Heavy Chain Ferritin siRNA Delivered by Cationic Liposomes Increases Sensitivity of Cancer Cells to Chemotherapeutic Agents

Abstract

Approximately half of all gliomas are resistant to chemotherapy, and new therapeutic strategies are urgently needed to treat this cancer. We hypothesized that disrupting iron homeostasis in glioma cells could block tumor growth, based on an acute requirement for high levels of iron to meet energy requirements associated with their rapid growth. Ferritin is best known as an intracellular iron storage protein, but it also localizes to tumor cell nuclei where it seems to protect DNA from oxidative damage and to promote transcription. In this study, we hypothesize that silencing the H-ferritin (heavy chain ferritin) gene could increase tumor sensitivity to chemotoxins. To test this hypothesis, H-ferritin siRNA was delivered to several human cancer cell lines by using cationic liposomes (C-liposome). H-ferritin siRNA decreased protein expression by 80% within 48 hours, and this decrease was associated with more than 50% decrease in the LD 50 for DNA-alkylating agent carmustine (BCNU), which is commonly used to treat glioma in clinic. In a subcutaneous mouse model of human glioma, intratumoral injections of liposomes containing H-ferritin siRNA reduced the effective dose of BCNU needed for tumor suppression by more than 50%. A plasmid supercoil relaxation assay showed that H-ferritin specifically and directly protected DNA from BCNU treatment. H-ferritin siRNA additionally seemed to increase apoptosis in glioma cells in vitro upon H-ferritin knockdown. Overall, our results illustrate how silencing H-ferritin can effectively sensitize tumors to chemotherapy and also show the ability of C-liposomes to serve as a novel in vivo delivery tool for siRNAs. Cancer Res; 71(6); 2240-9. Ó2011 AACR.

Role of H-Ferritin in Radiosensitivity of Human Glioma Cells

“…In order to apply this knowledge to cancer biology and tumor growth we developed a nanotechnology platform to produce an acute decrease in ferritin expression. Using this approach we previously demonstrated that a significant but transient change in iron homeostasis in cancer cells following exposure to ferritin siRNA is coupled with an increased sensitivity to chemotherapeutic agent [1] and we expand this finding in the present study to radiosensitivity in both in vitro and in vivo models. The results of this study demonstrate that reducing expression of H-ferritin in human glioblastoma cells disrupts iron homeostasis, promotes oxidative stress, alters DNA repair mechanisms, and increases cell sensitivity to radiation.…”

“…Nuclear ferritin is composed mainly of H-ferritin and is believed participate in the transcriptional regulation and DNA protection [42]. In our previous study, we found that H-ferritin is capable of protecting naked DNA in the presence of BCNU, a DNA alkylating chemotherapeutic agent [1]. In the current study we extend this idea to radiation (Figure 4).…”

“…Liposomes in the size range of 50-100 nm were formulated using DC-cholesterol: DOPE lipid and characterized as described in our previous report and complexed with H-ferritin siRNA for transfection [1]. Briefly, DC-cholesterol: DOPE lipid (Avanti) was solubilized in 1:1 (v/v) chloroform: methanol mixture and rotary evaporated to form a thin film followed by reconstitution in Phosphate Buffered Saline (PBS) and extrusion in a lipex extruder (Northern Lipids Inc.) utilizing 50 and 100 nm membranes, both for 10 times.…”

“…In both cases complexed liposomes in a total volume of 50μL were injected intratumorally on a weekly basis. We have previously shown that this dosing regimen will decrease ferritin expression in the tumors [1]. Mice were exposed to 4 Gy of whole body γ-radiation two days after liposomal injection.…”

“…Elevated iron levels in both serum and tissue from tumor-bearing individuals are observed [9]. The robust iron uptake by cancer cells must be regulated to keep the cells viable and this concept supports a rationale for our approach targeting iron management proteins, such as ferritin, [1] in cancer treatment.…”