Development of polymeric drug delivery vehicles for targeted cancer therapy

The aim of my Ph. D. thesis is to generalize a method for targeted anti-cancer drug delivery. Hydrophilic polymer-drug conjugates involve complicated synthesis; drug-encapsulated polymeric nanoparticles limit the loading capability of payloads. This thesis introduces the concept of nanoconjugates to overcome difficulties in synthesis and formulation. Drugs with hydroxyl group are able to initiate polyester synthesis in a regio- and chemo- selective way, with the mediation of ligand-tunable Zinc catalyst. Herein, three anti-cancer drugs are presented to demonstrate the high efficiency and selectivity in the method (Chapter 2-4). The obtained particles are stable in salt solution, releasing drugs over weeks in controlled manner. With the conjugation of aptamer, particles are capable to target prostate cancer cells in vitro. These results open the gateway to evaluate the in vivo efficacy of nanoconjugates for target cancer therapy (Chapter 5). Mechanism study of the polymerization leads to the discovery of chemosite selective synthesis of prodrugs with acrylate functional groups. Functional copolymer-drug conjugates will expand the scope of nanoconjugates (Chapter 6). Liposome-aptamer targeting drug delivery vehicle is well studied to achieve reversible cell-specific delivery of non-hydoxyl drugs e.g. cisplatin (Chapter 7). New monomers and polymerization mechanisms are explored for polyester in order to synthesize nanoconjugates with variety on properties (Chapter 8). Initial efforts to apply this type of prodrugs will be focused on the preparation of hydrogels for stem cell research (Chapter 9).

Evaluation of Adoptive T Cell Therapy and Oncolytic Virotherapy for Treatment of Brain Tumors

Adoptive immunotherapy and oncolytic virotherapy are two promising strategies for treating primary and metastatic malignant brain tumors. We demonstrate the ability of adoptively transferred tumor-specific T cells to rapidly mediate the clearance of established brain tumors in several mouse models. Similar to the clinical situation, tumor recurrences are frequent and result from immune editing of tumors. T cells can eliminate antigen-expressing tumor cells but are not effective against antigen loss variant (ALV) cancer cells that multiply and repopulate a tumor. We show that the level of tumor antigen present affects the success of adoptive T cell therapy. When high levels of antigen are present, tumor stromal cells such as microglia and macrophages present tumor peptide on their surface. As a result, T cells directly eliminate cancer cells and cross-presenting stromal cells and indirectly eliminate ALV cells. We were able to show the first direct evidence of tumor antigen cross-presentation by CD11b+ stromal cells in the brain using soluble, high-affinity T cell receptor monomers. Strategies that target brain tumor stroma or increase antigen shedding from tumor cells leading to increased crosspresentation by stromal cells may improve the clinical success of T cell adoptive therapies. We evaluated one potential strategy to complement adoptive T cell therapy by characterizing the oncolytic effects of myxoma virus (MYXV) in a syngeneic mouse brain tumor model of metastatic melanoma. MYXV is a rabbit poxvirus with strict species tropism for European rabbits. MYXV can also infect mouse and human cancer cell lines due to signaling defects in innate antiviral mechanisms and hyperphosphorylation of Akt. MYXV kills B16.SIY melanoma cells in vitro, and intratumoral injection of virus leads to robust, selective and transient infection of the tumor. We observed that virus treatment recruits innate immune cells iii to the tumor, induces TNFα and IFNβ production in the brain, and results in limited oncolytic effects in vivo. To overcome this, we evaluated the safety and efficacy of co-administering 2C T cells, MYXV, and neutralizing antibodies against IFNβ. Mice that received the triple combination therapy survived significantly longer with no apparent side effects, but eventually relapsed. Based on these findings, methods to enhance viral replication in the tumor and limit immune clearance of the virus will be pursued. We conclude that myxoma virus should be further explored as a vector for transient delivery of therapeutic genes to a tumor to enhance T cell responses.

Soybean lunasin mediates colon carcinogenesis by inducing apoptosis and preventing outgrowth of metastasis

Colorectal cancer (CRC) is the third most common cancer worldwide. Various factors such as age, lifestyle and dietary patterns affect the risk of having CRC. Epidemiological studies showed a chemopreventive effect of soy consumption against CRC. However, which component(s) of soybean is associated with this reduced risk is not yet fully delineated. The objective of this research was to evaluate the anti-colon cancer potential of lunasin isolated from defatted soybean flour using in vitro and in vivo models of CRC. Lunasin was isolated from defatted soybean flour by a combination of different chromatographic and ultrafiltration techniques. The anti-colon cancer potential of lunasin was determined using different human colon cancer cell lines in vitro and a CRC liver metastasis model in vivo. Lunasin caused cytotoxicity to different human colon cancer cells with an IC50 value of 13.0, 21.6, 26.3 and 61.7 µM for KM12L4, RKO, HCT-116 and HT-29 human colon cancer cells, respectively. This cytotoxicity correlated with the expression of the α5 integrin on human colon cancer cells with a correlation coefficient of 0.78. The mechanism involved in the cytotoxic effect of lunasin was through cell cycle arrest and induction of the mitochondrial pathway of apoptosis. In KM12L4 human colon cancer cells, lunasin caused a G2/M phase arrest increasing the percentage of cells at G2/M phase from 12% (PBS-treated) to 24% (treated with 10 µM lunasin). This arrest was attributed to the capability of lunasin to increase the expression of cyclin dependent kinase inhibitors p21 and p27. At 10 µM, lunasin increased the expression of p21 and p27 in KM12L4 colon cancer cells by 2.2- and 2.3-fold, respectively. Flow cytometric analysis showed that lunasin at 10 µM increased the percentage of cells undergoing apoptosis from 13.6% to 24.7%. This is further supported by fluorescence microscopic analysis of KM12L4 cells treated with 10 µM lunasin showing chromatin condensation and DNA fragmentation. The mechanism involved is through modification of proteins involved in the mitochondrial pathway of apoptosis in KM12L4 cells as 10 µM lunasin reduced the expression of the anti-apoptotic Bcl-2 protein by 2-fold and increased the expression of the pro-apoptotic proteins Bax, cytochrome c and nuclear clusterin by 2.2-, 2.1- and 2.3- fold, respectively. This led to increased expression and activity of the executioner of apoptosis, caspase-3 by 1.8- and 2.3-fold, respectively. This pro-apoptotic property of lunasin can be attributed to its capability to internalize into the cytoplasm and nucleus of colon cancer cells 24 h and 72 h after treatment, respectively. In addition, lunasin mediated metastasis of colon cancer cells in vitro by inhibiting the focal adhesion kinase activation thereby reducing expression of extracellular regulated kinase and nuclear factor kappa B and finally inhibiting migration of colon cancer cells. In KM12L4 colon cancer cells, 10 µM lunasin resulted in the reduction of phosphorylation of focal adhesion kinase and extracellular regulated kinase by 2.5-fold, resulting in the reduced nuclear translocation of p50 and p65 NF-κB subunits by 3.8- and 1.4-fold, respectively. In an in vivo model of CRC liver metastasis, daily intraperitoneal administration of lunasin at 4 mg/kg body weight resulted in the inhibition of KM12L4 liver metastasis as shown by the reduction of the number of liver metastases from 28 (PBS-treated) to 14 (lunasin-treated, P = 0.047) and reduction in tumor burden as measured by liver weight/body weight from 0.13 (PBS-treated) to 0.10 (lunasin-treated, P = 0.039). Moreover, lunasin potentiated the anti-metastatic effect of the chemotherapeutic drug oxaliplatin given at 5 mg/kg body weight twice per week. Lunasin and oxaliplatin combination resulted in a more potent inhibition of outgrowth of KM12L4 cell metastases to the liver reducing the number of liver metastases by 6-fold and reducing the tumor burden in the liver by 3-fold when compared to PBS-treated group. This can be attributed by the capability of lunasin and oxaliplatin to reduce expression of proliferating cell nuclear antigen in liver-tumor tissue as measured by immunohistochemical staining. The results of this research for the first time demonstrated the anti-colon cancer potential of lunasin isolated from defatted soybean flour which might contribute to the chemopreventive effect of soybean in CRC as seen in different epidemiological studies. In conclusion, lunasin isolated from defatted soybean flour mediated colon carcinogenesis by inducing apoptosis and preventing outgrowth of metastasis. We suggest that the results of this research serve as a basis for further study on the chemopreventive effect of lunasin against CRC and a possible adjuvant role for lunasin in therapy of patients with metastatic CRC.