HEAVY CHAIN FERRITIN SIRNA DELIVERED BY CATIONIC LIPOSOMES ENHANCES RADIATION AND CHEMOTHERAPEUTIC EFFICACY FOR TREATMENT OF GLIOMA
Open Access
- Author:
- Liu, Xiaoli
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 25, 2010
- Committee Members:
- James Robert Connor, Dissertation Advisor/Co-Advisor
James Robert Connor, Committee Chair/Co-Chair
Ira Joseph Ropson, Committee Member
Kathryn F Lanoue, Committee Member
Achuthamangalam B Madhankumar, Committee Member
Alistair J Barber, Committee Member - Keywords:
- siRNA
liposomes
ferritin - Abstract:
- Approximately fifty percent of gliomas especially glioblastoma multiforme(GBM)are resistant to radiation and chemotherapy. Therefore, efforts to develop new mechanisms to enhance the efficacy of current treatment strategies are clearly warranted. Glioma cells, similar to many other malignant tumor cells, have a robust appetite for iron to support their rapid growth and high metabolic rate. With elevated levels of iron uptake, a mechanism to protect cells from augmented iron induced oxidative damage is needed. This protection is likely provided by the intracellular iron storage protein ferritin. Ferritin has been detected in the cytosol, mitochondria and nucleus of almost all malignant tumor cells. Overexpression of H-ferritin in nuclei of glioma cells appear to protect DNA from iron induced oxidative damage and promote transcription. Overexpression of H-ferritin in nuclei of breast cancer cells and cervical cancer cells was also found to be associated with drug resistance. These findings lead to the central hypothesis of my thesis, silencing of H-ferritin by siRNA enhances the radiation and chemotherapeutic efficacy for treatment of glioma. It is anticipated that silencing of H-ferritin using gene therapy will decrease resistance to chemo/radiation therapy. To deliver a therapeutic dose of siRNA, I had to first develop a delivery system and chose cationic liposomes. Thus, there were three specific aims for my thesis: Aim1: development and characterization of cationic liposomes described in chapter 2. Aim2: investigation of whether silencing of heavy chain ferritin using gene therapy enhances radiation and chemotherapeutic efficacy for the treatment of glioma (illustrated in chapter 3.I). Aim 2 was further expanded to investigate the treatment of breast cancer, as well as malignant peripheral nerve sheath tumors. Aim3: examination of the potential roles of H-ferritin in the progression of malignant cancers (illustrated in chapter 3.II). A number of cell lines; human glioma U251 and U87 cells, neurofibrama #215 sNF96.2 cells, and breast cancer MCF-7 cells were selected for our in vitro models, and athymic nude mice were selected as our in vivo model. Cationic liposomes (C-liposomes) were prepared and characterized for use as a gene delivery vehicle. The cytotoxicity of C-liposomes was tested in vitro and in vivo. SiRNA was incorporated into C-liposomes to form C-liposomes:siRNA complexes. The uptake of C-liposomes:siRNA into glioma cells was confirmed, and the mechanism of uptake was identified. Radiation and chemotherapeutic efficacy were assessed after H-ferritin was down regulated. The potential role of H-ferritin in biological pathways was investigated by evaluating the effects on DNA conformation, the DNA repair related protein MGMT, DNA synthesis (by evaluating incorporation of BrdU and 3H-thymidine), and apoptotic activities. The C-liposomes exhibited safe, applicable, and stable traits, and its uptake into glioma cells depended on the endosomal pathway. Immunoblotting analysis demonstrated that siRNA delivered in this manner, decreased H-ferritin protein expression by 90% in U251 cells, and 70% in MCF-7 cells within 48 hours. This decrease in H-ferritin expression was associated with a decrease in the LD50 for, nitrosoureas carmustine (BCNU), from concentrations greater than 100 µM to 40 ìM in the U251 cells, 30 µM in MCF-7 cells, and 18 µM in MPNST cells. In addition, knockdown of H-ferritin was associated with a 50% increase in cell death of U251 cells using 20 Gy of radiation in vitro. The in vivo efficacy of siRNA delivered by C-liposomes was tested in an athymic nude mouse subcutaneous glioma tumor model. Intratumoral injections of C-liposomes containing H-ferritin siRNA reduced the required effective dose of BCNU for tumor suppression by more than 50%. Moreover, intratumoral injections of C-liposomes containing H-ferritin siRNA with 4Gy radiation suppressed tumor progression by more than 90% compared with C-liposomes containing non-specific siRNA. The interaction of H-ferritin with DNA in the presence of BCNU was investigated in vitro by supercoil relaxation assays. These assays demonstrated that H-ferritin maintained DNA in a relaxed form, which would promote DNA transcription even in the presence of radiation and chemotoxins. In addition to the protective function on H-ferritin on DNA, the anti tumor effect of H-ferritin siRNA was associated with activation of the apoptotic caspase-3 pathway. However, H-ferritin did not appear to facilitate DNA repair through the MGMT pathway. In conclusion, our study demonstrated that the vulnerability of cancer cells to radiation and chemotherapy can be increased by reducing H-ferritin expression. The importance of ferritin in cancer cell biology suggests that efficient delivery of siRNA could be a viable therapeutic option. As importantly, we have demonstrated that siRNA can be delivered effectively by C-liposomes in an in vivo tumor model.