Anti-breast Carcinoma Research Articles

Olive leaf extract green-formulated manganese nanoparticles: Characterization and antioxidant, cytotoxicity and anti-breast carcinoma properties

Olives are rich in many plant compounds; Antioxidants in olives protect against the occurrence of many cardiovascular diseases, blood pressure, diabetes, various types of cancers and tissue damage caused by free radicals. These antioxidant compounds include oluropine, hydroxytyrosol, tyrosol, oleanolic acid, and quercetin. Recent studies have focused on the therapeutic efficacies of manganese nanoparticles mediated by medicinal plants. The study investigated the characteristics of green manganese nanoparticles formulated using olive leaf extract on breast carcinoma cells. The nanoparticles were analyzed using FE-SEM, XRD, FT-IR, and UV–Vis techniques. The formation of manganese nanoparticles was confirmed by observing bands at 219 and 290 nm. The Mn-O vibration can be identified by the peaks observed at 516 and 610 cm−1. The peaks at 72.69, 60, 53.26, 44.97, 40.25, 34.83, and 28.48 corresponding to manganese nanoparticles (444), (440), (510), (420), (400), (222), and (220) diffraction planes, indicate the manganese nanoparticles formation. The MTT assay was employed to evaluate the anti-breast carcinoma effects (on CAMA-1 and MCF-7 cells) as well as the cytotoxicity effects (on HUVEC cell line) of manganese NPs. Results from the DPPH test showed that MnNPs and BHT at doses of 231 and 84 μg/ml were able to eliminate 50 % of radicals. The findings from the MTT experiment indicate that over 3 days, the cancer cells percentage that survived decreased proportionally to the increase in nanoparticle concentration. Furthermore, the data demonstrate that the cancer cells survival rate decreased with the rise in nanoparticle concentration, ultimately reaching a point where no viable cells were observed at a concentration of 1000 μg/ml. The most effective cytotoxicity was observed at a concentration of 1000 μg/ml. The MTT findings indicate that the IC50 values are 173 and 256 µg/ml, representing the concentration of nanoparticles at which half of the CAMA-1 and MCF-7 breast carcinoma cells in the culture medium are eradicated. Analysis of the gene expression levels of PIK3, AKT, and mTOR, linked to the mTOR pathway, in addition to Bax, Bcl2, and Caspase-3, demonstrates that copper nanoparticles efficiently control PC14 cell apoptosis and proliferation by regulating the PI3K-Akt-mTOR signaling pathway. The mTOR pathway could potentially be related to the suppression of the cell cycle and the initiation of apoptosis due to nanoparticles.

The application of silver nanoparticles green-formulated by Origanum majorana leaf extract as a hydrazine sensor and treatment of human breast cancer by following the P53 and STAT3 signaling pathways

Silver nanoparticles, because of their large absorption surface and small size, are considered to be intelligent magnetic particles. The advancements in nanotechnology have revolutionized cancer treatment, with silver nanoparticles playing a crucial role in this field. In a recent experiment, the effects of Ag nanoparticles formulated from Origanum majorana on breast cancer cells were investigated. It was found that these nanoparticles induce apoptosis through the signal transducer and activator of transcription 3 and P53 signaling pathways. The nanoparticle characterization was conducted using FE-SEM, XRD, and UV–Vis techniques. Furthermore, the Ag nanoparticles exhibited significant antioxidant activity by preventing 50% of DPPH at 183 μg/mL. The MTT assay revealed the anti-breast carcinoma properties of silver NPs on MCF-7, T-47D, and SkBr3 cells. The outcomes demonstrated that as the nanoparticle concentration increased, the cancer cells survival percentage reduced for 3 days. The most effective anticancer effect was observed at 1000 μg/ml. MTT findings indicate that a concentration of nanoparticles at IC50 = 97, 186, and 180 μg/ml effectively targets 50% of MCF-7, T-47D, and SkBr3 breast carcinoma cells. The presence of silver NPs induces cell apoptosis, which is with the Bax markers regulation and the pro-apoptotic cleaved caspase-8 upregulation, while the anti-apoptotic Bcl-2 marker is downregulated. Besides, silver NPs inhibit the formation of colonies. Molecular pathway analysis of breast cells treated with silver NPs reveals an increase in p53 expression, while the total and phosphorylated STAT3 expression is inhibited, suggesting that p53 and STAT3 have a notable role in the remedial efficacies of silver NPs on breast carcinoma cells. Additionally, the Ag nanoparticles exhibit high sensitivity in the hydrazine electrochemical detection, a potentially carcinogenic substance, with a detection limit of 0.25 μM. The developed sensor, utilizing Ag nanoparticles, shows great promise for the environmental monitoring of hydrazine due to its excellent catalytic performance and simple preparation process. Based on clinical research, recent silver nanoparticles have emerged as a viable option for breast cancer treatment.

Quercetin inhibits the metabolism of arachidonic acid by inhibiting the activity of CYP3A4, thereby inhibiting the progression of breast cancer

BackgroundRecent years have witnessed impressive growth in applying natural medicine in tumor treatment. Saffron is reported to elicit an inhibitory property against BC. Herein, we sought to explore the specific components and mechanistic basis of saffron’s anti-breast carcinoma (BC) function.MethodsBioinformatics analysis was employed to analyze saffron components' anti-BC activity and screen the corresponding target genes involved in BC. Then, the roles of the main saffron ingredient quercetin in the activity of BC cells were examined using CCK-8, MTS, flow cytometry, colony formation, Transwell, and Gelatin zymogram assays. Additionally, the interactions among Quercetin, EET, and Stat3 were assessed by immunofluorescence and Western blot, and LC–MS/MS determined the levels of AA, EETs, and CYP3A. Finally, BC xenograft mouse models were established to verify the anti-BC function of Quercetin in vivo.ResultsQuercetin, the main active component of saffron, inhibited BC progression. Quercetin suppressed BC cell growth, migration, and invasion and inhibited CYP3A4 expression and activity in BC. Mechanistically, Quercetin down-regulated CYP3A4 to block the nuclear translocation of Stat3 by decreasing the metabolization of AA to EETs, thereby alleviating BC. Moreover, exogenously added EETs counteracted the anti-tumor effect of Quercetin on BC. Quercetin also inhibited the tumor growth of tumor-bearing nude mice.ConclusionQuercetin could inhibit the activity of CYP3A to down-regulate AA metabolites EETs, consequently hampering p-Stat3 and nuclear translocation, thus impeding BC development.

Anti-breast carcinoma effects of green synthesized tin nanoparticles from Calendula officinalis leaf aqueous extract inhibits MCF7, Hs 319.T, and MCF10 cells proliferation

In this study, tin nanoparticles were green synthesized using the aqueous extract of Calendula officinalis. Different techniques such as FE-SEM, XRD, FT-IR, and EDS analysis were used to characterize SnNPs@ C. officinalis. A 21.87 nm was obtained for the crystal size of the tin nanoparticles using XRD analysis. The FE-SEM images show a spherical morphology for SnNPs@ C. officinalis with a range size of 21.63–64.07 for the synthetic nanoparticles. The anti-breast cancer effects of biologically synthesized SnNPs@ C. officinalis against breast cancer cell lines were assessed. The anti-breast cancer properties of the SnNPs@ C. officinalis could significantly remove the MCF7, Hs 319.T, and MCF10 cancer cell lines in a time and concentration-dependent manner by MTT assay. The antioxidant activity of SnNPs@ C. officinalis was determined by the DPPH method. The SnNPs@ C. officinalis showed the highest antioxidant activity according to the IC50 value. It seems the anti-human breast cancer effect of recent nanoparticles is due to their antioxidant effects.

Berberine alkaloids inhibit the proliferation and metastasis of breast carcinoma cells involving Wnt/β-catenin signaling and EMT

Berberine alkaloids belong to the class of isoquinoline alkaloids that have been shown to possess anticancer potential, berberine exhibits inhibitory effects on breast cancer development. However, the exact mechanisms of action for anti-breast carcinoma of the alkaloids, including epiberberine, berberrubine and dihydroberberine are still unclear. MTT assay, colony formation, wound healing and transwell invasion assays detected these alkaloids suppressed proliferation, migration and invasion of breast cancer cells. Hoechst and Annexin V-FITC/PI staining were used to analyze the apoptosis of breast cancer cells. Western blotting investigated the changes noted in the expression levels of the key proteins involved in the Wnt/β-catenin signaling pathway and epithelial to mesenchymal transition (EMT). The results showed that inhibited the proliferation of breast cancer cells. Berberine alkaloids inhibited the cell cycle at G2/M phase in MCF-7 cells, but in MDA-MB-231 cells berberine alkaloids arrested the cell cycle in G0/G1 and G2/M phases. By decreasing β-catenin expression, increasing GSK-3β expression and decreasing N-cadherin expression, increasing E-cadherin expression, which proved that epiberberine, berberrubine and dihydroberberine inhibited of metastasis of breast cancer cells through Wnt signaling pathway and reversed EMT except berberine. Furthermore, berberine alkaloids exert their anti-breast cancer effects through the synergistic action of intrinsic and extrinsic pathways of apoptosis. These findings highlight the different effects of different berberine alkaloids on breast cancer cells and confirm that berberine alkaloids may be potentially used in the treatment of breast cancer.

Anti-breast carcinoma effects of green synthesized nickel nanoparticles from Alhagi ‎sparsifolia leaf aqueous extract: A pre-clinical trial study ‎

IntroductionMany nanoparticles have pharmacological and biochemical properties, including antioxidant and anti-‎inflammatory properties, which appear to be involved in anticarcinogenic and antimutagenic activities. In the recent study, nickel nanoparticles were green-synthesized using the Alhagi sparsifolia leaf aqueous ‎extract. ‎Material and methodsThe synthesized Ni nanoparticles‎ were characterized by analytical techniques including SEM, UV-Vis., and FT-‎IR. The nanoparticles were formed in a spherical shape in the average size of 16.19 nm. ‎ResultsIn the antioxidant test, the IC50 of Ni nanoparticles‎ and BHT against DPPH free radicals were 316 and 231 ‎‎µg/mL, respectively. In the cellular and molecular part of the recent study, the treated cells with Ni ‎nanoparticles‎ were assessed by MTT assay for 48h about the cytotoxicity and anti-human breast cancer ‎properties on normal (HUVEC) and breast cancer cell lines i.e. lobular carcinoma of breast (UACC-3133), ‎inflammatory carcinoma of the breast (UACC-732), and metastatic carcinoma (MDA-MB-453). The viability of ‎malignant breast cell line reduced dose-dependently in the presence of Ni nanoparticles‎. The IC50 of Ni ‎nanoparticles‎ were 477, 548, and 605 µg/mL against lobular carcinoma of breast (UACC-3133), inflammatory ‎carcinoma of the breast (UACC-732), and metastatic carcinoma (MDA-MB-453) cell lines, respectively. ‎ConclusionsAfter clinical study, nickel nanoparticles containing Alhagi sparsifolia leaf aqueous extract may be used to ‎formulate a new chemotherapeutic drug or supplement to treat the several types of human breast cancer. ‎

Decorated silver nanoparticles on biodegradable magnetic chitosan/starch composite: Investigation of its cytotoxicity, antioxidant and anti-human breast cancer properties

Nanotechnology is the field to yield modern systems, tools, and materials by controlling at the atomic and molecular levels using the features that appear on those surfaces. Applications for nanotechnology in medical diagnostics, food, medicine, biotechnology, environment, energy, chemistry, physics, etc, introduce this technology as an interdisciplinary and cross-sectoral context. In the current years, conjugated bio-nanomaterials involving magnetic core are a promising platform in new generation biological composites. In this regard, we report herein a novel Ag NPs supported chitosan-starch composite functionalized Fe3O4, a dual core-shell type nanoparticle (Fe3O4@CS-Starch/Ag). In the oncological part of the recent study, the treated cells with Fe3O4@CS-Starch/Ag nanocomposite were assessed by MTT assay for 48 h about the cytotoxicity and anti-breast carcinoma properties on normal (HUVEC) and breast carcinoma cell lines i.e. Hs 578Bst, Hs 319. T, UACC-3133, UACC-732, and MDA-MB-453. The post-synthetically modified biogenic material was analyzed in detail over several physicochemical methods like, field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDS), energy dispersive X-ray analysis (EDX) elemental mapping, transmission electron microscopy (TEM), vibrating-sample magnetometer (VSM), X-ray diffraction analysis (XRD), and inductively coupled plasma-atomic emission spectrometry (ICP-AES) analysis. The viability of breast carcinoma cell lines reduced dose-dependently in Fe3O4@CS-Starch/Ag nanocomposite. The IC50 of Fe3O4@CS-Starch/Ag nanocomposite was 183, 207, 194, 279, and 252 µg/mL against Hs 578Bst, Hs 319. T, UACC-3133, UACC-732, and MDA-MB-453 cell lines, respectively.

Introducing a novel chemotherapeutic drug formulated with anthraflavic acid for treating human breast carcinoma and type 2 diabetes mellitus.

Molecular docking as a versatile theoretical method was used to investigate the biological activities of anthraflavic acid in the presence of α-amylase. The outcomes revealed that anthraflavic acid has a considerable binding affinity to the enzyme with a docking score of -7.913 kcal/mol. These outcomes were further evaluated with free binding energy calculations, and it was concluded that anthraflavic acid could be a potential inhibitor for α-amylase. Anthraflavic acid was explored in anti-human breast carcinoma tests. The in vitro cytotoxic and anti-breast carcinoma effects of biologically synthesized anthraflavic acid against MCF-7, CAMA-1, SK-BR-3, MDA-MB-231, AU565 [AU-565], and Hs 281.T cancer cell lines were assessed. In the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, the anti-breast carcinoma properties of anthraflavic acid could significantly kill the MCF-7, CAMA-1, SK-BR-3, MDA-MB-231, AU565 [AU-565], and Hs 281.T cancer cell lines in a time- and concentration-dependent manner. Also, we used human umbilical vein endothelial cells (HUVECs) to determine the cytotoxicity potentials of anthraflavic acid using MTT assay. The IC50 values of anthraflavic acid were 159, 193, 253, 156, 241, and 218 μg/ml against MCF-7, CAMA-1, SK-BR-3, MDA-MB-231, AU565 [AU-565], and Hs 281.T cancer cell lines. It seems the anti-human breast carcinoma effect of recent nanoparticles is due to their antioxidant effects.

Microwave Irradiation Synthesis and Anti-breast Carcinoma of 6-ethoxy-2-(2-methoxy- benzylideneamino)benzothiazole and its Metal Complexes

Efficient and clean synthesis of Schiff base as a new ligand, 6-ethoxy-2-(2-methoxy benzylideneamino)benzothiazole has been synthesized in equimolar reaction of 2-amino-6-ethoxy-benzothiazole with 2-methoxy benzaldehyde using microwave technique. The prepared Schiff base was reacted with some transition metal ions Ni(II), Cu(II), Pd(II), Ag(I) and Au(III) in equimolar ratio (M:L, 1:1) using microwave technique. The stereochemistry and the bonding characteristics of the ligand and its complexes were achieved based on elemental analysis, FT-IR, UV-Vis., 1HNMR and ESR as well as Thermo-Gravimetric Analysis (TGA). The thermal dehydration and decomposition of Ni(II), Cu(II) and Ag(I) complexes were studied kinetically using the integral method applying the Coats–Redfern and Horowitz Metzger equations. The reactivity of ligand and its Au(III) complex were studied against breast carcinoma cell. The antimicrobial activity of ligand and its Ag(I) complex studied against the bacterial (positive and negative) grams and fungal strains.

Abstract 2117: TRAIL-induced apoptosis in TRAIL-resistant breast carcinoma

Abstract Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), the optimized form of the endogenous death ligand TRAIL, shows therapeutic potential for cancer due to its ability to induce apoptosis in cancer cells independent of p53, while exhibiting minimal toxicity to normal cells. Despite this, a majority of breast cancers display resistance to rhTRAIL treatment due to up-regulation of pro-apoptotic proteins, down-regulation of anti-apoptotic proteins, and/or up-regulation of death receptors (DR) 4 and 5. To overcome rhTRAIL resistance, natural compounds have been investigated as sensitizing agents. This study considered the application of the naturally occurring flavonol Quercetin (Q). Q has been shown to have the ability to up-regulate DR5 and down-regulate anti-apoptotic proteins in cancer cells, and thereby, making Q a favorable choice to be employed as a sensitizing agent. The intention of this study was to ascertain the capacity of Q to sensitize rhTRAIL-resistant triple negative breast cancer BT-20 cells and hormone-dependent breast cancer MCF-7 cells to rhTRAIL-induced apoptosis and elucidate the underlying mechanism for Q’s sensitization. Q demonstrated the ability to intensify rhTRAIL’s pro-apoptotic effects in the breast cancer BT-20 and MCF-7 cell lines as detected through Annexin V/PI assays followed by FACS analysis. In comparison to single agent treatments, the cotreatment of Q and rhTRAIL enhanced the induction of the extrinsic pathway of apoptosis as marked by PARP cleavage (a hallmark of apoptosis), activation of caspase 8, and activation of the executioner caspases 3 and 7. The mechanism for Q’s augmentation in breast cancer was determined to be through the down-regulation of c-FLIPL (caspase 8 inhibitor) in a dose-dependent manner. Furthermore, Q promoted the ubiquitination of c-FLIPL facilitating the proteasome-mediated degradation of c-FLIPL in breast cancer. An additional mechanism for Q’s sensitization was displayed in breast cancer BT-20 cells. Q up-regulated DR5 membrane and protein expression in breast cancer BT-20 cells in a dose-dependent manner. RT-PCR analysis revealed that Q’s influence on DR5 expression occurred at the transcriptional level in those breast cancer cells. Thus, these data suggest that the cotreatment of Q and rhTRAIL possesses the therapeutic potential to be an effective anti-breast carcinoma regimen. Citation Format: Jasmine M. Manouchehri, Michael Kalafatis. TRAIL-induced apoptosis in TRAIL-resistant breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2117. doi:10.1158/1538-7445.AM2017-2117

Salvianolic acid B protects against myocardial damage caused by nanocarrier TiO2; and synergistic anti-breast carcinoma effect with curcumin via codelivery system of folic acid-targeted and polyethylene glycol-modified TiO2 nanoparticles.

Targeted delivery by the folate ligand is an effective way to enhance an anti-breast carcinoma effect, due to its high affinity for the folate receptor, which is overexpressed in many tumor cells. In this study, we firstly synthesized a folic acid (FA)-targeted and polyethylene glycol (PEG)-modified TiO2 nanocarrier. Then, an FA-PEG-TiO2 nanoparticle (NP) codelivery system loaded with curcumin and salvianolic acid B were prepared by emulsion evaporation–solidification at low temperature. The obtained folate-targeted NPs (FA-NPs) showed more cytotoxicity on MCF7 cells and MDA-MB-231 cells than a nontargeted NP group. Apart from a synergistic anti-breast cancer effect with curcumin, salvianolic acid B protects the cardiovascular system from oxidative injury by the TiO2 nanocarrier. With coumarin 6 as a fluorescent probe to observe cellular uptake of NPs, the results of in vitro cellular uptake demonstrated FA-NPs exhibited higher cellular uptake and accumulation in MCF7 cells and MDA-MB-231 cells than nontargeted NPs. Then, in vivo biodistribution of NPs was further qualitatively and quantitatively confirmed by in vivo imaging. More importantly, the animal study further suggested that FA-NPs had significantly stronger antitumor effects via receptor-mediated targeted delivery. Consequently, FA-PEG-TiO2 NPs loaded with curcumin and salvianolic acid B could be a promising drug-delivery system to treat breast cancer.

Isolation and characterization of the antibreast carcinoma cell growth components of Vernonia amygdalina extracts

Vernonia amygdalina (VA) is widely used for medicinal and food purposes in tropical Africa. Many health benefits (antioxidant, antimicrobial, anticancer activities and more) of VA extracts have been reported. The mechanisms of actions have also been described. We have previously reported that VA extracts elicited growth inhibitory activities in human estrogen receptor-positive (ER(+)) cells (MCF-7 cells) and ductal carcinoma cells (BT-549) in vitro. The active components in the organic solvent (chloroform)-extracted VA have been previously determined. However, the active components in the ethanolic extracts of VA have not been previously studied. Hence, the objectives of this study are to isolate and characterize the active components of the ethanolic extracts of VA using liquid-liquid extraction, thin layer chromatography and column techniques. Fractionation of the ethanolic extracts of VA yielded three fractions named A1, A2 and A3, and A2 retained the DNA synthesis-inhibitory activity of the extracts. Subsequent fractionation of A2 yielded fraction A2B whose activity was 16 and three times more potent than the ethanolic fraction and fraction A2, respectively. The treatment of cells with 100 μg/mL of either the ethanolic VA extracts, fraction A2 or fraction A2B resulted in a 23% (P < 0.01), 86% (P < 0.0001) and 97% (P < 0.0001) inhibition of DNA synthesis compared with vehicle-treated controls, respectively. Further purification of A2B by high-speed countercurrent chromatography and confirmed by spectroscopic analysis revealed that the major active components of A2B (65% by weight) were steroid glucosides.

Bioactivities of water-soluble polysaccharides from Jisongrong mushroom: anti-breast carcinoma cell and antioxidant potential

Polysaccharides were extracted from Jisongrong mushroom. Jisongrong polysaccharides was a water-soluble compound. Its molecular weight was about 7.4 × 10 4 Da. HPLC analysis showed that this polysaccharides was composed of mannose, ribose, rhamnose, glucose, xylose, galactose and arabinose in the molar contents of 3.3, 17.3, 6.0, 12.4, 98.4 and 2.1 μM, respectively. In the range of 2800–3500 cm −1, peaks at 2958 and 3407 cm −1 belonging to –C–H of –CH 2 groups, were observed in polysaccharides. When Jisongrong polysaccharides were orally administrated to rats for 2 months, level of lipid peroxidation products and activities of antioxidant enzymes in the blood were significantly decreased and enhanced compared to control rats. Moreover, Jisongrong polysaccharides still markedly inhibited cancer cells proliferation. These results suggested that Jisongrong polysaccharides possess a strong antioxidant and antitumour activities.

Immunohistochemical Differentiation of Extra‐Ocular Sebaceous Carcinoma from Other Skin Cancers

We performed an immunohistochemical study using routinely processed formalin-fixed and paraffin-embedded tissue specimens from 26 cases of extra-ocular sebaceous carcinoma (EOSC) and eight easily available antibodies. They were polyclonal anti-carcinoembryonic antigen (CEA) antibody, monoclonal anti-CEA antibody, anti-breast carcinoma associated antigen-225 antibody (CU18), anti-CA15.3 antibody (CA15.3), anti-CD15 antibody (CD15), anti-breast carcinoma associated antigen antibody (B6.2), anti-gross cystic disease fluid antigen-15 antibody (GCDFP15) and anti-Thomsen-Friedenreich antigen antibody (TFA). Squamous cell carcinoma, porocarcinoma, syringomatous carcinoma, malignant clear cell hidradenoma, apocrine adenocarcinoma, and extramammary Paget's disease with underlying adenocarcinoma were used as controls. EOSC was positive for CU18 and CA15.3 in most cases, and for CD15 in a few cases. Squamous cell carcinoma of the skin was positive for CA15.3 in only one case. Porocarcinoma, syringomatous carcinoma and malignant clear cell hidradenoma were positive for CEA, CU18, CA15.3, and B6.2 in most cases. Apocrine adenocarcinoma and extramammary Paget's disease with underlying adenocarcinoma were positive for CEA, CU18, CD15, GCDFP15, CA15.3, and B6.2 in most cases. TFA was positive not only in EOSC but also in other skin cancers. Immunohistochemical examinations using these seven of eight antibodies except for TFA and routinely processed formalin-fixed and paraffin-embedded tissue specimens are beneficial in differentiating EOSC from other skin cancers.

Cloning and Expression of cDNAs Encoding the Variable Domains of the Antibreast Carcinoma Antibody Mc5

Mc5, a murine monoclonal antibody that binds to human breast epithelial mucin (BEM), has been shown to be a promising reagent in the diagnosis of breast cancer. We have cloned cDNAs encoding both variable regions of Mc5 (VL and VH) as well as the CL and CH1 constant regions. Mc5 is an IgG1, kappa antibody. We have constructed an IgG1, kappa human/mouse chimeric antibody (by inserting the murine VH and VL-encoding cDNAs into plasmids encoding human constant domains), and expressed it in SP2/0-Ag14 mouse myeloma cells. The affinity of chimeric Mc5 (chMc5) for BEM is 4.4 x 10(8) M-1. Mc5 binds BEM with an affinity constant of 2.8 x 10(8) M-1. Purified chMc5 and purified Mc5 gave similar competition curves when tested against either 125I-labeled Mc5 or 125I-labeled chMc5 for binding to BEM in a competition radioimmunodetection format. Additionally, chMc5 used in breast carcinoma tissue staining stained as well as the original Mc5.

Purification of anti-epithelial mucin monoclonal antibodies by epitope affinity chromatography

Monoclonal antibodies against the protein core of epithelial mucins have been found to react with the immunodominant sequence P D T R P A P (Burchell et al., 1989; Price et al., 1990a). Two immunoadsorbent matrices were prepared by linking the peptide A P T R P P A P G to CNBr-activated Sephorose and by linking the peptide C A P D T R P A P G to activated thiol-Sepharose, so that each immunoadsorbent contained the immunodopinant motif. Anti-epithelial mucin antibodies (anti-breast carcinoma antibodies, anti-purified mucin antibodies and anti-human milk fat globule antibodies) were examined for reactivity with these preparations. The initial tests indicated that the substituted CNBr-activated Sepharose displayed lower non-specific antibody binding and this matrix was selected for further investigation. The anti-mucin antibodies were shown to react specifically with this affinity matrix and irrelevant antibodies failed to bind. A Sepharose-peptide immunoadsorbent column was examined for its capacity to purify several of these anti-mucin antibodies and it was determined that this procedure was highly efficient - purified IgG and IgM antibodies could be isolated from either hybridoma tissue culture supernatants or ascitic fluids. The capacity of the column was in excess of 40 mg antibody protein per ml of gel for the IgG3 antibody, C595 (anti-urinary mucin) and at least 10 mg antibody protein per ml of gel for the IgM antibody, NCRC-11 (anti-breast carcinoma). The procedure described permits the efficient purification of anti-mucin antibodies and provides a product which would be suitable for further investigations requiring highly immunoreactive antibodies (e.g., for radioimmunotherapy or immunoscintigraphy in patients with malignant disease).

Immunological and structural features of the protein core of human polymorphic epithelial mucin

The protein core of high mol. wt polymorphic epithelial mucin (PEM—approximately 400 kDa glycoprotein) which is associated with breast carcinomas, consists of a repeating 20 amino acid peptide motif [Gendler el al. (1988) J. biol. Chem. 263, 12,820–12,823]. Monoclonal antibodies C595 (anti-urinary mucin) and NCRC-11 (anti-breast carcinoma cells), and other antibodies against human milk fat globule membranes, were found to recognize determinants present within this 20 amino acid peptide. A model of the peptide was developed based on hydropathicity and structure prediction calculations and these indicated that the repeated structure is dominated by a hydrophilic domain of seven amino acids, extending into two flanking beta turns. NMR analysis of the 20 amino acid peptide was undertaken to probe the secondary structure. Epitope mapping experiments involving solid phase synthesis of overlapping heptapeptides in the repeat unit identified the minimum structures for antibody binding as Arg-Pro-Ala-Pro and Arg-Pro-Ala for the C595 and NCRC-11 antibodies, respectively. These determinants were found within the predicted hydrophilic turn region domain of the peptide. The epitopes for six other PEM-reactive monoclonal antibodies were also determined to reside within the predicted hydrophilic turn domain. This evidence is in accord with the disposition of this region of the PEM peptide core being at the exterior of the glycoprotein where it would be accessible to antibody recognition and binding events.

Polymorphic epithelial mucin from the sera of advanced breast cancer patients – isolation and partial characterisation

The anti-breast carcinoma monoclonal antibody (MAb), NCRC-11 defines a polymorphic epithelial mucin (PEM) which is elevated in the circulation of advanced breast carcinoma patients. Here we describe the purification and partial characterisation of this component from patients' sera and its use in the production of a second generation MAb, C568 (IgM). Pooled sera was fractionated by immunoaffinity and size-exclusion chromatography and the purity of preparations assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Serum-derived PEM shows a similar pattern of electrophoretic mobility to PEM isolated from primary breast tumour tissue and migrates as several bands in 4% SDS polyacrylamide gels (Mr greater than 400,000). The epitope expression of PEMs isolated from either source is also similar, with both bearing topographically distinct determinants for several anti-mucin MAbs. The immunoreactivities of antibodies C568 and NCRC-11 were unaffected by boiling, reduction and alkylation, or by enzyme desialylation of PEM. Periodate oxidation and proteolytic digestion have suggested that the antigenic determinant for C568 is carbohydrate in nature whilst that of NCRC-11 is peptidic. In accord with the mucinous nature of the molecule, serum-derived PEM is susceptible to reductive beta-elimination, elutes in the void volume of a Sepharose CL-4B column and has a buoyant density of 1.45 g ml-1.

C595 – a monoclonal antibody against the protein core of human urinary epithelial mucin commonly expressed in breast carcinomas

Urinary mucins which express determinants for the anti-breast carcinoma monoclonal antibody, NCRC-11 (IgM), closely resemble the mammary mucins found in milk fat globules and carcinomas. An IgG3 monoclonal antibody, C595, was prepared against urinary mucins isolated on a NCRC-11 antibody affinity column, and this 'second generation' antibody was shown to have a very similar pattern of reactivity to the original NCRC-11 antibody. By immunohistology, the profile of reactivity of both antibodies with tumour and normal tissue specimens was virtually identical. Both antibodies reacted with epithelial mucins isolated from breast tumours or normal urine using an NCRC-11 antibody affinity column, although the antibodies were unreactive with other antigen preparations. Heterologous immunoradiometric assays ('sandwich' tests) confirmed that NCRC-11 and C595 epitopes were co-expressed on the same molecule. C595 antibodies inhibited the binding of radiolabelled NCRC-11 antibodies to antigen, suggesting that the two epitopes were in close topographical proximity. The protein core of the mammary mucins has recently been shown to consist predominantly of a repeated 20 amino acid sequence (Gendler et al., 1988). Peptides with this complete sequence and small fragments were synthesised, and the C595 antibody was found to recognise an epitope within this repeat. The ability to identify and synthesise monoclonal antibody-defined determinants, as well as those in the adjacent or overlapping sequences within the protein core of epithelial mucins, is viewed as a strategy for facilitating the production of antibodies of new and novel specificity to complement the panels of existing anti-breast cancer reagents.

Breast cancer imaging with In-111 human IgM monoclonal antibodies: preliminary studies.

Detection of specific tumor sites was studied with scintigraphy and radiolabeled human IgM monoclonal antibodies (MoAbs). Ten patients with metastatic breast cancer received an infusion of one of three indium-111-labeled anti-breast carcinoma MoAbs. The time of infusion ranged from 30 minutes to 2 hours. Three patients received YBB-190 at total doses of 2, 4.25, or 11 mg, four patients received YBM-209 at total doses of 1 mg (n = 1) or 20 mg (n = 3), and three patients each received 22 mg of YBY-088. Imaging was performed immediately after infusion and at 4, 24, 48, 72, 120, and 144 hours. Many presumed sites of metastatic disease were imaged in three of the four patients who received 20 mg of YBM-209 and in two of the three patients who received YBY-088. Tumor was not detected in any of the patients who received YBB-190, in the patient who received a 1-mg dose of YBM-209, or in the patient who received YBY-088 and in whom a biopsy of tumor tissue failed to demonstrate target antigen. The authors conclude that In-111-labeled human IgM MoAbs can target human breast cancer, but antigen expression and antibody dose determine successful immunoscintigraphy.