Clostridium as a Cancer Therapy

This student page has not been curated.

A photomicrograph of Clostridium botulinum bacteria.The bacterium C. botulinum causes the rare, but serious paralytic illness Botulism. [1]

Clostridium is one of the largest prokaryotic genera and consists of a diverse range of obligatory anaerobic bacteria. There are over 100 species of Clostridium, including common free-living bacteria and pathogens. Clostridium bacteria are Gram-positive and rod-shaped. All of the members of the genus, with the exception of Clostridium perfringens, are motile and flagellated and form oval or spherical endospores that distend from the cell.

Most Clostridium species are nonpathogenic, and many are harmless and can be found in the soil. Some species have applications in bioremediation and wastewater treatment. Nonpathogenic species include Clostridium acetobutylicum and Clostridium beijerinckii, both of which have industrial applications. These solventogenic clostridia are used in acetone, butanol and isopropanol fermentation.

The ability of Clostridium bacteria to produce endospores that are highly resistant to harsh environmental conditions and can withstand high temperature, disinfectants, and low-energy radiation, makes many pathogenic Clostridium bacteria a major health concern and of clinical significance. Several pathogens-producing potent toxins part of this genera include C. tetani, C. botulinum, C. difficile, and C. perfringens. C. tetani is the causative agent of tetanus, and Clostridium botulinum is related to food poisoning and causes botulism. Other members of the Clostridium genus include Clostridium perfringens which can infect wounds and cause gas gangrene, and Clostridium difficile which can cause diarrhea and more serious intestinal conditions such as colitis. Clostridium difficile is one of the most common causes of infection of the large bowel. Another species, Clostridium novyi has been associated with an outbreak of serious illness and death amongst intravenous drug users. Despite pathogenic properties, research has shown that Clostridium novyi seems to be very promising in the fight against cancer as a bacteriolytic anti-cancer agent. It has been well known that severe bacterial infections can sometimes cure cancer patients once the bacterium’s pathogenicity has already been eliminated.

Anticancer Properties

Species of Clostridium bacteria are notable for their ability to selectively target and lyse tumor cells growing in hypoxic environments due to their anaerobic nature. Hypoxia is a major cause of resistance to ionizing radiation and the dose required to kill hypoxic cells is up to 3 times higher compared with the same amount of well-oxygenated cells. Cancerous tumors marked by uncontrolled growth and low oxygen levels that can provide a niche for anaerobic bacteria such as Clostridium, and intravenous administration of spores from nonpathogenic strains of Clostridium bacteria can be used to target hypoxic or necrotic solid tumors. With Clostridium-directed tumor therapy, advantages include tumor selectivity and safety of using nonpathogenic clostridia in cancer treatment.

Current Forms of Cancer Treatments

Traditional cancer therapies
Current and traditional cancer therapies involve regulating of cell division and growth most commonly at the genetic level. Conventional methods involve delivering tumor suppressor genes to cancer cells, delivering genes that activate toxic products to kills tumor cells and their neighbors, introducing defective genes that cause cell death, and directly attacking tumor cells and harnessing the immune responses to tumor antigens. However, the primary problem with such traditional methods is the lack of tumor specificity. Disadvantages of both viral and non-viral gene delivery methods include but are not limited to low transfection transduction levels, immune responses that may hinder repeated administrations of the vector, potential random integrations from retroviruses that could have carcinogenic effects, and a preferential infection of only non-dividing cells. Most drugs and current treatments are effective against rapidly dividing tumor cells but not against hypoxic regions in which cell proliferation is decreased.

Bacteria-directed cancer therapies
The introduction of bacterial applications in cancer therapy is not an entirely novel idea as studies have shown that bacterial infections can sometimes cure cancer patients once the bacterium’s pathogenicity has already been eliminated. Bacteria-directed cancer treatment has advantages over the traditional cancer therapies. Some nonrecombinant bacteria already exert inherent antitumor activities, and recombinant bacteria can be used as vectors to produce protein of therapeutic interest in tumor environments as an alternative to gene therapy. Through the bacterial-directed protein delivery system, there will be an efficient distribution of vector throughout the tumor mass with sufficient transfection levels and transient gene expression. This method prevents the random insertion of foreign DNA into the genome and the transfection of tumor cells with therapeutic genes that may instead enhance antitumor properties. The bacteria can then be inactivated at any moment during therapy by administering antibiotics.

Difficulties in delivering anticancer chemotherapeutics to the poorly vascularized, hypoxic regions of tumors can be overcome with bacteria-based tumor specific therapies using anaerobic bacteria. Anaerobic bacteria can specifically target the hypoxic or necrotic regions of the tumors and either directly exert innate oncolytic effects or act as a vector in introducing locally expressed therapeutic proteins.

Research and Studies

Clostridium novyi is a highly mobile spore-forming organism that is extremely sensitive to oxygen. Injection of C novyi takes advantage of the hypoxic nature of rapidly growing tumors and thus serves as a selective tumor killer.

To create a nonpathogenic strain of the target bacteria, researchers at Johns Hopkins University engineered the bacteria to get rid of its toxin gene. This way, once injected into the host tissue, the host would survive, but the cancerous tumors would be targeted. When mice were injected with the engineered nonpathogenic strain of C. novyi, the bacteria were found sporulating within the tumors within 16 hours with no migration to other healthy organs of the body. 100 million spores can be injected into a cancer patient causing mass proliferation of the bacteria in the malignant tumor but no harm to the host. It was found that pathogenic species from the genus Clostridium may be responsible for contamination of the materials used for illicit drug use involving injection. C. novyi type A has been targeted as one of these species in an outbreak that involved high mortality rates among drug users who injected heroin extravascularly. 16S rDNA sequence data was used to identify C. novyi as the pathogen responsible for these mortalities. The 2.55 Mb genomic sequence of C. novyi has recently been determined. From this determination, a new type of transposition and 139 genes which do not have homologs in other bacteria have been identified. Knowing C. novyi’s genomic sequence has allowed detection of transcripts in vitro and infections of tumors in vivo. From this, it was found that C. novyi-NT spores contain mRNA, and that the spores’ transcripts are very different from those transcripts found in the bacterium’s vegetative form.

[[ |thumb|300px|right|Effect of C. novyi-NT plus external beam radiation on various transplanted tumor models after the indicated treatments. a, HCT116; b, HuCC-T1; c, B16; d, HT29. Irradiated mice received 2 Gy/day for 5 days. Spores were administered after the third dose of radiation. Tumor growth curves are color-coded: light blue, untreated control; purple, C. novyi-NT spores alone; green, radiation (10 Gy) alone; red, radiation (10 Gy) plus C. novyi-NT spores. Each group contained at least six mice. Student's t test was performed by using the linear regression slope for each individual mouse. Each arm was compared with the combination of C. novyi-NT plus 10 Gy, and all were statistically significant in a–c. The P values for comparison between irradiation alone and irradiation plus C. novyi-NT were as follows: a, P < 0.0001; b, P < 0.05; and c, P < 0.05. Chetan Bettegowda et al. PNAS 2003;100:15083-15088 Copyright © 2003, The National Academy of Sciences[2]]]

Previous attempts to treat advanced cancers using sterile filtrates of C. histolyticum produced proteolytic enzymes that degraded cancerous tissues without affecting normal tissue. Spores germinated in tumors after inoculation of C. histolyticum spores into mice with transplanted sarcomas and observed lysis of tumor tissues. Intravenously injected spores of nonpathogenic strains of Clostridium species in rodent tumor models with transplanted tumors resulted in softening of tumors and tumor cell death primarily in the tumor center. However, tumor regrowth occurred overtime from the remaining outer rim of viable cells. To specifically attack the remaining cancer cells at the tumor periphery, combination therapies have been studied to improve the therapeutic outcomes.

C. sporogenes was transformed with the E. coli codA gene encoding cytosine deaminase. When spores of this recombinant strain were administered to mice bearing SCCVII tumors followed by 5-fluorocytosine injection, a significant antitumor effect was observed. C. sporogenes was also engineered to produce nitroreductase by combining it with the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid administered 4 h after injection of spores. These recombinants showed a fourfold increase in tumor colonization and in combination with the respective prodrugs, complete tumor reduction was observed.

Administration of C. novyi-NT spores to nude mice with various human xenografts worked in combination with different amounts of radiotherapy. The best result was observed with brachytherapy, as a single dose of spores combined with this treatment resulted in 100% cure in mice. When immunocompetent tumor-bearing mice were treated with C. novyi-NT spores alone, only 30% of the animals exhibited complete tumor regression.

Clostridia is still currently under investigation for cancer therapy uses in humans. Recently, Clostridium species have been engineered to produce proteins of interest in antitumor therapy. Although Clostridia is not yet completely efficient to sufficiently control tumor growth, recent combination treatments have improved in preclinical antitumor responses. Combination of clostridial spores administration and vascular targeting agents increase tumor colonization and concentrations of degradative enzymes and therapeutic proteins in the tumor and also reduce the probability of incomplete tumor cell death or tumor regrowth. Ongoing studies are searching for the best combinations or recombinant cancer therapies using a bacterial approach.

Other bacterial treatments

Other anerobic bacterial species under investigation for cancer therapy include Bifodobacterium, Streptococcus pyogenes, and Salmonella Typhimurium . Streptococcus pyogenes and Salmonella typhimurium have been used with some success in clinical trials.

References

1. Barbé, S., Van Mellaert, L., & Anné, J. (2006). The use of clostridial spores for cancer treatment. Journal of applied microbiology, 101(3), 571-578.[3]
2. Bettegowda, C., Huang, X., Lin, J., Cheong, I., Kohli, M., Szabo, S. A., ... & Zhou, S. (2006). The genome and transcriptomes of the anti-tumor agent Clostridium novyi-NT. Nature biotechnology, 24(12), 1573-1580. [4]
3. Cheong, I., Huang, X., Bettegowda, C., Diaz, L. A., Kinzler, K. W., Zhou, S., & Vogelstein, B. (2006). A bacterial protein enhances the release and efficacy of liposomal cancer drugs. Science, 314(5803), 1308-1311.[5]
4. Connell, H. C. (1935). The study and treatment of cancer by proteolytic enzymes: preliminary report. Canadian Medical Association journal, 33(4), 364.[6]
5. Dang, L. H., Bettegowda, C., Huso, D. L., Kinzler, K. W., & Vogelstein, B. (2001). Combination bacteriolytic therapy for the treatment of experimental tumors. Proceedings of the National Academy of Sciences, 98(26), 15155-15160.[7]
6. Lemmon, M. J., Van Zijl, P., Fox, M. E., Mauchline, M. L., Giaccia, A. J., Minton, N. P., & Brown, J. M. (1997). Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment. Gene therapy, 4(8), 791-796.[8]
7. Liu, S. C., Minton, N. P., Giaccia, A. J., & Brown, J. M. (2002). Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis. Gene therapy, 9(4), 291-296.[http://www.nature.com/gt/journal/v9/n4/pdf/3301659a.pdf
8. Malmgren, R. A., & Flanigan, C. C. (1955). Localization of the vegetative form of Clostridium tetani in mouse tumors following intravenous spore administration. Cancer research, 15(7), 473-478.[9]
9. Minton, N. P. (2003). Clostridia in cancer therapy. Nature Reviews Microbiology, 1(3), 237-242.[10]
10. Patyar, S., Joshi, R., Byrav, D. S., Prakash, A., Medhi, B., & Das, B. K. (2010). Review Bacteria in cancer therapy: a novel experimental strategy. J Biomed Sci, 17(1), 21-30.[11]
11. Pawelek, J. M., Low, K. B., & Bermudes, D. (2003). Bacteria as tumour-targeting vectors. The lancet oncology, 4(9), 548-556.[12]
12. Plomp, M., McCaffery, J. M., Cheong, I., Huang, X., Bettegowda, C., Kinzler, K. W., ... & Malkin, A. J. (2007). Spore coat architecture of Clostridium novyi NT spores. Journal of bacteriology, 189(17), 6457-6468.[13]
13. Roberts, N. J., Zhang, L., Janku, F., Collins, A., Bai, R. Y., Staedtke, V., ... & Zhou, S. (2014). Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses. Science translational medicine, 6(249), 249ra111-249ra111. [14]
14. Ryan, R. M., Green, J., & Lewis, C. E. (2006). Use of bacteria in anti-cancer therapies. Bioessays, 28(1), 84-94.[15]
15. Thiele, E. H., Arison, R. N., & Boxer, G. E. (1964). Oncolysis by clostridia. III. Effects of clostridia and chemotherapeutic agents on rodent tumors. Cancer research, 24(2 Part 1), 222-233.[16]
16. Van Mellaert, L., Barbé, S., & Anné, J. (2006). Clostridium spores as anti-tumour agents. TRENDS in Microbiology, 14(4), 190[17]

Edited by Anh Tran, a student of Nora Sullivan in BIOL168L (Microbiology) in The Keck Science Department of the Claremont Colleges Spring 2014.