IKKα has been implicated as a key regulator of oncogenesis and driver of the metastatic process; therefore is regarded as a promising therapeutic target in anticancer drug development. In spite of the progress made in the development of IKK inhibitors, no potent IKKα inhibitor(s) have been identified. Our multistep approach of molecular modeling and direct binding has led to the identification of plant flavone apigenin as a specific IKKα inhibitor. Here we report apigenin, in micro molar range, inhibits IKKα kinase activity, demonstrates anti-proliferative and anti-invasive activities in functional cell based assays and exhibits anticancer efficacy in experimental tumor model. We found that apigenin directly binds with IKKα, attenuates IKKα kinase activity and suppresses NF-ĸB/p65 activation in human prostate cancer PC-3 and 22Rv1 cells much more effectively than IKK inhibitor, PS1145. We also showed that apigenin caused cell cycle arrest similar to knockdown of IKKα in prostate cancer cells. Studies in xenograft mouse model indicate that apigenin feeding suppresses tumor growth, lowers proliferation and enhances apoptosis. These effects correlated with inhibition of p-IKKα, NF-ĸB/p65, proliferating cell nuclear antigen and increase in cleaved caspase 3 expression in a dose-dependent manner. Overall, our results suggest that inhibition of cell proliferation, invasiveness and decrease in tumor growth by apigenin are mediated by its ability to suppress IKKα and downstream targets affecting NF-ĸB signaling pathways.
prostate cancer; apigenin; NF-ĸB signaling; therapeutic target; cell cycle
Docetaxel (DTX) chemotherapy remains a standard-of-care for metastatic castration-resistant prostate cancer (CRPC). DTX modestly increases survival, yet results in frequent occurrence of side-effects and resistant disease. An alternate chemotherapy with greater efficacy and minimal side-effects is needed. Acquisition of metabolic aberrations promoting increased survival and metastasis in CRPC cells include constitutive activation of Akt, loss of adenosine monophosphate-activated protein kinase (AMPK) activity due to Ser-485/491 phosphorylation, and over-expression of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG-CoAR). We report that combination of simvastatin (SIM) and metformin (MET), within pharmacological dose range (500nM to 4µM SIM and 250µM to 2mM MET), significantly and synergistically reduces C4-2B3/B4 CRPC cell viability and metastatic properties with minimal adverse effects on normal prostate epithelial cells. Combination of SIM and MET decreased Akt Ser-473 and Thr-308 phosphorylation and AMPKα Ser-485/491 phosphorylation, increased Thr-172 phosphorylation and AMPKα activity as assessed by increased Ser-79 and Ser-872 phosphorylation of acetyl-CoA carboxylase and HMG-CoAR, respectively; decreased HMG-CoAR activity, and reduced total cellular cholesterol and its synthesis in both cell lines. Studies of C4-2B4 orthotopic NCr-nu/nu mice further demonstrated that combination of SIM and MET (3.5–7.0µg/g body weight SIM and 175–350µg/g body weight MET) daily by oral gavage over 9-week period significantly inhibited primary ventral prostate tumor formation, cachexia, bone metastasis, and biochemical failure more effectively than 24µg/g body weight DTX intraperitoneally-injected every three weeks, 7.0µg/g/day SIM, or 350µg/g/day MET treatment alone, with significantly less toxicity and mortality than DTX, establishing combination SIM and MET as a promising chemotherapeutic alternative for metastatic CRPC.
Simvastatin; metformin; synergy; prostate cancer
Aberrant Nuclear Factor-κappaB (NF-κB) activation due to rapid IκBα turnover and high basal IκBα kinase (IKK) activity has been frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits anti-proliferative, anti-inflammatory and anti-carcinogenic activities by inhibiting NF-κB pathway, through a mechanism not fully understood. We found that apigenin feeding in microgram doses (bioavailable in humans) inhibited prostate tumorigenesis in TRAMP mice by interfering with NF-κB signaling. Apigenin feeding to TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate and completely abolished metastasis, which correlated with inhibition of NF-κB activation and binding to the DNA. Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation. The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding. These events correlated with the induction of apoptosis in tumor cells, as evident by increased cleaved caspase-3 labeling index in the dorsolateral prostate. Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it’s phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.
Dysfunction of the apoptotic pathway in prostate cancer cells confers apoptosis resistance towards various therapies. A novel strategy to overcome resistance is to directly target the apoptotic pathway in cancer cells. Apigenin, an anticancer agent, selectively toxic to cancer cells induces cell cycle arrest and apoptosis through mechanisms which are not fully explored. In the present study we provide novel insight into the mechanisms of apoptosis induction by apigenin. Treatment of androgen-refractory human prostate cancer PC-3 and DU145 cells with apigenin resulted in dose-dependent suppression of XIAP, c-IAP1, c-IAP2 and survivin protein levels. Apigenin treatment resulted in significant decrease in cell viability and apoptosis induction with the increase of cytochrome C in time-dependent manner. These effects of apigenin were accompanied by decrease in Bcl-xL and Bcl-2 and increase in the active form of Bax protein. The apigenin-mediated increase in Bax was due to dissociation of Bax from Ku70 which is essential for apoptotic activity of Bax. Apigenin treatment resulted in the inhibition of class I histone deacetylases and HDAC1 protein expression, thereby increasing the acetylation of Ku70 and the dissociation of Bax resulting in apoptosis of cancer cells. Furthermore, apigenin significantly reduced HDAC1 occupancy at the XIAP promoter, suggesting that histone deacetylation might be critical for XIAP downregulation. These results suggest that apigenin targets inhibitor of apoptosis proteins and Ku70–Bax interaction in the induction of apoptosis in prostate cancer cells and in athymic nude mouse xenograft model endorsing its in vivo efficacy.
Prostate cancer; Apigenin; Inhibitor of apoptosis proteins; Apoptosis; Ku70; Histone deacetylase
Oxidative stress plays an important role in the development of various human diseases. Aqueous chamomile extract is used as herbal medicine, in the form of tea, demonstrated to possess antiinflammatory and antioxidant properties. We demonstrate the cytoprotective effects of chamomile on hydrogen peroxide (H2O2)-induced cellular damage in macrophage RAW 264.7 cells. Pretreatment of cells with chamomile markedly attenuated H2O2-induced cell viability loss in a dose-dependent manner. The mechanisms by which chamomile-protected macrophages from oxidative stress was through the induction of several antioxidant enzymes including NAD (P)H:quinone oxidoreductase, superoxide dismutase, and catalase and increase nuclear accumulation of the transcription factor Nrf2 and its binding to antioxidant response elements. Furthermore, chamomile dose-dependently reduced H2O2-mediated increase in the intracellular levels of reactive oxygen species. Our results, for the first time, demonstrate that chamomile has protective effects against oxidative stress and might be beneficial to provide defense against cellular damage.
chamomile; macrophages; phase II enzymes; oxidative stress; antioxidant defense
Apigenin (4′,5,7,-trihydroxyflavone), an anticancer agent, selectively toxic to cancer cells induces cell cycle arrest and apoptosis through mechanisms that have not been fully elucidated. Our studies indicate that apigenin-mediated growth inhibitory responses are due to inhibition of class I histone deacetylases (HDACs) in prostate cancer cells. Treatment of PC-3 and 22Rv1 cells with apigenin (20–40μM) resulted in the inhibition of HDAC enzyme activity, specifically HDAC1 and HDAC3 at the protein and message level. Apigenin-mediated HDAC inhibition resulted in global histone H3 and H4 acetylation, as well as localized hyperacetylation of histone H3 on the p21/waf1 promoter. A corresponding increase was observed in p21/waf1 and bax protein and mRNA expression after apigenin exposure, consistent with the use of HDAC inhibitor, trichostatin A. The downstream events demonstrated cell cycle arrest and induction of apoptosis in both cancer cells. Studies of PC-3 xenografts in athymic nude mice further demonstrated that oral intake of apigenin at doses of 20 and 50μg/mouse/day over an 8-week period resulted in a marked reduction in tumor growth, HDAC activity, and HDAC1 and HDAC3 protein expression at both doses of apigenin. An increase in p21/waf1 expression was observed in apigenin-fed mice, compared to the control group. Furthermore, apigenin intake caused a significant decrease in bcl2 expression with concomitant increase in bax, shifting the bax/bcl2 ratio in favor of apoptosis. Our findings confirm for the first time that apigenin inhibits class I HDACs, particularly HDAC1 and HDAC3 and its exposure results in reversal of aberrant epigenetic events that promote malignancy.
prostate cancer; apigenin; epigenetics; histone modification; chromatin remodeling
High-fat diet (HFD) is considered as a major risk factor for benign prostatic diseases and cancer in the Western world. Studies have shown an association between oxidative stress and prostatic diseases. NF-κB has been implicated in stress response and is deregulated in prostrate disorders; therefore, we sought to determine whether HFD could induce oxidative stress in the prostate which could contribute to prostatic diseases.
Transgenic NF-κB-Luc-Tag mice were either fed with regular diet (RD) or HFD for 12 weeks. Serial, non-invasive molecular imaging was performed to study NF-κB activation in the whole body, and in various organs including thymus, spleen, and prostate. Western blotting was used to determine the expression of NF-κB, its upstream and downstream targets in the prostate.
Two-fold increase in whole body NF-κB activity in vivo and 2–3 fold up-regulated prostate NF-κB activity ex vivo were observed after HFD intake compared with RD controls. HFD-induced NF-κB activity was elevated remarkably in the abdominal cavity, thymus, spleen, and prostate with increase in prostrate weight. In the prostrate, an increase in the protein expression of gp91phox, p22phox, and p47phox NADPH oxidase subunits was observed suggesting the involvement of HFD in causing oxidative stress. Nuclear extracts from the prostrate tissue showed an increased expression of p65/RelA that corresponded with elevated cytosolic levels of p-IκBα, along with increased expression of downstream targets of NF-κB, nitric oxide synthase and cyclooxygenase-2.
Our findings suggest that HFD-mediated oxidative stress and deregulation of NADPH oxidase leads to NF-κB activation in the prostrate.
oxidative stress; high-fat diet; prostate; obesity; NF-κB
Forkhead box, class “O” (FoxO) transcription factors are involved in multiple signaling pathways and possess tumor suppressor functions. Loss of PTEN and activation of PI3K/Akt is frequently observed in prostate cancer, which may potentially inactivate FoxO activity. We therefore investigated the role of FoxO transcription factors in prostate cancer progression, in particular FoxO3a, in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, which mimics progressive forms of human disease.
Prostate cancer progression in TRAMP mice was followed from 8–28 weeks. Expression patterns of Akt, FoxO1a, FoxO3a, FoxO4 and their phosphorylated form, DNA binding activity and downstream signaling molecules during different stages of disease progression were examined by immunoblotting, immunoprecipitation, enzyme-linked immunoabsorbant assay (ELISA), and immunohistochemistry. Inhibition of FoxO3a activity was attained by using FoxO3a peptide treatment to TRAMP mice.
In TRAMP mice, FoxO3a activity is negatively regulated by Akt/PKB through posttranslational modification. Progressive increase in Akt activation during prostate cancer progression led to increase phosphorylation of FoxO3a and binding with 14-3-3, which potentially affected its transcriptional activity in age-specific manner. Furthermore, blocking FoxO3a activity resulted in accelerated prostate cancer progression in these mice, which was associated with the loss of cell cycle control and increased proliferation and survival markers.
Restoration of FoxO3a activity represents an attractive therapeutic target in the chemoprevention and possibly in inhibition of progression of prostate cancer.
Forkhead transcription factors; prostate cancer; PI3K/Akt; PTEN; TRAMP; cell cycle
Deregulation of IGF signaling plays an important role in prostate cancer and contributes to invasion and metastasis. We determined the effect of apigenin, a plant flavone, on IGF signaling and its downstream targets in TRAMP mice.
Mice received p.o. apigenin at 20 and 50µg/day dose for 20 weeks. ELISA, Western blotting and immunohistochemistry were performed to examine the IGF-axis and its regulated pathway in response to apigenin intake.
Increased serum levels of IGF-I, VEGF, uPA and concomitant decrease in IGFBP3 were observed; p-Akt (Ser473), p-ERK1 (T202/Y204) and p-ERK2 (T185/Y187) expression increased in the dorso-lateral prostate of TRAMP mice during the course of cancer progression as a function of age. P.o. administration of apigenin resulted in substantial reduction in the levels of IGF-I and increase in the levels of IGFBP-3 in the serum and the dorso-lateral prostate. This modulation of IGF/IGFBP-3 was associated with an inhibition of p-Akt and p-ERK1/2. Apigenin intake resulted in marked inhibition of VEGF, uPA, MMP-2 and MMP-9 which coincided with tumor growth inhibition and complete absence of metastasis in TRAMP mice.
Our results indicate that apigenin effectively suppressed prostate cancer progression in TRAMP mice by attenuating IGF-I/IGFBP-3 signaling and inhibiting angiogenesis and metastasis.
insulin-like growth factor; prostate cancer; apigenin; angiogenesis; metastasis
While studies of alternative pre-mRNA splicing regulation have typically focused on RNA-binding proteins and their target sequences within nascent message, it is becoming increasingly evident that mRNA splicing, RNA polymerase II (pol II) elongation and chromatin structure are intricately intertwined. The majority of introns in higher eukaryotes are excised prior to transcript release in a manner that is dependent on transcription through pol II. As a result of co-transcriptional splicing, variations in pol II elongation influence alternative splicing patterns, wherein a slower elongation rate is associated with increased inclusion of alternative exons within mature mRNA. Physiological barriers to pol II elongation, such as repressive chromatin structure, can thereby similarly impact splicing decisions. Surprisingly, pre-mRNA splicing can reciprocally influence pol II elongation and chromatin structure. Here, we highlight recent advances in co-transcriptional splicing that reveal an extensive network of coupling between splicing, transcription and chromatin remodeling complexes.
alternative pre-mRNA splicing; chromatin; transcription elongation; RNA polymerase II
Protection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms.
The cytoprotective effect of chamomile was examined on H2O2-induced cellular stress in RAW 264.7 murine macrophages.
RAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H2O2. Treatment with 50 μM H2O2 for 6 h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10-20 μg/mL for 16 h followed by H2O2 treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus.
These molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties.
chamomile; macrophages; phase II enzymes; oxidative stress; antioxidant defense; inflammation
Signal transducer and activator of transcription (Stat)-3 and nuclear factor-kappa B (NF-κB) are important signaling pathways constitutively activated during inflammation. We previously reported that high-fat diet (HFD) intake induces oxidative stress in the prostate through elevated expression of NADPH oxidase subunits causing NF-κB activation. We sought to determine whether Stat-3 is involved in the activation of NF-κB in the prostate as a result of HFD feeding, leading to inflammation.
C57BL/6 mice were either fed with regular diet (RD) or HFD for 4 and 8 weeks. Plasma cytokine levels were determined by multiplex analysis. Western blotting was performed to determine the expression of NF-κB, Stat-3, Akt, PDK1, PKCε and their phosphorylated forms along with pathologic evaluation of the prostate. Immunoprecipitation and electrophoretic mobility shift assay were conducted to study the association between Stat-3 and NF-κB.
C57BL/6 mice fed with HFD showed a significant increase in the plasma levels of IL-1β, IL-6, IL-17 and TNFα after 4 and 8 weeks of feeding, compared with RD controls. HFD feeding elevated the intraprostatic expression of IL-6 and caused activation of PKCε and Akt, the upstream kinase regulating Stat-3 and NF-κB. Nuclear extracts from the prostates of mice fed with HFD exhibited constitutively activated levels of Stat-3 and NF-κB/p65. Increased association between the activated forms of Stat-3 and NF-κB/p65 was observed in the nucleus as a result of HFD feeding, a finding that was accompanied by morphologic evidence of increased intraprostatic inflammation.
Our findings suggest that HFD activates Stat-3 and NF-κB/p65 in the prostate, and their interaction is associated with increased inflammation in the prostate.
inflammation; high-fat diet; prostate; Stat-3; NF-κB
Disease aggressiveness remains a critical factor to the progression of prostate cancer. Transformation of epithelial cells to mesenchymal lineage, associated with the loss of E-cadherin, offers significant invasive potential and migration capability. Recently, Special AT-rich binding protein (SATB1) has been linked to tumor progression. SATB1 is a cell-type restricted nuclear protein, which functions as a tissue-specific organizer of DNA sequences during cellular differentiation. Our results demonstrate that SATB1 plays significant role in prostate tumor invasion and migration and its nuclear localization correlates with disease aggressiveness. Clinical specimen analysis showed that SATB1 was predominantly expressed in the nucleus of high-grade tumors compared to low-grade tumor and benign tissue. A progressive increase in the nuclear levels of SATB1 was observed in cancer tissues compared to benign specimens. Similarly, SATB1 protein levels were higher in a number of prostate cancer cells viz. HPV-CA-10, DU145, DUPro, PC-3, PC-3M, LNCaP and C4-2B, compared to non-tumorigenic PZ-HPV-7 cells. Nuclear expression of SATB1 was higher in biologically aggressive subclones of prostate cancer cells with their respective parental cell lines. Furthermore, ectopic SATB1 transfection conferred increased cell motility and invasiveness in immortalized human prostate epithelial PZ-HPV-7 cells which correlated with the loss of E-cadherin expression. Consequently, knockdown of SATB1 in highly aggressive human prostate cancer PC-3M cells inhibited invasiveness and tumor growth in vivo along with increase in E-cadherin protein expression. Our findings demonstrate that SATB1 has ability to promote prostate cancer aggressiveness through epithelial-mesenchymal transition.
Epigenetic silencing of gluthathione-S-transferase pi (GSTP1) is recognized as being a molecular hallmark of human prostate cancer. We investigated the effects of green tea polyphenols (GTP) on GSTP1 re-expression and further elucidated its mechanism of action and long-term safety, compared with nucleoside-analog inhibitor of DNA methyltransferase (DNMT), 5-aza-2′deoxycitidine. Exposure of human prostate cancer LNCaP cells to 1–10μg/mL of GTP for 1–7 days caused a concentration- and time-dependent re-expression of GSTP1 which correlated with DNMT1 inhibition. Methyl-specific-PCR and sequencing revealed extensive demethylation in the proximal GSTP1 promoter and regions distal to the transcription factor binding sites. GTP exposure in a time-dependent fashion diminished the mRNA and protein levels of MBD1, MBD4 and MeCP2; HDAC 1–3 and increased the levels of acetylated histone H3 (LysH9/18) and H4. Chromatin immunoprecipitation assays demonstrated that cells treated with GTP have reduced MBD2 association with accessible Sp1 binding sites leading to increased binding and transcriptional activation of the GSTP1 gene. Exposure of cells to GTP did not result in global hypomethylation, as demonstrated by methyl-specific PCR for LINE-1 promoter; rather, GTP promotes maintenance of genomic integrity. Furthermore, exposure of cells to GTP did not cause activation of the pro-metaststic gene S100P, a reverse response noted after exposure of cells to 5-aza-2′deoxycitidine. Our results, for the first time, demonstrate that GTP has dual potential to alter DNA methylation and chromatin modeling, the two global epigenetic mechanisms of gene regulation, and their lack of toxicity makes them excellent candidates for the chemoprevention of prostate cancer.
prostate cancer; GSTP1; chemoprevention; DNA methylation; histone modification; epigenetics
Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we aimed to investigate the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and to explore its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1β , IL-6 and TNFα-induced NO levels in RAW 264.7 macrophages. Chamomile caused reduction in LPS-induced iNOS mRNA and protein expression. In RAW 264.7 macrophages, LPS-induced DNA binding activity of RelA/p65 was significantly inhibited by chamomile, an effect that was mediated through the inhibition of IKKβ , the upstream kinase regulating NF-κ B/Rel activity, and degradation of inhibitory factor-κ B. These results demonstrate that chamomile inhibits NO production and iNOS gene expression by inhibiting RelA/p65 activation and supports the utilization of chamomile as an effective anti-inflammatory agent.
inflammation; nitric oxide; chamomile; nuclear factor-κ B; macrophages; pro-inflammatory cytokines
Forkhead box transcription factor FOXO3A is an important regulator of cellular function, is thought to act as a tumor suppressor. We studied whether alterations in FOXO3A activity occur in prostate tumorigenesis. Our studies demonstrate that FOXO3A activity is negatively regulated by Akt/PKB through posttranslational modifications. In prostate cancer cells, Akt activation causes increased accumulation of FOXO3A and its binding chaperone protein 14-3-3 in the cytosol. Higher levels of FOXO3A in the cytosol correlated with phosphorylation at Ser253, which accounted for its nuclear exclusion. Dominant negative Akt approach in PC-3 cells increased FOXO3A accumulation in the nucleus, causing upregulation of the downstream target, MnSOD. Conversely, stable DU145-Akt over-expressing cells exhibited decreased FOXO3A levels in the nucleus. Similar findings were noted in prostate tumor specimens, in which marked cytoplasmic accumulation of FOXO3A and 14-3-3 in prostate tumors was observed with increasing Gleason grade, in contrast to exclusively nuclear accumulation in benign prostate cells. These findings correlate with decreased FOXO3A DNA binding activity along with down modulation of FOXO3A transcriptional activity with increasing tumor grade. Our findings demonstrate that tumor associated alterations and redistribution of FOXO3A are frequent events in the etiology of prostate cancer.
Forkhead transcription factor; prostate cancer; tumor suppressor; Akt/PKB; 14-3-3
Apigenin, a naturally occurring plant flavone, abundantly present in common fruits and vegetables is recognized as a bioactive flavonoid shown to possess anti-inflammatory, antioxidant and anticancer properties. Epidemiologic studies suggest that a diet rich in flavones is related to a decreased risk of certain cancers, particularly cancers of the breast, digestive tract, skin, prostate and certain hematological malignancies. It has been suggested that apigenin may be protective in other diseases that are affected by oxidative process such as cardiovascular and neurological disorders, although more research needs to be conducted in this regard. Human clinical trials examining the effect of supplementation of apigenin on disease prevention have not been conducted although there is considerable potential for apigenin to be developed as a cancer chemopreventive agent.
apigenin; chemoprevention; flavonoids; apoptosis; cell cycle
Development of chemoresistance in androgen-refractory prostate cancer cells is partly due to constitutive activation of Rel/NF-κB transcription factors that regulate several cell survival and anti-apoptotic genes. In this study we examined whether betulinic acid (BetA), a pentacyclic triterpene from the bark of white birch, is effective in inhibiting NF-κB expression in androgen-refractory human prostate cancer cells exhibiting high constitutive NF-κB expression. Treatment of PC-3 cells with BetA inhibited DNA binding and reduced nuclear levels of the NF-κB/p65. BetA-mediated NF-κB inhibition involved decreased IKK activity and phosphorylation of IκBα at serine 32/36 followed by its degradation. Reporter assays revealed that NF-κB inhibition by BetA is transcriptionally active. These effects were found to correlate with a shift in Bax/Bcl-2 ratio and cleavage of poly(ADP)ribose polymerase more towards apoptosis. BetA also inhibited TNFα-induced activation of NF-κB via the IκBα pathway, thereby sensitizing the cells to TNFα-induced apoptosis. Our studies demonstrate that BetA effectively inhibits constitutive NF-κB activation and supports the rationale for targeting NF-κB through combination protocols with BetA in androgen-refractory prostate cancer.
transcription factor; betulinic acid; IκBα; IKK; prostate cancer
Deregulation of insulin-like growth factor (IGF)-I/IGF-IR signaling has been implicated in the development and progression of prostate cancer. Agents that can suppress the mitogenic activity of the IGF/IGF-IR growth axis may be of preventive or therapeutic value. We have previously demonstrated that apigenin, a plant flavone, modulates IGF signaling through upregulation of IGFBP-3. In this study, we investigated the mechanism(s) of apigenin action on the IGF/IGF-IR signaling pathway. Exposure of human prostate cancer DU145 cells to apigenin markedly reduced IGF-I-stimulated cell proliferation and induced apoptosis. Apigenin inhibited IGF-I-induced activation of IGF-IR and Akt in DU145 cells. Similar growth inhibitory and apoptotic responses were observed in PC-3 cells, which constitutively over-express this pathway. This effect of apigenin appears to be due partially to reduced autophosphorylation of IGF-IR. Inhibition of p-Akt by apigenin resulted in decreased phosphorylation of GSK-3β along with decreased expression of cyclin D1 and increased expression of p27/kip1. In vivo administration of apigenin to PC-3 tumor xenografts inhibited tumor growth, resulted in IGF-IR inactivation and dephosphorylation of Akt and its downstream signaling. These results suggest that inhibition of cell proliferation and induction of apoptosis by apigenin are mediated, at least in part, by its ability to inhibit IGF/IGF-IR signaling and the PI3K/Akt pathway.
prostate cancer; apigenin; IGF-I; IGF-IR; PI3K-Akt; glycogen synthase kinase-3
Inappropriate activation of phosphatidylinositol 3-kinase–Akt signaling contributes to the development of several human malignancies. Modulation of Akt activity is a strategy that may be valuable in chemopreventive and chemotherapeutic regimens. We have previously demonstrated that apigenin, a plant flavone, causes decreased survival in human prostate cancer cells. However, the molecular mechanism underlying this observation remains elusive. In the present study, we investigated the mechanisms of apigenin action on human prostate cancer PC-3 cells, which possess constitutively active Akt. Treatment of PC-3 cells with apigenin (5–40 μM) resulted in significant dose- and time-dependent decrease in Akt phosphorylation at Serine473. Apigenin-mediated dephosphorylation of Akt resulted in inhibition of its kinase activity, which was confirmed by reduced phosphorylation of proapoptotic proteins BAD and glycogen synthase kinase-3, essential downstream targets of Akt. Hypophosphorylation of BAD resulted in reduced interaction with 14-3-3β protein after 20 μM apigenin exposure to PC-3 cells for 24 h. Inactivation of Akt seems to be associated with downregulation of insulin-like growth factor receptor 1 protein level and inhibition of its autophosphorylation upon apigenin treatment. Exposure to apigenin significantly induced caspase-9 activity and decreased the survival of PC-3 cells in a dose-dependent manner. Furthermore, Serine473 phosphorylation of ectopically expressed Akt in DU145 cells was significantly reduced upon 20 μM apigenin treatment. In vivo, apigenin intake through gavage resulted in inactivation of Akt and induction of apoptosis in PC-3 tumors. These results suggest that Akt inactivation and dephosphorylation of BAD is a critical event, at least in part, in apigenin-induced decreased cell survival and apoptosis.
Our previous findings have shown that systemic administration of IL-1β induces up-regulation of NF-κB in mouse prostate tissue that may be responsible for leukocyte extravasation into prostate stroma. It has been hypothesized that NF-κB plays a role in the development of prostatitis, and that NF-κB activation might provide chemoattractive signals for leukocyte extravasation in the prostate.
IL-1β was administrated intravenously, alone or with dexamethasone, to separate groups of C57BL/6J mice. Expression of NF-κB, chemoattractant receptors, and IL-17F in the two groups of mouse prostates were evaluated and compared.
IL-1β administration up-regulated NF-κB/p65 activity in the mouse prostate. IL-1β administration promoted extravasation and accumulation of CD45+ mononuclear cells but not neutrophils in the mouse prostate stroma. IL-1β administration provided earlier (4 h) CXCR1/IL-8RA receptor expression in mouse prostate as a first signal, inducing capillary homing, adhesion and initial extravasation of mononuclear cells into the prostate tissue. IL-1β administration also induced relatively late (24 h) up-regulation of VCAM1 in the endothelial cells of microvessels and of IL-17F in prostate epithelium and in stromal infiltrating leukocytes. Concomitant administration of dexamethasone, a NF-κB inhibitor, resulted in significantly down-regulated IL-1β-induced NF-κB/p65 activity, as well as reduced expression of chemokine receptors and IL-17F in mouse prostate.
Systemic IL-1β administration induces aberrant NF-κB/p65 activity, which may be critical in the development of prostatitis through its role in the production of chemoattractant signals promoting extravasation and stromal accumulation of mononuclear cells and initiation of a new wave of pro-inflammatory signals favorable to chronic inflammation.
NF-κB; Prostate; IL-1β; CXCR4; IL-17F; Mouse
Inappropriate activation of PI3K/Akt signaling contributes to the development of several human malignancies. Modulation of Akt activity is a strategy that may be valuable in chemopreventive and chemotherapeutic regimens. We have previously demonstrated that apigenin, a plant flavone, causes decreased survival in human prostate cancer cells. However, the molecular mechanism underlying this observation remains elusive. In the present study, we investigated the mechanism(s) of apigenin action on human prostate cancer PC-3 cells, which possess constitutively active Akt. Treatment of PC-3 cells with apigenin (5–40µM) resulted in significant dose- and time- dependent decrease in Akt phosphorylation at Serine473. Apigenin-mediated dephosphorylation of Akt resulted in inhibition of its kinase activity, which was confirmed by reduced phosphorylation of pro-apoptotic proteins BAD and glycogen synthase kinase-3, essential downstream targets of Akt. Hypophosphorylation of BAD resulted in reduced interaction with 14-3-3β protein after 20µM apigenin exposure to PC-3 cells for 24 h. Inactivation of Akt seems to be associated with downregulation of insulin-like growth factor receptor 1 protein level and inhibition of its autophosphorylation upon apigenin treatment. Exposure to apigenin significantly induced caspase-9 activity and decreased the survival of PC-3 cells in a dose-dependent manner. Furthermore, Serine473 phosphorylation of ectopically expressed Akt in DU145 cells was significantly reduced upon 20µM apigenin treatment. In vivo, apigenin intake through gavage resulted in inactivation of Akt and induction of apoptosis in PC-3 tumors. These results suggest that Akt inactivation and dephosphorylation of BAD is a critical event, at least in part, in apigenin-induced decreased cell survival and apoptosis.
prostate cancer; apigenin; PI3K-Akt; BAD; glycogen synthase kinase-3
Apigenin, a plant flavone, potentially activates wild-type p53 and induces apoptosis in cancer cells. We conducted detailed studies to understand its mechanism of action. Exposure of human prostate cancer 22Rv1 cells, harboring wild-type p53, to growth-suppressive concentrations (10–80 μM) of apigenin resulted in the stabilization of p53 by phosphorylation on critical serine sites, p14ARF-mediated downregulation of MDM2 protein, inhibition of NF-κB/p65 transcriptional activity, and induction of p21/WAF-1 in a dose- and time-dependent manner. Apigenin at these doses resulted in ROS generation, which was accompanied by rapid glutathione depletion, disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. Interestingly, we observed accumulation of a p53 fraction to the mitochondria, which was rapid and occurred between 1 and 3 h after apigenin treatment. All these effects were significantly blocked by pretreatment of cells with the antioxidant N-acetylcysteine, p53 inhibitor pifithrin-α, and enzyme catalase. Apigenin-mediated p53 activation and apoptosis were further attenuated by p53 antisense oligonucleotide treatment. Exposure of cells to apigenin led to a decrease in the levels of Bcl-XL and Bcl-2 and increase in Bax, triggering caspase activation. Treatment with the caspase inhibitors Z-VAD-FMK and DEVD-CHO partially rescued these cells from apigenin-induced apoptosis. In vivo, apigenin administration demonstrated p53-mediated induction of apoptosis in 22Rv1 tumors. These results indicate that apigenin-induced apoptosis in 22Rv1 cells is initiated by a ROS-dependent disruption of the mitochondrial membrane potential through transcriptional-dependent and -independent p53 pathways.
Reactive oxygen species; Apoptosis; p53; Prostate cancer; Apigenin; Free radicals
Aberrant nuclear factor-κB (NF-κB) activation has been implicated in the pathogenesis of several human malignancies. In this study, we determined whether NF-κB is constitutively activated in human prostate adenocarcinoma, and, if so, whether increased NF-κB activation and its binding to DNA influence tumor progression. Using tissue samples obtained during transurethral prostatic resection and paraffin-embedded sections of benign and cancer specimens, we determined the nuclear expression of NF-κB/p65 and NF-κB/p50, cytoplasmic expression of IκBα, its phosphorylation, and expression of NF-κB-regulated genes, specifically Bcl2, cyclin D1, matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF). A progressive increase in the expression of NF-τB/p65 (but not of p50) was observed in cancer specimens compared to benign tissue, which correlated with increasing levels of IτBα and its phosphorylation. NF-τB DNA-binding activity increased with increasing tumor grade and the binding complex mainly consisted of NF-τB/p65-p50 heterodimers. Immunohistochemical analysis showed enhanced nuclear staining for NF-τB/p65 in both high-grade (P < .0001) and low-grade (P < .003) cancer specimens, compared to benign tissue. The nuclear levels of NF-τB/p65 correlated with concurrent increase in cytosolic levels of IτBα along with NF-τB-dependent expression of Bcl2, cyclin D1, MMP-9, and VEGF. These results demonstrate that NF-τB/p65 is constitutively activated in human prostate adenocarcinoma and is related to tumor progression due to transcriptional regulation of NF-τB-responsive genes.
Nuclear factor-κB; NF-κB-responsive genes; prostate cancer; IκB; Rel A