Celecoxib substituted biotinylated poly(amidoamine) G3 dendrimer as potential treatment for temozolomide resistant glioma therapy and anti-nematode agent.

Glioblastoma multiforme (GBM) is a one of the most widely diagnosed and difficult to treat type of central nervous system tumors. Resection combined with radiotherapy and temozolomide (TMZ) chemotherapy prolongs patients' survival only for 12 - 15 months after diagnosis. Moreover, many patients develop TMZ resistance, thus important is search for a new therapy regimes including targeted drug delivery. Most types of GBM reveal increased expression of cyclooxygenase-2 (COX-2) and production of prostaglandin E2 (PGE2), that are considered as valuable therapeutic target. In these studies, the anti-tumor properties of the selective COX-2 inhibitor celecoxib (CXB) and biotinylated third generation of the poly(amidoamine) dendrimer substituted with 31 CXB residues (G3BC31) on TMZ -resistant U-118 MG glioma cell line were examined and compared with the effect of TMZ alone including viability, proliferation, migration and apoptosis, as well as the cellular expression of COX-2, ATP level, and PGE2 production. Confocal microscopy analysis with the fluorescently labeled G3BC31 analogue has shown that the compound was effectively accumulated in U-118 MG cells in time-dependent manner and its localization was confirmed in lysosomes but not nuclei. G3BC31 reveal much higher cytotoxicity for U-118 MG cells at relatively low concentrations in the range of 2-4 µM with compared to CBX alone, active at 50-100 µM. This was due to induction of apoptosis and inhibition of proliferation and migration. Observed effects were concomitant with reduction of PGE2 production but independent of COX-2 expression. We suggest that investigated conjugate may be a promising candidate for therapy of TMZ-resistant glioblastoma multiforme, although applicable in local treatment, since our previous study of G3BC31 did not demonstrate selectivity against glioma cells compared to normal human fibroblasts. However, it has to be pointed that in our in vivo studies conducted with model organism, Caenorhabditis elegans indicated high anti-nematode activity of G3BC31 in comparison with CXB alone that confirms of usefulness of that organism for estimation of anti-cancer drug toxicity.

The Effect of Biotinylated PAMAM G3 Dendrimers Conjugated with COX-2 Inhibitor (celecoxib) and PPARγ Agonist (Fmoc-L-Leucine) on Human Normal Fibroblasts, Immortalized Keratinocytes and Glioma Cells in Vitro.

Glioblastoma multiforme (GBM) is the most malignant type of central nervous system tumor that is resistant to all currently used forms of therapy. Thus, more effective GBM treatment strategies are being investigated, including combined therapies with drugs that may cross the blood brain barrier (BBB). Another important issue considers the decrease of deleterious side effects of therapy. It has been shown that nanocarrier conjugates with biotin can penetrate BBB. In this study, biotinylated PAMAM G3 dendrimers substituted with the recognized anticancer agents cyclooxygenase-2 (COX-2) inhibitor celecoxib and peroxisome proliferator-activated receptor γ (PPARγ) agonist Fmoc-L-Leucine (G3-BCL) were tested in vitro on human cell lines with different p53 status: glioblastoma (U-118 MG), normal fibroblasts (BJ) and immortalized keratinocytes (HaCaT). G3-BCL penetrated efficiently into the lysosomal and mitochondrial compartments of U-118 MG cells and induced death of U-118 MG cells via apoptosis and inhibited proliferation and migration at low IC50 = 1.25 µM concentration, considerably lower than either drug applied alone. Comparison of the effects of G3-BCL on expression of COX-2 and PPARγ protein and PGE2 production of three different investigated cell line phenotypes revealed that the anti-glioma effect of the conjugate was realized by other mechanisms other than influencing PPAR-γ expression and regardless of p53 cell status, it was dependent on COX-2 protein level and high PGE2 production. Similar G3-BCL cytotoxicity was seen in normal fibroblasts (IC50 = 1.29 µM) and higher resistance in HaCaT cells (IC50 = 4.49 µM). Thus, G3-BCL might be a good candidate for the targeted, local glioma therapy with limited site effects.

Synthesis and Different Effects of Biotinylated PAMAM G3 Dendrimer Substituted with Nimesulide in Human Normal Fibroblasts and Squamous Carcinoma Cells.

Squamous cell carcinoma (SCC) remains a main cause of mortality in patients with neck and head cancers, with poor prognosis and increased prevalence despite of available therapies. Recent studies have identified a role of cyclooxygenases, particularly inducible isoform cyclooxygenase-2 (COX-2) and its metabolite prostaglandin E2 (PGE2) in cancer cell proliferation, and its inhibition become a target for control of cancer development, particularly in the view of recognized additive or synergic action of COX-2 inhibitors with other forms of therapy. Nimesulide (N), the selective COX-2 inhibitor, inhibits growth and proliferation of various types of cancer cells by COX-2 dependent and independent mechanisms. In the presented study, the conjugates of biotinylated third generation poly(amidoamine) dendrimer (PAMAM) with covalently linked 18 (G3B18N) and 31 (G3B31N) nimesulide residues were synthesized and characterized by NMR spectroscopy. Biological properties of conjugates were evaluated, including cytotoxicity, proliferation, and caspase 3/7 activities in relation to COX-2/PGE2 axis signaling in human normal fibroblast (BJ) and squamous cell carcinoma (SCC-15). Both conjugates exerted a selective cytotoxicity against SCC-15 as compared with BJ cells at low 1.25-10 µM concentration range and their action in cancer cells was over 250-fold stronger than nimesulide alone. Conjugates overcome apoptosis resistance and sensitized SCC-15 cells to the apoptotic death independently of COX-2/PGE2 axis. In normal human fibroblasts the same concentrations of G3B31N conjugate were less effective in inhibition of proliferation and induction of apoptosis, as measured by caspase 3/7 activity in a manner depending on increase of PGE2 production by either COX-1/COX-2.

Evaluation of the localization and biological effects of PAMAM G3 dendrimer-biotin/pyridoxal conjugate as HaCaT keratinocyte targeted nanocarrier.

Recognition of the molecular mechanisms of keratinocyte participation in normal skin homeostasis and in pathogenesis may lead to creation of more effective tools for topical application of cosmetics, cosmeceutics and drugs to a particular location within the skin for prevention and therapy of many skin disorders and diseases. For this purpose, the PAMAM G3 dendrimer with amide linkages of 9 biotin molecules and 10 molecules of pyridoxal phosphate (BC-PAMAM) was constructed, and its biological properties and cellular uptake and localization were investigated in the HaCaT keratinocytes. BC-PAMAM is nontoxic for HaCaT cells, as estimated by two assays (Neutral Red and tetrazolium salt reduction, XTT), and revealed low apoptosis induction at up to 50 µM concentration. Fluorescent labeled BC-PAMAM accumulates in HaCaT cells with high efficiency in a concentration-dependent manner. Its mitochondrial localization, estimated as Mander's colocalization coefficient, is substantially lower than the native PAMAM, and that correlates with its cytotoxicity. The only undesirable, but significant inhibitory effect on cell mobility, evaluated by the wound healing test, was observed at 10 µM BC-PAMAM. The important anti-inflammatory action of BC-PAMAM was clearly documented by decreased production of total IL-1α, assayed with an ELISA test with unstimulated and stimulated by bacterial antigens (LPS and GroEL) HaCaT cells. Thus, it is expected that the biotin pyridoxal phosphate conjugated PAMAM may be considered as a potential carrier for safe delivery of vitamins and drugs into the epidermis.