ABS 046: Anti-Proliferative Effects of Berberine on A-673 Ewing Sarcoma Cells: A Dose- and Time-Dependent Analysis

Nicolas Caputo ¹, Chad Barber Ph.D ¹

¹ California Lutheran University

The Van Wickle Journal (2026) Volume 2, ABS046

Introduction: Cancer is one of the leading causes of death globally, a burden expected to grow as populations increase and cancer-risk lifestyle factors rise (Torre et al., 2016). The landscape of cancer treatment has evolved considerably, from oncological surgery and radiation therapy to modern modalities including chemotherapy, immunotherapy, and targeted therapy. Despite these advances, treatment-related toxicity continues to significantly impact patient quality of life. Chemotherapeutic agents such as doxorubicin, while effective, carry severe side effects including cardiotoxicity leading to congestive heart failure (Singal & Iliskovic, 1998). Addressing these limitations has renewed interest in natural bioactive compounds capable of acting as chemosensitizers, agents that can reprogram cancer cell resistance mechanisms and allow existing treatments to remain effective while reducing systemic harm (Guo et al., 2020).

Berberine (BBR) is from the class of benzyl tetra isoquinoline alkaloids isolated from the roots and stems of plants such as Berberis vulgaris and B. aristata (Meng et al., 2018). Beyond its well-documented anti-inflammatory, antifungal, and antidiabetic properties, BBR has demonstrated meaningful anti-cancer activity across multiple studies (Silva et al., 2016). Its mechanisms include induction of apoptosis via caspase-3 and caspase-9 activation (Wang et al., 2020), increased cytochrome C expression (Li et al., 2015), AMPK pathway activation (Yang & Huang, 2013), and inhibition of VEGF-driven angiogenesis (Tsang et al., 2015). BBR has also shown promise as a chemosensitizer alongside doxorubicin, synergistically inhibiting cell viability in A549 and HeLa cells (Tong et al., 2012). Despite these findings, BBR's effects on Ewing sarcoma remain understudied. This study investigates the anti-proliferative and cytotoxic effects of berberine chloride on A-673 human Ewing sarcoma cells in vitro across a range of concentrations and time points.

Methods: A-673 human Ewing sarcoma cells (CRL-1598, ATCC) were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO₂ incubator. Cells were seeded at 100,000 cells per well and organized into six treatment groups: an untreated control, a DMSO vehicle control, and four BBR-treated groups at concentrations of 10, 20, 40, and 80 µM. Cell proliferation and viability were assessed at 24, 48, and 72 hours using hemocytometry with trypan blue exclusion, enabling visual distinction between live and dead cells. To evaluate apoptosis, Annexin V-FITC/PI staining was performed and samples were analyzed using a BD Accuri C6 flow cytometer at 72 hours, with appropriate unstained, single-stain, and vehicle controls established for accurate gating.

Results: BBR produced a dose- and time-dependent decrease in A-673 cell proliferation and viability across all tested concentrations, with the most pronounced reductions observed at 40 and 80 µM, particularly at 48 and 72 hours. Despite the significant decline in viable cell counts, Annexin V-FITC/PI staining and flow cytometry analysis at 72 hours did not reveal a statistically significant increase in apoptotic populations, indicating that the observed cell death was occurring through a mechanism other than classical apoptosis.

Discussion: These results demonstrate that BBR exerts meaningful anti-cancer activity against A-673 Ewing sarcoma cells, suppressing proliferation and viability in a dose- and time-dependent manner. The absence of apoptotic signaling at 72 hours suggests an alternative mode of cell death, potentially necrosis, autophagic death, or mitotic catastrophe, warranting further mechanistic investigation. These findings support BBR's candidacy as a complementary agent in cancer treatment. Future directions include additional flow cytometry trials to better characterize the cell death pathway, testing across additional cancer cell lines, and evaluation in primary tissue models including canine tumor specimens.