RESUMO
Two of the successful gene-directed enzyme prodrug therapies include herpes simplex virus-thymidine kinase (HSV1-TK) enzyme-ganciclovir prodrug and the Escherichia coli nitroreductase (NTR) enzyme-CB1954 prodrug strategies; these enzyme-prodrug combinations produce activated cytotoxic metabolites of the prodrugs capable of tumor cell death by inhibiting DNA synthesis and killing quiescent cells, respectively. Both these strategies also affect significant bystander cell killing of neighboring tumor cells that do not express these enzymes. We have developed a dual-combination gene strategy, where we identified HSV1-TK and NTR fused in a particular orientation can effectively kill tumor cells when the tumor cells are treated with a fusion HSV1-TK-NTR gene- along with a prodrug combination of GCV and CB1954. In order to determine whether the dual-system demonstrate superior therapeutic efficacy than either HSV1-TK or NTR systems alone, we conducted both in vitro and in vivo tumor xenograft studies using triple negative SUM159 breast cancer cells, by evaluating the efficacy of cell death by apoptosis and necrosis upon treatment with the dual HSV1-TK genes-GCV-CB1954 prodrugs system, and compared the efficiency to HSV1-TK-GCV and NTR-CB1954. Our cell-based studies, tumor regression studies in xenograft mice, histological analyses of treated tumors and bystander studies indicate that the dual HSV1-TK-NTR-prodrug system is two times more efficient even with half the doses of both prodrugs than the respective single gene-prodrug system, as evidenced by enhanced apoptosis and necrosis of tumor cells in vitro in culture and xenograft of tumor tissues in animals.
Assuntos
Neoplasias da Mama/terapia , Proteínas de Escherichia coli/genética , Terapia Genética , Nitrorredutases/genética , Proteínas Recombinantes de Fusão/genética , Timidina Quinase/genética , Animais , Apoptose/efeitos dos fármacos , Aziridinas/administração & dosagem , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Escherichia coli/genética , Proteínas de Escherichia coli/administração & dosagem , Ganciclovir/administração & dosagem , Genes Reporter/genética , Humanos , Camundongos , Nitrorredutases/administração & dosagem , Pró-Fármacos/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Simplexvirus/genética , Timidina Quinase/administração & dosagem , Transplante HeterólogoRESUMO
Volume changes among the unfolded (U), native (N), and molten globule (MG) conformations of horse heart ferricytochrome c have been measured. U to N (pH 2 to pH 7) was determined in the absence of added salt to be -136 +/- 5 mL/mol protein. U to MG (pH 2, no added salt to pH 2, 0.5 M KCl) yielded + 100 +/- 6 mL/mol. MG to N was broken into two steps, N to NClx at pH 7 by addition of buffered KCl to buffered protein lacking added salt (NClx = N interacting with an unknown number, X, of chloride ions), and MG to NClx by jumping MG at pH 2 in 0.5 M KCl to pH7 at the same salt concentration. The delta V of N to NClx was -30.9 +/- 1.4 mL/mol protein, whereas MG to NClx entailed a delta V of -235 +/- 6 mL/mol. Within experimental error, the results add up to zero for a complete thermodynamic cycle. We believe this to be the first volumetric cycle to have been measured for the conformational transitions of a protein. The results are discussed in terms of hydration contributions from deprotonation of the protein, other hydration effects, and the formation and/or enlargement of packing defects in the protein's tertiary structure during the steps of folding.