Combined inhibitory effects of green tea polyphenols and selective cyclooxygenase-2 inhibitors on the growth of human prostate cancer cells both in vitro and in vivo.

PURPOSE: Cyclooxygenase-2 (COX-2) inhibitors hold promise for cancer chemoprevention; however, recent toxicity concerns suggest that new strategies are needed. One approach to overcome this limitation is to use lower doses of COX-2 inhibitors in combination with other established agents with complementary mechanisms. In this study, the effect of (-)epigallocatechin-3-gallate (EGCG), a promising chemopreventive agent from green tea, was tested alone and in combination with specific COX-2 inhibitors on the growth of human prostate cancer cells both in vitro and in vivo. EXPERIMENTAL DESIGN: Human prostate cancer cells LNCaP, PC-3, and CWR22Rnu1 were treated with EGCG and NS398 alone and in combination, and their effect on growth and apoptosis was evaluated. In vivo, athymic nude mice implanted with androgen-sensitive CWR22Rnu1 cells were given green tea polyphenols (0.1% in drinking water) and celecoxib (5 mg/kg, i.p., daily, 5 days per week), alone and in combination, and their effect on tumor growth was evaluated. RESULTS: Combination of EGCG (10-40 micromol/L) and NS-398 (10 micromol/L) resulted in enhanced (a) cell growth inhibition; (b) apoptosis induction; (c) expression of Bax, pro-caspase-6, and pro-caspase-9, and poly(ADP)ribose polymerase cleavage; (d) inhibition of peroxisome proliferator activated receptor gamma; and (e) inhibition of nuclear factor-kappaB compared with the additive effects of the two agents alone, suggesting a possible synergism. In vivo, combination treatment with green tea polyphenols and celecoxib resulted in enhanced (a) tumor growth inhibition, (b) lowering of prostate-specific antigen levels, (c) lowering of insulin-like growth factor-I levels, and (d) circulating levels of serum insulin-like growth factor binding protein-3 compared with results of single-agent treatment. CONCLUSIONS: These data suggest synergistic and/or additive effects of combinatorial chemopreventive agents and underscore the need for rational design of human clinical trials.     

Synergistic inhibition of ovarian cancer cell growth by combining selective PI3K/mTOR and RAS/ERK pathway inhibitors

BackgroundOvarian cancer is the major cause of death from gynaecological malignancy with a 5 year survival of only ～30% due to resistance to platinum and paclitaxel-based first line therapy. Dysregulation of the phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) and RAS/extracellular signal-regulated kinase (ERK) pathways is common in ovarian cancer, providing potential new targets for 2nd line therapy.MethodsWe determined the inhibition of proliferation of an extensive panel of ovarian cancer cell lines, encompassing all the major histotypes, by the dual PI3K/mTOR inhibitor PF-04691502 and a MEK inhibitor, PD-0325901. In addition, we analysed global gene expression, mutation status of key PI3K/mTOR and RAS/ERK pathway members and pathway activation to identify predictors of drug response.ResultsPF-04691502 inhibits proliferation of the majority of cell lines with potencies that correlate with the extent of pathway inhibition. Resistant cell lines were characterised by activation of the RAS/ERK pathway as indicated by differential gene expression profiles and pathway activity analysis. PD-0325901 suppressed growth of a subset of cell lines that were characterised by high basal RAS/ERK signalling. Strikingly, using PF-04691502 and PD-0325901 in combination resulted in synergistic growth inhibition in 5/6 of PF-04691502 resistant cell lines and two cell lines resistant to both single agents showed robust synergistic growth arrest. Xenograft studies confirm the utility of combination therapy to synergistically inhibit tumour growth of PF-04691502-resistant tumours in vivo.ConclusionsThese studies identify dual targeted inhibitors of PI3K/mTOR in combination with inhibitors of RAS/ERK signalling as a potentially effective new approach to treating ovarian cancer.     

Potentiation of tumor response to radiation or chemoradiation by selective cyclooxygenase-2 enzyme inhibitors

Cyclooxygenase-2 (COX-2) is an enzyme expressed primarily in pathologic states, such as inflammatory disorders and cancer, where it mediates prostaglandin production. Its overexpression is associated with more aggressive biologic tumor behavior and adverse patient outcome. Increasing evidence shows that agents that selectively inhibit COX-2 enhance tumor response to radiation or chemotherapeutic agents. This article gives an overview of some of this evidence. In addition, we describe new results showing that celecoxib, a selective COX-2 inhibitor, enhanced response of A431 human tumor xenografts in nude mice to radiation by an enhancement factor (EF) of 1.43 and to the chemotherapeutic agent docetaxel by an EF of 2.07. Celecoxib also enhanced tumor response when added to the combined docetaxel plus radiation treatment (EF = 2.13). Further experiments showed that selective COX-2 inhibitors enhanced tumor cell sensitivity to ionizing radiation, involving inhibition of cellular repair from radiation damage and cell cycle redistribution as mechanisms for some cell types. The results show that selective COX-2 inhibitors have the potential to improve tumor radiotherapy or radiochemotherapy, and this therapeutic strategy is currently under clinical testing.     

Reduction in the risk of human breast cancer by selective cyclooxygenase-2 (COX-2) inhibitors

Background  

Epidemiologic and laboratory investigations suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) have chemopreventive effects against breast cancer due to their activity against cyclooxygenase-2 (COX-2), the rate-limiting enzyme of the prostaglandin cascade.     

Esculetin inhibits cell proliferation through the Ras/ERK1/2 pathway in human colon cancer cells

Esculetin, a phenolic compound, has been shown to inhibit the growth of colon tumors in animal studies. However, the roles of signaling pathways and cell cycle regulation in the esculetin-induced inhibition of cancer cell growth, remain to be elucidated. The present study suggests a novel mechanism for the Ras/ERK1/2 pathway in esculetin-treated human colon cancer HCT116 cells. The treatment of cells with esculetin resulted in significant growth inhibition and G1 phase cell cycle arrest, which led to the down-regulation of cyclin and cyclin-dependent kinase (CDK) expressions. This G1 phase cell cycle arrest was associated with the up-regulation of p27KIP expression. In addition, ERK1/2 was activated by esculetin. The pre-treatment of cells with the MEK1/2-specific inhibitor, PD98059, blocked the p27KIP expression induced by esculetin. Blockage of the ERK1/2 function consistently prevented the inhibition of cell proliferation and decreased G1 phase cell cycle protein levels. Furthermore, Ras activation was increased by the esculetin treatment. Transient transfection of the dominant negative Ras (RasN17) mutant gene abolished both the ERK1/2 activity and p27KIP expression induced by esculetin. Finally, the overexpression of RasN17 suppressed the esculetin-induced reduction in cell proliferation and cell cycle proteins. In conclusion, these results indicate that the Ras/ERK1/2 pathway is mediated by the p27KIP1 induction, leading to a reduction in cyclin/CDK complexes in the esculetin-induced inhibition of colon cancer cell growth. Overall, these findings indicate that the molecular action of esculetin has therapeutic potential for the treatment of colon malignancies.     

尼美舒利与顺铂联合应用对肺癌A549细胞增生的协同抑制效应

 目的 探讨尼美舒利（Aulin）联合化疗药物顺铂（Cis）对肺癌A549细胞生长的协同抑制效应。方法 分别采用MTT比色法、RT-PCR观察Aulin与Cis联合应用对A549细胞生长及PCNA、bcl-2的mRNA水平变化的影响；荧光显微镜、流式细胞仪和DNA琼脂糖凝胶电泳观察Aulin与Cis联合应用对A549细胞诱导凋亡的作用及细胞周期的影响。结果 Aulin协同Cis抑制A549细胞增生，抑制率最低64.9 %，最高82.9 %，呈时间、剂量依赖性方式； 联合干预组PCNA、bcl-2的mRNA表达水平均显著降低。Aulin协同Cis诱导A549细胞凋亡；并改变细胞周期分布，增加G0／G1期细胞比例。结论 Aulin以时间、剂量依赖性方式协同Cis抑制A549细胞增生；并改变细胞周期分布，以剂量依赖性方式协同诱导其凋亡。     

Inhibition of the Raf/MEK/ERK pathway up-regulates expression of the coxsackievirus and adenovirus receptor in cancer cells

Recombinant adenoviruses are presently being tested clinically as a new strategy for the treatment of cancer. An important determining factor for the successful entry of such adenoviruses into target cells is expression of the coxsackievirus and adenovirus receptor (CAR) at the cell surface. Recent observations suggest that expression of this receptor, which physiologically participates in formation of cell-cell adhesions, is frequently reduced in highly malignant cancer cells. This raises the possibility that those tumors representing the greatest therapeutic challenge might be the least susceptible to infection with therapeutic adenoviruses. We explored the role of the Raf-MEK-ERK pathway on CAR expression in a panel of cancer cells because this pathway is frequently up-regulated in cancer cells and is known to down-regulate cell-cell adhesion molecules. We found that disruption of signaling through the Raf-MEK-ERK pathway by inhibition of MEK up-regulated CAR expression, which was accompanied by increased representation of the protein at the cell surface. After Raf-MEK-ERK inhibition, adenovirus entry into cells was increased and cell killing by replication competent adenoviruses was enhanced in a CAR-dependent manner. Conversely, induction of Raf-1 resulted in reduction and disruption of CAR expression at the cell surface. We conclude that loss of CAR expression in cancer cells is, at least in part, mediated through the Raf-MEK-ERK signal transduction pathway and that pharmacological restoration of CAR at the cell surface could improve adenovirus-based treatments of cancer.     

HMGB1-mediated autophagy modulates sensitivity of colorectal cancer cells to oxaliplatin via MEK/ERK signaling pathway

In the present study, we examined the mechanisms of oxaliplatin-induced drug resistance in human colorectal cancer cell lines HT29 and HCT116. Our results demonstrate a significant autophagy expression in CRC cells after an oxaliplatin treatment. Administration of oxaliplatin to human CRC cells significantly enhanced the expression of HMGB1, which regulated the autophagy response and negatively regulate the cell apoptosis. Moreover, a decreased oxaliplatin -induced autophagy response and an increased apoptosis level were detected in stable CRC cells harboring HMGB1 shRNA. Then we noted that HMGB1 significantly induced extracellular signal-regulated kinase (ERK)/Extracellular signal-regulated kinase kinase (MEK) phosphorylation. Taken together, these data suggest that HMGB1-mediated autophagy modulates sensitivity of colorectal cancer cells to oxaliplatin via MEK/ERK signaling pathway.     

Antiproliferative effects of selective cyclooxygenase-2 inhibitor modulated by nimotuzumab in estrogen-dependent breast cancer cells

Breast cancer is the most common malignancy in women, and many breast cancer patients fail conventional treatment strategies of chemotherapy, radiation, and antiestrogen therapy. Research into the molecular pathways and biomarkers involved in the development of breast cancer should yield information that will guide therapeutic decisions. Epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) are involved in the carcinogenesis of breast cancer and exist tight crosstalk with estrogen receptor (ER) pathway. Combination of EGFR and COX-2 inhibitors, therefore, could be an effective strategy for reducing cell growth in estrogen-dependent breast cancer. In order to verify the effects of EGFR and COX-2 inhibitors, breast cancer cells MCF-7 and SKBR-3 were characterized for receptors status and then treated with respective inhibitors (nimotuzumab and celecoxib) alone and in combination. Both cell lines were sensitive to celecoxib, but not to nimotuzumab. However, combination of two drugs demonstrated synergistic effects on cell killing. Moreover, association of two drugs resulted in SKBR-3 cells, a further G0/G1 phase arrest than one drug alone. Downregulation of p-EGFR, p-Akt, p-mTOR, and amplified in breast cancer 1 (AIB1) were observed in both cell lines, and upregulation of E-cadherin was only found in MCF-7, after treatment with single agent or in combination. These studies suggest that nimotuzumab and celecoxib exert synergistic antiproliferation effects in breast cancer, which partly correlates with ER status. Due to Akt/mTOR, EMT and AIB1 pathways participate in this process, therefore, E-cadherin and AIB1 may be considered as possible biomarkers to predict response in ER-positive breast cancer cells treated with EGFR and COX-2 inhibitors.     

Pro-apoptotic role of the MEK/ERK pathway in ursodeoxycholic acid-induced apoptosis in SNU601 gastric cancer cells

Ursodeoxycholic acid (UDCA) has been regarded as a suppressor of gastrointestinal cancer, but the mechanisms underlying its antitumor effects are not fully understood. Previously, we reported the antitumor effect of UDCA by demonstrating that UDCA induces apoptosis of gastric cancer cells. Bile acids are known to activate the ERK pathway and ERK is a representative oncogenic kinase in cancer cells. Here, we investigated the role of ERK in UDCA-induced gastric cancer cell apoptosis. We found that UDCA enhanced the phosphorylation of ERK1/2 and MEK1/2. The prevention of MEK by the pharmacologic inhibitors PD98059 and U0126, resulted in decreased UDCA-induced apoptosis as shown by the reduction of apoptotic body formation, caspase-8 activity, and caspase-3, -6 and PARP cleavage, indicating that ERK exerts pro-apoptotic activity upon exposure to UDCA. In addition, U0126 reduced UDCA-triggered TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2/DR5) expression. In gene silencing studies, we observed that RNA interference of ERK2 decreased apoptosis and reduced DR5 overexpression. Lipid raft disrupting agent, methyl-β-cyclodextrin, blunted the phosphorylation of ERK1/2, indicating that ERK activation is regulated in a lipid raft-dependent manner. On the other hand, tumor-promoting bile acid, deoxycholic acid (DCA), also phosphorylated ERK in SNU601 cells. However, the DCA-triggered ERK pathway exerted anti-apoptotic function in the cells. Suppression of the ERK pathway enhanced DCA-induced apoptosis, and ERK activation was observed to be lipid raft-independently controlled. These results indicated that UDCA and DCA may cause differential responses in gastric cancer cells through the ERK signaling molecule. Thus, ERK activation may be a possible mechanism by which UDCA and DCA represent differential activities in gastrointestinal cancer.     

Enhanced proliferation of human hepatoma cells by PAR-2 agonists via the ERK/AP-1 pathway

To investigate the expression and role of PAR-2 in the proliferation of the human hepatoma cell line HepG2, PAR-2 protein and mRNA expression were evaluated by immuno-histochemistry, immunofluorescence and RT-PCR analysis. The signaling pathways downstream of PAR-2 activation that lead to hepatoma cell proliferation were analyzed. The results showed that PAR-2 is expressed in human hepatoma cells and PAR-2 mRNA expression was found to be upregulated in cells treated with trypsin or SLIGKV-NH2 (P<0.001). The proliferation rate of HepG2 cells treated with trypsin or SLIGKV-NH2 was significantly increased (P<0.001). The percentage of S?phase, G2/M phase and the proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH2 were significantly elevated (P<0.001). The proliferative responses of HepG2 to trypsin and SLIGKV-NH2 were associated with the upregulation of c-fos and PCNA, which were significantly blocked by PD98059 pretreatment. In conclusion, our results indicate that PAR-2 enhances proliferation of human hepatoma cells possibly via the ERK/AP-1 pathway.     

Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors

We provide evidence that cyclooxygenase (COX)-2-derived prostaglandins contribute to tumor growth by inducing newly formed blood vessels (neoangiogenesis) that sustain tumor cell viability and growth. COX-2 is expressed within human tumor neovasculature as well as in neoplastic cells present in human colon, breast, prostate, and lung cancer biopsy tissue. COX-1 is broadly distributed in normal, as well as in neoplastic, tissues. The contribution of COX-2 to human tumor growth was indicated by the ability of celecoxib, an agent that inhibits the COX-2 enzyme, to suppress growth of lung and colon tumors implanted into recipient mice. Mechanistically, celecoxib demonstrated a potent antiangiogenic activity. In a rat model of angiogenesis, we observe that corneal blood vessel formation is suppressed by celecoxib, but not by a COX-1 inhibitor. These and other data indicate that COX-2 and COX-2-derived prostaglandins may play a major role in development of cancer through numerous biochemical mechanisms, including stimulation of tumor cell growth and neovascularization. The ability of celecoxib to block angiogenesis and suppress tumor growth suggests a novel application of this anti-inflammatory drug in the treatment of human cancer.     

Sunitinib enhances antitumor effects against chemotherapy-resistant bladder cancer through suppression of ERK1/2 phosphorylation

Bladder cancer patients who are refractory to chemotherapy have a poor prognosis. Furthermore, additional chemotherapies provide little benefit to patients who have relapsed after an initial response. Recently, it was reported that several molecular pathways are implicated in bladder carcinogenesis, including the epidermal growth factor receptor (EGFR) pathway, the vascular endothelial growth factor (VEGF) pathway and the Ras-MAPK pathway. We hypothesized that sunitinib would be effective in bladder cancer as it is an oral inhibitor of multiple receptor tyrosine kinases, including VEGF receptors, platelet derived growth factor (PDGF) receptors and stem cell factor receptor (c-KIT), and is a standard first-line treatment of advanced clear cell renal carcinoma. In the present study, the antiproliferative effects of sunitinib were clearly demonstrated in KK47, KK47/DDP20 and KK47/ADR cell lines in vitro due to the suppression of ERK1/2 phosphorylation. In a mouse model, the antitumor effects of sunitinib were again clearly seen. Also, treatment with sunitinib decreased the abundance of regulatory T cells (Tregs). However, cytotoxic T lymphocyte (CTL) activity was not induced sufficiently as compared with an IFN-α-treated group. Our results suggested that sunitinib was effective in chemotherapy-resistant bladder cancer patients. On the other hand, these findings provided the rationale for combination therapy with sunitinib and immune-based cancer therapy for advanced malignancies to prevent the occurrence of rebound phenomena.     

Combined effects of cyclooxygenase-1 and cyclooxygenase-2 selective inhibitors on intestinal tumorigenesis in adenomatous polyposis coli gene knockout mice

As with cyclooxygenase (COX)-2, genetic disruption of COX-1 gene or pharmacologic inhibition of its activity has been shown to decrease the number of intestinal polyps in Apc gene-deficient mice. The present study was designed to investigate the combined effects of COX-1 and COX-2 selective inhibitors on spontaneous polyp formation in APC1309 female mice. The animals were treated with 300 or 600 ppm mofezolac (a COX-1 selective inhibitor) alone, 200 or 400 ppm nimesulide (a COX-2 selective inhibitor) alone, 300 ppm mofezolac plus 200 ppm nimesulide, 600 ppm mofezolac plus 400 ppm nimesulide, or 10 ppm indomethacin (a dual-COX inhibitor) in the diet from 7 weeks of age for 4 weeks. Percentage inhibition of polyp area in the intestine was 17% with 600 ppm mofezolac alone and 25% with 400 ppm nimesulide alone, their sum of 42% being almost equal to the 37% observed for the combination treatment. Administration of 300 ppm mofezolac plus 200 ppm nimesulide also significantly decreased polyp area in the intestine by 30%. Moreover, the numbers of polyps more than 2.5 mm in diameter were markedly decreased by combined treatment of both COX inhibitors. With 10 ppm indomethacin, the dual inhibitor, polyp area was also clearly reduced by 46%. Our results indicate that COX-1 and COX-2 may to some extent contribute to polyp formation independently and inhibitor combination treatment thus has particular potential for chemoprevention of colon carcinogenesis.