Synthesis and Properties of α-Mangostin and Vadimezan Conjugates with Glucoheptoamidated and Biotinylated 3rd Generation Poly(amidoamine) Dendrimer, and Conjugation Effect on Their Anticancer and Anti-Nematode Activities.

α-Mangostin and vadimezan are widely studied potential anticancer agents. Their biological activities may be improved by covalent bonding by amide or ester bonds with the third generation poly(amidoamine) (PAMAM) dendrimer, substituted with α-D-glucoheptono-1,4-lactone and biotin. Thus, conjugates of either ester- (G3gh4B5V) or amide-linked (G32B12gh5V) vadimezan, and equivalents of α-mangostin (G3gh2B5M and G32B12gh5M, respectively), were synthesized, characterized and tested in vitro against cancer cells: U-118 MG glioma, SCC-15 squamous carcinoma, and BJ normal human fibroblasts growth, as well as against C. elegans development. α-Mangostin cytotoxicity, stronger than that of Vadimezan, was increased (by 2.5-9-fold) by conjugation with the PAMAM dendrimer (with the amide-linking being slightly more effective), and the strongest effect was observed with SCC-15 cells. Similar enhancement of toxicity resulting from the drug conjugation was observed with C. elegans. Vadimezan (up to 200 µM), as well as both its dendrimer conjugates, was not toxic against both the studied cells and nematodes. It showed an antiproliferative effect against cancer cells at concentrations ≥100 µM. This effect was significantly enhanced after conjugation of the drug with the dendrimer via the amide, but not the ester bond, with G32B12gh5V inhibiting the proliferation of SCC-15 and U-118 MG cells at concentrations ≥4 and ≥12 µM, respectively, without a visible effect in normal BJ cells. Thus, the drug delivery system based on the PAMAM G3 dendrimer containing amide bonds, partially-blocked amino groups on the surface, larger particle diameter and higher zeta potential can be a useful tool to improve the biological properties of transported drug molecules.

Synthesis of Biotinylated PAMAM G3 Dendrimers Substituted with R-Glycidol and Celecoxib/Simvastatin as Repurposed Drugs and Evaluation of Their Increased Additive Cytotoxicity for Cancer Cell Lines.

Recent achievement in anticancer therapy considers the application of repurposed drugs in optimal combinations with the use of specific carriers for their targeted delivery. As a result, new optimized medications with reduced side effects can be obtained. In this study, two known anticancer drugs, celecoxib and/or simvastatin, were conjugated covalently with PAMAM G3 dendrimer and tested in vitro against human squamous carcinoma (SCC-15-15) and glioblastoma (U-118 MG) cells, as well as normal human fibroblasts (BJ). The obtained conjugates were also substituted with biotin and R-glycidol to increase their affinity for cancer cells and were characterized with NMR spectroscopy and dynamic light scattering technique. Conjugates furnished with two celecoxib and four simvastatin residues revealed the very high effectiveness and dramatically decreased the SCC-15 and U-118 MG cell viability at very low concentrations with IC50 equal to about 3 µM. Its action was 20-50-fold stronger than that of either drug alone or as a mixture. Combined conjugate revealed also additive action since it was 2-8-fold more effective than conjugates with either single drug. The combined conjugate revealed rather low specificity since it was also highly cytotoxic for BJ cells. Despite this, it may be concluded that biotinylated and R-glycidylated PAMAM G3 dendrimers substituted with both celecoxib and simvastatin can be considered as a new perspective anticancer agent, effective in therapy of malignant, incurable glioblastomas.

Biotin Transport-Targeting Polysaccharide-Modified PAMAM G3 Dendrimer as System Delivering α-Mangostin into Cancer Cells and C. elegans Worms.

The natural xanthone α-mangostin (αM) exhibits a wide range of pharmacological activities, including antineoplastic and anti-nematode properties, but low water solubility and poor selectivity of the drug prevent its potential clinical use. Therefore, the targeted third-generation poly(amidoamine) dendrimer (PAMAM G3) delivery system was proposed, based on hyperbranched polymer showing good solubility, high biocompatibility and low immunogenicity. A multifunctional nanocarrier was prepared by attaching αM to the surface amine groups of dendrimer via amide bond in the ratio 5 (G32B12gh5M) or 17 (G32B10gh17M) residues per one dendrimer molecule. Twelve or ten remaining amine groups were modified by conjugation with D-glucoheptono-1,4-lactone (gh) to block the amine groups, and two biotin (B) residues as targeting moieties. The biological activity of the obtained conjugates was studied in vitro on glioma U-118 MG and squamous cell carcinoma SCC-15 cancer cells compared to normal fibroblasts (BJ), and in vivo on a model organism Caenorhabditis elegans. Dendrimer vehicle G32B12gh at concentrations up to 20 µM showed no anti-proliferative effect against tested cell lines, with a feeble cytotoxicity of the highest concentration seen only with SCC-15 cells. The attachment of αM to the vehicle significantly increased cytotoxic effect of the drug, even by 4- and 25-fold for G32B12gh5M and G32B10gh17M, respectively. A stronger inhibition of cells viability and influence on other metabolic parameters (proliferation, adhesion, ATP level and Caspase-3/7 activity) was observed for G32B10gh17M than for G32B12gh5M. Both bioconjugates were internalized efficiently into the cells. Similarly, the attachment of αM to the dendrimer vehicle increased its toxicity for C. elegans. Thus, the proposed α-mangostin delivery system allowed the drug to be more effective in the dendrimer-bound as compared to free state against both cultured the cancer cells and model organism, suggesting that this treatment is promising for anticancer as well as anti-nematode chemotherapy.

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.

Alvaxanthone, a Thymidylate Synthase Inhibitor with Nematocidal and Tumoricidal Activities.

With the aim to identify novel inhibitors of parasitic nematode thymidylate synthase (TS), we screened in silico an in-house library of natural compounds, taking advantage of a model of nematode TS three-dimensional (3D) structure and choosing candidate compounds potentially capable of enzyme binding/inhibition. Selected compounds were tested as (i) inhibitors of the reaction catalyzed by TSs of different species, (ii) agents toxic to a nematode parasite model (C. elegans grown in vitro), (iii) inhibitors of normal human cell growth, and (iv) antitumor agents affecting human tumor cells grown in vitro. The results pointed to alvaxanthone as a relatively strong TS inhibitor that causes C. elegans population growth reduction with nematocidal potency similar to the anthelmintic drug mebendazole. Alvaxanthone also demonstrated an antiproliferative effect in tumor cells, associated with a selective toxicity against mitochondria observed in cancer cells compared to normal cells.

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.

Antitumor and anti-nematode activities of α-mangostin.

α-Mangostin, one of the major xanthones isolated from pericarp of mangosteen (Garcinia mangostana Linn), exhibits a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial as well as anticancer, both in in vitro and in vivo studies. In the present study, α-mangostin' anti-cancer and anti-parasitic properties were tested in vitro against three human cell lines, including squamous carcinoma (SCC-15) and glioblastoma multiforme (U-118 MG), compared to normal skin fibroblasts (BJ), and in vivo against Caenorhabditis elegans. The drug showed cytotoxic activity, manifested by decrease of cell viability, inhibition of proliferation, induction of apoptosis and reduction of adhesion at concentrations lower than 10 µM (the IC50 values were 6.43, 9.59 and 8.97 µM for SCC-15, U-118 MG and BJ, respectively). The toxicity, causing cell membrane disruption and mitochondria impairment, was selective against squamous carcinoma with regard to normal cells. Moreover, for the first time anti-nematode activity of α-mangostin toward C. elegans was described (the LC50 = 3.8 ±â€¯0.5 µM), with similar effect exerted by mebendazole, a well-known anthelmintic drug.

Biotinylated PAMAM G3 dendrimer conjugated with celecoxib and/or Fmoc-l-Leucine and its cytotoxicity for normal and cancer human cell lines.

Tumors still remain one of the main causes of mortality due to the lack of effective anti-cancer therapy. Recently it has been shown, that overexpression of inducible cyclooxygenase-2 (COX-2) and decrease of peroxisome proliferator-activated receptor γ (PPARγ) expression accompany many malignances, therefore, it has been proposed, that COX-2 inhibitors and PPARγ agonists are potential candidates for anticancer therapy and their synergistic, antineoplastic action has been described. In the present study a COX-2 inhibitor (celecoxib) and/or PPARγ agonist (Fmoc-l-Leucine) were conjugated with the biotinylated G3 PAMAM dendrimer to form a three different constructs targeted to cells with increased biotin uptake. All conjugates were characterized by the NMR spectroscopy. Investigation of three types of human cells: normal skin fibroblasts (BJ), immortalized keratinocytes (HaCaT) and cancer lines: glioblastoma (U-118 MG) and squamous cell carcinoma (SCC-15) revealed similar biotin labeled ATTO590 accumulation (after 24 h), except for SCC-15 with significantly lower loading. Constitutive expression of COX-2 protein was confirmed in all tested cells with significantly higher levels (2-2.5 times) in both cancer lines. Comparison of cytotoxicity of the new synthetized dendrimers clearly documented the highest cytotoxicity of the G31B16C15L dendrimer conjugated with both drugs (1: 1) as compared with drugs alone and single conjugates. Additive effects of construct with both compounds were shown for fibroblasts and both cancer cell lines in the order BJ > U-118 MG > SCC-15 with IC50 in the range: 0.69, 1.44 and 2.22 µM, respectively and lowest cytotoxicity in HaCaT cells (IC50 = 2.88). Our results showed, that biotinylated G3 PAMAM dendrimers substituted with COX-2 inhibitor, celecoxib, and PPARγ agonist, Fmoc-l-Leucine (1:1) may be a good candidate for local therapy of glioblastoma but not a skin cancer. Since the effect of PPARγ agonists on COX-2 expression vary depending upon the cell type, specificity of used agonist and the presence of other environmental factors, it is necessary to carefully evaluate the response of chosen drugs on the target cells.