EFFECT OF PTEROPODINE AND BETA-SITOSTEROL ON THE IMMUNE RESPONSE IN BALB/c MICE AND CYTOTOXICITY IN HepG2 TUMOR CELLS.

Abstract

INTRODUCTION: Beta-sitosterol (BS) and pteropodine (PT) are plant-derived phytopharmaceuticals with pharmacological activities of high biomedical interest, including immunostimulatory, antimutagenic, antioxidant, and anti-inflammatory properties. Consequently, they are likely to possess immunomodulatory activity and selective cytotoxicity, which substantiates the interest in evaluating their action in a murine model and their cytotoxic effect on tumor cell lines.

OBJECTIVE: To evaluate the cytotoxic effect exerted by PT and BS on tumor cells, as well as their activity on the immune response against thymus-dependent antigens in BALB/c mice.

METHODOLOGY: BALB/c mice (10–12 weeks old, 30–35 g) were used. Oral doses of BS (100, 200, and 300 mg/kg) and PT were administered. Sheep red blood cells (SRBC) at 10% were used as a positive immunization control, and mineral oil was used as a negative control. For the immune response, the Jerne plaque assay modified by Cunningham was performed on spleen cells to measure the production of hemolytic plaque-forming cells (PFC-IgM; n=5–10 per group). For the cytotoxicity study, 3T3 (normal fibroblasts) and HepG2 (hepatoma) cell lines were evaluated, determining the percentage of cytotoxicity at different incubation times (12 to 72 hours) via microscopic analysis and the MTT reduction assay (n=3). Statistical analysis was performed using Student's t-test.

RESULTS: Basal administration of BS presented a significant dual effect dependent on sex: it caused a dose-dependent increase of up to 98% (568 ± 11.0 PFC/million; p < 0.05) in female mice, whereas in males it generated an inhibition of the basal response compared to the control (p < 0.05). Furthermore, the evaluation of the immunization timeline schedule with BS (combining sexes) demonstrated that co-administration ("During") with the antigen significantly potentiates the cellular immune response, achieving a 133% increase (257 ± 11.0 PFC/million; p < 0.05) with the 300 mg/kg dose compared to the SRBC positive control. The negative control group showed no PFC-IgM formation. In the in vitro assays, both compounds demonstrated a safe profile and low cytotoxicity in the normal cell line 3T3 (mortality < 22%). In the HepG2 tumor line, BS showed a moderate time-dependent response (up to 69% at 48 h), confirming its viability to activate indirect host pathways.

CONCLUSIONS: The study demonstrated relevant biological activities for both compounds. The immunomodulatory potential of beta-sitosterol was confirmed, characterized by a sex-differential response and an optimization of IgM antibody production when co-administered with the antigen. Likewise, both phytopharmaceuticals evidenced an absence of severe cytotoxicity in normal cells, validating their biological safety and establishing a rationale for future antitumor regimens.