咖啡酸苯乙酯诱导人大肠癌细胞凋亡的研究

目的 :探讨咖啡酸苯乙酯 (caffeicacid phenethylester ,CAPE )诱导大肠癌HCT116细胞发生凋亡的作用 ,为CAPE用于大肠癌的临床治疗提供依据。方法 :采用MTT法、HE染色、AnnexinV FITC/PI双染色流式细胞术和末端脱氧核苷酸标记法(TUNEL) ,检测不同浓度CAPE作用后HCT116细胞增殖活性和细胞凋亡的变化。结果 :2 5、5 0、10 0mg/L的CAPE处理HCT116细胞 2 4h后 ,抑制率分别为12 2 0 %、2 2 44 %、3 7 86% ,细胞增殖明显受到抑制 ,呈量效依赖性 ;凋亡率分别为( 10 2 0± 0 66) %、 ( 16 60± 0 61) %、( 2 5 5 3± 3 2 7) % ,均显著高于对照组 ( 5 47± 0 93 ) % ,P <0 0 1。HE染色呈现典型的凋亡细胞形态特征。TUNEL法标记见明显的凋亡阳性细胞。结论 :CAPE可诱导体外培养的大肠癌细胞发生凋亡     

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.     

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.     

Inhibitory effect of caffeic acid phenethyl ester on the growth of C6 glioma cells in vitro and in vivo

Caffeic acid phenethyl ester (CAPE), a component of honeybee propolis, has been reported to hold various biochemical responses. In the preliminary study, we found that CAPE inhibited the growth of C6 glioma cells in a dose dependent and time dependent manner as shown by the results of trypan blue dye exclusion assay and cell proliferation assay. In addition, the cell number percentage of the G0/G1 phase increased to 85% after the treatment with 50 microM of CAPE for 24h. After treatment with CAPE (50 microM) for 6h, it demonstrated that the protein level of hyperphosphorylated pRb decreased, and cyclin dependent kinase inhibitors p21, p27, and p16 were marked up-regulated. The association of CDK2 and cyclin E that affects the CDK2 activity decreased. When C6 cells were grown as xenografts in nude mice, treatment with CAPE (1-10mg/kg; ip) induced a significant dose dependent decrease in tumor growth by evaluating tumor volume and tumor weight. Histochemical and immunohistochemical analysis revealed that CAPE treatment significantly reduced the number of mitotic cells and proliferating cell nuclear antigen (PCNA)-positive cells in C6 glioma. These results suggest that CAPE presents an antitumor potential for glioma by inhibiting the growth of tumor cells.     

Anti‐inflammatory effects of caffeic acid phenethyl ester (CAPE), a nuclear factor‐κB inhibitor,on Helicobacter pylori‐induced gastritis in Mongolian gerbils

Nuclear factor‐κB (NF‐κB) plays a major role in host inflammatory responses and carcinogenesis and as such is an important drug target for adjuvant therapy. In this study, we examined the effect of caffeic acid phenethyl ester (CAPE), an NF‐κB inhibitor, on Helicobacter pylori (H. pylori)‐induced NF‐κB activation in cell culture and chronic gastritis in Mongolian gerbils. In AGS gastric cancer cells, CAPE significantly inhibited H. pylori‐stimulated NF‐κB activation and mRNA expression of several inflammatory factors in a dose‐dependent manner, and prevented degradation of IκB‐α and phosphorylation of p65 subunit. To evaluate the effects of CAPE on H. pylori‐induced gastritis, specific pathogen‐free male, 6‐week‐old Mongolian gerbils were intragastrically inoculated with H. pylori, fed diets containing CAPE (0–0.1%) and sacrificed after 12 weeks. Infiltration of neutrophils and mononuclear cells and expression of NF‐κB p50 subunit and phospho‐IκB‐α were significantly suppressed by 0.1% CAPE treatment in the antrum of H. pylori‐infected gerbils. Labeling indices for 5′‐bromo‐2′‐deoxyuridine both in the antrum and corpus and lengths of isolated pyloric glands were also markedly reduced at the highest dose, suggesting a preventive effect of CAPE on epithelial proliferation. Furthermore, in the pyloric mucosa, mRNA expression of inflammatory mediators including tumor necrosis factor‐α, interferon‐γ, interleukin (IL)‐2, IL‐6, KC (IL‐8 homologue), and inducible nitric oxide synthase was significantly reduced. These results suggest that CAPE has inhibitory effects on H. pylori‐induced gastritis in Mongolian gerbils through the suppression of NF‐κB activation, and may thus have potential for prevention and therapy of H. pylori‐associated gastric disorders. © 2009 UICC     

Differential gene expression in epidermis of mice sensitive and resistant to phorbol ester skin tumor promotion

Previous data from two-stage carcinogenesis studies in mouse skin demonstrated that genetic control of susceptibility to skin tumor promotion by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), in crosses between susceptible DBA/2J and resistant C57BL/6J mice is a multigenic trait. Utilizing a cDNA microarray approach, we compared global gene expression profiles in the epidermis of these two mouse strains treated with TPA or vehicle (acetone). Gene expression in the epidermis was analyzed after the treatment to identify global effects of TPA, as well as potential candidate genes that modify susceptibility to skin tumor promotion. DBA/2J and C57BL/6J mice were treated topically four times with 3.4 nmol TPA or acetone over a 2-wk period, and RNA was extracted from epidermis 6 h after the final treatment. Labeled cDNA generated from each group was hybridized to commercial cDNA microarrays (Agilent) containing more than 8000 targets. More than 450 genes were significantly influenced, directly or indirectly, by TPA treatment in the epidermis of either strain. Notably, 44 genes exhibited differential expression between the tumor promotion sensitive and resistant mouse strains. Several genes that were differentially expressed in DBA/2J versus C57BL/6J epidermis after TPA treatment were located in chromosomal regions linked to TPA promotion susceptibility. Three genes, Gsta4, Nmes1 (MGC58382), and Serpinb2, located within promotion susceptibility loci Psl1 (chr 9), Psl2 (chr 2), and Psl3 (chr 1), respectively, were identified in this analysis as potential candidates for modifiers of susceptibility to skin tumor promotion by TPA.     

Regulation of the differentiation-related gene Drg-1 during mouse skin carcinogenesis.

Differentiation-related gene-1 (Drg-1) has been identified as a gene whose expression is increased in several processes related to differentiation, but its function is currently unknown. In this report, we show that Drg-1 was expressed in keratinocytes, this expression being rapidly increased as a result of induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) or the presence of an activating form of Ha-ras. Induction by TPA occurred both in cultured cell lines and primary keratinocytes as well as in mouse skin after a single TPA application. Overexpression of Drg-1 was also observed in TPA-induced hyperplastic skin. In agreement, mouse skin papillomas and carcinomas also overexpressed Drg-1. In addition, Drg-1 was induced when keratinocytes were forced to differentiate by calcium switch or serum starvation. Analysis of the expression of Drg-1 during the keratinocyte cell cycle demonstrated relatively high levels of Drg-1 mRNA in G(0), which increased in early G(1) and decreased afterwards in late G(1)/S. In situ analysis showed an accumulation of Drg-1 in the suprabasal layers of the skin, as well as in the more differentiated areas of mouse skin papillomas. These results suggest that, in addition to being upregulated during keratinocyte differentiation, the Drg-1 gene might have a complex function in skin tumorigenesis.     

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     

有机酸诱导肿瘤细胞凋亡研究进展

与传统的杀伤肿瘤细胞的方式相比，通过诱导肿瘤细胞凋亡进行肿瘤治疗的方法具有明显的优越性。有机酸类化合物是能够通过各种途径诱导肿瘤细胞凋亡的重要药物之一，对于寻找高效低毒的诱导肿瘤细胞凋亡的药物的研究具有重要的意义。本文就饱和脂肪酸、不饱和脂肪酸、含苯环的脂肪酸、维生素类酸、稠环类酸等五类有机酸诱导肿瘤细胞的凋亡以及其诱导机制的国内外研究现状进行简要综述。     

Eugenol precludes cutaneous chemical carcinogenesis in mouse by preventing oxidative stress and inflammation and by inducing apoptosis

The present study was designed to investigate the protective efficacy of eugenol against skin cancer and probe into the mechanistic aspects. Skin tumors were initiated by applying 160 nmol DMBA and promoted by twice weekly applications of 8.5 nmol TPA for 28 wk. All mice developed tumors by 13 wk of promotion. However, in mice pretreated with 30 µL eugenol, no tumors were detected until 8 wk (following anti‐initiation protocol) and until 14 wk (following antipromotion protocol) of tumor promotion. PCNA and TUNEL immunohistochemistry of tumors revealed eugenol to ameliorate cell proliferation and elevate apoptosis respectively. The effect of eugenol was assessed on specific stages of carcinogenesis. Initiation with DMBA led to a significant upregulation of p53 expression with a concomitant increase in p21^WAF1 levels in epidermal cells indicating induction of damage to the DNA. However, pretreatment with eugenol led to overexpression of these genes, which probably helped stimulate apoptosis of the initiated cells. To ascertain the molecular mechanisms implicated in the antitumor promoting activity of eugenol, its effect was investigated on markers of tumor promotion and inflammation: ODC activity and iNOS and COX‐2 expression, and on levels of proinflammatory cytokines (IL‐6, TNF‐α, and PGE₂). Eugenol markedly inhibited all. Eugenol also inhibited the upstream signaling molecule: NF‐κB, which regulates the expression of these genes. TPA‐induced depletion of cutaneous GSH and antioxidant enzymes armory was also precluded by eugenol. From these results, it could be concluded that eugenol markedly protects against chemically induced skin cancer and acts possibly by virtue of its antiproliferative, anti‐inflammatory, and antioxidant activities. © 2009 Wiley‐Liss, Inc.     

Guanine nucleotide depletion triggers cell cycle arrest and apoptosis in human neuroblastoma cell lines

Mycophenolic acid (MPA) specifically inhibits inosine-5'-monophosphate dehydrogenase, the first committed step toward GMP biosynthesis. In its morpholinoethyl ester pro-drug form it is one of the most promising immunosuppressive drugs recently developed. The aim of the present study was to investigate the in vitro effects of MPA, at concentrations readily attainable during immunosuppressive therapy, on 3 human neuroblastoma cell lines (LAN5, SHEP and IMR32). Mycophenolic acid (0.1-10 microM) caused a decrease of intracellular levels of guanine nucleotides, a G(1) arrest and a time- and dose-dependent death by apoptosis. These effects, associated with an up-regulation of p53, p21 and bax, a shuttling of p53 protein into the nucleus and a down-regulation of bcl-2, survivin and p27 protein, were reversed by the simultaneous addition of guanine or guanosine and were more evident using nondialysed serum containing hypoxanthine. These results suggest that in neuroblastoma cell lines clinically attainable concentrations of mycophenolic acid deplete guanine nucleotide pools triggering G(1) arrest and apoptosis through p53-mediated pathways, indicating a potential role of its morpholinoethyl ester pro-drug in the management of patients with neuroectodermal tumors.     

Inhibition of proliferation and induction of apoptosis in soft tissue sarcoma cells by interferon-alpha and retinoids.

Uncontrolled proliferation and a defect of apoptosis constitute crucial elements in the development and progression of tumours. Among many other biological response modifiers known to influence these mechanisms, the efficacy of retinoids and interferons in the treatment of various malignant entities is currently matter of discussion. In the present study, we have investigated the effects of 9-cis-retinoic acid (9cRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (tRA) and interferon-alpha on proliferation and apoptosis of human soft tissue sarcoma (STS) cell lines HTB-82 (rhabdomyosarcoma), HTB-91 (fibrosarcoma), HTB-92 (liposarcoma), HTB-93 (synovial sarcoma) and HTB-94 (chondrosarcoma) in relation to p53 genotype as well as p53 expression. HTB-91, HTB-92 and HTB-94 STS cells exhibited mutant p53, whereas wild-type p53 was found in HTB-93 STS cells, and a normal p53 status in HTB-82 STS cells, carrying a silent point mutation only. Interferon-alpha, irrespective of p53 status, inhibited the proliferation of all five cell lines dose- and time-dependently. Similarly, 9cRA, 13cRA and tRA decreased the proliferation of HTB-82 and HTB-93 STS cells, whereas the proliferation of p53-mutated HTB-91, HTB-92 and HTB-94 STS cells remained unchanged. Furthermore, only 9cRA and tRA were capable of inducing apoptosis in HTB-82 and HTB-93 STS cells, whereas HTB-91, HTB-92 and HTB-94 STS cells did not undergo apoptosis under the influence of 9cRA or tRA. Retinoic acid receptor (RAR)-alpha and RAR-beta mRNA were not detectable by Northern blot analysis in the five STS cell lines, whereas mRNA for the universal retinoic acid receptor, RAR-gamma, was expressed in all STS cell lines indicating that retinoid resistance was not associated with a lack of RAR expression. Apoptosis was not induced by interferon-alpha or 13cRA in any of the five STS cell lines tested. Our results indicate that within the panel of tested STS cell lines, inhibition of proliferation and induction of apoptosis result from different mechanisms which differ in their dependence upon the presence of intact p53.     

Resveratrol induces colon tumor cell apoptosis independently of p53 and precede by epithelial differentiation, mitochondrial proliferation and membrane potential collapse.

Resveratrol, a polyphenol present in wine and grapes, can inhibit tumor cell growth in vitro and tumorigenesis in vivo. Some of its effects have been linked to activation of the p53 tumor suppressor; however, p53 is frequently mutated in tumors, particularly in the common and often therapy-resistant colon cancers. Using the human wild-type p53-expressing HCT116 colon carcinoma cell line and HCT116 cells with both p53 alleles inactivated by homologous recombination, we show in the current study that resveratrol at concentrations comparable to those found in some foods can induce apoptosis independently of p53. The cell death is primarily mitochondria-mediated and not receptor-mediated. No cells survived in cultures continuously exposed to 100 microM resveratrol for 120 hr. When compared with 5-FU, resveratrol stimulated p53 accumulation and activity only weakly and with delayed kinetics and neither the increased levels nor the activity affected apoptosis detectably. The apoptosis agonist Bax was overproduced in response to resveratrol regardless of p53 status, yet the kinetics of Bax expression were influenced by p53. Remarkably, apoptosis was preceded by mitochondrial proliferation and signs of epithelial differentiation. Thus, resveratrol triggers a p53-independent apoptotic pathway in HCT116 cells that may be linked to differentiation.