α-Tocopheryl succinate sensitizes established tumors to vaccination with nonmatured dendritic cells

Purpose: Dendritic cells (DCs) are considered potential candidates for cancer immunotherapy due to their ability to process and present antigens to T cells and stimulate immune responses. However, DC-based vaccines have exhibited minimal effectiveness against established tumors in mice and human cancer patients. The use of appropriate adjuvants can enhance the efficacy of DC-based cancer vaccines in treating established tumors. Methods: In this study we have employed -tocopheryl succinate (-TOS), a nontoxic esterified analogue of vitamin E, as an adjuvant to enhance the effectiveness of DC vaccines in treating established murine Lewis lung (3LL) carcinomas. Results: We demonstrate that locally or systemically administered -TOS in combination with nonmatured DCs injected intratumorally (i.t.) or subcutaneously (s.c.) significantly inhibits the growth of preestablished 10-day tumors (mean tumor volume of 77.5 ± 17.8 mm³ on day 30 post–tumor injection) as compared to -TOS alone (mean tumor volume of 471 ± 68 mm³ on day 30 post–tumor injection). Additionally, the adjuvant effect of -TOS was superior to that of cyclophosphamide (CTX). The mean tumor volume on day 28 post–tumor injection in mice treated with CTX+DCs was 611 ± 94 mm³ as compared to 105 ± 36 mm³ in mice treated with -TOS+DCs. Analysis of purified T lymphocytes from mice treated with -TOS+DC revealed significantly increased secretion of IFN- as compared to T cells from the various control groups. Conclusion: This study demonstrates the potential usefulness of -tocopheryl succinate, an agent nontoxic to normal cell types, as an adjuvant to augment the effectiveness of DC-based vaccines in treating established tumors.Abbreviations AO acridine orange - CTX cyclophosphamide - DC dendritic cell - dUTP deoxyuridine triphosphate - FACS fluorescence-activated cell sorter - FBS fetal bovine serum - FITC fluorescein isothiocyanate - GM-CSF granulocyte-macrophage colony-stimulating factor - IFN- interferon-gamma - IL-4 interleukin-4 - NaS sodium succinate - OCT optimal cutting temperature - PBS phosphate-buffered saline - PI propidium iodide - Tdt terminal deoxynucleotidyl transferase - TNF- tumor necrosis factor alpha - -TOS -tocopheryl succinateSupported by grants 1 RO1 CA94111-02 from the NIH and DAMD 17010126 from the DOD.     

α-Tocopheryl succinate causes mitochondrial permeabilization by preferential formation of Bak channels

Mitocans are drugs selectively killing cancer cells by destabilizing mitochondria and many induce apoptosis via generation of reactive oxygen species (ROS). However, the molecular events by which ROS production leads to apoptosis has not been clearly defined. In this study with the mitocan α-tocopheryl succinate (α-TOS) the role of the Bcl-2 family proteins in the mechanism of malignant cell apoptosis has been determined. Exposure of several different cancer cell lines to α-TOS increased expression of the Noxa protein, but none of the other proteins of the Bcl-2 family, an event that was independent of the cellular p53 status. α-TOS caused a profound conformational change in the pro-apoptotic protein, Bak, involving oligomerization in all cell types, and this also applied to the Bax protein, but only in non-small cell lung cancer cells. Immunoprecipitation studies indicated that α-TOS activates the two BH1-3 proteins, Bak or Bax, to form high molecular weight complexes in the mitochondria. RNAi knockdown revealed that Noxa and Bak are required for α-TOS-induced apoptosis, and the role of Bak was confirmed using Bak- and/or Bax-deficient cells. We conclude that the major events induced by α-TOS in cancer cells downstream of ROS production leading to mitochondrial apoptosis involve the Noxa-Bak axis. It is proposed that this represents a common mechanism for mitochondrial destabilization activated by a variety of mitocans that induce accumulation of ROS in the early phases of apoptosis.     

KHC-4 inhibits β-catenin expression in prostate cancer cells

ABSTRACT

KHC-4 is a 2-phenyl-4-quinolone analogue that exhibits anticancer activity. Aberrant activation of β-catenin signaling contributes to prostate cancer development and progression. Therefore, targeting β-catenin expression could be a useful approach to treating prostate cancer. We found that KHC-4 can inhibit β-catenin expression and its signaling pathway in DU145 prostate cancer cells. Treatment with KHC-4 decreased total β-catenin expression and concomitantly decreased β-catenin levels in both the cytoplasm and nucleus of cells. KHC-4 treatment also inhibited β-catenin expression and that of its target proteins, PI3K, AKT, GSK3β and TBX3. We monitored the stability of β-catenin with the proteasomal inhibitor, MG132, in DU145 cells and found that MG132 reversed KHC-4-induced proteasomal β-catenin degradation. We verified CDK1/β-catenin expression in KHC-4 treated DU145 cells. We found that roscovitine treatment reversed cell proliferation by arresting the cell cycle at the G2/M phase and β-catenin expression caused by KHC-4 treatment. We suggest that KHC-4 inhibits β-catenin signaling in DU145 prostate cancer cells.     

Do neuroendocrine cells in human prostate cancer express androgen receptor?

The presence of androgen receptors (AR) in neuroendocrine cells was investigated in benign tissue of 10 prostatectomy specimens, in 12 prostatic adenocarcinomas with focal neuroendocrine differentiation and in 1 case of a pure neuroendocrine small cell carcinoma of the prostate. Neuroendocrine cells were defined by their reactivity with an antibody to chromogranin A. Monoclonal antibody F39.4 directed against the amino-terminal domain of the AR molecule was used to detect AR. AR and chromogranin A were simultaneously visualized with a double immunofluorescence technique. The results indicate that chromogranin positive cells in both benign and malignant prostatic tissue lack detectable expression of AR. No effect of endocrine therapy was noted. These results are in agreement with the hypothesis that prostatic neuroendocrine tumour cells represent an androgen insensitive cell population, which incidentally may expand to replace the androgen-sensitive tumour cell population during androgen ablation therapy.     

α-Tocopheryl succinate stabilizes the structure of tumor vessels by inhibiting angiopoietin-2 expression

α-Tocopheryl succinate (TS) is a tocopherol derivative and has multifaceted anti-cancer effects; TS not only causes cancer cell-specific apoptosis but also inhibits tumor angiogenesis. Although TS has the potential to be used as a well-tolerated anti-angiogenic drug, it is still unclear which step of the angiogenic process is inhibited by TS. Here, we show that TS inhibits the expression of angiopoietin (Ang)-2, which induces destabilization of vascular structure in the initial steps of the angiogenic process. In mouse melanoma cells, TS treatment decreased mRNA and extracellular protein levels of Ang-2; however, the mRNA level of Ang-1, which stabilizes the vascular structure, remained unchanged. Furthermore, aorta ring and Matrigel plug angiogenesis assays indicated that the conditioned medium from TS-treated cells (CM-TS) inhibited neovascularization and blood leakage from the existing blood vessels, respectively. Following immunohistochemical staining of the vessels treated with CM-TS, imaging studies showed that the vascular endothelial cells were highly packed with pericytes. In conclusion, we found that TS inhibits Ang-2 expression and, consequently, stabilizes the vascular structure during the initial step of tumor angiogenesis.     

α-Santalol, a derivative of sandalwood oil, induces apoptosis in human prostate cancer cells by causing caspase-3 activation

The anticancer effects of α-santalol, a major component of sandalwood oil, have been reported against the development of certain cancers such as skin cancer both in vitro and in vivo. The primary objectives of the current study were to investigate the cancer preventive properties of α-santalol on human prostate cancer cells PC-3 (androgen independent and P-53 null) and LNCaP (androgen dependent and P-53 wild-type), and determine the possible mechanisms of its action. The effect of α-santalol on cell viability was determined by trypan blue dye exclusion assay. Apoptosis induction was confirmed by analysis of cytoplasmic histone-associated DNA fragmentation using both an apoptotic ELISA kit and a DAPI fluorescence assay. Caspase-3 activity was determined using caspase-3 (active) ELISA kit. PARP cleavage was analyzed using immunoblotting. α-Santalol at 25-75 μM decreased cell viability in both cell lines in a concentration and time dependent manner. Treatment of prostate cancer cells with α-santalol resulted in induction of apoptosis as evidenced by DNA fragmentation and nuclear staining of apoptotic cells by DAPI. α-Santalol treatment also resulted in activation of caspase-3 activity and PARP cleavage. The α-santalol-induced apoptotic cell death and activation of caspase-3 was significantly attenuated in the presence of pharmacological inhibitors of caspase-8 and caspase-9. In conclusion, the present study reveals the apoptotic effects of α-santalol in inhibiting the growth of human prostate cancer cells.     

eIF2 kinases mediate β-lapachone toxicity in yeast and human cancer cells

β-lapachone (β-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with β-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in β-lap treated cells. β-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitocondrial NADH dehydrogenase Nde2p was found to be resistant to β-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by β-lap were abolished in the nde2Δ mutant. Amino acid biosynthesis genes were also induced in β-lap treated cells, suggesting that β-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, β-lap treatment increased phosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase. eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to β-lap and to elicit checkpoint responses. Remarkably, β-lap treatment increased phosphorylation of eIF2α in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of β-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses.     

Distinct mechanisms account for β-amyloid toxicity in PC12 and differentiated PC12 neuronal cells

Whether reactive oxygen species (ROS) mediate beta-amyloid (A beta) neurotoxicity remains controversial. Naive PC12 cells (PC12) and nerve growth factor-differentiated PC12 cells (dPC12) were used to study the role of ROS in cell death induced by A beta(25-35). The viability of PC12 and dPC12 cells decreased by 30-40% after a 48-hour exposure to 20 microM A beta(25-35). Microscopic examination showed that A beta(25-35) induced necrosis in PC12 cells and apoptosis in dPC12 cells. Vitamin E (100 microM) and other antioxidants protected PC12 cells, but not dPC12 cells, against the cytotoxic effect of A beta(25-35). Since H(2)O(2) has been proposed to be involved in A beta toxicity, the effects of H(2)O(2) on PC12 and dPC12 cells were studied. Differentiated PC12 cells appeared to be significantly more resistant to H(2)O(2) than naive PC12 cells. These data suggest that ROS may mediate A beta(25-35) toxicity in PC12 cells but not in dPC12 cells. Because the intracellular levels of ROS were elevated during the differentiation of PC12 cells, the baseline levels of ROS in these two model cell types may determine the intracellular mediators for A beta(25-35) toxicity. Therefore, the protective effects of antioxidants against A beta may depend upon the redox state of the cells.     

Stem cells: The root of prostate cancer?

The cancer stem cell (CSC) model states that tumors contain a reservoir of self-renewing cells that maintain the heterogeneous cell population of the tumor. These cells appear to be resistant to therapy and can therefore survive to repopulate the tumor during progression to therapy resistant disease. The biology of CSCs is still not definitive since it is difficult to isolate them from solid tumors and analyze their characteristics in vitro. Another challenge is to correlate these characteristics with tumor development and progression in vivo. Using the prostate CSC as a model, this review presents the CSC hypothesis, reviews the origin, identification and functions of prostate CSCs, and discusses the clinical implications and therapeutic challenges CSCs have for cancer therapy.     

Vinpocetine attenuates the metabolic dysfunction induced by amyloid β-peptides in PC12 cells

The cytoprotective effect of vinpocetine [14-ethoxycarbonyl-(3α,16α-ethyl)-14,15-eburnamine] was investigated on PC12 cells treated with the amyloid β-peptides (Aβ) for 24 hours. Vinpocetine was shown to protect cells from the inhibition in redox status induced by exposure to Aβ_25–35 and Aβ_1–40, the maximal protection being achieved at a vinpocetine concentration of 40 μM. At this concentration, vinpocetine blocked the inhibition of the mitochondrial respiratory chain complexes II–III and IV and completely abolished the depletion of pyruvate levels induced by toxic concentrations of Aβ peptides. Furthermore, the accumulation of ROS in cells exposed to Aβ_25–35 and Aβ_1–40 evaluated using the fluorescent probe 2′,7′-dichlorofluorescin (DCF), was reduced in the presence of 40 μM vinpocetine. Taken together, the data presented herein demonstrate that vinpocetine protects cells from Aβ toxicity, preventing the generation of oxidative stress due to the excessive accumulation of ROS. This study suggests that vinpocetine can exert neuroprotective properties which might be of importance and contribute to its clinical efficacy in the treatment of Alzheimer's disease or other neurodegenerative disorders in which oxidative stress is involved.     

α-Tocopheryl phosphate: uptake, hydrolysis, and antioxidant action in cultured cells and mouse

α-Tocopheryl phosphate (α-TP), a water-soluble analogue of α-tocopherol, is found in humans, animals, and plants. α-TP is resistant to both acid and alkaline hydrolysis and may exert its own function in this form in vivo. In this study, the uptake, hydrolysis, and antioxidant action of α-TP were measured using α-TP with a deuterated methyl group, CD(3), at position 5 of the chroman ring (α-TP(CD3)). The hydrolysis of α-TP(CD3) was followed by measuring α-tocopherol containing the CD(3) group, α-T(CD3), in comparison to unlabeled α-tocopherol, α-T(CH3). α-TP(CD3) was incubated with cultured cells, and the intracellular α-T(CD3) formed was measured with HPLC-ECD and GC-MS. α-TP(CD3) was also administered to mice for 4 weeks by mixing in the diet, and α-T(CD3) was measured in plasma, liver, brain, heart, and testis to compare with endogenous unlabeled α-T(CH3). It was found that α-TP(CD3) was taken in and hydrolyzed readily to α-T(CD3) in cultured cells and in mice. The hydrolysis of α-TP(CD3) in cell culture medium was not observed. α-TP protected primary cortical neuronal cells from glutamate-induced cytotoxicity, and α-TP given to mice reduced the levels of lipid peroxidation products in plasma and liver. These results suggest that α-TP is readily hydrolyzed in vivo to α-T, which acts as an antioxidant, and that α-TP may be used as a water-soluble α-T precursor in intravenous fluids, in eye drops, or as a dietary supplement.     

Metafectene is superior to lipofectamine in the transfection of Gsα prostate cancer cells

Transfection efficiency of the novel reagent metafectene has not been compared with that of lipofectamine in the published English literature. We used these agents to transfect two prostate cancer cell lines, PC3 and G_sα, with a deoxyribonucleic acid (DNA) expression vector that generates double-stranded ribonucleic acid (RNA) for RNA interference (RNAi). Cotransfection of the green fluorescent protein (GFP) reporter gene revealed that the mean (± standard deviation) transfection efficiencies with lipofectamine were 5.8±0.4% for PC3 cells and 3.6±1.5% for G_sα cells. Mean transfection efficiency with metafectene declined to 0.1±0% for PC3 cells but improved to 54.6±5.5% for G_sα cells. With G_sα cells, metafectene transfection of GFP plasmid alone yielded 46.9% positive cells, and cotransfection with CD44v9 expression vector yielded 45.9% positive cells. The visual impact of the transfected RNAi construct was detectable at the protein level 4 to 6 d posttransfection and was more dramatic after using metafectene than after using lipofectamine. Thus, in vitro, metafectene transfection efficiency was sufficient to allow us to assess the functional significance of our RNAi construct, suggesting metafectene as an excellent candidate for RNAi-mediated anticancer gene therapy.     

Effect of thapsigargin on Ca²+ fluxes and viability in human prostate cancer cells

Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca2?](i) levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca2?-sensitive fluorescent probe. Thapsigargin at concentrations between 10?nM and 10 μM increased [Ca2?](i) in a concentration-dependent fashion. The Ca2? signal was reduced partly by removing extracellular Ca2? indicating that Ca2? entry and release both contributed to the [Ca2?](i) rise. This Ca2? influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca2? channels or by modulation of protein kinase C activity. In Ca2?-free medium, pretreatment with the endoplasmic reticulum Ca2? pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca2? release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca2?](i) rise, suggesting that thapsigargin released Ca2? from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca2?](i) rise. At concentrations of 1-10 μM, thapsigargin induced cell death that was partly reversed by chelation of Ca2? with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca2?](i) rises by causing phospholipase C-independent Ca2? release from the endoplasmic reticulum and Ca2? influx via phospholipase A2-sensitive Ca2? channels. Thapsigargin also induced cell death via Ca2?-dependent pathways and Ca2?-independent apoptotic pathways.     

NKX3.1 potentiates TNF-α/CHX-induced apoptosis of prostate cancer cells through increasing caspase-3 expression and its activity

NKX3.1, a prostate-specific homeobox gene, plays an important role in prostate cancer and usually functions as tumor suppressor gene. Previously we have demonstrated that forced expression of NKX3.1 reduced cell growth and invasion in prostate cancer cell line PC-3. Presently, we investigated the effect of NKX3.1 on the sensitivity of the prostate cancer cells to apoptosis inducer tumor necrosis factor-α (TNF-α) and cycloheximide (CHX). PC-3 cells were transfected with NKX3.1 expression plasmid (pcDNA3.1-NKX3.1) and LNCaP cells were transfected with siRNA expression plasmid (pRNAT-RNAi1) targeting NKX3.1. The cell morphology and apoptotic rate were analyzed by Hoechst 33342 staining and Flow Cytometry in absence or presence of TNF-α and CHX. The activity of caspase-3 was determined using DEVD-pNA as substrate. Simultaneously, the effect of NKX3.1 on caspase-3 expression was detected using RT-PCR and Western blot. The results showed that ectopic expression of NKX3.1 promoted TNF-α/CHX-induced apoptosis in PC-3 cells, whereas knockdown of NKX3.1 protected LNCaP cells from apoptosis induced by TNF-α/CHX. The pro-apoptosis activity of NKX3.1 might partially contribute to its elevation of caspase-3 expression and activity. Manipulating NKX3.1 expression should be a promising therapeutic strategy for treating both androgen-dependent and androgen-independent prostate cancer.