5-fluorouracil, often abbreviated 5-Fu, has been a cornerstone of colorectal cancer treatment for decades. It works by interfering with DNA synthesis, forcing rapidly dividing cells to self-destruct. Yet its clinical performance remains constrained by two persistent problems: limited efficacy in many patients and a spectrum of serious side effects that can force dose reductions or treatment interruptions. These limitations have driven an intensive search for compounds that could sensitize tumors to 5-Fu, allowing lower doses to achieve the same or better effect. The new study positions a bacterial fermentation product as a candidate for exactly that role.

The research team, led by scientists at the Research Institute for Gastroenterology and Liver Diseases at Shahid Beheshti University of Medical Sciences in Tehran, took a systematic approach to comparing three different preparations of L. reuteri, a strain catalogued as IBRC-M 10755. They tested live bacteria, heat-killed bacteria, and the cell-free supernatant, the liquid fraction left behind after the bacteria are removed, which contains the bacterium’s secreted metabolites, proteins, and other molecules. Each preparation was applied to SW-480 cells, a well-established laboratory model of colorectal cancer, both alone and in combination with 5-fluorouracil across concentrations ranging from 1 to 100 micromolar, over 24 to 48 hours.

The results revealed a striking hierarchy among the three preparations. Live L. reuteri, tested at multiplicities of infection of 1, 10, and 100, had no effect on cancer cell viability at any concentration. Heat-killed bacteria at the highest multiplicity of infection, 100, reduced viability by up to 85 percent. The cell-free supernatant, tested at total protein concentrations of 1, 10, 25, and 50 micrograms per milliliter, cut viability by up to 78 percent at 25 micrograms per milliliter and 65 percent at 50 micrograms per milliliter, with statistical significance relative to untreated cells. The pattern suggests that whatever anticancer activity the bacterium possesses resides largely in what it secretes rather than in the living organism itself.

To understand how the supernatant killed the cancer cells, the researchers turned to the molecular machinery of apoptosis. Using quantitative real-time PCR, they measured the expression of five genes that act as regulators and executors of programmed cell death: Bax, a pro-apoptotic member of the Bcl-2 family; Bcl-2, its anti-apoptotic counterpart; and the initiator and executioner caspases 8, 9, and 3. Treatment with live bacteria, heat-killed bacteria at the highest dose, and the supernatant at 25 to 50 micrograms per milliliter all significantly increased the expression of Bax, caspase-3, caspase-8, and caspase-9. The simultaneous engagement of both the extrinsic pathway, signaled through caspase-8, and the intrinsic pathway, signaled through caspase-9, points to a broad activation of the cell’s suicide program rather than a single narrow trigger.

Flow cytometry provided independent confirmation. Cells stained with fluorescent markers that distinguish early apoptotic cells from late apoptotic and necrotic ones showed a dose-dependent increase in apoptosis following supernatant treatment, rising in step with the concentration of total protein applied. This dose-response relationship is an important technical detail, because it indicates a genuine pharmacological effect rather than random toxicity. It also aligns the gene expression data with the functional outcome: cells exposed to higher concentrations of the bacterial supernatant not only talked the apoptotic talk at the RNA level but actually walked the walk, dying at measurably higher rates.

The most clinically intriguing result emerged when the supernatant was combined with 5-fluorouracil. Co-treatment with the cell-free supernatant and a relatively low 10 micromolar dose of the drug produced greater expression of apoptosis-related genes and a higher apoptosis rate than either agent achieved alone. In practical terms, the bacterial metabolites appeared to lower the threshold at which the cancer cells capitulated to the chemotherapy. If this synergy could be reproduced in a living tumor, it might eventually allow oncologists to achieve therapeutic effects with reduced 5-Fu exposure, potentially sparing patients some of the drug’s harsh side effects, which include bone marrow suppression and gastrointestinal toxicity.

The study also examined the inflammatory dimension of the interaction. 5-fluorouracil is known to provoke inflammatory signaling, and chronic inflammation in the tumor microenvironment can undermine treatment. When SW-480 cells received the combined treatment, the drug-induced elevation of messenger RNA for two key inflammatory cytokines, tumor necrosis factor alpha and interleukin-1 beta, was significantly blunted. However, the researchers noted an important caveat: when they measured the actual secreted protein levels of these cytokines by enzyme-linked immunosorbent assay, the reduction was not consistently reflected. This discordance between mRNA and protein levels is a familiar challenge in molecular biology and underscores that transcriptional changes do not always translate cleanly into changes in secreted signaling molecules.

The authors are careful to frame their findings as preliminary. Because the work was conducted entirely in vitro, on a single cell line in culture dishes, it cannot speak to how the supernatant would behave in the far more complex environment of a human tumor, where immune cells, blood supply, and drug metabolism all intervene. The researchers explicitly call for formal combination-index analysis, a mathematical framework for quantifying whether two agents are truly synergistic, additive, or antagonistic, as well as for validation in animal models before any therapeutic claims can be made. The study was funded by the Iran National Science Foundation and the Research Institute for Gastroenterology and Liver Diseases, and the authors declare no competing interests.

Even with those caveats, the study contributes a well-characterized data point to an increasingly active field exploring how probiotic derivatives might complement cancer chemotherapy. The observation that the cell-free supernatant outperformed both live and heat-killed bacteria is particularly noteworthy, because a cell-free preparation would avoid the safety concerns associated with administering live microorganisms to immunocompromised cancer patients. If subsequent work identifies the specific molecules in the L. reuteri supernatant responsible for sensitizing SW-480 cells to 5-fluorouracil, the door could open to standardized, purified adjunct therapies built from the chemistry of the gut microbiome. For now, the message is one of cautious optimism: a familiar probiotic’s secretions, in a petri dish at least, appear to help a fifty-year-old chemotherapy drug finish the job it was designed to do.

Subject of Research: Probiotic-derived cell-free supernatant as an adjunct to 5-fluorouracil chemotherapy in colorectal cancer cells

Article Title: Cell-free supernatant of Limosilactobacillus reuteri enhances 5-fluorouracil-induced apoptosis in SW-480 colorectal cancer cells: an in vitro study

Article References: Saeedi Niasar, M., Sadeghi, A., Tarzemani, S., Safarpour, H., Alipour, S., Ghazvinian, Z., Yazdani, E., & Raeisi, H. (2026). Cell-free supernatant of Limosilactobacillus reuteri enhances 5-fluorouracil-induced apoptosis in SW-480 colorectal cancer cells: an in vitro study. BMC Complementary Medicine and Therapies. https://doi.org/10.1186/s12906-026-05611-z

Image Credits: AI Generated

DOI: 10.1186/s12906-026-05611-z

Keywords: Limosilactobacillus reuteri, 5-fluorouracil, colorectal cancer, apoptosis, cell-free supernatant, probiotics, SW-480 cells, chemotherapy, caspases, Bax, Bcl-2, in vitro study