The use of protein as a carrier of methotrexate for experimental cancer chemotherapy. V. Alternative method for preparation of serum albumin-methotrexate derivative

An alternative method for the preparation of human serum albumin-methotrexate derivative (HSA-MTX) using N-hydroxysuccinimide ester of methotrexate (MTX) was compared with that using the water-soluble carbodiimide (WSC) assistance. The prepared derivative was tested to find the advantages and drawbacks of this method. The N-hydroxysuccinimide ester method is more laborious than that using WSC but some reasons speak in favor of this method. The formation of protein-protein conjugates during the reaction was negligible and under specific conditions more MTX molecules could be bound to protein than by the WSC method. However, some disadvantages of this method were found: A laborious preparation, a small degree of denaturation of protein during the synthesis and, moreover, the binding of MTX to protein did not always take place through NH2-groups of protein but through some other groups (--OH, --SH) as well. These couplings were not so stable as an amidic (or peptidic) bond. In water environment they were hydrolyzed and MTX was slowly released. If there is no difference in a small fraction of protein-protein conjugates, the WSC-assisted method possesses evident advantages over the active MTX ester one, mainly because of the simple preparation of the stable derivative.     

The use of protein as a carrier of methotrexate for experimental cancer chemotherapy. III. Human serum albumin-methotrexate derivative, its preparation and basic testing

The preparation and a more detailed characterization of human serum albumin-methotrexate derivative (HSA-MTX) is described. The synthesis of the derivative was performed by means of 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (in methoiodide form) (WSC). Results of many experiments showed that, on the average, about 26 molecules of methotrexate (MTX) were coupled to one molecule of human serum albumin (HSA). The relative molecular weight of the formed derivative was estimated by gel chromatography on Sepharose 6B or on high-pressure liquid chromatography (HPLC) on SWK column, respectively. From the obtained data it follows that a considerable part of the HSA-MTX derivative formed protein-protein conjugate (up to about 4 X Mr HSA), nevertheless the derivative retains its good solubility as a native albumin. In order to eliminate the possibility of influencing the cytostatic activity of the derivative with byproducts of its synthesis, human serum albumin-folic acid derivative (HSA-FA) was prepared and tested by the same method. All demonstrated experiments proved that MTX was the only compound possessing the cytostatic activity. During the experimental therapy of Gardner lymphosarcoma (LSG) the following was found: (1) The intratumorous application of the drug was the most effective way of administration. (2) Any type of administration of the HSA-MTX derivative exerted a better effect than the same way of administration of free MTX. (3) The comparison of two (repeated) administrations of both drugs showed clearly that the HSA-MTX derivative was more efficient than free MTX. After HSA-MTX derivative treatment all animals survived without tumor. (4) For the estimation of the toxicity of the HSA-MTX derivative, three times and five times repeated intraperitoneal administration was performed. It was concluded that although the derivative was more toxic than free MTX, its therapeutic activity was better. After the elimination of the toxic manifestation of the HSA-MTX derivative by a suitable arrangement of drug doses, five times higher efficacy of the derivative was reached, as compared with free MTX. (5) The therapy by the HSA-FA derivative did not exhibit any therapeutic effect. The reason why HSA was used as a macromolecular carrier for cytostatics is discussed.     

Alpha-melanocyte-stimulating hormone stimulates protein kinase C activity in murine B16 melanoma.

The effect of alpha-melanocyte-stimulating hormone (alpha-MSH) on protein kinase C activity and distribution was investigated in murine B16 F1 melanoma cells. alpha-MSH was found to induce an increased association of protein kinase C (PKC) activity with the particulate fraction of the cells, with an associated loss of enzyme activity from the soluble fraction. The peak response to alpha-MSH occurred between 20 and 60 min of incubation time, and enzyme activities redistributed to those seen in the control cells over the following 12 to 24 h. The average response to alpha-MSH (1 nmol/l) was an approximate 2.5-fold increase in the percentage of enzyme activity associated with the membrane within 1 h of exposure to alpha-MSH; the particulate enzyme activity represented 19.2 +/- 4.4% of total activity in the absence of alpha-MSH and 50.7 +/- 4.7% (means +/- S.E.M., n = 9, P less than 0.005) in the presence of alpha-MSH (1 nmol/l). Cells which had a relatively small percentage of their PKC activity on the membrane initially were significantly (P less than 0.01) more responsive to alpha-MSH stimulation than cells which initially had a relatively large percentage of PKC activity on the membrane. The association of PKC activity with the membrane showed some evidence of being dose-related to alpha-MSH. This is the first report, to the best of our knowledge, of alpha-MSH activating PKC.     

Oligosaccharide modification by swainsonine treatment inhibits pulmonary colonization by B16-F10 murine melanoma cells.

Oligosaccharide moieties of cell-surface glycoconjugates are thought to be involved in recognition events associated with tumor metastasis and invasion. Using swainsonine (SW), an inhibitor of Golgi alpha-mannosidase II that results in the formation of hybrid-type oligosaccharides on N-linked glycoproteins, we have tested the hypothesis that specific glycan structures are required for pulmonary colonization by tumor cells. B16-F10 murine melanoma cells were treated with SW in growth medium and then injected intravenously into syngeneic C57BL/6 mice. This treatment resulted in dramatic inhibition of colonization, but it had no effect on B16-F10 viability or on cellular tumorigenicity after subcutaneous implantation. SW-treated radiolabeled B16-F10 cells were cleared from the lungs at a greater rate than control cells, suggesting that one effect of treatment is to alter tumor cell retention in the target organ. Our results implicate specific glycan structures in pulmonary colonization and offer a potential approach for identification of specific macromolecules involved in tumor cell-organ recognition during metastasis.     

Nuclear factor kappa B： A marker of chemotherapy for human stage Ⅳ gastric carcinoma

AIM： To detect the nuclear factor kappa B （NF-κB） condition in human stage IV gastric carcinoma patients and to explore the correlation between NF-κB activation and survival of these patients after chemotherapy. METHODS： Expression of NF-κB-p65 was determined by immunohistochemical analysis. Activity of NF-κB DNA-binding in carcinoma tissue was detected by electrophoretic mobility shift assay. Kaplan-Meier survival analysis was performed to show the relation between NF-κB and progression-free survival （PFS） or overall survival （OS） of the patients. RESULTS： The positive expression rate of NF-κB-p65 in 60 gastric cancer tissue samples was 76.7% （46160）. The expression of NF-κB-p65 was reduced in adjacent carcinoma and normal tissue samples. Electrophoretic mobility shift assay （EMSA） analysis showed a strong activation of NF-κB in cancer tissue samples. A survival difference was found in NF-κB-p65 positive and negative patients. NF-κB-p65 expression was negative in cancer tissue samples （n = 14）. PFS was 191.40 ± 59.88 d and 152.93 ±16.99 d, respectively, in patients with positive NF-κB-p65 expression （n = 46） （P = 0.4028）. The survival time of patients with negative and positive NF-κB-p65 expression was 425.16 ±61.61 d and 418.85 ±42.98 d, respectively （P = 0.7303）. Kaplan-Meier analysis showed no significant difference in PFS or OS. The 46 patient tissue which positive NF-κB-p65 expression was found in the tissue samples from the 46 patients whose PFS and OS were 564.89 ± 75.94 d and s 352.37 ±41.32 d, respectively （P = 0.0165）. CONCLUSION： NF-κB is activated in gastric carcinoma tissue, which is related to the OS after chemotherapy.     

Influence of collagen substrata on glycosaminoglycan production by B16 melanoma cells.

A cloned metastatic murine melanoma cell line exhibited similar growth characteristics when propagated on either type I collagen, type IV collagen, or plastic. However, cells grown on both types of collagen exhibited an altered cellular morphology and on type IV collagen only, an increased substrate adhesiveness, relative to those maintained on a plastic substratum. Incorporation of [3H]glucosamine and [35S]sulfate into glycosaminoglycans (GAGs) of cells grown on collagen substrates was 20% and 40% less, respectively, than cells grown on plastic, whereas degradation of cell-associated [35S]sulfate-labeled GAGs was similar in cells grown on collagen or plastic. Although the composition of GAGs was similar in all cultures, consisting of approximately 60% chondroitin and 40% heparin or heparan sulfate, the degree of sulfation of the heparin or heparan sulfate molecules was markedly decreased in cultures grown on collagen. The results indicate that the composition of the extracellular matrix influences the biological behavior of B16 melanoma cells, in part by altering the amount and nature of the GAG molecules produced.     

Three-year monitoring of serum p53 antibody during chemotherapy and surgery for stage IV rectal cancer

The overexpression of mutant p53 stimulates serum p53 antibody production in patients with colorectal carcinoma even in superficial tumors. Although the short-term perioperative monitoring of serum p53 antibody titers is reported to be useful in predicting tumor recurrence and patient survival in colorectal carcinoma, the clinical utility of the long-term monitoring of serum p53 antibody titers in patients with colorectal cancer remains unknown. Here, we report the 3-year monitoring of serum p53 antibody titers in a 60-year-old man with rectal cancer, clinical stage IV (T2N2M1b, lung and liver metastases), who was treated with chemotherapy and surgery. Screening tests for CEA (29.4 ng/ml), CA19-9 (41.1 U/ml), and serum p53 antibody (2170 U/ml) were positive before treatment. After chemotherapy with mFOLFOX6 + bevacizumab (B-mab), CEA and CA19-9 decreased to the normal range. However, serum p53 antibody titer remained positive (283 U/ml). After low anterior resection, the serum p53 antibody titer still remained positive (63.4 U/ml). Serum p53 antibody titer significantly changed and was associated with treatment response and tumor recurrence. In the last 6 months of the patient’s life, serum p53 antibody titer gradually decreased, which possibly reflects the modification of the patient’s immune response to p53 antigens.     

The nature of the vitamin B12 binding protein in human serum

It has been known for some years that the vitamin B₁₂ circulating in the plasma is bound to protein. When this plasma binding capacity is exceeded, by the oral or intravenous administration of large doses of the vitamin, the excess unbound vitamin is rapidly excreted in the urine (Mollin and Ross, 1953). The exact nature of this binding protein has yet to be identified. In normal subjects and in patients with myeloproliferative disorders it migrates electrophoretically with the speed of an α₁-globulin, and is present in that fraction of the serum proteins which is not precipitated by 0.6 M-perchloric acid, the seromucoid fraction (Weinstein, Weissman and Watkin, 1959). This serum fraction contains a number of antigenically distinct proteins (Burtin, 1964), two of which have been identified respectively as Orosomucoid and the α₁-acid–glycoprotein of Schultze (Hardwicke and St. Cyr, 1961). In this paper the binding of vitamin B₁₂ to the constituent fractions of a seromucoid, obtained by (NH₄)₂ SO₄ fractionation of serum, has been investigated by chromatography on DEAE-cellulose. Binding into this seromucoid fraction of the serum proteins is confirmed, in normals and in myeloproliferative disorders, but this binding appears to be onto two separable serum proteins which are neither the Orosomucoid nor any other recognized α₁-glycoprotein.     

The experimental chemotherapy of larval alveolar echinococcosis. The search for an optimal regimen of albendazole use]

The antiechinococcal activity of albendazole resynthesized at the E. I. Martsinovski? Institute of Medical Parasitology and Tropical Medicine was studied on infection models in rats and mouse in different experimental modifications. The efficiency of the therapy was determined in relation to the dose of the drug and its routes administrations, to the single or intermittent daily dose, to the presence or absence of intervals in the treatment regimen, to dosage forms. The trials indicated that albendazole was most active against larval alveolar echinococcosis of mice or cotton rats when it was used with their feed, i.e. through the gastrointestinal tract.     

Cloning and expression of a cDNA encoding human sterol carrier protein 2.

We report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP2). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP2 amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP2. The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A)+ RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP2 gene in the human genome or that the SCP2 gene is very large. Coexpression of the SCP2 cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP2 plays a role in regulating steroidogenesis, among other possible functions.     

Antigenic domains of the HIV-1 vif protein as recognized by human sera and murine monoclonal antibodies.

To analyze the vif antibody response in individuals infected with the human immunodeficiency virus type 1 (HIV-1) and to determine antigenic epitopes on the vif protein, 104 HIV-1+ sera were screened for reactivity with a recombinant vif protein; 30 (28.8%) of these sera recognized the recombinant vif protein in immunoblot and were employed, together with 17 HIV-1/vif-negative control sera, in an enzyme immunoassay (EIA)-based epitope scanning assay with 183 overlapping decapeptides that covered the complete amino acid sequence of the HIV-1 vif protein (strain BH10). Of the 30 HIV-1/vif+ sera, 87% reacted with decapeptides comprising the two following epitopes: IEWRKKRY (vif amino acids 87-94) or DRWNKPQ (vif amino acids 172-178). The two epitopes were 89% and 100% conserved among different HIV-1 strains and their antigenicity could be confirmed by computer-assisted predictions of vif antigenic determinants. All the sera reactive with recombinant vif protein and with vif peptides originated from patients in CDC stages III or IV. Two murine anti-vif monoclonal antibodies reacted only with the seven C-terminal amino acids of the vif protein (SHTMNGH), which were not recognized by any of the human sera. Our results may be useful for further studies of vif seroreactivity and for the production of anti-vif mono- or polyclonal antibodies using vif peptides.     

Acyl-acyl carrier protein as a source of fatty acids for bacterial bioluminescence

Pulse-chase experiments with [³H]tetradecanoic acid and ATP showed that the bioluminescence-related 32-kDa acyltransferase from Vibrio harveyi can specifically catalyze the deacylation of a ³H-labeled 18-kDa protein observed in extracts of this bacterium. The 18-kDa protein has been partially purified and its physical and chemical properties strongly indicate that it is fatty acyl-acyl carrier protein (acyl-ACP). Both this V. harveyi [³H]acylprotein and [³H]palmitoyl-ACP from Escherichia coli were substrates in vitro for either the V. harveyi 32-kDa acyltransferase or the analogous enzyme (“34K”) from Photobacterium phosphoreum. TLC analysis indicated that the hexane-soluble product of the reaction is fatty acid. Phosphate ions and, to a lesser extent, organic alcohols stimulated the rate of acyl-protein cleavage. No significant cleavage of either E. coli or V. harveyi tetradecanoyl-ACP was observed in extracts of these bacteria unless the 32-kDa or 34K acyltransferase was present. Since these enzymes are believed to be responsible for the supply of fatty acids for reduction to form the aldehyde substrate of luciferase, the above results suggest that long-chain acyl-ACP is the source of fatty acids for bioluminescence.     

Monoclonal antibody against angiotensin-converting enzyme: its use as a marker for murine, bovine, and human endothelial cells.

A monoclonal antibody has been prepared against rat angiotensin-converting enzyme (ACE). By selection for antibody binding to endothelial cells of bovine rather than rat origin we have obtained a reagent that has broad cross-species binding properties and that can at the same time serve as a useful marker for the surface of endothelial cells. The IgM-producing clone that we have established, alpha-ACE 3.1.1, has been grown in ascites form to yield ascites fluid that binds selectively to immobilized ACE at a greater than 1:10,000 dilution. By use of enzyme-linked immunosorbent assays, immunofluorescence histology, and flow cytometry, we have demonstrated the presence of ACE on endothelial cells of murine, bovine, and human origin. By means of a fluorescence-activated cell sorter (FACS-IV) we have been able to selectively isolate viable endothelial cells from a mixture of endothelial cells and fibroblasts. We believe the antibody will be useful not only for the selection and in vitro cultivation of endothelial cells but also as a tool for the identification and pharmacological study of ACE.     

Development of Apolipoprotein B Antisense Molecules as a Therapy for Hyperlipidemia

As new studies demonstrate that lower levels of low-density lipoprotein cholesterol (LDL-C) reduce cardiovascular disease, and as goals for LDL-C in high-risk individuals are reduced further and further, reaching those goals becomes more difficult for a significant percentage of the population. New therapeutic approaches to lower LDL-C would, therefore, be advantageous, particularly in those who are most likely to suffer cardiovascular disease—associated morbidity and mortality. Mouse and human genetic models suggest that decreasing hepatic apolipoprotein B (apoB) production may be a therapeutic approach for the treatment of dyslipidemia. Because antisense oligonucleotides naturally distribute to the liver and can specifically inhibit synthesis of proteins from their messenger RNAs, antisense oligonucleotides represent a potential approach for decreasing the biosynthesis of apoB, and thereby, the production of both very low density lipoprotein (VLDL) and LDL. Newly developed apoB antisense approaches have produced results in animal models and humans, providing proof of concept regarding reductions in LDL-C concentrations. Surprisingly, despite prior experience with inhibitors of microsomal triglyceride transfer protein, which also inhibits the secretion of VLDL, apoB antisense-mediated reduction in VLDL secretion does not appear to cause marked steatosis. The mechanisms whereby two different approaches for inhibiting apoB and triglyceride secretion have different effects on hepatic triglycerides are currently being examined.     

The use of intermittent human parathyroid hormone as a treatment for osteoporosis

Parathyroid hormone (PTH), given intermittently, is an anabolic agent. PTH has been demonstrated to increase bone mass and reduce vertebral and nonvertebral fractures, and has been approved for use in the US and Europe. PTH is a genetically engineered 34 amino acid protein with the designation teriparatide (recombinant DNA origin) or recombinant human PTH 1-34. A recombinant DNA preparation with all 84 amino acids of the native PTH molecule is in clinical trials. These PTH preparations are selfadministered daily injections, and it is approved for women and men at high risk for fractures, including patients with prevalent fractures, low bone mass, and multiple risk factors. PTH is likely to be used most frequently in patients who fracture on therapy, but can be used in high-risk treatment-naíve patients. Previous treatment with alendronate appears to impair the anabolic response of PTH preparations. Patients who have Paget’s disease, prior radiation therapy to the skeleton, as well as children and young adults with open epiphyses, are at higher risk for osteosarcoma and should not be given PTH. Patients with hypercalcemia and hyperparathyroidism also should not receive the drug.     

Quantitative determination of immunoglobulin G specific for group B streptococcal beta C protein in human maternal serum.

The beta C protein of group B streptococci (GBS) elicits antibody that is protective against GBS challenge in animals and is considered to be a potential component of a GBS conjugate vaccine. We developed a quantitative enzyme-linked immunosorbent assay to measure beta-specific serum immunoglobulin G (IgG) levels and used it to compare beta-specific IgG in a group of mothers of neonates with invasive type Ib/beta GBS disease and a group of mothers colonized with Ib/beta strains whose neonates remained well. beta-Specific IgG concentrations from these 2 groups were similar. To investigate differences in beta-specific antibody in animals and humans, protein fragments were generated that corresponded to major regions within the beta C protein. A single major region was predominantly recognized in human and rabbit serum samples. Thus, in contrast to immunized animals, no relationship was seen between levels of naturally acquired human beta-specific IgG and protection from neonatal disease. This difference was not explained by a major difference in epitope specificity.