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Published on: February 6, 2015
Cytotoxic gene therapy for human breast cancer in vitro
Shauna Levy1, Beilan Zhou, Nikiforos Ballian
1The Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas 77030, USA. cbrunica@bcm.tmc.edu
Background:
Transcription factor PDX-1 is expressed by human pancreatic and breast cancers. Although cytotoxicity of PDX-1-directed RIP-TK/GCV gene therapy to pancreatic cancer cells has been demonstrated, the efficacy of this treatment in breast cancer cells is unknown. The purpose of this study was to determine the expression of PDX-1 and its effect on RIP activation in two human breast cancer cell lines, AU565 and T47D. We also investigated the efficacy of RIP-TK/GCV gene therapy and examined whether exogenous PDX-1 to would enhance its cytotoxic effect.
Materials And Methods:
RT-PCR was used to determine PDX-1 expression. Gene constructs RSVLacZ and RIPLacZ were used for transient transfection and LacZ expression was determined using reporter assays. T47D cells were also transfected with adenoviral vectors. Cells were transfected with RIP-TK and the suboptimal level of GCV was determined for each cell line. Following GCV treatment, cytotoxicity was measured using MTS assays. The effect of exogenous PDX-1 on LacZ expression and RIP-TK cytotoxicity was determined.
Results:
PDX-1 mRNA was expressed in human breast cancer cells and activated the RIP. Exogenous PDX-1 enhanced LacZ expression in AU565 cells but not in T47D cells. Adenoviral transfection was more efficient in T47D cells than non-viral transfection. RIP-TK treatment was cytotoxic to AU565 and T47D cells and this effect was enhanced by exogenous PDX-1 with both transfection methods.
Conclusions:
RIP-TK/GCV therapy is cytotoxic to human breast cancer cells and exogenous PDX-1 enhances cytotoxicity. In vivo studies are necessary to determine the tumor specificity and efficacy of this treatment.
Insights
PDX-1 (Programmed cell death protein 1) gene therapy using RIP-TK/GCV is effective against human breast cancer cells. Adding more PDX-1 boosts the therapy's cancer-killing power, showing promise for future treatments.
Area of Science:
- Oncology
- Molecular Biology
- Gene Therapy
Background:
- PDX-1 (Programmed cell death protein 1) is a transcription factor found in pancreatic and breast cancers.
- Previous studies showed PDX-1-directed RIP-TK/GCV gene therapy is effective against pancreatic cancer.
- The efficacy of this therapy in breast cancer remains uninvestigated.
Purpose of the Study:
- To determine PDX-1 expression and its role in RIP activation in AU565 and T47D human breast cancer cell lines.
- To evaluate the efficacy of RIP-TK/GCV gene therapy in these cell lines.
- To assess if adding exogenous PDX-1 enhances the cytotoxic effect of the therapy.
Main Methods:
- PDX-1 expression was analyzed using RT-PCR.
- Transient transfection with RSVLacZ and RIPLacZ constructs.
- Reporter assays for LacZ expression.
- Adenoviral vector transfection in T47D cells.
- Cytotoxicity assessed via MTS assays after GCV treatment.
- Effect of exogenous PDX-1 on LacZ expression and RIP-TK cytotoxicity.
Main Results:
- PDX-1 mRNA was detected in human breast cancer cells, activating RIP.
- Exogenous PDX-1 enhanced LacZ expression in AU565 cells but not T47D cells.
- Adenoviral transfection proved more efficient in T47D cells.
- RIP-TK treatment demonstrated cytotoxicity in both cell lines, enhanced by exogenous PDX-1.
Conclusions:
- RIP-TK/GCV gene therapy exhibits cytotoxicity against human breast cancer cells.
- Exogenous PDX-1 enhances the cytotoxic effects of this gene therapy.
- Further in vivo studies are required to confirm tumor specificity and overall efficacy.
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