Emilia Peuhu

Targeting of Porous Hybrid Silica Nanoparticles to Cancer Cells

Mesoporous silica nanoparticles functionalized by surface hyperbranching polymerization of poly(ethylene imine), PEI, were further modified by introducing both fluorescent and targeting moieties, with the aim of specifically targeting cancer cells. Owing to the high abundance of folate receptors in many cancer cells as compared to normal cells, folic acid was used as the targeting ligand. The internalization of the particles in cell lines expressing different levels of folate receptors was studied. Flow cytometry was used to quantify the mean number of nanoparticles internalized per cell. Five times more particles were internalized by cancer cells expressing folate receptors as compared to the normal cells expressing low levels of the receptor. Not only the number of nanoparticles internalized per cell, but also the fraction of cells that had internalized nanoparticles was higher. The total number of particles internalized by the cancer cells was, therefore, about an order of magnitude higher than the total number of particles internalized by normal cells, a difference high enough to be of significant biological importance. In addition, the biospecifically tagged hybrid PEI-silica particles were shown to be noncytotoxic and able to specifically target folate receptor-expressing cancer cells also under coculture conditions.

Targeted intracellular delivery of hydrophobic agents using mesoporous hybrid silica nanoparticles as carrier systems.

Targeted nanoparticle-mediated intracellular delivery is demonstrated using two hydrophobic fluorophores as model drug cargo. The presented hybrid carrier system exhibits both cancer cell-targeting ability and capacity to retain a hydrophobic agent with subsequent specific release into the endosomal compartment. Furthermore, the incorporated agent is shown to be able to escape from the endosomes into the cytoplasm, making the particles promising candidates as carriers for targeted drug delivery for cancer treatment.

Cancer‐Cell‐Specific Induction of Apoptosis Using Mesoporous Silica Nanoparticles as Drug‐Delivery Vectors

Targeted delivery of the chemotherapeutic agent methotrexate (MTX) to cancer cells using poly(ethyleneimine)-functionalized mesoporous silica particles as drug-delivery vectors is reported. Due to its high affinity for folate receptors, the expression of which is elevated in cancer cells, MTX serves as both a targeting ligand and a cytotoxic agent. Enhanced cancer-cell apoptosis (programmed cell death) relative to free MTX is thus observed at particle concentrations where nonspecific MTX-induced apoptosis is not observed in the nontargeted healthy cell line, while corresponding amounts of free drug affect both cell lines equally. The particles remain compartmentalized in endo-/lysosomes during the time of observation (up to 72 h), while the drug is released from the particle only upon cell entry, thereby inducing selective apoptosis in the target cells. As MTX is mainly attached to the particle surface, an additional advantage is that the presented carrier design allows for adsorption (loading) of additional drugs into the pore network for therapies based on a combination of drugs.

The antitumor lignan Nortrachelogenin sensitizes prostate cancer cells to TRAIL-induced cell death by inhibition of the Akt pathway and growth factor signaling

Prostate cancer cells frequently develop resistance toward androgen-deprivation and chemotherapy. To identify new approaches to treat androgen-dependent prostate cancer, we have performed a structure-activity analysis of lignan polyphenols for cancer cell specific sensitization to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand that has ability to induce tumor-specific cell death. In this study, we report that the lignan nortrachelogenin (NTG) is the most efficient of the 27 tested lignan compounds in sensitizing prostate cancer cells to TRAIL-induced apoptosis. Importantly, pretreatment with NTG does not sensitize a non-malignant prostate cell line to TRAIL-induced cell death. The structural comparison of lignans reveals that the dibenzylbutyrolactone skeleton is required for the apoptosis-sensitizing activity, while substitutions at the aromatic rings do not seem to play a critical role in this lignan function. Our study also characterizes the cellular effects and molecular mechanisms involved in NTG anticancer activity. We previously reported that specific lignans inhibit the Akt survival-signaling pathway in concert with TRAIL sensitization. While NTG is also shown to be a effective inhibitor of Akt signaling, in this study we further demonstrate that NTG potently inhibits tyrosine kinase (RTK) activation in response to growth factors, such as insulin and insulin-like growth factor I (IGF-I). Our results identify NTG as a novel agent for prostate cancer therapy with ability to inhibit Akt membrane localization and activity as well as the activation of growth factor receptors (GFRs), thereby efficiently synergizing with TRAIL exposure.

PP2A Inhibitor PME-1 Drives Kinase Inhibitor Resistance in Glioma Cells

Glioblastoma multiforme lacks effective therapy options. Although deregulated kinase pathways are drivers of malignant progression in glioblastoma multiforme, glioma cells exhibit intrinsic resistance toward many kinase inhibitors, and the molecular basis of this resistance remains poorly understood. Here, we show that overexpression of the protein phosphatase 2A (PP2A) inhibitor protein PME-1 drives resistance of glioma cells to various multikinase inhibitors. The PME-1-elicited resistance was dependent on specific PP2A complexes and was mediated by a decrease in cytoplasmic HDAC4 activity. Importantly, both PME-1 and HDAC4 associated with human glioma progression, supporting clinical relevance of the identified mechanism. Synthetic lethality induced by both PME-1 and HDAC4 inhibition was dependent on the coexpression of proapoptotic protein BAD. Thus, PME-1-mediated PP2A inhibition is a novel mechanistic explanation for multikinase inhibitor resistance in glioma cells. Clinically, these results may inform patient stratification strategies for future clinical trials with selected kinase inhibitors in glioblastoma multiforme. Cancer Res; 76(23); 7001-11. ©2016 AACR.

Novel action modality of the diterpenoid anisomelic acid causes depletion of E6 and E7 viral oncoproteins in HPV-transformed cervical carcinoma cells.

Cervical cancer, the second most common malignancy among women, is mainly caused by human papilloma virus (HPV) infection. In HPV-positive cervical cancer cells, the activity of p53 and the induction of p21 are inhibited by the HPV oncoproteins E6 and E7. Therefore, blocking the activity of E6 and E7 would serve as an important therapeutic target in these cancer cells. In this study, anisomelic acid (AA), a natural compound belonging to the same diterpenoid family of bioactive compounds as taxol, was found to deplete the E6 and E7 proteins in HPV-positive cervical cancer cells. Consequently, p53 and the p53-responsive gene, p21, were dramatically induced, leading to G2/M-phase cell cycle arrest. AA-mediated cell cycle arrest and p21 expression were canceled when p53 was down-regulated by p53-shRNA. AA also induced p53-independent intrinsic apoptosis by depletion of the cellular inhibitor of apoptosis protein 2 (cIAP2) whose proteosomal degradation is inhibited by E6. The in ovo chick embryo chorioallantoic membrane (CAM) assay showed that anisomelic acid inhibited the tumor growth of the cervical cancer SiHa cells. AA is revealed to hold a novel action modality based on specific targeting of the HPV oncoproteins, which restores p53-mediated growth arrest and induces apoptosis by terminating E6-mediated cIAP2 stabilization.

Molecular targets for the protodynamic action of cis-urocanic acid in human bladder carcinoma cells

Backgroundcis-urocanic acid (cis-UCA) is an endogenous amino acid metabolite capable of transporting protons from the mildly acidic extracellular medium into the cell cytosol. The resulting intracellular acidification suppresses many cellular activities. The current study was aimed at characterizing the molecular mechanisms underlying cis-UCA-mediated cytotoxicity in cultured cancer cells.Methods5367 bladder carcinoma cells were left untreated or treated with cis-UCA. Cell death was assessed by measuring caspase-3 activity, mitochondrial membrane polarization, formation and release of cytoplasmic histone-associated DNA fragments, and cellular permeabilization. Cell viability and metabolic activity were monitored by colorimetric assays. Nuclear labelling was used to quantify the effects of cis-UCA on cell cycle. The activity of the ERK and JNK signalling pathways was studied by immunoblotting with specific antibodies. Phosphatase activity in cis-UCA-treated cells was determined by assay kits measuring absorbance resulting from the dephosphorylation of an artificial substrate. All statistical analyses were performed using the two-way Students t-test (p < 0.05).ResultsHere we report that treatment of the 5637 human bladder carcinoma cells with 2% cis-UCA induces both apoptotic and necrotic cell death. In addition, metabolic activity of the 5637 cells is rapidly impaired, and the cells arrest in cell cycle in response to cis-UCA. Importantly, we show that cis-UCA promotes the ERK and JNK signalling pathways by efficiently inhibiting the activity of serine/threonine and tyrosine phosphatases.ConclusionsOur studies elucidate how cis-UCA modulates several cellular processes, thereby inhibiting the proliferation and survival of bladder carcinoma cells. These anti-cancer effects make cis-UCA a potential candidate for the treatment of non-muscle invasive bladder carcinoma.

Phosphopeptide enrichment with stable spatial coordination on a titanium dioxide coated glass slide

Recent advances in phosphoproteomics have established powerful tools to analyze phosphorylation events. However, their spatial localization is lost due to sample homogenization procedures prior to the analysis. Imaging mass spectrometry (IMS) has emerged as a method to visualize the spatial distribution of molecules in tissue samples, but its application is still limited to relatively abundant molecules. Due to low phosphorylation stoichiometry, direct detection and imaging of protein phosphorylation by MS has not been achieved yet. Therefore we have developed a novel phosphopeptide enrichment strategy as a potential tool for in situ affinity imaging MS (AIMS). A specific type of titanium dioxide (TiO2)-coated glass slides was designed and validated with casein tryptic digests for their ability to selectively retain phosphopeptides while maintaining their spatial coordination.