Co-application of ricin A chain and a recombinant adenovirus expressing ricin B chain as a novel approach for cancer therapy
Abstract
Aim: To develop a novel ricin-based approach for the safe and effective therapy of cancer.
Methods: The ricin A chain (RTA) was expressed in Escherichia coliin the form of a 6×His-tagged fusion protein and purified with Ni2+-NTA affinity resin. A replication-deficient ricin B chain (RTB)-expression adenovirus green fluorescence protein (AdGFP-RTB) was constructed. RTA and AdGFP-RTB were tested for cytotoxicity either individually or in combination in human cell lines HEK293, HeLa, SMMC7721, and HL7702. Cell viability was determined with trypan blue staining or MTT assay.
Results: The expression and release of RTB, as well as the entry of RTA into AdGFP-RTB-infected cells were confirmed. When RTA and AdGFP-RTB was used individually, neither was toxic to the cells. When they were applied together, significant cell death was observed in all of the cell lines tested. The cell-killing effect correlated with the amount of RTA protein used, with cell mortality at about 60% at 4.8 μg RTA in combination with AdGFP-RTB at 100 pfu/cell. No major cell killing was seen when RTA was used in combination with a control adenovirus AdGFP. The treatment of healthy HeLa cells with the virus-free supernatant from AdGFP-RTB/RTA-treated HeLa cells resulted in cell death, suggesting the formation of RTA/RTB complex, and a potential by-stander effect.
Conclusion: The new approach was successful in vitro. Further modifications of the adenovirus vector, as well as an in vivo study are needed to confirm its potential in cancer therapy.
Keywords:
Methods: The ricin A chain (RTA) was expressed in Escherichia coliin the form of a 6×His-tagged fusion protein and purified with Ni2+-NTA affinity resin. A replication-deficient ricin B chain (RTB)-expression adenovirus green fluorescence protein (AdGFP-RTB) was constructed. RTA and AdGFP-RTB were tested for cytotoxicity either individually or in combination in human cell lines HEK293, HeLa, SMMC7721, and HL7702. Cell viability was determined with trypan blue staining or MTT assay.
Results: The expression and release of RTB, as well as the entry of RTA into AdGFP-RTB-infected cells were confirmed. When RTA and AdGFP-RTB was used individually, neither was toxic to the cells. When they were applied together, significant cell death was observed in all of the cell lines tested. The cell-killing effect correlated with the amount of RTA protein used, with cell mortality at about 60% at 4.8 μg RTA in combination with AdGFP-RTB at 100 pfu/cell. No major cell killing was seen when RTA was used in combination with a control adenovirus AdGFP. The treatment of healthy HeLa cells with the virus-free supernatant from AdGFP-RTB/RTA-treated HeLa cells resulted in cell death, suggesting the formation of RTA/RTB complex, and a potential by-stander effect.
Conclusion: The new approach was successful in vitro. Further modifications of the adenovirus vector, as well as an in vivo study are needed to confirm its potential in cancer therapy.