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 (C max ) 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.
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 anticancer 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.
We have developed a specific technique for imaging cancer in vivo using Cy5.5-labeled factor VIIa (fVIIa), clotting-deficient FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-tissue factor (TF) antibody. FVIIa is the natural ligand for TF. We took advantage of the fact that vascular endothelial cells (VECs) in cancer, but not normal tissue, aberrantly express TF due to its induction by vascular endothelial growth factor (VEGF). Under physiological conditions, TF is expressed by stromal cells and outer blood vessel layers (smooth muscle and adventitia), but not by VECs. We hypothesized that labeled fVIIa or anti-TF antibodies could be used to image the tumor vasculature in vivo. To test this, Cy5.5-labeled fVIIa, FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-TF antibody were developed and administered to athymic nude mice carrying xenografts including glioma U87EGFRviii, pancreatic cancer ASPC-1 and Mia PaCa-2, and squamous cell carcinoma KB-V1. Cy5.5 labeled with these targeting proteins specifically localized to the tumor xenografts for at least 14 days but unconjugated Cy5.5 did not localize to any xenografts or organs. This method of imaging TF in the tumor VECs may be useful in detecting primary tumors and metastases as well as monitoring in vivo therapeutic responses.
Objectives are to examine the efficacy of new synthetic curcumin analogs EF31 in head and neck squamous cell carcinoma in vitro and in vivo, and study their pharmacokinetic and toxicologic effects in vivo. The synthesis of EF31 was described for the first time. Solubility of EF24, EF31 was compared using nephelometric analysis. Human head and neck squamous cell carcinoma Tu212 xenograft tumors were established in athymic nude mice and treated with EF31 i.p. once daily five days a week for about 5 – 6 weeks. The long term effect of EF31 on the NF-κB signaling system in the tumors was examined by Western blot analysis. EF31 at 25 mg/kg, i.p. inhibited tumor growth almost completely. Solubility of EF24 and EF31 are <10, 13 μg/mL or <32, 47 μM, respectively. The serum chemistry profiles of treated mice were within the limits of normal, it revealed a linear increase of Cmax. EF31 decreased the level of phosphorylation of NF-κB p65. In conclusion, the novel synthetic curcumin analogs EF31 is efficacious in inhibiting the growth of Tu212 xenograft tumors and may be useful for treating head and neck squamous cell carcinoma. The long term EF31 treatment inhibited NF-kB p65 phosphorylation in xenografts, implicating downregulation of cancer promoting transcription factors such as angiogenesis and metastasis.
The nuclear factor-κB (NF-κB) 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-κB 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-κB signaling pathway through direct action on IκB 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-κB, with an IC50 value of 1.3 μM compared with curcumin, with an IC50 value of 13 μM; 3) EF24 effectively inhibits tumor necrosis factor (TNF)-α-induced IκB 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-α-induced NF-κB signaling by EF24 extends the therapeutic application of EF24 to other NF-κB-dependent diseases, including inflammatory diseases such as rheumatoid arthritis.
We previously reported a novel drug delivery system, drug-linker-Phe-Phe-Arg-methylketone-(FFR-mk)-factor VIIa (fVIIa). The method utilizes tissue factor (TF), which is aberrantly and abundantly expressed on many cancer cells. The advantage of this delivery system is its ability to furnish a potent anti-cancer drug specifically to the tumor vasculature and cancer cells. In this paper, we describe the synthesis of paclitaxel (PTX)-Phe-Phe-Arg-chloromethyl ketone (FFR-ck), followed by coupling with fVIIa to form PTX-FFR-mk-fVIIa. FFRck was separately linked to the OH groups at the C2′ or C7 positions of PTX (C2′- or C7-PTX-FFRck), the C2′ analog exhibiting better activity against human head and neck squamous KB 3-1 cells. The activity order against PTX-sensitive KB 3-1 cells is C2′-PTX-FFRmk-fVIIa > PTX >C2′-PTX-FFRck. The C2′-complex shows an IC50 of 12 nM against the PTX-sensitive cell line and 130 nM against PTX-resistant cells. a
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 p‑hydroxycinnamic acid (HCA), which is a botanical factor, possesses anticancer effects on cloned human pancreatic cancer MIA PaCa‑2 cells that possess resistance to radiation therapy in vitro. Proliferation of MIA PaCa‑2 cells was suppressed after culture with HCA (10‑1,000 nM). Such an effect was also noted in human pancreatic cancer Pt45P1 cells. In the MIA PaCa‑2 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 factor‑κB (NF‑κB), extracellular signal‑regulated kinase (ERK), protein kinase C, phosphatidylinositol 3‑kinase (PI3K) or nuclear transcription activity. Moreover, HCA was found to stimulate cell death in the MIA PaCa‑2 and Pt45P1 cells in vitro. The anticancer effects of HCA on MIA PaCa‑2 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.
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.
Purpose: Curcumin, a keto-enol constituent of turmeric, has in vitro and in vivo antitumor activity. However, in vivo potency is low due to poor oral absorption. The mono-carbonyl analog, 3,5-bis[(2-fluorophenyl)methylene]-4- piperidinone acetate (EF-24, NSC 716993), exhibited broad-spectrum activity in the NCI anticancer cell line screen and potent antiangiogenesis activity in a HUVEC cell migration assay. The purpose of this study was to characterize the preclinical pharmacology of EF-24 in mice. Methods: EF-24 plasma stability, protein binding, pharmacokinetics, and metabolism were characterized utilizing an LC/MS/MS assay.
Results: An LC/MS/MS assay incorporated protein precipitation with methanol, reverse-phase HPLC separation under gradient elution using an aqueous methanol mobile phase containing 0.1 % formic acid, and positive electrospray ionization detection of the m/z 312 > 149 transition for EF-24. The assay was linear over the range 7.8-1,000 nM. Plasma protein binding was >98 % with preferential binding to albumin. EF-24 plasma disposition in mice after i.v. administration of a 10 mg/kg dose was best fit to a 3-compartment open model. The terminal elimination half-life and plasma clearance values were 73.6 min and 0.482 L/min/kg, respectively. EF-24 bioavailability was 60 and 35 % after oral and i.p. administration, respectively. NADPH-dependent metabolism of EF-24 loss in liver microsomal preparations yielded several metabolites consistent with EF-24 hydroxylation and reduction.
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-MB-231 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.