Aim:To develop a novel ricin-based approach for the safe and effective therapyof cancer.Methods:The ricin A chain (RTA) was expressed in Escherichia coli inthe form of a 6×His-tagged fusion protein and purified with Ni~2+-NTA affinityresin.A replication-deficient ricin B chain (RTB)-expression adenovirus greenfluorescence protein (AdGFP-RTB) was constructed.RTA and AdGFP-RTB weretested for cytotoxicity either individually or in combination in human cell linesHEK293,HeLa,SMMC7721,and HL7702.Cell viability was determined with trypanblue staining or MTT assay.Results:The expression and release of RTB,as wellas the entry of RTA into AdGFP-RTB-infected cells were confirmed.When RTAand AdGFP-RTB was used individually,neither was toxic to the cells.When theywere applied together,significant cell death was observed in all of the cell linestested.The cell-killing effect correlated with the amount of RTA protein used,withcell mortality at about 60% at 4.8 μg RTA in combination with AdGFP-RTB at 100pfu/cell.No major cell killing was seen when RTA was used in combination with acontrol 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 ofthe adenovirus vector,as well as an in vivo study are needed to confirm its poten-tial in cancer therapy. 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 coli inthe form of a 6 × His-tagged fusion protein and purified with Ni ~ 2 + -NTA affinityresin.A replication-deficient ricin B chain (RTB) -expression adenovirus greenfluorescence protein (AdGFP-RTB) was constructed.RTA and AdGFP-RTB weretested for cytotoxicity either individually or in combination in human cell lines HEK293, HeLa, SMMC7721, and HL7702.Cell viability was determined with trypanblue staining or MTT assay. Results: The expression and release of RTB, as wellas 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 theywe applied together, significant cell death was observed in all of the cell lines that had been marked. 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 a t 100 pfu / cell. No major cell killing was seen when RTA was used in combination with acontrol 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. Confluence: The new approach was successful in vitro. Future modifications of the adenovirus vector, as well as an in vivo study are needed to confirm its poten- tial in cancer therapy .