alpha-chymotrypsin and Breast-Neoplasms

The purpose of present study is to examine the mechanism of the 5'-AMP-activated protein kinase (AMPK) activation induced by proteasome inhibitors. AMPK activation and ubiquitin proteasome system (UPS) inhibition have gained great attention as therapeutic strategies for the treatment of certain types of cancers. While AMPK serves as a master regulator of cellular metabolism, UPS regulates protein homeostasis. However, the relationship between these two important pathways is not very clear. We observe that proteasome inhibition leads to AMPK activation in human breast cancer cells. siRNA transfection, western blotting, qPCR, and proteasomal inhibition assays were used to study the mechanism of proteasome inhibitor-induced AMPK activation using human triple-negative breast cancer, lung, and cervical cancer cell lines. We report that a variety of proteasome inhibitors activate AMPK in all the tested different cancer cell lines. Our data using liver kinase B1-deficient cancer cells suggest that proteasome inhibitor-induced AMPK activation is primarily mediated by Calcium/Calmodulin-dependent kinase kinase β (CaMKKβ). This hypothesis is supported by that pharmacological or genetic inhibition of CaMKKβ leads to a decrease in proteasome inhibitor-induced AMPK activation. Additionally, the AMPK-activating function of the FDA-approved proteasome inhibitor bortezomib depends on an increase in intracellular calcium levels as calcium chelation abrogates its induced AMPK activation. Finally, bortezomib-mediated upregulation in CaMKKβ levels is due to its enhanced protein synthesis. These data suggest that proteasome inhibitors indirectly activate AMPK in human cancer cells primarily via Ca(2+)-CaMKKβ-dependent pathway.

Topics: AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Bortezomib; Breast Neoplasms; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Cell Line, Tumor; Chymotrypsin; Enzyme Activation; Female; Gene Knockdown Techniques; Humans; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Serine-Threonine Kinases; RNA, Small Interfering; Signal Transduction

Changes in the proteasome chymotrypsin-like activity in mammary and thyroid carcinomas in comparison with the adjacent tissue were studied at stages T(1-4)N(0-3)M(0) and T(2-3)N(0-1)M(0), respectively. The activities changed in a wave-like manner over the course of mammary carcinoma growth in cases with and without metastases. The minimum increment of the activity in the tumor was recorded during the T(2)N(0) stage in the absence of local metastases. The increment of the activity reached the peak in N(1) tumors of the same size with metastases. The activities in the tumor and adjacent tissues virtually did not differ during the T(3-4)N(1-3) stages. The time course of proteasome activity changes in thyroid tumors of the studied stages was similar to that in mammary carcinoma. The results can be used for development of methods for evaluating the aggressiveness of mammary and thyroid tumors.

Topics: Boronic Acids; Bortezomib; Breast; Breast Neoplasms; Chymotrypsin; Drug Resistance, Neoplasm; Female; Humans; Neoplasm Metastasis; Neoplasm Staging; Proteasome Endopeptidase Complex; Pyrazines; Thyroid Gland; Thyroid Neoplasms

Protease inhibitors play a protective role against pathogenic microorganisms and herbivorous insects. The two predominant protease inhibitors of soybean seeds are the Kunitz trypsin inhibitor (KTI) and Bowman-Birk protease inhibitor (BBI). In this study, we report that soybean seeds incubated in warm water release large amounts of proteins into the surrounding media. Two-dimensional gel electrophoresis analysis of the seed exudates resulted in the separation of 93 distinct protein spots out of which 90 spots were identified by LC-MS/MS. The basic 7S globulin and the BBI are the two predominant proteins found in the soybean seed exudates. In addition to 7S and 11S seed storage proteins, others known to protect the seeds against pathogens and pests including KTI, peroxidase, α-galactosidase, and endo-1.3-β-glucanase were also identified in the seed exudates. Soybean seed exudate obtained by incubating the seeds in warm water was also able to inhibit the growth of human breast cancer cell line MCF-7. Since soybean seeds release large amounts of enzymatically active BBI when immersed in warm water, our procedure could be exploited as a simplified alternative method for the preparation of BBI concentrate which is being used as a cancer chemoprotective agent.

Topics: Anticarcinogenic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chymotrypsin; Glycine max; Hot Temperature; Humans; Plant Proteins; Seeds; Trypsin Inhibitor, Bowman-Birk Soybean; Trypsin Inhibitors; Water

The soybean-derived protease inhibitor, Bowman-Birk inhibitor (BBI), is currently showing great promise as a novel cancer chemopreventive agent. In contrast to the wealth of research conducted on this compound, the anticancer effects of protease inhibitors isolated from other leguminous sources have received limited attention. In the current study, 7 protease inhibitor concentrates (PICs) were isolated from various leguminous sources (including soybean) and characterized. The effects of PICs on the proliferation of breast and prostate cancer cells were investigated in vitro. Chickpea PIC significantly inhibited the viability of MDA-MB-231 breast cancer and PC-3 and LNCaP prostate cancer cells at all concentrations tested (25-400 μg/ml). In addition, kidney bean (200, 400 μg/ml), soybean (50, 100 μg/ml), and mungbean (100, 200 μg/ml) PICs inhibited LNCaP cell viability. These findings suggest that leguminous PICs may possess similar anticancer properties to that of soybean BBI and deserve further study as possible chemopreventive agents.

Topics: Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chymotrypsin; Cicer; Drug Discovery; Fabaceae; Female; Humans; Male; Molecular Weight; Osmolar Concentration; Peptides; Plant Extracts; Plant Proteins; Prostatic Neoplasms; Protease Inhibitors; Serine Proteinase Inhibitors

Estrogen receptor-alpha (ERalpha) is a major therapeutic target of hormonal therapies in breast cancer, and its expression in tumors is predictive of clinical response. Protein levels of ERalpha are tightly controlled by the 26S proteasome; yet, how the clinical proteasome inhibitor, bortezomib, affects ERalpha regulation has not been studied. Bortezomib selectively inhibits the chymotrypsin-like activity of the proteasome. Unlike other laboratory proteasome inhibitors, bortezomib failed to stabilize ERalpha protein at a dose exceeding 90% inhibition of the chymotrypsin-like activity. Unexpectedly, however, chronic bortezomib exposure caused a reduction of ERalpha levels in multiple ER+ breast cancer cell lines. This response can be explained by the fact that bortezomib induced a dramatic decrease in ERalpha mRNA because of direct transcriptional inhibition and loss of RNA polymerase II recruitment on the ERalpha gene promoter. Bortezomib treatment resulted in promoter-specific changes in estrogen-induced gene transcription that related with occupancy of ERalpha and RNA polymerase II (PolII) on endogenous promoters. In addition, bortezomib inhibited estrogen-dependent growth in soft agar. These results reveal a novel link between proteasome activity and expression of ERalpha in breast cancer and uncover distinct roles of the chymotrypsin-like activity of the proteasome in the regulation of the ERalpha pathway.

Topics: Blotting, Western; Boronic Acids; Bortezomib; Breast Neoplasms; Cell Line, Tumor; Chymotrypsin; Dose-Response Relationship, Drug; Estrogen Receptor alpha; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Promoter Regions, Genetic; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Reverse Transcriptase Polymerase Chain Reaction; RNA Polymerase II; Signal Transduction

A dimeric 70-kDa chymotrypsin inhibitor with substantial N-terminal sequence homology to serine protease inhibitors was isolated from Acacia confusa seeds. The chymotrypsin inhibitor was purified using a protocol that entailed ion exchange chromatography on Q-Sepharose, SP-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. The chymotrypsin inhibitor was unadsorbed on both Q-Sepharose and SP-Sepharose. Its chymotrypsin inhibitory activity was stable from pH 3 to 10 and from 0 to 50 degrees C. It exerted antiproliferative activity toward breast cancer MCF-7 cells with an IC(50) of 10.7+/-4.2 microM. It inhibited HIV-1 reverse transcriptase with an IC(50) of 8+/-1.5 microM. It was devoid of antifungal activity toward a variety of fungal species. The distinctive features of the chymotrypsin inhibitor included dimeric nature, a high molecular mass, lack of trypsin inhibitory activity, highly potent HIV-1 reverse transcriptase inhibitory activity, specific antitumor activity and relatively high pH-stability.

Topics: Acacia; Adsorption; Amino Acid Sequence; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chromatography; Chymotrypsin; Dimerization; Dose-Response Relationship, Drug; Female; Fungi; Hep G2 Cells; HIV Reverse Transcriptase; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Molecular Structure; Phytotherapy; Plant Proteins; Reverse Transcriptase Inhibitors; Seeds; Sepharose; Sequence Homology; Temperature

Wobe Mugos(®) is an enzyme preparation containing the proteases trypsin and papain from the pancreatic calf and commonly used in complementary medicine. From non-randomized studies, its multiple favorable effects including the reduction of adverse events from radiotherapy and chemotherapy in oncology patients have been reported.. Patients with invasive breast cancer receiving adjuvant or palliative chemotherapy between 2005 and 2006 and who were scheduled for at least two further cycles of this specific chemotherapy were included in this pilot study. A specific toxicity of at least grade 2 using the NCI common toxicity criteria which occurred during the preceeding cycle and was relevant to the patient was recorded. This specific toxicity, e.g. grade 2 emesis, was again evaluated after two analogously administered further chemotherapy cycles in which Wobe Mugos(®) had been coadministered. The hypothesis was that specific toxicites of individual patients will be reduced by this enzyme therapy. The majority of the 57 consecutive patients received palliative chemotherapy. Peroral enzyme therapy was coadministered with two uncracked coated tablets three times daily on all days of a chemotherapy cycle except on the day of chemotherapy administration.. Tolerability was good. Positive and neutral effects on toxicity parameters were observed in 11 and 42 patients, respectively, and a negative influence in 4 women.. We observed only a marginal influence of Wobe Mugos(®) in patients with breast cancer who had experienced at least a grade 2 toxicity in the preceding cycle and who received two further identical cycles of this chemotherapy in conjunction with the enzyme preparation. Randomized studies on homogenous patient populations are necessary.

Topics: Administration, Oral; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Chymotrypsin; Drug Administration Schedule; Drug Combinations; Female; Humans; Palliative Care; Papain; Peptide Hydrolases; Pilot Projects; Treatment Outcome; Trypsin

A trypsin inhibitor with a molecular mass of about 19 kDa was isolated from seeds of Chinese black soybean Glycine max cv. "Small Glossy Black". It was isolated using a protocol that comprised ion exchange chromatography on Q-Sepharose, SP-Sepharose and DEAE-cellulose. It was adsorbed on all three ion exchangers. It inhibited trypsin with an IC(50) of 19 microM and chymotrypsin with an IC(50) of 14.3 microM. Its trypsin inhibitory activity was stable in the pH range pH 3-pH 13 and in the temperature range 0 degree C-60 degrees C. The trypsin inhibitor was inhibited by dithiothreitol (from 5 to 25 mM) in a dose-dependent manner. It exhibited an N-terminal sequence highly homologous to Kunitz-type trypsin inhibitors. It inhibited HIV-1 reverse transcriptase with an IC(50) of 0.16 microM, and suppressed proliferation of MCF-7 breast cancer cells with an IC(50) of 4.3 microM and HepG2 hepatoma cells with an IC(50) higher than 25 microM. The trypsin inhibitor lacked antifungal activity and mitogenic activity towards mouse splenocytes.

Topics: Animals; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Proliferation; Chromatography, Ion Exchange; Chymotrypsin; Dithiothreitol; Glycine max; HIV Reverse Transcriptase; Humans; Liver Neoplasms; Mice; Plant Extracts; Reverse Transcriptase Inhibitors; Seeds; Trypsin Inhibitors

Proteasome inhibition provides an attractive approach to cancer therapy and may have application in the treatment of breast cancer. However, results of recent clinical trials to evaluate the effect of the proteasome inhibitor Bortezomib (Velcade, also called PS-341) in metastatic breast cancer patients have shown limited activity when used as a single agent. This underscores the need to find new and more efficacious proteasome inhibitors. In this study, we evaluate the efficacy of the novel proteasome inhibitor BU-32 (NSC D750499-S) using in vitro and in vivo breast cancer models.. We have recently synthesized a novel proteasome inhibitor (BU-32) and tested its growth inhibitory effects in different breast cancer cells including MCF-7, MDA-MB-231, and SKBR3 by in vitro cytotoxicity and proteasomal inhibition assays. The apoptotic potential of BU32 was tested using flow cytometry and analyzing cell cycle regulatory proteins. In vivo tumor xenograft studies for solid tumor as well as tumor metastasis were conducted using MDA-MB-231-GFP cells.. We report for the first time that BU-32 exhibits strong cytotoxicity in a panel of cell lines: MDA-MB-231 (IC50 = 5.8 nM), SKBR3 (IC50 = 5.7 nM) and MCF-7 cells (IC50 = 5.8 nM). It downregulates a wide array of angiogenic marker genes and upregulates apoptotic markers, including Bid and Bax. Incubation of MDA-MB-231 cells with BU-32 results in the accumulation of cell cycle inhibitor proteins p21 and p27 and stabilization of the tumor suppressor protein p53. Studies in in vivo solid tumor and metastasis models show significant effect with a 0.06 mg/kg dose of BU-32 and marked reduction in tumor burden in the skeleton.. We have shown that BU-32 is effective in cultured breast cancer cells and in breast cancer xenografts. The results suggest its potential benefit in breast cancer treatment.

Topics: Animals; Apoptosis; Bone Neoplasms; Boronic Acids; Bortezomib; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Chymotrypsin; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; NF-kappa B; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Pyrazoles; Xenograft Model Antitumor Assays

The most abundant and biologically active green tea catechin, (-)-epigallocatechin-3-gallate or (-)-EGCG, has been shown to act as a proteasome inhibitor and tumor cell death inducer. However, (-)-EGCG is unstable under physiologic conditions and has poor bioavailability. Previously, in an attempt to increase the stability of (-)-EGCG, we introduced peracetate protections to its reactive hydroxyl groups and showed that this peracetate-protected (-)-EGCG [Pro-EGCG (1); formerly named compound 1] could be converted into (-)-EGCG under cell-free conditions. In the current study, we provide evidence that when cultured human breast cancer MDA-MB-231 cells were treated with Pro-EGCG (1), (-)-EGCG was not only converted but also accumulated, accompanied by enhanced levels of proteasome inhibition, growth suppression, and apoptosis induction, compared with cells treated with natural (-)-EGCG. To investigate the potential use of Pro-EGCG (1) as a novel prodrug that converts to a cellular proteasome inhibitor and anticancer agent in vivo, MDA-MB-231 tumors were induced in nude mice, followed by treatment with Pro-EGCG (1) or (-)-EGCG for 31 days. Results of this in vivo study showed a significant inhibition of breast tumor growth by Pro-EGCG (1), compared with (-)-EGCG, associated with increased proteasome inhibition and apoptosis induction in tumor tissues. In conclusion, we have shown that Pro-EGCG (1) increases the bioavailability, stability, and proteasome-inhibitory and anticancer activities of (-)-EGCG in human breast cancer cells and tumors, suggesting its potential use for cancer prevention and treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Catechin; Cell Growth Processes; Cell Line, Tumor; Chromatography, High Pressure Liquid; Chymotrypsin; Female; Humans; Mice; Mice, Nude; Peracetic Acid; Prodrugs; Protease Inhibitors; Xenograft Model Antitumor Assays

Disulfiram (DSF), a member of the dithiocarbamate family capable of binding copper and an inhibitor of aldehyde dehydrogenase, is currently being used clinically for the treatment of alcoholism. Recent studies have suggested that DSF may have antitumor and chemosensitizing activities, although the detailed molecular mechanisms remain unclear. Copper has been shown to be essential for tumor angiogenesis processes. Consistently, high serum and tissue levels of copper have been found in many types of human cancers, including breast, prostate, and brain, supporting the idea that copper could be used as a potential tumor-specific target. Here we report that the DSF-copper complex potently inhibits the proteasomal activity in cultured breast cancer MDA-MB-231 and MCF10DCIS.com cells, but not normal, immortalized MCF-10A cells, before induction of apoptotic cancer cell death. Furthermore, MDA-MB-231 cells that contain copper at concentrations similar to those found in patients, when treated with just DSF, undergo proteasome inhibition and apoptosis. In addition, when administered to mice bearing MDA-MB-231 tumor xenografts, DSF significantly inhibited the tumor growth (by 74%), associated with in vivo proteasome inhibition (as measured by decreased levels of tumor tissue proteasome activity and accumulation of ubiquitinated proteins and natural proteasome substrates p27 and Bax) and apoptosis induction (as shown by caspase activation and apoptotic nuclei formation). Our study shows that inhibition of the proteasomal activity can be achieved by targeting tumor cellular copper with the nontoxic compound DSF, resulting in selective apoptosis induction within tumor cells.

Topics: Animals; Apoptosis; Breast Neoplasms; Chymotrypsin; Copper; Disulfiram; Female; Humans; Mice; Neoplasm Transplantation; Proteasome Inhibitors; Transplantation, Heterologous

Epidemiological studies have suggested that increased soy consumption is associated with reduced cancer occurrence. Genistein, a soy isoflavone, has been reported to inhibit the growth of human tumor cells although the involved molecular mechanisms are not clearly defined. Here we report that genistein inhibits the proteasomal chymotrypsin-like activity in vitro and in vivo. Computational docking studies suggest that the interaction of genistein with the proteasomal beta 5 subunit is responsible for inhibition of the chymotrypsin-like activity. Inhibition of the proteasome by genistein in prostate cancer LNCaP and breast cancer MCF-7 cells is associated with accumulation of ubiquitinated proteins and three known proteasome target proteins, the cyclin-dependent kinase inhibitor p27(Kip1), inhibitor of nuclear factor-kappa B (I kappa B-alpha), and the pro-apoptotic protein Bax. Genistein-mediated proteasome inhibition was accompanied by induction of apoptosis in these solid tumor cells. Finally, genistein induced proteasome inhibition and apoptosis selectively in simian virus 40-transformed human fibroblasts, but not in their parental normal counterpart. Our results suggest that the proteasome is a potential target of genistein in human tumor cells and that inhibition of the proteasome activity by genistein might contribute to its cancer-preventive properties.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Transformation, Viral; Chymotrypsin; Cysteine Endopeptidases; Female; Fibroblasts; Genistein; Humans; Isoflavones; Male; Multienzyme Complexes; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The proteasome is emerging as a target for cancer therapy because small molecule inhibitors of its catalytic activity induce apoptosis in both in vitro and in vivo models of human malignancies and are proving to have efficacy in early clinical trials. To further elucidate the mechanism of action of these inhibitors, their impact on signaling through the p44/42 mitogen-activated protein kinase (MAPK) pathway was studied. Proteasome inhibition with either carbobenzoxy-leucyl-leucyl-phenylalaninal or lactacystin led to a loss of dually phosphorylated, activated p44/42 MAPK in A1N4-myc human mammary and MDA-MB-231 breast carcinoma cells in a dose- and time-dependent fashion. This correlated with an induction of the dual specificity MAPK phosphatases (MKP)-1 and -2, and blockade of MKP induction using either actinomycin D or Ro-31-8220 significantly decreased loss of activated p44/42 MAPK. Inhibition of p44/42 MAPK signaling by use of the MAPK kinase inhibitors PD 98059 or U0126, or by use of a dominant negative MAPK construct, enhanced proteasome inhibitor-mediated apoptosis. Conversely, activation of MAPK by epidermal growth factor, or use of a mutant MAPK resistant to MKP-mediated dephosphorylation, inhibited apoptosis. These studies support a role for inactivation of signaling through the p44/42 MAPK pathway in proteasome inhibitor-mediated apoptosis.

Topics: Apoptosis; Breast Neoplasms; Butadienes; Chymotrypsin; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dactinomycin; Enzyme Inhibitors; Female; Flavonoids; Humans; Kinetics; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Nitriles; Oligopeptides; Proteasome Endopeptidase Complex; Tumor Cells, Cultured

[corrected] To evaluate the impact of postoperative treatment with an oral enzyme (OE) preparation given complementary to an antineoplastic therapy in patients with breast cancer.. The design of this epidemiological study was a retrolective cohort analysis with parallel groups. Design and conduct of the study were performed to current standards for prospective, controlled clinical trials. A cohort of 2,339 breast cancer patients undergoing surgical intervention and radio-, chemo- or hormonal therapy were studied in 216 centres. Of the 2,339 patients, 1,283 received complementary treatment with OE and 1,056 did not receive OE. Patients with other complementary medications were excluded and the final analysis was performed with the data from 649 patients, of whom 239 (37%) were additionally treated with OE (test group) and 410 (63%) without OE (control group). The median follow-up time for the test group was 485 days and for the control group 213 days. The primary endpoint of the study was to determine whether complementary treatment with OE can reduce typical disease- or therapy-associated signs and symptoms (gastrointestinal symptoms, mental symptoms, dyspnoea, headache, tumour pain, cachexia, skin disorders, infections, and side effects associated with the antineoplastic therapy) in patients with breast cancer. Imbalances for causal effects (covariates) were adjusted for by means of the propensity score. Outcome analysis was performed by estimating the linear regression between change in symptom score and propensity score with all data and using this regression line to calculate the change in symptom score which would be expected for each patient. Tumour-associated events (recurrence, metastasis, and death) were evaluated in terms of the number of events observed and time to event. The safety of treatment with OE was analysed in terms of the number and severity of adverse events, their duration, treatment and outcome.. For all symptoms except tumour pain, the adjusted mean improvement in symptom scores was larger in the test group than in the control group. The adjusted difference was statistically significant for all symptoms, except tumour pain and infections. The results show that the typical disease- and therapy-associated signs and symptoms in patients on complementary therapy with OE during postoperative treatment were significantly less. For 75% of the test group and 55% of the control group the physician recorded "no signs and symptoms". A clear reduction in the side effects of radiotherapy and chemotherapy was documented in 74% of the test group and 55% of the control group. Analysis of survival, recurrence, and metastasis demonstrated a reduced number of events in the test group. There was evidence of a beneficial influence of OE on time to event, although the median observation time was too short in these breast cancer patients to draw definite conclusions. The safety component was judged in 98% of the test group and 76% of the control group as "very good" or "good". In the total sample of 2,339 patients, the rate of OE-associated adverse reactions was 3.2%. All side effects were mild to moderate gastrointestinal symptoms.. Complementary treatment of breast cancer patients with OE improves the quality of life by reducing signs and symptoms of the disease and the side effects of adjuvant antineoplastic therapies. This epidemiological retrolective cohort analysis provides evidence that the patients may also gain benefit by a prolongation of the time to event for cancer recurrence, metastasis and survival. OE was generally well tolerated.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Breast Neoplasms; Chemotherapy, Adjuvant; Chymotrypsin; Cohort Studies; Drug Combinations; Endopeptidases; Female; Humans; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Papain; Postoperative Care; Quality of Life; Radiotherapy, Adjuvant; Retrospective Studies; Treatment Outcome; Trypsin

We determined the amino acid and radiolabel sequences of tryptic [32P]phosphopeptides of the purified human estrogen receptor (hER) from MCF-7 cells and Sf9 cells. Serine 118 was identified as a site that was phosphorylated independently of estradiol-binding in MCF-7 cells. Proline is on the carboxy terminus of serine 118, which suggests that the serine-proline may be a consensus phosphorylation site motif for either the mitogen-activated protein (MAP) kinase or p34cdc2 kinase. MAP kinase selectively phosphorylated the recombinant hER in vitro on serine 118 independent of estradiol-binding, whereas p34cdc2 did not phosphorylate the hER. We demonstrated previously that serine 167 of the hER was phosphorylated in an estradiol-dependent manner. We therefore compared the consequence of hER phosphorylation at serine 118 by MAP kinase and phosphorylation at serine 167 by casein kinase II on the receptor's affinity for specific DNA binding. The binding of the hER to an estrogen response element was not altered by phosphorylation with MAP kinase at serine 118 but was significantly increased when phosphorylated at serine 167 by casein kinase II. These data suggest that phosphorylation of the hER by MAP kinase(s) pathways may influence receptor action by a mechanism other than the estradiol-dependent phosphorylation of hER by casein kinase II.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Casein Kinase II; Cell Line; Chymotrypsin; DNA; DNA-Binding Proteins; Female; Humans; Molecular Sequence Data; Peptide Fragments; Phosphates; Phosphopeptides; Phosphorus Radioisotopes; Phosphorylation; Protein Serine-Threonine Kinases; Receptors, Estrogen; Recombinant Proteins; Serine; Spodoptera; Substrate Specificity; Transfection; Trypsin; Tumor Cells, Cultured

We have examined the synthesis of the protease inhibitors alpha 1-antitrypsin (alpha 1-AT) and alpha 1-antichymotrypsin (alpha 1-ACHY) by variants of the MCF-7 human breast cancer cell line. Spent medium from MCF-7 203P cells, grown in the absence of serum, was found to contain immunoreactive alpha 1-AT and alpha 1-ACHY by Western blotting. In the presence of 10(-8) M estradiol, levels of both inhibitors were increased 3- to 6-fold. Incubation of spent medium with [125I]trypsin or [125I]chymotrypsin resulted in the formation of stable 75- and 90-kDa complexes identical to the complexes formed between these proteases and the protease inhibitors in plasma, showing the release of active protease inhibitors by MCF-7 cells in culture. Immunoprecipitation of 35S-labeled proteins from the medium of cells grown in the presence of [35S]methionine yielded comparable results, confirming hormonally sensitive synthesis of both protease inhibitors. Northern blot analysis suggests that stimulation of estradiol occurs at the level of transcription. Tetradecanoyl phorbol acetate (50 ng/ml) also stimulated alpha 1-AT and alpha 1-ACHY synthesis 2- to 4-fold, suggesting the involvement of protein kinase-C. Comparison studies with MCF-7 cell sublines ML, BK, 203P, and 300P (a variant spontaneously appearing after 100 passages of 203P) show a wide variation in synthesis of alpha 1-AT and alpha 1-ACHY proteins; sublines 203P and 300P synthesize both inhibitors, the ML subline synthesizes detectable amounts only of alpha 1-ACHY, while no detectable synthesis of either inhibitor was seen in the BK subline. Similar results were obtained for protease inhibitor mRNA transcription by Northern blotting, although low levels of alpha 1-AT mRNA transcription by the ML subline and of alpha 1-AT and alpha 1-ACHY mRNA transcription by the BK subline could be detected.

Topics: alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Blotting, Western; Breast Neoplasms; Chymotrypsin; Estradiol; Humans; Immunosorbent Techniques; Kinetics; Nucleic Acid Hybridization; Tetradecanoylphorbol Acetate; Transcription, Genetic; Trypsin; Tumor Cells, Cultured

The proteolytic fragments of the nuclear estrogen receptor in the MCF-7 cell line were characterized following limited digestion with chymotrypsin and trypsin. Nuclei were isolated from cells previously exposed to 10 nM [3H]estradiol. The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei. The molecular weights (Mr) were calculated from the sedimentation coefficients determined on a sucrose gradient and from the Stokes radii estimated by gel filtration. Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 151,000. This receptor form was degraded by chymotrypsin to a receptor of Mr = 33,000 and by trypsin to a receptor of Mr = 60,000. Digestion of intact nuclei with chymotrypsin solubilized a receptor form of Mr = 62,000 which dissociated in 0.4 M KCl to a receptor of Mr = 32,000. Digestion of intact nuclei with trypsin followed by micrococcal nuclease solubilized a receptor form of Mr = 75,000 which was further dissociated by 0.4 M KCl to a receptor form of Mr = 60,000. The ability of the receptor forms to bind DNA was tested using DNA-cellulose column chromatography. About 40% of the micrococcal nuclease solubilized receptor form, compared to about 7% of the chymotrypsin degraded receptor and to about 13% of the trypsin degraded receptor forms, all bound to the column and could be eluted by high salt concentrated buffer. We conclude that the nuclear estrogen receptor in the MCF-7 cell line can be partially degraded either in the micrococcal nuclease hydrolysate or in intact nuclei by chymotrypsin or trypsin generating protein moieties, probably receptor fragments of Mr = 33,000 and 60,000 respectively. Both fragments retain their estradiol binding domain and it may be hypothesized that the heavier fragment retains its chromatin binding domain.

Topics: Breast Neoplasms; Cell Line; Cell Nucleus; Centrifugation; Chymotrypsin; DNA; DNA-Binding Proteins; Humans; Micrococcal Nuclease; Molecular Weight; Peptide Fragments; Receptors, Estrogen; Trypsin

The synthesis of an active proteinase inhibitor, gp 66, by human breast epithelial cells is reported. This glycoprotein is identical to serum alpha 1-antichymotrypsin, which inhibits proteinases that cleave at hydrophobic residues. Immunohistological studies show the in vivo expression on normal secretory and ductal epithelial cells and on primary and metastatic adenocarcinomas. Immunoaffinity-purified gp 66 from MCF-7 culture supernatants is an active inhibitor of chymotrypsin as determined in a fluorogenic enzyme assay and can form stable 88 kDa enzyme-inhibitor complexes. The synthesis of a functional inhibitor may represent the epithelial cell's attempt to stabilize its extracellular milieu.

Topics: alpha 1-Antichymotrypsin; Breast; Breast Neoplasms; Cell Adhesion; Cell Line; Chymotrypsin; Epithelium; Female; Fluorescent Antibody Technique; Glycoproteins; Humans; Isoelectric Point; Molecular Weight

alpha 1-Antichymotrypsin (Achy) is an antiprotease of the acute inflammation phase, which is also released by MCF7 human breast cancer cells in culture. Using a fluorimetric assay with the synthetic substrate L-Seryl-L-Tyrosyl-2-N-naphthylamide, we have shown that a medium conditioned by MCF7 cells treated by estradiol inhibits the activity of alpha-chymotrypsin. This inhibition increased when physiological concentrations of estradiol were added to the cells for 2 days. It was due to an increased production of Achy and not to a direct effect of estradiol on alpha-chymotrypsin activity as shown by double immunoprecipitation with an antiserum against human alpha 1-antichymotrypsin. An increased accumulation by estradiol of an antigen located in the cytoplasm of MCF7 cells, which was revealed by immunoperoxidase staining with antibodies to Achy, also indicated that estradiol increased the production of Achy in these cells. Similar immunostaining was observed in a breast cancer tissue. Most of the estrogen regulated 60-68 kDa protein secreted by T47D cells (Chalbos et al. (1982) J. Clin. Endocrinol. Metab. 55, 276-283) was also specifically immunoprecipitated by the antibodies to Achy. Thus, alpha 1-antichymotrypsin is the first protein to be identified which is induced by estradiol and secreted by breast cancer cells.

Topics: alpha 1-Antichymotrypsin; Breast Neoplasms; Cell Line; Chymotrypsin; Estradiol; Female; Humans; Immunoenzyme Techniques

Topics: alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Breast; Breast Neoplasms; Chymotrypsin; Epithelium; Female; Histocytochemistry; Humans; Immunoenzyme Techniques; Muramidase; Protease Inhibitors

Breast carcinoma with multinucleated reactive stromal giant cells is a rare histological type. The number of published cases, to which we add two new cases, is too small for it to be possible to reach a conclusion as to whether this type of complaint, for which the authors put forward morphological criteria, should or should not be considered as an anatomoclinical entity. The diagnosis can be made as soon as cytological examination has shown a large number of multinucleated giant cells associated with carcinomatous cells. Ultrastructural study confirms the macrophagic origin of the giant cells but does not show any macrophagic activity.

Topics: alpha 1-Antichymotrypsin; Breast Neoplasms; Carcinoma; Chymotrypsin; Female; Humans; Immunoenzyme Techniques; Middle Aged; Muramidase

Malignant and uninvolved human breast tissues were maintained in organ culture for 3 to 6 days. Under these conditions, the three-dimensional glandular architecture is maintained with the least disruption of tissue integrity. The biosynthesis and release of glycoproteins were studied by using the incorporation of [14C]glucosamine and [14C]leucine by the breast surgical specimens. Five major families of labeled glycoproteins were identified from culture supernatants using two-dimensional gel electrophoresis. Quantitative immunoprecipitation established that 16 to 30% of the total of labeled glycoproteins were recognized as normal serum components. Two of these glycoproteins were antigenically related to normal human serum components as demonstrated with crossed immunoelectrophoresis. Evidence was obtained for the active synthesis of alpha 1-antichymotrypsin and alpha 1-acid glycoprotein by human breast epithelial cells. alpha 1-Antichymotrypsin accounted for 0.9 to 7.8% of the biosynthetically labeled glycoproteins from organ culture supernatants. This component was 11.9% of the glycoproteins released by a monolayer culture of the established breast carcinoma cell line, MCF-7. alpha 1-Acid glycoprotein made up 0.7 to 3.1% of the labeled glycoproteins. alpha 1-Antichymotrypsin is a known neutral serine proteinase inhibitor with a particularly strong affinity for cathepsin G. alpha 1-Acid glycoprotein may function primarily as a potent immunomodulator by suppressing lymphoblastogenesis. These glycoproteins may thus have regulatory roles in the proteolytic modification of breast tissue and represent the tissue's own protecting shield against invading leukocytes.

Topics: alpha 1-Antichymotrypsin; Breast; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Chymotrypsin; Female; Humans; Immunoelectrophoresis, Two-Dimensional; Molecular Weight; Organ Culture Techniques; Orosomucoid

The cellular localization of alpha-1-antichymotrypsin (alpha 1-ACT) was studied immunohistochemically using rabbit HRP-labeled Fab' against human alpha 1-ACT. alpha 1-ACT was found in cell nuclei of carcinomas of the stomach, liver, breast, pancreas and leiomyosarcoma and in cell nuclei of lymphoid cells infiltrated into the stomach carcinoma mass. alpha 1-ACT was not found in carcinoma cells of the colon, uterus, rectum or esophagus, or in lymphoid cells infiltrated into the rectal carcinoma mass or into inflammatory regions such as gastric ulcers or appendicitis. Further, alpha 1-ACT was not found in normal cells around the carcinoma mass or in normal tissues.

Topics: alpha 1-Antichymotrypsin; Breast Neoplasms; Carcinoma; Cell Nucleus; Chymotrypsin; Humans; Immunologic Techniques; Leiomyosarcoma; Liver Neoplasms; Lymphoid Tissue; Neoplasms; Pancreatic Neoplasms; Stomach Neoplasms; Trypsin Inhibitors

The synthesis and release of glycoproteins were studied in organ cultures of human breast surgical specimens and in established breast epithelial cell lines, MCF-7 and MDA-MB-231. Biosynthesis was monitored by the incorporation of 14C-glucosamine. Labeled macromolecules in the culture supernatants were analyzed by biochemical and immunological techniques. One to 8% of the labeled glycoproteins from benign breast and infiltrating ductal carcinoma specimens was precipitated by antibodies produced against human serum alpha-1-antichymotrypsin. Twelve percent of the total glycoproteins from the culture supernatants of the MCF-7 cell line was identified as alpha-1-antichymotrypsin. Both the normal serum and the human breast epithelia-derived proteinase inhibitor can be resolved into similar subclasses by two-dimensional gel electrophoresis. MDA-MD-231 and MCF-7 cells which were extensively washed with EDTA, serum-free medium, and phosphate-buffered saline retain this proteinase inhibitor on their cell surfaces. Three to 4% of the total cell-surface iodinated components was immunoprecipitated by these specific antibodies. Since alpha-1-antichymotrypsin is a potent inhibitor of neutral proteinases such as cathepsin G, the demonstration of its synthesis by benign and malignant breast epithelial cells is of considerable interest. This glycoprotein may represent the epithelia's own protective shield of cell surface components and the cell's attempt to moderate the effects of invading leukocytes. In addition, it may play a regulatory role in the maintenance of three-dimensional glandular structures.

Topics: Adenocarcinoma; alpha 1-Antichymotrypsin; Breast; Breast Neoplasms; Cell Line; Chymotrypsin; Epithelium; Female; Humans; Molecular Weight; Organ Culture Techniques; Protease Inhibitors

Topics: alpha 1-Antitrypsin; alpha-Macroglobulins; Antithrombins; Breast Neoplasms; Caseins; Chromatography, Affinity; Chymotrypsin; Female; Humans; In Vitro Techniques; Orosomucoid; Protease Inhibitors

Topics: Blood Proteins; Breast Neoplasms; Carcinoma; Chymotrypsin; Cyclophosphamide; Female; Hematocrit; Hemoglobins; Humans; Kininogens; Kinins; Leukocyte Count; Leukocytes; Time Factors

Topics: Adult; Aged; Breast Neoplasms; Carcinoma; Cholecystitis; Chymotrypsin; Colorimetry; Duodenal Ulcer; Female; Hepatitis; Humans; Intestinal Diseases; Liver Cirrhosis; Lung Diseases; Lung Diseases, Obstructive; Male; Middle Aged; Neoplasm Metastasis; Pancreatitis; Pregnancy; Respiratory Tract Infections; Trypsin Inhibitors

Topics: Breast Neoplasms; Chymotrypsin; Enzymes; Female; Humans; Injections, Intramuscular; Mastectomy; Postoperative Complications

Topics: Breast; Breast Neoplasms; Carcinoma; Chymotrypsin; Humans