Antitumor effects of human interferon-alpha 2b secreted by recombinant bacillus Calmette-Guérin vaccine on bladder cancer cells

Abstract

Objective: Our objective was to construct a recombinant bacillus Calmette-Guérin vaccine (rBCG) that secretes human interferon-alpha 2b (IFNα-2b) and to study its immunogenicity and in vitro antitumor activity against human bladder cancer cell lines T24 and T5637. Methods: The signal sequence BCG Ag85B and the gene IFNα-2b were amplified from the genome of BCG and human peripheral blood, respectively, by polymerase chain reaction (PCR). The two genes were cloned in Escherichia coli-BCG shuttle-vector pMV261 to obtain a new recombinant plasmid pMV261-Ag85B-IFNα-2b. BCG was transformed with the recombinant plasmid by electroporation and designated rBCG-IFNα-2b. Mononuclear cells were isolated from human peripheral blood (PBMCs) and stimulated with rBCG-IFNα-2b or wild type BCG for 3 d, and then cultured with human bladder cancer cell lines T24 and T5637. Their cytotoxicities were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: BCG was successfully transformed with the recombinant plasmid pMV261-Ag85B-IFNα-2b by electroporation and the recombinant BCG (rBCG-IFNα-2b) was capable of synthesizing and secreting cytokine IFNα-2b. PBMC proliferation was enhanced significantly by rBCG-IFNα-2b, and the cytotoxicity of PBMCs stimulated by rBCG-IFNα-2b to T24 and T5627 was significantly stronger in comparison to wild type BCG. Conclusions: A recombinant BCG, secreting human , was constructed successfully and was superior to control wild type BCG in inducing immune responses and enhancing cytotoxicity to human bladder cancer cell lines T24 and T5637. This suggests that rBCG-IFNα-2b could be a promising agent for bladder cancer patients in terms of possible reductions in both clinical dosage and side effects of BCG immunotherapy.

Enhanced Control of Bladder-Associated Tumors Using Shrimp Anti-Lipopolysaccharide Factor (SALF) Antimicrobial Peptide as a Cancer Vaccine Adjuvant in Mice

“…The LIVE/DEAD ® Cell-Mediated Cytotoxicity Kit (L-7010, Invitrogen, New York, NY, USA) was used to analyze the toxicity of cytotoxic T-lymphocytes (CTLs). The CTL assay was performed on MBT-2 cells as previously described, with some modifications [ 38 ]. Briefly, cells were grown in RPMI 1640 (Gibco-BRL, Paisley, UK) supplemented with 10% fetal bovine serum (FBS; Dutscher, Brumath, France), 1 mM sodium pyruvate (Sigma, St. Louis, MO, USA), 100 UI/mL penicillin, 0.1 mg/mL streptomycin, and 1% glucose (complete medium).…”

Biosystems Engineering of Prokaryotes with Tumor-Killing Capacities

“…[48]. More recent studies have also used recombinant strains of bacillus Calmette-Guérin (rBCG) [49], S. enterica serovar typhimurium LB5000 and attenuated S. typhimurium SL3261 [50], and Listeria monocytogenes [51] as live cancer vaccines.…”

Applications of bacillus Calmette–Guerin and recombinant bacillus Calmette–Guerin in vaccine development and tumor immunotherapy

“…bcl-2 and p53) [103]. The BCG-ΔleuD/Ag85B was more cytotoxic against human bladder carcinoma cell line 5637 than BCG [178;192]. However, there is no evidence that the BCG-ΔleuD/Ag85B induces stronger immune responses than the original BCG.…”