1,10-Phenanthroline promotes copper complexes into tumor cells and induces apoptosis by inhibiting the proteasome activity

Indole-3-acetic acid and indole-3-propionic acid, two potent natural plant growth hormones, have attracted attention as promising prodrugs in cancer therapy. Copper is known to be a cofactor essential for tumor angiogenesis. We have previously reported that taurine, l-glutamine, and quinoline-2-carboxaldehyde Schiff base copper complexes inhibit cell proliferation and proteasome activity in human cancer cells. In the current study, we synthesized two types of copper complexes, dinuclear complexes and ternary complexes, to investigate whether a certain structure could easily carry copper into cancer cells and consequently inhibit tumor proteasome activity and induce apoptosis. We observed that ternary complexes binding with 1,10-phenanthroline are more potent proteasome inhibitors and apoptosis inducers than dinuclear complexes in PC-3 human prostate cancer cells. Furthermore, the ternary complexes potently inhibit proteasome activity before induction of apoptosis in MDA-MB-231 human breast cancer cells, but not in nontumorigenic MCF-10A cells. Our results suggest that copper complexes binding with 1,10-phenanthroline as the third ligand could serve as potent, selective proteasome inhibitors and apoptosis inducers in tumor cells, and that the ternary complexes may be good potential anticancer drugs.     

Protein kinase C epsilon mediates the induction of P-glycoprotein in LNCaP prostate carcinoma cells.

P-glycoprotein (P-gp) mediates drug resistance. Protein kinase C (PKC) expression correlates with drug resistance in several types of cancer. We determined whether PKC signals the induction of P-gp in LNCaP human prostate cancer cells, and identified a specific isozyme involved, in a model of aspirin-induced P-glycoprotein expression. An inhibitor of PKC activity, and a specific peptide inhibitor of PKC epsilon translocation, suppressed the induction of P-gp. The PKC activator ingenol, but not OAG, induced P-gp expression in a dose-dependent manner. Based on our results, we conclude that PKC epsilon mediates the induction of P-gp. Accordingly, PKC epsilon is activated and translocates from the membrane fraction to the cytoskeleton fraction in aspirin-treated cells. The findings of this study point to PKC epsilon as a signalling molecule for the induction of P-gp in LNCaP prostate cancer cells.     

Inhibition of 5-lipoxygenase triggers apoptosis in prostate cancer cells via down-regulation of protein kinase C-epsilon

Previous studies have shown that human prostate cancer cells constitutively generate 5-lipoxygenase (5-LOX) metabolites from arachidonic acid, and inhibition of 5-LOX blocks production of 5-LOX metabolites and triggers apoptosis in prostate cancer cells. This apoptosis is prevented by exogenous metabolites of 5-LOX, suggesting an essential role of 5-LOX metabolites in the survival of prostate cancer cells. However, downstream signaling mechanisms which mediate the survival-promoting effects of 5-LOX metabolites in prostate cancer cells are still unknown. Recently, we reported that MK591, a specific inhibitor of 5-LOX activity, induces apoptosis in prostate cancer cells without inhibition of Akt, or ERK, two well-characterized regulators of pro-survival mechanisms, suggesting the existence of an Akt and ERK-independent survival mechanism in prostate cancer cells regulated by 5-LOX. Here, we report that 5-LOX inhibition-induced apoptosis in prostate cancer cells occurs via rapid inactivation of protein kinase C-epsilon (PKCε), and that exogenous 5-LOX metabolites prevent both 5-LOX inhibition-induced down-regulation of PKCε and induction of apoptosis. Interestingly, pre-treatment of prostate cancer cells with diazoxide (a chemical activator of PKCε), or KAE1-1 (a cell-permeable, octa-peptide specific activator of PKCε) prevents 5-LOX inhibition-induced apoptosis, which indicates that inhibition of 5-LOX triggers apoptosis in prostate cancer cells via down-regulation of PKCε. Altogether, these findings suggest that metabolism of arachidonic acid by 5-LOX activity promotes survival of prostate cancer cells via signaling through PKCε, a pro-survival serine/threonine kinase.     

Differential effects on growth, cell cycle arrest, and induction of apoptosis by resveratrol in human prostate cancer cell lines.

Epidemiologic studies have suggested that nutrition plays an important role in carcinogenesis and that 30% of cancer morbidity and mortality can potentially be prevented with proper adjustment of diets. Resveratrol, a polyphenol present in red wines and a variety of human foods, has recently been reported to exhibit chemopreventive properties when tested in a mouse skin cancer model system. In this study, we investigated the effects of resveratrol on growth, induction of apoptosis, and modulation of prostate-specific gene expression using cultured prostate cancer cells that mimic the initial (hormone-sensitive) and advanced (hormone-refractory) stages of prostate carcinoma. Androgen-responsive LNCaP and androgen-nonresponsive DU-145, PC-3, and JCA-1 human prostate cancer cells were cultured with different concentrations of resveratrol (2. 5 x 10(-5)-10(-7) M). Cell growth, cell cycle distribution, and apoptosis were determined. Addition of 2.5 x 10(-5) M resveratrol led to a substantial decrease in growth of LNCaP and in PC-3 and DU-145 cells, but only had a modest inhibitory effect on proliferation of JCA-1 cells. Flow cytometric analysis showed resveratrol to partially disrupt G1/S transition in all three androgen-nonresponsive cell lines, but had no effect in the androgen-responsive LNCaP cells. In difference to the androgen-nonresponsive prostate cancer cells however, resveratrol causes a significant percentage of LNCaP cells to undergo apoptosis and significantly lowers both intracellular and secreted prostate-specific antigen (PSA) levels without affecting the expression of the androgen receptor (AR). These results suggest that resveratrol negatively modulates prostate cancer cell growth, by affecting mitogenesis as well as inducing apoptosis, in a prostate cell-type-specific manner. Resveratrol also regulates PSA gene expression by an AR-independent mechanism.     

Inhibition of arachidonate 5-lipoxygenase triggers prostate cancer cell death through rapid activation of c-Jun N-terminal kinase

Previously, we reported that inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in both androgen-sensitive (LNCaP) and androgen-refractory (PC3) human prostate cancer cells within hours of treatment [Proc. Natl. Acad. Sci. USA 95 (1998) 13182-13187]. Apoptosis was prevented by exogenous 5(S)-HETE, a product of 5-lipoxygenase, indicating a role of this eicosanoid as an essential survival/anti-apoptotic factor for prostate cancer cells. However, nothing was clearly known about details of the underlying molecular mechanisms or events mediating the induction of fulminating apoptosis in these cells. This report documents the fact that inhibition of arachidonate 5-lipoxygenase induces rapid activation of c-Jun N-terminal kinase (JNK) in human prostate cancer cells which is prevented by the 5-lipoxygenase metabolite, 5(S)-HETE. Activation of JNK is unaffected by the cell-permeable tetra-peptide inhibitors of caspase 8 or caspase 3 (IETD-FMK and DEVD-FMK), though these inhibitors effectively blocked apoptosis triggering, suggesting that activation of JNK is independent or upstream of caspase activation. Both 5-lipoxygenase inhibition-induced activation of JNK and induction of apoptosis are prevented by curcumin, an inhibitor of JNK-signaling pathway. Apoptosis is also blocked by SP600125, a specific inhibitor of JNK activity, indicating that JNK activity is required for the induction of apoptosis in these cells. These findings suggest that the metabolites of arachidonate 5-lipoxygenase promote survival of prostate cancer cells involving down-regulation of stress-activated protein kinase.     

Polyethylene glycol induces apoptosis in HT-29 cells: potential mechanism for chemoprevention of colon cancer

Recent experimental evidence suggests that polyethylene glycol (PEG) is a highly effective chemopreventive agent against colon cancer; however, the mechanism(s) remain largely unexplored. To further elucidate this issue, we evaluated the effect of PEG on two human colon cancer cell lines. PEG treatment resulted in a dose- and time-dependent reduction in cell number without alteration in markers of cell proliferation. However, there was a dramatic and specific, concentration-dependent induction of apoptosis, with 50 mM PEG rendering approximately half the cells apoptotic. This corresponded with a 17-fold induction in the expression of the pro-apoptotic protein, prostate apoptosis response-4. Our data suggest that induction of apoptosis may be responsible, at least in part, for the ability of PEG to prevent experimental colon cancer.     

The effect of chelerythrine on cell growth, apoptosis, and cell cycle in human normal and cancer cells in comparison with sanguinarine

We compared the effects of chelerythrine (CHE) and sanguinarine (SA) on human prostate cancer cell lines (LNCaP and DU-145) and primary culture of human gingival fibroblasts. CHE and SA treatment of cell lines for 24 h resulted in (1) inhibition of cell viability in a dose-dependent manner in all tested cells (as evaluated by MTT test and bromodeoxyuridine incorporation assay); (2) dose-dependent increase in DNA damage in all tested cells (as evaluated by DNA comet assay); (3) changes in apoptosis (assessed by western blot analysis and TUNEL assay); and (4) significant induction of cyclin kinase inhibitors p21^Waf1/Cip1 and p27^Kip1 in prostate cancer cells (identified by western blot analysis). Our study demonstrates that CHE had significant cytotoxic effect, independent of p53 and androgen status, on human prostate cancer cell lines. Normal gingival fibroblasts and DU-145 cells were more sensitive to the treatment with both alkaloids than were LNCaP cells. CHE and SA may be prospective natural molecules for use in the treatment of prostate cancer owing to their involvement in apoptosis and cell cycle regulation.     

Caspase-dependent apoptosis induced by two synthetic halogenated flavanones, 3′,7-dichloroflavanone and 3′,6-dichloroflavanone,on human breast and prostate cancer cells

The destruction of cancer cells with chemotherapeutic agents is normally achieved through apoptosis. We previously introduced two synthetic halogenated flavanone derivatives, 3^′,7-dichloroflavanone (3′-7 DCF) and 3^′,6-dichloroflavanone (3′-6 DCF), as potential apoptosis-inducing agents. In the current study, we investigated the ability of these compounds in triggering intrinsic or/and extrinsic pathway of apoptosis in breast and prostate cancer cells. Also, the synergistic effect of 3′-7 DCF with TLR3 (Toll-like receptor 3) agonist in apoptosis induction was evaluated on PC3 and LNCaP human prostate cancer cells. The involved pathway of apoptosis in the treated cells was delineated by caspase-3 activity assay, PARP-1 (poly(ADP-ribose)polymerase-1) cleavage, and procaspase-9 cleavage as markers of the intrinsic pathway and procaspase-8 cleavage as the marker of the extrinsic pathway. With the exception of the normal cells, treatment of all cell lines with both 3′-7 DCF and 3′-6 DCF triggered the cleavage of procaspase-8 and procaspase-9. These results indicate that the intrinsic and the extrinsic pathways of apoptosis are the mechanisms of the toxicity of flavanones in these cancer cell lines. However, the cytoxicity of the compound 3′-7 DCF was not synergistic with TLR3 agonist. Interestingly, the activation of caspases-9 preceeded that of caspase-8 suggesting that the intrinsic pathway is the primary reason for apoptosis induction by the flavanones.     

Pardaxin-induced apoptosis enhances antitumor activity in HeLa cells

Pardaxin, a pore-forming antimicrobial peptide that encodes 33 amino acids was isolated from the Red Sea Moses sole, Pardachirus mamoratus. In this study, we investigated its antitumor activity in human fibrosarcoma (HT-1080) cells and epithelial carcinoma (HeLa) cells. In vitro results showed that the synthetic pardaxin peptide had antitumor activity in these two types of cancer cells and that 15 μg/ml pardaxin did not lyse human red blood cells. Moreover, this synthetic pardaxin inhibited the proliferation of HT1080 cells in a dose-dependent manner and induced programmed cell death in HeLa cells. DNA fragmentation and increases in the subG1 phase and caspase 8 activities suggest that pardaxin caused HeLa cell death by inducing apoptosis, but had a different mechanism in HT1080 cells.     

Uptake and activation of eicosapentaenoic acid are related to accumulation of triacylglycerol in Ramos cells dying from apoptosis

The present study investigates the mechanism behind induction of cell death by eicosapentaenoic acid (EPA) in leukemia cells. The PUFA-sensitive cell lines Raji and Ramos, which die by necrosis and apoptosis upon incubation with EPA respectively, had 2- to 3-fold higher uptake rate of EPA than the PUFA-resistant U-698 cell line. Furthermore, Ramos cells contained more lipid bodies and 3-fold more triacylglycerol than U-698 cells after 24 h incubation with 60 microm EPA. The mechanism behind the increased rate of EPA uptake in the PUFA-sensitive cell lines was examined by comparing the expression of 6 different fatty acid binding proteins (FABPs) and 3 acyl-CoA synthetases (ACSs) in U-698 and Ramos cells. Moreover, enzymatic activity of ACS and acyl-CoA:1,2-diacylglycerol acyltransferase (ADGAT) was investigated. The protein expression level of CD36 and p-FABPpm, the mRNA level of FABP, liver-FABP, heart-FABP, intestinal-FABP, ACS1, ACS2, and enzymatic ADGAT activity were similar in the two cell lines. However, an mRNA signal observed with a probe for ACS3 was 1.7 times higher in Ramos than in U-698 cells, and lysate from Ramos cells had a higher capacity to activate EPA to EPA-CoA than U-698 cell lysate.In conclusion, the present findings indicate that cellular uptake, activation and incorporation of EPA into lipids may be related to induction of cell death in leukemia cell lines.     

Juglone,isolated from Juglans mandshurica Maxim,induces apoptosis via down-regulation of AR expression in human prostate cancer LNCaP cells

Juglone is a natural compound which has been isolated from Juglans mandshurica Maxim. Recent studies have shown that juglone had various pharmacological effects such as anti-viral, anti-bacterial and anti-cancer. However, its anti-cancer activity on human prostate cancer LNCaP cell has not been examined. Thus, the current study was designed to elucidate the molecular mechanism of apoptosis induced by juglone in androgen-sensitive prostate cancer LNCaP cells. MTT assay was performed to examine the anti-proliferative effect of juglone. Occurrence of apoptosis was detected by Hoechst 33342 staining and flow cytometry in LNCaP cells treated with juglone for 24 h. The result shown that juglone inhibited the growth of LNCaP cells in a dose-dependent manner. Morphological changes of apoptotic body formation after juglone treatment were observed by Hoechst 33342 staining. This apoptotic induction was associated with loss of mitochondrial membrane potential, and caspase-3, -9 activation. Moreover, we found that juglone significantly inhibited the expression levels of androgen receptor (AR) and prostate-specific antigen (PSA) in a dose-dependent manner, as well as abrogated up-regulation of AR and PSA genes with and/or without dihydrotestosterone (DHT). Take together, our results demonstrated that juglone might induce the apoptosis in LNCaP cell via down-regulation of AR expression. Therefore, our results indicated that juglone may be a potential candidate of drug for androgen-sensitive prostate cancer.     

Phellinus Linteus Extract Sensitizes Advanced Prostate Cancer Cells to Apoptosis in Athymic Nude Mice

Phellinus linteus (PL) mushroom possesses anti-tumor property. We previously reported that the treatment with PL caused cultured human prostate cancer cells to undergo apoptosis. To further studying the mechanisms of PL-mediated apoptosis, we performed xenograft assay, together with in vitro assays, to evaluate the effect of PL on the genesis and progression of the tumors formed from the inoculation of prostate cancer PC3 or DU145 cells. After the inoculation, nude mice were injected with PL every two days for 12 days. Although PL treatment did not prevent the formation of the inoculated tumors, the growth rate of the tumors after PL treatment was dramatically attenuated. We then tested the effect of PL on the tumors 12 days after the inoculation. After inoculated tumors reached a certain size, PL was administrated to the mice by subcutaneous injection. The histochemistry or immunochemistry analysis showed that apoptosis occurred with the activation of caspase 3 in the tumors formed by inoculating prostate cancer DU145 or PC3 cells. The data was in a good agreement with that from cultured cells. Thus, our in vivo study suggests that PL not only is able to attenuate tumor growth, but also to cause tumor regression by inducing apoptosis.     

Flavonoid ampelopsin inhibits the growth and metastasis of prostate cancer in vitro and in mice

The objective of this study was to evaluate the chemopreventive effect of a novel flavonoid, ampelopsin (AMP) on the growth and metastasis of prostate cancer cells. AMP showed the more potent activity in inhibiting the proliferation of androgen-sensitive LNCaP and, to less extent, androgen-independent PC-3 human prostate cancer cell lines in vitro, primarily by induction of apoptosis associated with down-regulation of bcl-2. On the other hand, AMP showed much less activity in inhibiting the proliferation of normal prostate epithelial cells than that of prostate cancer cell lines. AMP also inhibited the migration and invasion of PC-3 cells in vitro associated with down-regulation of CXCR4 expression. In the animal study using an orthotopic prostate tumor model, AMP (150 and 300 mg/kg body weight) inhibited the growth of PC-3 tumors and lymph node and lung metastases in a dose-dependent manner. Compared to the control mice, mice treated with AMP at 300 mg/kg BW had reduced final tumor weight by 49.2% (P<0.05), lymph node metastases by 54.5% (P?=?0.3) and lung metastases by 93% (P<0.05), but had no apparent alteration on food intake or body weight. The in vivo anti-growth and anti-metastasis activities of AMP were associated with induction of apoptosis and inhibition of proliferation of prostate cancer cells, reduction of prostate tumor angiogenesis, and reduction of CXCR4 expression. Our results provide supporting evidence to warrant further investigation to develop AMP as a novel efficacious and safe candidate agent against progression and metastasis of prostate cancer.     

Apoptosis in prostate carcinogenesis

Development of effective therapeutic modalities for the treatment of human cancer relies heavily upon understanding the molecular alterations that result in initiation and progression of the tumorigenic process. Many of the molecular changes identified in human prostate tumorigenesis so far play key roles in apoptosis regulation. Apoptosis represents a universal and exquisitely efficient cellular suicide pathway. Since the therapeutic goal is to trigger tumor-selective apoptotic cell death (without clinically significant effects on the host), elucidation of the mechanisms underlying apoptosis deregulation will lead to the identification of specific cellular components for targeting therapeutic interventions. As our understanding of its vital role in the development and growth of the prostate gland has expanded, numerous genes that encode apoptotic regulators have been identified that are severely impaired in prostate cancer cells. In addition, the expression of apoptotic modulators within prostatic tumors appears to correlate with tumor sensitivity to traditional therapies such as hormonal ablation and radiotherapy. No strict correlation between apoptosis induction and a patient's long-term prognosis has emerged, perhaps due to the fact that the ability to achieve initial remission alone does not adequately predict long-term outcome. This review will encompass the known molecular changes intimately involved in the apoptotic pathway which have potential prognostic value in disease progression, as well as therapeutic significance in the enhancement of the apoptotic response to novel and established treatment strategies for the treatment of androgen-dependent and androgen-independent prostatic tumors. The main focus will be on the role of the transforming growth factor-beta (TGF-beta) signaling pathway, bcl-2 and the bcl-2 family members, the caspase cascade (apoptosis executioners), and the Fas pathway in induction and regulation of apoptosis following therapeutic stimuli for the management of advanced prostate cancer.     

Selective apoptosis induction by the cancer chemopreventive agent N-(4-hydroxyphenyl)retinamide is achieved by modulating mitochondrial bioenergetics in premalignant and malignant human prostate epithelial cells

Prostate tumorigenesis is coupled with an early metabolic switch in transformed prostate epithelial cells that effectively increases their mitochondrial bioenergetic capacity. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) inhibits prostate cancer development in vivo, and triggers reactive oxygen species (ROS)-dependent prostate cancer cell apoptosis in vitro. The possibility that 4HPR-induced ROS production is associated with mitochondrial bioenergetics and required for apoptosis induction in transformed prostate epithelial cells in vitro would advocate a prospective mechanistic basis for 4HPR-mediated prostate cancer chemoprevention in vivo. We investigated this tenet by comparing and contrasting 4HPR’s effects on premalignant PWR-1E and malignant DU-145 human prostate epithelial cells. 4HPR promoted a dose- and/or time-dependent apoptosis induction in PWR-1E and DU-145 cells, which was preceded by and dependent on an increase in mitochondrial ROS production. In this regard, the PWR-1E cells were more sensitive than the DU-145 cells, and they consumed roughly twice as much oxygen as the DU-145 cells suggesting oxidative phosphorylation was higher in the premalignant cells. Interestingly, increasing the [Ca²⁺] in the culture medium of the PWR-1E cells attenuated their proliferation as well as their mitochondrial bioenergetic capacity and 4HPR’s cytotoxic effects. Correspondingly, the respiration-deficient derivatives (i.e., ρ⁰ cells lacking mitochondrial DNA) of DU-145 cells were markedly resistant to 4HPR-induced ROS production and apoptosis. Together, these observations implied that the reduction of mitochondrial bioenergetics protected PWR-1E and DU-145 cells against the cytotoxic effects of 4HPR, and support the concept that oxidative phosphorylation is an essential determinant in 4HPR’s apoptogenic signaling in transformed human prostate epithelial cells.     

Activation of Notch pathway is linked with epithelial-mesenchymal transition in prostate cancer cells

Notch signaling has been reported to play an essential role in tumorigenesis. Several studies have suggested that Notch receptors could be oncoproteins or tumor suppressors in different types of human cancers. Emerging evidence has suggested that Notch pathway regulates cell growth, apoptosis, cell cycle, and metastasis. In the current study, we explore whether Notch-1 could regulate the cell invasion and migration as well as EMT (epithelial-mesenchymal transition) in prostate cancer cells. We found that overexpression of Notch-1 enhanced cell migration and invasion in PC-3 cells. However, downregulation of Notch-1 retarded cell migration and invasion in prostate cancer cells. Importantly, we observed that overexpression of Notch-1 led to EMT in PC-3 cells. Notably, we found that EMT-type cells are associated with EMT markers change and cancer stem cell phenotype. Taken together, we concluded that downregulation of Notch-1 could be a promising approach for inhibition of invasion in prostate cancer cells, which could be useful for the treatment of metastatic prostate cancer.     

Disparity of apoptotic response in human breast cancer cells lines MCF-7 and MDA-MB-231 after infection with recombinant adenovirus encoding the VP2 gene of infectious bursal disease virus

Recombinant adenovirus encoding the VP2 gene of infectious bursal disease virus (ADV-VP2) has shown potent anti-tumour effects due to its capability of apoptotic induction in cancer cells. In the present study, human breast cancer cells MCF-7 and MDA-MB-231 were infected with ADV-VP2. The expression of VP2 protein was registered 4 hours post-infection, particularly in MCF-7 cells. Multiple time-point DNA ladder assay demonstrated that ADV-VP2 infected MDA-MB-231 and MCF-7 cells endured apoptosis as early as 8- and 12- hours post-infection, respectively. Apoptosis induction in both MDA-MB-231 and MCF-7 cells, albeit different start points, lasted until 36-hours post-infection. The induction of apoptosis by ADV-VP2 was further shown by the TUNEL assay, with dark brown discoloration of apoptotic cells. The present study also explored the different stages of apoptosis by Annexin V/PI double staining flow cytometry quantification. Treated MCF-7 and MDA-MB-231 cells, respectively detected 25.58 ± 9.02% and 14.51 ± 3.12% of early apoptotic cells, 6.09 ± 4.06% and 77.12 ± 5.09% of late apoptotic cells. Results revealed that there were significant differences in the number of cells of both types which underwent early and late apoptosis. Significant differences were also observed among viable and apoptotic cells which have been post treated with ADV-VP2. The apoptotic effects of ADV-VP2 on human breast cancer cell lines were consistently demonstrated by three apoptosis detection methods. Therefore, a cancer vaccine basing on gene therapy could be developed in the near future using the present construct.