Phloridzin Docosahexaenoate Inhibits Spheroid Formation by Breast Cancer Stem Cells and Exhibits Cytotoxic Effects against Paclitaxel-Resistant Triple Negative Breast Cancer Cells.

The eradication of cancer stem cells (CSCs) is vital to successful cancer treatment and overall disease-free survival. CSCs are a sub-population of cells within a tumor that are defined by their capacity for continuous self-renewal and recapitulation of new tumors, demonstrated in vitro through spheroid formation. Flavonoids are a group of phytochemicals with potent anti-oxidant and anti-cancer properties. This paper explores the impact of the flavonoid precursor phloridzin (PZ) linked to the ω-3 fatty acid docosahexaenoate (DHA) on the growth of MCF-7 and paclitaxel-resistant MDA-MB-231-TXL breast cancer cell lines. Spheroid formation assays, acid phosphatase assays, and Western blotting were performed using MCF-7 cells, and the cell viability assays, Annexin-V-488/propidium iodide (PI) staining, and 7-aminoactinomycin D (7-AAD) assays were performed using MDA-MB-231-TXL cells. PZ-DHA significantly reduced spheroid formation, as well as the metabolic activity of MCF-7 breast cancer cells in vitro. Treatment with PZ-DHA also suppressed the metabolic activity of MDA-MB-231-TXL cells and led to apoptosis. PZ-DHA did not have an observable effect on the expression of the drug efflux transporters ATP-binding cassette super-family G member 2 (ABCG2) and multidrug resistance-associated protein 1 (MRP1). PZ-DHA is a potential treatment avenue for chemo-resistant breast cancer and a possible novel CSC therapy. Future pre-clinical studies should explore PZ-DHA as a chemo-preventative agent.

Investigating Potential Applications of the Fish Anti-Microbial Peptide Pleurocidin: A Systematic Review.

The anti-microbial peptide (AMP) pleurocidin is found in winter flounder (Pseudopleuronectes americanus), an Atlantic flounder species. There is promising evidence for clinical, aquaculture, and veterinary applications of pleurocidin. This review provides an overview of the current literature available on pleurocidin to guide future research directions. By fully elucidating pleurocidin's mechanism of action and developing novel treatments against pathogenic microbes, populations of flatfish and humans can be protected. This review consulted publications from PubMed and Environment Complete with search terms such as "pleurocidin", "winter flounder", and "antimicrobial". The fish immune system includes AMPs as a component of the innate immune system. Pleurocidin, one of these AMPs, has been found to be effective against various Gram-positive and Gram-negative bacteria. More investigations are required to determine pleurocidin's suitability as a treatment against antibiotic-resistant pathogens. There is promising evidence for pleurocidin as a novel anti-cancer therapy. The peptide has been found to display potent anti-cancer effects against human cancer cells. Research efforts focused on pleurocidin may result in novel treatment strategies against antibiotic-resistant bacteria and cancer. More research is required to determine if the peptide is a suitable candidate to be developed into a novel anti-microbial treatment. Some of the microbes susceptible to the peptide are also pathogens of fish, suggesting its suitability as a therapeutic treatment for fish species.

Review: Examining the Natural Role of Amphibian Antimicrobial Peptide Magainin.

Host defense peptides (HDPs) are a group of antimicrobial peptides (AMPs) that are crucial components of the innate immune system of many different organisms. These small peptides actively kill microbes and prevent infection. Despite the presence of AMPs in the amphibian immune system, populations of these organisms are in decline globally. Magainin is an AMP derived from the African clawed frog (Xenopus laevis) and has displayed potent antimicrobial effects against a wide variety of microbes. Included in this group of microbes are known pathogens of the African clawed frog and other amphibian species. Arguably, the most deleterious amphibious pathogen is Batrachochytrium dendrobatidis, a chytrid fungus. Investigating the mechanism of action of magainin can help understand how to effectively fight off infection. By understanding amphibian AMPs' role in the frog, a potential conservation strategy can be developed for other species of amphibians that are susceptible to infections, such as the North American green frog (Rana clamitans). Considering that population declines of these organisms are occurring globally, this effort is crucial to protect not only these organisms but the ecosystems they inhabit as well.

MDA-MB-231 Breast Cancer Cells Resistant to Pleurocidin-Family Lytic Peptides Are Chemosensitive and Exhibit Reduced Tumor-Forming Capacity.

Direct-acting anticancer (DAA) peptides are cytolytic peptides that show promise as novel anticancer agents. DAA peptides bind to anionic molecules that are abundant on cancer cells relative to normal healthy cells, which results in preferential killing of cancer cells. Due to the mechanism by which DAA peptides kill cancer cells, it was thought that resistance would be difficult to achieve. Here, we describe the generation and characterization of two MDA-MB-231 breast cancer cell-line variants with reduced susceptibility to pleurocidin-family and mastoparan DAA peptides. Peptide resistance correlated with deficiencies in peptide binding to cell-surface structures, suggesting that resistance was due to altered composition of the cell membrane. Peptide-resistant MDA-MB-231 cells were phenotypically distinct yet remained susceptible to chemotherapy. Surprisingly, neither of the peptide-resistant breast cancer cell lines was able to establish tumors in immune-deficient mice. Histological analysis and RNA sequencing suggested that tumorigenicity was impacted by alternations in angiogenesis and extracellular matrix composition in the peptide-resistant MDA-MB-231 variants. Collectively, these data further support the therapeutic potential of DAA peptides as adjunctive treatments for cancer.

Thy-1 (CD90) Signaling Preferentially Promotes RORγt Expression and a Th17 Response.

Thy-1 (CD90) is a glycosylphosphatidylinositol-anchored protein (GPI-AP) with signaling properties that is abundant on mouse T cells. Upon antibody-mediated crosslinking, Thy-1 provides a T cell receptor (TcR)-like signal that is sufficient to drive CD4+ T cell proliferation and differentiation into effector cells when costimulatory signals are provided by syngeneic lipopolysaccharide-matured bone marrow-derived dendritic cells. In this study, we investigated the impact of Thy-1 signaling on the production of the T helper (Th) cell subset-associated cytokines, interferon (IFN) γ, interleukin (IL)-4 and IL-17A, as well as the in vitro polarization of highly purified resting CD4+ T cells into Th1, Th2, and Th17 cells. Although CD8+ T cells expressed more Thy-1 than CD4+ T cells, both T cell populations were equally responsive to Thy-1 stimulation. In contrast to TcR stimulation of CD3+ T cells, which favored IFNγ and IL-4 production, Thy-1 signaling favored IL-17 synthesis, indicating a previously unidentified difference between the consequences of Thy-1 and TcR signal transduction. Moreover, Thy-1 signaling preferentially induced the Th17-associated transcription factor RORγt in CD4+ T cells. As with TcR signaling, Thy-1 stimulation of CD4+ T cells under the appropriate polarizing conditions resulted in Th1, Th2 or Th17 cell induction; however, Thy-1 stimulation induced nearly 7- and 2-fold more IL-4 and IL-17A, respectively, but only slightly more IFNγ. The ability to provide a TcR-like signal capable of promoting T helper cell differentiation and cytokine synthesis was not common to all GPI-APs since cross-linking of Ly6A/E with mitogenic mAb did not promote substantial production of IFNγ, IL-4 or IL-17, although there was a substantial proliferative response. The preferential induction of RORγt and Th17 cytokine synthesis as a consequence of Thy-1 signaling suggests a default T helper cell response that may enhance host defense against extracellular pathogens.

Docosahexaenoic acid-acylated phloridzin, a novel polyphenol fatty acid ester derivative, is cytotoxic to breast cancer cells.

Docosahexaenoic acid-acylated phloridzin (PZ-DHA), a novel polyphenol fatty acid ester derivative, was synthesized through a regioselective acylation reaction with the aim of increasing the bioactivity of phloridzin (PZ) and docosahexaenoic acid (DHA). In this study, PZ-DHA's cytotoxic activity was explored using in vitro and in vivo models of mammary carcinoma. PZ-DHA was selectively cytotoxic for mammary carcinoma (MDA-MB-231, MDA-MB-468, 4T1, MCF-7 and T-47D) cells compared to non-malignant human mammary epithelial cells (HMEC and MCF-10A) and fibroblasts by MTS assay and Annexin-V-FLUOS/propidium iodide staining. Flow cytometric analysis of Oregon Green 488- and Ki-67-stained MDA-MB-231 cells showed antiproliferative activity of PZ-DHA at a subcytotoxic concentration. PZ-DHA also arrested MDA-MB-231 cell division at the G2/M phase and down-regulated expression of cyclin B1 and cyclin-dependent kinase 1 (CDK1). PZ-DHA-induced apoptosis in MDA-MB-231 cells was confirmed by caspase 3/7 activation in a luminescence assay and DNA fragmentation by TUNEL staining. Moreover, MDA-MB-231 xenograft growth in non-obese diabetic severe combined immunodeficient mice was suppressed by intra-tumoral administration of PZ-DHA. This study shows that PZ-DHA is selectively cytotoxic to breast cancer cells in vitro and in vivo, suggesting that further investigations of PZ-DHA are warranted as a potential treatment for breast cancer.

Apigenin inhibits the inducible expression of programmed death ligand 1 by human and mouse mammary carcinoma cells.

Programmed death ligand 1 (PD-L1) is expressed by many cancer cell types, as well as by activated T cells and antigen-presenting cells. Constitutive and inducible PD-L1 expression contributes to immune evasion by breast cancer (BC) cells. We show here that the dietary phytochemical apigenin inhibited interferon (IFN)-γ-induced PD-L1 upregulation by triple-negative MDA-MB-468 BC cells, HER2(+) SK-BR-3 BC cells, and 4T1 mouse mammary carcinoma cells, as well as human mammary epithelial cells, but did not affect constitutive PD-L1 expression by triple-negative MDA-MB-231 BC cells. IFN-ß-induced expression of PD-L1 by MDA-MB-468 cells was also inhibited by apigenin. In addition, luteolin, the major metabolite of apigenin, inhibited IFN-γ-induced PD-L1 expression by MDA-MB-468 cells. Apigenin-mediated inhibition of IFN-γ-induced PD-L1 expression by MDA-MB-468 and 4T1 cells was associated with reduced phosphorylation of STAT1, which was early and transient at Tyr701 and sustained at Ser727. Apigenin-mediated inhibition of IFN-γ-induced PD-L1 expression by MDA-MB-468 cells also increased proliferation and interleukin-2 synthesis by PD-1-expressing Jurkat T cells that were co-cultured with MDA-MB-468 cells. Apigenin therefore has the potential to increase the vulnerability of BC cells to T cell-mediated anti-tumor immune responses.

4T1 Murine Mammary Carcinoma Cells Enhance Macrophage-Mediated Innate Inflammatory Responses.

Tumor progression and the immune response are intricately linked. While it is known that cancers alter macrophage inflammatory responses to promote tumor progression, little is known regarding how cancers affect macrophage-dependent innate host defense. In this study, murine bone-marrow-derived macrophages (BMDM) were exposed to murine carcinoma-conditioned media prior to assessment of the macrophage inflammatory response. BMDMs exposed to 4T1 mammary carcinoma-conditioned medium demonstrated enhanced production of pro-inflammatory cytokines tumor necrosis factor α, interleukin-6, and CCL2 in response to lipopolysaccharide (LPS) while production of interleukin-10 remained unchanged. The increased LPS-induced production of pro-inflammatory cytokines was transient and correlated with enhanced cytokine production in response to other Toll-like receptor agonists, including peptidoglycan and flagellin. In addition, 4T1-conditioned BMDMs exhibited strengthened LPS-induced nitric oxide production and enhanced phagocytosis of Escherichia coli. 4T1-mediated augmentation of macrophage responses to LPS was partially dependent on the NFκB pathway, macrophage-colony stimulating factor, and actin polymerization, as well as the presence of 4T1-secreted extracellular vesicles. Furthermore, peritoneal macrophages obtained from 4T1 tumor-bearing mice displayed enhanced pro-inflammatory cytokine production in response to LPS. These results suggest that uptake of 4T1-secreted factors and actin-mediated ingestion of 4T1-secreted exosomes by macrophages cause a transient enhancement of innate inflammatory responses. Mammary carcinoma-mediated regulation of innate immunity may have significant implications for our understanding of host defense and cancer progression.

Pleurocidin-family cationic antimicrobial peptides mediate lysis of multiple myeloma cells and impair the growth of multiple myeloma xenografts.

Abstract Multiple myeloma is a common hematological malignancy that urgently requires new approaches to treatment, since the disease is not curable using current chemotherapeutic regimens. The aim of this study was to determine whether human and mouse multiple myeloma cells are killed by the pleurocidin-like cationic antimicrobial peptides NRC-03 and NRC-07, previously shown to be active against breast cancer cells. We demonstrate here that NRC-03 and NRC-07 bound to and rapidly killed multiple myeloma cells by causing extensive membrane damage, as well as DNA cleavage. NRC-03 showed greater binding to multiple myeloma cells and a more potent cytotoxic effect than NRC-07. In addition, intratumoral injections of NRC-03 impaired the growth of multiple myeloma xenografts in immune-deficient mice. We conclude that NRC-03 warrants further investigation for its possible use in the treatment of multiple myeloma.

Induction of CD4(+)CD25(+)Foxp3(-) regulatory T cells by Thy-1 stimulation of CD4(+) T cells.

Thy-1 (CD90) on mouse T cells has been reported to have both T-cell activating and regulatory roles. In this study, we show that monoclonal antibody (mAb)-mediated crosslinking of Thy-1 on CD4(+) mouse T-cells-induced regulatory T (T(reg)) cells that expressed CD25, CD39 and glucocorticoid-induced tumor necrosis factor receptor family-related gene, but not CD73, CD122 or Foxp3. The proliferation of CD4(+) T(responder) cells in response to anti-CD3/anti-CD28mAb-coated T-cell expander beads or syngeneic dendritic cells and soluble anti-CD3mAb was inhibited by Thy-1-induced T(reg) cells, in spite of elevated IL-2 levels in the co-cultures. Interestingly, stimulation with T-cell expander beads caused Thy-1-induced T(reg) cells to synthesize large amounts of interleukin-2 (IL-2). IL-10 was also elevated in co-cultures of activated T(responder) cells and Thy-1-induced T(reg) cells. However, mAb-mediated neutralization of IL-10 did not restore T(responder)-cell proliferation to control levels, which excluded IL-10 as a potential mediator of Thy-1-induced T(reg)-cell suppressor function. In addition, Thy-1-induced T(reg) cells did not inhibit IL-2-dependent proliferation of CTLL-2 cells, suggesting that IL-2 receptor signaling remained intact in the presence of Thy-1-induced T(reg) cells. We suggest that T(reg) cells induced by Thy-1 ligation in vivo may contribute to the maintenance of T-cell homeostasis.