en Cancer Cancer Original Article Original Article <span style="font-family:Tahoma;"><span style="font-size:12px;"><span style="line-height:107%">Cancer is the second leading cause of death worldwide, surpassed only by cardiovascular diseases. This study investigated the anticancer effects of recombinant Clostridium &alpha;-toxin on breast cancer, both in vitro and in vivo. The entire coding sequence of a codon-optimized &alpha;-toxin was designed, cloned into the pET28a (+) vector, and expressed as recombinant &alpha;-toxin in Escherichia coli (E. coli) BL 21(DE3) cells transformed with the recombinant plasmid. The recombinant &alpha;-toxin was purified using Ni&sup2;⁺ affinity chromatography, and its accuracy and purity were confirmed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The anticancer effects of purified &alpha;-toxin were then assessed in vitro and animal models against MCF-7 breast cancer cells. Protein analysis confirmed the presence of a 48 kDa band corresponding to the recombinant &alpha;-toxin. Additionally, the IC₅₀ values of &alpha;-toxin against MCF-7 cells at 24, 48, and 72 h were 407.3&plusmn;2.392 &mu;g/mL, 287.3&plusmn;5.411 &mu;g/mL, and 258.1&plusmn;4.671 &mu;g/mL, respectively. In vivo, results demonstrated a significant reduction in mean cancer nodule size following &alpha;-toxin treatment (p<0.001). These findings suggest that &alpha;-toxin may serve as a promising candidate for breast cancer therapy.</span></span></span> α-toxin, Clostridium, breast cancer, in vitro, in vivo 404 416 http://ijmcmed.org/browse.php?a_code=A-10-8053-1&slc_lang=en&sid=1 Nahid Rezaei Khozani nahidrezaei653@gmail.com 100319475328460032764 100319475328460032764 Yes Department of Microbiology and Immunology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran. Mohammad Shayesteh Pour shayestehpour_m@kaums.ac.ir 100319475328460032765 100319475328460032765 No Department of Bacteriology and Virology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran. Mina Yekani Mina_yekani71@yahoo.com 100319475328460032766 100319475328460032766 No Dental and Periodontal Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Seyed Hossein Hejazi hejazi@med.mui.ac.ir 100319475328460032767 100319475328460032767 No Skin Diseases and Leishmaniasis Research Center, Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran. Mahmood Saffari Saffari_m@kaums.ac.ir 100319475328460032768 100319475328460032768 No Department of Microbiology and Immunology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran.