Antiproliferative effect of diallyl disulfide (DADS) on prostate cancer cell line LNCaP

Garlic has been used throughout the world to treat coughs, toothache, earache, dandruff, hypertension, hysteria, diarrhoea, dysentery, diptheria, vaginitis and many other conditions. Garlic contains a complex mixture of oil and water-soluble organosulfur compounds. Diallyl disulfide (DADS), an oil-soluble constituent of garlic seems to be effective in reducing tumour cells originating from colon, lung and skin. Hence our present study focuses on the dose-dependent effect of DADS on an androgen-dependent prostate cancer cell line. Various concentrations of DADS ranging from 25 to 100 microM were given to LNCaP cells and the activity of lactate dehydrogenase (LDH) prostatic acid phosphatase (PAcP) and the level of prostate specific antigen were studied. DADS reduced the secretory activity of LNCaP cells with the gradual increase in dosage. DADS was found to act as a good antiproliferative agent, which was confirmed by proliferation assay. DADS also induced apoptosis and nuclear segmentation in the higher doses.     

Effect of diallyl trisulfide derivatives on the induction of apoptosis in human prostate cancer PC-3 cells

The effects of five derivatives of diallyl trisulfide (DATS) were investigated on apoptosis in prostate cancer PC-3 cells, including dibutenyl trisulfide (DBTS), bis(2-methylallyl) trisulfide (2-M-DATS), dipentenyl trisulfide (DPTS), bis(3-methylbut-2-enyl) trisulfide (3-M-DBTS), and dihexenyl trisulfide (DHTS). Our present study demonstrated that DATS derivatives can suppress proliferation of PC-3 cells in a dose- and time-dependent manner, and that a change in the DATS structure could have an impact on its biological activity in the following order: 2-M-DATS > DBTS ≈ DPTS ≈ DATS > 3-M-DBTS ≈ DHTS. Typical apoptotic nuclei were shown by Hoechst 33342 staining with 80 μM concentrations of DATS derivatives for 24 h. And flow cytometric analysis and DNA fragmentation assay also demonstrated that DATS derivatives induced apoptosis in PC-3 cells. Meanwhile, experimental results showed that DBTS, 2-M-DATS, and DPTS cause G2-M phase cell cycle arrest. Furthermore, a series of apoptosis-associated features were observed, which include a notable decrease in the expression of procaspases-3, up-regulation of pro-apoptotic proteins Bax expression, and down-regulation of anti-apoptotic proteins Bcl-2 expression in PC-3 cells. All of the evidences above indicate that DATS derivatives suppressed proliferation of PC-3 cells which was associated with the induction of apoptosis regulated by Bax/Bcl-2.     

Inhibition of angiogenic differentiation of human umbilical vein endothelial cells by diallyl disulfide (DADS)

Angiogenesis is a crucial step in the growth and metastasis of cancers. The activation of endothelial cells and their further behaviour are very critical during angiogenesis. We analyzed the effect of diallyl disulfide (DADS) on angiogenesis in in vitro models using human umbilical vein endothelial cells (HUVECs). DADS significantly inhibited endothelial cell migration, invasion and tube formation. (3)H-thymidine proliferation assay clearly showed the inhibitory effect of DADS on the proliferation of HUVECs in vitro. The role of metalloproteinases has been shown to be important in angiogenesis; therefore, zymography was performed to determine whether DADS affected protease activity. Gelatin zymographic analysis showed the inhibitory effect of DADS on the activation of matrix metalloproteinases-MMP-2 and MMP-9. These findings suggest that DADS acts as an angiogenesis inhibitor by inhibiting the activation of matrix metalloproteinases during endothelial morphogenesis.     

Effects of dihydroxy gymnemic triacetate (DGT) on expression of apoptosis associated proteins in human prostate cancer cell lines (PC3)

Abstract

Prostate cancer is the most common malignancies among men. The present study is aimed at the investigation of dihydroxy gymnemic triacetate (DGT) from Gymnema sylvestre on mitochondrial apoptotic pathway and cell cycle arrest. Treatment of DGT resulted in a dose-dependent inhibition of growth of PC-3 cells. The cell cycle arrest was observed at the G2/M phase and accumulation of apoptotic cells was observed in DGT-treated prostate cancer cell lines. The occurrence of apoptosis in these cells was observed by DNA fragmentation. These events were associated with increased levels of pro-apoptotic proteins Bax, Bad and reduced levels of the antiapoptotic proteins Bcl-2, Bcl-xL and Mcl-1. DGT also induces the activation of caspase-9 and caspase-3. The above results, clearly, suggest that DGT induces apoptosis by the intrinsic pathways which could be very useful for the treatment of prostate cancer.     

Quercetin regulates insulin like growth factor signaling and induces intrinsic and extrinsic pathway mediated apoptosis in androgen independent prostate cancer cells (PC-3)

Progression of prostate cancer is facilitated by growth factors that activate critical signaling cascades thereby promote prostate cancer cell growth, survival, and migration. To investigate the effect of quercetin on insulin-like growth factor signaling and apoptosis in androgen independent prostate cancer cells (PC-3), IGF-IR, PI-3K, p-Akt, Akt, cyclin D1, Bad, cytochrome c, PARP, caspases-9 and 10 protein levels were assessed by western blot analysis. Mitochondrial membrane potency was detected by rhodamine-123 staining. Quercetin induced caspase-3 activity assay was performed for activation of apoptosis. Further, RT-PCR was also performed for Bad, IGF-I, II, IR, and IGFBP-3 mRNA expression. Quercetin significantly increases the proapoptotic mRNA levels of Bad, IGFBP-3 and protein levels of Bad, cytochrome C, cleaved caspase-9, caspase-10, cleaved PARP and caspase-3 activity in PC-3 cells. IGF-IRβ, PI3K, p-Akt, and cyclin D1 protein expression and mRNA levels of IGF-I, II and IGF-IR were decreased significantly. Further, treatment with PI3K inhibitor (LY294002) and quercetin showed decreased p-Akt levels. Apoptosis is confirmed by loss of mitochondrial membrane potential in quercetin treated PC-3 cells. This study suggests that quercetin decreases the survival of androgen independent prostate cancer cells by modulating the expression of insulin-like growth factors (IGF) system components, signaling molecules and induces apoptosis, which could be very useful for the androgen independent prostate cancer treatment.     

Quercetin downregulates matrix metalloproteinases 2 and 9 proteins expression in prostate cancer cells (PC-3)

Background: Cancer metastasis, involving multiple processes and various cytophysiological changes, is a primary cause of cancer death and may complicate the clinical management, even lead to death. Quercetin is a flavonoid and widely used as an antioxidant and recent studies have revealed its pleiotropic anticancer and antiproliferative capabilities. Gelatinases A and B (matrixmetalloproteinases 2 and 9) are enzymes known to involve in tumor invasion and metastases. In this study, we observed the precise involvement of quercetin role on these proteinases expression and activity. Design and methods: PC-3 cells were treated with quercetin at various concentrations (50 and 100 μM), for 24 h period and then subjected to western blot analysis to investigate the impact of quercetin on matrix metalloproteinase-2 (MMP-2) and 9 (MMP-9) expressions. Conditioned medium and cell lysate of quercetin-treated PC-3 cells were subjected to western blot analysis for proteins expression of MMP-2 and MMP-9. Gelatin zymography was also performed in quercetin treated PC-3 cells. Results: The results showed that quercetin treatment decreased the expressions of MMP-2 and MMP-9 in dose-dependent manner. The level of pro-MMP-9 was found to be high in the 100 μM quercetin-treated cell lysate of PC-3 cells, suggesting inhibitory role of quercetin on pro-MMP-9 activation. Gelatin zymography study also showed the decreased activities of MMP-2 and MMP-9 in quercetin treated cells. Conclusion: Hence, we speculated that inhibition of metastasis-specific MMPs in cancer cells may be one of the targets for anticancer function of quercetin, and thus provides the molecular basis for the development of quercetin as a novel chemopreventive agent for metastatic prostate cancer.     

The role of reactive oxygen species (ROS) production on diallyl disulfide (DADS) induced apoptosis and cell cycle arrest in human A549 lung carcinoma cells

Diallyl disulfide (DADS), an oil soluble constituent of garlic (Allium sativum), has been reported to cause antimutagentic and anticarcinogenic effects in vitro and in vivo by modulating phases I and II enzyme activities. In recent years, several studies suggested that the chemopreventive effects of DADS can also be attributed to induction of cell cycle arrest and apoptosis in cancer cells. In the present study, we reported that DADS-induced cell cycle arrest at G2/M and apoptosis in human A549 lung cancer cells in a time- and dose-dependent manner. Additionally, a significant increase of intracellular reactive oxygen species (ROS) was induced in A549 cells less than 0.5 h after DADS treatment, indicating that ROS may be an early event in DADS-modulated apoptosis. Treatment of A549 cells with N-acetyl cysteine (NAC) completely abrogated DADS-induced cell cycle arrest and apoptosis. The result indicated that oxidative stress modulates cell proliferation and cell death induced by DADS.     

Transport of methotrexate into PC-3 human prostate cancer cells

Recombinant BmRad51 and BmDmc1, silkworm homologs of the Escherichia coli RecA proteins catalyzing the homologous DNA pairing, were purified from E. coli cells carrying expression vectors. These possessed different enzymatic properties in the joint molecule formation between single-stranded circular DNA and homologous linear double-stranded DNA. The requirement of single-stranded circular DNA for the efficient reaction was twofold higher in BmRad51 than in BmDmc1. Although able to mediate the joint molecule formation independently, a complex of the two enzymes formed prior to single-stranded DNA binding was found to have augmented efficiency of the pairing reaction.     

Secretion of endogenous kallikreins 2 and 3 by androgen receptor-transfected PC-3 prostate cancer cells

Androgen independent PC-3 cells lack androgen receptor (AR) expression and do not produce kallikrein 2 (hK2) or 3 (prostate-specific antigen, PSA). In this paper, we examined the ability of androgens to stimulate PSA and hK2 production in AR transfected PC-3 cells (PC-3(AR)) and compared this to LNCaP cells. PSA and hK2 were measured in the culture medium and cell lysates using an ELISA-based immunofluorometric assay. Only androgens were able to induce PSA and hK2 secretion in PC-3(AR) cells in a dose- and time-dependent manner depending on the level of AR present. The level of androgen-induced PSA and hK2 secretion in PC-3(AR) cells was approximately 1.5 and 0.9% that induced in LNCaP cells, respectively. Insulin-like growth factor-I (IGF-I), which has been shown to activate AR in the absence of ligand, did not activate PSA secretion in the absence of androgen, but further increased the dihydrotestosterone-induced PSA secretion in PC-3(AR) cells. The lack of PSA and hK2 production in parental PC-3 cells is thus a result of their lack of AR expression. PSA and/or hK2 production in PC-3(AR) cells can thus serve as an endogenous reporter system to investigate AR action or to screen putative endocrine disrupters.     

Pipernonaline from Piper longum Linn. induces ROS-mediated apoptosis in human prostate cancer PC-3 cells

The antiproliferation effects of pipernonaline, a piperine derivative, were investigated on human prostate cancer PC-3 cells. It inhibited growth of androgen independent PC-3 and androgen dependent LNCaP prostate cells in a dose-dependent (30–90 μM) and time-dependent (24–48 h) manner. The growth inhibition of PC-3 cells was associated with sub-G₁ and G₀/G₁ accumulation, confirmed by the down-regulation of CDK2, CDK4, cyclin D1 and cyclin E, which are correlated with G₁ phase of cell cycle. Pipernonaline up-regulated cleavage of procaspase-3/PARP, but did not change expression of proapoptotic bax and antiapoptotic bcl-2 proteins. Its caspase-3 activation was confirmed by the caspase-3 assay kit. In addition, pipernonaline caused the production of reactive oxygen species (ROS), increase of intracellular Ca²⁺, and mitochondrial membrane depolarization, which these phenomena were reversed by N-acetylcysteine, a ROS scavenger. The results suggest that pipernonaline exhibits apoptotic properties through ROS production, which causes disruption of mitochondrial function and Ca²⁺ homeostasis and leads to its downstream events including activation of caspase-3 and cleavage of PARP in PC-3 cells. This is the first report of pipernonaline toward the anticancer activity of prostate cancer cells, which provides a role for candidate agent as well as the molecular basis for human prostate cancer.     

Gene expression profiles in the PC-3 human prostate cancer cells induced by NKX3.1

NKX3.1, a prostate-specific gene, plays an important role in prostate development and carcinogenesis. However, its precise function has not been established. In present study, we transfected the NKX3.1 eukaryotic expression plasmid (pcDNA3.1-NKX3.1) into human prostate cancer cells PC-3, which lack of NKX3.1 expression, and established stable transfectants. Then, we investigated the influence of NKX3.1 on the cell growth, cell migration and colony formation efficiency. The results showed that restoration of NKX3.1 expression inhibited proliferation and invasion activities of PC-3 cells. Further, a cDNA microarray containing 22,000 human genes was used to identify the gene expression differences. The results showed that there were 1,953 genes showing more than a two-fold difference in expression. Subsequent ontological analysis revealed that a large proportion of the classified genes were related to cell growth, cell signal and cell invasion. Finally, the expression of Caspase-3, Bcl-2, P27, Cdk6 and AMACR, randomly selected genes from microarray data, was validated by RT-PCR and western blot. Collectively, our results first analyzed the gene expression profile in PC-3 cells induced by NKX3.1 and indicated that NKX3.1 might exert its function by regulating the expression of relative genes.     

A new proteasome inhibitor, TP-110, induces apoptosis in human prostate cancer PC-3 cells

Proteasome inhibitors are useful in the treatment of cancer. Recently, we found a new proteasome inhibitor, TP-110, derived from tyropeptin A produced by Kitasatospora sp. Here we report that TP-110 induces apoptosis in human prostate cancer PC-3 cells. TP-110 showed strong cytotoxicity to PC-3 cells (IC(50)=0.05 muM). It increased the number of cells in the G(2)-M phase and increased the accumulated amounts of the p21 and p27 proteins, which are negative regulators of cell cycle progression. Furthermore, it induced apoptosis along with chromatin condensation and DNA fragmentation in PC-3 cells, and TP-110-induced apoptosis appeared to be associated with caspase activation. Additionally, TP-110 inhibited not only the degradation of IkappaB and the nuclear translocation of nuclear factor-kappaB (NF-kappaB), but also the DNA binding activity of NF-kappaB. These results indicate that TP-110 shows a strong growth inhibition and apoptosis in PC-3 cells.     

Combination of 2-methoxyestradiol (2-ME2) and eugenol for apoptosis induction synergistically in androgen independent prostate cancer cells

Lack of effective treatment options for the management of hormone refractory prostate cancer (PCA) reinforce the great need to develop novel compounds that act singly or in combination. 2-Methoxyestradiol (2-ME₂) is an endogenous estrogenic metabolite that has been reported to work as an antiproliferative agent in various tumor models including prostate. Recently conducted clinical trial in hormone refractory prostate cancer (HRPC) patients concluded that 2-ME₂ was safe and well tolerated. However this study identified bioavailability of 2-ME₂ as a limiting factor. Here we report the ability of a combination of 2-ME₂ and eugenol (4-allyl-2-methoxyphenol) as an approach for enhancing anticancerous activities in prostate cancer cells. Combining 2-ME₂ with eugenol (i) inhibited growth of prostate cancer cells and induced apoptosis at lower concentrations than either single agent alone; (ii) analysis of the data using combination index (CI) showed CI values of 0.4 indicating strong synergistic interaction; (iii) increased population of cells G₂/M phase by 4.5-fold (p = 0.01); (iv) significantly reduced expression of antiapoptotic protein Bcl-2 and enhanced expression of proapoptotic protein Bax. Combination induced apoptosis was not affected in PC-3 cells that over-express or lack Bcl-2 but was associated with loss of mitochondrial membrane potential. Since 2-ME₂ was well tolerated in phase II trail in patients with HRPC; and eugenol is consumed by humans in the form of spices, the combination of 2-ME₂ with eugenol may offer a new clinically relevant treatment regimen. Combining these agents may allow ameliorating any adverse effects of either 2-ME₂ or eugenol alone by reducing their individual concentrations should these two agents be developed for human use.     

5,8,11,14-Eicosatetraynoic acid-induced destruction of mitochondria in human prostate cells (PC-3)

Summary Culturing human prostate PC-3 cells for 4, 24, or 72 h in the presence of 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of arachidonic acid metabolism and cholesterol biosynthesis, markedly altered the morphology and reduced the number of mitochondria in the treated cells. Using quantitative electron microscopic morphometry, we documented changes in the number, form, area, matrix density, and integrity of the cristae and limiting membranes of mitochondria in cells cultured with ETYA. The inhibition of cholesterol synthesis or the substitution of ETYA for polyunsaturated fatty acids in the inner membrane may participate in the disruption of the mitochondria, which resembles the morphologic sequelae of oxidative stress. If sufficiently extensive, these changes could contribute to the inhibition of cellular proliferation by ETYA.     

Gossypol induces apoptosis in human PC-3 prostate cancer cells by modulating caspase-dependent and caspase-independent cell death pathways

The rate of gossypol-induced apoptosis does not correlate very well with the same dose of gossypol-induced cell growth inhibition, indicating an anti-proliferative effect of gossypol. Using a co-immunoprecipitation assay, it was observed that the level of Bcl-X(L) protein bound to Bax was clearly lower than that of Bcl-2 protein at 5 micro M of gossypol treatment, and the level of Bim protein bound to Bcl-X(L) was lowered at 20 micro M of gossypol treatment for 24 h, implicating that gossypol inhibits the heterodimerization of Bcl-X(L) with Bax and Bim. Gossypol-induced apoptosis is partly suppressed by as low as 0.5 micro M, but not abolished by as high as 50 micro M of a broad range caspase inhibitor, Z-VAD-FMK, suggesting that gossypol-induced apoptosis is both caspase-dependent and -independent. Furthermore, the release of apoptosis inducing factor (AIF), which triggers caspase-independent apoptosis, from mitochondria to cytosol was observed in PC-3 cells exposed to gossypol treatment. In conclusion, gossypol inhibits the proliferation and induces apoptosis in PC-3 cells. Gossypol-induced apoptosis is, at least, through inhibiting the heterodimerization of Bcl-X(L)/Bcl-2 with pro-apoptosis molecules, followed by a caspase-dependent and -independent process which involves the release of AIF from the mitochondria to cytosol.     

Effects of androgen receptor and androgen on gene expression in prostate stromal fibroblasts and paracrine signaling to prostate cancer cells

The androgen receptor (AR) is expressed in a subset of prostate stromal cells and functional stromal cell AR is required for normal prostate developmental and influences the growth of prostate tumors. Although we are broadly aware of the specifics of the genomic actions of AR in prostate cancer cells, relatively little is known regarding the gene targets of functional AR in prostate stromal cells. Here, we describe a novel human prostate stromal cell model that enabled us to study the effects of AR on gene expression in these cells. The model involves a genetically manipulated variant of immortalized human WPMY-1 prostate stromal cells that overexpresses wildtype AR (WPMY-AR) at a level comparable to LNCaP cells and is responsive to dihydrotestosterone (DHT) stimulation. Use of WPMY-AR cells for gene expression profiling showed that the presence of AR, even in the absence of DHT, significantly altered the gene expression pattern of the cells compared to control (WPMY-Vec) cells. Treatment of WPMY-AR cells, but not WPMY-Vec control cells, with DHT resulted in further changes that affected the expression of 141 genes by 2-fold or greater compared to vehicle treated WPMY-AR cells. Remarkably, DHT significantly downregulated more genes than were upregulated but many of these changes reversed the initial effects of AR overexpression alone on individual genes. The genes most highly effected by DHT treatment were categorized based upon their role in cancer pathways or in cell signaling pathways (transforming growth factor-β, Wnt, Hedgehog and MAP Kinase) thought to be involved in stromal-epithelial crosstalk during prostate or prostate cancer development. DHT treatment of WPMY-AR cells was also sufficient to alter their paracrine potential for prostate cancer cells as conditioned medium from DHT-treated WPMY-AR significantly increased growth of LNCaP cells compared to DHT-treated WPMY-Vec cell conditioned medium.     

PC-1在前列腺癌细胞中促进c-myc基因的表达

前列腺癌相关基因PC-1(Prostate and colon gene1)是属于癌基因D52家族成员,具有促进前列腺癌细胞雄激素非依赖性生长的功能。为了研究PC-1发挥这种生物功能的分子机制,文章在PC-1高表达的LNCaP-pc-1及对照LNCaP-zero细胞中,利用RT-PCR和Western blotting等方法检测c-myc基因表达;提取两细胞胞质和胞核蛋白,利用Western blotting分析c-myc上游调节蛋白β-catenin变化;利用c-Myc蛋白抑制剂10058-F4作用前列腺癌细胞C4-2,Western blotting检测PC-1蛋白表达变化。发现PC-1促进c-myc基因表达,并促进β-catenin入核;c-Myc蛋白抑制剂10058-F4可抑制PC-1的表达。结果表明:PC-1在前列腺癌中促进c-myc基因的表达,并且这种促进作用可能是通过Wnt/β-catenin信号通路实现的。同时,PC-1与c-Myc蛋白间可相互促进,进一步促进前列腺癌细胞雄激素非依赖性生长。     

Lysophosphatidic acid inhibits serum deprivation-induced autophagy in human prostate cancer PC-3 cells

Lysophosphatidic acid (LPA) is a platelet-enriched bioactive lysophospholipid. By binding to its cognitive G protein-coupled receptors, which are encoded by endothelial differentiation genes (edgs), LPA regulates various cellular activities including proliferation, survival, and migration. Currently, little is known about the influences of LPA on autophagy, a pivotal mechanism for cell survival during conditions of starvation. Herein we present data indicating that LPA attenuates starvation-induced autophagy, by monitoring the percentage of LC3-II, an autophagy indicator, in human prostate PC-3 cells. In addition, by using cells stably expressing EGFP-LC3, LPA is shown to inhibit the formation of autophagosomes in serum-starved conditions. Our results suggest that in these conditions, LPA inhibits autophagy, which might facilitate early cancer development.     

Alpha-tomatine synergises with paclitaxel to enhance apoptosis of androgen-independent human prostate cancer PC-3 cells in vitro and in vivo

Alpha (α)-tomatine, a major saponin found in tomato has been shown to inhibit the growth of androgen-independent prostate cancer PC-3 cells. The effects of α-tomatine in combination with the chemotherapeutic agent paclitaxel against PC-3 cells were investigated in the present study. Combined treatment with a sub-toxic dose of α-tomatine and paclitaxel significantly decreased cell viability with concomitant increase in the percentage of apoptotic PC-3 cells. The combined treatment, however, had no cytotoxic effect on the non-neoplastic prostate RWPE-1 cells. Apoptosis of PC-3 cells was accompanied by the inhibition of PI3K/Akt pro-survival signaling, an increase in the expression of the pro-apoptotic protein BAD but a decrease in the expressions of anti-apoptotic proteins, Bcl-2 and Bcl-xL. Results from a mouse xenograft model showed the combined treatment completely suppressed subcutaneous tumor growth without significant side effects. Consistent with its in vitro anti-cancer effects, tumor materials from mice showed increased apoptosis of tumor cells with reduced protein expression of activated PI3K/Akt. These results suggest that the synergistic anti-cancer effects of paclitaxel and α-tomatine may be beneficial for refractory prostate cancer treatment.     

Delta9-tetrahydrocannabinol induces apoptosis in human prostate PC-3 cells via a receptor-independent mechanism

During hypoxia of isolated cardiomyocytes, Ca2+ entry into mitochondria may occur via the Na/Ca exchanger, the normal efflux pathway, and not the Ca-uniporter, the normal influx route. If this is the case, then depletion of myocyte Na+ should inhibit Ca2+ uptake, and collapse of the mitochondrial membrane potential (delta psi(m)) would inhibit the uniporter. To test these hypotheses, isolated rat myocytes were exposed to metabolic inhibition, to mimic hypoxia, and [Ca2+]m and [Ca2+]c determined by selective loading of indo-1 into these compartments. Delta psi(m) was determined using rhodamine 123. Following metabolic inhibition, [Ca2+]m was significantly lower in Na-depleted cells than controls (P<0.001), [Ca2+]c was approximately the same in both groups, and mitochondria depolarised completely. Thus Na-depletion inhibited mitochondrial Ca2+ uptake, suggesting that Ca2+ entry occurred via Na/Ca exchange, and the collapse of delta psi(m) during metabolic inhibition is consistent with inactivity of the Ca-uniporter.