Curcumin (diferuloylmethane) inhibits constitutive and IL-6-inducible STAT3 phosphorylation in human multiple myeloma cells

Numerous reports suggest that IL-6 promotes survival and proliferation of multiple myeloma (MM) cells through the phosphorylation of a cell signaling protein, STAT3. Thus, agents that suppress STAT3 phosphorylation have potential for the treatment of MM. In the present report, we demonstrate that curcumin (diferuloylmethane), a pharmacologically safe agent in humans, inhibited IL-6-induced STAT3 phosphorylation and consequent STAT3 nuclear translocation. Curcumin had no effect on STAT5 phosphorylation, but inhibited the IFN-alpha-induced STAT1 phosphorylation. The constitutive phosphorylation of STAT3 found in certain MM cells was also abrogated by treatment with curcumin. Curcumin-induced inhibition of STAT3 phosphorylation was reversible. Compared with AG490, a well-characterized Janus kinase 2 inhibitor, curcumin was a more rapid (30 min vs 8 h) and more potent (10 micro M vs 100 micro M) inhibitor of STAT3 phosphorylation. In a similar manner, the dose of curcumin completely suppressed proliferation of MM cells; the same dose of AG490 had no effect. In contrast, a cell-permeable STAT3 inhibitor peptide that can inhibit the STAT3 phosphorylation mediated by Src blocked the constitutive phosphorylation of STAT3 and also suppressed the growth of myeloma cells. TNF-alpha and lymphotoxin also induced the proliferation of MM cells, but through a mechanism independent of STAT3 phosphorylation. In addition, dexamethasone-resistant MM cells were found to be sensitive to curcumin. Overall, our results demonstrated that curcumin was a potent inhibitor of STAT3 phosphorylation, and this plays a role in the suppression of MM proliferation.     

Differential cross-talk of estrogen and growth factor receptors in two human mammary tumor cell lines

Cultured human mammary MCF7 and T47D tumor cell lines were used to test the interference of the partial antiestrogen 4′-hydroxytamoxifen (4-OH-TAM) and the pure antiestrogen ZM 182780 with growth factor (IGF-I, heregulin) signaling pathways. Growth of both cell lines was stimulated by IGF-I (20 ng/ml) or heregulin (3 nM). ZM 182780 effectively blocked growth factor induced as well as basal proliferation of MCF7 cells while the compound was ineffective in interfering with growth factor mitogenic activity in T47D cells. On both cell lines the IGF-I or heregulin- induced proliferation was enhanced further by 4-OH-TAM. This synergism could be inhibited dose-dependently by ZM 182780. When cells were grown in the presence of estradiol plus growth factors, the antiestrogenic potencies of both compounds and the efficacy of ZM 182780 were unaffected, while the efficacy of 4-OH-TAM was reduced. Our data show cell type specific cross-talk between the receptor for estrogen and that for IGF-I or heregulin, which is different in MCF7 and T47D cells, respectively. In MCF7 cells with demonstrable cross-talk, a clear superiority exists for a pure antiestrogen over a partial agonist in interfering with growth factor mitogenic activity.     

Activation of phosphatidylinositol-3-kinase by insulin is mediated by both A and B human insulin receptor types.

Activation of a phosphatidylinositol-3-kinase (PI-3-kinase) is one of the earliest consequences of insulin binding to the receptor. The human insulin receptor exists in two isoforms which differ in the length of the alpha-subunit (HIR-A = 719 aa, HIR-B = 731 aa). To test whether both isoforms transduce an insulin signal on PI-3-kinase we used rat-1-fibroblasts expressing HIR-A or HIR-B. We found that insulin stimulates 32P incorporation into PIP through both HIR-A and HIR-B to a similar extent (approx. 8-10 fold).     

Study on the growth inhibition of human multiple myeloma cells by an IL-6Ralpha mutant

DM650, a soluble human IL-6Ralpha mutant with mutation of C277D/H280I, was previously shown to exhibit an antagonism to IL-6, which resulted in growth-inhibition of human multiple myeloma cell line AF-10 autocrining as the growth-stimulating factor. We investigated here the nature of the growth inhibition by examining cell apoptosis. Flow-cytometric analysis of the DNA fragmentation demonstrated that 7.2% of the AF-10 cells were apoptotic after 24 h of treatment with DM650. The constitutive gene expression of bcl-2 in AF-10 cells indicated that apoptosis suppressed by IL-6 was independent of bcl-2 regulation. The altered gene expression of c-myc and p53 suggested that a novel apoptosis pathway, other than that suppressed by IL-6, might be triggered by a complex of DM650 and IL-6.     

Definition of receptor binding sites on human interleukin-11 by molecular modeling-guided mutagenesis.

Interleukin-11 (IL-11) belongs to the interleukin-6 (IL-6)-type subfamily of long-chain helical cytokines including IL-6, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M, and cardiotrophin-1, which all share the glycoprotein gp130 as a signal transducing receptor component. IL-11 acts on cells expressing gp130 and the IL-11 receptor (IL-11R) alpha-subunit (IL-11Ralpha). The structural epitopes of IL-11 required for the recruitment of the individual receptor subunits have not yet been defined. Based on the structure of CNTF, a three-dimensional model of human IL-11 was built. Using this model, 10 surface exposed amino acid residues of IL-11 were selected for mutagenesis using analogies to the well-characterized receptor recruitment sites of IL-6, CNTF, and LIF. The respective mutants of human IL-11 were expressed as soluble fusion proteins in bacteria. Their biological activities were determined on HepG2 and Ba/F3-130-11alpha cells. Several mutants with substantially decreased bioactivity and one hyperagonistic mutant were identified and further analyzed with regard to recruitment of IL-11Ralpha and gp130. The low-activity mutant I171D still binds IL-11Ralpha but fails to recruit gp130, whereas the hyperagonistic variant R135E more efficiently engages the IL-11R subunits. The low-activity mutants R190E and L194D failed to bind to IL-11Ralpha. These findings reveal a common mechanism of receptor recruitment in the family of IL-6-type cytokines and offer considerable perspectives for the rational design of IL-11 antagonists and hyperagonists.     

BALB/C mouse 3T3 fibroblasts expressing human estrogen receptor: effect of estradiol on cell growth

Mouse 3T3 fibroblasts (clone A31) were stably transfected with human estrogen receptor (hER). Among the four sublines expressing functional hER at approximately 10(4) estrogen binding sites/cell, three retained a non-transformed morphology and growth characteristics while the fourth displayed a transformed phenotype (criss-cross growth, lack of density arrest, reduced dependence on exogenous growth factors). Estradiol (E2) had no effect on the growth of the three non-transformed hER expressing sublines. In contrast, low concentrations (1 to 20 nM) of E2 strongly inhibited the proliferation of the subline with transformed phenotype and high (100 nM) concentrations were toxic in these cells.     

The rational designed antagonist derived from the complex structure of interleukin-6 and its receptor affectively blocking interleukin-6 might be a promising treatment in multiple myeloma

Human interleukin-6 is involved in the maintenance and progression of several diseases such as multiple myeloma (MM), rheumatoid arthritis, or osteoporosis. Our previous work demonstrated that an interleukin-6 antagonist peptide (named PT) possessed potential bioactivity to antagonize the function of hIL-6 and could efficiently induce the growth arrest and apoptosis of XG-7 and M1 cells in a dose-dependent manner. In this study, the theoretical interaction of the peptide PT with its receptor was analyzed further more with molecular docking and molecular dynamics methods. The theoretical studies showed that PT possessed very high affinity to interleukin-6R and offered a practical means of imposing long-term blockade of interleukin-6 activity in vivo. According to the theoretical results, the biological evaluation of PT was researched on two different cells models with more sensitive approaches: (1) The antagonist activity of PT was studied on the interleukin-6 dependent MM cells (XG-7) cultured with interleukin-6. In the other interleukin-6 dependent MM cells (SKO-007), they survived themselves by auto/paracrine without the exogenous interleukin-6, and also could be antagonized by PT. The therapeutic value of PT only limited on the interleukin-6 dependent category in MM. (2) Myeloid leukemia M1 cells were induced for growth arrest and apoptosis in response to interleukin-6. The results supported our previous findings and showed that PT could be evaluated by protecting the cells from interleukin-6 induced apoptosis. In conclusion, PT could induce interleukin-6-dependent XG-7 and SKO-007 cells to apoptosis while inhibit interleukin-6-stimulated apoptosis in M1 cells.     

Structural and functional studies on the human interleukin-6 receptor. Binding, cross-linking, internalization, and degradation of interleukin-6 by fibroblasts transfected with human interleukin-6-receptor cDNA

A cDNA coding for the human interleukin-6 receptor (IL-6-R) has been expressed stably in murine NIH/3T3 fibroblasts. Transfected cells exhibited a single class of binding sites for 125I-labeled recombinant human interleukin-6 (125I-rhIL-6) (Kd = 440 pM, 20,000 receptors per cell). Affinity cross-linking of 125I-rhIL-6 to the IL-6-R-expressing NIH/3T3 cells led to the detection of three 125I-rhIL-6-containing protein complexes with molecular masses of 100, 120, and 200 kDa suggesting a complex organization of the IL-6-R in the plasma membrane. IL-6 added to the transfected NIH/3T3 cells exerted growth inhibition. This anti-growth effect was observed by the measurement of cell numbers and ornithine decarboxylase mRNA expression. IL-6-R overexpressing fibroblasts internalized 125I-rhIL-6. Intracellular limited proteolysis of IL-6 could be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A possible implication of skin fibroblasts in the catabolism of IL-6 is discussed.     

A molecular model for the differential activation of STAT3 and STAT6 by the herpesviral oncoprotein tip

Constitutive STAT signaling provides growth promoting signals in many forms of malignancy. We performed molecular modeling and molecular dynamics studies of the interaction between the regulatory Src homology 2 (SH2) domains of STAT3 and 6 with phosphorylated peptides of the herpesviral oncoprotein Tip, which facilitates Src kinase mediated STAT-activation and T cell proliferation. The studies give insight into the ligand binding specificity of the STAT SH2 domains and provide the first model for the differential activation of STAT3 or STAT6 by two distinct regions of the viral Tip protein. The biological relevance of the modeled interactions was then confirmed by activation studies using corresponding recombinant oncoproteins, and finally by respective recombinant viruses. The functional data give experimental validation of the molecular dynamics study, and provide evidence for the involvement of STAT6 in the herpesvirus induced T cell proliferation.