Development of drug conjugates in cancer therapy and evaluation of dual siRNA silencing effect on breast cancer growth and invasion

Abstract

The objective of this dissertation is to present various approaches for treatment cancer, which is the leading cause of death worldwide. Compared to other disease, cancer has many unique biological characteristics that can be exploited for its therapy. In chapter 1 and 2, its molecular characteristics and microenvironments, as well as the corresponding therapeutic strategies, are summarized. In chapter 3, we developed a peptide drug conjugate to specifically deliver TGX-221 to HER2 overexpressed prostate cancer cells. TGX-221 is a highly potent phosphoinositide 3- kinases β (PI3Kβ) inhibitor that holds great promise as a novel chemotherapy agent for prostate cancer. However, poor solubility and lack of targetability limit its therapeutic applications. The peptide drug conjugate was proven to be gradually cleaved by PSA to release TGX-D1 (TGX- 221 analogue). Both the peptide drug conjugate and its cleaved products demonstrate a comparable activity to the parent drug, TGX-D1. Moreover, cellular uptake of the peptide drug conjugate and its cleaved product SL-TGX were significantly higher in prostate cancer cells compared to the parent drug. The high cellular uptake of dipeptide drug conjugate SL-TGX might be mediated by peptide transporters in prostate cancer cells. However, the expression of peptide transporters in prostate cancer cell lines has not been reported before. Therefore, in Chapter 4, the expression profile and functional activity of peptide transporters were investigated in the prostate cancer cell lines LNCaP, PC-3 and DU145. Peptide transporter 1 (PEPT1) is found overexpressed in PC-3 cells, and peptide transporter 2 (PEPT2) is upregulated in LNCaP cells. We also developed another approach to enhance water solubility and targetability of hydrophobic drugs. In Chapter 5, we developed a polymer-rapamycin conjugate using a novel, linear and PEG based multiblock copolymer (Mw ~ 32 kDa). Rapamycin has demonstrated potent anti-tumor activity in preclinical and clinical studies. However, the clinical development of its formulations has been hampered due to its poor solubility and undesirable distribution in vivo. The polymer-rapamycin conjugate provided enhanced solubility in water compared with free rapamycin and shows a profound activity against a panel of human cancer cell lines. This polymer-rapamycin conjugate also presented high drug loading capacity (wt% ~ 28%) when GlyGlyGly was used as a linker. The uptake study further indicated that the lysosome is the major site of intracellular localization of polymer drug conjugate. Thus, these preclinical data suggested that polymer rapamycin conjugate is a novel anti-cancer agent that holds great promising for treatment of a wide variety of tumors. Macromolecules such as siRNA can also be used as anticancer drugs. In chapter 6, we designed nine HER2 siRNAs and ten VEGF siRNAs and identified potent siRNA that can silence the target gene up to 75-83%. The most potent HER2 and VEGF siRNAs were used to conduct functional studies in HER2 positive breast cancer cells. Combination of HER2 and VEGF siRNAs demonstrated synergistic silencing effect on VEGF. Both HER2 siRNA and VEGF siRNA showed significant inhibition on cell migration and proliferation. HER2 siRNA also demonstrated dramatic suppression of cell spreading and adhesion to ECM, as well as induction of apoptosis. Dual silencing of HER2 and VEGF led to significant cell morphology change and substantial suppression on migration, spreading, cell adhesion, and proliferation.