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Jafari B, Reza Bahrami A, Matin MM. Targeted bacteria-mediated therapy of mouse colorectal cancer using baicalin, a natural glucuronide compound, and E. coli overexpressing β-glucuronidase. Int J Pharm 2023:123099. [PMID: 37271252 DOI: 10.1016/j.ijpharm.2023.123099] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2023] [Revised: 05/27/2023] [Accepted: 05/29/2023] [Indexed: 06/06/2023]
Abstract
The side effects of common chemotherapeutic drugs that damage healthy tissues account for one of the most important problems in cancer research that needs careful addressing. Bacterial-Directed Enzyme Prodrug Therapy (BDEPT) is a promising strategy that uses bacteria to direct a converting enzyme to the tumor site and activate a systemically injected prodrug selectively within the tumor; so that the side effects of the therapy would significantly decrease. In this study, we evaluated the efficacy of baicalin, a natural compound, as a glucuronide prodrug in association with an engineered strain of Escherichia coli DH5α harboring the pRSETB-lux/βG plasmid in a mouse model of colorectal cancer. E. coli DH5α-lux/βG was designed to emit luminescence and overexpress the β-glucuronidase. Unlike the non-engineered bacteria, E. coli DH5α-lux/βG showed the ability to activate baicalin, and the cytotoxic effects of baicalin on the C26 cell line were increased in the presence of E. coli DH5α-lux/βG. Analyzing the tissue homogenates of mice bearing C26 tumors inoculated with E. coli DH5α-lux/βG indicated the specific accumulation and multiplication of bacteria in the tumor tissues. While both baicalin and E. coli DH5α-lux/βG could inhibit tumor growth as monotherapy, an enhanced inhibition was observed when animals were subjected to combination therapy. Moreover, no significant side effects were observed after histological investigation. The results of this study indicate that baicalin has the capability of being used as a suitable prodrug in the BDEPT, however further research is required before it can be applied in the clinic.
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Affiliation(s)
- Bahareh Jafari
- Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Ahmad Reza Bahrami
- Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran; Industrial Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Maryam M Matin
- Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran; Novel Diagnostics and Therapeutics Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.
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2
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Quintero D, Carrafa J, Vincent L, Kim HJ, Wohlschlegel J, Bermudes D. Co-Expression of a Chimeric Protease Inhibitor Secreted by a Tumor-Targeted Salmonella Protects Therapeutic Proteins from Proteolytic Degradation. J Microbiol Biotechnol 2018; 28:2079-2094. [PMID: 30661346 PMCID: PMC6883771 DOI: 10.4014/jmb.1807.08036] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022]
Abstract
Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from an individual colony of E. coli using a culture-based inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14-amino-acid, disulfide-bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of anticancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.
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Affiliation(s)
- David Quintero
- Department of Biology, California State University Northridge, Northridge, CA 91330-8303, USA
- Interdisciplinary Research Institute for the Sciences (IRIS), California State University, College of Science and Math, California State University, Northridge, Northridge, CA 91330-8303
| | - Jamie Carrafa
- Department of Biology, California State University Northridge, Northridge, CA 91330-8303, USA
| | - Lena Vincent
- Department of Biology, California State University Northridge, Northridge, CA 91330-8303, USA
- Current Address, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Hee Jong Kim
- Department of Biological Chemistry, David Geffen School of Medicine at the University of California at Los Angeles, 615 Charles E. Young Drive South, Los Angeles, CA 90095, USA
| | - James Wohlschlegel
- Department of Biological Chemistry, David Geffen School of Medicine at the University of California at Los Angeles, 615 Charles E. Young Drive South, Los Angeles, CA 90095, USA
| | - David Bermudes
- Department of Biology, California State University Northridge, Northridge, CA 91330-8303, USA
- Interdisciplinary Research Institute for the Sciences (IRIS), California State University, College of Science and Math, California State University, Northridge, Northridge, CA 91330-8303
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3
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Fritz SE, Henson MS, Greengard E, Winter AL, Stuebner KM, Yoon U, Wilk VL, Borgatti A, Augustin LB, Modiano JF, Saltzman DA. A phase I clinical study to evaluate safety of orally administered, genetically engineered Salmonella enterica serovar Typhimurium for canine osteosarcoma. Vet Med Sci 2016; 2:179-190. [PMID: 29067193 PMCID: PMC5645873 DOI: 10.1002/vms3.32] [Citation(s) in RCA: 48] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
We conducted a prospective phase I study to evaluate safety of an orally administered Salmonella encoding IL‐2 (SalpIL2) in combination with amputation and adjuvant doxorubicin for canine appendicular osteosarcoma. Efficacy was assessed as a secondary measure. The first dose of SalpIL2 was administered to 19 dogs on Day 0; amputation was done after 10 days with chemotherapy following 2 weeks later. SalpIL2 was administered concurrent with chemotherapy, for a total of five doses of doxorubicin and six doses of SalpIL2. There were six reportable events prior to chemotherapy, but none appeared due to SalpIL2. Dogs receiving SalpIL2 had significantly longer disease‐free interval (DFI) than a comparison group of dogs treated with doxorubicin alone. Dogs treated using lower doses of SalpIL2 also had longer DFI than dogs treated using the highest SalpIL2 dose. The data indicate that SalpIL2 is safe and well tolerated, which supports additional testing to establish the potential for SalpIL2 as a novel form of adjuvant therapy for dogs with osteosarcoma.
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Affiliation(s)
- Sara E Fritz
- Animal Cancer Care and Research ProgramUniversity of MinnesotaSt. PaulMinnesotaUSA.,Department of Veterinary Clinical SciencesCollege of Veterinary MedicineUniversity of MinnesotaSt. PaulMinnesotaUSA
| | - Michael S Henson
- Animal Cancer Care and Research ProgramUniversity of MinnesotaSt. PaulMinnesotaUSA.,Department of Veterinary Clinical SciencesCollege of Veterinary MedicineUniversity of MinnesotaSt. PaulMinnesotaUSA.,Masonic Caner CenterUniversity of MinnesotaMinneapolisMinnesotaUSA
| | - Emily Greengard
- Masonic Caner CenterUniversity of MinnesotaMinneapolisMinnesotaUSA.,Department of Pediatric Hematology/OncologySchool of Medicine and Masonic Children's HospitalUniversity of MinnesotaMinneapolisMinnesotaUSA
| | - Amber L Winter
- Animal Cancer Care and Research ProgramUniversity of MinnesotaSt. PaulMinnesotaUSA.,Clinical Investigation CenterCollege of Veterinary MedicineUniversity of MinnesotaSt. PaulMinnesotaUSA
| | - Kathleen M Stuebner
- Animal Cancer Care and Research ProgramUniversity of MinnesotaSt. PaulMinnesotaUSA.,Clinical Investigation CenterCollege of Veterinary MedicineUniversity of MinnesotaSt. PaulMinnesotaUSA
| | - Una Yoon
- Department of Veterinary Clinical SciencesCollege of Veterinary MedicineUniversity of MinnesotaSt. PaulMinnesotaUSA
| | - Vicki L Wilk
- Department of Veterinary Clinical SciencesCollege of Veterinary MedicineUniversity of MinnesotaSt. PaulMinnesotaUSA
| | - Antonella Borgatti
- Animal Cancer Care and Research ProgramUniversity of MinnesotaSt. PaulMinnesotaUSA.,Department of Veterinary Clinical SciencesCollege of Veterinary MedicineUniversity of MinnesotaSt. PaulMinnesotaUSA.,Masonic Caner CenterUniversity of MinnesotaMinneapolisMinnesotaUSA
| | - Lance B Augustin
- Department of SurgeryDivision of Pediatric SurgerySchool of MedicineUniversity of MinnesotaMinneapolisMinnesotaUSA
| | - Jaime F Modiano
- Animal Cancer Care and Research ProgramUniversity of MinnesotaSt. PaulMinnesotaUSA.,Department of Veterinary Clinical SciencesCollege of Veterinary MedicineUniversity of MinnesotaSt. PaulMinnesotaUSA.,Masonic Caner CenterUniversity of MinnesotaMinneapolisMinnesotaUSA.,Center for ImmunologyUniversity of MinnesotaMinneapolisMinnesotaUSA
| | - Daniel A Saltzman
- Masonic Caner CenterUniversity of MinnesotaMinneapolisMinnesotaUSA.,Department of SurgeryDivision of Pediatric SurgerySchool of MedicineUniversity of MinnesotaMinneapolisMinnesotaUSA.,Center for ImmunologyUniversity of MinnesotaMinneapolisMinnesotaUSA
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Abstract
Attenuated Salmonella vaccines can be administered orally to deliver recombinant antigens to mucosal surfaces inducing a protective immune response against a variety of targeted pathogens. A number of exciting new approaches and technologies for attenuated Salmonella vaccines have been developed recently. However, a disconnect remains between results obtained with mice in preclinical studies and results obtained in human clinical trials. This is due to an incomplete understanding of Salmonella Typhi interactions with human hosts and inadequate animal models available for study. In this review, the authors describe recent progress in identifying important differences underlying S. Typhi-host interactions, the development of novel approaches to vaccine design and six recent clinical trials evaluating Salmonella-vectored vaccines.
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Affiliation(s)
- Kenneth L Roland
- The Biodesign Institute, Arizona State University, 1001 S. McAllister Avenue, Tempe, AZ 85287-5401, USA
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Guan GF, Zhao M, Liu LM, Jin CS, Sun K, Zhang DJ, Yu DJ, Cao HW, Lu YQ, Wen LJ. Salmonella typhimurium mediated delivery of Apoptin in human laryngeal cancer. Int J Med Sci 2013; 10:1639-48. [PMID: 24155656 PMCID: PMC3805922 DOI: 10.7150/ijms.6960] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/20/2013] [Accepted: 08/30/2013] [Indexed: 01/26/2023] Open
Abstract
An effective cancer therapeutic should target tumours specifically with limited systemic toxicity. Here, we transformed an attenuated Salmonella typhimurium (S. typhimurium) with an Apoptin expressing plasmid into a human laryngeal carcinoma cell line. The expression of the inserted gene was measured using fluorescence and immunoblotting assays. The attenuated S. typhimurium-mediated Apoptin significantly decreased cytotoxicity and strongly increased cell apoptosis through the activation of caspase-3. The process was mediated by Bax, cytochrome c and caspase-9. A syngeneic nude murine tumour model was used to determine the anti-tumour effects of the recombinant bacteria in vivo. Systemic injection of the recombinant bacteria with and without re-dosing caused significant tumour growth delay and reduced tumour microvessel density, thereby extending host survival. Our findings indicated that the use of recombinant Salmonella typhimurium as an Apoptin expression vector has potential cancer therapeutic benefits.
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Affiliation(s)
- Guo-fang Guan
- 1. Department of Otolaryngology, Head and Neck Surgery, The Second Hospital of Jilin University, Changchun 130041, P. R. China
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Inhibitory effects of the attenuated Salmonella typhimurium containing the IL-2 gene on hepatic tumors in mice. J Biomed Biotechnol 2012. [PMID: 23193368 PMCID: PMC3502055 DOI: 10.1155/2012/946139] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
To observe the inhibitory effects of an attenuated S. typhimurium strain carrying IL-2 gene (TPI) on hepatoma cell line (HepG2) and transplanted tumors in mice. TPI, TPG (an attenuated S. typhimurium strain carrying green fluorescent protein gene), and TP (an attenuated S. typhimurium strain) strains were transfected into HepG2 cells. At 48h after transfecting, the transfection rate was 82.58 ± 1.74%. The expression level of IL-2 was (99.5 ± 12.2) ng/1 × 106 cells. Compared with TPG, TP, and normal mouse groups, the proportion of CD4+ T and CD8+ T cells in the blood from the TPI group was higher, the levels of IgM and IgG1 were significantly increased, and the proliferation activity of splenic lymphocyte was significantly stronger. The transplanted tumor weight in the TPI group was significantly smaller than that in the other two groups. The infiltration of lymphocytes increased in the tumor from TPI group mice. TPI was effectively transfected into cancer cells, which expressed the protein of interest. Oral administration of TPI prolonged survival of mice transplanted with hepatoma cell tumours.
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Sorenson BS, Banton KL, Augustin LB, Leonard AS, Saltzman DA. Antioxidant oils and Salmonella enterica Typhimurium reduce tumor in an experimental model of hepatic metastasis. Onco Targets Ther 2011; 4:59-69. [PMID: 21691578 PMCID: PMC3116794 DOI: 10.2147/ott.s17081] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2011] [Indexed: 01/30/2023] Open
Abstract
Fruit seeds high in antioxidants have been shown to have anticancer properties and enhance host protection against microbial infection. Recently we showed that a single oral dose of Salmonella enterica serovar Typhimurium expressing a truncated human interleukin-2 gene (SalpIL2) is avirulent, immunogenic, and reduces hepatic metastases through increased natural killer cell populations in mice. To determine whether antioxidant compounds enhance the antitumor effect seen in SalpIL2-treated animals, we assayed black cumin (BC), black raspberry (BR), and milk thistle (MT) seed oils for the ability to reduce experimental hepatic metastases in mice. In animals without tumor, BC and BR oil diets altered the kinetics of the splenic lymphocyte response to SalpIL2. Consistent with previous reports, BR and BC seed oils demonstrated independent antitumor properties and moderate adjuvant potential with SalpIL2. MT oil, however, inhibited the efficacy of SalpIL2 in our model. Based on these data, we conclude that a diet high in antioxidant oils promoted a more robust immune response to SalpIL2, thus enhancing its antitumor efficacy.
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Affiliation(s)
- Brent S Sorenson
- Department of Surgery, University of Minnesota Medical School, Minneapolis, MN, USA
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