Apoptosis In Human Prostate Cancer Cells Research Articles

In Vitro Effect and Mechanism of Action of Ergot Alkaloid Dihydroergocristine in Chemoresistant Prostate Cancer Cells.

Chemoresistance is a major obstacle in the treatment of prostate cancer (PCa). It is imperative to develop novel strategies for overcoming chemoresistance and improving clinical outcomes. We evaluated the in vitro activity and mechanism of action of dihydroergocristine (DHECS), an ergot alkaloid approved for the treatment of dementia, in PCa cells. The in vitro effects of DHECS on PCa cell cycle and viability were determined by flow cytometry and colorimetric assay. The effects of DHECS on PCa cell signaling were evaluated by quantitative PCR, western blot analysis and reporter assay. DHECS was effective in inducing cell cycle arrest and apoptosis in human PCa cells. Of particular interest, DHECS demonstrated high potency against chemoresistant PCa cells. At the molecular level, DHECS affected multiple factors implicated in the regulation of cancer cell cycle and programmed cell death, including p53, mouse double minute 2 homolog (MDM2), retinoblastoma protein (RB), p21, E2F transcription factor 1 (E2F1), survivin, myeloid cell leukemia 1 (Mcl-1) and poly ADP ribose polymerase (PARP). Furthermore, DHECS may function through dopamine receptor-mediated effects on 5'-AMP-activated protein kinase (AMPK) and nuclear factor kappa B (NF-ĸB). DHECS has the potential to be repurposed as a novel anticancer agent for the management of chemoresistant PCa.

Formononetin induces apoptosis of PC-3 human prostate cancer cells via regulating long noncoding RNA H19 and the mitochondrial apoptosis pathway

Background: Prostate cancer is a life-threating disease with high incidence and mortality in male. Formononetin, the main active component of some natural products, has been hypothesized as a promising anticancer agent in previous studies. Objectives: We investigated the toxic effects and potential molecular mechanism of formononetin in PC-3 prostate cancer cells to further understand the pharmacological effects of formononetin and provide more references for intensive research. Materials and Methods: PC-3 cells were incubated with different doses of formononetin for 24 h or 48 h. After that, cell viability was measured by Cell Counting Kit-8, and apoptosis was analyzed by Hoechst 33258 stains. The expression levels of tumor-related factors such as long noncoding RNA (LncRNA) H19, Bax, and Bcl-2 were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot methods. Subsequently, PC-3 cells were infected with a lentiviral vector to overexpress or knock down H19, and then, the expression of insulin-like growth factor-1 receptor (IGF-1R) mRNA was measured by RT-qPCR. Results: Formononetin significantly inhibited the viability of PC-3 cells and promoted apoptosis in a time-dose-dependent manner. We observed that the expressions of lncRNA H19 and Bcl-2 were significantly downregulated compared with the untreated group, while an opposite pattern was observed for Bax. According to the results of gene interaction experiments, IGF-1R may be a downstream target of H19 in PC-3 cells. Conclusion: Our results present evidence that formononetin induced apoptosis of PC-3 cells by regulating lncRNA H19 and the mitochondrial apoptosis pathway. Furthermore, we put forward the hypothesis that formononetin has an interference effect on the H19/IGF-1R pathway, which remains to be further confirmed.

Exploration of association of telmisartan with calf thymus DNA using a series of spectroscopic methodologies and theoretical calculation

Telmisartan (TMST) can block selectively and irreversibly an angiotensin II receptor type 1 (AT1), but has no effect on other receptor systems and also induce human prostate cancer cell apoptosis. In this work, domain specific association of TMST with calf thymus DNA was explored through multi-spectral methodologies and theoretical calculation. The results of absorption spectroscopy revealed that TMST interacting with ct-DNA formed the TMST–ct-DNA complex through non-covalent interaction and the association constant (Ka) was of 2.71 × 103 M−1 at 298 K. Based on the thermodynamic analysis, it can be inferred that the interacting TMST with ct-DNA was an enthalpy-driven, spontaneous, and exothermic process due to the ΔG0 < 0, ΔH0 < 0, and |ΔH0|>T|ΔS0| under the studied temperature ranges. The domain interacting forces for interacting TMST with ct-DNA were van der Waals forces and hydrogen bonding interaction due to the ΔH0 < 0 and ΔS0 < 0. Meanwhile, the electrostatic and hydrophobic interactions also played a supporting function. Additionally, from experiments and theoretical calculation (molecular docking), it was also confirmed that TMST interacted to a minor groove of the C-G rich region of ct-DNA and resulted in the slight alteration in the secondary structure of ct-DNA whereas the obvious alteration in the geometry of TMST after binding to DNA, indicating that the flexibility of TMST contributed to stabilizing the TMST–ct-DNA complex.

Effects of interferons and double-stranded RNA on human prostate cancer cell apoptosis.

Prostate cancer is the second most commonly diagnosed cancer among men in the United States. Prostate cancer therapy is severely hampered by lack of response and development of resistance to conventional chemotherapeutic drugs in patients. Therefore, the development and discovery of new drugs have become an urgent clinical need. Interferons (IFNs), a family of pleiotropic cytokines, exert antitumor activities due to their anti-proliferative, immunomodulatory and proapoptotic functions. Here, we report that pretreatment of prostate cancer PC-3 cells with IFNs sensitized these cells to double-stranded RNAs (dsRNAs)-induced apoptosis. The enhancement effect of IFN treatment was dependent on IFN subtypes, in particular, IFN γ. In comparison with IFN α or β, IFN γ treatment remarkably augmented apoptosis in PC-3 cells induced with polyinosinic:polycytidylic acid (poly I:C), a synthesized form of dsRNA. We demonstrated that IFN-signaling was necessary for these effects by using mutant cell lines. Transfection of 2-5A, the activator of RNase L, or silencing of dsRNA-dependent protein kinase R (PKR) by siRNA did not have any significant impact on this event, suggesting that neither RNase L nor PKR was involved in poly I:C/IFN γ-induced apoptosis in the cells. Further investigation of the apoptotic pathway revealed that Bak, a pro-apoptotic member of the Bcl-2family, was synergistically up-regulated by IFN γ and poly I:C, whereas other members of the family were not affected. Knocking down of Bak demonstrated its contribution to poly I:C/IFN γ-induced apoptosis in the cells. We believeour findings will precipitate the design of novel therapeutic strategies for prostate cancer.

Abstract 5724: Capsaicin and lycopene in combination reduce proliferation and induce apoptosis in prostate cancer cells via TRPV6 mediated phenomenon

Abstract Introduction and Hypothesis: Prostate cancer (PCa) is the most common internal malignancy and the second most frequent cause of cancer death in Canadian men. Capsaicin recently investigated for its anti-cancer properties, is the active compound found in chilli peppers. Well-established as a pain reliever, capsaicin acts mainly on the transient receptor potential vanilloid (TRPV)-1 and TRPV6 receptor. The TRPV1 receptor is a non-selective cation channel that induces an inflow of cations Ca2+ and Na+ when activated. Similarly, activation of the TRPV6 receptor facilitates Ca2+ entry across the plasma membrane. Both TRPV1 and TRPV6 receptors are expressed in PCa tissue. Our present study aims to investigate the chemopreventive effect of capsaicin in combination with lycopene, an antioxidant found in tomatoes. We have found that PCa cells treated with capsaicin and lycopene alone and combined, cause a significant reduction in proliferation and an induction of apoptosis. Mechanistic studies examining this relationship have not been well defined. We hypothesize that capsaicin and lycopene in combination reduce proliferation and induce apoptosis via the TRPV6 and TRPV1 receptor in PCa cells in vitro. Materials and Methods: Two human PCa cell lines (LNCaP (AR+) and PC3 (AR-)) were analyzed. Cells were treated with capsaicin alone, or in combination with lycopene. Cells were incubated for up to 24 hours and proliferation assessed using MTS assay. Alterations in TRPV6, TRPV1, PSA, cell-regulatory molecules, and apoptotic markers were assessed by Western blot analysis. Results: There was a significant (P&amp;lt; 0.05) decrease in the proliferation of LNCaP and PC3 cells treated with capsaicin alone and in combination with lycopene (p&amp;lt; 0.001). Western blot analysis revealed a reduction (2-fold change) in PSA expression in LNCaP cells when treated with capsaicin alone. Interestingly this treatment resulted in the up-regulation in cell cycle marker p27 as well as cleaved PARP, in a time-dependent manner, demonstrating that the cells are undergoing cell cycle arrest and apoptosis. Further, we established that there is an up-regulation (3-fold change) of TRPV6 expression and an increase in TRPV1 expression with the treatment of capsaicin and lycopene alone and in combination. A plausible mechanism of action for this combination of capsaicin and lycopene is currently being investigated. Conclusions: We have shown for the first time that the TRPV6 receptor may play an important role in capsaicin and lycopene mediated cell-cycle arrest and apoptosis in human PCa cells. These studies may eventually help identify patients likely to benefit from the use of capsaicin in combination with lycopene. Ultimately these strategies may have a more meaningful impact on PCa morbidity and mortality than other therapeutic strategies currently in use. Funding: CIHR grant to VV. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5724.

Involvement of endoplasmic reticulum stress and activation of MAP kinases in beta-lapachone-induced human prostate cancer cell apoptosis.

Beta-lapachone, an o-naphthoquinone, induces various carcinoma cells to undergo apoptosis, but the mechanism is poorly understood. In the present study, we found that the beta-lapachone-induced apoptosis of DU145 human prostate carcinoma cells was associated with endoplasmic reticulum (ER) stress, as shown by increased intracellular calcium levels and induction of GRP-78 and GADD-153 proteins, suggesting that the endoplasmic reticulum is a target of beta-lapachone. Beta-Lapachone-induced DU145 cell apoptosis was dose-dependent and accompanied by cleavage of procaspase-12 and phosphorylation of p38, ERK, and JNK, followed by activation of the executioner caspases, caspase-7 and calpain. However, pretreatment with the general caspase inhibitor, z-VAD-FMK, or calpain inhibitors, including ALLM or ALLN, failed to prevent beta-lapachone-induced apoptotic cell death. Blocking the enzyme activity of NQO1 with dicoumarol, a known NQO1 inhibitor, or preventing an increase in intracellular calcium levels using BAPTA-AM, an intracellular calcium chelator, substantially inhibited MAPK phosphorylation, abolished the activation of calpain, caspase-12 and caspase-7, and provided significant protection of beta-lapachone-treated cells. These findings show that beta-lapachone-induced ER stress and MAP kinase phosphorylation is a novel signaling pathway underlying the molecular mechanism of the anticancer effect of beta-lapachone.

Fisetin, a novel dietary flavonoid, causes apoptosis and cell cycle arrest in human prostate cancer LNCaP cells

Novel dietary agents for prevention and therapy of prostate cancer (PCa) are desired. The aim of this study was to determine the effect of fisetin, a tetrahydroxyflavone, on inhibition of cell growth and induction of apoptosis in human PCa cells. Treatment of fisetin (10-60 microM, 48 h) was found to result in a decrease in the viability of LNCaP, CWR22Rupsilon1 and PC-3 cells but had only minimal effects on normal prostate epithelial cells as assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide assay. Treatment of LNCaP cells with fisetin also resulted in G(1)-phase arrest that was associated with a marked decrease in the protein expression of cyclins D1, D2 and E and their activating partner cyclin-dependent kinases 2, 4 and 6 with concomitant induction of WAF1/p21 and KIP1/p27. Fisetin treatment also resulted in induction of apoptosis, poly (ADP-ribose) polymerase (PARP) cleavage, modulation in the expressions of Bcl-2 family proteins, inhibition of phosphatidyl inositol 3-kinase and phosphorylation of Akt at Ser(473) and Thr(308). There was also induction of mitochondrial release of cytochrome c into cytosol, downregulation of X-linked inhibitor of apoptosis protein and upregulation of second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI on treatment of cells with fisetin. Treatment of cells with fisetin also resulted in significant activation of caspases-3, -8 and -9. Pretreatment of cells with caspase inhibitor (Z-VAD-FMK) blocked fisetin-induced activation of caspases. These data provide the first evidence that fisetin could be developed as an agent against PCa.

Expression of p53 enhances selenite-induced superoxide production and apoptosis in human prostate cancer cells.

Although the anticancer effects of selenium have been shown in clinical, preclinical, and laboratory studies, the underlying mechanism(s) remains unclear. Our previous study showed that sodium selenite induced LNCaP human prostate cancer cell apoptosis in association with production of reactive oxygen species, alteration of cell redox state, and mitochondrial damage. In the present study, we showed that selenite-induced apoptosis was superoxide mediated and p53 dependent via mitochondrial pathways. In addition, we also showed that superoxide production by selenite was p53 dependent. Our study showed that wild-type p53-expressing LNCaP cells were more sensitive to selenite-induced apoptosis than p53-null PC3 cells. Selenite treatment resulted in high levels of superoxide production in LNCaP cells but only low levels in PC3 cells. LNCaP cells also showed sequential increases in levels of phosphorylated p53 (serine 15), total p53, Bax, and p21(Waf1) proteins following selenite treatment. The effects of selenite were suppressed by pretreatment with a synthetic superoxide dismutase mimic or by knockdown of p53 via RNA interference. LNCaP cells treated with selenite also showed p53 translocation to mitochondria, cytochrome c release into the cytosol, and activation of caspase-9. On the other hand, restoration of wild-type p53 expression in PC3 cells increased cellular sensitivity to selenite and resulted in increased superoxide production, caspase-9 activation, and apoptosis following selenite treatment. These results suggest that selenite induces apoptosis by producing superoxide to activate p53 and to induce p53 mitochondrial translocation. Activation of p53 in turn synergistically enhances superoxide production and apoptosis induced by selenite.

Combination therapy with vitamins C plus E inhibits survivin and human prostate cancer cell growth.

We postulated that combinations of C and E vitamins modulate the antioxidant network and blocks survivin gene expression in androgen-responsive and non-responsive human prostate cancer cell (HPCC) lines. ALVA-101 and DU-145 cell growth and apoptosis were estimated using the Cell Titer 96 AQ and cell death detection ELISA. Reverse transcription-polymerase chain reaction, Western blot and electrophoretic mobility shift assay were used to quantify survivin mRNA, protein, nuclear factor kappa B (NF-kappaB), and activator protein-1 (AP-1). All the tested combinations of vitamins C and E (25-100 microM, 72 hr) reduced cell growth (4-83%). Vitamin C enhanced the growth suppressive effect of vitamin E. Apoptosis was enhanced (25-45%) (vitamins = 100 microM, 24 hr). Survivin mRNA was decreased (26-29%) (vitamins = 250 microM, 24 hr), and survivin protein was decreased (>90%) (vitamins = 100 microM, 72 hr). NF-kappaB and AP-1 activities were increased (vitamins = 100 microM, 24 hr). The combinations of vitamins C and E are potent inducers of apoptosis in HPCC and suppressers of surviving, an antiapoptotic factor.

Induction of CDK Inhibitors (p21WAF1and p27Kip1) and Bak in the β-Lapachone-Induced Apoptosis of Human Prostate Cancer Cells

beta-Lapachone, a novel anti-neoplastic drug, induces various cancer cells to undergo apoptosis. In a previous report, we showed that beta-lapachone-induced apoptosis of HL-60 cells is mediated by oxidative stress. However, in the present study, we found that beta-lapachone-induced apoptosis of human prostate cancer (HPC) cells may be independent of oxidative stress. In contrast to the 10-fold beta-lapachone-induced increase in H(2)O(2) production seen in HL-60 cells, only a 2- to 4-fold increase was observed in HPC cells. N-acetyl-L-cysteine (NAC), a thiol antioxidant, inhibited the apoptosis in DU145 cells after 12 h exposure to beta-lapachone. Nonetheless, NAC, along with other antioxidants, failed to exert similar effect in HPC cells subjected to beta-lapachone treatment for 24 h. Under this premise, we suggest that the oxidative stress may not play a crucial role in beta-lapachone-mediated HPC cell apoptosis. Here we demonstrate that damage to genomic DNA is the trigger for the apoptosis of HPC cells induced by beta-lapachone. According to our results, beta-lapachone stimulates DNA dependent kinase expression and poly(ADP-ribose) polymerase cleavage in advance of significant morphological changes. beta-Lapachone promotes the expression of cyclin-dependent kinase (cdk) inhibitors (p21(WAF1) and p27(Kip1)), induces bak expression, and subsequently stimulates the activation of caspase-7 but not of caspase-3 or caspase-8 during the apoptosis of HPC cells. Taken together, these results suggest that the signaling pathway involving the beta-lapachone-induced apoptosis of HPC cell may be by DNA damage, induction of cdk inhibitors (p21 and p27), and then subsequent stimulation of caspase-7 activation.

Intracellular Fas ligand expression causes Fas-mediated apoptosis in human prostate cancer cells resistant to monoclonal antibody-induced apoptosis.

Several laboratories have attempted with little success to induce Fas-mediated apoptosis in prostate cancer (PCa) cells, using different external Fas agonists, i.e., anti-Fas antibodies and membrane-bound FasL. The present study confirms these earlier results using the anti-Fas antibody CH-11 in five human PCa cell lines (PPC-1, LNCaP, PC-3, TSU-Pr1, and DU145). However, intracellular murine FasL expression induced Fas-mediated apoptosis in all CH-11-resistant cell lines. Adenovirus (AdGFPFasL(TET)) was used to deliver a Murine FasL-GFP fusion gene into human PCa cells resulting in 70-98% apoptosis at 48 h as determined by the MTS assay. DU145 and PPC-1 cells treated with AdGFPFasL(TET) stained positive for the TUNEL assay, indicating that cell death was via apoptosis. Using immunofluorescent microscopy, Fas and GFPFasL colocalized to the same intracellular compartment. The anti-Fas neutralizing antibody ZB-4 was unable to block AdGFPFasL(TET)-mediated cell death, suggesting that intracellular FasL may ligate Fas within the Golgi and/or endoplasmic reticulum. This is the first evidence suggesting that these two molecules interact prior to cell surface presentation. Collectively, these findings indicate that intracellular GFPFasL expression is superior to CH-11 at inducing Fas-mediated apoptosis in human PCa cells and may allow use of AdGFPFasL(TET) for PCa gene therapy.