Ashley L. Hilchie

Curcumin-induced apoptosis in ovarian carcinoma cells is p53-independent and involves p38 mitogen-activated protein kinase activation and downregulation of Bcl-2 and survivin expression and Akt signaling†

New cytotoxic agents are urgently needed for the treatment of advanced ovarian cancer because of the poor long‐term response of this disease to conventional chemotherapy. Curcumin, obtained from the rhizome of Curcuma longa, has potent anticancer activity; however, the mechanism of curcumin‐induced cytotoxicity in ovarian cancer cells remains a mystery. In this study we show that curcumin exhibited time‐ and dose‐dependent cytotoxicity against monolayer cultures of ovarian carcinoma cell lines with differing p53 status (wild‐type p53: HEY, OVCA429; mutant p53: OCC1; null p53: SKOV3). In addition, p53 knockdown or p53 inhibition did not diminish curcumin killing of HEY cells, confirming p53‐independent cytotoxicity. Curcumin also killed OVCA429, and SKOV3 cells grown as multicellular spheroids. Nuclear condensation and fragmentation, as well as DNA fragmentation and poly (ADP‐ribose) polymerase‐1 cleavage in curcumin‐treated HEY cells, indicated cell death by apoptosis. Procaspase‐3, procaspase‐8, and procaspase‐9 cleavage, in addition to cytochrome c release and Bid cleavage into truncated Bid, revealed that curcumin activated both the extrinsic and intrinsic pathways of apoptosis. Bax expression was unchanged but Bcl‐2, survivin, phosphorylated Akt (on serine 473), and total Akt were downregulated in curcumin‐treated HEY cells. Curcumin also activated p38 mitogen‐activated protein kinase (MAPK) without altering extracellular signal‐regulated kinase 1/2 activity. We conclude that p53‐independent curcumin‐induced apoptosis in ovarian carcinoma cells involves p38 MAPK activation, ablation of prosurvival Akt signaling, and reduced expression of the antiapoptotic proteins Bcl‐2 and survivin. These data provide a mechanistic rationale for the potential use of curcumin in the treatment of ovarian cancer.

Curcumin-induced apoptosis in PC3 prostate carcinoma cells is caspase-independent and involves cellular ceramide accumulation and damage to mitochondria.

Curcumin, the principal curcuminoid of tumeric, has potent anticancer activity. To determine the mechanism of curcumin-induced cytotoxicity in prostate cancer cells, we exposed PC3 prostate carcinoma cells to 25 to 100 μ M curcumin for 24 to 72 h. Curcumin treatment of PC3 cells caused time- and dose-dependent induction of apoptosis and depletion of cellular reduced glutathione (GSH). Exogenous GSH and its precursor N-acetyl-cysteine, but not ascorbic acid (AA) or ebselen, decreased curcumin accumulation in PC3 cells and also prevented curcumin-induced DNA fragmentation. The failure of AA and ebselen to protect PC3 cells from curcumin-induced apoptosis argued against the involvement of reactive oxygen species; rather, GSH-mediated inhibition of curcumin-induced cytotoxicity was due to reduced curcumin accumulation in PC3 cells. Curcumin-treated PC3 cells showed apoptosis-inducing cellular ceramide accumulation and activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK). Caspase-3, caspase-8, and caspase-9 were activated, and cytochrome c and apoptosis-inducing factor (AIF) were released from mitochondria following curcumin treatment. Interestingly, curcumin-induced apoptosis was not prevented by p38 MAPK, JNK, or caspase inhibition. We conclude that curcumin-induced cytotoxicity was due to cellular ceramide accumulation and damage to mitochondria that resulted in apoptosis mediated by AIF and other caspase-independent processes.

Piperine, a dietary phytochemical, inhibits angiogenesis

Angiogenesis plays an important role in tumor progression. Piperine, a major alkaloid constituent of black pepper, has diverse physiological actions including killing of cancer cells; however, the effect of piperine on angiogenesis is not known. Here we show that piperine inhibited the proliferation and G(1)/S transition of human umbilical vein endothelial cells (HUVECs) without causing cell death. Piperine also inhibited HUVEC migration and tubule formation in vitro, as well as collagen-induced angiogenic activity by rat aorta explants and breast cancer cell-induced angiogenesis in chick embryos. Although piperine binds to and activates the cation channel transient receptor potential vanilloid 1 (TRPV1), its effects on endothelial cells did not involve TRPV1 since the antiproliferative effect of piperine was not affected by TRPV1-selective antagonists, nor did HUVECs express detectable TRPV1 mRNA. Importantly, piperine inhibited phosphorylation of Ser 473 and Thr 308 residues of Akt (protein kinase B), which is a key regulator of endothelial cell function and angiogenesis. Consistent with Akt inhibition as the basis of piperines action on HUVECs, inhibition of the phosphoinositide-3 kinase/Akt signaling pathway with LY-294002 also inhibited HUVEC proliferation and collagen-induced angiogenesis. Taken together, these data support the further investigation of piperine as an angiogenesis inhibitor for use in cancer treatment.

Generation of a hematologic malignancy-selective membranolytic peptide from the antimicrobial core (RRWQWR) of bovine lactoferricin.

Cationic antimicrobial peptides such as bovine lactoferricin (LfcinB) constitute an important innate defense mechanism against many microbial pathogens. LfcinB also binds to and selectively kills human cancer cells via a mechanism that involves reactive oxygen species (ROS) generation and caspase activation. The antimicrobial core of LfcinB consists of only six amino acids (RRWQWR), referred to in this study as LfcinB6. Although free LfcinB6 is devoid of cytotoxic activity against cancer cells, we show here that adding a cell-penetrating hepta-arginine sequence via a glycine-glycine linker to LfcinB6 generates a peptide (MPLfcinB6) that is selectively cytotoxic for human T-leukemia and B-lymphoma cells. Flow cytometric analysis of propidium iodide and fluorescein isothiocyanate-dextran uptake by MPLfcinB6-treated cancer cells revealed extensive damage to the cell membrane, which was confirmed by scanning electron microscopy. MPLfcinB6-induced cytotoxicity was also associated with sequential ROS production and mitochondrial membrane permeabilization; however, neither ROS nor caspase activation caused by the loss of mitochondrial membrane integrity was essential for peptide-mediated cell death. We conclude that MPLfcinB6 selectively kills human T-leukemia and B-lymphoma cells by causing extensive and irreparable damage to the cell membrane.

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.

Assessment of Antimicrobial (Host Defense) Peptides as Anti-Cancer Agents

Cationic antimicrobial (host defense) peptides (CAPs) are able to kill microorganisms and cancer cells, leading to their consideration as novel candidate therapeutic agents in human medicine. CAPs can physically associate with anionic membrane structures, such as those found on cancer cells, causing pore formation, intracellular disturbances, and leakage of cell contents. In contrast, normal cells are less negatively-charged and are typically not susceptible to CAP-mediated cell death. Because the interaction of CAPs with cells is based on charge properties rather than cell proliferation, both rapidly dividing and quiescent cancer cells, as well as multidrug-resistant cancer cells, are targeted by CAPs, making CAPS potentially valuable as anti-cancer agents. CAPs often exist as families of peptides with slightly different amino acid sequences. In addition, libraries of synthetic peptide variants based on naturally occurring CAP templates can be generated in order to improve upon their action. High-throughput screens are needed to quickly and efficiently assess the suitability of each CAP variant. Here we present the methods for assessing CAP-mediated cytotoxicity against cancer cells (suspension and adherent) and untransformed cells (measured using the tritiated thymidine-release or MTT assay), and for discriminating between cell death caused by necrosis (measured using lactate dehydrogenase- or (51)Cr-release assays), or apoptosis and necrosis (single-stranded DNA content measured by flow cytometry). In addition the clonogenic assay, which assesses the ability of single transformed cells to multiply and produce colonies, is described.