ROS-mediated activation of AKT induces apoptosis via pVHL in prostate cancer cells

Reactive oxygen species (ROS) play a central role in oxidative stress, which leads to the onset of diseases, such as cancer. Furthermore, ROS contributes to the delicate balance between tumor cell survival and death. However, the mechanisms by which tumor cells decide to elicit survival or death signals during oxidative stress are not completely understood. We have previously reported that ROS enhanced tumorigenic functions in prostate cancer cells, such as transendothelial migration and invasion, which depended on CXCR4 and AKT signaling. Here, we report a novel mechanism by which ROS facilitated cell death through activation of AKT. We initially observed that ROS enhanced the expression of phosphorylated AKT (p-AKT) in 22Rv1 human prostate cancer cells. The tumor suppressor PTEN, a negative regulator of AKT signaling, was rendered catalytically inactive through oxidation by ROS, although the expression levels remained consistent. Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased, and contains a target site for p-AKT phosphorylation. pVHL and p-AKT associated in vitro, and knockdown of pVHL rescued HIF1α expression and the cells from apoptosis. Collectively, our study suggests that in the context of oxidative stress, p-AKT facilitated apoptosis by inducing pVHL function.     

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.     

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.     

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.     

Analgesic activity of Piper longum Linn. root

Piper longum root, commonly called Kandantippili, is traditionally used to treat rheumatism, insomnia, palsy and epilepsy. But a scientific study on its central actions is not available. This study screens P. longum root for opioid type analgesia using rat tail-flick method and for NSAID type analgesia using acetic-acid writhing method. Pentazocine (ip) and ibuprofen (oral) are used as respective drug controls. An aqueous suspension of P. longum root powder is given orally to mice and rat in doses of 200, 400 and 800 mg/kg. The delay in reaction time for thermal stimulus in rats and the number of writhings to chemical stimulus in mice are determined in each group. The results are analysed statistically. The 400 and 800 mg/kg doses of P. longum show significant NSAID type of analgesia (P < 0.001). Both Ibuprofen (40 mg/kg) and P. longum (800 mg/kg) show 50% protection against writhing. The delay in reaction time to thermal stimulus was less than 6% for different doses of P. longum as against 100% for pentazocine. This indicates that P. longum root has weak opioid but potent NSAID type of analgesic activity.     

Hispolon induces apoptosis in human gastric cancer cells through a ROS-mediated mitochondrial pathway

Severe side effects and complications such as gastrointestinal and hematological toxicities because of current anticancer drugs are major problems in the clinical management of gastric cancer, which highlights the urgent need for novel effective and less toxic therapeutic approaches. Hispolon, an active polyphenol compound, is known to possess potent antineoplastic and antiviral properties. In this study, we investigated the efficacy of hispolon in human gastric cancer cells and explored the cell death mechanism. Hispolon induced ROS-mediated apoptosis in gastric cancer cells and was more toxic toward gastric cancer cells than toward normal gastric cells, suggesting greater susceptibility of the malignant cells. The mechanism of hispolon-induced apoptosis was that hispolon abrogated the glutathione antioxidant system and caused massive ROS accumulation in gastric cancer cells. Excessive ROS caused oxidative damage to the mitochondrial membranes and impaired the membrane integrity, leading to cytochrome c release, caspase activation, and apoptosis. Furthermore, hispolon potentiated the cytotoxicity of chemotherapeutic agents used in the clinical management of gastric cancer. These results suggest that hispolon could be useful for the treatment of gastric cancer either as a single agent or in combination with other anticancer agents.     

Depletion of the histone chaperone tNASP inhibits proliferation and induces apoptosis in prostate cancer PC-3 cells

Background  

NASP (Nuclear Autoantigenic Sperm Protein) is a histone chaperone that is present in all dividing cells. NASP has two splice variants: tNASP and sNASP. Only cancer, germ, transformed, and embryonic cells have a high level of expression of the tNASP splice variant. We examined the consequences of tNASP depletion for prostate cancer PC-3 cells.     

Thiosulfinates from Allium tuberosum L. induce apoptosis via caspase-dependent and -independent pathways in PC-3 human prostate cancer cells

This study was aimed to evaluate the apoptotic effects of thiosulfinates purified from Allium tuberosum L. on PC-3 human prostate cancer cells, and to elucidate detailed apoptosis mechanisms. Thiosulfinates significantly decrease viable cell numbers in dose- and time-dependent manners by apoptotic cell death via DNA fragmentation, chromatin condensation, and an increased sub-G1 phase. Apoptosis induced by thiosulfinates is associated with the activation of initiator caspase-8 and -9, and the effector caspase-3. In this study, thiosulfinates stimulated Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. Thiosulfinates decreased the expression of the anti-apoptotic protein Bcl-2 and increased the expression of the pro-apoptotic protein Bax. Thiosulfinates also increased the expression of AIF, a caspase-independent mitochondrial apoptosis factor, in PC-3 cells. These results indicate that thiosulfinates from A. tuberosum L. inhibit cell proliferation and induce apoptosis in PC-3 cells, which may be mediated via both caspase-dependent and -independent pathways.     

Ginkgetin induces apoptosis via activation of caspase and inhibition of survival genes in PC-3 prostate cancer cells

Ginkgetin is a natural biflavonoid isolated from leaves of Ginkgo biloba L. Though it was known to have anti-inflammatory, anti-influenza virus, anti-fungal activity, osteoblast differentiation stimulating activity and neuro-protective effects, the underlying antitumor mechanism of ginkgetin still remains unclear. Thus, in the present study, anti-cancer mechanism of ginkgetin was elucidated in human prostate cancer PC-3 cells. Ginkgetin suppressed the viability of PC-3 cells in a concentration-dependent manner and also significantly increased the sub-G1 DNA contents of cell cycle in PC-3 cells. Ginkgetin activated caspase-3 and attenuated the expression of survival genes such as Bcl-2, Bcl-x_L, survivin and Cyclin D1 at protein and mRNA levels. Consistently, pan-caspase inhibitor Z-DEVD-fmk blocked sub G1 accumulation and cleavages of PRAP and caspase 3 induced by ginkgetin in PC-3 cells. Overall, these findings suggest that ginkgetin induces apoptosis in PC-3 cells via activation of caspase 3 and inhibition of survival genes as a potent chemotherapeutic agent for prostate cancer treatment.     

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.     

The vanilloid capsaicin induces IL-6 secretion in prostate PC-3 cancer cells

Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a constituent of green and red peppers, has been linked with suppression of tumorigenesis through a mechanism that is not well understood. In the present study, we examined the effects of capsaicin on the production of the cytokine interleukin (IL)-6 by PC-3 cells at both protein and mRNA levels which were evaluated by ELISA and real-time PCR, respectively. Capsaicin-treated PC-3 cells increased the synthesis and secretion of IL-6 which was abrogated by the transient receptor potential vanilloid receptor subtype 1 (TRPV1) antagonist capsazepine, as well as by inhibitors of PKC-α, phosphoinositol-3 phosphate kinase (PI-3K), Akt and extracellular signal-regulated protein kinase (ERK). We analyzed the role of capsaicin in the tumor necrosis factor (TNF)-α secretion by PC-3 cells which was increased at shorter times than IL-6 production. Furthermore, incubation of PC-3 cells with an anti-TNF-α antibody blocked the capsaicin-induced IL-6 secretion. These results raise the possibility that capsaicin-mediated IL-6 increase in prostate cancer PC-3 cells is regulated at least in part by TNF-α secretion and signaling pathway involving Akt, ERK and PKC-α activation.     

Methylseleninic acid induces apoptosis of human bladder cancer cells through the ROS-mediated mitochondrial pathway

As the most common selenium derivative, methylseleninic acid (MSA) has attracted wide attention. Its apoptotic induction ability and the possible molecular mechanism in human bladder cancer (BC) J82 and T24 cells were investigated in the present study. We found that the survival of J82 and T24 cells were inhibited in a dose-dependent manner after MSA treatment. Propidium iodide (PI) staining and Annexin V-fluorescein isothiocyanate/PI double staining clarified that MSA stocked cells at G₂/M phase and caused apoptosis in J82 and T24 cells. Further, typical morphological features of apoptotic cells were also observed. Accumulation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential were also detected by dichlorodihydrofluorescein diacetate and Rhodamin123 staining. Meanwhile, pretreatment with N-acetylcysteine, an ROS scavenging agent, found that the apoptosis of BC cells induced by MSA was related to the production of ROS. Western blot analysis results showed that MSA interrupted Bax/Bcl-2 balance, stimulated cytochrome c release into the cytoplasm, activated caspase-9 and caspase-3, and finally induced the apoptosis of the BC cells. These findings demonstrated that MSA was able to induce apoptosis in J82 and T24 cells through ROS-mediated mitochondrial apoptosis.     

Cholesterol regulates prostasome release from secretory lysosomes in PC-3 human prostate cancer cells

Prostasomes are vesicles secreted by epithelial cells of the prostate gland. However, little is known about the mechanism and the regulation of prostasome secretion. Since endocytic organelles may be involved in prostasome release, PC-3-derived prostasomes were investigated by Western blot analysis for the presence of marker proteins normally associated with these organelles. Prostasomes secreted by PC-3 cells contain clathrin, Tsg101, Hrs, Rab11, Rab5, LAMP-1, LAMP-2, LAMP-3/CD63, and annexin II. Moreover, electron microscopy of PC-3 cells revealed the presence of characteristic multivesicular body-like secretory lysosomes containing vesicles with the same size-distribution as released prostasomes. Ultrastructural immunogold labelling showed that LAMP-1, LAMP-2 and LAMP-3/CD63 were associated with these vesicles. In addition, we have investigated whether cholesterol plays a role in prostasome release by the human prostate cancer cell line PC-3. Interestingly, prostasome release was significantly increased when the cholesterol levels of PC-3 cells were reduced by the cholesterol-sequestering agent methyl-beta-cyclodextrin (MBCD), or by treatment with lovastatin and mevalonate. In conclusion, these studies indicate that cholesterol plays an important role in the release of prostasomes by the human prostate cancer PC-3 cells, and suggest that prostasomes may be released after fusion of secretory lysosomes with the plasma membrane.     

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.     

Extracellular matrix protein fibronectin induces matrix metalloproteinases in human prostate adenocarcinoma cells PC-3

Abstract

Studies on interaction of tumor cells with ECM components showed increased extracellular protease activity mediated by the family of matrix metalloproteinases (MMPs). Here we studied the effect of human prostate adenocarcinoma PC-3 cells–fibronectin (FN) interaction on MMPs and the underlying signaling pathways. Culturing of PC-3 cells on FN-coated surface upregulated MMP-9 and MMP-1. This response is abrogated by the blockade of α₅ integrin. siRNA and inhibitor studies indicate possible involvement of phosphatidyl-inositol-3-kinase (PI-3K), focal adhesion kinase (FAK) and nuclear factor-kappaB (NF-κB) in FN-induced upregulation of MMPs. FN treatment also enhanced phosphorylation of FAK, PI3K, protein kinase B (PKB or Akt), nuclear translocation of NF-κB, surface expression of CD-44, and cell migration. Our findings indicate that, binding of PC-3 cells to FN, possibly via α_5β1 integrin, induces signaling involving FAK, PI-3K, Akt, NF-κB followed by upregulation of MMP-9 and MMP-1. CD-44 may have role in modulating MMP-9 activity.     

Seleno-short-chain chitosan induces apoptosis in human non-small-cell lung cancer A549 cells through ROS-mediated mitochondrial pathway

Seleno-short-chain chitosan (SSCC) is a synthesized chitosan derivative. In this study, antitumor activity and underlying mechanism of SSCC on human non-small-cell lung cancer A549 cells were investigated in vitro. The MTT assay showed that SSCC could inhibit cell viability in a dose- and time-dependent manner, and 200 μg/ml SSCC exhibited significantly toxic effects on A549 cells. The cell cycle assay showed that SSCC triggered S phase cell cycle arrest in a dose- and time-dependent manner, which was related to a downregulation of S phase associated cyclin A. The DAPI staining and Annexin V-FITC/PI double staining identified that the SSCC could induce A549 cells apoptosis. Further studies found that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) by DCFH-DA and Rhodamin 123 staining, respectively. Meanwhile, free radical scavengers N-acetyl-l-cysteine (NAC) pretreatment confirmed that SSCC-induced A549 cells apoptosis was associated with ROS generation. Furthermore, real-time PCR and western blot assay showed that SSCC up-regulated Bax and down-regulated Bcl-2, subsequently incited the release of cytochrome c from mitochondria to cytoplasm, activated the increase of cleaved-caspase 3 and finally induced A549 cells apoptosis in vitro. In general, the present study demonstrated that SSCC induced A549 cells apoptosis via ROS-mediated mitochondrial apoptosis pathway.     

Profiling of microRNAs in exosomes released from PC-3 prostate cancer cells

Exosomes are small extracellular vesicles released to the extracellular milieu through fusion of multivesicular bodies with the plasma membrane. These vesicles contain microRNAs and might therefore be vehicles transferring genetic information between cells. The aim of this study was to investigate whether there was a sorting of microRNAs into exosomes in the prostate cancer cell line PC-3. In addition, microRNAs in PC-3 cells and in the non-cancerous prostate cell line RWPE-1 were compared. Exosomes were isolated from the conditioned media from PC-3 cells by ultracentrifugation and inspected by electron microscopy. Total RNA was isolated and microRNAs were analyzed by microarray analysis and real time RT-PCR. MicroRNA microarray analysis revealed that the microRNA profile of PC-3 released exosomes was similar to the profile of the corresponding parent cells. Nevertheless, a sorting of certain microRNAs into exosomes was observed, and low number microRNAs (microRNAs with a low number in their name) were found to be underrepresented in these vesicles. Moreover, the miRNA profile of PC-3 cells resembled the miRNA profile of RWPE-1 cells, though some miRNAs were found to be differently expressed in these cell lines. These results show that exosomes from PC-3 cells, in agreement with previous reports from other cell types, contain microRNAs. Furthermore, this study supports the idea that there is a sorting of microRNAs into exosomes and adds a new perspective by pointing at the underrepresentation of low number miRNAs in PC-3 released exosomes.     

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.     

Facile synthetic nano-curcumin encapsulated Bio-fabricated nanoparticles induces ROS-mediated apoptosis and migration blocking of human lung cancer cells

Lung cancer is one of the major life-threatening cancers, with a higher mortality rate and morbidity. Curcumin acts as a potential anticancer agent but it shows less aqueous solubility so its clinical application is limited. In this present study, we developed curcumin-surface decorated Zinc Oxide nanoparticles ZnO@Cur to enhance its aqueous formulation and improve the anti-cancer activity of curcumin. The synthesized ZnO@Cur confirmed by various spectroscopies (UV–vis and fluorescence) and electroscopic techniques (Powder XRD, SEM, TEM, and DLS). ZnO@Cur dramatically suppressed the proliferation of A549 and HEL-299 cells and showed low toxicity in MTT assays. Compared with free ZnO and curcumin, ZnO@Cur significantly inhibited the migration ability of A549 cells. Moreover, the induction of apoptosis in A549 cells by ZnO@Cur was resulted by AO-EB staining. ZnO@Cur activated to promote intracellular ROS overproduction and induced apoptosis. ZnO@Cur shows excellent biocompatibility compared to free ZnO and Curcumin, the present study explained that ZnO@Cur as a safe and hopeful strategy for chemotherapeutics of lung cancer therapy and deserve for further clinical evaluations.