-small cell lung carcinoma (NSLC). The arginase activity and ornithine level in tumor tissues were 1.89 +/- 1.28 U/mg protein and 42.32 +/- 25.82 nmol/mg protein, respectively versus 0.67 +/- 0.19 U/mg protein and 10.12 +/- 3.69 nmol/mg protein for normal tissues (p < 0.01).
MUC1 is a glycoprotein and its expression is altered in breast cancer. Mucin protects epithelia from the external hostile environment. The expression of mucin changes when epithelia come in contact with toxic agents such as ethanol. Previously, we characterized the expression and regulation of tracheo-bronchial mucin (TBM) gene. In the present study, we studied the effect of ethanol on the gene encoding mammary gland mucin MUC1 and observed that ethanol regulates MUC1 expression at the transcription level. Ethanol enhanced the expression of MUC1 mRNA in a dose- and time-dependent manner in MCF-7 cells. At 100 mM concentration (a concentration reported to be present in alcoholics), ethanol induced a three to five-fold increase in mucin transcription as determined by nuclear run on analysis. This concentration of ethanol does not affect the half-life of MUC1 mRNA.
Carnitine has two main functions, i.e., transporting long-chain fatty acids into the mitochondrial matrix for beta-oxidation to provide cellular energy and modulating the rise in intramitochondrial acyl-CoA/CoA ratio, which relieves the inhibition of many intramitochondrial enzymes involving glucose and amino acid catabolism. The present study examined the acid soluble carnitine (ASCAR) acid insoluble carnitine (AICAR) and total carnitine (TCAR) concentrations of 50 human brain tumor tissues and 11 normal brain tissues. The ASCAR levels significantly higher in gliomas and meningiomas than brain, however similar to brain in metastatic adenocarcinomas. AICAR levels were lower than brain in all tumors with the exception of a medullablastoma. TCAR levels were similar to brain in all tumor types. Decreased AICAR levels may be due to increased utilization of lipids or enhanced phospholipid and cholesterol synthesis which is need for increased membrane synthesis or formation of eicosanoids. Also decreased concentrations may be a reflection of camitine and its acylesters role in preserving the physiologic membrane structure function from oxidative damage.
The urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) are very similar serine proteases with the same physiological function, the activation of plasminogen. An increased amount or activity of uPA but not tPA has been detected in human cancers. The PAs are weak proteolytic enzymes, but they activate plasminogen to plasmin, a strong proteolytic enzyme largely responsible for the malignant properties of cancers. It has been shown recently that the administration of uPA inhibitors can reduce tumor size. Inhibitors of uPA could therefore be used as anti-cancer and anti-angiogenesis agents. It has been found that amiloride competitively inhibits the catalytic activity of uPA but not tPA. Modification of this chemical could therefore produce a new class of uPA specific inhibitors and a new class of anti-cancer agents. The X-ray structure of the uPA complex with amiloride is not known. There are structural differences in the specificity pocket of uPA and tPA. However, the potential energy of binding amiloride is lower outside this cavity in the case of tPA. A region responsible for binding amiloride to tPA has been proposed as the loop B93-B101, reached in negatively charged amino acids present in tPA but not uPA.
Free-radical-mediated damages may play an important role during metastasis. To investigate their relevance in the metastatic process MDA levels, glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities, and selenium, zinc and copper contents were determined in plasma and erythrocytes from 20 cancerous patients with metastasis and 30 age-matched controls. Significantly higher concentrations of MDA in plasma as well as in erythrocytes were found comparing to the control group. In both plasma and erythrocytes, GPX activity and selenium and zinc levels were significantly lower in patients than in controls. However, SOD activity in erythrocytes and copper levels in both plasma and erythrocytes were significantly higher in patients. The impaired antioxidant system may favor accumulation of free radicals which may induce the process of metastasis. On the other hand, it is possible that the antioxidant system is impaired as a consequence of abnormality in the antioxidative metabolisms due to the cancer process.
The expression of mdm-2 oncoprotein (p90) was determined in a human breast tumor xenograft line (GI-101) that was derived from a 57 year old female cancer patient with recurrent, infiltrating ductal adenocarcinoma (Stage IIIa, T3N2MX). Immunoprecipitation coupled western blot analysis of the primary tumors that have been obtained from xenograft implanted athymic nude mice, using mdm-2 (Ab-1) mouse monoclonal antibody, primarily revealed high level expression of a 90 kD full length mdm-2 protein. In the GI-101 tumor the level of full length mdm-2 (p90) protein expression increased with the increase in the size of the tumor (100 to 2,000 mm(3)) and a maximum expression was detected in 2,000 mm(3) size tumors. In addition to the expression in the primary site, a significantly high level expression of mdm-2 protein (p90) was detected in the lung and liver tissues also, which are the known metastatic sites for GI-101 xenograft tumors. However, the level of mdm-2 protein expression was undetectable in the lung and liver tissues obtained from control mice. A cell line (GI-101A) derived from the GI-101 xenograft tumor also showed a high level expression of mdm-2 protein after several generations of cell passage. When the GI-101A cells were treated with DES (Diethylstilbestrol) the mdm-2 protein expression increased after 10 min treatment and reached a peak level at 40 min. Interestingly, DES (10 and 20 microM) treatment increased the total cell number also after 96 hr treatment compared to the non-treated cells. It appears that mdm-2 (p90) may have a significant role in supporting the tumor cell growth as well as the metastatic process of the GI-101A cells.
Native fluorescence emission and excitation spectra of SV40 infected human keratinocytes, A431 and SCC324 carcinoma cells, and normal human keratinocytes were measured and compared. A difference in the intracellular metabolic state of NADH was found between the normal cells and the cancer or virus-transformed cells. The observed difference, namely an increased proportion of bound, mitochondrial NADH in the cancer and virus-infected cells, manifests as a blue spectral shift in the emission spectra.
Cell-matrix interactions have important effects on phenotypic features, such as morphology, differentiation and cell growth. Several papers have suggested that when cell-matrix interaction is interrupted, cells grow as multicellular spheroids and eventually undergo apoptosis. We found that when ET(−), a laminin non-adherent colon cancer cell line, was cultured on poly-2-hydroxyethyl methacrylate (HEMA) coated plastic, the cells floated as cellular aggregates of spheroids or as single cells. Some of the single cells contained large intracytoplasmic lumens (ICL) and appeared similar to signet ring cells. These ICL were lined by a layer of short microvilli. The number of the cell did not increase when cultured on poly-HEMA. Another type of single cells, usually without ICL, demonstrated the characteristics of apoptotic cells by histologic examination. Acridine orange staining, flow cytometry and electron microscopy confirmed the apoptotic nature of those cells. On immunohistochemical staining for proliferating cell nuclear antigen, spheroids of cells and single cells with ICL were immunoreactive, while most of the single cells without ICL were negative. These results suggest that multicellular aggregates and formation of ICL were induced by the adaptation of ET(−) colon cancer cells in a harmful environment caused by reduced adhesiveness, and these changes might be related to cell survival.
UNLABELLED:The diagnostic value of ferritin in pleural effusions or ascites was studied in 151 samples from 147 patients (four patients had both kind of effusions). Samples (99 pleural effusions, 52 ascites) were evaluated in 4 groups: benign transudate (27 cases), benign nontuberculous exudate (26 cases), tuberculous exudate (47 cases) and malignant exudate (51 cases). Median ferritin levels in effusions were 67 ng/ml, 805 ng/ml, 889 ng/ml, 998 ng/ml and median effusion/serum (E/S) ratios were 0.7. 2.0, 4.9, 3.2 respectively. There was a significant difference between the concentrations of ferritin in malignant (51 cases) and nonmalignant effusions (100 cases) (p < 0.001), but the specificity and positive predictive value were low (43% and 45% respectively). Ferritin levels in transudate group were significantly lower than those in the others (p < 0.001). However, ferritin concentrations in three exudate groups were similar (p > 0.05). When compared the all inflammatory effusions (malignant, tuberculous, nontuberculous inflammatory exudates) with noninflammatory effusions (transudate and exudate), we determined a significant difference (p < 0.001). CONCLUSIONS:1) Elevated ferritin concentration in effusions is significant indicators of exudates; 2) It is not good a parameter to discriminate the malignant effusions from the benign ones; 3) They can be useful in the differential diagnosis of the inflammatory exudations from the noninflammatory ones.
In this investigation, we report a relationship between the terbium (Tb3+) binding protein and the cytotoxicity of cisplatin in human head and neck cancer cells. In the FaDu cell line, the cytotoxic action of cisplatin was shown to be approximately six times more potent than the cytotoxicity of Tb3+. When cisplatin was combined with 80 microM Tb3+, the IC20 and IC50 values for cisplatin were reduced by 70% and 24%, respectively. The IC80 value, however, was increased by 124%. The results suggest that the cytotoxicity of cisplatin is enhanced by Tb3+ at low cisplatin concentrations. In agreement with previous studies, calcium and cisplatin were found to be mixed-type and noncompetitive inhibitors, respectively, of the Tb3+ -FaDu intensity. These findings imply that the receptor binding of Tb3+ can modulate the cytotoxic activity of cisplatin.
Studies have described the protective role of vitamin C (ascorbic acid) in certain types of cancer. In this study, we report the effects of vitamin C treatment of two androgen independent prostate cancer cell lines from human (PC3) and rat (Mat-Ly-Lu or MLL) sources. In vitro treatment of PC3 and MLL with sodium ascorbate acid (0-10 mM) resulted in a decrease in cell viability and thymidine incorporation into DNA. These effects of vit. C were dose and time dependent. Ascorbate induced these changes through the production of hydrogen peroxide since addition of catalase (100-300 units/ml), an enzyme that degrades hydrogen peroxide, inhibited the effects of ascorbate on these cell lines. In contrast, superoxide dismutase, an enzyme that dismutates superoxide and generates hydrogen peroxide did not prevent ascorbate-induced changes emphasizing the involvement of reactive oxygen species (ROS) in cellular damage. That singlet oxygen scavengers such as sodium azide and hydroquinone, hydroxyl radical scavengers such as D-mannitol and DL-alpha-tocopherol did not counteract the effects of ascorbate on thymidine incorporation suggests that these free radicals are not involved in cellular damage. In conclusion, these results suggest that vitamin C inhibits tumor growth by virtue of producing reactive oxygen species. These results suggest that ascorbate is a potent anticancer agent for prostate cancer cells.
A controversy exists for many years about the role of sex hormone binding globulin (SHBG) in the uptake of estradiol by the cells. Using the estradiol-sensitive human breast carcinoma cell line MCF-7 and SHBG isolated from human serum by a new method, we observed a strong inhibition of estradiol uptake. The inhibition was higher when the concentration of the hormone was low. On the other hand, there seemed to be a lag period in inhibition when the concentrations of SHBG were very low, followed by an exponential increase, when the concentration exceeded a critical value. The inhibitory activity was higher when SHBG was added before or along with estradiol in the cell culture, as well as when the incubation period was elongated, while was dramatically minimized by the presence of dihydrotestosterone. Despite the inhibition of estradiol uptake caused by SHBG, the distribution of the hormone in various cell components remained practically the same. In conclusion, all indications from experimental data seem to suggest a simple deprivative mechanism being responsible for the inhibitory activity of SHBG on estradiol uptake by MCF-7 cells in culture.
