Bromelain: biochemistry, pharmacology and medical use.

Bromelain is a crude extract from the pineapple that contains, among other components, various closely related proteinases, demonstrating, in vitro and in vivo, antiedematous, antiinflammatory, antithrombotic and fibrinolytic activities. The active factors involved are biochemically characterized only in part. Due to its efficacy after oral administration, its safety and lack of undesired side effects, bromelain has earned growing acceptance and compliance among patients as a phytotherapeutical drug. A wide range of therapeutic benefits has been claimed for bromelain, such as reversible inhibition of platelet aggregation, angina pectoris, bronchitis, sinusitis, surgical traumas, thrombophlebitis, pyelonephritis and enhanced absorption of drugs, particularly of antibiotics. Biochemical experiments indicate that these pharmacological properties depend on the proteolytic activity only partly, suggesting the presence of nonprotein factors in bromelain. Recent results from preclinical and pharmacological studies recommend bromelain as an orally given drug for complementary tumor therapy: bromelain acts as an immunomodulator by raising the impaired immunocytotoxicity of monocytes against tumor cells from patients and by inducing the production of distinct cytokines such as tumor necrosis factor-a, interleukin (Il)-1beta, Il-6, and Il-8. In a recent clinical study with mammary tumor patients, these findings could be partially confirmed. Especially promising are reports on animal experiments claiming an antimetastatic efficacy and inhibition of metastasis-associated platelet aggregation as well as inhibition of growth and invasiveness of tumor cells. Apparently, the antiinvasive activity does not depend on the proteolytic activity. This is also true for bromelain effects on the modulation of immune functions, its potential to eliminate burn debris and to accelerate wound healing. Whether bromelain will gain wide acceptance as a drug that inhibits platelet aggregation, is antimetastatic and facilitates skin debridement, among other indications, will be determined by further clinical trials. The claim that bromelain cannot be effective after oral administration is definitely refuted at this time.

Tributyrin enhances the cytotoxic activity of interleukin-2/interleukin-12 stimulated human natural killer cells against LS 174T colon cancer cells in vitro.

Tributyrin has been shown to be cytostatic to tumor cells by inducing differentiation and apoptosis. On the other hand, immunological NK cells can kill tumor cells, particularly when stimulated with interleukin-2 (IL-2) and/or interleukin-12(IL-12). However, little is known about whether and how both antitumor mechanisms act together, although in vivo such an interaction must exist. Here we demonstrate in vitro, that pretreatment of human LS 174T colon cancer cells with nontoxic concentrations of tributyrin augments the sensitivity to spontaneous NK cell activity two-fold. However, when NK cells have been activated with an optimized combination of IL-2 and IL-12, the immunocytotoxicity increases up to five-fold (from 14% to 70%), versus a 3.8-fold increase against untreated cancer cells. These effects are accompanied by increased IFN-gamma secretion and decreased TGF-beta1 secretion. Tributyrin is found to be a potent inducer of ICAM-1, LFA-3 and Fas on target cells corresponding to an increase of the FasL expression by IL-2/IL-12 on the effector cells. Our data suggest a synergistic link between induction of tumor cell differentiation and immunological defense mechanisms that may provide a rational basis for the improvement of clinical protocols, especially for colon cancer.

Bromelain reversibly inhibits invasive properties of glioma cells.

Bromelain is an aqueous extract from pineapple stem that contains proteinases and exhibits pleiotropic therapeutic effects, i.e., antiedematous, antiinflammatory, antimetastatic, antithrombotic, and fibrinolytic activities. In this study, we tested bromelain's effects on glioma cells to assess whether bromelain could be a potential contributor to new antiinvasive strategies for gliomas. Several complementary assays demonstrated that bromelain significantly and reversibly reduced glioma cell adhesion, migration, and invasion without affecting cell viability, even after treatment periods extending over several months. Immunohistochemistry and immunoblotting experiments demonstrated that alpha3 and beta1 integrin subunits and hyaluronan receptor CD44 protein levels were reduced within 24 hours of bromelain treatment. These effects were not reflected at the RNA level because RNA profiling did not show any significant effects on gene expression. Interestingly, metabolic labelling with 35-S methionine demonstrated that de novo protein synthesis was greatly attenuated by bromelain, in a reversible manner. By using a transactivating signaling assay, we found that CRE-mediated signaling processes were suppressed. These results indicate that bromelain exerts its antiinvasive effects by proteolysis, signaling cascades, and translational attenuation.

Effects of differentiation inducers on cell phenotypes of cultured nontransformed and immortalized mammary epithelial cells: a comparative immunocytochemical analysis.

We examined the effects of the differentiation-inducing agents 1, 25-dihydroxyvitamin D(3) (calcitriol), phorbol myristate acetate (PMA), the cytokine interferon-gamma (IFN-gamma), and the tumor necrosis factor-alpha (TNF-alpha) alone and in combination with calcitriol on cell growth and differentiation parameters of cultured nontransformed human mammary epithelial cell (HMEC) lines, the chemically transformed HMEC line H184 A1N4, and the human mammary carcinoma cell line CAL51. Cell differentiation was phenotyped by semiquantitative immunocytochemistry using a panel of 15 monoclonal antibodies against marker molecules representing epithelial cell differentiation, cell-cell adhesion processes and malignancy. Cell proliferation of HMEC and H184 A1N4, but not of CAL51 cells was reduced by the agents. Cell phenotypes were analyzed by examining the expression of cytokeratins (pan CK and CK19), the epithelial mucin (MUC1), isoactin, and the blood group-related H type 2 carbohydrate antigen. HMEC and H184 A1N4 cells showed characteristics of basal cells, whereas CAL51 cells resembled a lumenal phenotype. The cell-cell adhesion molecules E-cadherin, intracellular adhesion molecule (ICAM-1), and the tumor markers carcinoembryonic antigen (CEA) and CD44v6 were expressed on all 3 mammary cell lines, with moderate differences. With respect to effects on cell phenotypes, HMEC were sensitive to PMA and IFN-gamma resulting in an increased expression of MUC1, CEA and ICAM-1 molecules. H184 A1N4 cells responded to TNF-alpha in combination with calcitriol by increased expression of pan CK, MUC1, and decreased H type 2 antigen expression, suggesting a transition towards a lumenal phenotype. Furthermore, CEA, ICAM-1 and CD44v6 were increased by TNF-alpha plus calcitriol. In contrast, CAL51 cells were overall less sensitive to differentiation induction attempts; only TNF-alpha stimulated MUC1, isoactin and epithelial cell adhesion molecule (Ep-CAM) expression.

Effects of oral bromelain administration on the impaired immunocytotoxicity of mononuclear cells from mammary tumor patients.

The protease bromelain from pineapple was suggested for adjuvant therapy of malignant diseases. We studied immunological effects of an orally applied bromelain drug on 16 breast cancer patients in comparison with healthy donors. Bromelain was applied for 10 days with a daily dose of 3000 F.I.P. units and the immunocytotoxicity of blood monocytes and lymphocytes against the leukemic K562 and MDA-MB-231 mammary carcinoma target cells was determined in vitro. In addition, the expression of the cell surface markers CD44, CD16, CD11a and CD62L on lymphocytes and the secretion of IL-2 and IL-1beta from monocytes was measured. Patients leukocytes expressed lower bMAK-, MAK-, NK- and LAK-cell activities, compared with those from healthy donors. Orally applied bromelain increased the reduced bMAK- and MAK-cell activity of patients monocytes about 2-fold. When the patients were classified on the basis of bromelain effects on the monocytic cytotoxicity into bromelain responders and nonresponders, about 40% of the patients responded to bromelain with an increase of cytotoxicity from 7.8% to 54% (bMAK-cell activity) and from 16% to 47% (MAK-cell activity). Bromelain was less effective on the higher cytotoxicity of monocytes from healthy donors, but stimulated the secretion of IL-1beta from monocytes. In contrast, patient monocytes secreted no detectable IL-1beta, before, during and after bromelain treatment. Bromelain had no effects on the impaired patients NK- and LAK-cell activity, but reduced the LAK-cell activity of healthy donors. No IL-2 was found in the supernatants of untreated and treated lymphocytes from healthy donors. Bromelain reduced the expression of CD44, but weakly increased CD11a and CD62L expression on patient lymphocytes, whereas CD16 remained unchanged. In vitro bromelain application to lymphocytes had similar effects, with greater reduction rates of CD44 and CD16 expression. As to coagulation parameters in plasma of healthy donors, the activated partial thromboplastin time was increased from 38 to 46 sec, leaving prothrombin time and plasminogen unchanged. These data suggest, that orally applied bromelain stimulates the deficient monocytic cytotoxicity of mammary tumor patients, which may partially explain its proposed antitumor activity.

Bromelain proteases reduce human platelet aggregation in vitro, adhesion to bovine endothelial cells and thrombus formation in rat vessels in vivo.

The thiol protease, bromelain, an extract from pineapple stem, was suggested to have antithrombotic and anticoagulant activities in vivo. We studied the effects of bromelain on cell size distribution of isolated human platelets in vitro by Coulter Counter measurements. Preincubation of platelets with bromelain (10 micrograms/mL) completely prevented the thrombin (0.2 U/mL) induced platelet aggregation. Papain was less active in preventing platelet aggregation. In vitro, bromelain (0.1 microgram/mL) reduced the adhesion of bound, thrombin stimulated, fluorescent labeled platelets to bovine aorta endothelial cells. In addition, preincubation of platelets with bromelain, prior to thrombin, activation, reduced the platelet adhesion to the endothelial cells to the low binding value of unstimulated platelets. On the basis of mass concentrations, the proteases papain and trypsin were as effective as bromelain. Using a laser thrombosis model, the in vivo effects of orally and intraveneously applied bromelain on thrombus formation in rat mesenteric vessels were studied. Bromelain, orally applied at 60 mg/kg body weight, inhibited the thrombus formation in a time dependent manner, the maximum being after 2 hours in 11% of arterioles and 6% of venoles. Intravenous application at 30 mg/kg was slightly more active in reducing thrombus formation in arterioles (13%) and venoles (5%), suggesting that orally applied bromelain is biologically active. These results may help to explain some of the clinical effects observed after bromelain treatment in patients with thrombosis and related diseases.

Prothymosin alpha1 antagonizes the inhibitory effects of transforming growth factor-beta1 on the adhesion of peripheral blood lymphocytes to human umbilical vein endothelial cells.

We investigated the in vitro effects of both prothymosin alpha1 (Pro alpha1) and transforming growth factor-beta1 (TGF-beta1) on the adhesion of peripheral blood lymphocytes (PBL) and on the expression of the adhesion molecules, endothelial-leukocyte adhesion molecule-1 (ELAM-1), intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on human umbilical vein endothelial cells (HUVECs). TGF-beta1 moderately but significantly decreased PBL-binding to interleukin-1 (IL-1)- stimulated HUVECs. However, Pro alpha1 in combination with TGF-beta1 completely restored the TGF-beta1 mediated effects. Other thymic peptides tested were ineffective. On HUVECs, TGF-beta1 diminished ICAM-1 and VCAM-1 expression, leaving ELAM-1 unchanged. Pro alpha1 in combination with TGF-beta1 showed no significant effects on ELAM-1 expression, but antagonized the TGF-beta1-induced decrease of ICAM-1 and VCAM-1 expression on activated HUVECs.

Tributyrin induces growth inhibitory and differentiating effects on HT-29 colon cancer cells in vitro.

Tributyrin (TB) is a prodrug of butyrate known to induce tumor cells to differentiate. We examined its effects on cell growth, viability, cellular morphology and differentiation of HT-29 colon cancer cells in vitro, as reflected by the expression of CEA, E-cadherin and the induction of alkaline phosphatase activity. TB, applied in a stable emulsion, inhibited tumor cell proliferation in a reversible and dose-dependent manner (0.5-4 mM) with significant morphological changes. The IC50 value of TB was 1 mM after 6 days. For comparison, sodium butyrate, applied in equimolar concentration, inhibited cell growth with an IC50 value of 2.2 mM. TB treatment at concentrations of 0.5 mM and 2 mM resulted in an increase of the doubling times by 18% and 160%, respectively, without any effects on cell viability. By a colorimetric immunoassay, 1.5 mM TB induced the expression of both CEA and E-cadherin by about 260% and 100%, respectively. Furthermore, the activity of the brush border enzyme alkaline phosphatase was enhanced in a dose-dependent manner, up to 60-fold at the maximum of 2 mM TB. Our results show that TB is more active than butyrate in suppressing cell growth and concomitantly promoting differentiation of HT-29 colon cancer cells. Hence it may be a promising candidate for clinical therapeutic protocols and merits further investigation.

Isolation and characterization of two forms of an acidic bromelain stem proteinase.

Two forms of an acidic bromelain proteinase isolated from crude bromelain, an extract from pineapple stem, were found by a two-step FPLC purification procedure. The basic main components were removed by cation exchange chromatography and the breakthrough fraction was further resolved by anion exchange chromatography into 15 protein fractions, only two of which, called SBA/a and SBA/b, were proteolytically active. These components were characterized by electrospray mass spectroscopy (ESMS), isoelectric focusing, N-terminal amino acid sequence analysis, monosaccharide analysis, and enzymatic parameters. The molecular masses of SBA/a and SBA/b were determined by ESMS to be 23,550 and 23,560, respectively. The isoelectric points (pI) of the two bands of SBA/a were 4.8 and 4.9; SBA/b focused as a single band at pI = 4.8. Partial N-terminal amino acid sequences (11 residues) were identical to SBA/a and SBA/b and identical with those of stem bromelain, the basic main proteinase of the pineapple stem, and fruit bromelain, the acidic main proteinase of the pineapple fruit. Both components are highly glycosylated; hydrolysis of SBA/a yielded about twofold more monosaccharide per protein than SBA/b. The comparison of the catalytic properties of SBA/a with those of SBA/b revealed no relevant differences in the hydrolysis of three peptidyl-NH-Mec substrates and in the inhibition profiles using chicken cystatin and E-64, indicating that these components can be considered as two forms of a single enzyme. Both forms are scarcely inhibited by chicken cystatin and slowly inactivated by E-64, hence are nontypical cysteine proteinases of the papain superfamily.

Docetaxel treatment of HT-29 colon carcinoma cells reinforces the adhesion and immunocytotoxicity of peripheral blood lymphocytes in vitro.

In vitro effects of docetaxel on the human colon carcinoma cell line HT-29 were studied with respect to the expression of different adhesion and surface marker molecules, the adhesion and immunocytotoxicity of peripheral blood lymphocytes and the secretion of IFN-gamma and TNF-alpha. Docetaxel, in a low concentration range (1-3x10-9 M), increased the expression of the adhesion molecules LFA-3, ICAM-1, CD44s, CD44v6, CD15, CD13 and VLA-4/5/6 on the tumor cells. Unstimulated and interleukin-2 (IL-2) activated killer (LAK) cells showed a better adherence to docetaxel treated HT-29 cells than to untreated cells. In neutralization experiments, anti-LFA-3, -CD44v6, -CD15, -VLA -4 and anti-CD13 mAb reduced the lymphocyte adhesion to untreated and docetaxel treated cells at different degrees, while CEA mAb increased the adhesion. Unstimulated and IL-2 activated lymphocytes exhibited significantly higher cytotoxicities against docetaxel treated cells than against untreated HT-29 cells. Unstimulated and IL-2 stimulated lymphocytes secreted more TNF-alpha and IFN-gamma when cocultured with docetaxel treated HT-29 cells than with untreated cells. These results suggest, that the increased lymphocyte mediated cytotoxicity against docetaxel treated HT-29 colon carcinoma cells may reflect an immunological process coupled with induction of differentiation, that may contribute to the clinically known cytostatic effects of the drug.

Expression and modulation of the Lewis x antigen (CD15) on the T cell line Molt-4.

The T cell lines Molt-4 and H9 exhibited a characteristic distribution of the cell adhesion molecule Lewis x (CD15, lacto-N-fucopentanose III) showing an unusually broad peak by flow cytometry ranging from cells without CD15 to cells with high expression. The cytokines IL-1, IL-2, IFN-beta, IFN-gamma, and TNF-alpha, known to activate T cells, did not affect CD15 expression. However, phorbol myristate acetate and the thymic peptide extract Thymex-L were able to enhance both the number of CD15-positive cells and the median fluorescence. The effects of both inducers were dose- and time-dependent. An additive or synergistic effect was not seen. These data suggest that phorbol esters and distinct thymic peptides can modulate the expression of the cell surface antigen CD15.

Prothymosin alpha 1 effects in vitro on chemotaxis, cytotoxicity and oxidative response of neutrophils from melanoma, colorectal and breast tumor patients.

Immunoregulatory effects of thymic peptides on functions of polymorphonuclear leukocytes (PMNs) are poorly investigated. We studied the effects of prothymosin alpha 1 (Pro alpha 1) on PMNs from patients with colorectal tumors, breast tumors and melanoma (total n = 37) in comparison with healthy donors (n = 18), with respect to chemotaxis, cytotoxicity against HCT-116 colon tumor cells, oxidative response (chemiluminescence reaction) as well as expression of surface marker molecules. We found that Pro alpha 1 was equally effective in stimulating the chemotactic activity of PMNs from tumor patients and healthy donors (43% increase). PMNs from tumor patients, especially with breast tumor, showed a significant enhancement of cytotoxicity against the tumor target cells in comparison with healthy donors. With respect to the PMNs cytotoxicity, only about 50% of the colorectal tumor patients and healthy donors responded to Pro alpha 1 and FMLP. As to the oxidative response of PMNs, elevated levels were found only among colorectal tumor patients. Pro alpha 1 significantly increased the oxidative response in breast and colorectal tumor patients by 55% and 25%, respectively. Pro alpha 1 decreased the expression of CD16 on PMNs of healthy donors, but not that of CD11a, CD11b, CD11c, CD13, CD14, CD15 and CD32. Therefore, we suggest, that Pro alpha 1 may improve some PMN functions of tumor patients, associated with the proposed role in host-tumor interaction.

Prothymosin alpha 1 effects, in vitro, on the antitumor activity and cytokine production of blood monocytes from colorectal tumor patients.

Recent animal studies demonstrate that prothymosin alpha 1 (ProT alpha) enhances the antitumor response by stimulation of mononuclear phagocyte functions. The present study was aimed at characterizing the in vitro effects by ProT alpha on blood monocytes from human colon cancer patients. Purified peripheral blood monocytes were studied in terms of tumor cytostatic ability and cytokine production after incubation with ProT alpha or interferon (rIFN-gamma) and transforming growth factor-beta (TGF beta), used as reference substances. SW620 colon carcinoma cells were used as tumor target cells in growth inhibition experiments. The level of baseline growth inhibitory activity of unstimulated patient's monocytes was significantly lower than that of normal monocytes. The defective antitumor activity of patient monocytes was associated with a higher production of the inhibitory monokines prostaglandin E2 (PGE2) and TGF beta. The stimulation of monocytes by ProT alpha and/or rIFN-gamma elevated the average antitumor activity in all donor groups. The ProT alpha-induced increase was associated with a significantly higher monocytic secretion of IL-1 beta and TNF-alpha. Moreover, the concentrations of TGF beta and PGE2 in the culture supernatants decreased significantly, when patient's monocytes were treated with ProT alpha and/or rIFN-gamma. Additionally, ProT alpha enhanced the diminished antitumor activity of TGF beta-treated normal monocytes. These results suggest that ProT alpha selectively regulates distinct functions of blood monocytes, the effect of this cytokine varying with the parameter and donor population examined. These data provide a rational and biological endpoint for further studies with ProT alpha as an activator of mononuclear function in colon cancer.

Prothymosin alpha1 effects on IL-2-induced expression of LFA-1 on lymphocytes and their adhesion to human umbilical vein endothelial cells.

Prothymosin alpha1 (Pro alpha1) is known to stimulate in vitro and in vivo natural killer (NK) and lymphokine (IL-2)-activated killer (LAK) cells against tumor cells. In this process, LAK cells first adhere to endothelial cells in vivo, raising the question whether Pro alpha1 affects this interaction as well. The binding ability of peripheral blood lymphocytes (PBL) to human umbilical vein endothelial cells (HUVEC) was increased by incubation with IL-2 in a concentration-dependent manner, reaching a maximal value at 20U/ml IL-2. Although Pro alpha1 alone was without any stimulating effect, it significantly increased PBL binding to unstimulated HUVECs in combination with suboptimal IL-2 (5 and 10 U/ml). The combination of Pro alpha1 (1 microg/ml) and 5 U/ml or 10 U/ml IL-2 is as effective as 10 U/ml or 20 U/ml IL-2 alone. This Pro alpha1 effect on IL-2-activated lymphocytes was found to be augmented on IL-1 or tumor necrosis factor (TNF)-alpha-activated endothelial cells. Analyzing the effect of Pro alpha1 on IL-2-activated lymphocytes by flow cytometry revealed an increase of CD16, CD56, and CD18 surface marker expression, whereas CD3, CD11a/b, CD49d, and CD54 were not affected. In conclusion, Pro alpha1 functions as a mediator of the adhesion of IL-2-activated lymphocytes to HUVECs.

Docetaxel enhances the expression of E-cadherin and carcinoembryonic antigen (CEA) on human colon cancer cell lines in vitro.

The in vitro effects of docetaxel on the human colon carcinoma cell lines-HT-29 and SW620 were studied by measuring the expression of the differentiation markers E-cadherin and CEA with a recently developed immunohistochemical assay. Docetaxel was shown to increase the expression of E-cadherin and CEA in the low concentration range of 3 x 10(-10) M - 3 x 10(-9) M for HT-29 cells and 0.6 - 3 x 10(-9) M for SW620 cells. Docetaxel strongly increased CEA (270%) and E-cadherin (160%) expression in HT-29 cells, compared with both marker expression in SW620 cells (60%). E-cadherin and CEA expression were found to be inversely correlated to the antiproliferating potential of docetaxel, which inhibits the cell proliferation of HT-29 cells at an IC50 of 6 x 10(-10) M and of SW620 cells at 8 x 10(-10) M. The data suggest, that in addition to its antiproliferative activity docetaxel also exhibits a strong differentiation inducing capacity at concentrations < 10(-9) M in vitro. These docetaxel effects were more pronounced on the differentiated HT-29 cells than on the poorly differentiated SW620 cells.

Interleukin-2-activated killer cell activity in colorectal tumor patients: evaluation of in vitro effects by prothymosin alpha1.

The effects of prothymosin alpha1 (Pro alpha1) in combination with interleukin-2 (IL-2) on peripheral blood lymphocytes from 50 colorectal tumor patients at different stages were studied with respect to immunocytotoxicity, adhesion to cultured SW620 colon carcinoma cells, secretion of cytokines and expression of adhesion and surface marker molecules. On average, the patients showed lower natural killer (NK) cell activity than healthy donors, which was associated with a lower adhesion capacity to the tumor target cells. The NK cell activity of the patients was inversely related to the tumor stage. The generation of lymphokine(IL-2)-activated killer (LAK) cell activity was found to be comparable on lymphocytes from healthy individuals and patients and was not correlated to tumor stage. Pro alpha1 stimulated patients' LAK cell activity only, primarily at the early stage (Dukes A/B). The Pro alpha1 effect was associated with an increased adhesion of lymphocytes to tumor target cells and an increased secretion of the deficient IL-2-induced IFN gamma secretion. No significant effects on the low level of TNF alpha secretion was noted. By flow cytometry, Pro alpha1 in combination with IL-2 augmented the expression of the NK cell markers CD56, CD16/56, the subset CD3/16/56 and CD25 on lymphocytes of the patients. In contrast, Pro alpha1 was equally effective by increasing the expression of CD18 and CD11a on lymphocytes from the patients and from normal controls. In conclusion, Pro alpha1, in combination with IL-2, can partially normalize lymphocyte deficiencies of colon cancer patients in vitro. This potential might provide an experimental basis for applying Pro alpha1 or related thymic peptides in selected immunotherapies against colorectal tumors.

Preclinical studies with prothymosin alpha1 on mononuclear cells from tumor patients.

The immunomodulating potential of the thymic protein prothymosin alpha1 (Pro alpha1) on the lymphocyte and monocyte directed antitumor reactions of melanoma and colorectal tumor patients in comparison with healthy controls were studied in vitro. On average, tumor patients showed lower NK- and LAK-cell activities than healthy controls, being associated with a lower adhesion capacity to tumor target cells. The NK-cell activity of the tumor patients was inversely related to the tumor stage. Pro alpha1 stimulated the impaired patients LAK-cell activity only at an early stage of disease. The Pro alpha1 effects were associated with an increased adhesion of lymphocytes to tumor target cells and an increased secretion of deficient IFN-gamma and IL-2 secretion. By flow cytometry, Pro alpha1 in combination with IL-2 increased the NK-cell marker CD56, CD16/56 and CD25 as well as CD18/11a adhesion molecule expression. Monocytes from tumor patients showed deranged tumoristatic activities compared with healthy controls. Pro alpha1 elevated the mean of the antitumor activity, when applied alone or in combination with rIFN-gamma. In the presence of IFN-gamma, Pro alpha1 stimulated the adhesion of monocytes to cultured tumor cells, mainly by increasing CD54 expression. Pro alpha1 stimulated alone or in combination with IFN-gamma the TNF-alpha and IL-1beta secretion by monocytes and decreased the high PGE2 and TGF-beta level, especially in the patients groups. Perspectively, this may provide the basis for applying Pro alpha1 in selected antitumor immunotherapy protocols.

A rapid and sensitive fluorometric screening assay using YO-PRO-1 to quantify tumour cell invasion through Matrigel.

A new quantitative assay for the study of tumour cell invasion in vitro is described. Employing the novel fluorescent dye YO-PRO-1, cells that penetrate Matrigel-coated transwells are counted on the basis of dye-bound cellular nucleic acid content. Following transmigration, the cells in the lower compartments are lysed by freezing in water. After a brief incubation with YO-PRO-1, nucleic acid or DNA content is measured as fluorescence intensity in 96-well microplates and quantitated by a cell- or DNA-calibration curve. Using standard curves, a linear relationship between fluorescence intensity and cell number was found in the range tested (from 100 to 80 000 cells). The mean relative intra- and inter-assay variability of the cell quantitation in this range was 3.5 and 4.2%, respectively. When applied to Matrigel invasion studies, as few as 400 cells could be counted. The quantitation could be performed within 3 h. HCT 116, MDA MB 231 and HT 29 cells were investigated as examples of tumour cells with different invasive abilities in the 48-h Matrigel invasion assay. Using YO-PRO-1, 6.5 +/- 0.6% invasive HCT 116 cells and 52.6 +/- 4.5% MDA MB 231 cells (percentage of the inoculated cell population) were measured. HT 29 cells were practically non-invasive. These results were confirmed by visual scoring of DAPI-stained nuclei. In conclusion, the main advantages of the assay are its sensitive, reproducible and rapid quantitation of tumour cell invasion in vitro and the applicability to extended sample numbers by measuring in 96-well microplates.