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Purpose <p>Despite therapeutic advances, breast cancer treatment remains limited by non-selective toxicity, highlighting the need for targeted agents. We previously introduced DT386-BR2 as a selective cytotoxic fusion protein. This study aimed to produce soluble DT386-BR2 using an intein-mediated purification system and to evaluate its cytotoxicity and antitumor efficacy in vitro and in a nude mouse xenograft model.</p> Methods <p>The gene encoding DT386-BR2 was cloned into the pTWIN-1 vector and expressed in <i>E. coli</i> BL21 (DE3). Following induction with IPTG (1 mM, 15&#xa0;°C, 24&#xa0;h), the protein was purified using the IMPACT system. Cytotoxicity against MCF-7 breast cancer cells was determined via MTT assay. In vivo efficacy and safety were assessed in nude mice bearing MCF-7 xenografts. Tumor and vital organs were examined histopathologically using H&amp;E and TUNEL staining.</p> Results <p>Soluble DT386-BR2 was successfully expressed and purified. The protein exhibited a potent cytotoxic effect on MCF-7 cells, with an IC50 of 199 nM. In vivo, DT386-BR2 significantly reduced tumor volume compared with controls (<i>P</i> &lt; 0.05), without evidence of metastasis or abnormalities in heart, lungs, kidneys, or spleen. Histological analysis, however, revealed moderate hepatic necrosis in treated mice.</p> Conclusion <p>DT386-BR2 effectively suppressed breast tumor growth in vitro and in vivo, with limited off-target toxicity aside from hepatic effects. These findings highlight DT386-BR2 as a promising targeted therapeutic candidate for breast cancer and warrant further optimization and preclinical safety studies.</p>

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Intein-Mediated Production and Anti-tumor Evaluation of DT386-BR2 in Animal Model of Human Breast Cancer

  • 期刊论文

  • 发表日期：

    2026年1月12日

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  • Sajad Maghareh-Dehkordi,
  • Taha Safi Ghahderijani,
  • Melika Kolahdoozan,
  • Fatemeh Moazen,
  • Ardeshir Talebi,
  • Fatemeh Shafiee,
  • Ali Jahanian-Najafabadi

摘要

Purpose

Despite therapeutic advances, breast cancer treatment remains limited by non-selective toxicity, highlighting the need for targeted agents. We previously introduced DT386-BR2 as a selective cytotoxic fusion protein. This study aimed to produce soluble DT386-BR2 using an intein-mediated purification system and to evaluate its cytotoxicity and antitumor efficacy in vitro and in a nude mouse xenograft model.

Methods

The gene encoding DT386-BR2 was cloned into the pTWIN-1 vector and expressed in E. coli BL21 (DE3). Following induction with IPTG (1 mM, 15 °C, 24 h), the protein was purified using the IMPACT system. Cytotoxicity against MCF-7 breast cancer cells was determined via MTT assay. In vivo efficacy and safety were assessed in nude mice bearing MCF-7 xenografts. Tumor and vital organs were examined histopathologically using H&E and TUNEL staining.

Results

Soluble DT386-BR2 was successfully expressed and purified. The protein exhibited a potent cytotoxic effect on MCF-7 cells, with an IC50 of 199 nM. In vivo, DT386-BR2 significantly reduced tumor volume compared with controls (P < 0.05), without evidence of metastasis or abnormalities in heart, lungs, kidneys, or spleen. Histological analysis, however, revealed moderate hepatic necrosis in treated mice.

Conclusion

DT386-BR2 effectively suppressed breast tumor growth in vitro and in vivo, with limited off-target toxicity aside from hepatic effects. These findings highlight DT386-BR2 as a promising targeted therapeutic candidate for breast cancer and warrant further optimization and preclinical safety studies.