Luteolin-Treated 4T1 Cell-Derived Exosomes as Novel Antiproliferative Agents In Vitro and In Vivo
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Heidari, Pardis
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Faculty of Veterinary Sciences, SR.C., Islamic Azad University, Tehran, Iran
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Hosseini, Razieh
Department of Veterinary Basic Sciences, SR.C., Islamic Azad University, Tehran, Iran, Tel: +98 9173059160; Fax: +98 21 44865166; E-mail: razieh.hosseini@iau.ac.ir, Hosseini_945@yahoo.com
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Nasihatgar , Nika
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Faculty of Veterinary Sciences, SR.C., Islamic Azad University, Tehran, Iran
Abstract: Background: Breast cancer is the most widespread malignancy among women worldwide. Luteolin, a flavonoid, has demonstrated anti-cancer effects by triggering apoptosis in tumor cells. Exosomes are gaining much attention for cancer therapeutic approaches due to multitude of beneficial effects. This study is aimed to investigate the possible potential of exosomes derived from luteolin-treated 4T1 cells to ameliorate tumor in comparison to luteolin treatment only.
Methods: In this study, 4T1 cell culture was exposed to luteolin. Following exosome extraction, they were characterized using field emission scanning electron microscopy, dynamic light scattering and western blot analysis. MTT assay was performed in order to evaluate cell viability after exposure to different concentrations of luteolin and exosomes. An in vivo breast cancer model was induced via subcutaneous injection of 4T1 cells to the BALB/C mice. After 14 days, tumor volume was measured, and expression of RhoA and ERK mRNAs were quantified by Real Time PCR.
Results: The MTT assay demonstrated that exosomes from luteolin-treated 4T1 cells at a concentration of 320 μg/μl reduced cell viability by approximately 70% in a dose-dependent manner. Tumor volume in the exosome-treated group decreased by 57% relative to the tumor group, while the luteolin-treated group demonstrated a 39% reduction. Furthermore, RhoA gene expression was substantially downregulated in the exosome-treated group, and exosomes were more effective than luteolin in reducing ERK gene expression.
Conclusion: Exosomes derived from luteolin-treated 4T1 cells effectively suppress breast cancer cell growth by reducing 4T1 cell viability and by decreasing tumor volume and downregulating tumor-associated genes RhoA and ERK. These results propose a novel therapeutic strategy for breast cancer, highlighting the promising potential of exosomes as an efficient drug delivery system.
Introduction :
Breast cancer is the leading cause of tumor-related morbidity and mortality among women globally 1. Epidemiological evidence suggests that approximately 90–95% of cancer cases are associated with lifestyle and environmental factors, while only 5–10% are attributable to inherited genetic abnormalities 2. Chemotherapy and radiotherapy remain the mainstay treatment modalities for breast cancer; however, they are frequently associated with significant adverse effects that negatively impact patients’ quality of life 3. Despite substantial advances in technology and pharmaceutical interventions over the past two decades, cancer remains a major global health challenge 4. Consequently, there is a growing effort to develop alternative therapeutic strategies for patients who experience both physical
and psychological complications as a result of conventional treatments.
Natural products and isolated bioactive compounds derived from medicinal plants have been shown to possess both cancer-preventive and therapeutic properties 3. Among these, flavonoids—a diverse class of polyphenolic compounds—have attracted considerable interest in cancer research, particularly in the context of breast malignancies. Flavonoids have been reported to induce apoptosis, inhibit cell viability and proliferation, and modulate critical signaling pathways involved in breast cancer progression 5,6. Luteolin (3′, 4′, 5, 7-tetrahydroxyflavone), a naturally occurring flavonoid abundant in various fruits and vegetables, is widely recognized for its anticancer properties 7. Its thera-peutic effects are primarily attributed to its ability to induce apoptosis in malignant cells and to inhibit tumor cell proliferation in both in vitro and in vivo models 8. Furthermore, luteolin has been shown to induce cell cycle arrest at different stages, thereby effectively limi-ting cancer cell proliferation and metastatic potential 9,10.
Exosomes are small extracellular vesicles, typically 30–150 nm in diameter, that play a crucial role in intercellular communication. They transport a wide range of bioactive molecules, including proteins, nucleic acids, and lipids 11. Moreover, exosomes exhibit several advantageous properties, such as high biocompatibility, low immunogenicity, structural stability, favorable pharmacokinetics, efficient biodistribution, and effec-tive cellular uptake, which make them promising can-didates for anticancer therapies 12. Indeed, exosomes can be engineered to deliver specific cargos that target oncogenic signaling pathways, thereby enhancing their therapeutic efficacy in cancer treatment 13,14. Relying on the aforementioned facts, this study explores the combined impact of luteolin and exosome on prevention of breast cancer progression. In this regard, anti-cancer effect of exosomes derived from luteolin-treated 4T1 cells was evaluated both in vitro using MTT assay and in vivo using tumor volume measurement and expression of RhoA and ERK genes.
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