Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1

Prostate cancer (PCa) patients receiving the androgen ablation therapy ultimately develop recurrent castration-resistant prostate cancer (CRPC) within 1–3 years. Treatment with caffeic acid phenethyl ester (CAPE) suppressed cell survival and proliferation via induction of G1 or G2/M cell cycle arrest in LNCaP 104-R1, DU-145, 22Rv1, and C4–2 CRPC cells. CAPE treatment also inhibited soft agar colony formation and retarded nude mice xenograft growth of LNCaP 104-R1 cells. We identified that CAPE treatment significantly reduced protein abundance of Skp2, Cdk2, Cdk4, Cdk7, Rb, phospho-Rb S807/811, cyclin A, cyclin D1, cyclin H, E2F1, c-Myc, SGK, phospho-p70S6kinase T421/S424, phospho-mTOR Ser2481, phospho-GSK3α Ser21, but induced p21^Cip1, p27^Kip1, ATF4, cyclin E, p53, TRIB3, phospho-p53 (Ser6, Ser33, Ser46, Ser392), phospho-p38 MAPK Thr180/Tyr182, Chk1, Chk2, phospho-ATM S1981, phospho-ATR S428, and phospho-p90RSK Ser380. CAPE treatment decreased Skp2 and Akt1 protein expression in LNCaP 104-R1 tumors as compared to control group. Overexpression of Skp2, or siRNA knockdown of p21^Cip1, p27^Kip1, or p53 blocked suppressive effect of CAPE treatment. Co-treatment of CAPE with PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or Bcl-2 inhibitor ABT737 showed synergistic suppressive effects. Our finding suggested that CAPE treatment induced cell cycle arrest and growth inhibition in CRPC cells via regulation of Skp2, p53, p21^Cip1, and p27^Kip1.     

Monepantel induces autophagy in human ovarian cancer cells through disruption of the mTOR/p70S6K signalling pathway

We have recently shown that the novel anthelmintic drug monepantel (MPL) inhibits growth, proliferation and colony formation, arrests the cell cycle and induces cleavage of PARP-1 in ovarian cancer cell lines. Here we report on the mechanism behind the anticancer properties of MPL. The cytotoxic effect of MPL on ovarian cancer cells (OVCAR-3 and A2780) was investigated employing a panel of tests used for the detection of apoptosis and autophagy. Apoptosis and autophagy were defined by caspase activity, DNA-laddering, Annexin-V and acridine orange (AO) staining. Autophagy markers such as LC3B, SQSTM1/p62 and mammalian target of rapamycin (mTOR) pathway related proteins were assessed by western blotting and ELISA techniques. MPL did not activate caspases 3 or 8, nor did it alter the percentage of Annexin V positive stained cells. Failure to cause DNA laddering and the inability of z-VAD-fmk to block the MPL antiproliferative effects led to the ruling out of apoptosis as the mechanism behind MPL-induced cell death. On the other hand, accumulation of acidic vacuoles with distinct chromatin morphology and an increase in punctuate localization of green fluorescent protein-LC3B, and MPL-induced changes in the expression of SQSTM1/p62 were all indicative of MPL-induced autophagy. Consistent with this, we found inhibition of mTOR phosphorylation leading to suppression of the mTOR/p70S6K signalling pathway. Our findings provide the first evidence to show that MPL triggers autophagy through the deactivation of mTOR/p70S6K signalling pathway.     

Ionizing radiation activates Erb-B receptor dependent Akt and p70 S6 kinase signaling in carcinoma cells

In this study we have investigated the effects of low dose ionizing radiation (2 Gy) on p70 S6 kinase and Akt signaling with respect to Erb-B receptors in both the A431 squamous and the MDA-MB-231 mammary carcinoma cell lines. Ionizing radiation caused a 2-3-fold increase in p70 S6 kinase activity that was blocked pharmacologically using an EGFR inhibitor (AG1478) alone, or in combination with an Erb-B2 inhibitor (AG825). These results suggested that both EGFR and Erb-B2 receptors could initiate radiation-induced activation of p70 S6K. EGFR dependent Erb-B3 signaling also contributed to p70 S6 kinase activity through recruitment and activation of PI3K, which has been shown to regulate p70 S6 kinase activity. Furthermore, inhibition of the EGFR blocked IR stimulated increases in protein translation, a biologic consequence of p70 S6 kinase activation. We also report that ionizing radiation stimulated Akt activity that was partially independent of PI3K activity, but dependent on Erb-B2 function. Erb-B2 inhibition also correlated with enhanced apoptosis following IR exposure, suggesting an important role for Erb-B2 in cell survival. Together this work demonstrates that the Erb-B receptor tyrosine kinase network stimulates cytoprotective p70 S6 kinase and Akt activity in response to clinically relevant doses of ionizing radiation.     

Ninjurin1 suppresses metastatic property of lung cancer cells through inhibition of interleukin 6 signaling pathway

Nerve injury‐induced protein 1 (Ninjurin1, Ninj1) is a cell surface molecule that can mediate homophilic adhesion and promote neurite outgrowth from cultured dorsal root ganglion (DRG) neurons. Interestingly, Ninj1 overexpressed in human cancer; however, its role in metastasis is not clear. This study showed that inhibition of Ninj1 promotes lung cancer metastasis through interleukin 6 (IL‐6)/STAT3 signaling. Ninj1 levels were relatively low in highly motile lung cancer cells. While inhibition of Ninj1 enhanced cell migration in lung cancer cells, overexpression of Ninj1 significantly suppressed it. We found that inhibition of Ninj1 significantly increased expression and secretion of IL‐6 in A549 cells. We also found that inhibition of IL‐6 decreased intercellular adhesion molecule 1 (ICAM‐1) expression. In addition, inhibition of Ninj1 significantly increased cell motility and invasiveness of lung cancer cells. In an in vivo model, we found that Ninj1 suppression did not affect tumor growth but induced significant increase in incidence of lung metastasis, and sizes and number of tumor nodules. Taken together, our data clearly demonstrate that Ninj1 suppresses migration, invasion and metastasis of lung cancer via inhibition of the IL‐6 signaling pathway in vitro and in vivo.     

AKT1 amplification regulates cisplatin resistance in human lung cancer cells through the mammalian target of rapamycin/p70S6K1 pathway

Cisplatin [cis-diaminodichloroplatinum (II) (CDDP)] is one of the most widely used and effective therapeutic agents for many kinds of cancers. However, its efficiency is limited due to development of drug resistance. In this study, we showed that CDDP resistance was associated with AKT1 overexpression and gene amplification in human lung cancer cells that acquired the drug resistance. We showed that AKT1 forced expression in the cells was sufficient to render the cells CDDP resistant, and that AKT1 inhibition by its dominant negative mutant reversed the CDDP-resistant cells to be CDDP sensitive. These results show that AKT1 activity is essential for regulating CDDP resistance in cultured lung cancer cells. To study whether these results were correlated with human lung cancer tumors, we randomly selected tumor samples from human lung cancer patients to study the correlation of AKT activation and CDDP resistance in clinical tumor samples. We showed that AKT activation was highly related to CDDP chemosensitivity in human tumor tissues. Our results further showed that AKT1 induced lung cancer cells to become resistant to CDDP through the mammalian target of the rapamycin (mTOR) signaling pathway. These studies conclude that AKT amplification and the mTOR pathway play an important role in human lung cancer cells acquiring CDDP resistance, which represents a new mechanism for acquiring CDDP resistance and a potential novel therapeutic target for overcoming CDDP resistance in human cancer in the future.     

Gallic acid suppresses the migration and invasion of PC-3 human prostate cancer cells via inhibition of matrix metalloproteinase-2 and -9 signaling pathways

Epidemiological studies have demonstrated that a natural diet or consumption of fruits or vegetables can decrease the risk of cancer development. Cancer cells can migrate to and invade other organs or tissues that cause more difficulty to treat them and this also results in the need for treatments targeting multiple cellular pathways. Gallic acid (GA) has been demonstrated to possess multiple biological activities including anticancer function. However, no report exist on GA inhibited invasion and migration of human prostate cancer cells. We investigated the effects of migration and invasion in GA-treated PC-3 human prostate cancer cells with a series of in?vitro experiments. Boyden chamber transwell assay was used to examine the migration and invasion of PC-3 cells. Western blotting, real-time PCR and gelatin zymography were used for determining the protein levels, gene expression and enzyme activities of matrix metalloproteinase-2 (MMP-2) and -9 in?vitro. Results indicated that GA inhibited the invasion and migration of PC-3 cells and these effects are dose-dependent. GA inhibited the protein levels of MMP-2 and -9, son of sevenless homolog?1 (SOS1), growth factor receptor-bound protein 2 (GRB2), protein kinase C (PKC) and nuclear factor-κ B (NF-κB) p65, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), p38, p-AKT (Thr308) and p-AKT (Ser473), but it promoted the levels of phosphatidylinositol 3-kinase (PI3K) and AKT in PC-3 cells. GA also reduced the enzyme activities of MMP-2 and -9 in the examined cells. Moreover, the down-regulation of focal adhesion kinase (FAK) and Ras homolog gene family, member A (Rho A) mRNA expression levels, and up-regulation of the tissue inhibitor of metalloproteinase-1 (TIMP1) gene levels occurred in GA-treated PC-3 cells after 24?h treatment. Based on these observations, we suggest that GA might modulate through blocking the p38, JNK, PKC and PI3K/AKT signaling pathways and reducing the NF-κB protein level, resulting in the inhibition of MMP-2 and -9 of PC-3 human prostate cancer cells.     

Antiproliferation and radiosensitization of caffeic acid phenethyl ester on human medulloblastoma cells

Purpose: To investigate antiproliferative and radiosensitizing effects of caffeic acid phenethyl ester (CAPE) on medulloblastoma (MB) Daoy cells. Methods and materials: Daoy cells were treated with CAPE in different concentrations and assessed for cell viability, apoptosis, cell cycles, cyclin B1 expressions, radiosensitization and chemosensitization. Human astroglia SVGp12 cells were treated with CAPE to present the possible protection or complication effects in normal tissues. Results: CAPE inhibited the growth of Daoy cells in a time- and concentration-dependent manner in MTT and Trypan blue exclusion assays. Flow cytometry revealed that CAPE significantly decreased G2/M fraction, and increased the S phase fraction. Western blot demonstrated a down-regulated cyclin B1 protein expression. Pretreatment with CAPE markedly decreased the viability of irradiated Daoy cells. The sensitizer enhancement ratios (SERs) were increased in CAPE-treated Daoy cells. CAPE in doxorubicin and cisplatin did not show chemosensitizing effect. Conclusions: These findings demonstrate the antiproliferative and radiosensitizing effects of CAPE for Daoy cells, which might bring improvement to the treatment of MB. For clinical application, in vivo models are expected.     

Akt promotes cisplatin resistance in human ovarian cancer cells through inhibition of p53 phosphorylation and nuclear function

Resistance to cisplatin-based chemotherapy is a major cause of treatment failure in human ovarian cancer. Wild-type TP53 status is often, but not always, associated with cisplatin sensitivity, suggesting that additional factors may be involved. Overexpression/activation of the phosphatidylinositol-3-kinase/Akt pathway is commonly observed in ovarian cancer, and Akt activation is a determinant of chemoresistance in ovarian cancer cells, an effect that may be due, in part, to its inhibitory actions on p53-dependent apoptosis. To that end, we examined the role and regulation of p53 in chemosensitive ovarian cancer cells, as well as in their chemoresistant counterparts, and investigated if and how Akt influences this pathway. Cisplatin induced apoptosis in chemosensitive, but not chemoresistant cells, and this was inhibited by downregulation of p53. Cisplatin upregulated PUMA in a p53-dependent manner, and the presence of PUMA was necessary, but not sufficient for cisplatin-induced apoptosis. p53 was phosphorylated on numerous N-terminal residues, including Ser15, Ser20, in response to cisplatin in chemosensitive, but not chemoresistant cells. Furthermore, activation of Akt inhibited the cisplatin-induced upregulation of PUMA, and suppressed cisplatin-induced p53 phosphorylation, while inhibition of Akt increased total and phospho-p53 contents and sensitized p53 wild-type, chemoresistant cells to cisplatin-induced apoptosis. Finally, mutation of Ser15 and/or Ser20, but not of Ser37, to alanine significantly attenuated the ability of p53 to facilitate CDDP-induced apoptosis, and this was independent of PUMA expression. These results support the hypothesis that p53 is a determinant of CDDP sensitivity, and suggest that Akt contributes to chemoresistance, in part, by attenuating p53-mediated PUMA upregulation and phosphorylation of p53, which are essential, but independent determinants of sensitivity to CDDP-induced apoptosis.     

PTPRJ通过Src/PI3K/Akt信号通路促进前列腺癌细胞的黏附、迁移和侵袭

目的:探究PTPRJ基因表达对前列腺癌DU145细胞黏附、迁移和侵袭的影响以及可能的调控机制。方法:实时荧光定量PCR、Western blot检测PTPRJ在前列腺肿瘤组织和细胞系中的表达;用携带PTPRJ特异shRNA的重组慢病毒(LV-shPTPRJ)感染沉默PTPRJ表达;MTT检测细胞黏附力,Transwell检测细胞迁移和侵袭;实时荧光定量PCR、Western blot检测信号通路分子mRNA和蛋白表达。结果:与正常前列腺组织和细胞相比,PTPRJ在前列腺肿瘤组织和PC-3、DU145细胞系中表达升高（P＜0.05）;与对照组相比,沉默PTPRJ后前列腺癌DU145细胞黏附、迁移和侵袭能力显著下降（P＜0.01）、信号通路蛋白pY418Src、p-PI3K和p-Akt表达水平均显著降低（P＜0.05）;SC79激活PI3K/Akt可逆转PTPRJ下调对DU145细胞黏附和侵袭的影响;沉默PTPRJ下调裸鼠瘤体组织中pY418Src、p-PI3K和p-Akt表达（P＜0.05）。结论:PTPRJ可能通过激活Src/PI3K/Akt信号通路来促进DU145细胞的黏附、迁移和侵袭,预示PTPRJ可能成为前列腺癌治疗的潜在靶点。     

Wild-type p53 suppresses growth of human prostate cancer cells containing mutant p53 alleles

Evidence supporting a broad role for the inactivation of the p53 gene in human tumorigenesis has been provided by studies showing that the p53 gene is mutated in many human cancers. In this study, we report on the mutational status of the p53 gene in prostate cancer cells and provide functional evidence that the wild-type p53 gene may have a role in suppressing prostatic tumorigenesis. Sequence analysis of exons 5-8 of the p53 gene reveals that three of five prostate cancer cell lines (TSUPr-1, PC3, DU145) contain mutations which alter the amino acid sequence of this most highly conserved portion of the gene. One of two primary prostatic cancer specimens examined also contained a mutation in this region. Transfection of the wild-type p53 gene versus a mutated p53 gene into two cell lines with p53 mutations results in reduced colony formation. Wild-type p53 gene expression is apparently incompatible with continued growth of these tumor cells inasmuch as none of the colonies which formed after wild-type transfections retain the transfected p53 sequences. Immunocytochemical data indicate that prostate carcinoma cells expressing the transfected wild-type p53 gene are growth arrested because they exhibit a reduced level of thymidine incorporation into DNA. This study is the first report of p53 gene mutations in prostate cancer cells and suggests a functional role for the p53 gene in suppressing prostatic tumorigenesis.     

Preferential cytotoxicity of caffeic acid phenethyl ester analogues on oral cancer cells

As part of our previous search for new compounds with improved biological activities including antibiotic, antiviral, anti-inflammatory, and tumor growth inhibition activities, we synthesized some caffeic acid phenethyl ester (CAPE)-like compounds from commercially available caffeic acid. Nine chemicals were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on the growth of buccal mucosal fibroblast (BF), oral submucosus fibroblast (OSF), neck metastasis of Gingiva carcinoma (GNM), and tongue squamous cell carcinoma (TSCCa) cells. CAPE and its ethyl analogue show significant cytotoxicity on OSF, GNM, and TSCCa cells, but not on BF cells. The results suggest that CAPE-like compounds may be potential chemotherapy agents against oral cancer.     

Caffeic acid phenethyl ester decreases cholangiocarcinoma growth by inhibition of NF‐κB and induction of apoptosis

Caffeic acid phenethyl ester (CAPE) inhibits the growth of tumor cells and is a known inhibitor of nuclear factor kappa beta (NF‐κB), which is constitutively active in cholangiocarcinoma (CCH) cells. We evaluated the effects of CAPE on CCH growth both in vitro and in vivo. Inhibition of NF‐κB DNA‐binding activity was confirmed in nuclear extracts treated with CAPE at 50, 40 and 20 μM. CAPE decreases the expression of NF‐κB1 (p50) and RelA (p65). CAPE decreased the growth of a number of CCH cells but not normal cholangiocytes. Cell cycle decrease was seen by a decrease in PCNA protein expression and the number of BrdU‐positive cells treated with CAPE at 20 μM compared to vehicle. Inhibition of growth and increased cell cycle arrest of Mz‐ChA‐1 cells by CAPE were coupled with increased apoptosis. Bax expression was increased, whereas Bcl‐2 was decreased in cells treated with CAPE compared to vehicle. In vivo studies were performed in BALB/c nude (nu/nu) mice implanted subcutaneously with Mz‐ChA‐1 cells and treated with daily IP injections of DMSO or CAPE (10 mg/kg body weight in DMSO) for 77 days. Tumor growth was decreased and tumor latency was increased 2‐fold in CAPE compared to vehicle‐treated nude mice. In tumor samples, decreased CCH growth by CAPE was coupled with increased apoptosis. CAPE both in vivo and in vitro decreases the growth of CCH cells by increasing apoptosis. These results demonstrate that CAPE might be an important therapeutic tool in the treatment of CCH. © 2009 UICC     

��-Tocotrienol suppresses prostate cancer cell proliferation and invasion through multiple-signalling pathways

Tocotrienol-rich fraction (TRF) has demonstrated antiproliferative effect on prostate cancer (PCa) cells. To elucidate this anticancer property in PCa cells, this study aimed, first, to identify the most potent isomer for eliminating PCa cells; and second, to decipher the molecular pathway responsible for its activity. Results showed that the inhibitory effect of γ-tocotrienol was most potent, which resulted in induction of apoptosis as evidenced by activation of pro-caspases and the presence of sub-G₁ cell population. Examination of the pro-survival genes revealed that the γ-tocotrienol-induced cell death was associated with suppression of NF-κB, EGF-R and Id family proteins (Id1 and Id3). Meanwhile, γ-tocotrienol treatment also resulted in the induction of JNK-signalling pathway and inhibition of JNK activity by a specific inhibitor (SP600125) was able to partially block the effect of γ-tocotrienol. Interestingly, γ-tocotrienol treatment led to suppression of mesenchymal markers and the restoration of E-cadherin and γ-catenin expression, which was associated with suppression of cell invasion capability. Furthermore, a synergistic effect was observed when cells were co-treated with γ-tocotrienol and Docetaxel. Our results suggested that the antiproliferative effect of γ-tocotrienol act through multiple-signalling pathways, and demonstrated for the first time the anti-invasion and chemosensitisation effect of γ-tocotrienol against PCa cells.