{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T09:50:52Z","timestamp":1791798652253,"version":"4.3.4"},"reference-count":31,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T00:00:00Z","timestamp":1789603200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Mol. Biosci."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Colorectal cancer is a leading cause of cancer-related mortality worldwide, underscoring the need for effective therapies with lower toxicity. Probiotic-derived metabolites (postbiotics) have emerged as promising anticancer components with immunomodulatory and antiproliferative effects.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Cell-Free Supernatant (CFS) from\n                      <jats:italic>Lactobacillus rhamnosus<\/jats:italic>\n                      GG, produced under microaerophilic conditions and characterized by GC-MS, was tested against HT-29 colorectal adenocarcinoma cells. Cytotoxicity was evaluated by MTT assay, apoptosis following Annexin V-FITC\/PI flow cytometry, and cell cycle distribution.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Growth under microaerophilic conditions yielded a sterile CFS that was subsequently profiled by GC-MS. Cytotoxicity was assessed by MTT assay, while apoptosis and cell-cycle distribution were evaluated by flow cytometry. GC-MS showed more than 65 chromatographic peaks, and 28 major compounds were tentatively identified by NIST-library matching; diketopiperazines accounted for approximately 45% of the total identified peak area. The study-strain CFS reduced HT-29 cell viability in a concentration-dependent manner, with an estimated IC50 of 14.2 micrograms\/mL expressed as total lyophilized solids. Annexin V-FITC\/PI flow cytometry showed 47.6 \u00b1 2.9% early apoptotic, 2.3 \u00b1 0.8% late apoptotic, 2.3 \u00b1 0.6% necrotic, and 47.8 \u00b1 3.2% viable cells after treatment. Treated cells were distributed as 47.3% G1, 38.9% S, 3.22% G2\/M, and 2.60% sub-G1; because reliable quantitative control data were unavailable, phase-specific arrest could not be established.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>The study-strain CFS showed concentration-dependent in vitro cytotoxic and pro-apoptotic activity against HT-29 cells. The activity may be associated with the partial metabolite profile identified by GC-MS, but specific active compounds and molecular pathways were not established. The cell-cycle findings are descriptive because reliable quantitative control data were unavailable. Further studies using processed-medium controls, non-transformed colonic cells, additional cancer cell lines, orthogonal viability assays, mechanistic markers, and activity-guided fractionation are required.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fmolb.2026.1868859","type":"journal-article","created":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T04:29:17Z","timestamp":1789619357000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["In vitro cytotoxic and pro-apoptotic effects of Lactobacillus rhamnosus GG cell-free supernatant on HT-29 colorectal cancer cells"],"prefix":"10.3389","volume":"13","author":[{"given":"F. Fareedhul","family":"Fahmitha","sequence":"first","affiliation":[{"name":"PG and Research Department of Microbiology, Sacred Heart College (Autonomous)","place":["Tirupattur, Tamil Nadu, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manal Mowaffaq","family":"Mohammed","sequence":"additional","affiliation":[{"name":"Department of Biology, College of Education for Pure Sciences, University of Al-Hamdaniya","place":["Al-Hamdaniya, Iraq"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Prithviraj","family":"Karak","sequence":"additional","affiliation":[{"name":"Department of Physiology, Bankura Christian College","place":["Bankura, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Estabraq Ali","family":"Hameed","sequence":"additional","affiliation":[{"name":"Scientific Affairs Department, University of Al-Hamdaniya","place":["Nineveh, Iraq"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Basim Mohammed","family":"Hanon","sequence":"additional","affiliation":[{"name":"Department of Microbiology, College of Veterinary Medicine, University of Wasit","place":["Wasit, Iraq"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Israa M.","family":"Essa","sequence":"additional","affiliation":[{"name":"Department of Public Health, College of Veterinary Medicine, University of Basrah","place":["Basra, Iraq"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hasanain A. J.","family":"Gharban","sequence":"additional","affiliation":[{"name":"Department of Internal and Preventive Veterinary Medicine, College of Veterinary Medicine, University of Wasit","place":["Kut, Iraq"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"K.","family":"Gayathri","sequence":"additional","affiliation":[{"name":"PG and Research Department of Microbiology, Sacred Heart College (Autonomous)","place":["Tirupattur, Tamil Nadu, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"P.","family":"Saranraj","sequence":"additional","affiliation":[{"name":"PG and Research Department of Microbiology, Sacred Heart College (Autonomous)","place":["Tirupattur, Tamil Nadu, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M.","family":"Manigandan","sequence":"additional","affiliation":[{"name":"PG and Research Department of Microbiology, Sacred Heart College (Autonomous)","place":["Tirupattur, Tamil Nadu, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Afsona","family":"Parveen","sequence":"additional","affiliation":[{"name":"School of Allied and Healthcare Sciences, Centurion University of Technology and Management","place":["Jatni, Odisha, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sayandeep K.","family":"Das","sequence":"additional","affiliation":[{"name":"Department of Pathology, Shri B. M. Patil Medical College Hospital and Research Centre, BLDE (Deemed to be University)","place":["Vijayapura, Karnataka, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,17]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"5983","DOI":"10.1080\/10408398.2021.1895061","article-title":"The biological activities of postbiotics in gastrointestinal disorders","volume":"62","author":"Abbasi","year":"2022","journal-title":"Crit. Rev. Food Sci. Nutr."},{"key":"B2","doi-asserted-by":"publisher","first-page":"337","DOI":"10.1126\/science.1198469","article-title":"Induction of colonic regulatory T cells by indigenous Clostridium species","volume":"331","author":"Atarashi","year":"2011","journal-title":"Science"},{"key":"B3","doi-asserted-by":"publisher","first-page":"e3096","DOI":"10.31661\/gmj.v12i.3096","article-title":"Anticancer activity of postbiotic mediators derived from Lactobacillus rhamnosus GG and Lactobacillus reuteri on acute lymphoblastic leukemia cells","volume":"12","author":"Banakar","year":"2023","journal-title":"Galen. Med. J."},{"key":"B4","doi-asserted-by":"publisher","first-page":"603","DOI":"10.3389\/fphar.2017.00603","article-title":"LactobacillusrhamnosusGG: an overview to explore the rationale of its use in cancer","volume":"8","author":"Banna","year":"2017","journal-title":"Front. Pharmacol."},{"key":"B5","doi-asserted-by":"publisher","first-page":"1484","DOI":"10.1007\/s12602-022-10002-0","article-title":"Exploration of antidiabetic, cholesterol-lowering, and anticancer upshot of probiotic bacterium Pediococcuspentosaceus OBK05 strain of buttermilk","volume":"15","author":"Bhukya","year":"2023","journal-title":"Probiotics Antimicrob. Proteins"},{"key":"B6","doi-asserted-by":"publisher","first-page":"1608897","DOI":"10.3389\/fcimb.2025.1608897","article-title":"Biotherapeutic potential of different fractions of cell-free supernatants from Lactobacillusrhamnosusagainst Pseudomonas aeruginosa","volume":"15","author":"Bianchi","year":"2025","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"B7","doi-asserted-by":"publisher","first-page":"S1","DOI":"10.1097\/mcg.0000000000001170","article-title":"Thirty years of LactobacillusrhamnosusGG: a review","volume":"53","author":"Capurso","year":"2019","journal-title":"J. Clin. Gastroenterol."},{"key":"B8","doi-asserted-by":"publisher","first-page":"304","DOI":"10.1111\/j.1365-2672.2010.04885.x","article-title":"Apoptosis-inducing effect of diketopiperazine disulfides produced by Aspergillus sp. KMD 901 isolated from marine sediment on HCT116 colon cancer cell lines","volume":"110","author":"Choi","year":"2011","journal-title":"J. Applied Microbiology"},{"key":"B9","doi-asserted-by":"publisher","first-page":"245","DOI":"10.34172\/bi.2021.32","article-title":"Cell cycle arrest and anti-cancer potential of probiotic Lactobacillusrhamnosusagainst HT-29 cancer cells","volume":"11","author":"Dehghani","year":"2021","journal-title":"BioImpacts"},{"key":"B10","doi-asserted-by":"publisher","first-page":"1391039","DOI":"10.3389\/fmicb.2024.1391039","article-title":"Antimicrobial and antibiofilm effects of cyclic dipeptide-rich fraction from Lactobacillus plantarum loaded on graphene oxide nanosheets","volume":"15","author":"Dezaki","year":"2024","journal-title":"Front. Microbiol."},{"key":"B11","doi-asserted-by":"publisher","first-page":"66","DOI":"10.3390\/microorganisms7030066","article-title":"Probiotics and colon cancer","volume":"7","author":"Drago","year":"2019","journal-title":"Microorganisms"},{"key":"B12","doi-asserted-by":"publisher","first-page":"501","DOI":"10.3748\/wjg.v22.i2.501","article-title":"Gut microbiota imbalance and colorectal cancer","volume":"22","author":"Gagni\u00e8re","year":"2016","journal-title":"World J. Gastroenterol."},{"key":"B13","doi-asserted-by":"publisher","first-page":"12827","DOI":"10.3390\/ijms222312827","article-title":"The MTT assay: utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis","volume":"22","author":"Ghasemi","year":"2021","journal-title":"Int. J. Mol. Sci."},{"key":"B14","doi-asserted-by":"publisher","first-page":"1732","DOI":"10.3390\/cancers14071732","article-title":"Colorectal cancer: a review of carcinogenesis, global epidemiology, current challenges, risk factors, preventive and treatment strategies","volume":"14","author":"Hossain","year":"2022","journal-title":"Cancers"},{"key":"B15","doi-asserted-by":"publisher","first-page":"111565","DOI":"10.1016\/j.cbi.2025.111565","article-title":"The combined application of cis-cyclo (L-Phe-L-Pro) with antimicrobial proline-based 2, 5-diketopiperazines significantly enhances anti-breast cancer activity by selectively targeting cancer stem cells","volume":"417","author":"Liu","year":"","journal-title":"Chemico-Biological Interact."},{"key":"B16","doi-asserted-by":"publisher","first-page":"e2408007","DOI":"10.4014\/jmb.2408.08007","article-title":"Optimization of the culture conditions of lactic acid bacteria for antimicrobial activity and mass production of Cyclic dipeptides","volume":"35","author":"Liu","year":"","journal-title":"J. Microbiology Biotechnology"},{"key":"B17","first-page":"113","article-title":"HT29 cell line","volume-title":"The Impact of Food Bioactives on Health: In Vitro and Ex Vivo Models","author":"Mart\u00ednez-Maqueda","year":"2015"},{"key":"B18","doi-asserted-by":"publisher","first-page":"7630","DOI":"10.1128\/JB.01287-09","article-title":"Complete genome sequence of the probiotic LactobacillusrhamnosusATCC 53103","volume":"191","author":"Morita","year":"2009","journal-title":"J. Bacteriol."},{"key":"B19","doi-asserted-by":"publisher","first-page":"736343","DOI":"10.3389\/fmicb.2021.736343","article-title":"Cell-free supernatant of Odoribactersplanchnicus isolated from human feces exhibits anti-colorectal cancer activity","volume":"12","author":"Oh","year":"2021","journal-title":"Front. Microbiol."},{"key":"B20","doi-asserted-by":"publisher","first-page":"872","DOI":"10.22088\/IJMCM.BUMS.14.3.872","article-title":"Evaluation of Lactobacillus delbrueckii supernatant effects on cell cycle regulation, apoptosis induction, and gene expression in HT-29 colorectal cancer cells","volume":"14","author":"Pahlaviani","year":"2025","journal-title":"Int. J. Mol. Cell. Med."},{"key":"B21","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1385\/1-59259-811-0:301","article-title":"Analysis of cell cycle by flow cytometry","volume-title":"Checkpoints Controls Cancer Methods in Molecular Biology","author":"Pozarowski","year":"2004"},{"key":"B22","doi-asserted-by":"publisher","first-page":"1787","DOI":"10.1080\/10408398.2020.1765310","article-title":"Molecular mechanisms of postbiotics in colorectal cancer prevention and treatment","volume":"61","author":"Rad","year":"2021","journal-title":"Crit. Rev. Food Sci. Nutr."},{"key":"B23","doi-asserted-by":"publisher","first-page":"195","DOI":"10.1186\/s12967-023-04036-3","article-title":"Lactobacillus rhamnosusGG cell-free supernatant as a novel anti-cancer adjuvant","volume":"21","author":"Salemi","year":"2023","journal-title":"J. Transl. Med."},{"key":"B24","doi-asserted-by":"publisher","first-page":"S7","DOI":"10.1186\/1475-2859-13-S1-S7","article-title":"Towards a better understanding of LactobacillusrhamnosusGG-host interactions","volume":"13","author":"Segers","year":"2014","journal-title":"Microb. Cell Factories"},{"key":"B25","doi-asserted-by":"publisher","first-page":"110","DOI":"10.1080\/01635581.2019.1615514","article-title":"Antigenotoxicity and cytotoxic potentials of metabiotics extracted from isolated probiotic, LactobacillusrhamnosusMD 14 on Caco-2 and HT-29 human colon cancer cells","volume":"72","author":"Sharma","year":"2020","journal-title":"Nutr. Cancer"},{"key":"B26","doi-asserted-by":"publisher","first-page":"2679","DOI":"10.3390\/cancers12092679","article-title":"Adjuvant chemotherapy for stage III colon cancer","volume":"12","author":"Taieb","year":"2020","journal-title":"Cancers"},{"key":"B27","doi-asserted-by":"publisher","first-page":"1823","DOI":"10.1042\/BCJ20160510","article-title":"Introduction to the human gut microbiota","volume":"474","author":"Thursby","year":"2017","journal-title":"Biochem. Journal"},{"key":"B28","doi-asserted-by":"publisher","first-page":"38","DOI":"10.3390\/cancers11010038","article-title":"Gut Microbiota and cancer: from pathogenesis to therapy","volume":"11","author":"Vivarelli","year":"2019","journal-title":"Cancers"},{"key":"B29","doi-asserted-by":"publisher","first-page":"22","DOI":"10.1038\/s41392-020-0116-z","article-title":"Comprehensive review of targeted therapy for colorectal cancer","volume":"5","author":"Xie","year":"2020","journal-title":"Sig Transduct. Target Ther."},{"key":"B30","doi-asserted-by":"publisher","first-page":"2639","DOI":"10.1021\/acs.jafc.9b06909","article-title":"Effect of Lactobacillus acidophilus CICC 6074 S-layer protein on colon cancer HT-29 cell proliferation and apoptosis","volume":"68","author":"Zhang","year":"2020","journal-title":"J. Agricultural Food Chemistry"},{"key":"B31","doi-asserted-by":"publisher","first-page":"898240","DOI":"10.3389\/fbioe.2022.898240","article-title":"Anti-gastric cancer activity of the cell-free culture supernatant of serofluid dish and Lactiplantibacillus plantarum YT013","volume":"10","author":"Zhang","year":"2022","journal-title":"Front. Bioeng. Biotechnol."}],"updated-by":[{"DOI":"10.3389\/fmolb.2026.2005518","type":"correction","label":"Correction","source":"publisher","updated":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T00:00:00Z","timestamp":1791763200000}}],"container-title":["Frontiers in Molecular Biosciences"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmolb.2026.1868859\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T09:36:21Z","timestamp":1791797781000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmolb.2026.1868859\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,17]]},"references-count":31,"alternative-id":["10.3389\/fmolb.2026.1868859"],"URL":"https:\/\/doi.org\/10.3389\/fmolb.2026.1868859","relation":{},"ISSN":["2296-889X"],"issn-type":[{"value":"2296-889X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,17]]},"article-number":"1868859"}}