Page 484 - AWSAR 2.0

460 || AWSAR Awarded Popular Science Stories - 2019
These significant drawbacks are major limitations that increase human morbidity and mortality. We tried to address this problem using a bacteria Salmonella typhimurium for targeting and killing colon cancer cells.
S. typhimurium could overcome the aforementioned limitations of existing therapy by virtue of the following properties: (1) the ability to reach target tumors in the intestine even if administered by the oral route; (2) preferential growth in a hypoxic tumor environment; (3) Bacteria, being motile, can actively swim and spread in tumor tissues, and hence they have good cancer cell penetration; (4) low toxicity in normal cells; and (5) ease in genetic manipulations.
After registering myself for Doctor of Philosophy (PhD) in Microbiology, I started to work on colon cancer cells using Caco- 2 cell line in the Animal Tissue Culture Lab, Scientific Research Centre (SRC), Mulund, Mumbai. Caco-2 cells are
obtained from human epithelial
colon carcinoma and are used
as a model for studies on the
human intestinal tract. Caco-2
cells are grown in tissue culture
flasks and plates using special
incubators, providing 5%
carbon dioxide with 95% air for
the proper growth of cells.
Salmonella is regarded as a bad bug of the intestine as it causes infections. Having stated this, the question arises How can the same Salmonella be used to kill tumor cells?
In order to achieve this, we
genetically manipulated Salmonella in such a way that it became suitable for killing cancer cells, but did not cause major infections. The present study was conducted to develop genetically engineered novel S. typhimurium strain, which was strongly invasive and
destructive only for colon cancer cells (Caco- 2). For this, S. typhimurium NCIM 2501 was mutagenized by exposing it to a mutagenic agent. The mutagenic agent changed the DNA structure of Salmonella in such a way that it stopped producing two amino acids on its own and became dependent (one of the prime reasons S. typhimurium got attracted toward tumor cells) on the external source (tumor cells, which produce these amino acids abundantly in comparison to normal intestinal cells). This phenomenon is called auxotrophy, and a new auxotrophic strain of S. typhimurium, rp2 (amino acid auxotroph), was developed. Mutations in S. typhimurium were induced by a mutagenic agent ethyl methanesulfonate (EMS). Freshly prepared EMS was added to the Salmonella typhimurium culture, and the reaction was set for 30 min to induce mutations. The EMS-treated culture was transferred to the nutrient broth to detect induced mutations.
Later, the mutations (which altered amino acid production) were identified by carrying out replica plating on minimal agar plates. Auxotroph rp2, mutated in proline and arginine genes (hence requiring proline and arginine from other sources for its growth), was identified. This strain penetrated and multiplied inside colon adenocarcinoma (Caco-2) cells and induced apoptosis (self-destruction of cancer cells). To summarize, a novel S. typhimurium strain rp2 was developed by inducing mutations. The strain was
selectively dependent on cancer cells for its nutritional requirements, got attracted to cancer cells, invaded them, multiplied, and killed cancer cells.
The next step in this study was to design a technique that could enable us to look inside
Radiotherapy (RT) and chemotherapy (CT) are two mainstay therapies in oncology. Although these therapies are widely used, a major limitation lies in the fact that hypoxic colorectal cancer cells are resistant to RT and CT (two times more resistant to ionizing radiations).