Human Prostate Carcinoma DU145 Cells Research Articles

Decreased intracellular chloride enhances cell migration and invasion via activation of the ERK1/2 signaling pathway in DU145 human prostate carcinoma cells

Our previous study revealed that changes of the intracellular Cl− concentration ([Cl−]i) affected cell proliferation in cancer cells. However, the role of Cl− on cell migration and invasion in cancer cells remains unanalyzed. Therefore, the aim of the present study is to investigate whether changes of [Cl−]i affects cell migration and invasion of cancer cells. In human prostate cancer DU145 cells, cell migration and invasion were enhanced by culturing in the low Cl− medium (replacement of Cl− by NO3−). We also found that DU145 cells in the low Cl− condition caused significant transient ERK1/2 activation followed by an increase of MMP-1 mRNA levels. Inhibition of ERK1/2 activation in the low Cl− condition reduced enhancement of MMP-1 mRNA levels and decreased cell migration and invasion. These observations indicate that [Cl−]i plays important roles in metastatic function by regulating the ERK1/2 signaling pathway in human prostate cancer cells, and intracellular Cl− would be one of the key targets for anti-cancer therapy.

Xenografts on nude mouse diaphragm of human DU145 prostate carcinoma cells: mesothelium removal by outgrowths and angiogenesis

ABSTRACT Human prostate carcinoma DU145 cells, androgen-independent malignant cells, implanted in the athymic nu/nu male mouse, developed numerous tumors on peritoneal and retro-peritoneal organs whose growth aspects and vascular supply have yet to be investigated with fine structure techniques. A series of necropsies from moribund implanted mice diaphragms were examined with light, scanning, and transmission electron microscopy. DU145 xenografts installations, far away from the implanted site, were described as the smallest installation to large diaphragm outgrowths in moribund mice. Carcinomas did not show extracellular matrix and, reaching more than 0.15 mm in thickness, they revealed new structures in these outgrowths. Voids to be gland-like structures with mediocre secretion and, unexpectedly, intercellular spaces connected with fascicles of elongated DU145 cells that merged with a vascular supply originated from either the tumor cells and/or some perimysium vessels. In the largest carcinomas, most important vascular invasions coincidently accompanied the mouse lethality, similarly to human cancers. This androgen-independent model would be useful to study tumor outgrowth’s changes related to testing anticancer strategy, including anti-angiogenic therapies involving toxicity, simultaneously with those of other vital organs with combined biomolecular and fine structure techniques.

Three new compounds from the twigs and leaves of Nageia fleuryi Hickel

Two new diterpenoids, 12,15-di-O-acetylhypargenin B (1) and taiwanin F-12-O-β-D-glucopyranoside (2), one new monoterpenoid, (S)-7-methyl-3-methyleneoct-6-ene-1,2-diyl diacetate (3), together with eight known compounds (4−11), were obtained from the twigs and leaves of Nageia fleuryi Hickel. The structures of the new compounds were elucidated by extensive spectroscopic techniques including HR-ESI-MS and 1 D and 2 D NMR experiments. Spectroscopic data of the known compound 4 were provided for the first time. Compounds 1 and 11 exhibited strong inhibitory activity on LPS-stimulated production of NO in RAW 264.7 murine macrophages, while compounds 1, 3, and 5 showed significant quinone reductase inducing activity in Hepa 1c1c7 murine hepatoma cells. Moreover, compounds 7 and 8 showed inhibitory activity against the proliferation of the human prostate carcinoma DU145 cells.

Fluorescent Products of Anthocyanidin and Anthocyanin Oxidation.

We found that the oxidation of cyanidin with hydrogen peroxide, tert-butyl hydroperoxide, 2,2'-azobis(2-methylpropionamidine), 3-morpholinosydnonimine hydrochloride, sodium hypochlorite, Fe3+, and Fe2+ induced the appearance of a new fluorescence band, centered at 525-540 nm when excited at 470-490 nm. The intensity of this fluorescence was related to the oxidant concentration. The same fluorescence was induced by the oxidation of other anthocyanidins, an anthocyanin (cyanidin-3-O-glucoside) as well as anthocyanin-rich red cabbage extract, and blackcurrant and elderberry juices. Peroxidized blood plasma also induced the appearance of cyanidin fluorescence. We also showed that the formation of the fluorescent product of pelargonidin can be observed in a culture of the MRC-5 human fetal lung fibroblast cell line and the DU-145 human prostate carcinoma cell line. Our results suggest that this new anthocyanidin/anthocyanin fluorescence may be an indicator of oxidation, especially of food products, where these compounds are present or added as colorants, and can also be useful to detect oxidation in biomedical experiments.

Synergistic Impact of Xanthorrhizol and d-δ-Tocotrienol on the Proliferation of Murine B16 Melanoma Cells and Human DU145 Prostate Carcinoma Cells

Isoprenoids suppress the mevalonate pathway that provides prenyl groups for the posttranslational modification of growth-regulating proteins. We hypothesize that xanthorrhizol and d-δ-tocotrienol synergistically suppress the growth of murine B16 melanoma and human DU145 prostate carcinoma cells. Xanthorrhizol (0–200 µmol/L; half maximal inhibitory concentration [IC50] = 65 µmol/L) and d-δ-tocotrienol (0–40 µmol/L; IC50 = 20 µmol/L) each induced a concentration-dependent suppression of the proliferation of B16 cells and concurrent cell cycle arrest at the G1 phase. A blend of 16.25 µmol/L xanthorrhizol and 10 µmol/L d-δ-tocotrienol suppressed B16 cell proliferation by 69%, an impact greater than the sum of those induced by xanthorrhizol (15%) and d-δ-tocotrienol (12%) individually. The blend cumulatively reduced the levels of cyclin-dependent kinase four and cyclin D1, key regulators of cell cycle progression at the G1 phase. The expression of RAS and extracellular signal-regulated kinase (ERK1/2) in the proliferation-stimulating RAS-RAF-MEK-ERK pathway was downregulated by the blend. Xanthorrhizol also induced a concentration-dependent suppression of the proliferation of DU145 cells with concomitant morphological changes. Isobologram confirmed the synergistic effect of xanthorrhizol and d-δ-tocotrienol on DU145 cell proliferation with combination index values ranging 0.61-0.94. Novel combinations of isoprenoids with synergistic actions may offer effective approaches in cancer prevention and therapy.

The Effects of (11R)-13-(6-Nitroindazole)-11,13-Dihydroludartin on Human Prostate Carcinoma Cells and Mouse Tumor Xenografts

BackgroundThis study aimed to investigate the effects of the 6-nitroindazole compound and amino analog of ludartin, (11R)-13-(6-nitroindazole)-11,13-dihydroludartin (NDHL), on human prostate carcinoma cells in vitro and in mouse tumor xenografts in vivo.Material/MethodsDU-145 and LNCaP human prostate carcinoma cells were cultured with increasing concentrations of NDHL. Cell viability was measured using the MTT assay, and cell apoptosis was measured by fluorescence flow cytometry. Mouse tumor xenografts were created by implanting 2×106 of DU-145 cells subcutaneously in the left flank. On the second day following DU-145 cell implantation, the mice in the treatment groups were injected intraperitoneally with 2, 5, and 10 mg/kg of NDHL.ResultsTreatment of DU-145 and LNCaP cells with NDHL (range, 2.5–20.0 μM) significantly reduced cell proliferation in vitro (P<0.05). The proliferation rate of DU-145 and LNCaP cells was reduced to 27% and 24%, respectively, following treatment with 20.0 μM of NDHL. Treatment with NDHL significantly increased cell apoptosis and the formation of reactive oxygen species (ROS) formation in DU-145 cells at 48 h (P<0.05). NDHL significantly increased the proportion of DU-145 cells in the G1 phase of the cell cycle and significantly increased the expression of cyclin D1 and p21 (P<0.05). Treatment of the mice in the xenograft tumor model with NDHL significantly increased survival and suppressed tumor growth (P<0.02).ConclusionsNDHL inhibited cell proliferation, increased apoptosis, and caused cell cycle arrest in human prostate carcinoma cells in vitro and inhibited mouse tumor xenograft growth in vivo.

Lipophilized Epigallocatechin Gallate Derivative Exerts Anti-Proliferation Efficacy through Induction of Cell Cycle Arrest and Apoptosis on DU145 Human Prostate Cancer Cells.

Epigallocatechin gallate (EGCG) is the predominant tea polyphenol and it exhibits a hydrophilic character. The lipophilized EGCG derivative (LEGCG) was synthesized by enzymatic esterification of EGCG with lauric acid to enhance its bioactivity. The tetralauroyl EGCG was confirmed by high-performance liquid chromatography-tandem mass spectrometry and further identified as 3′, 5′, 3″, 5″-4-O-lauroyl EGCG by 1H and 13C nuclear magnetic resonance. The anti-proliferation effect of LEGCG on DU145 human prostate carcinoma cells was evaluated by MTT assay. In addition, the underlying molecular mechanism by which LEGCG exerts anti-proliferation efficacy was elucidated by flow cytometry and immunoblot analysis. Results suggested that LEGCG exhibited a dose-dependent anti-proliferation effect on DU145 cells by G0/G1 phase arrest and induction of apoptosis. LEGCG induced cell cycle arrest via p53/p21 activation, which down-regulated the cyclin D1 and CDK4 expression. In addition, LEGCG induced apoptosis by increasing the Bax/Bcl-2 ratio, the cytochrome c release, and the caspases cleavage on DU145 cells. The results provide theoretical support to prevent prostate cancer with LEGCG.

Synergistic Impact of Xanthorrhizol and d-δ-tocotrienol on the Proliferation of Murine B16 Melanoma Cells and Human DU145 Prostate Carcinoma Cells (P06-042-19)

ObjectivesXanthorrhizol, a sesquiterpene, and d-δ-tocotrienol, a vitamin E molecule, each suppresses the proliferation of a number of tumor cells. This study aims to examine the potentially synergistic effect of xanthorrhizol and d-δ-tocotrienol in tumor cells. MethodsMurine B16 melanoma and human DU145 prostate carcinoma cells were incubated for 48 h (B16) or 72 h (DU145) with xanthorrhizol or d-δ-tocotrienol before cell populations were determined by CellTiter 96â Aqueous One Solution. Cells incubated with the agents for 24 hours were stained with propidium iodide and analyzed for cell cycle using flow cytometry and MultiCycle AV. Isobologram and combination index (CI) were used to demonstrate their synergistic anti-proliferative impacts. ResultsXanthorrhizol (0–200 μmol/L) and d-δ-tocotrienol (0–40 μmol/L) each elicited a concentration-dependent suppression of the proliferation of B16 cells. A blend of 16.25 μmol/L xanthorrhizol and 10 μmol/L d-δ-tocotrienol achieved 69% (P < 0.05) growth suppression of B16 cells, exceeding the sum of individual effects. B16 cells incubated with 5 and 10 μmol/L d-δ-tocotrienol for 24-h had a concentration-dependent increase in the percentage of cells in the G1 phase with a concomitant decrease in the percentage of cells in the S phase. The G1/S ratio, an indicator of cell cycle arrest at the G1 phase, increased from 1.73 ± 0.05 (Control) to 2.01 ± 0.10 (5 μmol/L) and 2.73 ± 0.05 (10 μmol/L). A parallel pattern of concentration-dependent increase in the G1/S ratio was induced by xanthorrhizol at concentrations equivalent to 25% (16.25 μmol/L) and 50% (32.5 μmol/L) of its IC50 value. A blend of 5 μmol/L d-δ-tocotrienol and 16.25 μmol/L xanthorrhizol, each at no-effect concentrations, significantly increased the percentage of B16 cells in the G1 phase to 62.6 ± 0.6%. Isobologram and CI confirmed the synergistic effect of xanthorrhizol (50 and 100 μmol/L) and d-δ-tocotrienol (10 and 20 μmol/L) on the proliferation of DU145 cells. ConclusionsXanthorrhizol and d-δ-tocotrienol synergistically suppress tumor cell proliferation by inducing G1 arrest and may have potential in cancer prevention and therapy. Funding SourcesAmerican River Nutrition, Inc. and the University Assistantship Program and the Department of Nutrition of Georgia State University.

Dietary antioxidants as a source of hydrogen peroxide.

Studies of 54 antioxidants revealed that 27 of them, mainly polyphenols, generated hydrogen peroxide (H2O2) when added to Dulbecco's modified Eagle's medium (DMEM), other media used for culture of mammalian and yeast cells and phosphate-buffered saline. The most active antioxidants were: propyl gallate (PG), (-)-epigallocatechin gallate (EGCG) and quercetin (Q). Chelex treatment and iron chelators decreased H2O2 generation suggesting that transition metal ions catalyze antioxidant autoxidation and H2O2 production. Green tea also generated H2O2; tea prepared on tap water generated significantly more H2O2 than tea prepared on deionized water. Ascorbic acid decreased H2O2 production although it generated H2O2 itself, in the absence of other additives. Lemon added to the tea significantly reduced generation of H2O2. Hydrogen peroxide generated in the medium contributed to the cytotoxicity of PG, EGCG and Q to human prostate carcinoma DU-145 cells, since catalase increased the survival of the cells subjected to these compounds in vitro.

Effect of Levisticum officinale Hydroalcoholic Extract on DU-145 and PC-3 Prostate Cancer Cell Lines

: Levisticum officinale contains a variety of components used for its medicinal properties in traditional medicine. The previous studies showed that it had an anti-cancer effect on cell lines. The current study aims to evaluate the cytotoxic potential of Levisticum officinale hydroalcoholic extract (LOHE) on DU-145 and PC-3 human advanced prostate cancer cell lines. The anti-proliferative activity of LOHE was evaluated using the MTT assay. The results of MTT showed that the IC50 of DU-145 and PC-3 were 500 µg/mL and 400 µg/mL of LOHE, respectively, following 24 hours treatment. This study found that the hydroalcoholic extract of Levisticum officinale had beneficial cell death inducing effects on both the PC3 and DU145 human prostate carcinoma cell lines in time- and concentration-dependent fashion. Hence, LOHE may have remarkable potential for uses as new natural anticancer agent.

Synergistic Impact of d-δ-Tocotrienol and Geranylgeraniol on the Growth and HMG CoA Reductase of Human DU145 Prostate Carcinoma Cells

ABSTRACTThe growth-suppressive effect of d-δ-tocotrienol and geranylgeraniol is at least partially attributed to their impact on 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate-limiting enzyme in the mevalonate pathway that provides essential intermediates for the posttranslational modification of growth-related proteins including RAS. We hypothesize that these agents synergistically impact cell growth based on their complementary mechanisms of action with HMG CoA reductase. d-δ-tocotrienol (0–40 µmol/L; half maximal inhibitory concentration [IC50] = 15 µmol/L) and geranylgeraniol (0–100 µmol/L; IC50 = 60 µmol/L) each induced concentration-dependent suppression of the growth of human DU145 prostate carcinoma cells. Blends of the two agents synergistically suppressed the growth of DU145 cells, with combination index values ranging 0.67–0.75. While 7.5 µmol/L d-δ-tocotrienol and 30 µmol/L geranylgeraniol individually had no impact on cell cycle distribution in DU145 cells, a blend of the agents induced cell cycle arrest at the G1 phase. The synergistic downregulation of the expression of HMG CoA reductase by 7.5 µmol/L d-δ-tocotrienol and 30 µmol/L geranylgeraniol was accompanied by a reduction in membrane K-RAS protein. Our finding supports the cancer chemopreventive action of plant-based diets and their isoprenoid constituents. Properly formulated isoprenoids and derivatives may provide novel approaches in prostate cancer prevention and therapy.

Phthalate induced toxicity in prostate cancer cell lines and effects of alpha lipoic acid.

The effects of dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di-n-butyl phthalate, benzylbutyl phthalate, di-2-ethylhexyl phthalate were investigated on human prostate cancer cell lines DU145 and PC3 in vitro. Standards of dimethyl phthalate, diethyl phthalate, di-isobutyl phthalate, dibutyl phthalate, benzyl butyl phthalate, and di-ethyl hexyl phthalate were used. Alpha lipoic acid was used as antioxidant compound. DU145 and PC3 human prostate carcinoma cells were used. MTT assay were used for cytotoxicity assay. A low dose proliferative effect of phthalates in vitro was observed. With the hypothesis of the inhibition of aerobic glycolysis activity in cancer treatment, α-lipoic acid was applied to cells; where as a contrary to previous studies, no change in the cell proliferation was observed. In combination with ALA, at IC50 and lower doses, an increase of the cytotoxic effect was found for DIBP, DBP and BBP; while for DMP, DEP and DEHP, a decrease was observed for DU145 cells. In PC3 cells, a decrease was observed for DMP, DEP and DBPs; while no significant difference were observed for DEHP, DIBP and BBP. The present study demonstrates preliminary information regarding the low dose proliferative effects of phthalates in prostate cancer in vitro (Tab. 2, Fig. 2, Ref. 65).

Abstract B36: RENCA macrobead-induced AKT hyperphosphorylation leads to MEF2 activation and inhibition of the proliferation of human DU145 prostate carcinoma cells

Abstract We have previously reported that suppression of the isoforms of myocyte enhancer factor (MEF2) in target tumor cells in vitro reduces the demonstrated proliferation-inhibitory effect of agarose-agarose encapsulated mouse renal adenocarcinoma cells (RENCA macrobeads), suggesting that it is a critical component of a pathway by which the inhibitory action, now being evaluated also in Phase IIb human clinical trials, is mediated. Importantly, MEF2 originally described as active in myogenesis, has been found to be a key regulator of many developmental pathways and to have both pro- and anti-growth regulatory functions in murine and human tumor cells. Recent studies have supported a molecular intersection between components of the PI3K/Akt and the canonical Wnt signaling (carcinogenic) pathways, converging at the glycogen synthase kinase 3β (GSK3β) node. In skeletal and cardiac muscle cells, GSK3β regulates MEF2 activity indirectly through reciprocal regulation of the p38 mitogen-activated protein kinase (MAPK) pathway. To determine, therefore, whether PI3K/Akt is also involved in the anti-proliferative effect of the RENCA macrobeads, we evaluated signaling through PI3K/Akt in response to macrobead-conditioned media and examined post-translational modification of MEF2D in two human cell lines DU145 (prostate cancer) and MCF7 (breast adenocarcinoma). Using In-Cell Western analysis, we determined the expression of phospho-Akt T308 and S473 in parallel with total Akt in cells exposed to naïve or conditioned media. Nuclear extracts for phospho-MEF2D and total MEF2D were assessed using western blotting. In the DU145 cells, exposure to RENCA macrobead-conditioned medium resulted in Akt hyperphosphorylation (≥ 48 hours) in parallel with the increasing age and inhibitory capacity of macrobeads. Further, the growth inhibition of DU145 cells exposed to RENCA conditioned medium (36-40% at 12-16 weeks of age; 45-48% at 20-24 weeks of age) was accompanied by de-phosphorylation of MEF2D at Serine 444, supporting a role for increased MEF2D transcriptional activity in the inhibitory effect. In the MCF7 cells, which are less sensitive to RENCA macrobead inhibition (8-12% at 12-16 weeks of age; 17-21% at 20-24 weeks of age), Akt phosphorylation diminished one hour following exposure to conditioned media, and MEF2D remained phosphorylated at Serine 444. These findings support the hypothesis that RENCA macrobeads, as biological cell systems with the in vitro and in vivo (model and clinical) capability of inhibiting cancer cell proliferation and tumor growth, achieve this effect, at least in part, by signaling through Akt to differentially regulate MEF2D. Citation Format: Prithy C. Martis, Melissa A. Laramore, Atira T. Dudley, Barry H. Smith, Lawrence S. Gazda. RENCA macrobead-induced AKT hyperphosphorylation leads to MEF2 activation and inhibition of the proliferation of human DU145 prostate carcinoma cells. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B36.

Synergistic Impact of d‐δ‐Tocotrienol and Geranylgeraniol on the Growth and HMG CoA Reductase of Human DU145 Prostate Carcinoma Cells

d‐δ‐Tocotrienol and geranylgeraniol suppress the growth of diverse types of tumors in vitro and in vivo. Their growth‐suppressive effect is at least partially attributed to their impact on 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase, the rate‐limiting enzyme in the mevalonate pathway that provides essential intermediates for the posttranslational modification of growth‐related proteins including RAS and nuclear lamins. d‐δ‐Tocotrienol blocks the processing and nuclear localization of sterol regulatory element binding protein 2, the transcriptional factor for HMG CoA reductase, while d‐δ‐tocotrienol and geranylgeraniol collaboratively enhance the proteasome‐mediated degradation of HMG CoA reductase. We hypothesize that these agents have synergistic inhibitory impact on cell growth based on their complementary mechanisms of action with HMG CoA reductase. d‐δ‐Tocotrienol (0 – 40 μmol/L) and geranylgeraniol (0 – 100 μmol/L) each induced concentration‐dependent suppression of the growth of human DU145 prostate carcinoma cells as measured by CellTiter 96® AQueous One Solution. Blends of the two agents synergistically suppressed the growth of DU145 cells, with combination index values in the range of 0.67–0.75. While 7.5 μmol/L d‐δ‐tocotrienol and 30 μmol/L geranylgeraniol individually had no impact on cell cycle distribution in DU145 cells, a blend of the agents at these concentrations induced cell cycle arrest at the G1 phase. The synergistic downregulation of the expression of HMG CoA reductase by 7.5 μmol/L d‐δ‐tocotrienol and 30 μmol/L geranylgeraniol was accompanied by a reduction in membrane RAS protein induced by the blend. Our finding lends further support to the cancer chemopreventive action of plant‐based diets and their isoprenoid constituents. Properly formulated isoprenoid combinations and their derivatives with complementary mechanisms may provide novel approaches in prostate cancer therapy.Support or Funding InformationTDA FFRP, TWU REP, and American River Nutrition, Inc.

Silibinin Combination with Arsenic Strongly Inhibits Survival and Invasiveness of Human Prostate Carcinoma Cells

Effects of silibinin, a naturally occurring flavanone, on prostate carcinoma (PCa) cells in presence of arsenic are not known. Arsenic is clinically approved for leukemia treatment; however, studies are not enough to support its role in the management of solid tumors. In the present study, we observed that silibinin (100 µM) modulated the oxidative status of human PCa DU145 cells exposed to arsenic (0.5 or 5 µM) and inhibited cell growth and survival by primarily inducing autophagy and apoptosis. The silibinin–arsenic combination also inhibited the growth, survival, and clonogenic potential of 22Rv1 PCa cells. Silibinin with 0.5 or 5 µM arsenic induced G1 or G2/M phase arrest, respectively, and decreased the protein levels of CDK2, -4, and -6 and cyclin D1, D3, and E and increased CDK inhibitors p21 and p27. Arsenic alone increased cyclin B1 level and Cdc2 kinase activity which were reduced in silibinin combination. Cell motility and invasiveness along with expression of MMP-2 and vimentin were suppressed. Together, these in vitro findings suggest that in presence of arsenic, silibinin strongly inhibits tumorigenic and metastatic potential of PCa cells.

Synergistic Impact of d ‐δ‐Tocotrienol and Geranylgeraniol in Human DU145 Prostate Carcinoma Cells

The mevalonate pathway produces essential intermediates for the post-translational prenylation and dolichylation of growth-associated proteins including Ras, nuclear lamins and growth factor receptors. Dysregulation and overexpression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of this pathway, in malignant cells supports tumor growth and survival. d-δ-Tocotrienol, a vitamin E molecule with farnesyl side chain, and geranylgeraniol, a diterpene, accelerate the ubiquitination and proteasome-mediated degradation of HMG CoA reductase via complementary and collaborative mechanisms. We evaluated the impacts of these two isoprenoids in human DU145 prostate carcinoma cells. d-δ-Tocotrienol (IC50 = 15 μmol/L) and geranylgeraniol (IC50 = 60 μmol/L) individually induced concentration-dependent suppression of the viability of DU145 cells as measured by CellTiter 96® AQueous One Solution. Isobologram using CompuSyn software demonstrated the synergistic impact of the two agents in combination. Blends of d-δ-tocotrienol (7.5 μmol/L) and geranylgeraniol (30 μmol/L), each at half of their respective IC50 concentrations, cumulatively suppressed the level of membrane HMG CoA reductase and putatively as a consequence of reduced prenylation, that of membrane Ras protein. Synergistic impact of diverse isoprenoids on the mevalonate pathway and tumor cell viability may offer a potential therapeutic or preventive approach to prostate cancer. Supported by TWU REP and American River Nutrition, Inc.

Abstract 2452: Silibinin inhibits lipid metabolism by primarily targeting the master regulator sterol response element binding protein 1 (SREBP1) in prostate cancer cells

Abstract Aberrant lipid metabolism is a major factor in growth, progression and invasion/metastasis of prostate tumors. Prostate cancer (PCa) cells are unique in terms of their low glycolytic rate and weak expression of glucose transporters. Concomitantly, PCa cells overexpress fatty acid synthase (FASN) which is considered a metabolic oncogene. As glucose consumption is not elevated in PCa, a metabolic switch to lipogenesis and fatty acid oxidation provides energy and cellular building blocks for abnormal cell growth and proliferation. In the present study, we evaluated the effect and related molecular mechanisms of a natural flavonoid silibinin in inhibiting lipid metabolism in PCa cells. Results showed that silibinin (30-90 µM) inhibited lipid and cholesterol accumulation specifically in human prostate carcinoma LNCaP and DU145 cells but not in non-neoplastic prostate epithelial PWR-1E cells. This inhibition by silibinin was mediated via a decrease in the expression of key enzymes regulating fatty acid and cholesterol synthesis including FASN, acetyl CoA carboxylase, acetyl CoA synthetase and HMG CoA reductase. Since most of these lipogenic enzymes are transcriptionally controlled by SREBP, we next analyzed the effect of silibinin on this master transcriptional regulator of lipogenesis. Silibinin inhibited SREBP1 (as well as SREBP2) protein expression without affecting its mRNA level in PCa cells suggesting a post-translational mode of regulation. SREBP1 activation involves proteolytic cleavage of precursor protein (125 kDa), generating a 68 kDa fragment that translocates to the nucleus and binds to sterol response element on target genes. Confocal and Immunoblot analyses confirmed that silibinin inhibited the proteolytic cleavage and subsequent nuclear translocation of SREBP1. Importantly, silibinin activated AMPK that phosphorylates SREBP1 (at Ser-372 site) inhibiting its nuclear translocation and transcriptional activity. Moreover, AMPK inhibition, through Compound C, reversed silibinin-mediated inhibition of nuclear SREBP1 and lipid accumulation. The central role of SREBP1 in silibinin-mediated effects on lipid accumulation and cell growth inhibition was also established by using a specific synthetic SREBP inhibitor, fatostatin. Parallely, we also observed that silibinin attenuates fatty acid uptake through down regulating LDL receptor expression. Furthermore, silibinin also significantly reduced AMACR (α-methylacyl-CoA racemase) expression in PCa cells, suggesting that it could also negatively regulate fatty acid oxidation. Overall, we found that silibinin targets SREBP1 and metabolically starves PCa cells by reducing de novo lipogenesis and lipolysis leading to a strong growth inhibition. Considering the critical role of lipid metabolism in prostate carcinogenesis, non-toxic silibinin could be useful in PCa prevention and treatment. Citation Format: Dhanya K. Nambiar, Gagan Deep, Rana P. Singh, Chapla Agarwal, Rajesh Agarwal. Silibinin inhibits lipid metabolism by primarily targeting the master regulator sterol response element binding protein 1 (SREBP1) in prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2452. doi:10.1158/1538-7445.AM2014-2452

Poloxamer surfactant preserves cell viability during photoacoustic delivery of molecules into cells.

Efficient intracellular delivery of molecules is needed to modulate cellular behavior for laboratory and medical applications, but is often limited by trade-offs between achieving high intracellular delivery and maintaining high cell viability. Here, we studied photoacoustic delivery of molecules into cells by exposing DU145 human prostate carcinoma cells to nanosecond laser pulses in the presence of carbon black nanoparticles. Under strong laser exposure conditions, less than 30% of cells were viable and exhibited uptake. Addition of poloxamer surfactant at those laser exposure conditions increased cell viability to almost 90%, with intracellular uptake in >80% of cells. This remarkable increase in efficiency of intracellular delivery and cell viability may be attributed to enhanced cell membrane resealing by poloxamer surfactant after photoacoustic delivery. While F-68 poloxamer was effective, the larger, more-hydrophobic F-127 poloxamer provided the best results. There was no significant protective effect from addition of Ca(2+) , BAPTA-AM, ATP, fetal bovine serum or glycine betaine, which were expected to promote active cell membrane repair mechanisms and other active intracellular protective processes. We conclude that poloxamer surfactant preserves cell viability during photoacoustic delivery of molecules into cells, thereby enabling highly efficient intracellular delivery.

Pro-oxidant Treatment of Human Prostate Carcinoma (DU145) Induces Autoschizis Cell Death: Autophagosomes Build up out of Injured Endomembranes and Mitochondria

One hour after pro-oxidative treatment by either ascorbate (VC), menadione (VK3), or VC: VK3 combination followed by 24-h incubation in culture medium, DU145 human prostate carcinoma cells developed ultrastructural-dependent organelle damage with the sequence Sham > VC > VK3 > VC: VK3. Along the nuclear alterations and the cytoplasm self-excisions reducing cell size, other induced injuries concerned mitochondria and endomembranes that associated with lysosomes. Damaged organelles surrounded by specialized endoplasmic membranes formed autophagosomes out of phagophores that also captured pieces of glycogen-rich cytoplasm. Most autophagosomes amassed in the diminished-size perikarya and corroborated the enhanced cytotoxicity of the VC: VK3 treatment. These accumulations did not initiate cell death, instead were merely signs of excessive “recycling” of damaged organelles. These features may reflect that high lysosomal activities provided foodstuffs in an ultimate strategy of survival of the tumor cells already devastated by reactive oxidative species (ROS) energetic sites. As such they became transient markers preceding cell death induced to occur by autoschizis and not by apoptosis or other cell deaths. This report could provide more support for the usage of this vitamin combination named APATONE as inexpensive potent adjuvant or treatment in prostate cancers.

Magnetic resonance imaging contrast of iron oxide nanoparticles developed for hyperthermia is dominated by iron content

Purpose: Magnetic iron oxide nanoparticles (MNPs) are used as contrast agents for magnetic resonance imaging (MRI) and hyperthermia for cancer treatment. The relationship between MRI signal intensity and cellular iron concentration for many new formulations, particularly MNPs having magnetic properties designed for heating in hyperthermia, is lacking. In this study, we examine the correlation between MRI T2 relaxation time and iron content in cancer cells loaded with various MNP formulations. Materials and methods: Human prostate carcinoma DU-145 cells were loaded with starch-coated bionised nanoferrite (BNF), iron oxide (Nanomag® D-SPIO), Feridex™, and dextran-coated Johns Hopkins University (JHU) particles at a target concentration of 50 pg Fe/cell using poly-D-lysine transfection reagent. T2-weighted MRI of serial dilutions of these labelled cells was performed at 9.4 T and iron content quantification was performed using inductively coupled plasma mass spectrometry (ICP-MS). Clonogenic assay was used to characterise cytotoxicity. Results: No cytotoxicity was observed at twice the target intracellular iron concentration (∼100 pg Fe/cell). ICP-MS revealed highest iron uptake efficiency with BNF and JHU particles, followed by Feridex and Nanomag-D-SPIO, respectively. Imaging data showed a linear correlation between increased intracellular iron concentration and decreased T2 times, with no apparent correlation among MNP magnetic properties. Conclusions: This study demonstrates that for the range of nanoparticle concentrations internalised by cancer cells the signal intensity of T2-weighted MRI correlates closely with absolute iron concentration associated with the cells. This correlation may benefit applications for cell-based cancer imaging and therapy including nanoparticle-mediated drug delivery and hyperthermia.