ABSTRACT
According to the cancer stem cell (CSC) theory, a small subset of cells with stem cell-like characteristics is responsible for tumor initiation, progression, and recurrence. CD44+/CD24- phenotype is assumed to be one of the main characteristics of the breast CSCs. We developed an MDA-MB-231 cell line overexpressing cell surface HER2 antigen for the evaluation of targeting efficiency of anti-HER2 nanobody (Nb)-conjugated polyamidoamine (PAMAM) polyplexes. Apoptosis-inducing tBid gene under control of CXCR1 promoter was delivered by this nanoparticle. Cellular uptake study showed higher uptake of Nb-targeted PAMAM carriers compared to non-targeted nanoparticles after 6 h of incubation. Gene expression analysis showed a significant rise in the expression of tBid in both MDA-MB-231/HER2+ and MDA-MB-231 compared to the two other cell lines. The same effect was observed after transfection with Nb-conjugated polyplexes within MDA-MB-231/HER2+ cell line compared to non-conjugated PAMAM polyplexes. We confirmed the killing efficiency of the gene construct in both MDA-MB-231/HER2+ and MDA-MB-231 cell lines by caspase 3 activity assay. These findings suggest that imposing pre-entry and post-entry restrictions on tBid killer gene might be a promising approach to specifically target the breast CSCs.
Subject(s)
Antineoplastic Agents, Immunological , Breast Neoplasms , Dendrimers , Drug Delivery Systems/methods , Receptor, ErbB-2/antagonists & inhibitors , Single-Domain Antibodies , Transcription, Genetic/drug effects , Antineoplastic Agents, Immunological/chemistry , Antineoplastic Agents, Immunological/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Dendrimers/chemistry , Dendrimers/pharmacology , Female , HEK293 Cells , Humans , Receptor, ErbB-2/metabolism , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/pharmacologyABSTRACT
BACKGROUND: Cancerous cells proliferate as fast as possible without a proper surveillance system. This rapid cell division leads to enormous mutation rates, which help a tumor establish. OBJECTIVES: This study evaluated the potential of inducing apoptosis using Noxa and Puma in a hepatocarcinoma cell line. METHODS: The current study generated two recombinant lentiviruses, pLEX-GCN and pLEX-GCP, bearing Noxa and Puma, respectively. Transduction of both genes to hepatocarcinoma (HepG2) was verified using fluorescent microscopic analysis, western blotting, and quantitative real-time polymerase chain reaction (PCR). To evaluate the potential of Noxa and Puma to initiate apoptosis, a caspase-9 real-time, MTT assay, and a 4', 6-diamidino-2-phenylindole (DAPI) reagent were performed to stain apoptotic cells. RESULTS: The data verified successful transduction to HepG2 and HEK293T. Higher relative expression of Noxa and Puma rather than the untransduced cell line showed these genes are expressed more in HepG2 in comparison to HEK293T. The results of the real-time PCR, MTT assay, and DAPI reagent illustrated that higher cells initiated apoptosis following Puma transduction rather than Noxa. CONCLUSIONS: In this approach, the suicide gene was transferred to transformed cells and ignited apoptosis to exterminate them. Puma is a more potent killer gene and has higher capabilities to start intrinsic apoptosis pathway.
