Enhancing the therapeutic effect via elimination of hepatocellular carcinoma stem cells using Bmi1 siRNA delivered by cationic cisplatin nanocapsules.

Resistance of hepatocellular carcinoma (HCC) to systemic chemotherapy is partially due to presence of drug-resistant cancer stem cells. Bmi1 protein is essential for survival and proliferation of HCC cancer stem cells (CSCs). Here, we report that Bmi1 siRNA (Bmi1siR) loaded in cationic nanocapsules of cisplatin (NPC) eliminated stem cells in situ HCC in mice. NPC/Bmi1siR was fabricated via electrostatic complexation of Bmi1 siRNA to NPCs, which had cores composed of cisplatin and were coated with cationic lipids. In vivo, NPC/Bmi1siR showed higher anti-tumor activity in HCC bearing mice compared with cisplatin or NPC. Critically, both flow cytometry (FACS) analysis in vitro and histological examination in vivo revealed that side population or CD133+ HCC cells were dramatically decreased by NPC/Bmi1siR treatment, suggesting that HCC CSCs were eliminated. Altogether, our results suggest that drug resistance of HCC can be overcome by co-delivering Bmi1 siRNA with cisplatin in cationic nanocapsules.

Epigenetic Regulation of Bmi1 by Ubiquitination and Proteasomal Degradation Inhibit Bcl-2 in Acute Myeloid Leukemia.

Bmi1 is associated with advanced prognosis of acute myeloid leukemia (AML), and polyethylenimine (PEI)-stabilized Bmi1 siRNA-entrapped human serum albumin (HSA) nanocarriers (PEI@HSANCs) were used to protect siRNA from degradation and also to control epigenetic regulation-based AML therapy. The nanoform increased the transfection efficiency of Bmi1 siRNA through caveolae-mediated endocytosis and enhanced Bax translocation into the mitochondria. It enhanced the caspase 3-mediated apoptosis through the Bax activation and Bcl-2 inhibition. The molecular analysis reveals the downregulation of polycomb proteins, Bmi1 and EzH2, along with inhibition of H3K27me3 and H2AK119ub1. The signaling cascade revealed downregulation of Bmi1 through ubiquitin-mediated degradation and is reversed by a proteasome inhibitor. Further mechanistic studies established a crucial role of transcription factor, C-Myb and Bmi1, as its direct targets for maintenance and progression of AML. Chromatin immunoprecipitation (ChIP) assay confirmed Bmi1 as a direct target of C-Myb as it binds to promoter sequence of Bmi1 between -235 to +43 and -111 to +43. The in vivo studies performed in the AML xenograft model evidence a decrease in the population of leukemic stem cells marker (CD45+) and an increase in the myeloid differentiating marker expression (CD11b+) in the bone marrow after the Bmi1 siRNA nanoconjugated therapy. Activation of apoptotic pathways and withdrawal of epigenetic repression through a ubiquitin proteasomal pathway potentiating a novel antileukemic therapy were established.

Co-delivery of Bmi1 small interfering RNA with ursolic acid by folate receptor-targeted cationic liposomes enhances anti-tumor activity of ursolic acid in vitro and in vivo.

Overexpression of Bmi1 gene is an important feature of cancer stem cell in various human tumors. Therefore, Bmi1 gene can be a potential target for small interfering RNA (siRNA) mediated cancer therapy. Ursolic acid (UA) as a natural product plays a pivotal role in anti-tumor field, although its performance is limited by low bioavailability and poor hydrophilicity. A folate receptor-targeted cationic liposome system was designed for the purpose of investigating the relationship between Bmil siRNA and UA. The folate receptor-targeted cationic liposomes co-delivering UA and Bmi1 siRNA (FA-UA/siRNA-L) were fabricated by electrostatic interaction between folate UA liposome (FA-UA-L) and Bmi1 siRNA. Tumor growth is inhibited by FA-UA/siRNA-L in vitro and in vivo and this inhibition is contributed by a synergistic anti-tumor effect of UA and Bmi1 siRNA. The western blot measurement of apoptosis-protein and cancer stem cell (CSC) marked-protein demonstrated that UA led to activation-induced tumor cell death and Bmi1 siRNA resulted in inhibition of cancer stem cells. Overall, these results indicate that Bmi1 as a regulating gene for cancer stem cell is an effective target for cancer treatment using siRNA and co-delivery of UA and Bmi1 siRNA using folate-targeted liposomes is a promising strategy for improved anti-tumor effect.

Co-delivery of doxorubicin and Bmi1 siRNA by folate receptor targeted liposomes exhibits enhanced anti-tumor effects in vitro and in vivo.

Bmi1 gene overexpression is found in various human tumors and has been shown as a potential target for gene treatment. However, siRNA-based treatments targeting Bmi1 gene have been restricted to limited delivery, low bioavailability and hence relatively reduced efficacy. To overcome these barriers, we developed a folate receptor targeted co-delivery system folate-doxorubicin/Bmi1 siRNA liposome (FA-DOX/siRNA-L). The FA-DOX/siRNA-L was prepared through electrostatic interaction between folate doxorubicin liposome (FA-DOX-L) and Bmi1 siRNA. In vitro and in vivo studies showed that FA-DOX/siRNA-L inhibited tumor growth by combinatory role of Bmi1 siRNA and doxorubicin (DOX). Co-delivery of Bmi1 siRNA and DOX by FA-DOX/siRNA-L showed significantly higher efficacy than sole delivery of either DOX or Bmi1 siRNA. Real-time PCR and western blot analysis showed that FA-DOX/siRNA-L silenced the expression of Bmi1 gene. In addition, higher accumulation of the siRNA and DOX in tumor cells indicated that folate ligand displayed tumor targeting effect. These results suggest that Bmi1 is an effective therapeutic target for siRNA based cancer treatment that can be further improved by co-delivery of DOX through targeted liposomes.