- Open Access
The small molecule curcumin analog FLLL32 induces apoptosis in melanoma cells via STAT3 inhibition and retains the cellular response to cytokines with anti-tumor activity
© Bill et al; licensee BioMed Central Ltd. 2010
Received: 17 May 2010
Accepted: 25 June 2010
Published: 25 June 2010
We characterized the biologic effects of a novel small molecule STAT3 pathway inhibitor that is derived from the natural product curcumin. We hypothesized this lead compound would specifically inhibit the STAT3 signaling pathway to induce apoptosis in melanoma cells.
FLLL32 specifically reduced STAT3 phosphorylation at Tyr705 (pSTAT3) and induced apoptosis at micromolar amounts in human melanoma cell lines and primary melanoma cultures as determined by annexin V/propidium iodide staining and immunoblot analysis. FLLL32 treatment reduced expression of STAT3-target genes, induced caspase-dependent apoptosis, and reduced mitochondrial membrane potential. FLLL32 displayed specificity for STAT3 over other homologous STAT proteins. In contrast to other STAT3 pathway inhibitors (WP1066, JSI-124, Stattic), FLLL32 did not abrogate IFN-γ-induced pSTAT1 or downstream STAT1-mediated gene expression as determined by Real Time PCR. In addition, FLLL32 did not adversely affect the function or viability of immune cells from normal donors. In peripheral blood mononuclear cells (PBMCs), FLLL32 inhibited IL-6-induced pSTAT3 but did not reduce signaling in response to immunostimulatory cytokines (IFN-γ, IL 2). Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-γ production when cultured with K562 targets as compared to vehicle (DMSO).
These data suggest that FLLL32 represents a lead compound that could serve as a platform for further optimization to develop improved STAT3 specific inhibitors for melanoma therapy.
Malignant melanoma is the most deadly form of skin cancer, and its incidence is rising faster than that of any other cancer. The prognosis for patients with metastatic disease is poor, and even the most effective therapies produce an overall response rate of only 10-15%. Therefore, novel approaches for treating this disease are urgently needed.
Activation of signal transducer and activator of transcription-3 (STAT3) in melanoma tumors is associated with poor prognosis [1–3]. This transcription factor can promote cell proliferation and angiogenesis, inhibit apoptosis, and drive invasion and metastasis [1–3]. Constitutive STAT3 phosphorylation is mediated by several upstream kinases (e.g. Jak2, Src) and is thought to be a key component of the oncogenic process [4, 5]. Despite its necessity in early embryogenesis, STAT3 appears to be largely dispensable in most normal adult cell and tissue types [6, 7]. These data suggest that STAT3 inhibition represents a rational approach to therapy for this disease.
Emerging data suggest that natural products may represent effective candidate molecules for drug discovery. Curcumin, 1,7-bis(4-hydroxy-3methoxyphenyl)-1,6-heptadien-3,5-dione, is one such candidate  based on its chemopreventative and therapeutic properties in experimental models including melanoma and its ability to inhibit a variety of targets including STAT3 [9–11]. Administration of curcumin has been shown to be safe in humans [12, 13], however its clinical utility is somewhat limited due to the poor bioavailability and target selectivity. The lack of selectivity is due to the numerous molecular targets with which curcumin is known to interact. Therefore, efforts are underway by our group and others to design and synthesize novel curcumin analogs to focus its inhibitory activity toward the STAT3 pathway . Indeed prior studies by our group have shown that despite its direct pro-apoptotic effects on human melanoma cells, curcumin inhibits the cellular response to clinically relevant cytokines . These data suggest that structural analogs of curcumin which retain the ability to inhibit the STAT3 oncogenic signaling pathways while leaving the STAT1 tumor suppressor pathway, and immune effector function intact could be most useful for cancer therapy.
In the present report we have characterized the biologic activity of the FLLL32 curcumin analog on human melanoma and immune effector cells. Our data indicate that FLLL32 can inhibit STAT3 phosphorylation and promote caspase-dependent apoptosis of human melanoma cells at concentrations 10-fold lower than curcumin . FLLL32 displayed a greater specificity for STAT3 than curcumin or other comparable inhibitors. This compound did appear to alter the activation of other structurally similar STAT proteins, as interferon-induced STAT1 signaling and gene expression were intact in the presence of FLLL32. Treatment of PBMCs with FLLL32 also eliminated basal and IL-6 induced pSTAT3. In contrast, FLLL32 did not adversely affect the response of PBMCs to stimulation with IFN-γ and IL 2 or the viability and cytotoxicity of NK cells. These data suggest that FLLL32 represents a promising lead compound that can be optimized further for development as a therapeutic agent in melanoma.
Materials and methods
Cell Culture and Reagents
A375, HT144 and Hs294T human melanoma, and the K562 leukemia cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and 1106 MEL, 1259 MEL, MEL-39 and F01 human melanoma cell lines were provided by Dr. Soldano Ferrone (University of Pittsburgh, Pittsburgh, PA) and cultured as described . Melanoma cell lines were authenticated via karyotype analysis in the Molecular Cytogenetics Core Laboratory of The Ohio State University. The radial growth phase WM 1552c and vertical growth phase WM 793b human melanoma cell lines were provided by Dr. M. Herlyn (Wistar Institute, Philadelphia, PA) and cultured as described . Primary cultures from patients with recurrent cutaneous melanomas were cultured as previously described . Tetramethylrhodamine ethyl ester perchlorate (TMRE) was purchased from Invitrogen (Carlsbad, CA). The pan-caspase inhibitor (Z VAD-FMK), control (Z-FA-FMK) and recombinant human IFN-γ were purchased from R & D Systems, Inc. (Minneapolis, MN). Recombinant human interleukin-6 (IL 6) was purchased from Peprotech, Inc. (Rocky Hill, NJ). Recombinant human IL-2 (specific activity = 107 U/mg) was purchased from Hoffmann-La Roche Pharmaceuticals (Nutley, NJ). The JSI-124 and Stattic inhibitors were purchased from Calbiochem (Gibbstown, NJ). WP1066 was synthesized in the laboratory of Dr. P-K Li . FLLL32 and curcumin were synthesized, purified and evaluated for purity as previously described [16, 20, 21].
Peripheral Blood Mononuclear Cell Isolation
Peripheral blood mononuclear cells (PBMCs) were isolated from source leukocytes of healthy donors (American Red Cross, Columbus, OH) via density gradient centrifugation using Ficoll-Paque (Amersham, Pharmacia Biotech, Bjorkgatan, Sweeden) as described . NK cells were enriched from source leukocytes by negative selection with Rosette Sep reagents (Stem Cell Technologies, Inc., Vancouver, British Columbia, Canada).
Lysates were prepared from melanoma cell lines or PBMCs and assayed for protein expression by immunoblot analysis as previously described with antibodies (Ab) to STAT1 (BD Biosciences), Survivin (Novus Biologicals, Littleton, CO), pSTAT1, STAT3, pSTAT3, pSTAT5, STAT5, pJAK2, JAK2, PARP, Cyclin D1, Caspase-3, Caspase-8, Caspase-9, phosphorylated and total Akt (pAkt), Src (pSrc), p38 MAPK (p-p38 MAPK), ERK (pERK) (Cell Signaling Technology, Danvers, MA), or β-actin (Sigma) . Following incubation with the appropriate horseradish-peroxidase-conjugated secondary Ab, immune complexes were detected using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).
Annexin V/Propidium Iodide Staining
Phosphatidyl serine exposure was assessed in tumor cells by flow cytometry using APC-Annexin V and propidium iodide (PI; BD Pharmingen, San Diego, CA) as described . Analyses were performed utilizing at least 10,000 events.
STAT3 DNA binding assays
STAT3 DNA binding was measured with the Pierce LightShift Chemiluminescent EMSA kit used according to manufacturer's instructions (Thermo Fisher Scientific Inc, Rockford, IL). Nuclear protein was collected using the NucBuster™ Protein Extraction kit (EMD Chemicals Inc, Gibbstown, NJ). Binding reactions using equal amounts of nuclear protein were incubated for 20 minutes at room temperature with DNA probes. A biotinylated STAT3 binding sequence in the human survivin promoter (sense 5'-GAGACTCAGTTTCAAATAAATAAATAAAC-3') was purchased from Operon Biotechnologies (Huntsville, AL). Reactions with biotinylated target DNA only and nuclear protein with biotinylated target DNA and excess unlabelled target DNA to compete for binding were included. STAT3 specificity was confirmed by incubation with 6μg of anti-STAT3 Ab (Santa Cruz Biotechnology Inc, Santa Cruz, CA) to interfere with the protein-DNA complex. Following electrophoresis, DNA was transferred to a nylon membrane, cross-linked and detected by chemiluminescence.
Flow Cytometric Assay of Mitochondrial Membrane Potential
The mitochondrial membrane potential (ΔΨm) was assayed using 150 nM TMRE in regular medium at 37ºC for 15 minutes and by subsequent flow cytometric analysis as described .
Real Time PCR
Real-time PCR was used to evaluate the expression of the IFN-γ stimulated gene (IRF1) as described [25, 26] with pre-designed primer/probe sets (Assays On Demand; Applied Biosystems, Foster City, CA) and 2X TaqMan Universal PCR Master Mix (Applied Biosystems) per manufacturer's recommendations. Primer/probe sets for 18s rRNA (Applied Biosystems) were used to normalize expression values (housekeeping gene). Data were acquired and analyzed using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems).
ELISPOT Assay for Granzyme B and IFN-γ
To measure granzyme B (GrB) and IFN-γ secretion, ELISPOT experiments were conducted using MultiScreen 96-well plates (Millipore, Bedford, MA) and biotinylated monoclonal anti-human GrB or IFN-γ detecting Ab (Mabtech) as described . Freshly isolated NK cells (effectors) were incubated overnight in IL-2-containing media (1 nM) with either 5μM FLLL32 or DMSO. Effector cells were then co-incubated in triplicate with K562 cells as targets at an effector:target ratio of 10:1 for four hours. Targets and effectors cultured alone were used as controls. Spots were visualized and counted using the ImmunoSpot Imaging Analyzer (Cellular Technology Ltd, Cleveland, OH).
The 4-parameter logistic or Hill model  was the assumed dose-response relationship for FLLL32 concentration and proportion of apoptotic cells. Nonlinear least squares regression was used to estimate the parameters. ELISPOT data were compared between groups using a two-sample t-test. All analyses were performed in Statistical Analysis System (version 9.2; SAS Institute). P-values were considered significant at the 0.05 level and all tests were two-sided.
FLLL32 induces apoptosis in human melanoma cell lines
The pro-apoptotic effects of FLLL32 (Figure 1A) were examined by flow cytometry following Annexin V/PI staining of a panel of metastatic human melanoma cell lines with basal STAT3 phosphorylation (A375, Hs294T, FO1, HT144, MEL-39) and the pSTAT3 negative 1106 MEL and 1259 MEL cell lines . Dose-response studies revealed consistent induction of apoptosis in pSTAT3-positive metastatic human melanoma cell lines following a 48 hour treatment with FLLL32 as compared to DMSO (vehicle) treated cells (Figure 1B). The pSTAT3-positive A375 cell line was particularly sensitive to the pro-apoptotic effects of FLLL32 (IC50 = 1.3μM at 48 hours). Similar data were obtained in multiple pSTAT3 positive human melanoma cell lines (IC50 range = 1.9 -- 2.8 at 48 hours). The pSTAT3 negative 1106 MEL and 1259 MEL cell lines were poorly sensitive to FLLL32 (Figure 1B). FLLL32 was more potent than curcumin at inducing apoptosis (Figure 1C). Consistent with prior studies from our group, a 10-fold greater concentration of curcumin was required to achieve the same degree of apoptosis at the 48 hour time point . FLLL32-induced apoptosis was also confirmed in pSTAT3+ human melanoma cell lines derived from other disease phenotypes, including the WM 1552c radial growth phase and WM 793b vertical growth phase lines following treatment with FLLL32 (data not shown).
FLLL32 inhibits STAT3 phosphorylation and gene expression in human melanoma cell lines
FLLL32 induced cell death is caspase-dependent
IFN-γ induced STAT1 signaling and gene expression are not inhibited by FLLL32
Effects of FLLL32 on immune effector cells
We have characterized the biologic activity of the curcumin analog, FLLL32 on melanoma and immune effector cells. The present study has demonstrated that the FLLL32 small molecule can inhibit STAT3 signal transduction and induce caspase-dependent, pro-apoptotic effects against human melanoma cell lines and primary melanoma cultures at micromolar concentrations. In contrast to curcumin and other STAT3 pathway inhibitors, IFN-γ-induced STAT1 phosphorylation was not altered in the presence of FLLL32. This compound did not inhibit the viability of PBMCs, NK cells or their cellular responsiveness to clinically relevant cytokines. These data show that FLLL32 represents a novel small molecule curcumin analog with STAT3 pathway specificity that will be considered as a lead compound for further drug development in melanoma.
FLLL32 represents a structural analog of curcumin when locked into its diketone tautomeric form. A number of favorable biologic properties resulting from these modifications have been characterized in this study. First, FLLL32 was ten-fold more potent than curcumin at inducing apoptosis of melanoma cells . Second, FLLL32 did not appear to have toxic effects on either normal PBMCs or NK cells. Third, FLLL32 was designed to specifically target the oncogenic STAT3 pathway, while leaving the STAT1 pathway intact. Data from the present report indicate that in terms of in vitro specificity, FLLL32 was superior to other STAT3 pathway inhibitors or to curcumin. In fact, prior studies from our group have demonstrated that curcumin inhibited the phosphorylation of numerous STAT proteins in response to clinically relevant cytokines including IFN-γ (STAT1), IFN-γ (STAT1) and IL 2 (STAT5) . These inhibitory effects of curcumin were observed in both melanoma cell lines and in PBMCs from healthy donors. As a result, design of the FLLL32 analog was focused on maximizing the target specificity for STAT3 over other STAT proteins. The present data support the STAT3 specificity of the FLLL32 lead compound, although they do not conclusively exclude that FLLL32 could modulate the phosphorylation of other unidentified kinases.
Numerous early generation small molecule STAT3 inhibitors (e.g. Stattic, STA-21, LLL12, S32 M2001, S3I-201) have been reported to induce apoptosis via inhibition of STAT3 activation and/or dimerization [33, 37], while siRNA specific for the SH2 coding region of STAT3 could induce apoptosis in prostate cancer cells in vitro and in nude mice bearing human xenograft tumors . Finally, studies have also shown that platinum complexes can promote anti-tumor activity by virtue of their ability to inhibit STAT3 . Collectively, these studies provide precedent for targeting STAT3 as a means of inducing tumor cell apoptosis. However, the specificity of many existing inhibitory strategies for STAT3 and not other STAT proteins (e.g. STAT1) or oncogenic pathways has not been validated in biological systems. An attractive aspect of FLLL32 was its specificity and activity at micromolar concentrations. Data from the present study suggest that FLLL32 represents a unique molecule that can be optimized further for inhibition of the STAT3 pathway.
STAT3 can promote immune tolerance in the setting of cancer and thus represents an attractive target to enhance immunotherapy (Reviewed in ). Recent studies from our group and others have demonstrated that the presence of constitutively active STAT3 in melanoma cells is correlated with reduced responsiveness to cytokines which act via STAT1 signal transduction . These data suggest that the balance between pSTAT1 and pSTAT3 may influence cellular responsiveness to immunostimulatory cytokines and ultimately immune-mediated tumor regression [17, 40]. Data from this report also shows that FLLL32 inhibited IL-6 induced STAT3 phosphorylation within PBMCs. Of note, elevated levels of IL 6 are associated with poor prognosis in melanoma, and contribute to the generation of immunosuppressive lymphoid cell populations . Finally, our studies indicate that FLLL32-mediated inhibition of STAT3 does not alter production of granzyme b or IFN γ by NK cells from normal donors when cultured with K562 targets, or their viability when cultured with IL-2. These properties are of importance based on recent murine studies showing the Jak2 inhibitor WP1193 can augment immunotherapy with IFN-α , and STAT3 siRNA-CpG oligodeoxynucleotides can elicit anti-tumor immune responses . Together these data suggest that STAT3 pathway inhibition could be investigated further as a potential means by which to overcome immune tolerance and augment responsiveness to standard or experimental immune-based therapies.
Despite its improved STAT3 specificity, the FLLL32 analog retains some structural properties of its parent compound, curcumin which as expected, limit its solubility and bioavailability (data not shown). Therefore, our group is pursuing additional structural modifications or formulation approaches to further improve upon the bioavailability of this small molecule, in light of its potent and specific in vitro activity. The present results provide evidence that the FLLL32 curcumin analog represents a promising lead compound on which to base the further development of STAT3-specific inhibitors against melanoma. The ability of FLLL32 to specifically inhibit the STAT3 pathway while retaining the cellular response to cytokines with anti-tumor activity is a particular advantage that will be optimized in future pre-clinical studies.
This work was supported by NIH Grants K22CA134551 (Lesinski), 1R21CA141434-01A1 (Lesinski), P01CA95426 (Caligiuri), P30CA16058 (Caligiuri), CA84402, K24CA93670 (Carson); The Valvano Foundation for Cancer Research (Lesinski), and Grant # IRG-67-003-44 from the American Cancer Society (Benson and Fuchs). The project described was supported by Award Number UL1RR025755 from the National Center for Research Resources, funded by the Office of the Director, National Institutes of Health (OD) and supported by the NIH Roadmap for Medical Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. We also thank the OSU CCC Analytical Cytometry, Nucleic Acid, Molecular Cytogenetics, Pharmacoanalytical and Biostatistics Shared Resources. We also thank Dr. Mitch Phelps, Dr. Cheryl London and Dr. Samuel Kulp for their guidance associated with this project.
- Kortylewski M, Jove R, Yu H: Targeting STAT3 affects melanoma on multiple fronts. Cancer Metastasis Rev. 2005, 24: 315-327. 10.1007/s10555-005-1580-1View ArticlePubMedGoogle Scholar
- Niu G, Bowman T, Huang M, Shivers S, Reintgen D, Daud A, Chang A, Kraker A, Jove R, Yu H: Roles of activated Src and Stat3 signaling in melanoma tumor cell growth. Oncogene. 2002, 21: 7001-7010. 10.1038/sj.onc.1205859View ArticlePubMedGoogle Scholar
- Xie TX, Huang FJ, Aldape KD, Kang SH, Liu M, Gershenwald JE, Xie K, Sawaya R, Huang S: Activation of stat3 in human melanoma promotes brain metastasis. Cancer Res. 2006, 66: 3188-3196. 10.1158/0008-5472.CAN-05-2674View ArticlePubMedGoogle Scholar
- Sellers LA, Feniuk W, Humphrey PP, Lauder H: Activated G protein-coupled receptor induces tyrosine phosphorylation of STAT3 and agonist-selective serine phosphorylation via sustained stimulation of mitogen-activated protein kinase. Resultant effects on cell proliferation. J Biol Chem. 1999, 274: 16423-16430. 10.1074/jbc.274.23.16423View ArticlePubMedGoogle Scholar
- Zhang Y, Turkson J, Carter-Su C, Smithgall T, Levitzki A, Kraker A, Krolewski JJ, Medveczky P, Jove R: Activation of Stat3 in v-Src-transformed fibroblasts requires cooperation of Jak1 kinase activity. J Biol Chem. 2000, 275: 24935-24944. 10.1074/jbc.M002383200View ArticlePubMedGoogle Scholar
- Akira S: Roles of STAT3 defined by tissue-specific gene targeting. Oncogene. 2000, 19: 2607-2611. 10.1038/sj.onc.1203478View ArticlePubMedGoogle Scholar
- Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida N, Kishimoto T, Akira S: Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc Natl Acad Sci USA. 1997, 94: 3801-3804. 10.1073/pnas.94.8.3801PubMed CentralView ArticlePubMedGoogle Scholar
- Goel A, Kunnumakkara AB, Aggarwal BB: Curcumin as "Curecumin": from kitchen to clinic. Biochem Pharmacol. 2008, 75: 787-809. 10.1016/j.bcp.2007.08.016View ArticlePubMedGoogle Scholar
- Bush JA, Cheung KJ, Li G: Curcumin induces apoptosis in human melanoma cells through a Fas receptor/caspase-8 pathway independent of p53. Exp Cell Res. 2001, 271: 305-314. 10.1006/excr.2001.5381View ArticlePubMedGoogle Scholar
- Siwak DR, Shishodia S, Aggarwal BB, Kurzrock R: Curcumin-induced antiproliferative and proapoptotic effects in melanoma cells are associated with suppression of IkappaB kinase and nuclear factor kappaB activity and are independent of the B-Raf/mitogen-activated/extracellular signal-regulated protein kinase pathway and the Akt pathway. Cancer. 2005, 104: 879-890. 10.1002/cncr.21216View ArticlePubMedGoogle Scholar
- Zheng M, Ekmekcioglu S, Walch ET, Tang CH, Grimm EA: Inhibition of nuclear factor-kappaB and nitric oxide by curcumin induces G2/M cell cycle arrest and apoptosis in human melanoma cells. Melanoma Res. 2004, 14: 165-171. 10.1097/01.cmr.0000129374.76399.19View ArticlePubMedGoogle Scholar
- Cheng AL, Hsu CH, Lin JK, Hsu MM, Ho YF, Shen TS, Ko JY, Lin JT, Lin BR, Ming-Shiang W, Yu HS, Jee SH, Chen GS, Chen TM, Chen CA, Lai MK, Pu YS, Pan MH, Wang YJ, Tsai CC, Hsieh CY: Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer Res. 2001, 21: 2895-2900.PubMedGoogle Scholar
- Shoba G, Joy D, Joseph T, Majeed M, Rajendran R, Srinivas PS: Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers. Planta Med. 1998, 64: 353-356. 10.1055/s-2006-957450View ArticlePubMedGoogle Scholar
- Anand P, Thomas SG, Kunnumakkara AB, Sundaram C, Harikumar KB, Sung B, Tharakan ST, Misra K, Priyadarsini IK, Rajasekharan KN, Aggarwal BB: Biological activities of curcumin and its analogues (Congeners) made by man and Mother Nature. Biochem Pharmacol. 2008, 76: 1590-1611. 10.1016/j.bcp.2008.08.008View ArticlePubMedGoogle Scholar
- Bill MA, Bakan C, Benson DM, Fuchs J, Young G, Lesinski GB: Curcumin induces proapoptotic effects against human melanoma cells and modulates the cellular response to immunotherapeutic cytokines. Mol Cancer Ther. 2009, 8: 2726-2735. 10.1158/1535-7163.MCT-09-0377PubMed CentralView ArticlePubMedGoogle Scholar
- Lin L, Hutzen B, Zuo M, Ball S, Deangelis S, Foust E, Pandit B, Ihnat MA, Shenoy SS, Kulp S, Li PK, Li C, Fuchs J, Lin J: Novel STAT3 phosphorylation inhibitors exhibit potent growth-suppressive activity in pancreatic and breast cancer cells. Cancer Res. 70: 2445-2454.Google Scholar
- Lesinski GB, Trefry J, Brasdovich M, Kondadasula SV, Sackey K, Zimmerer JM, Chaudhury AR, Yu L, Zhang X, Crespin TR, Walker MJ, Carson WE: Melanoma cells exhibit variable signal transducer and activator of transcription 1 phosphorylation and a reduced response to IFN-alpha compared with immune effector cells. Clin Cancer Res. 2007, 13: 5010-5019. 3, 10.1158/1078-0432.CCR-06-3092View ArticlePubMedGoogle Scholar
- Satyamoorthy K, DeJesus E, Linnenbach AJ, Kraj B, Kornreich DL, Rendle S, Elder DE, Herlyn M: Melanoma cell lines from different stages of progression and their biological and molecular analyses. Melanoma Res. 1997, 7 (Suppl 2): S35-42.PubMedGoogle Scholar
- Priebe W, Donato N, Talpaz M, Szymanshi S, Fokt I, Levitki A: Preparation of benzyl cyanocinnamides and related compounds for treatment of cell proliferative diseases. pp. PCT Int. Appl.; 2005: PCT Int. Appl,Google Scholar
- Park BS, Kim JG, Kim MR, Lee SE, Takeoka GR, Oh KB, Kim JH: Curcuma longa L. constituents inhibit sortase A and Staphylococcus aureus cell adhesion to fibronectin. J Agric Food Chem. 2005, 53: 9005-9009. 10.1021/jf051765zView ArticlePubMedGoogle Scholar
- Venkateswarlu S, Ramachandra MS, Subbaraju GV: Synthesis and biological evaluation of polyhydroxycurcuminoids. Bioorg Med Chem. 2005, 13: 6374-6380. 10.1016/j.bmc.2005.06.050View ArticlePubMedGoogle Scholar
- Lesinski GB, Kondadasula SV, Crespin T, Shen L, Kendra K, Walker MJ, Carson WE: Multiparametric flow cytometric analysis of inter-patient variation in STAT1 phosphorylation following interferon alfa immunotherapy. J Natl Cancer Inst. 2004, 96: 1331-1342. 10.1093/jnci/djh252View ArticlePubMedGoogle Scholar
- Lesinski GB, Raig T, Guenterberg K, Brown L, Go M, Shah N, Lewis A, Quimper M, Hade E, Young G, Chaudhury AR, Ladner KJ, Guttridge DC, Bouchard P, Carson WE: IFN-alpha and bortezomib overcome Bcl-2 and Mcl-1 overexpression in melanoma cells by stimulating the extrinsic pathway of apopotsis. Cancer Res. 2008, 68: 8351-8360. 3, 10.1158/0008-5472.CAN-08-0426PubMed CentralView ArticlePubMedGoogle Scholar
- Li Y, Upadhyay S, Bhuiyan M, Sarkar FH: Induction of apoptosis in breast cancer cells MDA-MB-231 by genistein. Oncogene. 1999, 18: 3166-3172. 10.1038/sj.onc.1202650View ArticlePubMedGoogle Scholar
- Ramsauer K, Sadzak I, Porras A, Pilz A, Nebreda AR, Decker T, Kovarik P: p38 MAPK enhances STAT1-dependent transcription independently of Ser-727 phosphorylation. Proc Natl Acad Sci USA. 2002, 99: 12859-12864. 10.1073/pnas.192264999PubMed CentralView ArticlePubMedGoogle Scholar
- Zimmerer JM, Lesinski GB, Kondadasula SV, Karpa VI, Lehman A, Raychaudhury A, Becknell B, Carson WE: IFN-alpha-induced signal transduction, gene expression, and antitumor activity of immune effector cells are negatively regulated by suppressor of cytokine signaling proteins. J Immunol. 2007, 178: 4832-4845. 3,View ArticlePubMedGoogle Scholar
- Shafer-Weaver KA, Sayers T, Kuhns DB, Strobl SL, Burkett MW, Baseler M, Malyguine A: Evaluating the cytotoxicity of innate immune effector cells using the GrB ELISPOT assay. J Transl Med. 2004, 2: 31- 10.1186/1479-5876-2-31PubMed CentralView ArticlePubMedGoogle Scholar
- Hill A: The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J Physiol. 1910, 40: iv-vii.Google Scholar
- Sengupta TK, Talbot ES, Scherle PA, Ivashkiv LB: Rapid inhibition of interleukin-6 signaling and Stat3 activation mediated by mitogen-activated protein kinases. Proc Natl Acad Sci USA. 1998, 95: 11107-11112. 10.1073/pnas.95.19.11107PubMed CentralView ArticlePubMedGoogle Scholar
- Bromberg J: Stat proteins and oncogenesis. J Clin Invest. 2002, 109: 1139-1142.PubMed CentralView ArticlePubMedGoogle Scholar
- Bromberg JF, Horvath CM, Wen Z, Schreiber RD, Darnell JE: Transcriptionally active Stat1 is required for the antiproliferative effects of both interferon alpha and interferon gamma. Proc Natl Acad Sci USA. 1996, 93: 7673-7678. 10.1073/pnas.93.15.7673PubMed CentralView ArticlePubMedGoogle Scholar
- Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C, Darnell JE: Stat3 as an oncogene. Cell. 1999, 98: 295-303. 10.1016/S0092-8674(00)81959-5View ArticlePubMedGoogle Scholar
- Fletcher S, Turkson J, Gunning PT: Molecular approaches towards the inhibition of the signal transducer and activator of transcription 3 (Stat3) protein. ChemMedChem. 2008, 3: 1159-1168. 10.1002/cmdc.200800123PubMed CentralView ArticlePubMedGoogle Scholar
- Dunn GP, Koebel CM, Schreiber RD: Interferons, immunity and cancer immunoediting. Nat Rev Immunol. 2006, 6: 836-848. 10.1038/nri1961View ArticlePubMedGoogle Scholar
- Grimm EA, Mazumder A, Zhang HZ, Rosenberg SA: Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes. J Exp Med. 1982, 155: 1823-1841. 10.1084/jem.155.6.1823View ArticlePubMedGoogle Scholar
- Lotze MT, Grimm EA, Mazumder A, Strausser JL, Rosenberg SA: Lysis of fresh and cultured autologous tumor by human lymphocytes cultured in T-cell growth factor. Cancer Res. 1981, 41: 4420-4425.PubMedGoogle Scholar
- Lin L, Hutzen B, Li PK, Ball S, Zuo M, DeAngelis S, Foust E, Sobo M, Friedman L, Bhasin D, Cen L, Li C, Lin J: A novel small molecule, LLL12, inhibits STAT3 phosphorylation and activities and exhibits potent growth-suppressive activity in human cancer cells. Neoplasia. 12: 39-50.Google Scholar
- Turkson J, Zhang S, Palmer J, Kay H, Stanko J, Mora LB, Sebti S, Yu H, Jove R: Inhibition of constitutive signal transducer and activator of transcription 3 activation by novel platinum complexes with potent antitumor activity. Mol Cancer Ther. 2004, 3: 1533-1542.PubMedGoogle Scholar
- Yu H, Kortylewski M, Pardoll D: Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment. Nat Rev Immunol. 2007, 7: 41-51. 10.1038/nri1995View ArticlePubMedGoogle Scholar
- Wang W, Edington HD, Rao UN, Jukic DM, Land SR, Ferrone S, Kirkwood JM: Modulation of signal transducers and activators of transcription 1 and 3 signaling in melanoma by high-dose IFNalpha2b. Clin Cancer Res. 2007, 13: 1523-1531. 10.1158/1078-0432.CCR-06-1387View ArticlePubMedGoogle Scholar
- Tartour E, Dorval T, Mosseri V, Deneux L, Mathiot C, Brailly H, Montero F, Joyeux I, Pouillart P, Fridman WH: Serum interleukin 6 and C-reactive protein levels correlate with resistance to IL-2 therapy and poor survival in melanoma patients. Br J Cancer. 1994, 69: 911-913.PubMed CentralView ArticlePubMedGoogle Scholar
- Kong LY, Gelbard A, Wei J, Reina-Ortiz C, Wang Y, Yang EC, Hailemichael Y, Fokt I, Jayakumar A, Qiao W, Fuller GN, Overwijk WW, Priebe W, Heimberger AB: Inhibition of p-STAT3 enhances IFN-alpha efficacy against metastatic melanoma in a murine model. Clin Cancer Res. 16: 2550-2561.Google Scholar
- Kortylewski M, Swiderski P, Herrmann A, Wang L, Kowolik C, Kujawski M, Lee H, Scuto A, Liu Y, Yang C, Deng J, Soifer HS, Raubitschek A, Forman S, Rossi JJ, Pardoll DM, Jove R, Yu H: In vivo delivery of siRNA to immune cells by conjugation to a TLR9 agonist enhances antitumor immune responses. Nat Biotechnol. 2009, 27: 925-932. 10.1038/nbt.1564PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.