Background: Small cell lung and pancreatic cancer are two of the most lethal malignancies for which current therapeutic options involve combinations of cytotoxic chemotherapies. In both cases, the treatment results in only marginal survival advantage as compared to mono-therapies. Toxicity due to off-target effects and the development of resistance have compromised the effectiveness of chemotherapies. Hence, there is an urgent need for better-targeted approaches. The neuropeptide, gastrin-releasing peptide (GRP) and its receptor (GRPR) are implicated in an autocrine/paracrine growth factor loop to sustain the tumourigenesis of SCLC and PC. In addition, carbonic anhydrase IX (CAIX) is increasingly upregulated in SCLC and PC in response to hypoxia to help maintain the intracellular pH for cell survival. Thus, it was hypothesised that targeting these proteins represents a new therapy for both these cancers. This project investigated the action of Bu peptide and ureidosulfamate S4 to target GRPR and CAIX expressing cells, respectively and tested the feasibility of developing liposomal drug formulations to aid delivery. Approach: Bu peptide was made by new solid phase peptide synthesis (SPPS) approach. GRPR targeting bombesin, conjugated to carboxyfluorescein (CF-BBN) was used to study GRPR dependent peptide uptake. Liposomal formulations of S4 and Bu- peptide were prepared by thin film hydration method and characterised. Correlation of GRPR expression with the efficacy of the Bu-peptide against a number of SCLC and PC cell lines was investigated using western blotting, flow cytometry and CellTiter-Glo assays respectively. Scratch assays were used to assess the effect of the Bu peptide on cell migration. Cytotoxic action of the Bu peptide was studied and compared to conventional chemotherapeutic agents (etoposide and cisplatin). The expression of CAIX and sensitivity and effect on migration to CAIX inhibitor was assessed under normoxia and hypoxia (0.1% O2). Finally, to study the effect of the Bu peptide and S4 combination, viability and apoptosis were tested in selected responsive cell lines expressing both targets. Results: SPPS method was successfully developed yielding Bu peptide with good purity illustrating the final product withstood harsh HF cleavage conditions. S4 and Bu peptide were incorporated into PEGylated liposomes displaying acceptable characteristics in terms of size, morphology, storage stability and a slow drug release profile. The expression level of the GRPR protein strongly correlated with the cellular uptake of BBN-CF and to the sensitivity of all cell lines studied. Bu-peptide also significantly inhibited the migration of PC cell lines. Bu-peptide reduced cell viability by apoptosis as confirmed by the presence of DNA fragmentation (TUNEL) assay. Furthermore, Bu peptide induced apoptosis through caspase 3/7 activation. DMS 79 cells appeared to be sensitive to treatment with the Bu-peptide despite being relatively resistant to conventional chemotherapeutic agents namely, etoposide and cisplatin. Results also showed that cell lines expressed CAIX protein under normoxia and expression was upregulated under hypoxia. Cell line expressing CAIX exhibited cytotoxicity against Bu peptide and/or S4 in a concentration- and time-dependent manner with effects being more pronounced under normoxia. S4 had also significantly reduced cell migration especially under hypoxia. In addition, S4 inhibited cell growth via apoptosis and with higher levels of cell death being observed under normoxia, which correlated with the reduced cell viability mentioned above. The dual targeting action of S4 and Bu peptide on representative SCLC and PC cell lines indicated that this drug combination has strong synergistic effect to further decrease cell viability and increase apoptosis compared to single drug treatment. Conclusion: Targeting neuropeptide growth loops with Bu-peptide has therapeutic potential to treat SCLC and PC in cells express
Development of Nanoscale Drug Delivery System for Treatment of Cancers Expressing GRPR and CAIX Targets
Alghamdi, A. (Author). 31 Aug 2021
Student thesis: Phd