| BACKGROUND Bladder transitional cell carcinoma (BTCC) is the most common cancer of genitourinary tract in our country. Although approximately 70% of patients with BTCC are superficial at initial presentation, the recurrence and progression of tumor lesions are very frequent after surgical resection. Adjuvant intravesical Bacillus Camette-Gu e rin (BCG) instillation has been proven the most successful immunotherapy to date in the treatment and prophylaxis of superficial papillary bladder tumors and carcinoma in situ of the bladder. It is superior to intravesical chemotherapy with regard to the tumor recurrence and progression rate. Despite its successful use, two major problems exist: a significant proportion of patients do not respond to BCG therapy and side-effects are common. These limit its use in clinical practice. Therefore, current studies aim at developing new BCG strain to further improve its efficacy and reduce its dosage and side-effects. Recombinant BCG strains secreting cytokines has been considered a potentially useful agents.It is known that combination therapy with BCG plus interferon-alpha 2b (IFN α -2b) favours bladder cancer treatment both in clinic and animal models. However, IFN α -2b therapy has several shortcomings, such as high costs, repeated administration, short retention time inside the bladder and so on. Therefore, construction of recombinant BCG secreting human IFN α -2b could potentially cope with these shortcomings.OBJECTIVE This study was to establish a more effective anti-cancer immunomodulating agent by constructing recombinant BCG secreting human IFN α -2b (rBCG-IFN α -2b) and to study its immunogenicity and antitumor activity against human bladder cancer cells, T24 and T5637, in vitro.METHODS (1) Construction and identification of recombinant shuttle plasmid. BCG Ag85B signal sequence and IFN α -2b gene were amplified from the genome of BCG and of human peripheral blood by polymerase chain reaction (PCR), respectively. IFN α -2b gene was cloned in E.coli-BCG shuttle-vector pMV261 to get pMV261-IFN α -2b. A new recombinant plasmid pMV261-IFN α -2b was constructed by inserting BCG Ag85B signal sequence into pMV261- Ag85B-IFN α -2b. The recombinant plasmid pMV261-Ag85B-IFN α -2b was identified by restriction endonuclease digestion, PCR amplification and nucleotide sequencing respectively. (2) Construction of recombinant BCG secreting IFN α -2b. BCG was transformed with pMV261-Ag85B-IFN α -2b by electroporation, and designated as rBCG-IFN α -2b . The DNA and protein expressions of IFN α -2b gene in rBCG were determined by PCR and Western blotting respectively. Also the quantity of IFN α -2b protein secreted by rBCG in culture supernatants was determined by enzyme linked immunosorbent assay (ELISA). (3) The effect of rBCG-IFN α -2b on suppression of human bladder cancer cell lines in vitro. Mononuclear cells (PBMCs) were isolated from human peripheral blood and stimulated with rBCG-IFN α -2b or wild-type BCG for three days. Then the stimulated PBMCs were cultured with human bladder cancer cell lines, T24 and T5637. And their cytotoxic activity was measured by MTT reduction assay.RESULTS (1) By partial nucleotide sequencing, the DNA sequences of human IFN α -2b and BCG Ag85B were consistent with that in the GeneBank, and were correctly inserted into the shuttle expression vector pMV261 to construct recombinant plasmid pMV261-Ag85B-IFN α -2b. BCG was successfully transformed with thisrecombinant plasmid by electroporation and the recombinant BCG (rBCG-IFN α -2b) was capable of synthesizing and secreting cytokine IFN α -2b. The concentration of IFN α -2b in culture supernatants was quantified by ELISA and calculated to be approximately 301.45pg /ml. (2) PBMCs proliferation was enhanced significantly by rBCG-IFN α -2b. Compared with that of control wild-type BCG, cytotoxicity of PBMCs stimulated by rBCG-IFN α -2b to human bladder cancer cell lines, T24 and T5637, was significantly more efficient.CONCLUSIONS Recombinant BCG secreting human IFN α -2b (rBCG-IFN α -2b) was constructed successfully and the specific IFN α -2b protein can be expressed highly and steadily by rBCG vaccine. rBCG-IFN α -2b is superior to control wild-type BCG for induction of immune responses and cytotoxicity to human bladder cancer cell lines, T24 and T5637. This suggested that rBCG-IFN α -2b may be a promising agent for bladder cancer patient to reduce both clinical dosage and side effects of BCG for immunotherapy.
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