Sonia Aroui

Cytotoxicity, intracellular distribution and uptake of doxorubicin and doxorubicin coupled to cell-penetrating peptides in different cell lines: a comparative study.

One of the major obstacles which are opposed to the success of anticancer treatment is the cell resistance that generally develops after administration of commonly used drugs. In this study, we try to overcome the tumour cell resistance of doxorubicin (Dox) by developing a cell-penetrating peptide (CPP)-anticancer drug conjugate in aim to enhance its intracellular delivery and that its therapeutic effects. For this purpose, two cell-penetrating peptides, penetratin (pene) and tat, derived from the HIV-1 TAT protein, were chemically conjugated to Dox. The cytotoxicity, intracellular distribution and uptake were accessed in CHO cells (Chinese Hamster Ovarian carcinoma cells), HUVEC (Human Umbilical Vein Endothelial Cells), differentiated NG108.15 neuronal cell and breast cancer cells MCF7drug-sensitive or MDA-MB 231 drug-resistant cell lines. The conjugates showed different cell killing activity and intracellular distribution pattern by comparison to Dox as assessed respectively by MTT-based colorimetric cellular cytotoxicity assay, confocal fluorescence microscopy and FACS analysis. After treatment with 3 microM with Dox-CPPs for 2h, pene increase the Dox cytotoxicity by 7.19-fold in CHO cells, by 11.53-fold in HUVEC cells and by 4.87-fold in MDA-MB 231 cells. However, cytotoxicity was decreased in NG108.15 cells and MCF7. Our CPPs-Dox conjugate proves the validity of CPPs for the cytoplasmic delivery of therapeutically useful molecules and also a valuable strategy to overcome drug resistance.

Direct Peptide Interaction with Surface Glycosaminoglycans Contributes to the Cell Penetration of Maurocalcine

Maurocalcine (MCa), initially identified from a tunisian scorpion venom, defines a new member of the family of cell penetrating peptides by its ability to efficiently cross the plasma membrane. The initiating mechanistic step required for the cell translocation of a cell penetrating peptide implicates its binding onto cell surface components such as membrane lipids and/or heparan sulfate proteoglycans. Here we characterized the interaction of wild-type MCa and MCa K20A, a mutant analogue with reduced cell-penetration efficiency, with heparin (HP) and heparan sulfates (HS) through surface plasma resonance. HP and HS bind both to MCa, indicating that heparan sulfate proteoglycans may represent an important entry route of the peptide. This is confirmed by the fact that (i) both compounds bind with reduced affinity to MCa K20A and (ii) the cell penetration of wild-type or mutant MCa coupled to fluorescent streptavidin is reduced by about 50% in mutant Chinese hamster ovary cell lines lacking either all glycosaminoglycans (GAGs) or just HS. Incubating MCa with soluble HS, HP, or chondroitin sulfates also inhibits the cell penetration of MCa-streptavidin complexes. Analyses of the cell distributions of MCa/streptavidin in several Chinese hamster ovary cell lines show that the distribution of the complex coincides with the endosomal marker Lyso-Tracker red and is not affected by the absence of GAGs. The distribution of MCa/streptavidin is not coincident with that of transferrin receptors nor affected by a dominant-negative dynamin 2 K44A mutant, an inhibitor of clathrin-mediated endocytosis. However, entry of the complex is greatly diminished by amiloride, indicating the importance of macropinocytosis in MCa/streptavidin entry. It is concluded that (i) interaction of MCa with GAGs quantitatively improves the cell penetration of MCa, and (ii) GAG-dependent and -independent MCa penetration rely similarly on the macropinocytosis pathway.

Design of a disulfide-less, pharmacologically inert, and chemically competent analog of maurocalcine for the efficient transport of impermeant compounds into cells

Maurocalcine is a 33-mer peptide initially isolated from the venom of a Tunisian scorpion. It has proved itself valuable as a pharmacological activator of the ryanodine receptor and has helped the understanding of the molecular basis underlying excitation-contraction coupling in skeletal muscles. Because of its positively charged nature, it is also an innovative vector for the cell penetration of various compounds. We report a novel maurocalcine analog with improved properties: (i) the complete loss of pharmacological activity, (ii) preservation of the potent ability to carry cargo molecules into cells, and (iii) coupling chemistries not affected by the presence of internal cysteine residues of maurocalcine. We did this by replacing the six internal cysteine residues of maurocalcine by isosteric 2-aminobutyric acid residues and by adding an additional N-terminal biotinylated lysine (for a proof of concept analog) or an N-terminal cysteine residue (for a chemically competent coupling analogue). Additional replacement of a glutamate residue by alanyl at position 12 further improves the potency of these analogues. Coupling to several cargo molecules or nanoparticles are presented to illustrate the cell penetration potency and usefulness of these pharmacologically inactive analogs.

Doxorubicin coupled to penetratin promotes apoptosis in CHO cells by a mechanism involving c-Jun NH2-terminal kinase

Doxorubicin (Dox) has demonstrated potent activity in treating malignant lymphomas but its therapeutic efficacy is hampered by induction of cardiotoxicity. This side effect is related to the ability of the drug to generate reactive oxygen species in cells. Previously, we demonstrated that coupling Dox to penetratin (Pen), a cell penetrating peptide, represent a valuable strategy to overcome drug resistance in CHO cells. In the present study, we evaluated the consequences of the conjugation of Dox to Pen in term of apoptosis induction. When tested on CHO cells, Dox-Pen generated a typical apoptotic phenotype but at lower dose that needed for unconjugated Dox. Cell death induction was associated with chromatin condensation, caspase activation, Bax oligomerisation and release of cytochrome c. By using reactive oxygen species and c-jun NH2-terminal kinase (JNK) inhibitors, we prevented Dox- and Dox-Pen-induced CHO cell death. The chimeric soluble DR5 receptor that inhibits TRAIL induced cell death does not prevent Dox or Dox-Pen-induced cytotoxicity. These observations indicate that conjugation of Dox to cell penetrating peptide does not impair the ability of the drug to trigger cell death through activation of the intrinsic pathway involving c-Jun NH2-terminal kinase but could exhibit less toxic side effects and could warrant its use in clinic.

Naringin inhibits the invasion and migration of human glioblastoma cell via downregulation of MMP-2 and MMP-9 expression and inactivation of p38 signaling pathway

Gliomas are the most common and malignant primary brain tumors. They are associated with a poor prognosis despite the availability of multiple therapeutic options. Naringin, a common dietary flavonoid abundantly present in fruits and vegetables, is believed to possess strong anti-proliferative and anti-cancer properties. However, there are no reports describing its effects on the invasion and migration of glioblastoma cell lines. Our results showed that the treatment of U251 glioma cell lines with different concentrations of naringin inhibited the invasion and migration of these cells. In addition, we revealed a decrease in the levels of matrix metalloproteinases (MMP-2) and (MMP-9) expression as well as proteinase activity in U251 glioma cells. In contrast, the expression of tissue inhibitor of metalloproteinases (TIMP-1) and (TIMP-2) was increased. Furthermore, naringin treatment decreased significantly the phosphorylated level of p38. Combined treatment with a p38 inhibitor (SB203580) resulted in the synergistic reduction of MMP-2 and MMP-9 expressions correlated with an increase of TIMP-1 and TIMP-2 expressions and the anti-invasive properties. However, p38 chemical activator (anisomycin) could block these effects produced by naringin, suggesting a direct downregulation of the p38 signaling pathway. These data suggest that naringin may have therapeutic potential for controlling invasiveness of malignant gliomas by inhibiting of p38 signal transduction pathways.

Involvement of C-jun NH2-terminal kinase and apoptosis induced factor in apoptosis induced by deglycosylated bleomycin in laryngeal carcinoma cells.

In our previous studies, we demonstrated that the deglycosylation of bleomycin‐A2 (BLM‐A2) does not affect the capacity of this drug to induce cell death by apoptosis in a caspase‐independent manner in laryngeal cancer cells (HEp‐2), but suppresses the ability of BLM‐A2 to induce ROS formation. We have now investigated the consequence of BLM‐A2 deglycosylation in terms of the involvement of apoptotic pathways in HEp‐2 cells. Apoptosis induced by bleomycin‐A2 and deglyco‐BLM‐A2 is associated with the release of cytochrome c and AIF. Only Bax was oligomerized with BLM‐A2‐induced HEp‐2 cell death. BLM‐A2 and deglyco‐BLM‐A2‐induced apoptosis depended on JNK activation but was independent of death receptors expression. In contrast, both of these drugs would sensitize HEp‐2 cells to death receptor ligand‐induced cell death. These observations indicate that the deglycosylation of BLM does not impair the ability of the drug to trigger cell death through activation of the intrinsic pathway by the release of AIF responsible for mitochondrial permeability and chromatin condensation independent of caspases activation.

PTEN-regulated AKT/FoxO3a/Bim signaling contributes to Human cell glioblastoma apoptosis by platinum-maurocalcin conjugate

A previous report has shown that a chimera between a platinum complexing agent (1) and the cell penetrating peptide maurocalcin, synthesized with D-amino acids, (DMCa), termed Pt-1-DMCa, is a highly successful anticancer compound that works by targeting the intracellular redox system in glioblastoma (GBM) cells. However, the detailed cellular mechanism whereby the conjugate specifically kills tumor cells remains unclear. Herein, we show that Pt-1-DMCa induces apoptosis in Human U87 GBM cells through reactive oxygen species (ROS)-dependent modulation of the PI3K/AKT/FoxO3a signalling pathway. First, we found that Pt-1-DMCa treatment of these cells induces inhibition of AKT and nuclear accumulation of FoxO3a thereby facilitating transcription of the target genes Bim and PTEN. Modulation of the AKT/FoxO3a/Bim signaling pathway by RNA interference confirms that these signaling events are critical for Pt-1-DMCa-induced apoptosis of U87 GBM cells. Furthermore, we reveal that FoxO3a-mediated up-regulation of PTEN exerts an additional inhibitory effect on the AKT survival pathway. Thus, our results demonstrate that the conjugate can induce ROS-dependent FoxO3a-mediated apoptosis in U87 cells through PTEN-mediated inhibition of the PI3K/AKT survival axis. Our results help elucidate the molecular mechanisms underlying Pt-1-DMCa-induced cell death in U87 GBM cells and support a theoretical basis for future applications of the MCa peptide.

Anticancer properties of lipid and poly(ε-caprolactone) nanocapsules loaded with ferrocenyl-tamoxifen derivatives

We synthesized new tamoxifen derivatives as anticancer drug candidates and elaborated on convection‐enhanced delivery (CED) as a strategy for delivery.

The combination of Bleomycin with TRAIL agonists or PKC inhibitors sensitizes solid tumor cells to BLM-mediated apoptosis: new strategies to overcome chemotherapy resistance of tumors

In this study we evaluated the effects of low dose of bleomycin in an associative treatment strategy in solid tumor cells. For this purpose, Human and murine colon cancer (SW480, HCT8, and CT26), and murine melanoma (B16-F10) cells were treated with different agents including protein kinase C, and c-jun NH2-terminal kinase inhibitors, and tumor necrosis factor-related apoptosis-inducing ligand. Apoptosis was identified by morphological criteria. Reactive oxygen species are evaluated by flow cytometry. Our data showed that bleomycin (100 µM) induced apoptosis in all the four cell lines tested with a level ranging from 30 to 60%. However, at lower dose (25 µM), bleomycin was less efficient to trigger apoptosis. In contrast, when bleomycin (25 µM) was combined with the protein kinase C inhibitor chelerythrine, or tumor necrosis factor-related apoptosis-inducing ligand, it elicited more apoptotic cell death ranging from 40 to 75%, depending on the cell type, whereas when it was associated with the c-jun NH2-terminal kinase inhibitor SP600125, bleomycin displayed different cell death responses. If bleomycin and SP600125 enhanced apoptosis in two colon cancer cells, HCT-8, and CT26, they reduced to 50% apoptosis in the melanoma B16-F10 cells, and were not synergistic in the human colon cancer cells, SW480. This synergism seemed to rely partially to reactive oxygen species, because N-acetyl cysteine inhibited apoptosis in some cells and with some agents. These findings indicate that tumor necrosis factor-related apoptosis-inducing ligand, and protein kinase C inhibition can represent candidates for improved cancer chemotherapy.