Curcumin analogs: Their roles in pancreatic cancer growth and metastasis.

Curcumin is a polyphenolic constituent of turmeric that is known to have various molecular effects in preclinical models, leading to prevention and anticancer properties. In clinical trials, curcumin has failed to demonstrate activity against pancreatic cancer possibly due to its low bioavailability and potency. Using the curcumin molecular model, our group and others have synthesized several analogs with better bioavailability and higher potency in pancreatic cancer in vitro and xenograft models. This mini review summarizes some of the known molecular effects of curcumin analogs and their potential role as novel therapeutics for pancreatic cancer.

Novel synthetic curcumin analogs as potent antiangiogenic agents in colorectal cancer.

The transcription factor NF-κB plays a central role in angiogenesis in colorectal cancer (CRC). Curcumin is a natural dietary product that inhibits NF-κB. The objective of this study is to evaluate the antiangiogenic effects of curcumin and two potent synthetic analogues (EF31 and UBS109) in CRC. IC50 values for curcumin, EF31, and UBS109 were determined in the HCT116 and HT-29 cell lines. HUVEC tube formation, egg CAM assay, and matrigel plug assays revealed decreased angiogenesis in cell lines treated with curcumin, EF31, or UBS109. Curcumin and its analogues significantly inhibited VEGF-A synthesis and secretion in both cell lines in association with loss of HIF-1α, COX-2, and p-STAT-3 expression. Nuclear NF-κB expression was inhibited by curcumin, EF31, and UBS109. Transfection of p65-NF-κB in HCT116 and HT-29 cells resulted in increased expression of HIF-1α, COX-2, STAT-3, and VEGF-A. Treatment with curcumin, EF31, or UBS109 inhibited these effects in transfected cell lines. In mice carrying HCT116 and HT-29 cell xenografts, EF31 and UBS109 inhibited subcutaneous tumor growth and potentiated the effects of oxaliplatin and 5-FU. Tumors from treated animals revealed inhibition of HIF-1α, COX-2, p-STAT-3, and VEGF expression. Our findings suggest that inhibition of NF-κB leading to decreased transcription and expression of HIF-1α, COX-2, STAT-3, and VEGF is a rational approach for antiangiogenic therapy in CRC. The distinctive properties of EF31 and UBS109 make them promising therapeutic agents for development in CRC as single agents or as part of combination chemotherapy regimens. © 2016 Wiley Periodicals, Inc.

Survival of lung cancer patients is prolonged with higher regucalcin gene expression: suppressed proliferation of lung adenocarcinoma A549 cells in vitro.

Regucalcin plays a crucial role as a suppressor of transcription signaling, and its diminished expression or activity may play a key role in human carcinogenesis. Higher regucalcin expression has been demonstrated to prolong survival of the patients of pancreatic cancer, breast cancer, and hepatocellular carcinoma. Moreover, we investigated an involvement of regucalcin in human lung cancer. Human non-small cell lung cancer (NSCLC) accounts for over 80% in human lung cancer and is one of the leading causes of malignancy-related mortality with fewer than 16% patients surviving beyond 5 years. In this study, gene expression and survival data of 204 lung adenocarcinoma patients were obtained through the gene expression omnibus database (GSE31210) for outcome analysis. Gene expression data demonstrated that prolonged survival in lung cancer patients is associated with higher regucalcin gene expression. Overexpression of regucalcin suppressed the proliferation, cell death, and migration of human lung adenocarcinoma NSCLC A549 cells in vitro. Mechanistically, regucalcin induced G1 and G2/M phase cell cycle arrest of A549 cells through suppression of multiple signaling pathways including Ras, Akt, MAP kinase, and SAPK/JNK. Moreover, overexpression of regucalcin caused decreases in the oncogenes c-fos and c-myc and elevation of the tumor suppressers p53 and Rb. These findings suggest that regucalcin may play a potential role as a suppressor of human lung cancer, and that downregulation of regucalcin expression may predispose patients to development of lung cancer. Overexpression of regucalcin using gene delivery may constitute a novel therapeutic approach to treating lung cancer.

Curcumin analog UBS109 prevents bone marrow osteoblastogenesis and osteoclastogenesis disordered by coculture with breast cancer MDA-MB-231 bone metastatic cells in vitro.

UBS109 is a curcumin analog that possesses antitumor properties has been shown to stimulate osteoblastogenesis and suppress osteoclastogenesis in vitro. This study was undertaken to determine whether UBS109 might alleviate the inhibitory activity of breast cancer cells on osteoblastic mineralization and stimulatory effects on osteoclastogenesis. Mouse bone marrow cells were cocultured with breast cancer MDA-MB-231 bone metastatic cells in vitro. UBS109 stimulated osteoblastic mineralization and suppressed adipogenesis and osteoclastogenesis in bone marrow culture. Coculture with MDA-MB-231 cells suppressed osteoblastic mineralization and enhanced osteoclastogenesis in bone marrow culture. Effects that were reserved by UBS109 (50-200 nM). Mineralization in preosteoblastic MC3T3-E1 cells was suppressed by coculture with MDA-MB-231 cells, while MDA-MB-231 cells did not have effects on osteoclastogenesis of RAW267.4 cells in vitro. UBS109 (500 nM) revealed toxic effects on MDA-MB-231 bone metastatic cells. This study demonstrates that UBS109, which is an antitumor agent, reveals restorative effects on bone marrow cell differentiation disordered by coculture with breast cancer MDA-MB-231 bone metastatic cells in vitro. This in vitro model may be a useful tool to evaluate the mechanism of breast cancer cell bone metastasis.

Curcumin analogue UBS109 prevents bone loss in breast cancer bone metastasis mouse model: involvement in osteoblastogenesis and osteoclastogenesis.

Bone metastasis of breast cancer typically leads to osteolysis, which causes severe pathological bone fractures and hypercalcemia. Bone homeostasis is skillfully regulated through osteoblasts and osteoclasts. Bone loss with bone metastasis of breast cancer may be due to both activation of osteoclastic bone resorption and suppression of osteoblastic bone formation. This study was undertaken to determine whether the novel curcumin analogue UBS109 has preventive effects on bone loss induced by breast cancer cell bone metastasis. Nude mice were inoculated with breast cancer MDA-MB-231 bone metastatic cells (10(6) cells/mouse) into the head of the right and left tibia. One week after inoculation, the mice were treated with control (vehicle), oral administration (p.o.) of UBS109 (50 or 150 mg/kg body weight), or intraperitoneal administration (i.p.) of UBS109 (10 or 20 mg/kg body weight) once daily for 5 days per week for 7 weeks. After UBS109 administration for 7 weeks, hind limbs were assessed using an X-ray diagnosis system and hematoxylin and eosion staining to determine osteolytic destruction. Bone marrow cells obtained from the femurs and tibias were cultured to estimate osteoblastic mineralization and osteoclastogenesis ex vivo and in vitro. Remarkable bone loss was demonstrated in the tibias of mice inoculated with breast cancer MDA-MB-231 bone metastatic cells. This bone loss was prevented by p.o. administration of UBS109 (50 and 150 mg/kg body weight) and i.p. treatment of UBS109 (10 and 20 mg/kg) in vivo. Culture of bone marrow cells obtained from the bone tissues of mice with breast cancer cell bone metastasis showed suppressed osteoblastic mineralization and stimulated osteoclastogenesis ex vivo. These changes were not seen after culture of the bone marrow cells obtained from mice treated with UBS109. Moreover, UBS109 was found to stimulate osteoblastic mineralization and suppress lipopolysaccharide (LPS)-induced osteoclastogenesis in bone marrow cells obtained from normal nude mice in vitro. These findings suggest that the novel curcumin analogue UBS109 prevents breast cancer cell bone metastasis-induced bone loss by stimulating osteoblastic mineralization and suppressing osteoclastogenesis.

Bioactive flavonoid p-hydroxycinnamic acid stimulates osteoblastogenesis and suppresses adipogenesis in bone marrow culture.

The bioactive flavonoid p-hydroxycinnamic acid (HCA), which is an intermediate-metabolic substance in plants and fruits, is synthesized from tyrosine. The biological effect of HCA is poorly understood. Among cinnamic acid and its related compounds, HCA has a specific-anabolic effect on bone, being found to stimulate osteoblastogenesis and to inhibit osteoclastogenesis through the suppression of NF-κB signaling, thereby preventing bone loss. Bone marrow mesenchymal stem cells give rise to ostoblasts and adipocytes. HCA might therefore have effects on osteoblastogenesis and adipogenesis in bone marrow culture. This study demonstrates (1) that HCA has stimulatory effects on osteoblastogenesis and mineralization and suppressive effects on adipogenesis in mouse bone marrow culture and (2) that HCA depresses adipogenesis in mouse 3T3-L1 preadipocytes in vitro. Such effects of HCA might be involved in the differentiation of mesenchymal stem cells.

Curcumin analog cytotoxicity against breast cancer cells: exploitation of a redox-dependent mechanism.

A series of novel curcumin analogs, symmetrical dienones, were previously shown to possess cytotoxic, anti-angiogenic and anti-tumor activities. Analogs 1 (EF24) and 2 (EF31) share the dienone scaffold and serve as Michael acceptors. We propose that the anti-cancer effects of 1 and 2 are mediated in part by redox-mediated induction of apoptosis. In order to support this concept, 1 and 2 were treated with L-glutathione (GSH) and cysteine-containing dipeptides under mild conditions to form colorless water-soluble adducts, which were identified by LC/MS. Comparison of the cytotoxic action of 1, 2 and the corresponding conjugates, 1-(GSH)(2) and 2-(GSH)(2), illustrated that the two classes of compounds exhibit essentially identical cell killing capabilities. Compared with the yellow, somewhat light sensitive and nearly water insoluble compounds 1 and 2, the glutathione conjugates represent a promising new series of stable and soluble anti-tumor pro-drugs.

Inhibition of IkappaB kinase-nuclear factor-kappaB signaling pathway by 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24), a novel monoketone analog of curcumin.

The nuclear factor-kappaB (NF-kappaB) signaling pathway has been targeted for therapeutic applications in a variety of human diseases, includuing cancer. Many naturally occurring substances, including curcumin, have been investigated for their actions on the NF-kappaB pathway because of their significant therapeutic potential and safety profile. A synthetic monoketone compound termed 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24) was developed from curcumin and exhibited potent anticancer activity. Here, we report a mechanism by which EF24 potently suppresses the NF-kappaB signaling pathway through direct action on IkappaB kinase (IKK). We demonstrate that 1) EF24 induces death of lung, breast, ovarian, and cervical cancer cells, with a potency about 10 times higher than that of curcumin; 2) EF24 rapidly blocks the nuclear translocation of NF-kappaB, with an IC(50) value of 1.3 microM compared with curcumin, with an IC(50) value of 13 microM; 3) EF24 effectively inhibits tumor necrosis factor (TNF)-alpha-induced IkappaB phosphorylation and degradation, suggesting a role of this compound in targeting IKK; and 4) EF24 indeed directly inhibits the catalytic activity of IKK in an in vitro-reconstituted system. Our study identifies IKK as an effective target for EF24 and provides a molecular explanation for a superior activity of EF24 over curcumin. The effective inhibition of TNF-alpha-induced NF-kappaB signaling by EF24 extends the therapeutic application of EF24 to other NF-kappaB-dependent diseases, including inflammatory diseases such as rheumatoid arthritis.

Targeting tissue factor-expressing tumor angiogenesis and tumors with EF24 conjugated to factor VIIa.

Tissue factor (TF) is aberrantly expressed on tumor vascular endothelial cells (VECs) and on cancer cells in many malignant tumors, but not on normal VECs, making it a promising target for cancer therapy. As a transmembrane receptor for coagulation factor VIIa (fVIIa), TF forms a high-affinity complex with its cognate ligand, which is subsequently internalized through receptor-mediated endocytosis. Accordingly, we developed a method for selectively delivering EF24, a potent synthetic curcumin analog, to TF-expressing tumor vasculature and tumors using fVIIa as a drug carrier. EF24 was chemically conjugated to fVIIa through a tripeptide-chloromethyl ketone. After binding to TF-expressing targets by fVIIa, EF24 will be endocytosed along with the drug carrier and will exert its cytotoxicity. Our results showed that the conjugate inhibits vascular endothelial growth factor-induced angiogenesis in a rabbit cornea model and in a Matrigel model in athymic nude mice. The conjugate-induced apoptosis in tumor cells and significantly reduced tumor size in human breast cancer xenografts in athymic nude mice as compared with the unconjugated EF24. By conjugating potent drugs to fVIIa, this targeted drug delivery system has the potential to enhance therapeutic efficacy, while reducing toxic side effects. It may also prove to be useful for treating drug-resistant tumors and micro-metastases in addition to primary tumors.

Inhibition of breast cancer metastasis to the lungs with UBS109.

Synthetic monocarbonyl analogs of curcumin (MACs) are cytotoxic against several cancers including head and neck cancer, pancreatic cancer, colon cancer, and breast cancer. Mechanisms of action include depolarization of the mitochondrial membrane potential and inhibition of NF-κB, leading to apoptosis. We previously demonstrated that UBS109 (MAC), has preventive effects on bone loss induced by breast cancer cell lines. We determined whether UBS109 could inhibit and prevent lung metastasis, since lung metastasis of breast cancer is a major problem in addition to bone metastasis. A breast cancer lung metastasis (colonization) model was created by injection of breast cancer cells MDA-MB-231 into the tail vein of athymic nude mice, nu/nu. Animals were treated with vehicle or UBS109 at 5 or 15 mg/kg body weight by intraperitoneal injection once daily 5 days a week for 5 weeks. UBS109 at 15 mg/kg significantly inhibited lung metastasis/colonization as demonstrated by reduced lung weight consisting of tumor nodules. The body weight of animals treated with UBS109 15 mg/kg remained the same as in the other groups. UBS109 killed completely (100%) MDA-MB-231 breast cancer cells at 1.25 µM in a cytotoxicity assay in vitro. UBS109 15 mg/kg i.p. showed a maximal blood concentration (Cmax) of 432 ± 387 ng/mL at 15 min post injection. This is approximately 1.5 ng/ml in the blood of mice and equals 1.5 µM of UBS109. These in vitro and in vivo results are consistent with each other.

Synthesis of EF24-tripeptide chloromethyl ketone: a novel curcumin-related anticancer drug delivery system.

The blood coagulation cascade includes a step in which the soluble protein, factor VIIa (fVIIa), complexes with its transmembrane receptor, tissue factor (TF). The fVIIa/TF protein-protein complex is subsequently drawn into the cell by endocytosis. The observation that TF is aberrantly and abundantly expressed on many cancer cells offers an opportunity to specifically target those cells with an effective anticancer drug. Thus, we propose a new drug delivery system, drug-linker-Phe-Phe-Arg-mk-fVIIa, which can associate with TF on the surface of cancer cells, but release the cytotoxic agent in the cytoplasm. Synthetic procedures have been developed for the preparation of phenylalanine-phenylalanine-arginine chloromethyl ketone, (FFRck) followed by coupling with the cytotoxin EF24 and subsequently fVIIa to give EF-24-FFRmk-fVIIa. When breast cancer cells (MDA-MB-231) and human melanoma cells (RPMI-7951) are treated with the complex, the cells are arrested to a greater extent than EF24 alone by comparison with controls.

Increased regucalcin gene expression extends survival in breast cancer patients: Overexpression of regucalcin suppresses the proliferation and metastatic bone activity in MDA-MB-231 human breast cancer cells in vitro.

Human breast cancer is highly metastatic to bone and drives bone turnover. Breast cancer metastases cause osteolytic lesions and skeletal damage that leads to bone fractures. Regucalcin, which plays a pivotal role as an inhibitor of signal transduction and transcription activity, has been suggested to act as a suppressor of human cancer. In the present study, we compared the clinical outcome between 44 breast cancer patients with higher regucalcin expression and 43 patients with lower regucalcin expression. Prolonged relapse-free survival was identified in the patients with increased regucalcin gene expression. We further demonstrated that overexpression of full length, but not alternatively spliced variants of regucalcin, induces G1 and G2/M phase cell cycle arrest, suppressing the proliferation of MDA-MB-231 cells, a commonly used in vitro model of human breast cancer that metastasize to bone causing osteolytic lesions. Overexpression of regucalcin was found to suppress multiple signaling pathways including Akt, MAP kinase and SAPK/JNK, and NF-κB p65 and ß-catenin along with increased p53, a tumor suppressor, and decreased K-ras, c-fos and c-jun. Moreover, we found that co-culture of regucalcin-overexpressing MDA-MB-231 cells with mouse bone marrow cells prevented enhanced osteoclastogenesis and suppressed mineralization in mouse bone marrow cells in vitro. Taken together, the present study suggests that regucalcin may have important anticancer properties in human breast cancer patients. Mechanistically, these effects are likely mediated through suppression of multiple signaling pathways, upregulation of p53 and downregulation of oncogenes leading to anti-proliferative effects and reduced metastases to bone, a phenotype associated with poor clinical outcome.

Prolonged survival in pancreatic cancer patients with increased regucalcin gene expression: Overexpression of regucalcin suppresses the proliferation in human pancreatic cancer MIA PaCa-2 cells in vitro.

Approximately 90% of all pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). PDAC is a highly aggressive malignancy and is one of the deadliest. This poor clinical outcome is due to the prominent resistance of pancreatic cancer to drug and radiation therapies. Regucalcin plays a pivotal role as a suppressor protein in signal transduction in various types of cells including tumor tissues. We demonstrated that the prolonged survival is induced in PDAC patients with increased regucalcin gene expression using a dataset of PDAC obtained from GEO database (GSE17891) together with the clinical annotation data file. Moreover, overexpression of regucalcin with full length was demonstrated to suppress the proliferation, cell death and migration in human pancreatic cancer MIA PaCa-2 (K-ras mutated) cells that possess resistance to drug and radiation therapies. Suppressive effects of regucalcin on cell proliferation and death were not seen in the cells overexpressed with regucalcin cDNA alternatively spliced variants (deleted exon 4 or deleted exon 4 and 5). Regucalcin was suggested to induce G1 and G2/M phase cell cycle arrest in MIA PaCa-2 cells. Suppressive effects of regucalcin on cell proliferation were independent of cell death. Overexpression of regucalcin was found to suppress signaling pathways including Akt, MAP kinase and SAPK/JNK, to increase the protein levels of p53, a tumor suppresser, and to decrease K-ras, c-fos and c-jun, a oncogene, by suppressing signaling pathways that are related to signaling of K-ras. Regucalcin may play a potential role as a suppressor protein in human pancreatic cancer.

Prolonged survival in hepatocarcinoma patients with increased regucalcin gene expression: HepG2 cell proliferation is suppressed by overexpression of regucalcin in vitro.

Hepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide and ranks third in overall global cancer-related mortality rates. Importantly, in this study gene expression data demonstrate that prolonged survival in HCC patients is associated with increased regucalcin gene expression. Regucalcin has been shown to play a pivotal role as a transcription repressor and diminished expression or activity of regucalcin may play a key role in the development of human carcinogenesis. Indeed, overexpression of regucalcin suppressed the proliferation, cell death, and migration of human HCC HepG2 cells in vitro. Mechanistically, regucalcin induced G1 and G2/M phase cell cycle arrest of HepG2 cells through suppression of multiple signaling pathways including Ras, Akt, MAP kinase and SAPK/JNK and by increasing the tumor suppressors p53 and Rb. Furthermore, the oncogenes c-fos and c-myc were suppressed by overexpression of regucalcin, and overexpression of regucalcin caused an increase in p21 and a decrease in NF-κB p65 and ß-catenin. These findings suggest that regucalcin may play a potential role as a suppressor of human HCC, and that diminished expression of regucalcin may predispose patients to development of HCC. Overexpression of regucalcin may constitute a novel therapeutic approach to treating HCC.

Combination of tolfenamic acid and curcumin induces colon cancer cell growth inhibition through modulating specific transcription factors and reactive oxygen species.

Curcumin (Cur) has been extensively studied in several types of malignancies including colorectal cancer (CRC); however its clinical application is greatly affected by low bioavailability. Several strategies to improve the therapeutic response of Cur are being pursued, including its combination with small molecules and drugs. We investigated the therapeutic efficacy of Cur in combination with the small molecule tolfenamic acid (TA) in CRC cell lines. TA has been shown to inhibit the growth of human cancer cells in vitro and in vivo, via targeting the transcription factor specificity protein1 (Sp1) and suppressing survivin expression. CRC cell lines HCT116 and HT29 were treated with TA and/or Cur and cell viability was measured 24-72 hours post-treatment. While both agents caused a steady reduction in cell viability, following a clear dose/ time-dependent response, the combination of TA+Cur showed higher growth inhibition when compared to either single agent. Effects on apoptosis were determined using flow cytometry (JC-1 staining to measure mitochondrial membrane potential), Western blot analysis (c-PARP expression) and caspase 3/7 activity. Reactive oxygen species (ROS) levels were measured by flow cytometry and the translocation of NF-kB into the nucleus was determined using immunofluorescence. Results showed that apoptotic markers and ROS activity were significantly upregulated following combination treatment, when compared to the individual agents. This was accompanied by decreased expression of Sp1, survivin and NF-kB translocation. The combination of TA+Cur was more effective in HCT116 cells than HT29 cells. These results demonstrate that TA may enhance the anti-proliferative efficacy of Cur in CRC cells.

Inhibition of NF-κB translocation by curcumin analogs induces G0/G1 arrest and downregulates thymidylate synthase in colorectal cancer.

Cell cycle progression and DNA synthesis are essential steps in cancer cell growth and resistance. Thymidylate synthase (TS) is a therapeutic target for 5FU. Curcumin is a potent inhibitor of NF-κB. EF31 and UBS109 are potent synthetic analogues of curcumin. We tested the hypothesis that inhibition of NF-κB translocation by curcumin and its analogs EF31 and UBS109 can inhibit cell cycle progression and downregulate TS levels in colorectal cancer (CRC) cell lines. Two CRC cell lines (HCT116 and HT-29) were either untreated (control) or treated with IC50 concentrations of curcumin, EF31 UBS109 led to G0/G1 cell cycle arrest. Treatment with curcumin, EF31 or UBS109 inhibited NF-κB, downregulated survival pathways and inhibited cell cycle progression. Arrest in the G0/G1 phase was associated with downregulation of the transcription factor E2F-1 and its target gene TS. NF-κB over-expression induced E2F-1 and TS protein and mRNA levels in both cell lines. EF31 and UBS109 treatment significantly decreased tumor growth in compared to untreated tumors. EF31 and UBS109 are promising agents for the prevention and treatment of CRC.

Small molecule tolfenamic acid and dietary spice curcumin treatment enhances antiproliferative effect in pancreatic cancer cells via suppressing Sp1, disrupting NF-kB translocation to nucleus and cell cycle phase distribution.

Combination of dietary/herbal spice curcumin (Cur) and COX inhibitors has been tested for improving therapeutic efficacy in pancreatic cancer (PC). The objective of this study was to identify agent with low toxicity and COX-independent mechanism to induce PC cell growth inhibition when used along with Cur. Anticancer NSAID, tolfenamic acid (TA) and Cur combination were evaluated using PC cell lines. L3.6pl and MIA PaCa-2 cells were treated with Cur (5-25µM) or TA (25-100µM) or combination of Cur (7.5µM) and TA (50µM). Cell viability was measured at 24-72h posttreatment using CellTiter-Glo kit. While both agents showed a steady/consistent effect, Cur+TA caused higher growth inhibition. Antiproliferative effect was compared with COX inhibitors, Ibuprofen and Celebrex. Cardiotoxicity was assessed using cordiomyocytes (H9C2). The expression of Sp proteins, survivin and apoptotic markers (western blot), caspase 3/7 (caspase-Glo kit), Annexin-V staining (flow cytometry), reactive oxygen species (ROS) and cell cycle phase distribution (flow cytometry) was measured. Cells were treated with TNF-α, and NF-kB translocation from cytoplasm to nucleus was evaluated (immunofluorescence). When compared to individual agents, combination of Cur+TA caused significant increase in apoptotic markers, ROS levels and inhibited NF-kB translocation to nucleus. TA caused cell cycle arrest in G0/G1, and the combination treatment showed mostly DNA synthesis phase arrest. These results suggest that combination of Cur+TA is less toxic and effectively enhance the therapeutic efficacy in PC cells via COX-independent mechanisms.

Mitogen-actived protein kinase activation is an early event in melanoma progression.

PURPOSE: Melanoma is the most common cause of death from cutaneous malignancy, and is the cancer that is most rapidly rising in incidence. Because current therapeutic methods for metastatic melanoma are poorly efficacious, enhanced understanding of signal transduction in melanoma progression is warranted. Prior experimental studies in murine models and human tissues have shown a correlation among activation of mitogen activated protein kinase (MAPK) signaling, angiogenesis, and tumorigenesis. Because of these findings, we wanted to assess the role of MAPK signaling in melanoma progression and angiogenesis. EXPERIMENTAL DESIGN: We studied expression of phosphorylated (active) MAPK and two target genes known to be induced by MAPK signaling, tissue factor and vascular endothelial growth factor, in 131 melanocytic lesions, ranging from atypical nevi to metastatic melanoma. RESULTS: We observed little staining for activated (phosphorylated) MAPK and low amounts of angiogenesis in atypical nevi, but angiogenesis and MAPK activation were activated in radial growth melanoma and in later stage lesions. CONCLUSIONS: Our findings implicate MAPK activation as an early event in melanoma progression, and MAPK may be a potential target for pharmacologic intervention.

The flavonoid p-hydroxycinnamic acid exhibits anticancer effects in human pancreatic cancer MIA PaCa-2 cells in vitro: Comparison with gemcitabine.

Pancreatic cancer is a highly aggressive malignancy with a notoriously dismal prognosis. A major contributor to this poor clinical outcome is pancreatic cancer's prominent chemoresistance. The present study was undertaken to determine whether the flavonoid p­hydroxycinnamic acid (HCA), which is a botanical factor, possesses anticancer effects on cloned human pancreatic cancer MIA PaCa­2 cells that possess resistance to radiation therapy in vitro. Proliferation of MIA PaCa­2 cells was suppressed after culture with HCA (10­1,000 nM). Such an effect was also noted in human pancreatic cancer Pt45P1 cells. In the MIA PaCa­2 cells, HCA induced G1 and G2/M phase cell cycle arrest in the cells. The suppressive effects of HCA on proliferation were suggested to be mediated through the inhibition of various signaling pathways related to nuclear factor­κB (NF­κB), extracellular signal­regulated kinase (ERK), protein kinase C, phosphatidylinositol 3­kinase (PI3K) or nuclear transcription activity. Moreover, HCA was found to stimulate cell death in the MIA PaCa­2 and Pt45P1 cells in vitro. The anticancer effects of HCA on MIA PaCa­2 cells were exhibited at a lower concentration than gemcitabine, a potent cancer drug. The flavonoid HCA may be a useful tool in the therapy of human pancreatic cancer in vivo.

The flavonoid p-hydroxycinnamic acid mediates anticancer effects on MDA-MB-231 human breast cancer cells in vitro: Implications for suppression of bone metastases.

Tumor invasion into bone tissues is associated with osteoclast and osteoblast recruitment, resulting in the liberation of growth factors from the bone matrix, which can feed back to enhance tumor growth resulting in the vicious cycle of bone metastasis. Activated nuclear factor-κB (NF-κB) in breast cancer cells has been shown to play a crucial role in the osteolytic bone metastasis of breast cancer in stimulating osteoclastogenesis. The flavonoid p-hydroxycinnamic acid (HCA) mediates bone anabolic and anti-catabolic effects by stimulating osteoblastic bone formation and suppressing osteoclastic bone resorption. However, the capacity of HCA to ameliorate the negative effects of breast cancer on bone cells has not been investigated. The present study was undertaken to determine the anticancer effects of HCA on MDA-MB-231 human breast cancer bone metastatic cells in vitro models. Proliferation of MDA-MB­231 cells was suppressed by culture with HCA (10-1000 nM) due to G1 and G2/M phase cell cycle arrest. The suppressive effects of HCA were mediated through signaling pathways that are related to NF-κB, extracellular signal-regulated kinase (ERK), protein kinase C, calcium signaling, phosphatidylinositol 3-kinase (PI3K) and nuclear transcription activity. HCA was also found to induce death of confluent cancer cells. Furthermore, co-culture with MDA-MB-231 cells suppressed mineralization and stimulated osteoclastogenesis in bone marrow cells. These alterations were prevented by HCA (10-250 nM). The present study demonstrates that HCA possesses anticancer properties in MDA-MB-231 human breast cancer cells and alleviates the negative effects on osteoblastogenesis and osteoclastogenesis in vitro. HCA may have important applications in the treatment of breast cancer bone metastasis.