Selective Induction Of Apoptosis Research Articles

Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast

The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane.

Alteration of Plasma Membrane Organization by an Anticancer Lysophosphatidylcholine Analogue Induces Intracellular Acidification and Internalization of Plasma Membrane Transporters in Yeast

The lysophosphatidylcholine analogue edelfosine is a potent antitumor lipid that targets cellular membranes. The underlying mechanisms leading to cell death remain controversial, although two cellular membranes have emerged as primary targets of edelfosine, the plasma membrane (PM) and the endoplasmic reticulum. In an effort to identify conditions that enhance or prevent the cytotoxic effect of edelfosine, we have conducted genome-wide surveys of edelfosine sensitivity and resistance in Saccharomyces cerevisiae presented in this work and the accompanying paper (Cuesta-Marbán, Á., Botet, J., Czyz, O., Cacharro, L. M., Gajate, C., Hornillos, V., Delgado, J., Zhang, H., Amat-Guerri, F., Acuña, A. U., McMaster, C. R., Revuelta, J. L., Zaremberg, V., and Mollinedo, F. (January 23, 2013) J. Biol. Chem. 288,), respectively. Our results point to maintenance of pH homeostasis as a major player in modulating susceptibility to edelfosine with the PM proton pump Pma1p playing a main role. We demonstrate that edelfosine alters PM organization and induces intracellular acidification. Significantly, we show that edelfosine selectively reduces lateral segregation of PM proteins like Pma1p and nutrient H(+)-symporters inducing their ubiquitination and internalization. The biology associated to the mode of action of edelfosine we have unveiled includes selective modification of lipid raft integrity altering pH homeostasis, which in turn regulates cell growth.

Selective Induction of Tumor Cell Apoptosis by a Novel P450-mediated Reactive Oxygen Species (ROS) Inducer Methyl 3-(4-Nitrophenyl) Propiolate

Induction of tumor cell apoptosis has been recognized as a valid anticancer strategy. However, therapeutic selectivity between tumor and normal cells has always been a challenge. Here, we report a novel anti-cancer compound methyl 3-(4-nitrophenyl) propiolate (NPP) preferentially induces apoptosis in tumor cells through P450-catalyzed reactive oxygen species (ROS) production. A compound sensitivity study on multiple cell lines shows that tumor cells with high basal ROS levels, low antioxidant capacities, and p53 mutations are especially sensitive to NPP. Knockdown of p53 sensitized non-transformed cells to NPP-induced cell death. Additionally, by comparing NPP with other ROS inducers, we show that the susceptibility of tumor cells to the ROS-induced cell death is influenced by the mode, amount, duration, and perhaps location of ROS production. Our studies not only discovered a unique anticancer drug candidate but also shed new light on the understanding of ROS generation and function and the potential application of a ROS-promoting strategy in cancer treatment.

Induction of selective cytotoxicity and apoptosis in human T4-lymphoblastoid cell line (CEMss) by boesenbergin a isolated from boesenbergia rotunda rhizomes involves mitochondrial pathway, activation of caspase 3 and G2/M phase cell cycle arrest

BackgroundBoesenbergia rotunda (Roxb.) Schlecht (family zingiberaceae) is a rhizomatous herb that is distributed from north-eastern India to south-east Asia, especially in Indonesia, Thailand and Malaysia. Previous research has shown that the crude extract of this plant has cytotoxic properties. The current study examines the cytotoxic properties of boesenbergin A isolated from Boesenbergia rotunda.MethodsMTT assay was used to check the cytotoxicity of boesenbergin A. The morphological assessment of apoptosis was monitored using normal and fluorescence microscopy. The early and late phase of apoptosis was investigated using annexin V and DNA laddering assays, respectively. The mitochondrial membrane potential (MMP) was assessed by fluorescence microscopy. Human apoptosis proteome profiler assays were performed to investigate the mechanism of cell death. In addition, the protein levels of Bax, Bcl2 and HSP 70 were also analyzed using western blot. Assays of caspase =-3/7, -8 and =-9 were carried out in order to test for induction during treatment. Lastly, cell cycle progression was analyzed using flow cytometry.ResultsBoesenbergin A was found to have the highest toxicity towards CEMss cancer cells (IC50 = 8 μg/ml). The morphology of CEMss cells after treatment showed evidence of apoptosis that included blebbing and chromatin condensation. The annexin V assay revealed that early apoptosis is induced after treatment. The DNA laddering assay confirmed that DNA fragmentation had occurred during late apoptosis. The cell cycle analysis indicated that boesenbergin A was able to induce G2/M phase arrest in CEMss cells. The activity of caspases -3/7, -8 and -9 was increased after treatment which indicates both intrinsic and extrinsic pathways are induced during apoptosis. The involvement of mitochondria was established by increased mitochondrial membrane potential and up and down regulation of Bcl2 and Bax proteins as well as HSP70.ConclusionIn conclusion, the results demonstrated that boesenbergin A induced apoptosis of CEMss cells through Bcl2/Bax signaling pathways with the involvement of caspases and G2/M phase cell cycle arrest. The current findings warrant further research on boesenbergin A as a novel chemotherapeutic agent for leukemia intervention including studies in animal models.

Arginine modified PAMAM dendrimer for interferon beta gene delivery to malignant glioma

A xenograft brain tumor model was established by the subcutaneous injection of U87MG cells into nude mice to investigate the efficacy of a non-viral vector, arginine-modified polyamidoamine dendrimer (PAMAM-R), in delivering a therapeutic gene, human interferon beta (IFN-β). We used 4',6-diamidino-2-phenylindole staining, the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, and the caspase-3 activity assay to determine the induction of apoptosis upon transfection with the PAMAM-R/IFN-β gene polyplex in vitro. The polyplex was injected into xenograft brain tumors. Mice treated with PAMAM-R/pORF-IFN-β exhibited a significantly smaller tumor size than control mice and PAMAM-R/pORF treated mice. Hematoxylin/eosin staining and immunohistochemistry with the endothelial growth factor receptor antibody also revealed inhibition of tumor growth. Furthermore, reverse transcription polymerase chain reaction and the TUNEL assay also verified the expression of IFN-β and induction of apoptosis in vivo. These results indicate that the PAMAM-R/pORF-IFN-β polyplex is an effective therapeutic candidate for glioblastoma multiforme due to its selective induction of apoptosis in tumor cells.

Selective Induction of Apoptosis: Promising Therapy in Pancreatic Cancer

Pancreatic cancer is one of lethal and poor prognostic malignancies. Due to the absence of effective detecting methods, quite a number of efforts have been made to improve a survival advantage for treatment in patients with pancreatic cancer. Over the past decade, single-agent gemcitabine and gemcitabine-containing combinations were considered standard first-line therapies for advanced pancreatic cancer. Although these routine uses of chemotherapy failed to significantly improve survival benefit for most therapies, these trials provided insights into the molecular mechanisms involved in the development of pancreatic cancer and therefore opened up new therapeutic avenues. Apoptotic inducer as a therapeutic concept has been widely proposed and experimentally identified in some works. Some reviews have revealed that apoptosis-inducing was a promising therapy in cancers with the least side effects and more effectiveness. Apoptosis is a highly controlled physiological mechanism and proceeds through two major pathways for apoptosis-inducing. Some anticancer drugs kill cancer cells by inducing apoptosis via death receptor pathway; however, other chemotherapeutic drugs trigger apoptosis via mitochondrial pathway. In this review, we summarize briefly current chemotherapy in pancreatic cancer, describe the apoptotic mechanisms, and provide a novel therapeutic strategy by targeting Ras intermediate.

Quantitative proteomics analysis of BMS-214662 effects on CD34 positive cells from chronic myeloid leukaemia patients

Chronic myeloid leukaemia (CML) arises in a haemopoietic stem cell and is driven by the Bcr-Abl oncoprotein. Abl kinase inhibitors (protein tyrosine kinase inhibitors) represent standard treatment for CML and induce remission in the majority of patients with early disease, however these drugs do not target leukaemic stem cells (LSCs) effectively, thus preventing cure. Previously, we identified the farnesyl transferase inhibitor BMS-214662 as a selective inducer of apoptosis in LSCs of CML patients relative to normal controls; however, the mechanism underlying LSC-specific apoptosis remains unclear. To identify pathways involved in the favourable effects of BMS-214662 in CML, we employed a proteomic approach (based on iTRAQ) to analyse changes in protein expression in response to drug treatment in the nuclear and cytoplasmic fractions of CD34(+) CML cells. The study identified 88 proteins as altered after drug treatment, which included proteins known to be involved in nucleic acid metabolism, oncogenesis, developmental processes and intracellular protein trafficking. We found that expression of Ebp1, a negative regulator of proliferation, was upregulated in the nucleus of BMS-214662-treated cells. Furthermore, proteins showing altered levels in the cytosol, such as histones, were predominantly derived from the nucleus and BMS-214662 affected expression levels of nuclear pore complex proteins. Validation of key facets of these observations suggests that drug-induced alterations in protein localisation, potentially via loss of nuclear membrane integrity, contributes to the LSC specificity of BMS-214662, possibly via Ran proteins as targets.

The Biological Effects and Mechanisms of Interleukin-34 in the U937 Human Leukemic Cell Model.

Abstract 2155Interleukin-34 (IL-34) is a cytokine consisting of a 39kD homodimer employing the Macrophage Colony Stimulating Factor (MCSF/CSF-1) receptor; recently identified through functional proteomics experiments. Among its physiological activities, IL-34 has been shown to promote monocyte viability and proliferation; promote the differentiation of bone marrow cells into macrophage progenitors and induce pro-inflammatory cytokines: IL-1β, IL-6, TNF-α, and GM-CSF in human whole blood. All of the published work on IL-34 involves its effects on hematopoietic and osteoclast progenitors; however, it is not known whether IL-34 has the ability to induce any of these physiological responses in cancer cells. The purpose of this study is to evaluate the biological effects of IL-34 in the U937 leukemia cell line while deducing the signaling mechanisms associated with these effects. Thus far, we have evaluated IL-34 on U937 cells for cell growth and proliferation, induction of cytokines, migration, differentiation and apoptosis. Congruently, we are also exploring the JAK/STAT and PI3K/Akt signaling pathways for activation. In U937 cells, we have noted that while IL-34 does not induce cell growth or proliferation, there is a reduction in cells in the S phase of the cell cycle at 48hours, post treatment. Additionally, we note a differential induction of a number of Th1/Th2/Th17 and pro-inflammatory cytokines, namely IL-1α, IL-1b, IL-6, IL-8 and G-CSF. Our results also show that IL-34 influences the migration of monocytes by the possible induction of chemoattractant cytokines in media. Furthermore, we report that IL-34 is able to differentiate the human leukemic cellular model, U937, from monoblastic precursor cells towards monocyte- and macrophage-like cells. Accompanying this differentiation appears to be a selective induction of apoptosis in undifferentiated U937 cells, supported by the induction of cleaved Caspase-3. To our knowledge, this is the first report that IL-34 induces differentiation in human leukemic cellular model, let alone any cancer model. Exploration of the JAK/STAT signaling pathway has identified TYK2 and JAK 1 as mediators of the IL-34 signal through in U937 cells, while identification of its associated STAT proteins is currently ongoing. Interestingly, the evaluation of the PI3K/Akt pathway has demonstrated a reduction of phosphorylated Akt, suggesting an influence of PTEN, which is currently being evaluated. This research may provide insight into the influence of IL-34 in the cancer microenvironment as compared to the normal cell microenvironment, resulting in potential therapies targeting its mechanistic actions. Disclosures:No relevant conflicts of interest to declare.

Biomimetic Synthetic High Density Lipoprotein Nanostructures Target the SR-B1 Receptor and Differentially Manipulate Cellular Cholesterol Flux in Lymphoma Cells: A Novel Treatment Paradigm

Abstract 157We report a nanoparticle-enabled therapeutic approach to B cell lymphoma using synthetic, high-density lipoprotein nanoparticles (HDL-NP). Like natural HDLs, biomimetic HDL-NPs target scavenger receptor type B-1 (SR-B1), a high-affinity HDL receptor expressed by lymphoma cells. Functionally, and unlike natural HDL, a gold nanoparticle template used to control HDL-NP synthesis enables differential manipulation of cellular cholesterol flux through SR-B1. Recent evidence in lymphoblasts and myeloblasts from patients with acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML) demonstrates enhanced uptake of cholesterol through high-density lipoprotein (HDL) carriers, which may result in increased cell proliferation. We therefore hypothesized that by targeting SR-B1, we could manipulate cholesterol flux in lymphoma cells thereby targeting cellular signaling pathways that would lead to cell death and offer an innovative approach to the treatment of lymphoma and other cancers. Methods:To accomplish this, we developed a biomimetic spherical nanoparticle (HDL-NP) with surface chemical properties similar to natural HDL, including the ability to sequester cholesterol. Biomimetic HDL-NPs are synthesized using a 5 nm diameter gold (Au) nanoparticle (NP) as a size- and shape-restrictive template on which to assemble the surface chemical components of natural HDLs, including phospholipids and the HDL-defining apolipoprotein A1 (Apo A1). Importantly, the core AuNP template occupies the real estate in natural cholesterol-rich HDLs reserved for esterified cholesterol, which inherently limits the ability of HDL-NPs to deliver cholesterol. We incubated the HDL-NPs with various lymphoma cell lines, and similarly tested the HDL-NPs in a xenograft model. Results:We first examined gene expression profiles of diffuse large B-cell lymphoma (DLBCL), Burkitt Lymphoma (BL) and normal B cells from patient samples in a database generated using Affymetrix U133plus 2.0 arrays in order to establish the prevalence of SR-B1 expression. We compared the expression of SR-B1 in BL cases (n=20), and DLBCL cases (n=40) that were further subdivided as activated B-cell (ABC)-like DLBCL (n=20), and germinal center (GC)-like DLBCL (n=20) to normal naive (n=3) and memory (n=3) B cells obtained from healthy donors. We found that SR-B1 was expressed at two to four-fold higher levels in the lymphomas (ABC and GC) compared with normal B cells. Next, we determined the expression of SR-B1 in lymphoma cell lines and normal peripheral lymphocytes by immunoblotting, and we found that SR-B1 is expressed in multiple B cell lymphoma cell lines, but not in Jurkat, a T-cell line, and is not expressed by normal human lymphocytes. Incubation of HDL-NP with Ramos, LY-3 and SUDHL-4 resulted in a dose-dependent decrease in cell viability and apoptosis (Figure 1) of the Ramos and SUDHL-4 cells, less so in LY-3 cells, and not in the Jurkat line. This required the nanoparticle construct and could not be duplicated by individual components of that construct, and was reversible with addition of acetylated low-density lipoprotein, indicating that the SR-B1 receptor was targeted. Xenograft experiments with SCID beige mice (C.B-Igh-1b/GbmsTac-Prkdcscid-Lystbg N7) bearing Ramos and Jurkat flank tumor xenografts confirmed the activity of the HDL-NP (Figure 2). Conclusion:We report a template-directed and bio-functional therapeutic nanostructure that could shift the paradigm for treating lymphoma and other cancers. A combination of SR-B1 binding and manipulation of cholesterol flux is responsible for selective induction of apoptosis in B cell lymphoma. [Display omitted] [Display omitted] Disclosures:Thaxton:Aurasense: Employment, Equity Ownership.

Selective Induction of Apoptosis and Autophagy Through Treatment With Dandelion Root Extract in Human Pancreatic Cancer Cells

Pancreatic cancer has a 100% mortality rate; the aim of this study is to evaluate the efficacy of dandelion root extract (DRE) in inducing apoptosis and autophagy in aggressive and resistant pancreatic cancer cells. The effect of DRE was evaluated using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay. Apoptotic cell death was confirmed by nuclear condensation by Hoechst staining and externalization of phosphatidylserine to the outer leaflet of the plasma membrane by Annexin-V binding assay. Loss of mitochondrial membrane potential was observed using the JC-1 (5,5',6, 6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) dye. The induction of autophagy was detected using a monodansylcadaverine assay and this was confirmed by immunofluorescence for light chain 3-II. BxPC-3 and PANC-1 pancreatic cells were sensitive to aqueous DRE. This extract induces selective apoptosis in a dose- and time-dependent manner. Dandelion root extract caused the collapse of the mitochondrial membrane potential, leading to prodeath autophagy. Normal human fibroblasts were resistant at similar doses. We demonstrate that DRE has the potential to induce apoptosis and autophagy in human pancreatic cancer cells with no significant effect on noncancerous cells. This will provide a basis on which further research in cancer treatment through DRE can be executed.

The Antiangiogenic Compound Aeroplysinin-1 Induces Apoptosis in Endothelial Cells by Activating the Mitochondrial Pathway

Aeroplysinin-1 is a brominated metabolite extracted from the marine sponge Aplysina aerophoba that has been previously characterized by our group as a potent antiangiogenic compound in vitro and in vivo. In this work, we provide evidence of a selective induction of apoptosis by aeroplysinin-1 in endothelial cells. Studies on the nuclear morphology of treated cells revealed that aeroplysinin-1 induces chromatin condensation and nuclear fragmentation, and it increases the percentage of cells with sub-diploid DNA content in endothelial, but not in HCT-116, human colon carcinoma and HT-1080 human fibrosarcoma cells. Treatment of endothelial cells with aeroplysinin-1 induces activation of caspases-2, -3, -8 and -9, as well as the cleavage of apoptotic substrates, such as poly (ADP-ribose) polymerase and lamin-A in a caspase-dependent mechanism. Our data indicate a relevant role of the mitochondria in the apoptogenic activity of this compound. The observation that aeroplysinin-1 prevents the phosphorylation of Bad relates to the mitochondria-mediated induction of apoptosis by this compound.

Selective cytotoxicity and apoptosis induction in glioma cell lines by 5-oxygenated-6,7-methylenedioxycoumarins from Pterocaulon species

The coumarins 5-methoxy-6,7-methylenedioxycoumarin 1 5-(3-methyl-2-butenyloxy)-6,7-methylenedioxycoumarin 2 and 5-(2,3-dihydroxy-3-methylbutyloxy)-6,7-methylenedioxycoumarin 3 isolated from Pterocaulon species showed significant cytotoxicity against two glioma cells lines. Compound 1 presented IC50 values of 34.6 μM and 31.6 μM against human (U138-MG) and rat (C6) glioma cells, respectively, and this compound was at least two times more potent than compounds 2 and 3. This result could be explained by the planar conformation adopted by 1 through a non-classical hydrogen bond between a hydrogen of the methoxy and the oxygen of the methylenedioxy groups. Another important finding was that the cytotoxic effect induced by 1 in glioma cells was not observed in organotypic cultures, indicating a selective cytotoxicity for tumor cells.

Poster 61: Induction of Selective Apoptosis by Means of Anti-HER2 Antibody-Conjugated Gold Nanoparticles on Melanoma Cell

Poster 61: Induction of Selective Apoptosis by Means of Anti-HER2 Antibody-Conjugated Gold Nanoparticles on Melanoma Cell

Preferential induction of apoptotic cell death in melanoma cells as compared with normal keratinocytes using a non-thermal plasma torch

Selective induction of apoptosis in melanoma cells is optimal for therapeutic development. To achieve this goal, a non-thermal helium plasma torch was modified for use on cultured cells in a temperature-controlled environment. Melanoma cells were targeted with this torch (1) in parallel cultures with keratinocytes, (2) in co-culture with keratinocytes and (3) in a soft agar matrix. Melanoma cells displayed high sensitivity to reactive oxygen species generated by the torch and showed a 6-fold increase in cell death compared with keratinocytes. The extent of cell death was compared between melanoma cells and normal human keratinocytes in both short-term (5 min) co-culture experiments and longer assessments of apoptotic cell death (18–24 h). Following a 10 sec plasma exposure there was a 4.9-fold increase in the cell death of melanoma vs. keratinocytes as measured after 24 h at the target site of the plasma beam. When the treatment time was increased to 30 sec, a 98% cell death was reported for melanoma cells, which was 6-fold greater than the extent of cell death in keratinocytes. Our observations further indicate that this preferential cell death is largely due to apoptosis.. In addition, we report that this non-thermal plasma torch kills melanoma cells growing in soft agar, suggesting that the plasma torch is capable of inducing melanoma cell death in 3D settings. We demonstrate that the presence of gap junctions may increase the area of cell death, likely due to the “bystander effect” of passing apoptotic signals between cells. Our findings provide a basis for further development of this non-invasive plasma torch as a potential treatment for melanoma.

Selective induction of apoptosis by HMG-CoA reductase inhibitors in hepatoma cells and dependence on p53 expression

HMG-CoA-reductase inhibitors (statins) are widely used drugs to interfere with cholesterol biosynthesis. Besides this usage, evidence is increasing that statins might also be useful in therapy of viral infections or cancer. We investigated the effects of fluva-, simva-, atorva-, rosuva- and lovastatin on the viability of primary mouse and human hepatocytes as well as mouse (Hepa1-6) and human (Huh7, HepG2) hepatoma cell lines. Our results show selective cytotoxic effects of fluva-, simva- and lovastatin on hepatoma cells in comparison to primary hepatocytes. Using human hepatoma cells we found significant reduction of cell viability and induction of apoptosis in HepG2 cells, while statins did not affect Huh7 cells at concentrations not toxic for primary hepatocytes. Stable knockdown of endogenous p53, which is overexpressed in Huh7 cells, was able to restore susceptibility of Huh7 cells towards statin-induced toxicity. The anti-tumor effect of statins did not depend on a lack of cholesterol production, but was restored by supplementation of mevalonate or geranyl-geranyl pyrophosphate, prerequisites for prenylation of small G proteins. In conclusion, statins display a selective apoptotic effect on human hepatoma cells, with fluva-, simva- and lovastatin being both, most selective for tumor cells and most effective in inducing tumor cell apoptosis. Additionally, our results implicate that anti-tumor activity of statins requires cell proliferation and is reduced by p53 overexpression.

Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells. We have reported selective induction of apoptosis in cancer cells by the natural compound pancratistatin (PST). Recently, a novel PST analogue, a C-1 acetoxymethyl derivative of 7-deoxypancratistatin (JCTH-4), was produced by de novo synthesis and it exhibits comparable selective apoptosis inducing activity in several cancer cell lines. Recently, autophagy has been implicated in malignancies as both pro-survival and pro-death mechanisms in response to chemotherapy. Tamoxifen (TAM) has invariably demonstrated induction of pro-survival autophagy in numerous cancers. In this study, the efficacy of JCTH-4 alone and in combination with TAM to induce cell death in human breast cancer (MCF7) and neuroblastoma (SH-SY5Y) cells was evaluated. TAM alone induced autophagy, but insignificant cell death whereas JCTH-4 alone caused significant induction of apoptosis with some induction of autophagy. Interestingly, the combinatory treatment yielded a drastic increase in apoptotic and autophagic induction. We monitored time-dependent morphological changes in MCF7 cells undergoing TAM-induced autophagy, JCTH-4-induced apoptosis and autophagy, and accelerated cell death with combinatorial treatment using time-lapse microscopy. We have demonstrated these compounds to induce apoptosis/autophagy by mitochondrial targeting in these cancer cells. Importantly, these treatments did not affect the survival of noncancerous human fibroblasts. Thus, these results indicate that JCTH-4 in combination with TAM could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells.

Trends in the treatment of cutaneous T-cell lymphoma – critical evaluation and perspectives on vorinostat

Epigenetic modification with small molecule histone deacetylase inhibitors has been a promising new anti-neoplastic approach for various solid and hematological malignancies, particularly cutaneous T-cell lymphoma (CTCL). Oral vorinostat was the first histone deacetylase inhibitor approved to enter the clinical oncology market for treating CTCL patients who have progressive, persistent, or recurrent disease after failing two systemic therapies. In two phase II clinical trials, oral vorinostat was found to be safe and effective at a dose of 400 mg/day, with an overall response rate of 24%-30% in heavily pretreated patients with advanced CTCL, including those with large-cell transformed mycosis fungoides and Sezary syndrome. About half of CTCL patients receiving vorinostat also experienced substantial relief in pruritus and thus a marked improvement in quality of life. A subsequent follow-up study reported long-term safety and clinical benefits of vorinostat in patients with refractory CTCL, regardless of previous treat- ment failures. The most frequent side effects of vorinostat include gastrointestinal symptoms, fatigue, and thrombocytopenia. These adverse reactions are dose-related and reversible upon cessation of therapy. Preclinical studies have supported the therapeutic potential of vorinostat by demonstrating in vitro and in vivo anti-tumor activities against CTCL, including selective induction of apoptosis in malignant T cells, inhibition of angiogenesis, suppression of signal transducer and activator of transcription proteins, and up-regulation of pro-apoptotic proteins. Identification of biomarkers of response and resistance will help select CTCL patients most likely to benefit from treatment and guide the design of effective combination therapies.

Fluorescence Guided Resection and Photodynamic Therapy in Meningiomas

Meningiomas represent 30% of all intracranial neoplasms. They are predominantly slow growing, extra axial brain tumours arising from arachnoidal cells and usually have an excellent prognosis if completely removed. However, radical tumour removal near vital functional structures and areas of infiltration is not always possible and histologically "benign" meningiomas may exhibit aggressive behaviour like infiltration and early recurrence. Biological markers to make areas of infiltration visible by fluorescence might therefore have a significant impact on patient survival and quality of life. 5-Aminolevulinic acid (5-ALA) is a precursor in the cellular heme biosynthesis. The application of exogenous 5-ALA leads to an intracellular accumulation of protoporphyrin IX (PpIX), causing PpIX saturated cells to become fluorescent and photosensitive under light of an appropriate wavelength. 5-ALA induced PpIX fluorescence has the ability to define infiltration zones into dural structures and bone and mark residual meningioma tissue. It has the potential to facilitate meningioma resection and to individualize the extent of dural resection for each patient. Specific intracellular accumulation of PpIX can be used for photodynamic therapy (PDT). Induction of selective apoptosis, reduction of tumour vessel density and no risk of secondary carcinogenesis make PDT an ideal treatment option for meningiomas. Due to the variable potential for PpIX accumulation within different meningioma subtypes, further research is required to ensure sufficiently intense fluorescence to enable PDT in these cases.

Selective induction of apoptosis by HMG-CoA-reductase inhibitors in hepatoma cells is dependent on p53 expression

Background and Aims: HMG-CoA-reductase-inhibitors (statins) are widely used drugs to interfere with cholesterol biosynthesis. Besides this usage, evidence increases that statins might also be useful in therapy of viral infections or cancer. We investigated the effects of statins on primary mouse and human hepatocytes as well as mouse (Hepa1–6) and human (Huh7, HepG2, Hep3B) hepatoma cell lines. Methods: Effects of fluva-, simva-, atorva-, rosuva-, prava- and lovastatin on viability of primary mouse and human hepatocytes as well as mouse (Hepa1–6) and human (Huh7, HepG2, Hep3B) hepatoma cell lines were measured by MTT assay. Stable knockdown of p53 was performed by lentiviral shRNA transfer into Huh7 cells. Prenylation was restored by supplementation of geranyl-geranyl pyrophosphate (GGPP). Results: Fluva-, simva- and lovastatin selectively reduced mouse hepatoma cells viability. Human hepatoma cell lines HepG2 and Hep3B cell lines showed a significant reduction of cell viability, while statins did not reduce viability of Huh7 cells at concentrations not toxic for primary hepatocytes. Stable knockdown of endogenous p53, which is over represented in Huh7 cells in comparison to HepG2 and Hep3B cells, was able to induce susceptibility towards statin-induced toxicity in Huh7 cells. The anti-tumor effect of statins did not depend on a lack of cholesterol, but was restored by supplementation of GGPP, a prerequisite for prenylation of small G proteins. Conclusions: Certain statins display a selective apoptotic effect on human hepatoma cells, with lovastatin being both, most selective for tumor cells and most effective in inducing tumor cell apoptosis. Additionally, our results provide evidence that the p53 expression status seem to be crucial for statin effects on hepatoma cells.

Combination of TRAIL and actinomycin D liposomes enhances antitumor effect in non-small cell lung cancer

The intractability of non-small cell lung cancer (NSCLC) to multimodality treatments plays a large part in its extremely poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cytokine for selective induction of apoptosis in cancer cells; however, many NSCLC cell lines are resistant to TRAIL-induced apoptosis. The therapeutic effect can be restored by treatments combining TRAIL with chemotherapeutic agents. Actinomycin D (ActD) can sensitize NSCLC cells to TRAIL-induced apoptosis by upregulation of death receptor 4 (DR4) or 5 (DR5). However, the use of ActD has significant drawbacks due to the side effects that result from its nonspecific biodistribution in vivo. In addition, the short half-life of TRAIL in serum also limits the antitumor effect of treatments combining TRAIL and ActD. In this study, we designed a combination treatment of long-circulating TRAIL liposomes and ActD liposomes with the aim of resolving these problems. The combination of TRAIL liposomes and ActD liposomes had a synergistic cytotoxic effect against A-549 cells. The mechanism behind this combination treatment includes both increased expression of DR5 and caspase activation. Moreover, systemic administration of the combination of TRAIL liposomes and ActD liposomes suppressed both tumor formation and growth of established subcutaneous NSCLC xenografts in nude mice, inducing apoptosis without causing significant general toxicity. These results provide preclinical proof-of-principle for a novel therapeutic strategy in which TRAIL liposomes are safely combined with ActD liposomes.