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.

The Natural Alkaloid Piperlongumine Inhibits Metastatic Activity and Epithelial-to-Mesenchymal Transition of Triple-Negative Mammary Carcinoma Cells.

In this study, we determined the effect of low dose piperlongumine on the motility/invasive capacity and epithelial-to-mesenchymal transition (EMT) of MDA-MB-231 triple-negative breast cancer (TNBC) cells and the metastasis of 4T1 mouse mammary carcinoma cells. MTT assays measured the effect of piperlongumine on TNBC cell growth. Motility/invasiveness were determined by gap closure/transwell assays. Western blotting assessed ZEB1, Slug, and matrix metalloproteinase (MMP) 9 expression. Interleukin (IL) 6 was detected by ELISA. MMP2, E-cadherin, and miR-200c expression was determined by real-time quantitative polymerase chain reaction. Reactive oxygen species (ROS) were measured by flow cytometry. The orthotopic 4T1 mouse model of breast cancer was used to examine metastasis. Piperlongumine-treated MDA-MB-231 cells showed reduced motility/invasiveness, decreased MMP2 and MMP9 expression, increased miR-200c expression, reduced IL-6 synthesis, decreased expression of ZEB1 and Slug, increased E-cadherin expression, and epithelial-like morphology. Piperlongumine also inhibited transforming growth factor ß-induced ZEB1 and Slug expression. ROS accumulated in piperlongumine-treated cells, while changes in metastasis-associated gene expression were ablated by exogenous glutathione. Metastasis of 4T1 cells to the lungs of BALB/c mice was dramatically reduced in piperlongumine-treated animals. These findings reveal a previously unknown capacity of low dose piperlongumine to interfere with TNBC metastasis via an oxidative stress-dependent mechanism.

Chemopreventive Effect of Dietary Anthocyanins against Gastrointestinal Cancers: A Review of Recent Advances and Perspectives.

Anthocyanins are a group of dietary polyphenols, abundant mainly in fruits and their products. Dietary interventions of anthocyanins are being studied extensively related to the prevention of gastrointestinal (GI) cancer, among many other chronic disorders. This review summarizes the hereditary and non-hereditary characteristics of GI cancers, chemistry, and bioavailability of anthocyanins, and the most recent findings of anthocyanin in GI cancer prevention through modulating cellular signaling pathways. GI cancer-preventive attributes of anthocyanins are primarily due to their antioxidative, anti-inflammatory, and anti-proliferative properties, and their ability to regulate gene expression and metabolic pathways, as well as induce the apoptosis of cancer cells.

Cyanidin-3-O-Glucoside-Rich Haskap Berry Administration Suppresses Carcinogen-Induced Lung Tumorigenesis in A/JCr Mice.

In our previous study, we demonstrated that cyanidin-3-O-glucoside (C3G)-rich haskap (Lonicera caerulea L.) berry extracts can attenuate the carcinogen-induced DNA damage in normal lung epithelial cells in vitro. Here, the efficacy of lyophilized powder of whole haskap berry (C3G-HB) in lowering tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK)-induced lung tumorigenesis in A/JCr mice was investigated. Three weeks after daily oral administration of C3G-HB (6 mg of C3G in 0.2 g of C3G-HB/mouse/day), lung tumors were initiated by a single intraperitoneal injection of NNK. Dietary C3G-HB supplementation was continued, and 22 weeks later, mice were euthanized. Lung tumors were visualized through positron emission tomography (PET) and magnetic resonance imaging (MRI) 19 weeks after NNK injection. Dietary supplementation of C3G-HB significantly reduced the NNK-induced lung tumor multiplicity and tumor area but did not affect tumor incidence. Immunohistochemical analysis showed reduced expression of proliferative cell nuclear antigen (PCNA) and Ki-67 in lung tissues. Therefore, C3G-HB has the potential to reduce the lung tumorigenesis, and to be used as a source for developing dietary supplements or nutraceuticals for reducing the risk of lung cancer among high-risk populations.

The anti-malarial drug artesunate causes cell cycle arrest and apoptosis of triple-negative MDA-MB-468 and HER2-enriched SK-BR-3 breast cancer cells.

Breast cancer is the most prevalent cancer diagnosis in women, with triple-negative and human epidermal growth factor 2 (HER2)-enriched advanced breast cancers having the poorest prognoses. The morbidity and mortality associated with advanced disease, as well as the emergence of multi-drug resistant variants, highlights the urgency to develop novel therapeutic agents. Artesunate (ART) is a semi-synthetic derivative of artemisinin from the Chinese herb sweet wormwood. ART is widely used in the treatment of malaria and is well tolerated by patients. Importantly, ART also has anti-cancer activities and may therefore represent a less toxic alternative to conventional chemotherapy. In this study, we demonstrate a dose- and time-dependent inhibitory effect of ART on the growth of triple-negative MDA-MB-468 and HER2-enriched SK-BR-3 breast cancer cells, which was the result of both anti-proliferative and cytotoxic activities. ART inhibited breast cancer cell proliferation via a reactive oxygen species (ROS)-dependent G2/M arrest and ROS-independent G1 arrest. ART-treated MDA-MB-468 and SK-BR-3 cells also experienced apoptotic cell death, which was both ROS- and iron-dependent. ART-induced oxidative stress caused the loss of mitochondrial outer membrane integrity and damage to the cellular DNA of MDA-MB-468 and SK-BR-3 cells. In addition, exposure to low-dose ART sensitized MDA-MB-468 and SK-BR-3 cells to chemotherapeutic drugs. On the basis of our findings, we suggest that ART may have clinical utility in the treatment of triple-negative and HER2-enriched breast cancers.

DIBI, a novel 3-hydroxypyridin-4-one chelator iron-binding polymer, inhibits breast cancer cell growth and functions as a chemosensitizer by promoting S-phase DNA damage.

Breast cancer is a leading cause of cancer-related death in women; however, chemotherapy of breast cancer is often hindered by dose-limiting toxicities, demonstrating the need for less toxic approaches to treatment. Since the rapid growth and metabolism of breast cancer cells results in an increased requirement for iron, withdrawal of bioavailable iron using highly selective iron chelators has been suggested to represent a new approach to breast cancer treatment. Here we show that the recently developed iron-binding polymer DIBI inhibited the growth of five different breast cancer cell lines (SK-BR3, MDA-MB-468, MDA-MB-231, MCF-7, and T47D). In cultures of MDA-MB-468 breast cancer cells, which were most sensitive to DIBI-mediated growth inhibition, iron withdrawal was associated with increased expression of transferrin receptor 1 and ferritin H mRNA but decreased expression of ferroportin mRNA. MDA-MB-468 cells that were exposed to DIBI experienced double-strand DNA breaks during the S phase of the cell cycle. DNA damage was not mediated by reactive oxygen species (ROS) since DIBI-treated MDA-MB-468 cells exhibited a reduction in intracellular ROS. DIBI-treated MDA-MB-468 cells also showed increased sensitivity to growth inhibition by the chemotherapeutic drugs cisplatin, doxorubicin, and 4-hydroperoxy cyclophosphamide (active metabolite of cyclophosphamide). Combination treatment of MDA-MB-468 cells with DIBI and cisplatin caused greater DNA damage than either treatment alone, which was also associated with an increase in apoptotic cell death. Taken together, these findings suggest that DIBI-mediated iron withdrawal may enhance the effect of chemotherapeutic agents used in breast cancer treatment.

Apple Peel Flavonoid Fraction 4 Suppresses Breast Cancer Cell Growth by Cytostatic and Cytotoxic Mechanisms.

Many dietary flavonoids possess anti-cancer activities. Here, the effect of apple peel flavonoid fraction 4 (AF4) on the growth of triple-negative (MDA-MB-231, MDA-MB-468), estrogen receptor-positive (MCF-7), and HER2-positive (SKBR3) breast cancer cells was determined and compared with the effect of AF4 on normal mammary epithelial cells and dermal fibroblasts. AF4 inhibited breast cancer cell growth in monolayer cultures, as well as the growth of MCF-7 spheroids, without substantially affecting the viability of non-malignant cells. A sub-cytotoxic concentration of AF4 suppressed the proliferation of MDA-MB-231 cells by inhibiting passage through the G0/G1 phase of the cell cycle. AF4-treated MDA-MB-231 cells also exhibited reduced in vitro migration and invasion, and decreased Akt (protein kinase B) signaling. Higher concentrations of AF4 were selectively cytotoxic for MDA-MB-231 cells. AF4 cytotoxicity was associated with the intracellular accumulation of reactive oxygen species. Importantly, intratumoral administration of AF4 suppressed the growth of MDA-MB-231 xenografts in non-obese diabetic severe combined immunodeficient (NOD-SCID) female mice. The selective cytotoxicity of AF4 for breast cancer cells, combined with the capacity of sub-cytotoxic AF4 to inhibit breast cancer cell proliferation, migration, and invasion suggests that flavonoid-rich AF4 (and its constituents) has potential as a natural therapeutic agent for breast cancer treatment.

Myricetin-induced oxidative stress suppresses murine T lymphocyte activation.

A number of polyphenolic compounds present in fruits and vegetables have the capacity to modulate immune responses; however, the impact of the common plant-derived flavonoid myricetin on T lymphocyte function has not been investigated. We show that myricetin inhibited mouse T lymphocyte activation by bead-immobilized anti-CD3 and anti-CD28 monoclonal antibodies, as indicated by a dose-dependent reduction in cell proliferation and decreased synthesis of interferon-γ, interleukin (IL)-2, IL-4, and IL-17 associated with different T helper cell subsets. This effect was attributed to myricetin-induced reactive oxygen species (ROS) since myricetin caused hydrogen peroxide (H2 O2 ) to accumulate in cell-free culture medium and H2 O2 inhibited T cell proliferation and cytokine synthesis. In addition, the antioxidant N-acetyl cysteine restored the ability of myricetin-treated T lymphocytes to proliferate in response to a mitogenic stimulus. The presence of dendritic cells or bone marrow-derived macrophages negated the inhibitory effect of myricetin on T cell activation, and H2 O2 in T cell cultures that were treated with exogenous H2 O2 was reduced when antigen-presenting cells were also present. These findings suggest that antioxidant molecules produced by dendritic cells and macrophages protected T cells from myricetin-induced oxidative stress, and underscore the importance of considering immune cell interactions when evaluating the immunomodulatory activity of ROS-generating phytochemicals.

Mastoparan is a membranolytic anti-cancer peptide that works synergistically with gemcitabine in a mouse model of mammary carcinoma.

Anti-cancer peptides (ACPs) are small cationic and hydrophobic peptides that are more toxic to cancer cells than normal cells. ACPs kill cancer cells by causing irreparable membrane damage and cell lysis, or by inducing apoptosis. Direct-acting ACPs do not bind to a unique receptor, but are rather attracted to several different molecules on the surface of cancer cells. Here we report that an amidated wasp venom peptide, Mastoparan, exhibited potent anti-cancer activities toward leukemia (IC50~8-9.2µM), myeloma (IC50~11µM), and breast cancer cells (IC50~20-24µM), including multidrug resistant and slow growing cancer cells. Importantly, the potency and mechanism of cancer cell killing was related to the amidation of the C-terminal carboxyl group. Mastoparan was less toxic to normal cells than it was to cancer cells (e.g., IC50 to PBMC=48µM). Mastoparan killed cancer cells by a lytic mechanism. Moreover, Mastoparan enhanced etoposide-induced cell death in vitro. Our data also suggest that Mastoparan and gemcitabine work synergistically in a mouse model of mammary carcinoma. Collectively, these data demonstrate that Mastoparan is a broad-spectrum, direct-acting ACP that warrants additional study as a new therapeutic agent for the treatment of various cancers.

The "PepSAVI-MS" Pipeline for Natural Product Bioactive Peptide Discovery.

The recent increase in extensively drug-resistant bacterial pathogens and the associated increase of morbidity and mortality demonstrate the immediate need for new antibiotic backbones with novel mechanisms of action. Here, we report the development of the PepSAVI-MS pipeline for bioactive peptide discovery. This highly versatile platform employs mass spectrometry and statistics to identify bioactive peptide targets from complex biological samples. We validate the use of this platform through the successful identification of known bioactive peptides from a botanical species, Viola odorata. Using this pipeline, we have widened the known antimicrobial spectrum for V. odorata cyclotides, including antibacterial activity of cycloviolacin O2 against A. baumannii. We further demonstrate the broad applicability of the platform through the identification of novel anticancer activities for cycloviolacins by their cytotoxicity against ovarian, breast, and prostate cancer cell lines.

Contribution of reactive oxygen species to ovarian cancer cell growth arrest and killing by the anti-malarial drug artesunate.

Ovarian cancer is a leading cause of cancer-related death in women and the most lethal gynecological malignancy in the developed world. The morbidity and mortality of ovarian cancer underscore the need for novel treatment options. Artesunate (ART) is a well-tolerated anti-malarial drug that also has anti-cancer activity. In this study, we show that ART inhibited the in vitro growth of a panel of ovarian cancer cell lines, as well as the growth of ovarian cancer cells isolated from patients. Moreover, ART decreased tumor growth in vivo in a mouse model of ovarian cancer. ART-treated ovarian cancer cells showed a strong induction of reactive oxygen species (ROS) and reduced proliferation. ROS-dependent cell cycle arrest occurred in the G2/M phase whereas ROS-independent cell cycle arrest occurred in the G1 phase, depending on the concentration of ART to which ovarian cancer cells were exposed. The anti-proliferative effect of ART was associated with altered expression of several key cell cycle regulatory proteins, including cyclin D3, E2F-1, and p21, as well as inhibition of mechanistic target of rapamycin signaling. Exposure of ovarian cancer cells to higher concentrations of ART resulted in ROS-dependent DNA damage and cell death. Pretreatment of ovarian cancer cells with a pan-caspase inhibitor or ferroptosis inhibitor decreased but did not completely eliminate ART-mediated cytotoxicity, suggesting the involvement of both caspase-dependent and caspase-independent pathways of killing. These data show that ART has potent anti-proliferative and cytotoxic effects on ovarian cancer cells, and may therefore be useful in the treatment of ovarian cancer. © 2016 Wiley Periodicals, Inc.

[10]-Gingerol, a major phenolic constituent of ginger root, induces cell cycle arrest and apoptosis in triple-negative breast cancer cells.

The ginger rhizome is rich in bioactive compounds, including [6]-gingerol, [8]-gingerol, and [10]-gingerol; however, to date, most research on the anti-cancer activities of gingerols have focused on [6]-gingerol. In this study, we compared [10]-gingerol with [8]-gingerol and [6]-gingerol in terms of their ability to inhibit the growth of human and mouse mammary carcinoma cells. A colorimetric assay based on the enzymatic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide revealed that [10]-gingerol was more potent than [6]-gingerol and at least as potent as [8]-gingerol for the inhibition of triple-negative human (MDA-MB-231, MDA-MB-468) and mouse (4T1, E0771) mammary carcinoma cell growth. Further investigation of [10]-gingerol showed that it suppressed the growth of estrogen receptor-bearing (MCF-7, T47D) and HER2-overexpressing (SKBR3) breast cancer cells. The inhibitory effect of [10]-gingerol on the growth of MDA-MB-231 cells was associated with a reduction in the number of rounds of cell division and evidence of S phase-cell cycle arrest, as well as induction of apoptosis due to mitochondrial outer membrane permeabilization and the release of proapoptotic mitochondrial cytochrome c and SMAC/DIABLO into the cytoplasm. Surprisingly, killing of MDA-MB-231 cells by [10]-gingerol was not affected by a pan-caspase inhibitor (zVAD-fmk) or an anti-oxidant (N-acetylcysteine), suggesting that the cytotoxic effect of [10]-gingerol did not require caspase activation or the accumulation of reactive oxygen species. These findings suggest that further investigation of [10]-gingerol is warranted for its possible use in the treatment of breast cancer.

Piperine, a Pungent Alkaloid from Black Pepper, Inhibits B Lymphocyte Activation and Effector Functions.

Piperine has several well-documented anti-inflammatory properties; however, little is known regarding its effect on humoral immunity. In this study, we describe the immunosuppressive effect of piperine on B lymphocytes, which are integral to the humoral immune response. Mouse B cells were cultured in the absence or presence of non-cytotoxic concentrations (25, 50, and 100 µM) of piperine during T-dependent or T-independent stimulation. Piperine inhibited B cell proliferation by causing G0/G1 phase cell cycle arrest in association with reduced expression of cyclin D2 and D3. The inhibitory effect of piperine was not mediated through transient receptor potential vanilloid-1 ion channel (TRPV1) because piperine also inhibited the proliferation of B cells from TRPV1-deficient mice. Expression of class II major histocompatibility complex molecules and costimulatory CD40 and CD86 on B lymphocytes was reduced in the presence of piperine, as was B cell-mediated antigen presentation to syngeneic T cells. In addition, piperine inhibited B cell synthesis of interleukin (IL)-6 and IL-10 cytokines, as well as IgM, IgG2b, and IgG3 immunoglobulins. The inhibitory effect of piperine on B lymphocyte activation and effector function warrants further investigation for possible application in the treatment of pathologies related to inappropriate humoral immune responses. Copyright © 2017 John Wiley & Sons, Ltd.

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.

Structure-function relationships in histidine-rich antimicrobial peptides from Atlantic cod.

Gad-1 and Gad-2 are antimicrobial peptide (AMP) sequences encoded by paralogous genes. They are rich in histidine, which suggests that their activity might be pH-dependent. We examined their structure-function relationships with a view to learning how to improve AMP therapeutic ratios. Activity assays with Gram-negative bacteria and cancer cell lines demonstrate that Gad-2 is substantially more active at slightly acidic pH than it is at neutral pH. By contrast, the activity of Gad-1 at lower pH is similar to its activity at pH7. Circular dichroism spectra indicate that the greater functional plasticity of Gad-2 correlates with a greater structural plasticity; Gad-2's percent helicity varies dramatically with altered pH and lipid environment. Interestingly, Gad-2's highest levels of helicity do not correspond to the conditions where it is most active. High resolution solution NMR structures were determined in SDS micelles at pH5, conditions that induce an intermediate level of helicity in the peptides. Gad-1 is more helical than Gad-2, with both peptides exhibiting the greatest helical tendencies in their central region and lowest helicity in their N-termini. The high resolution structures suggest that maximum activity relies on the appropriate balance between an N-terminal region with mixed hydrophobic/hydrophilic structure features and an amphipathic central and C-terminal region. Taken together with previous studies, our results suggest that to improve the therapeutic ratio of AMPs, consideration should be given to including sequential histidine-pairs, keeping the overall charge of the peptide modest, and retaining a degree of structural plasticity and imperfect amphipathicity.

The Dietary Flavonoid Fisetin Causes Cell Cycle Arrest, Caspase-Dependent Apoptosis, and Enhanced Cytotoxicity of Chemotherapeutic Drugs in Triple-Negative Breast Cancer Cells.

Fisetin (3,3',4',7-tetrahydroxyflavone), a flavonoid found in a number of fruits and vegetables, has diverse biological activities, including cytotoxic effects on cancer cells. In this study, we investigated the effect of fisetin on triple-negative breast cancer (TNBC) cells. TNBC has a poorer prognosis than other types of breast cancer and treatment options for this disease are limited. Fisetin inhibited the growth of MDA-MB-468 and MDA-MB-231 TNBC cells, as well as their ability to form colonies, without substantially affecting the growth of non-malignant cells. In addition, fisetin inhibited the growth of estrogen receptor-bearing MCF-7 breast cancer cells and human epidermal growth factor receptor 2-overexpressing SK-BR-3 breast cancer cells. Fisetin inhibited TNBC cell division and induced apoptosis, which was associated with mitochondrial membrane permeabilization and the activation of caspase-9 and caspase-8, as well as the cleavage of poly(ADP-ribose) polymerase-1. Induction of caspase-dependent apoptosis by fisetin was confirmed by reduced killing of TNBC cells in the presence of the pan-caspase inhibitors Z-VAD-FMK and BOC-D-FMK. Decreased phosphorylation of histone H3 at serine 10 in fisetin-treated TNBC cells at G2/M phase of the cell cycle suggested that fisetin-induced apoptosis was the result of Aurora B kinase inhibition. Interestingly, the cytotoxic effect of cisplatin, 5-fluorouracil, and 4-hydroxycyclophosphamide metabolite of cyclophosphamide on TNBC cells was increased in the presence of fisetin. These findings suggest that further investigation of fisetin is warranted for possible use in the management of TNBC. J. Cell. Biochem. 117: 1913-1925, 2016. © 2016 Wiley Periodicals, Inc.

Piperine from black pepper inhibits activation-induced proliferation and effector function of T lymphocytes.

Piperine is a major alkaloid component of black pepper (Piper nigrum Linn), which is a widely consumed spice. Here, we investigated the effect of piperine on mouse T lymphocyte activation. Piperine inhibited polyclonal and antigen-specific T lymphocyte proliferation without affecting cell viability. Piperine also suppressed T lymphocyte entry into the S and G2 /M phases of the cell cycle, and decreased expression of G1 -associated cyclin D3, CDK4, and CDK6. In addition, piperine inhibited CD25 expression, synthesis of interferon-γ, interleukin (IL)-2, IL-4, and IL-17A, and the generation of cytotoxic effector cells. The inhibitory effect of piperine on T lymphocytes was associated with hypophosphorylation of Akt, extracellular signal-regulated kinase, and inhibitor of κBα, but not ZAP-70. The ability of piperine to inhibit several key signaling pathways involved in T lymphocyte activation and the acquisition of effector function suggests that piperine might be useful in the management of T lymphocyte-mediated autoimmune and chronic inflammatory disorders.

Piperine, an alkaloid from black pepper, inhibits growth of human colon cancer cells via G1 arrest and apoptosis triggered by endoplasmic reticulum stress.

Piperine, a piperidine alkaloid present in black pepper, inhibits the growth of cancer cells, although the mechanism of action is not well understood. In this study, we show that piperine (75-150 µM) inhibited the growth of several colon cancer cell lines but had little effect on the growth of normal fibroblasts and epithelial cells. Piperine inhibited HT-29 colon carcinoma cell proliferation by causing G1 phase cell cycle arrest that was associated with decreased expression of cyclins D1 and D3 and their activating partner cyclin-dependent kinases 4 and 6, as well as reduced phosphorylation of the retinoblastoma protein and up-regulation of p21/WAF1 and p27/KIP1 expression. In addition, piperine caused hydroxyl radical production and apoptosis that was partially dependent on the production of reactive oxygen species. Piperine-treated HT-29 cells showed loss of mitochondrial membrane integrity and cleavage of poly (ADP-ribose) polymerase-1, as well as caspase activation and reduced apoptosis in the presence of the pan-caspase inhibitor zVAD-FMK. Increased expression of the endoplasmic reticulum stress-associated proteins inositol-requiring 1α protein, C/EBP homologous protein, and binding immunoglobulin protein, and activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, as well as decreased phosphorylation of Akt and reduced survivin expression were also observed in piperine-treated HT-29 cells. Furthermore, piperine inhibited colony formation by HT-29 cells, as well as the growth of HT-29 spheroids. Cell cycle arrest and endoplasmic reticulum stress-associated apoptosis following piperine treatment of HT-29 cells provides the first evidence that piperine may be useful in the treatment of colon cancer.

Enhanced killing of breast cancer cells by a d-amino acid analog of the winter flounder-derived pleurocidin NRC-03.

Cationic antimicrobial peptides (CAPs) defend against pathogens and, in some cases, exhibit potent anticancer activities. We previously reported that the pleurocidin NRC-03 causes lysis of breast cancer and multiple myeloma cells. NRC-03 also reduces the EC50 of other cytotoxic compounds and prevents tumor growth in vivo. However, the therapeutic utility of NRC-03 may be limited by its susceptibility to degradation by proteases. The goal of this study was to characterize the anticancer activities of a d-amino acid analog of NRC-03 ([D]-NRC-03) that was predicted to be resistant to proteolytic degradation. Unlike NRC-03, [D]-NRC-03 was not degraded by human serum or trypsin and, in comparison to NRC-03, showed increased killing of breast cancer cells, including multidrug-resistant cells; however, [D]-NRC-03 was somewhat more cytotoxic than NRC-03 for several types of normal cells. Importantly, [D]-NRC-03 was more effective than NRC-03 in vivo since 4-fold less peptide was required for an equivalent inhibitory effect on the growth of breast cancer cell xenografts in immune-deficient mice. These findings demonstrate that a d-amino acid analog of NRC-03 overcomes a major limitation to the therapeutic use of NRC-03, namely peptide stability. Further modification of [D]-NRC-03 is required to improve its selectivity for cancer cells.

Inhibitory effect of iron withdrawal by chelation on the growth of human and murine mammary carcinoma and fibrosarcoma cells.

Since iron uptake is essential for cell growth, rapidly dividing cancer cells are sensitive to iron depletion. To explore the effect of iron withdrawal on cancer cell growth, mouse and human mammary carcinoma cells (4T1 and MDA-MB-468, respectively) and mouse and human fibrosarcoma cells (L929 and HT1080, respectively) were cultured in the absence or presence of DIBI, a novel iron-chelating polymer containing hydroxypyridinone iron-ligand functionality. Cell growth was measured by a colorimetric assay for cell metabolic activity. DIBI-treated 4T1, MDA-MB-468, L929 and HT1080 cells, as well as their normal counterparts, showed a dose- and time-dependent reduction in growth that was selective for human cancer cells and mouse fibrosarcoma cells. The inhibitory effect of DIBI on fibrosarcoma and mammary carcinoma cell growth was reversed by addition of exogenous iron in the form of iron (III) citrate, confirming the iron selectivity of DIBI and that its inhibitory activity was iron-related. Fibrosarcoma and mammary carcinoma cell growth inhibition by DIBI was associated with S-phase cell cycle arrest and low to moderate levels of cell death by apoptosis. Consistent with apoptosis induction following DIBI-mediated iron withdrawal, fibrosarcoma and mammary carcinoma cells exhibited mitochondrial membrane permeabilization. A comparison of DIBI to other iron chelators showed that DIBI was superior to deferiprone and similar to or better than deferoxamine for inhibition of fibrosarcoma and mammary carcinoma cell growth. These findings suggest that iron withdrawal from the tumor microenvironment with a selective and potent iron chelator such as DIBI may prevent or inhibit tumor progression.