Indomethacin derivatives as tubulin stabilizers to inhibit cancer cell proliferation

Cyclooxygenase (COX) inhibitor Indomethacin analogs exhibited more potent cancer cell growth inhibition and apoptosis inducing activities than the parental compound. The anti-proliferative mechanism investigation of the analogs revealed that they inhibited tubulin polymerization at high concentrations whereas enhanced polymerization at low concentrations. The two opposite activities might antagonize each other and impaired the anti-proliferative activity of the derivatives eventually. In this study, we further performed lead optimization based on the structure activity relationship (SAR) generated. One of the new Indomethacin derivatives compound 11 {2-(4-(benzyloxy)phenyl)-N-(1-(4-bromobenzoyl)-3-(2-((2-(dimethylamino)ethyl)amino)-2-oxoethyl)-2-methyl-1H-indol-5-yl)acetamide} inhibited the proliferation of a panel of cancer cell lines with IC₅₀s at the sub-micromole levels. Further study revealed that the compound only enhanced tubulin polymerization and was a tubulin stabilizer.     

纳米羟基磷灰石表面修饰及其转染细胞的研究

目的:探讨羟基磷灰石-聚乙烯亚胺(nHA-PEI 10KD)纳米颗粒的癌细胞基因转染效率.方法:通过透射电子显微镜(TEM)观察HA-PEI(10KD)纳米颗粒的形态及粒径,Zeta电位仪测定nHA-PEI和HA在酸、碱、中性环境中的电位,用琼脂糖凝胶电泳检测nHAP-PEI(10KD)与DNA结合的能力,MTT比色法检测nHAP-PEI(10KD)对nepG2细胞的毒性,选用增强型绿色荧光蛋白质粒pEGFP1与nHA-PEI结合后,分别转染真核细胞HepG2、Hela、SW620,计算其转染率.结果:nHA-PEI(10KD)分散程度好,粒径60-80nm,在PH7.2时,Zeta电位42.87mV,能转染实验中的细胞,转然效果最好的是HepG2细胞,其次Hela、SW620,转染率高于PEI(10KD)、nHA,但低于脂质体.结论:通过阳离子PEI修饰HA,可有效将增强型绿色荧光蛋白质粒转入HepG2细胞,HA-PEI(10KD)纳米颗粒复合物有望成为基因传递的有效栽体.     

Tumorigenic poxviruses up-regulate intracellular superoxide to inhibit apoptosis and promote cell proliferation

Tumorigenic leporipoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD1). The function of the orthologous myxoma virus M131R and Shope fibroma virus S131R gene products is uncertain, but they inhibit SOD1 activity by a process linked to binding its copper chaperone. Using a superoxide-sensitive dye (hydroethidine), we observed that virus infection increased intracellular superoxide levels in an M/S131R-dependent manner. To see whether this effect promotes infection, we deleted the Shope fibroma virus S131R gene and compared the clinical manifestations of wild-type and mutant virus infections in rabbits. S131RDelta virus produced significantly smaller fibroxanthosarcoma-like growths in vivo and, at a point where these growths were already receding, wild-type infections still showed extensive leukocyte infiltration, necrosis, and fibromatous cell proliferation. Coincidentally, whereas Jurkat cells are protected from mitochondria- and Fas-mediated apoptosis by wild-type myxoma virus in vitro, M131RDelta virus could not block Fas-initiated apoptosis as judged by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end labeling, and caspase 3 cleavage assays. These data suggest that tumorigenic poxviruses can modulate intracellular redox status to their advantage to stimulate infected cell growth and inhibit programmed cell death.     

Acylamido analogs of endocannabinoids selectively inhibit cancer cell proliferation

A series of amide derivatives of long-chain fatty acids has been studied for their effects on the proliferation of cancer cells in vitro. Fatty acids ranged from palmitic to higher polyunsaturated types containing 22 carbon atoms. The amino portions of the molecules included ammonia, ethanolamine, various amino acids and dopamine. Several cell lines were used as models and these included HTB-125 (normal human breast cells), HTB-126 (human breast cancer cells), HeLa (cervical cancer cells), WI-38 (human embryonic lung cells), RAW264.7 (mouse macrophage tumor cells) and RBL-2H3 (rat basophilic leukemia cells). The HTB lines were obtained from the same donor, so, could be considered a matched pair, that is, normal control versus cancer cells and thus, provide a model for testing specificity of action for the acylamido analogs. While many compounds were efficacious in inhibiting the proliferation of various cell lines, only two analogs showed a high degree of specificity in the matched HTB cell lines. N-palmitoyl dopamine and N-palmitoyl tyrosine each demonstrated complete specificity of action at a concentration of 10muM and were highly efficacious in both cases. No clear structure-activity pattern could be derived from these studies since the intensity of the inhibitory action seemed to depend on three factors, namely, the fatty acid, the amine group and the cell type.     

Antibodies to nonpolymorphic determinants of the Thy-1 molecule inhibit T cell proliferation

The effect of anti-Thy-1 monoclonal antibodies on murine mixed lymphocyte reactions and concanavalin A-induced mitogenesis were investigated. It is demonstrated that rat antibodies against nonpolymorphic determinants of the murine Thy-1 antigen inhibited cell proliferation in the absence of complement. In contrast, antibodies against polymorphic determinants of Thy-1 had no effect on T cell activation. Inhibition of T cell proliferation did not depend on the isotype of the blocking antibody, because both IgM and IgG antibodies against monomorphic determinants were inhibitory, whereas IgM or IgG antibodies against allotypic determinants were inactive. In addition, the blocking activity could not be attributed to the xenogeneic (rat) origin of the antibodies to nonpolymorphic Thy-1 determinants, because rat anti-Thy-1.2 antibodies had no effect on cell activation. Thus, the efficacy of anti-Thy-1 antibodies as T cell inhibitors was determined by the antibody specificity. The suppressive mechanism of anti-Thy-1 antibodies was effective throughout the entire course of mixed lymphocyte reactions. Addition of antibodies at any time point during the first 90 hr of a 120-hr mixed lymphocyte culture resulted in significant suppression of the proliferative response. However, in some cases an early enhancement preceded suppression of the response. The modulation of proliferative responses by anti-Thy-1 did not result from a nonspecific mitogenic effect of the antibodies on T lymphocytes, because no effects were observed when antibodies were added to responder cells alone. These results suggest that the Thy-1 molecule, or a molecule that is located on the cell membrane in close proximity to the Thy-1 antigen, is involved in the activation of T lymphocytes.     

Natural antisense mRNAs to hyaluronan synthase 2 inhibit hyaluronan biosynthesis and cell proliferation

We report the identification of a natural antisense mRNA of hyaluronan synthase 2 that we have chosen to designate as HASNT (for HA synthase 2 antisense) in human and mouse. HASNT is transcribed from the opposite strand of the HAS2 gene locus and is represented by several independent expressed sequence tags in human. Portions of the mouse Hasnt gene were identified through an exon-trapping approach. Sequence conservation is extremely low between human and mouse HASNT, and it is not clear whether these mRNAs contain functional open reading frames. HASNT has an alternate splice site in both human and mouse. This splice site is located at an identical position within the gene in both species and results in mRNAs of two different lengths. In each species, the antisense portion of the HASNT gene is complementary to the first exon of HAS2, which represents the 5'-untranslated region. To study the biological activity of HASNT, two human expressed sequence tag clones, representing long and short HASNT splice variants, were cloned into a tetracycline-inducible vector and were stably transfected into human osteosarcoma U2-OS Tet-on cells. The long and short HASNT-expressing cells had a reduction in HAS2 mRNA levels up to 94 and 86%, respectively, whereas hyaluronan biosynthesis was inhibited by 40 and 37%, respectively. Cell proliferation was reduced throughout the time frame of the experiment. Exogenous high molecular mass hyaluronan failed to rescue the suppressed cell proliferation, whereas adenoviral-mediated overexpression of hyaluronan synthase 3, which stimulated endogenous hyaluronan biosynthesis, was able to rescue. Collectively, our data suggest that natural antisense mRNAs of HAS2 are able to regulate HAS2 mRNA levels and hyaluronan biosynthesis in a cell culture model system and may have an important and novel regulatory role in the control of HAS2, HA biosynthesis, and HA-dependent cell functions in vivo.     

Adipose-derived stromal cells inhibit prostate cancer cell proliferation inducing apoptosis

Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferation of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa.     

Immunopathologic effects of silicone breast implants.

Silicone-gel breast implants have been associated with a myriad of autoimmune and connective tissue disorders by anecdotal reports and small observational series. To date, no prospective epidemiologic studies have been done to substantiate these observations, but an increasing body of literature is being developed and older studies are being recognized that point to immunotoxic or inflammatory effects of these breast implant components. The development of disease due to implants would depend on the interaction of genetic host factors so that only a few patients would potentially be at risk. Based on the example of other chemically mediated disorders, such as scleroderma in association with silica exposure, latency periods of more than 30 years before disease develops may be possible. Herein we review studies on silicone and immunity.     

Biodurability of retrieved silicone gel breast implants

This study analyzed the shells of single-lumen silicone gel breast implants within the general context of device durability in vivo. The investigation included the major types of gel-filled implants that were manufactured in the United States in a 30-year period. The implants analyzed were Cronin seamed (two explants and one control), Silastic 0 and Silastic I (18 explants and seven controls), and Silastic II (22 explants and 43 controls). The biodurability of the explants was investigated with measurements of the mechanical and chemical properties of the various types of silicone gel control and explanted shells, with implantation times ranging from 3 months to 32 years. The shell properties measured for the controls and explants included the stress-strain relationships, tensile strength, elongation, tear resistance, moduli, cross-link density, and amount of extractable material in the shell. In addition, the mechanical properties of shells that had been extracted with hexane were analyzed for both explants and control implants. The silicone gel explants investigated in this study included some of the oldest explants of the various major types that have been tested to date. For assessment of long-term implantation effects, the data obtained in this study were combined with all known data from other institutions on the various major types of gel implants. The study also addressed the failure mechanisms associated with silicone gel breast implants. The results of the study demonstrated that silicone gel implants have remained intact for 32 years in vivo and that degradation of the shell mechanical and chemical properties is not a primary mechanism for silicone gel breast implant failure.     

Antimitotic sulfonamides inhibit microtubule assembly dynamics and cancer cell proliferation

Several sulfonamides have antitumor activities and are currently undergoing clinical evaluation for the treatment of cancer. In this study, we have elucidated the antiproliferative mechanism of action of five indole sulfonamides. The indole sulfonamides inhibited the polymerization of microtubule protein into microtubules in vitro. In addition, three representative derivatives, ER-68378 (2), ER-68384 (4) and ER-68394 (5), suppressed the dynamic instability behavior at the plus ends of individual steady-state microtubules in vitro. The analogues inhibited HeLa cell proliferation with half-maximal inhibitory concentrations in the range of 6-17 microM. The compounds blocked cell cycle progression at mitosis. At their lowest effective antimitotic concentrations, they depolymerized the spindle microtubules and disorganized the chromosomes but did not affect the microtubules in interphase cells. However, at relatively high concentrations, interphase microtubules were also depolymerized by these sulfonamides. Furthermore, all five compounds were found to induce apoptosis in the cells in association with the phosphorylation of bcl-2. The results suggest that the indole sulfonamides inhibit cell proliferation at mitosis by perturbing the assembly dynamics of spindle microtubules and that they can kill cancer cells by inducing apoptosis through the bcl-2-dependent pathway.     

Imiquimod upregulates the opioid growth factor receptor to inhibit cell proliferation independent of immune function

The imidazoquinoline compounds imiquimod and resiquimod are low-molecular-weight immune response modifiers that have potent anti-viral and anti-tumor properties. The mechanism by which they exert their effects remains unclear. Using pancreatic and colorectal cancer cells, as well as squamous carcinoma cells of the head and neck in tissue culture, which eliminated the immune system and toll-like receptors, we show that the imidazoquinolines upregulate the Opioid Growth Factor receptor (OGFr), which in turn stimulates the interaction of the OGF-OGFr axis. This native, tonically active inhibitory pathway regulates cell proliferation by modulating cyclin dependent kinase inhibitors, resulting in a retardation of cells at the G(1)-S interface of the cell cycle. Neutralization of OGF or knockdown of OGFr by siRNA technology eliminates the inhibitory effects of imidazoquinolines on cell replication. This exciting new knowledge of the mechanism of imidazoquinolines has important physiological relevance, and allows strategies to be developed for the use of these agents that will enhance effectiveness as well as attenuate side-effects.     

NK cells inhibit T cell proliferation via p21-mediated cell cycle arrest

NK cells have been shown to influence immune responses via direct interaction with cells of the adaptive immune system, such as dendritic cells, B cells, and T cells. A role for NK cells in down-regulation of T cell responses has been implicated in several studies; however, the underlying mechanism of this suppression has remained elusive. In this study we show that dark Agouti rat NK cells inhibit syngeneic T cell proliferation via up-regulation of the cell cycle inhibitor, p21, resulting in a G0/G1 stage cell cycle arrest. The inhibition is cell-cell contact dependent, reversible, and Ag nonspecific. Interestingly, NK cells do not inhibit IL-2 secretion or IL-2R up-regulation and do not induce T cell death. Thus, our results show that NK cells do not affect early T cell activation events, but specifically inhibit T cell proliferation by direct interaction with T cells. Our findings suggest that NK cells may play an important role in maintaining immune homeostasis by directly regulating clonal expansion of activated T cells. This novel mechanism of T cell regulation by NK cells provides insight into NK cell-mediated regulation of adaptive immunity and provides a mechanistic link between NK cell function and suppression of T cell responses.     

S-nitrosothiols inhibit uterine smooth muscle cell proliferation independent of metabolism to NO and cGMP formation

S-nitrosothiols (RSNOs) are important mediators of nitric oxide (NO) biology. The two mechanisms that appear to dominate in their biological effects are metabolism leading to the formation of NO and S-nitrosation of protein thiols. In this study we demonstrate that RSNOs inhibit uterine smooth muscle cell proliferation independent of NO. The antiproliferative effects of NO on vascular smooth muscle are well defined, with the classic NO-dependent production of cGMP being demonstrated as the active pathway. However, less is known on the role of NO in mediating uterine smooth muscle cell function, a process that is important during menstruation and pregnancy. The RSNOs S-nitrosoglutathione and S-nitroso-N-acetyl pencillamine inhibited growth factor-dependent proliferation of human and rat uterine smooth muscle cells (ELT-3). Interestingly, these cells reduced RSNOs to generate NO. However, use of NO donors and other activators of the cGMP pathway failed to inhibit proliferation. These findings demonstrate the tissue-specific nature of responses to NO and demonstrate the presence of a RSNO-dependent but NO-independent pathway of inhibiting DNA synthesis in uterine smooth muscle cells.     

Amniotic membrane-derived cells inhibit proliferation of cancer cell lines by inducing cell cycle arrest

Cells derived from the amniotic foetal membrane of human term placenta have drawn particular attention mainly for their plasticity and immunological properties, which render them interesting for stem-cell research and cell-based therapeutic applications. In particular, we have previously demonstrated that amniotic mesenchymal tissue cells (AMTC) inhibit lymphocyte proliferation in vitro and suppress the generation and maturation of monocyte-derived dendritic cells. Here, we show that AMTC also significantly reduce the proliferation of cancer cell lines of haematopoietic and non-haematopoietic origin, in both cell-cell contact and transwell co-cultures, therefore suggesting the involvement of yet-unknown inhibitory soluble factor(s) in this 'cell growth restraint'. Importantly, we provide evidence that the anti-proliferative effect of AMTC is associated with induction of cell cycle arrest in G0/G1 phase. Gene expression analyses demonstrate that AMTC can down-regulate cancer cells' mRNA expression of genes associated with cell cycle progression, such as cyclins (cyclin D2, cyclin E1, cyclin H) and cyclin-dependent kinase (CDK4, CDK6 and CDK2), whilst they up-regulate cell cycle negative regulator such as p15 and p21, consistent with a block in G0/G1 phase with no progression to S phase. Taken together, these findings warrant further studies to investigate the applicability of these cells for controlling cancer cell proliferation in vivo.