Apoptosis: A Target for Anticancer Therapy.

Apoptosis, the cell's natural mechanism for death, is a promising target for anticancer therapy. Both the intrinsic and extrinsic pathways use caspases to carry out apoptosis through the cleavage of hundreds of proteins. In cancer, the apoptotic pathway is typically inhibited through a wide variety of means including overexpression of antiapoptotic proteins and under-expression of proapoptotic proteins. Many of these changes cause intrinsic resistance to the most common anticancer therapy, chemotherapy. Promising new anticancer therapies are plant-derived compounds that exhibit anticancer activity through activating the apoptotic pathway.

Cell-cycle Checkpoints and Aneuploidy on the Path to Cancer.

The cell cycle is a complex sequence of events through which a cell duplicates its contents and divides, and involves many regulatory proteins for proper cellular reproduction, including cyclin proteins and cyclin-dependent kinases, oncogenes and tumor-suppressor genes, and mitotic checkpoint proteins. Mutations of any of these regulatory mechanisms can lead to reproduction of cells carrying genetic mutations or abnormal numbers of chromosomes, resulting in genomic instability. Chromosomal instability, contributing to genomic instability, refers to abnormalities in the number of chromosomes, and leads to aneuploidy. The role of aneuploidy in cancer cell development is often disputed, as conflicting hypotheses and research make it unclear as to whether aneuploidy is a cause or consequence of cancer. Here, we present an overview of the importance of cell-cycle checkpoint regulation and chromosomal instability in the development of cancer, and discuss evidence for conflicting arguments for the role of aneuploidy in cancer, leading us to conclude that further investigation of this role would benefit our understanding of cancer development.

Update on Nanotechnology-based Drug Delivery Systems in Cancer Treatment.

The emerging field of nanotechnology meets the demands for innovative approaches in the diagnosis and treatment of cancer. The nanoparticles are biocompatible and biodegradable and are made of a core, a particle that acts as a carrier, and one or more functional groups on the core which target specific sites. Nanotech in drug delivery includes nanodisks, High Density Lipoprotein nanostructures, liposomes, and gold nanoparticles. The fundamental advantages of nanoparticles are: improved delivery of water-insoluble drugs, targeted delivery, co-delivery of two or more drugs for combination therapy, and visualization of the drug delivery site by combining imaging system and a therapeutic drug. One of the potential applications of nanotechnology is in the treatment of cancer. Conventional methods for cancer treatments have included chemotherapy, surgery, or radiation. Early recognition and treatment of cancer with these approaches is still challenging. Innovative technologies are needed to overcome multidrug resistance, and increase drug localization and efficacy. Application of nanotechnology to cancer biology has brought in a new hope for developing treatment strategies on cancer. In this study, we present a review on the recent advances in nanotechnology-based approaches in cancer treatment.

The Evolution, Functions and Applications of the Breast Cancer Genes BRCA1 and BRCA2.

BRCA1 and BRCA2 are both tumor suppressors whose mutations are the cause of most hereditary breast cancers. Both genes are highly involved in ensuring genome stability. BRCA1 homologs are found in the plant and animal kingdoms while BRCA2 homologs are additionally found in the fungi kingdom. The initial origin of both genes remains unknown, however it is expected that the common ancestors originated around 1.6 billion years ago prior to the kingdoms diverging. There has been a great amount of divergence between homologs that is not observed in other tumor suppressors with only functionally important domains conserved. This divergence continues today with evidence of primate BRCA1/2 evolution. Cancer-associated mutations have been found to occur at conserved sites, indicating that conserved sites are important for function. In this study, we present a review on the phylogenesis of BRCA1 and BRCA2.

Novel Nanoscale Delivery Particles Encapsulated with Anticancer Drugs, All-trans Retinoic Acid or Curcumin, Enhance Apoptosis in Lymphoma Cells Predominantly Expressing CD20 Antigen.

BACKGROUND: Mantle cell lymphoma (MCL), a B-cell lymphoma, pursues a relatively aggressive course, is resistant to long-term remission, and is associated with a poor prognosis. There is a pressing need for innovative treatment approaches against MCL. One such approach is targeted delivery of cytotoxic drugs to MCL cells. MATERIALS AND METHODS: In the current investigation, we pursued a strategy to employ retinoid-based or curcumin-based nanoscale delivery particles, called nanodisks (NDs), for targeted drug delivery to MCL cells (Granta), and human follicular lymphoma (HF-1) cells. The cells were incubated with NDs made of CD20 single-chain variable antibody fragment (scFv)/apolipoprotein A-1 fusion protein, and loaded with either all-trans retinoic acid (ATRA) or curcumin, and cell apoptosis was measured using flow cytometry. RESULTS AND CONCLUSION: At 10 µM, curcumin-ND induced cell death more effectively than ATRA-ND. Combination of curcumin-ND and ATRA-ND significantly enhanced the biological activity of these drugs against lymphoma cells compared to individual treatments.

Biomimetic, synthetic HDL nanostructures for lymphoma.

New therapies that challenge existing paradigms are needed for the treatment of cancer. We report a nanoparticle-enabled therapeutic approach to B-cell lymphoma using synthetic high density lipoprotein nanoparticles (HDL-NPs). HDL-NPs are synthesized using a gold nanoparticle template to control conjugate size and ensure a spherical shape. Like natural HDLs, biomimetic HDL-NPs target scavenger receptor type B-1, a high-affinity HDL receptor expressed by lymphoma cells. Functionally, compared with natural HDL, the gold NP template enables differential manipulation of cellular cholesterol flux in lymphoma cells, promoting cellular cholesterol efflux and limiting cholesterol delivery. This combination of scavenger receptor type B-1 binding and relative cholesterol starvation selectively induces apoptosis. HDL-NP treatment of mice bearing B-cell lymphoma xenografts selectively inhibits B-cell lymphoma growth. As such, HDL-NPs are biofunctional therapeutic agents, whose mechanism of action is enabled by the presence of a synthetic nanotemplate. HDL-NPs are active in B-cell lymphomas and potentially, other malignancies or diseases of pathologic cholesterol accumulation.

Curcumin nanodisk-induced apoptosis in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a pre-germinal center neoplasm characterized by cyclin D1 overexpression resulting from t(11;14)(q13;q32). Since MCL is incurable with standard lymphoma therapies, new treatment approaches are needed that target specific biologic pathways. In the present study, we investigated a novel drug delivery nanovehicle enriched with the bioactive polyphenol, curcumin (curcumin nanodisks; curcumin-ND). Cells treated with curcumin-ND showed a dose-dependent increase in apoptosis. This was accompanied by enhanced generation of reactive oxygen species (ROS). The antioxidant, N-acetylcysteine, inhibited curcumin-ND induced apoptosis, suggesting that ROS generation plays a role in curcumin action on MCL cells. Curcumin-ND decreased cyclin D1, pAkt, pIκBα, and Bcl(2) protein. In addition, enhanced FoxO3a and p27 expression as well as caspase-9, -3, and poly(ADP-ribose) polymerase (PARP) cleavage were observed. Curcumin-ND treatment led to enhanced G(1) arrest in two cultured cell models of MCL.

Curcumin nanodisks: formulation and characterization.

Nanodisks (NDs) are nanoscale, disk-shaped phospholipid bilayers whose edge is stabilized by apolipoproteins. In the present study, NDs were formulated with the bioactive polyphenol curcumin at a 6:1 phospholipid-to-curcumin molar ratio. Atomic force microscopy revealed that curcumin-NDs are particles with diameters <50 nm and the thickness of a phospholipid bilayer. When formulated in NDs, curcumin is water soluble and gives rise to a characteristic absorbance spectrum with a peak centered at 420 nm. Fluorescence spectroscopy of curcumin-NDs provided evidence of self-quenching. Incubation of curcumin-NDs with empty NDs relieved the self-quenching, indicating redistribution of curcumin between curcumin-loaded and empty NDs. In HepG2 cells, curcumin-NDs mediated enhanced cell growth inhibition as compared with free curcumin. In a cell culture model of mantle cell lymphoma, curcumin-NDs were a more potent inducer of apoptosis than free curcumin. The nanoscale size of the complexes, combined with their ability to solubilize curcumin, indicates NDs may have in vivo therapeutic applications. FROM THE CLINICAL EDITOR: Nanodisks (NDs), disk-shaped phospholipid bilayers stabilized by apolipoproteins, are shown entrap curcumin and improve its delivery to HepG2 and mantle cell lymphoma cells in culture. These novel nanocomplexes demonstrate interesting therapeutic application potentials.

Mitochondrial-mediated apoptosis in lymphoma cells by the diterpenoid lactone andrographolide, the active component of Andrographis paniculata.

PURPOSE: Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an herbal medicine used in Asia. It has been reported to have anti-inflammatory, antihypertensive, antiviral, and immune-stimulant properties. Furthermore, it has been shown to inhibit cancer cell proliferation and induce apoptosis in leukemia and solid tumor cell lines. EXPERIMENTAL DESIGN: We studied the Burkitt p53-mutated Ramos cell line, the mantle cell lymphoma (MCL) line Granta, the follicular lymphoma (FL) cell line HF-1, and the diffuse large B-cell lymphoma (DLBCL) cell line SUDHL4, as well as primary cells from patients with FL, DLBCL, and MCL. RESULTS: We found that andrographolide resulted in dose- and time-dependent cell death as measured by MTT. Andrographolide significantly increased reactive oxygen species (ROS) production in all cell lines. To determine mechanism of cell death, we measured apoptosis by Annexin V/propidium iodide in the presence and absence of the antioxidant N-acetyl-l-cysteine (NAC), the glutathione (GSH)-depleting agent buthionine sulfoxamine (BSO), or caspase inhibitors. We found that apoptosis was greatly enhanced by BSO, blocked by NAC, and accompanied by poly(ADP-ribose) polymerase cleavage and activation of caspase-3, caspase-8, and caspase-9. We measured BAX conformational change and mitochondrial membrane potential, and using mouse embryonic fibroblast (MEF) Bax/Bak double knockouts (MEF(Bax-/-/Bak-/-)), we found that apoptosis was mediated through mitochondrial pathways, but dependent on caspases in both cell lines and patient samples. CONCLUSIONS: Andrographolide caused ROS-dependent apoptosis in lymphoma cell lines and in primary tumor samples, which was enhanced by depletion of GSH and inhibited by NAC or the pan-caspase inhibitor Z-VAD-FMK. Further studies of diterpenoid lactones in lymphoma are warranted.

Motexafin gadolinium enhances p53-Mdm2 interactions, reducing p53 and downstream targets in lymphoma cell lines.

BACKGROUND: Loss of p53 renders cells more susceptible to acute oxidant stress induced by oxidant-generating agents such as motexafin gadolinium (MGd). We hypothesized that reactive oxygen species (ROS)-generating MGd results in low-level p53 expression, making cells more susceptible to oxidant stress. MATERIALS AND METHODS: Lymphoma cells were incubated with different concentrations of MGd with or without zinc (Zn) and ascorbate, and ROS, apoptosis, proteins, and oxidant genes were measured. RESULTS: MGd, with ascorbate and Zn, induced apoptosis in lymphoma cells. This was accompanied by reduction of p53 protein but not message, and by reduction of p53 downstream targets p21, glutathione peroxidase 1 (GPx1), and p53 up-regulated modulator of apoptosis (PUMA). p53 protein reduction was reversed by MG132, and nutlin-3. CONCLUSION: Our data are consistent with a pathway of cell death that is independent of p53-mediated induction of PUMA; the cellular response to reduce p53 represents a cell survival adjustment to ROS-mediated stress.

All trans retinoic acid nanodisks enhance retinoic acid receptor mediated apoptosis and cell cycle arrest in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is characterized by translocation t(11;14)(q13;q32), aggressive clinical behaviour, and poor patient outcomes following conventional chemotherapy. New treatment approaches are needed that target novel biological pathways. All trans retinoic acid (ATRA) is a key retinoid that acts through nuclear receptors that function as ligand-inducible transcription factors. The present study evaluated cell killing effects of ATRA-enriched nanoscale delivery particles, termed nanodisks (ND), on MCL cell lines. Results show that ATRA-ND induced cell death more effectively than naked ATRA (dimethyl sulphoxide) or empty ND. ATRA-ND induced reactive oxygen species (ROS) generation to a greater extent than naked ATRA. The antioxidant, N-acetylcysteine, inhibited ATRA-ND induced apoptosis. Compared to naked ATRA, ATRA-ND enhanced G1 growth arrest, up-regulated p21and p27, and down regulated cyclin D1. At ATRA concentrations that induced apoptosis, expression levels of retinoic acid receptor-alpha (RARalpha) and retinoid X receptor-gamma (RXRgamma) were increased. Compared to naked ATRA, ATRA-ND significantly stimulated transcriptional activity of RARA in a model carcinoma cell line. Furthermore, the RAR antagonist, Ro 41-5253, inhibited ATRA-ND induced ROS generation and prevented ATRA-ND induced cell growth arrest and apoptosis. In summary, incorporation of ATRA into ND enhanced the biological activity of this retinoid in cell culture models of MCL.

Blockade of the erbB2 receptor induces cardiomyocyte death through mitochondrial and reactive oxygen species-dependent pathways.

Overexpression of the receptor tyrosine kinase erbB2 (Her2 in humans) is correlated with a poor prognosis in breast and ovarian cancers. Treatment with trastuzumab (a monoclonal antibody against erbB2) improves survival; however, it also causes cardiomyopathy. We hypothesized that blockade of the erbB2 receptor induces cardiomyocyte death through a mitochondrial pathway that is dependent on the production of reactive oxygen species (ROS). We first showed that levels of erbB2 receptor are significantly decreased in an animal model of ischemic heart disease and in human ischemic cardiomyopathy. We treated neonatal rat cardiomyocytes with an inhibitory erbB2 antibody to study the mechanism behind the deleterious effects of erbB2 blockade. These cells displayed a dose-dependent increase in ROS production and cell death compared with control IgG-treated cells; these processes were reversed by the antioxidant, N-acetylcysteine. The effects of erbB2 antibody on both cell death and ROS production were also reversed by cyclosporine A and diazoxide, chemicals that regulate the pro- and anti-apoptotic channels in the mitochondria, respectively. Furthermore, mouse embryonic fibroblasts lacking Bax and Bak (proteins that mediate cell death through a mitochondrial pathway) were resistant to the deleterious effects of erbB2 antibody. These effects of erbB2 blockade appear to occur through a pathway involving AKT and PKC-alpha. Our results suggest that erbB2 plays a role in cardiomyocyte survival, and that the deleterious effects of trastuzumab on the heart occur through a mitochondrial pathway and is mediated by ROS production. Manipulation of redox signaling may be beneficial in cancer patients receiving trastuzumab.

Hypoxia inducible factor-alpha activation in lymphoma and relationship to the thioredoxin family.

Hypoxia inducible factors (HIFs) activate oncogenic pathways, while thioredoxins (Trx), including Trx1 and Trx reductases-1 and -2 (TrxR1 and TrxR2), promote HIF-alpha stabilization. In immunoblotting studies in lymphoma cell lines we found that Raji and SUDHL4 cells exhibited normoxic HIF-2alpha protein stabilization. Five cell lines showed increased TrxR1 expression, while only Namalwa, HF1 and SUDHL4 had Trx1 and TrxR2 activation. Tissue microarrays in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) identified different HIF expression among histological subgroups (e.g. 44% DLBCL vs. 11% of FL cases with moderate-to-high expression of HIF-1alpha and HIF-2alpha, P = 0.0017). These data demonstrate that HIF and the thioredoxin family are abnormally activated in lymphoma.

Parathyroid hormone stimulates phosphatidylethanolamine hydrolysis by phospholipase D in osteoblastic cells.

Parathyroid hormone (PTH) and phorbol-12,13-dibutyrate (PDBu) stimulate phospholipase D (PLD) activity and PC hydrolysis in UMR-106 osteoblastic cells {Singh, A.T., Kunnel, J.G., Strieleman, P.J., and Stern, P.H. (1999) Parathyroid Hormone (PTH)-(1-34), [Nle8,18,Tyr34]PTH-(3-34) Amide, PTH-(1-31) Amide, and PTH-Related Peptide-(1-34) Stimulate Phosphatidylcholine Hydrolysis in UMR-106 Osteoblastic Cells: Comparison with Effects of Phorbol 12,13-Dibutyrate, Endocrinology 140, 131-137}. The current studies were designed to determine whether ethanolamine-containing phospholipids, and specifically PE, could also be substrates. In cells labeled with 14C-ethanolamine, PTH and PDBu treatment decreased 14C-PE. In cells co-labeled with 3H-choline and 14C-ethanolamine, PTH and PDBu treatment increased both 3H-choline and 14C-ethanolamine release from the cells. Choline and ethanolamine phospholipid hydrolysis was increased within 5 min, and responses were sustained for at least 60 min. Maximal effects were obtained with 10 nM PTH and 50 nM PDBu. Dominant negative PLD1 and PLD2 constructs inhibited the effects of PTH on the phospholipid hydrolysis. The results suggest that both PC and PE are substrates for phospholipase D in UMR-106 osteoblastic cells and could therefore be sources of phospholipid hydrolysis products for downstream signaling in osteoblasts.

Role of protein kinase A, phospholipase C and phospholipase D in parathyroid hormone receptor regulation of protein kinase Calpha and interleukin-6 in UMR-106 osteoblastic cells.

Parathyroid hormone (PTH) stimulates both bone formation and resorption by activating diverse osteoblast signalling pathways. Upstream signalling for PTH stimulation of protein kinase C-alpha (PKCalpha) membrane translocation and subsequent expression of the pro-resorptive cytokine interleukin-6 (IL-6) was investigated in UMR-106 osteoblastic cells. PTH 1-34, PTH 3-34, PTHrP and PTH 1-31 stimulated PKCalpha translocation and IL-6 promoter activity. Pharmacologic intervention at the adenylyl cyclase (AC) pathway (forskolin, IBMX, PKI) failed to alter PTH 1-34- or PTH 3-34-stimulated PKCalpha translocation. The phosphoinositol-phospholipase C (PI-PLC) antagonist U73122 slightly decreased PTH 1-34-stimulated PKCalpha translocation; however, the control analogue U73343 acted similarly. Propranolol, an inhibitor of phosphatidic acid (PA) phosphohydrolase, decreased diacylglycerol (DAG) formation and attenuated PTH 1-34- and PTH 3-34-stimulated PKCalpha translocation and IL-6 promoter activity, suggesting a phospholipase D (PLD)-dependent mechanism. This is the first demonstration that PLD-mediated signalling leads to both PKC-alpha translocation and IL-6 promoter activation in osteoblastic cells.

Regulation of parathyroid hormone-stimulated phospholipase D in UMR-106 cells by calcium, MAP kinase, and small G proteins.

UNLABELLED: Signaling intermediates for PTH and phorbol activation of PLD in UMR-106 cells were determined. Calcium was required, and the effects of PTH, phorbol, and calcium were dependent on p42/44 MAP kinase and small G proteins, specifically RhoA, acting through Rho kinase. INTRODUCTION: Phospholipase D (PLD) plays a key signaling role in numerous cellular processes. PLD-stimulated hydrolysis of phosphatidylcholine (PC) generates phosphatidic acid, a source of diacylglycerol (DAG). We previously reported that parathyroid hormone (PTH) stimulates PLD activity in UMR-106 osteoblastic cells by a protein kinase C (PKC)-independent mechanism. The current study investigated the roles of calcium, MAP kinase, and small G proteins in PTH- and phorbol-12,13-dibutyrate (PDBu)-stimulated transphosphatidylation of ethanol, a reaction catalyzed by PLD. METHODS: UMR-106 cells were labeled with 3H-palmitic and treated in the presence of ethanol. Phosphatidylethanol was separated by thin-layer chromatography and detected by autoradiography, and the bands were scraped and counted. Statistical significance of the responses from three to nine replicates was determined by ANOVA and Tukey's post-test. RESULTS AND CONCLUSIONS: PTH and PDBu effects were attenuated by EGTA, BAPTA, nifedipine, and dantrolene, whereas ionomycin or 2X calcium increased basal PLD activity. PTH activated p42/p44 MAP kinase, and the effects of PTH, PDBu, and ionomycin on PLD, but not on calcium influx, were prevented by the MEK inhibitors PD98059 and U0126. Small G proteins were shown to be involved in the effects of PTH, PDBu, and ionomycin on PLD. Inhibition of ARF by brefeldin prevented the PLD activation by all three agonists. A nonselective Rho/Rac/cdc-42 inhibitor, Clostridium difficile toxin B, also inhibited the effects of all three agonists on PLD. More selective inhibition of RhoA with a dominant negative RhoA construct or by inhibiting geranylgeranyltransferase I antagonized the effects of PTH, PDBu, and ionomycin, as did inhibiting the downstream kinase, Rho kinase. The current results reveal the importance of calcium, MAP kinase, and small G proteins in PTH and PDBu stimulation of PLD activity in UMR-106 cells.