DRD1 and DRD4 are differentially expressed in breast tumors and breast cancer stem cells: pharmacological implications.

Background: Abnormal expression of dopamine receptors (DRs) has been described in multiple tumors, but their roles in breast cancer are inconclusive or contradictory since evidence of pro- and anti-tumoral effects have been reported. Herein, we analyzed the expression of DRs in breast cancer, especially in the subpopulation of cancer stem cells (CSCs), and evaluated the functional role of the receptors by pharmacological targeting. Methods: Expression of DRD1, DRD2, DRD3, DRD4 and DRD5 was investigated in human breast tumors and cancer cell lines using public databases. Correlation between gene expression and clinical outcome was studied by Kaplan-Mayer analyses. By flow cytometry, we assessed DRD1, DRD2, and DRD4 expression in cultures of MCF-7 (luminal) and MDA-MB-231 (triple-negative) cells. Using the previously reported SORE6 reporter system we examined the differential expression of DRD1, DRD2, and DRD4 in CSCs and tumor-bulk cells. The effect of pharmacological modulation of DRs on stemness and cell migration was studied by quantification of the reporter-positive fraction and wound healing assays, respectively. Results: DRD1, DRD2 and DRD4 transcripts were expressed in breast tumors. DRD4 was overexpressed compared to normal tissue and showed prognostic value. DRD1, DRD2 and DRD4 transcripts were also found in MCF-7 and MDA-MB-231 cells, but only DRD1 and DRD4 proteins were detected. DRD4 was underexpressed in CSCs compared to tumor-bulk cells, whereas DRD1 was found only in the CSCs fraction, suggesting that those receptors may have relevance in stemness control. Subtoxic concentrations of DRD1-targeting compounds did not induced significant changes in the CSCs pool. On the other hand, DRD4 inhibition by Haloperidol slightly increased the CSCs content but also reduced cell migration. Conclusions: Pharmacological modulation of DRD1 in MCF-7 or MDA-MB-231 cells seems to be irrelevant for stemness maintenance. DRD4 reduced expression in breast CSCs or its inhibition by Haloperidol favors CSCs-pool expansion. DRD4 inhibition can also reduce cell migration, indicating that DRD4 plays different roles in stem and non-stem breast cancer cells.

Relationship between the expression of complement inhibitory proteins and therapeutic efficacy of antibodies in breast cancer.

Complement regulatory proteins (mCRPs) CD55, CD46 and CD59 have been proposed as key elements in therapeutic resistance against cancer. mCRP-expressing tumor cells, in addition to hindering trastuzumab, pertuzumab and sacituzumab-govitecan therapeutic activity in breast cancer, can regulate biological processes that promote tumor progression. This review describes the structure of mCRPs and analyzes their expression using transcriptomic databases from breast cancer patients, in addition to collecting information on mCRPs interactions and signaling in tumor cells. Given that mCRPs are relevant targets, several strategies that have been explored for their inhibition and regulation in order to increase therapeutic efficacy and prevent cancer resistance and progression are described.

Se ha propuesto a las proteínas reguladoras de complemento (mCRP) CD55, CD46 y CD59 como piezas clave en la resistencia terapéutica contra el cáncer. Las células tumorales que expresan las mCRP, además de obstaculizar la actividad terapéutica de trastuzumab, pertuzumab y sacituzumab-govitecan en cáncer de mama, pueden regular procesos biológicos que promueven la progresión tumoral. Esta revisión describe la estructura de las mCRP y analiza su expresión a partir de bases de datos transcriptómicos de pacientes con cáncer de mama; también recopila información de interacciones y señalización de las mCRP en células tumorales. Dado que estas mCRP son dianas relevantes, se describen diversas estrategias para su inhibición y regulación para incrementar la eficacia terapéutica y evitar la resistencia y progresión del cáncer.

Relación entre la expresión de proteínas inhibidoras de complemento y la eficacia terapéutica de anticuerpos en cáncer de mama / Relationship between the expression of complement inhibitory proteins and therapeutic efficacy of antibodies in breast cancer

Resumen Se ha propuesto a las proteínas reguladoras de complemento (mCRP) CD55, CD46 y CD59 como piezas clave en la resistencia terapéutica contra el cáncer. Las células tumorales que expresan las mCRP, además de obstaculizar la actividad terapéutica de trastuzumab, pertuzumab y sacituzumab-govitecan en cáncer de mama, pueden regular procesos biológicos que promueven la progresión tumoral. Esta revisión describe la estructura de las mCRP y analiza su expresión a partir de bases de datos transcriptómicos de pacientes con cáncer de mama; también recopila información de interacciones y señalización de las mCRP en células tumorales. Dado que estas mCRP son dianas relevantes, se describen diversas estrategias para su inhibición y regulación para incrementar la eficacia terapéutica y evitar la resistencia y progresión del cáncer.

Abstract Complement regulatory proteins (mCRPs) CD55, CD46 and CD59 have been proposed as key elements in therapeutic resistance against cancer. mCRP-expressing tumor cells, in addition to hindering trastuzumab, pertuzumab and sacituzumab-govitecan therapeutic activity in breast cancer, can regulate biological processes that promote tumor progression. This review describes the structure of mCRPs and analyzes their expression using transcriptomic databases from breast cancer patients, in addition to collecting information on mCRPs interactions and signaling in tumor cells. Given that mCRPs are relevant targets, several strategies that have been explored for their inhibition and regulation in order to increase therapeutic efficacy and prevent cancer resistance and progression are described.

Bioassays for the Evaluation of Target Neutralization and Complement-Dependent Cytotoxicity (CDC) of Therapeutic Antibodies.

Therapeutic monoclonal antibodies (mAbs) are complex bioengineered proteins that require to be routinely characterized with robust and reliable bioassays. Infliximab was the first anti-TNFα mAb approved for use in humans and its use has revolutionized the treatment TNF-mediated inflammatory disorders. The mechanism of action (MOA) of infliximab involves its binding to soluble (s) and membrane (m) TNFα. Here, we describe two simple in vitro bioassays for the assessment of key activities of infliximab. First, a bioassay for TNFα neutralization, which evaluates the Fab binding to sTNFα and the consequent reduction in the activation of TNFα receptors and TNFα-induced expression of adhesion molecules on endothelial cells. A second bioassay evaluates the triggering of Complement-Dependent Cytotoxicity (CDC) in cells expressing mTNFα, which requires the interaction of infliximab-Fc with proteins of the complement system. In both cases, the biological responses are measured by flow cytometry, which is accessible for most laboratories. The methods reported here can be easily adapted to other therapeutic mAbs with similar MOA.

Reporter gene systems for the identification and characterization of cancer stem cells.

Cancer stem cells (CSCs) are tumor cells that share functional characteristics with normal and embryonic stem cells. CSCs have increased tumor-initiating capacity and metastatic potential and lower sensitivity to chemo- and radiotherapy, with important roles in tumor progression and the response to therapy. Thus, a current goal of cancer research is to eliminate CSCs, necessitating an adequate phenotypic and functional characterization of CSCs. Strategies have been developed to identify, enrich, and track CSCs, many of which distinguish CSCs by evaluating the expression of surface markers, the initiation of specific signaling pathways, and the activation of master transcription factors that control stemness in normal cells. We review and discuss the use of reporter gene systems for identifying CSCs. Reporters that are under the control of aldehyde dehydrogenase 1A1, CD133, Notch, Nanog homeobox, Sex-determining region Y-box 2, and POU class 5 homeobox can be used to identify CSCs in many tumor types, track cells in real time, and screen for drugs. Thus, reporter gene systems, in combination with in vitro and in vivo functional assays, can assess changes in the CSCs pool. We present relevant examples of these systems in the evaluation of experimental CSCs-targeting therapeutics, demonstrating their value in CSCs research.

Dopamine Receptors in Cancer: Are They Valid Therapeutic Targets?

The dopamine receptors (DRs) family includes 5 members with differences in signal transduction and ligand affinity. Abnormal DRs expression has been correlated multiple tumors with their clinical outcome. Thus, it has been proposed that DRs-targeting drugs-developed for other diseases as schizophrenia or Parkinson's disease-could be helpful in managing neoplastic diseases. In this review, we discuss the role of DRs and the effects of DRs-targeting in tumor progression and cancer cell biology using multiple high-prevalence neoplasms as examples. The evidence shows that DRs are valid therapeutic targets for certain receptor/disease combinations, but the data are inconclusive or contradictory for others. In either case, further studies are required to define the precise role of DRs in tumor progression and propose better therapeutic strategies for their targeting.

The Entry Blocker Peptide Produced in Chlamydomonas reinhardtii Inhibits Influenza Viral Replication in vitro.

This year, a respiratory virus caused an emergency pandemic alert in health services around the world, showing the need for biotechnological approaches to fight these diseases. The influenza virus is one of the main viral agents that generate pandemic outbreaks. Currently, the majority of co-circulating influenza A virus (IAV) strains are adamantine- and oseltamivir-resistant strains, and the challenge is to find new antivirals for more efficient treatments. The antiviral entry blocker (EB) peptide is a promising candidate for blocking the virus entry into cells. The aim of this research was to express the EB peptide in the microalgae Chlamydomonas reinhardtii and test its antiviral activity against IAV in vitro. The EB peptide nucleotide sequence was introduced into the nuclear genome of microalgae using Agrobacterium tumefaciens transformation. The EB peptide amount produced in transformed microalgae was 4.99 ± 0.067% of the total soluble protein. In hemagglutination inhibition assays using influenza A/H1N1 pdm and influenza A H1N1/Virginia/ATCC/2009 strains, we reported that the EB peptide extract from the microalgae showed 100-fold higher efficiency than the EB synthetic peptide. In addition, both the EB peptide extract and synthetic peptide inhibited viral replication in MDCK cells (IC50 = 20.7 nM and IC50 = 754.4 nM, respectively); however, the EB peptide extract showed a 32-fold higher antiviral effectiveness than the synthetic peptide against influenza A/H1N1 pdm. Extracts from untransformed and transformed microalgae and synthetic peptide did not show cytotoxic effect on MDCK cell monolayers. Thus, C. reinhardtii may be a fast, safe, and effective expression platform for production of peptides with significant antiviral activity and can be used as a prophylactic treatment to reduce viral propagation.

Transferon™, a peptide mixture with immunomodulatory properties is not immunogenic when administered with various adjuvants.

Transferon, a human dialyzable leukocyte extract (hDLE), is a biotherapeutic that comprises a complex mixture of low-molecular-weight peptides (< 10 kDa) and is used to treat diseases with an inflammatory component. Some biotherapeutics, including those composed of peptides, can induce anti-drug antibodies (ADA) that block or diminish their therapeutic effect. Nevertheless, few studies have evaluated peptide-derived drug immunogenicity. In this study, the immunogenicity of Transferon was examined in a murine model during an immunization scheme using the following adjuvants: Al(OH)3, incomplete Freund's adjuvant (IFA), or Titermax Gold. The inoculation scheme entailed three routes of administration (intraperitoneal, Day 1; subcutaneous, Day 7; and intramuscular, Day 14) using 200 µg Transferon/inoculation. Serum samples were collected on Day 21. Total IgG levels were quantitated by affinity chromatography, and specific antibodies against components of Transferon were analyzed by dot-blot and ELISA. Ovalbumin (OVA, 44 kDa) and peptides from hydrolyzed collagen (PFHC, < 17 kDa) were used as positive and negative controls, respectively, in the same inoculation scheme and analyses for Transferon. OVA, PFHC, and Transferon increased total IgG concentrations in mice. However, only IgG antibodies against OVA were detected. Based on the results, it is concluded that Transferon does not induce generation of specific antibodies against its components in this model, regardless of adjuvant and route of administration. These results support the safety of Transferon by confirming its inability to induce ADA in this animal model.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis.

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

Herpes murine model as a biological assay to test dialyzable leukocyte extracts activity.

Human dialyzable leukocyte extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that are released on disruption of peripheral blood leukocytes from healthy donors. DLEs improve clinical responses in infections, allergies, cancer, and immunodeficiencies. Transferon is a human DLE that has been registered as a hemoderivate by Mexican health authorities and commercialized nationally. To develop an animal model that could be used routinely as a quality control assay for Transferon, we standardized and validated a murine model of cutaneous HSV-1 infection. Using this model, we evaluated the activity of 27 Transferon batches. All batches improved the survival of HSV-1-infected mice, wherein average survival rose from 20.9% in control mice to 59.6% in Transferon-treated mice. The activity of Transferon correlated with increased serum levels of IFN-γ and reduced IL-6 and TNF-α concentrations. Our results demonstrate that (i) this mouse model of cutaneous herpes can be used to examine the activity of DLEs, such as Transferon; (ii) the assay can be used as a routine test for batch release; (iii) Transferon is produced with high homogeneity between batches; (iv) Transferon does not have direct virucidal, cytoprotective, or antireplicative effects; and (v) the protective effect of Transferon in vivo correlates with changes in serum cytokines.

Swine Dialyzable Spleen Extract as Antiviral Prophylaxis.

Worldwide, the most highly consumed meat is of porcine origin. The production and distribution of swine meat are affected by diverse health matters, such as influenza and diarrhea, which cause head losses and require the use of antibiotics and other drugs in hog farms. To stimulate newborn piglet immune responses and increase resistance to infections, we developed a spray-drying technique to produce dried swine dialyzable spleen extract (sDSE), an immunomodulator. Based on the size-exclusion ultra performance liquid chromatography quantitative analysis, it was possible to recover up to 58% of the product after the drying process. The biological activity of orally administered dried sDSE increased mouse survival and induced cytokine production in a herpes infection model.

A flow cytometry-based assay for the evaluation of antibody-dependent cell-mediated cytotoxicity (ADCC) in cancer cells.

The development of new therapeutic monoclonal antibodies (mAbs) for cancer therapy will rise in the following years. The evaluation of biological activity of mAbs is required during drug development and during drug production as quality control test. Antibody-dependent cell-mediated cytotoxicity (ADCC) is a desirable activity of anticancer mAbs. Here, we describe a flow cytometry-based method to quantify ADCC that combines the staining of cancer cells, effector cells, and dead cells, with specific dyes. This method is inexpensive, has low background, and avoids the use of radioisotopes.

Batch-to-batch reproducibility of Transferon™.

Human dialyzable leukocyte extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that modulate immune responses in various diseases. Due their complexity, standardized methods to identify their physicochemical properties and determine that production batches are biologically active must be established. We aimed to develop and validate a size exclusion ultra performance chromatographic (SE-UPLC) method to characterize Transferon™, a DLE that is produced under good manufacturing practices (GMPs). We analyzed an internal human DLE standard and 10 representative batches of Transferon™, all of which had a chromatographic profile characterized by 8 main peaks and a molecular weight range between 17.0 and 0.2kDa. There was high homogeneity between batches with regard to retention times and area percentages, varying by less than 0.2% and 30%, respectively, and the control chart was within 3 standard deviations. To analyze the biological activity of the batches, we studied the ability of Transferon™ to stimulate IFN-γ production in vitro. Transferon™ consistently induced IFN-γ production in Jurkat cells, demonstrating that this method can be included as a quality control step in releasing Transferon™ batches. Because all analyzed batches complied with the quality attributes that were evaluated, we conclude that the DLE Transferon™ is produced with high homogeneity.

Antimetastatic, antineoplastic, and toxic effects of 4-hydroxycoumarin in a preclinical mouse melanoma model.

PURPOSE: We have previously reported that in vitro treatment of B16-F10 melanoma cells with 4-hydroxycoumarin (4-HC) decreases their metastatic potential. However, the antimetastatic efficacy of 4-HC in vivo is unknown; therefore, we investigated the antimetastatic and antineoplastic effects of 4-HC in a mouse melanoma model. Based on the findings, the immunomodulatory and toxic effects of 4-HC were also studied. METHODS: Experimental metastasis assay was performed in C57BL/6 mice that received 4-HC before intravenous injection of B16-F10 cells. Antitumor and antimetastatic efficacy of 4-HC was assessed in mice implanted subcutaneously with melanoma cells. Possible immunostimulant and toxic effects of 4-HC were studied in healthy mice. RESULTS: 4-HC reduced the number of experimental lung metastases. Moreover, 4-HC diminished primary tumor growth and increased survival time in mice bearing melanoma tumors. Treatments also decrease spontaneous lung metastases in the same animals. Different to other coumarins, the antitumor effect of 4-HC seems to be unrelated to immunostimulation, since plasma concentrations of cytokines remained unchanged. In contrast, toxic histological changes in nephrons and bronchiolar epithelium and a pronounced anticoagulant effect were found in 4-HC treated animals. CONCLUSIONS: These results show that 4-HC not only exhibit antimetastatic effect in vivo, but also effectively reduces tumor growth and improves survival, even when it produce toxic effects. Although the molecular mechanism of 4-HC actions needs to be further defined, our data suggest that 4-HC may lead to the development of agents that could be used as adjuvants in the therapy of melanoma.

Reduced paxillin expression contributes to the antimetastatic effect of 4-hydroxycoumarin on B16-F10 melanoma cells.

BACKGROUND: 4-Hydroxycoumarin (4-HC) is a coumarin that lacks anticoagulant activity. 4-HC affects the cytoskeletal stability and decreases cell adhesion and motility of the melanoma cell line B16-F10. Together with integrins and other cytoskeletal proteins, paxillin participates in the regulation of cell adhesion and motility, acting as an adapter protein at focal adhesions. The present study determined the participation of paxillin in the reported effects of 4-HC and analyzed the role of paxillin in the formation of melanoma metastases. RESULTS: 4-HC decreased protein and mRNA levels of alpha- and beta-paxillin isoforms in B16-F10 cells. Paxillin downregulation correlated with an inadequate translocation of paxillin to focal adhesions and a reduced phosphotyr118-paxillin pool. Consequently, 4-HC altered paxillin-mediated signaling, decreasing the phosphorylation of FAK and the level of GTP-bound Rac-1. These results partially explain the mechanism of the previously reported effects of 4-HC. Additionally, we studied the effect of 4-HC on metastatic potential of B16-F10 cells through experimental metastasis assays. In vitro treatment of cells with 4-HC inhibited their capability to originate pulmonary metastases. 4-HC did not affect cell proliferation or survival, demonstrating that its antimetastatic effect is unrelated to changes on cell viability. We also studied the importance of paxillin in metastasis by transfecting melanoma cells with paxillin-siRNA. Transfection produced a modest reduction on metastatic potential, indicating that: i) paxillin plays a role as inducer of melanoma metastasis; and ii) paxillin downregulation is not sufficient to explain the antimetastatic effect of 4-HC. Therefore, we evaluated other changes in gene expression by differential display RT-PCR analysis. Treatment with 4-HC produced a downregulation of Adhesion Regulating Molecule-1 (ARM-1), which correlated with a decreased adhesion of melanoma cells to lung slides. CONCLUSION: This study shows that reduced paxillin expression is associated with the impaired cell adhesion and motility seen in 4-HC-treated cells and partially contributes to the antimetastatic effect of 4-HC. In contrast, the role of ARM-1 reduced expression in the effects of 4-HC is still to be clarified. The antimetastatic effect of 4-HC suggests that this compound, or others with similar mode of action, might be useful for the development of adjuvant therapies for melanoma.