Mechanisms of resistance and adaptation to thapsigargin in androgen-independent prostate cancer PC3 and DU145 cells

Cells with increasing resistance to the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG), ranging from 60-fold (PC3/TG(10) cells) to 1350-fold (PC3/TG(2000) cells), were derived from PC3 cells. SERCA2 is overexpressed in all PC3/TG cells but retains sensitivity to TG. siRNA-mediated downregulation of SERCA completely or partially reverses TG resistance in PC3/TG(10) or PC3/TG(2000) cells, respectively; thus SERCA overexpression mediates resistance in PC3/TG(10) cells but is not the only resistance mechanism in PC3/TG(2000) cells. By contrast, SERCA is not overexpressed in TG-resistant DU145/TG cells derived from DU145 cells. DU145/TG cells retain resistance while in PC3/TG cells resistance decreases upon removal of TG selection. The transport proteins PGP/BCRP/MRP1 and anti-apoptotic proteins Bcl2/Bcl(XL) are not involved in mediating resistance in either cell line. PARP and caspase 3 cleavage in response to other drugs demonstrate that the apoptotic pathways tested remain intact in these cells. Further, no cross-resistance occurs to other drugs. Thus, novel TG-specific resistance mechanisms are recruited by these cancer cells.     

Activation of polyamine catabolic enzymes involved in diverse responses against epibrassinolide-induced apoptosis in LNCaP and DU145 prostate cancer cell lines

Epibrassinolide (EBR) is a biologically active compound of the brassinosteroids, steroid-derived plant growth regulator family. Generally, brassinosteroids are known for their cell expansion and cell division-promoting roles. Recently, EBR was shown as a potential apoptotic inducer in various cancer cells without affecting the non-tumor cell growth. Androgen signaling controls cell proliferation through the interaction with the androgen receptor (AR) in the prostate gland. Initially, the development of prostate cancer is driven by androgens. However, in later stages, a progress to the androgen-independent stage is observed, resulting in metastatic prostate cancer. The androgen-responsive or -irresponsive cells are responsible for tumor heterogeneity, which is an obstacle to effective anti-cancer therapy. Polyamines are amine-derived organic compounds, known for their role in abnormal cell proliferation as well as during malignant transformation. Polyamine catabolism-targeting agents are being investigated against human cancers. Many chemotherapeutic agents including polyamine analogs have been demonstrated to induce polyamine catabolism that depletes polyamine levels and causes apoptosis in tumor models. In our study, we aimed to investigate the mechanism of apoptotic cell death induced by EBR, related with polyamine biosynthetic and catabolic pathways in LNCaP (AR+), DU145 (AR?) prostate cancer cell lines and PNT1a normal prostate epithelial cell line. Induction of apoptotic cell death was observed in prostate cancer cell lines after EBR treatment. In addition, EBR induced the decrease of intracellular polyamine levels, accompanied by a significant ornithine decarboxylase (ODC) down-regulation in each prostate cancer cell and also modulated ODC antizyme and antizyme inhibitor expression levels only in LNCaP cells. Catabolic enzymes SSAT and PAO expression levels were up-regulated in both cell lines; however, the specific SSAT and PAO siRNA treatments prevented the EBR-induced apoptosis only in LNCaP (AR+) cells. In a similar way, MDL 72,527, the specific PAO and SMO inhibitor, co-treatment with EBR during 24 h, reduced the formation of cleaved fragments of PARP in LNCaP (AR+) cells.     

Androgen regulation of aldehyde dehydrogenase 1A3 (ALDH1A3) in the androgen-responsive human prostate cancer cell line LNCaP

Previous gene array data from our laboratory identified the retinoic acid (RA) biosynthesis enzyme aldehyde dehydrogenase 1A3 (ALDH1A3) as a putative androgen-responsive gene in human prostate cancer epithelial (LNCaP) cells. In the present study, we attempted to identify if any of the three ALDH1A/RA synthesis enzymes are androgen responsive and how this may affect retinoid-mediated effects in LNCaP cells. We demonstrated that exposure of LNCaP cells to the androgen dihydrotestosterone (DHT) results in a 4-fold increase in ALDH1A3 mRNA levels compared with the untreated control. The mRNA for two other ALDH1A family members, ALDH1A1 and ALDH1A2, were not detected and not induced by DHT in LNCaP cells. Inhibition of androgen receptor (AR) with both the antiandrogen bicalutamide and small interfering RNA for AR support that ALDH1A3 regulation by DHT is mediated by AR. Furthermore, specific inhibition of the extracellular signal-regulated kinase and Src family of kinases with PD98059 and PP1 supports that AR's regulation of ALDH1A3 occurs by the typical AR nuclear-translocation cascade. Consistent with an increase in ALDH1A3 mRNA, DHT-treated LNCaP cells showed an 8-fold increase in retinaldehyde-dependent NAD(+) reduction compared with control. Lastly, treatment of LNCaP with all-trans retinal (RAL) in the presence of DHT resulted in significant up-regulation of the RA-inducible, RA-metabolizing enzyme CYP26A1 mRNA compared with RAL treatment alone. Taken together, these data suggest that (i) the RA biosynthesis enzyme ALDH1A3 is androgen responsive and (ii) DHT up-regulation of ALDH1A3 can increase the oxidation of retinal to RA and indirectly affect RA bioactivity and metabolism.     

Vesicle traffic through intercellular bridges in DU 145 human prostate cancer cells

We detected cell-to-cell communication via intercellular bridges in DU 145 human prostate cancer cells by fluorescence microscopy. Since DU 145 cells have deficient gap junctions, intercellular bridges may have a prominent role in the transfer of chemical signals between these cells. In culture, DU 145 cells are contiguous over several cell diameters through filopodial extensions, and directly communicate with adjacent cells across intercellular bridges. These structures range from 100 nm to 5 microm in diameter, and from a few microns to at least 50-100 microm in length. Time-lapse imagery revealed that (1) filopodia rapidly move at a rate of microns per minute to contact neighboring cells and (2) intercellular bridges are conduits for transport of membrane vesicles (1-3 microm in diameter) between adjacent cells. Immunofluorescence detected alpha-tubulin in intercellular bridges and filopodia, indicative of microtubule bundles, greater than a micron in diameter. The functional meaning, interrelationship of these membrane extensions are discussed, along with the significance of these findings for other culture systems such as stem cells. Potential applications of this work include the development of anti-cancer therapies that target intercellular communication and controlling formation of cancer spheroids for drug testing.     

Characterization of sphere-propagating cells with stem-like properties from DU145 prostate cancer cells

While accumulating evidence demonstrates the existence of prostate cancer stem cells (PCSCs), PCSCs have not been isolated and thoroughly characterized. We report here the enrichment and characterization of sphere-propagating cells with stem-like properties from DU145 PC cells in a defined serum-free medium (SFM). Approximately 1.25% of monolayer DU145 cells formed spheres in SFM and 26% of sphere cells formed secondary spheres. Spheres are enriched for cells expressing prostate basal and luminal cytokeratins (34βE12 and CK18) and for cancer stem cell markers, including CD44, CD24, and integrin α2β1. Upon culturing spheres under differentiating media conditions in the presence of 10% serum, cells positive for CD44 and CD24 were substantially reduced. Furthermore, spheres could be generated from the sphere-derived adherent cell cultures and xenograft tumors, demonstrating the stemness of DU145 spheres. We have maintained spheres for more than 30 passages within 1.5 years without noticeable loss of their “stemness”. Sphere cells possess self-renewal capacity, display significant increases in proliferation potential, and initiate xenograft tumors with enhanced capacity compared to monolayer DU145 cells. While EGF promoted the generation and maintenance of these stem-like cells, bFGF inhibited these events. Sphere cells proliferate slowly with a significant reduction in the activation of the PI3K-AKT pathway compared to monolayer DU145 cells. While knockdown of PTEN enhanced AKT activation, this did not affect the generation of primary spheres and the propagation of secondary spheres. Consistent with this observation, we were able to demonstrate the generation and propagation of spheres without the addition of external growth factors. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Restructuring dynamics of du 145 and LNCaP prostate cancer spheroids

Summary Neoplastic cells acquire multidrug resistance as they assemble into multicellular spheroids. Image analysis and Monte Carlo simulation provided an insight into the adhesion and motility events during spheroid restructuring in liquid-overlay culture of DU 145 and LNCaP human prostate cancer cells. Irregularly shaped, two-dimensional aggregates restructured through incremental cell movements into three-dimensional spheroids. Of the two cultures examined, restructuring was more pronounced for DU 145 aggregates. Motile DU 145 cells formed spheroids with a minimum cell overlay of 30% for 25-mers as estimated by simulation versus 5% for adhesive LNCaP cells in aggregates of the same size. Over 72 h, the texture ratio increased from 0.55±0.05 for DU 145 aggregates with projected areas exceeding 2000 μm² to a value approaching 0.75±0.02 (P<0.05). For LNCaP aggregates of comparable size, the increase in texture ratio was more modest, less than 15% during the same time period (P<0.05). Combined, these data suggest that motility events govern the overall rate of spheroid restructuring. This information has application to the chemosensitization of solid tumors and kinetic modeling of spheroid production.     

Antiandrogen-induced cell death in LNCaP human prostate cancer cells

Antiandrogens such as Casodex (Bicalutamide) are designed to treat advance stage prostate cancer by interfering with androgen receptor-mediated cell survival and by initiating cell death. Treatment of androgen sensitive, non-metastatic LNCaP human prostate cancer cells with 0-100 microM Casodex or 0-10 ng/ml TNF-alpha induces cell death in 20-60% of the cells by 48 h in a dose-dependent manner. In cells treated with TNF-alpha, this is accompanied by the loss of mitochondrial membrane potential (DeltaPsim) and cell adhesion. In contrast, cells treated with Casodex display loss of cell adhesion, but sustained mitochondrial dehydrogenase activity. Overexpression of Bcl-2 in LNCaP cells attenuates the induction of cell death by TNF-alpha but not Casodex, suggesting that mitochondria depolarization is not required for the induction of cell death by Casodex. While both TNF-alpha and Casodex-induced release of cytochrome c in LNCaP cell is predominantely associated with the translocation and cleavage of Bax, our data also suggest that Casodex induces cell death by acting on components downstream of decline of DeltaPsim and upstream of cytochrome c release. Furthermore, while induction of both caspase-3 and caspase-8 activities are observed in TNF-alpha and Casodex-treated cells, a novel cleavage product of procaspase-8 is seen in Casodex-treated cells. Taken together, these data support the hypothesis that Casodex induces cell death by a pathway that is independent of changes in DeltaPsim and Bcl-2 actions and results in an extended lag phase of cell survival that may promote the induction of an invasive phenotype after treatment.     

Induction of apoptosis by green tea catechins in human prostate cancer DU145 cells

Green tea catechins (GTCs) including (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG) and (-)-epicatechin (EC) were shown to suppress cell growth and induce apoptosis in various cell systems in addition to their chemo-preventive effect. In this study, except EC which was inactive, green tea extract (TE) and other 3 GTCs were found to suppress the growth and induce apoptosis in human prostate cancer DU145 cells largely through an increase in reactive oxygen species formation and mitochondrial depolarization. The conclusion was supported by the fact that the profiles for different GTCs in growth suppression, apoptosis induction, ROS formation and mitochondrial depolarization are in a similar order, i.e. ECG > EGCG > EGC > EC. Although the molecular mechanisms are still not clear, apoptosis induced by GTCs is not related to the members of BCL-2 family as EGCG did not alter the expression of BCL-2, BCL-X(L) and BAD in DU145 cells.     

Blueberry flavonoids inhibit matrix metalloproteinase activity in DU145 human prostate cancer cells.

Regulation of the matrix metalloproteinases (MMPs), the major mediators of extracellular matrix (ECM) degradation, is crucial to regulate ECM proteolysis, which is important in metastasis. This study examined the effects of 3 flavonoid-enriched fractions (a crude fraction, an anthocyanin-enriched fraction, and a proanthocyanidin-enriched fraction), which were prepared from lowbush blueberries (Vaccinium angustifolium), on MMP activity in DU145 human prostate cancer cells in vitro. Using gelatin gel electrophoresis, MMP activity was evaluated from cells after 24-hr exposure to blueberry fractions. All fractions elicited an ability to decrease the activity of MMP-2 and MMP-9. Of the fractions tested, the proanthocyanidin-enriched fraction was found to be the most effective at inhibiting MMP activity in these cells. No induction of either necrotic or apoptotic cell death was noted in these cells in response to treatment with the blueberry fractions. These findings indicate that flavonoids from blueberry possess the ability to effectively decrease MMP activity, which may decrease overall ECM degradation. This ability may be important in controlling tumor metastasis formation.     

Anti-proliferative effects of paeonol on human prostate cancer cell lines DU145 and PC-3

Paeonol (Pae) is the main active ingredient from the root bark of Paeonia moutan and the grass of Radix Cynanchi Paniculati. Numerous reports indicate that Pae effectively inhibits several types of cancer lines. In this study, we report that Pae hinders prostate cancer growth both in vivo and in vitro. Human prostate cancer lines DU145 and PC-3 were cultured in the presence of Pae. The xenograft tumor in mice was established by subcutaneous injection of DU145 cells. Cell growth was measured by MTT, and the apoptosis was detected by the flow cytometry. Expression of Bcl-2, Bax, Akt, and mTOR were tested by western blotting assay. DU145 and PC-3 showed remarkable sensitivity to Pae, and exposure to Pae induced dose-and time-dependent growth inhibitory responses. Moreover, treatment of Pae promoted apoptosis and enhanced activities of caspase-3, caspase-8, and caspase-9 in DU145. Further work demonstrated Pae reduced expression of Bcl-2 and increased expression of Bax in DU145. Interestingly, we observed that Pae significantly decreased phosphorylated status of Akt and mTOR, and inhibitory effects of Pae and PI3K/Akt inhibitor on DU145 proliferation were synergistic. Finally, we confirmed that oral administration of Pae to the DU145 tumor-bearing mice significantly lowered tumor cell proliferation and led to tumor regression. Pae possesses inhibitory effects on prostate cancer cell growth both in vitro and in vivo, and the anti-proliferative effect may be closely related to its activation of extrinsic and intrinsic apoptotic pathway and inhibition of the PI3K/Akt pathway.     

Dynamics of spheroid self-assembly in liquid-overlay culture of DU 145 human prostate cancer cells.

The in vitro self-assembly of multicellular spheroids generates highly organized structures in which the three-dimensional structure and differentiated function frequently mimic that of in vivo tissues. This has led to their use in such diverse applications as tissue regeneration and drug therapy. Using Smoluchowski-like rate equations, herein we present a model of the self-aggregation of DU 145 human prostate carcinoma cells in liquid-overlay culture to elucidate some of the physical parameters affecting homotypic aggregation in attachment-dependent cells. Experimental results indicate that self-aggregation in our system is divided into three distinct phases: a transient reorganization of initial cell clusters, an active aggregation characterized by constant rate coefficients, and a ripening phase of established spheroid growth. In contrast to the diffusion-controlled aggregation previously observed for attachment-independent cells, the model suggests that active aggregation in our system is reaction-controlled. The rate equations accurately predict the aggregation kinetics of spheroids containing up to 30 cells and are dominated by spheroid adhesive potential with lesser contributions from the radius of influence. The adhesion probability increases with spheroid size so that spheroid-spheroid adhesions are a minimum of 2.5 times more likely than those of cell-cell, possibly due to the upregulation of extracellular matrix proteins and cell-adhesion molecules. The radius of influence is at least 1.5 to 3 times greater than expected for spherical geometry as a result of ellipsoidal shape and possible chemotactic or Fr?hlich interactions. Brownian-type behavior was noted for spheroids larger than 30 microm in diameter, but smaller aggregates were more motile by as much as a factor of 10 for single cells. The model may improve spheroid fidelity for existing applications of spheroids and form the basis of a simple assay for quantitatively evaluating cellular metastatic potential as well as therapies that seek to alter this potential.     

Anti-proliferative effects of evodiamine on human prostate cancer cell lines DU145 and PC3

Prostate carcinoma is one of the most common malignant tumors and has become a more common cancer in men. Previous studies demonstrated that evodiamine (EVO) exhibited anti-tumor activities on several cancers, but its effects on androgen-independent prostate cancer are unclear. In the present study, the action mechanisms of EVO on the growth of androgen-independent prostate cancer cells (DU145 and PC3 cells) were explored. EVO dramatically inhibited the growth and elevated cytotoxicity of DU145 and PC3 cells. The flow cytometric analysis of EVO-treated cells indicated a block of G2/M phase and an elevated level of DNA fragmentation. The G2/M arrest was accompanied by elevated Cdc2 kinase activity, an increase in expression of cyclin B1 and phosphorylated Cdc2 (Thr 161), and a decrease in expression of phosphorylated Cdc2 (Tyr 15), Myt-1, and interphase Cdc25C. TUNEL examination showed that EVO-induced apoptosis was observed at 72 h. EVO elevated the activities of caspase 3, 8, and 9 in DU145 cells, while in PC3 cells only the activities of caspase 3 and 9 were elevated. EVO also triggered the processing of caspase 3 and 9 in both DU145 and PC3 cells. We demonstrate that roscovitine treatment result in the reversion of G2/M arrest in response to EVO in both DU145 and PC3. However, inhibitory effect of roscovitine on EVO-induced apoptosis could only be observed in DU145 rather than PC3. In DU145, G2/M arrest might be a signal for initiation of EVO-triggered apoptosis. Whereas EVO-triggered PC3 apoptosis might be independent of G2/M arrest. These results suggested that EVO inhibited the growth of prostate cancer cell lines, DU145 and PC3, through an accumulation at G2/M phase and an induction of apoptosis.     

A partner monoclonal antibody to Moab 730 kills 100% of DU145 and PC3 androgen-independent cancer cells

A number of therapeutic options are available for patients with prostate carcinoma till the time that the tumour is hormone dependent. However, no fully effective therapy is available for the treatment of androgen-independent prostate carcinomas. Antibodies directed at epitopes unique to or overexpressed on the cancer cells could be of therapeutic utility. A monoclonal antibody (Moab) 2C4 has been generated, which binds with cells of two androgenin-dependent prostate cancers, DU145 and PC3, and does not bind to peripheral blood leukocytes (PBLs) of healthy donors. This antibody, along with the previously developed Moab 730, kills 100% of both DU145 and PC3 cells in the presence of complement and does not have a deleterious effect on PBLs of healthy males. The anti-tumour action of the two antibodies prevents the establishment of DU145 cell tumour in nude mice in vivo. Moab 2C4 in combination with 730 has potential for use as therapy for androgen-independent cancers.     

Cytoprotective effect of arginine deiminase on taxol-induced apoptosis in DU145 human prostate cancer cells

We purified and partially sequenced a cytostatic protein from the ASC-17D Sertoli cell-conditioned media (rSCCM) showing a molecular weight of 90 kDa with homodimeric composition. N-terminal amino acid analysis revealed that the protein was homologous to the arginine deiminase (ADI) of Mycoplasma arginini. We found ADI enzyme activity in rSCCM and the abolishment of the growth inhibitory effect by the supplement of L-arginine. Thus, we confirmed that the cytostatic activity in rSCCM was due to the depletion of extracellular L-arginine by ADI. Apparent increase of cell death or DNA fragmentation was not observed in DU145 cells cultured in the presence of ADI. Incubation of DU145 cancer cells with taxol resulted in a marked DNA fragmentation, whereas pretreatment with ADI or cycloheximide protected the cells from taxol-induced apoptosis. Preincubation of the cells with ADI inhibited S35-methionine incorporation into protein synthesis in a dose dependent manner. These data suggest that ADI-induced arginine depletion may inhibit protein synthesis, and result in the protection of apoptotic cell death that requires new protein synthesis.     

鸦胆子苦醇抑制人前列腺癌DU145细胞生长及作用机制

中药鸦胆子是一种常用的抗肿瘤中草药,鸦胆子苦醇是来源于鸦胆子的主要成分。该研究探讨了鸦胆子苦醇(brusatol)对人前列腺癌DU145细胞的生长抑制及其作用机制。采用四甲基偶氮唑盐(MTT)法检测鸦胆子苦醇对不同细胞株的生长抑制情况,以及不同浓度的鸦胆子苦醇对DU145细胞的增殖抑制率;应用Hoechst 33258染色法观察鸦胆子苦醇处理DU145细胞后所发生的形态学变化;分别采用PI单染及AnnexinV-FITC双染法流式细胞术分析细胞周期分布个凋亡率的变化;以Western blot测定鸦胆子苦醇对MAPK信号通路相关蛋白表达的影响。结果表明:鸦胆子苦醇对人前列腺癌DU145细胞的抑制作用更为显著,并且可以时间和剂量依赖性地抑制人前列腺癌DU145细胞的生长,其半数有效抑制浓度IC50为(0.27±0.04)μmol·L-1;鸦胆子处理DU145细胞后,Hoechst 33258染色可见到明显的凋亡特征;细胞周期图中可见明显的亚二倍体峰,且随着作用时间的延长凋亡比例增加,FCM检测鸦胆子苦醇作用24 h后凋亡图中,可见凋亡的发生;Western blot检测表明鸦胆子苦醇处理后可使磷酸化的p38和JNK表达增加,使磷酸化的ERK表达降低。鸦胆子苦醇能显著抑制DU145细胞增殖,诱导DU145细胞凋亡。磷酸化的P38和JNK的表达增加,但磷酸化的ERK表达下降,这表明MAPK途径的活化可能是鸦胆子苦醇对DU145细胞生长抑制的作用机制之一。因此,鸦胆子苦醇是潜在的抗前列腺癌药物,有必要进一步在动物水平阐明其抗前列腺癌活性。     

AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells

Prostate cancer is the most highly diagnosed cancer in men worldwide. It is characterized by high proliferation, great invasion and metastatic potential. Sodium channel subtypes have been identified as highly expressed in different prostate cancer cell lines. In this study, we have screened the negatively charged fractions of Androctonus australis (Aa) scorpion venom to identify active peptides on DU145 prostate cancer cells proliferation. The most active compound was identified to be the sodium channel peptide AaHIV with an IC₅₀ value of 15 μM. At this concentration, AaHIV had low effect on the adhesion of DU145 cells to fibronectin. When compared to other Na⁺ channel Aa toxins, AaHIV was found to be 2 times more active than AaHI and AaHII on DU145 cells proliferation and slightly less active than AaHII on their adhesion. The three peptides are inactive on DU145 cells migration. AaHIV was found to be 16 times more active than veratridine, asteroidal alkaloid from plants of the lily family widely used as a sodium channel activator. Electrophysiological experiments showed that the AaHIV toxin activates Nav1.6 channel, suggesting that this sodium channel subtype is implicated in the proliferation of DU145 prostate cancer cells.     

Proteomic analysis of proteins regulated by TRPS1 transcription factor in DU145 prostate cancer cells

The aim of the present study was to identify proteins differentially regulated by TRPS1 in human prostate cancer cells in order to better understand the role of TRPS1 in prostate cancer development. The proteomes of androgen-independent DU145 prostate cancer cells, that do not express TRPS1 and of genetically engineered DU145 cells that stable and inducible express recombinant TRPS1 protein, were compared. Using two-dimensional electrophoresis followed by mass spectrometric analysis, 13 proteins that were differentially expressed between these two cell lines were identified. These proteins represent a dominant reduction of expression of antioxidant proteins, including superoxide dismutase, protein disulfide isomerase A3 precursor, endoplasmin precursor and annexin A2. Furthermore, regulation was observed for mitochondrion-associated proteins, glycolytic enzymes, a cytoskeleton-associated protein, a nuclear protein and proteins involved in apoptosis. Our data indicate that overexpression of TRPS1 protein is correlated with reduced protein expression of certain antioxidants. This suggests a possible involvement of TRPS1 in oxidative stress, and possibly in apoptosis in androgen-independent DU145 prostate cancer cells.