Development of an effective cell penetrating peptide: towards viable approaches to gene delivery and chemotherapy against cancer

Doctor of PhilosophyDepartment of ChemistryStefan H. BossmannCancer is not only the second leading cause of death worldwide, but this disease consists of more than 200 different and unique types of cancer, making it a major challenge to develop a cancer treatment that could be effective against multiple cancer types. Multiple alternatives for cancer treatment have been exploited, and the use of therapeutic peptides (molecules capable of acting as neurotransmitters, hormones, ion channel ligands, or anti-infective/anti-cancer drugs) is among the most promising approaches. For this reason, our study focuses on developing a peptide that could be used as a nanocarrier to reach cancerous cells in a targeted manner. Here, a peptide was modified by replacing an amino acid at a specific location that would modify the peptide structure that could facilitate cellular uptake. Four peptides containing microtube-associated sequences (MTAS) and/or nuclear localization signals (NLS) were modified and synthesized following the solid phase peptide synthesis (SPPS) protocol. After cellular uptake experiments, WTAS peptide (MTAS segment containing an amino acid replaced with Trp “W”), resulted in the most effective cell penetrating peptide. Fixed and live confocal studies demonstrated that WTAS was able to penetrate cells within a couple seconds after exposure, and it was further transported to the cell nucleus in the GL26 cancerous cell within a few minutes after penetrating the cell. Interestingly enough, WTAS seemed to lose its ability to penetrate the cell nucleus when it was tested on SIM-A9, a non-cancerous cell line. More studies must be conducted to clearly demonstrate whether WTAS is capable to penetrate cell nuclei in cancer cells only. Furthermore, WTAS was used to develop a second peptide that could be an improved anti-cancer therapeutic peptide. D-SA-K6L9-AS is a highly toxic therapeutic peptide that had been previously synthesized in our research group. For our next approach, following the SPPS procedure, we synthesized a longer version of both peptides, WTAS and D-SA-K6L9-AS to create WTAS-D-SA-K6L9-AS. After successfully synthesizing this peptide, it was characterized by HPLC and MS. Cytotoxicity effects were compared to those of D-SA-K6L9-AS alone on B16F10 and GL26 cell lines, and results demonstrated that toxicity levels did not change after the addition of the WTAS peptide. Also, confocal studies determined that WTAS-D-SA-K6L9-AS still had the ability to target the mitochondria after penetration of the cell and could also reach the cell nucleus in the GL26 cell line. This behavior reflects a unique characteristic common to both peptides. Lastly, fluorescence microscope experiments determined that WTAS-D-SA-K6L9-AS kills cells in the GL26 cell line via the necrosis pathway. WTAS was further used to develop a novel gene delivery nanocarrier composed of WTAS peptide as the primary nanocarrier and poly(β-amino ester) (PBAE) polymer as the secondary nanocarrier. PBAE polymer, a nontoxic and biodegradable polymer, was used to improve the stability of WTAS peptide while facilitating the transportation into cells. After assembling and characterizing the nanocarrier, cell cytotoxicity studies were determined in three cell lines, SIM-A9, B16F10, and GL26. Finally, cell transfection was achieved by utilizing the self-assembling PBAE-WTAS nanocarrier and plasmid DNA genetically modified to express GFP (green fluorescent protein). Results demonstrated effective transfection of the GL26 cell line within 48 hours after loading the cells with the nanocarrier. This nanocarrier can be optimized by including targeting reagents in conjunction with designer plasmids against various diseases

Early detection of non-small cell lung cancer in liquid biopsies by ultrasensitive protease activity analysis

Aim: A significant fraction of mortalities from non-small cell lung cancer could be prevented, if the cancer would be diagnosed earlier. Nanobiosensors for the ultrasensitive detection of active proteases in serum were designed to detect a significant protease activity signature of non-small cell lung cancer (stage I and higher).Methods: We determined the activity of nine protease biomarkers in the sera of non-small cell lung cancer patients and compared them with the protease activities of a control group of healthy human subjects using optical nanobiosensors. They consist of a central Fe/Fe3O4 core/shell nanoparticle with an attached Fluorescence resonance energy transfer-pair [tetrakis-carboxyphenyl porphyrin (TCPP) and cyanine 5.5]. TCPP is attached to the central nanoparticle via a protease-cleavable tether, whereas cyanine 5.5 is tethered permanently to the dopamine-layer surrounding the nanoparticle.Results: Based on the activity pattern of urokinase plasminogen activator, matrix metalloproteinases 1, 2, 3, 7, 9, and 13, and cathepsins B and L as well, non-small cell lung cancer could be detected at stage I by means of a liquid biopsy.Conclusion: This feasibility study, comprising 33 non-small cell lung cancer patients and 20 apparently healthy subjects, clearly demonstrated the feasibility of minimally invasive early diagnosis of non-small cell lung cancer, starting with stage I

Anticancer Activity of Novel Difluorinated Curcumin Analog and Its Inclusion Complex with 2-Hydroxypropyl-β-Cyclodextrin against Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is the primary reason for cancer-related deaths in the US. Genetic mutations, drug resistance, the involvement of multiple signaling pathways, cancer stem cells (CSCs), and desmoplastic stroma, which hinders drug penetrance, contribute to poor chemotherapeutic efficacy. Hence, there is a need to identify novel drugs with improved delivery to improve treatment outcomes. Curcumin is one such compound that can inhibit multiple signaling pathways and CSCs. However, curcumin’s clinical applicability for treating PDAC is limited because of its poor solubility in water and metabolic instability. Hence, we developed a difluorinated curcumin (CDF) analog that accumulates selectively in the pancreas and inhibits PDAC growth in vitro and in vivo. In the present work, we developed its 2-hydroxy-propyl-β-cyclodextrin (HCD) inclusion complex to increase its water solubility and hydrolytic stability. The CDFHCD inclusion complex was characterized by spectroscopic, thermal, and microscopic techniques. The inclusion complex exhibited increased aqueous solubility, hydrolytic stability, and antiproliferative activity compared to parent CDF. Moreover, CDF and CDFHCD inhibited colony and spheroid formation, and induced cell cycle and apoptosis in PDAC cell lines. Hence, CDFHCD self-assembly is an efficient approach to increase water solubility and anticancer therapeutic efficacy, which now warrants advancement towards a clinical proof of concept in PDAC patients

Selective targeting of IRAK1 attenuates low molecular weight hyaluronic acid-induced stemness and non-canonical STAT3 activation in epithelial ovarian cancer

Abstract Advanced epithelial ovarian cancer (EOC) survival rates are dishearteningly low, with ~25% surviving beyond 5 years. Evidence suggests that cancer stem cells contribute to acquired chemoresistance and tumor recurrence. Here, we show that IRAK1 is upregulated in EOC tissues, and enhanced expression correlates with poorer overall survival. Moreover, low molecular weight hyaluronic acid, which is abundant in malignant ascites from patients with advanced EOC, induced IRAK1 phosphorylation leading to STAT3 activation and enhanced spheroid formation. Knockdown of IRAK1 impaired tumor growth in peritoneal disease models, and impaired HA-induced spheroid growth and STAT3 phosphorylation. Finally, we determined that TCS2210, a known inducer of neuronal differentiation in mesenchymal stem cells, is a selective inhibitor of IRAK1. TCS2210 significantly inhibited EOC growth in vitro and in vivo both as monotherapy, and in combination with cisplatin. Collectively, these data demonstrate IRAK1 as a druggable target for EOC