Cytotoxic effect of famotidine in breast adenocarcinoma cells line.

Highlight key points

Famotidine, a widely used H2-receptor antagonist, is gaining attention for potential anticancer properties beyond its gastric indications.

Drug repurposing strategies offer a cost-effective and rapid pathway to identify new therapeutic uses for established medications.

In vitro evaluation of famotidine on breast adenocarcinoma cells provides foundational evidence supporting further mechanistic and preclinical investigations.

Breast cancer continues to represent a major global health burden and remains one of the most frequently diagnosed malignancies among women [1]. Standard treatment modalities include surgery, such as breast-conserving procedures or mastectomy, often followed by radiation therapy to minimize local recurrence [2]. Standard management typically involves a multimodal strategy, including surgical intervention, radiotherapy to reduce locoregional recurrence, and systemic treatments such as endocrine therapy, chemotherapy, and targeted biological agents [3]. Given the complexity of breast cancer, continued research into novel anti-cancer agents is essential to improve treatment outcomes and provide more effective, personalised therapies with fewer side effects.
Famotidine, a histamine H2-receptor antagonist primarily used for the treatment of peptic ulcer disease and gastroesophageal reflux, has shown biological properties beyond its conventional indications [4]. Recent studies suggest that H2-receptor antagonists may affect tumor biology through various mechanisms, including modulation of immune response, inhibition of angiogenesis, or interference with cellular signaling pathways involved in proliferation and survival [5, 6].
Earlier in vitro and clinical investigations have shown that famotidine may enhance antitumor immune responses especially when combined with interleukin-2 (IL-2) [7]. Famotidine treatment increased tumor-infiltrating lymphocytes significantly in colorectal cancer patients, indicating enhanced anti-tumor immune activity [8]. In metastatic kidney and melanoma cancers, famotidine improved responses when used alongside IL-2, likely by enhancing IL-2 receptor activity on immune cells [9].
However, the potential cytotoxic and antineoplastic effects of famotidine in human breast cancer models, remain insufficiently characterized and need further evidence [10, 11]. Our study aims to evaluate the cytotoxic properties of famotidine on human breast adenocarcinoma (MCF-7) cells in vitro.

MATERIALS AND METHODS

Cell Culture and Treatment
The human breast adenocarcinoma cell line MCF-7 was used in all experiments. Cells were routinely propagated in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Lonza, EU) supplemented with 10% fetal bovine serum (FBS) and a standard antibiotic mixture containing penicillin (100 U/mL) and streptomycin (100 μg/mL). Cultures were maintained at 37 °C in a humidified incubator with 5% CO2. For drug preparation, commercially available famotidine tablets were dissolved in sterile culture medium, and working concentrations ranging from 0.364 to 20 mg/mL were freshly prepared prior to treatment. As the routinely used culture medium for this cell line was employed, a cytotoxic effect is not expected; even if observed, a similar effect would be anticipated in the positive control.

Experimental Design and Drug Exposure
Once MCF-7 cultures reached approximately 90–95% confluence, cells were detached and seeded into 96-well plates at equal densities. After allowing 24 hours for cell attachment, varying concentrations of famotidine were applied to initiate the exposure period. Cells were incubated for an additional 24 hours under standard culture conditions. Parallel plates were prepared for metabolic viability assessment via MTT assay, for Live/Dead fluorescence staining, and for morphological observations using an inverted microscope.

MTT Viability Assay
At the end of the incubation period, the medium from each well was removed and cells were washed with phosphate-buffered saline (PBS) to eliminate residual drug. MTT reagent was then added to each well, and plates were incubated for two hours at 37 °C in the dark to allow intracellular formation. Dimethyl sulfoxide (DMSO) was subsequently introduced to solubilize the formazan crystals. Plates were gently agitated on a horizontal shaker to ensure complete dissolution, and absorbance was recorded at 540 nm using a microplate spectrophotometer. Viability percentages were calculated relative to untreated control wells [12].

Cytotoxicity
Cytotoxicity was quantified by comparing absorbance values to those of untreated cells (defined as 100% viability) and Triton X-100–treated cells (0% viability). Using these reference points, cytotoxicity ratios were computed for all famotidine concentrations.

Live/Dead Fluorescence Staining
To further characterize viability and membrane integrity, the Live/Dead™ Viability/Cytotoxicity Kit (Thermo Fisher Scientific) was employed. After a gentle PBS wash, cells were incubated with a freshly prepared staining solution containing calcein-AM and ethidium homodimer-1 for 30 minutes at room temperature in the dark. Excess dye was removed with PBS, and fluorescence images were captured immediately using an inverted fluorescence microscope. Viable cells emitted green fluorescence, while non-viable cells with compromised membranes displayed red fluorescence.

Patient Consent Information
Because this project utilized only commercially established cell lines, no human or animal subjects were involved, and ethical approval was not required.

Statistical Analysis
All data are presented as mean±standard deviation (SD). Statistical comparisons between groups were performed using ANOVA test in GraphPad Prism (version 6.00; GraphPad Software, San Diego, CA, USA).

RESULTS
Phase-contrast microscopy revealed dose-dependent morphological alterations in MCF-7 cells following 24-hour famotidine exposure (Fig. 1). At higher concentrations (20 and 10 mg/mL), cells exhibited reduced density, and increased detachment. Moderate changes were observed at 5 and 2.5 mg/mL, while lower concentrations (1.25 and 0.625 mg/mL) showed minimal morphological disruption compared to the positive control. The negative control, treated with Triton X-100, displayed complete cell lysis, confirming assay sensitivity.

Famotidine Reduces MCF-7 Cell Viability in a Dose-Dependent Manner
To evaluate the cytotoxic effect of famotidine on MCF-7 breast cancer cells, an MTT assay was performed after 24 hours of treatment with increasing concentrations of the drug. A dose-dependent increase in cytotoxicity was observed (Fig. 2A).
As shown in Figure 2B, cell viability decreased significantly with increasing concentrations of famotidine. While lower doses (0.312–1.125 mg/mL) showed moderate reductions in viability, higher concentrations (≥5 mg/mL) resulted in substantial cytotoxicity, with viability dropping below 20% at 10 and 20 mg/mL.

Live/Dead Assay Confirms Dose-Dependent Cytotoxicity of Famotidine
Cell viability was further assessed with a Live/Dead fluorescence assay, where live cells were stained green with calcein-AM and dead cells red with ethidium homodimer-1. As shown in Figure 3, control groups validated the staining: the positive control (untreated cells) exhibited predominantly green fluorescence, indicating high viability, whereas the negative control (Triton X-100-treated cells) showed extensive red fluorescence, confirming complete loss of viability.
Based on confocal microscopy observations, famotidine treatment resulted in a dose-dependent reduction in viable (green) cells accompanied by a corresponding increase in dead (red) cells. At lower concentrations (0.625–2.5 mg/mL), most cells remained viable with minimal red fluorescence. In contrast, from 5 mg/mL onward, red-stained cells became increasingly apparent. At concentrations of 10 and 20 mg/mL, cell death was pronounced, characterized by markedly reduced green fluorescence and widespread red signal.

DISCUSSION
The study provides evidence that famotidine may have cytotoxic effects on MCF-7 breast cancer cells in a dose-dependent manner. The dosing strategy was designed by stepwise halving from the highest selected concentration down to trace levels, allowing the assessment of concentration-dependent effects across a broad exposure range. Both the MTT assay and Live/Dead fluorescence staining demonstrated a progressive decline in cell viability as the drug concentration increased. Phase-contrast microscopy supported these findings by revealing characteristic morphological alterations, particularly at higher doses, where pronounced membrane disruption and cell detachment were observed.
Several pathways have been proposed to explain the antineoplastic actions of H2-receptor antagonists, including interference with histamine-dependent signaling, suppression of angiogenic processes, and modulation of immune activity [13, 14]. Also it has been suggested that H2 receptors are found in various cancer tissues and the blockage of this receptor could have anti-neoplastic properties [15]. Additionally, in colonic cell lines, famotidine has been shown to induce tumour infiltrating lymphocytes which causes the death of cancer cells by promoting perforin induced membrane lysis and cancer cell apoptosis [16]. Another study by Gholami-Javadie et al. [10] investigated the combined antineoplastic effects of cimetidine or famotidine with doxorubicin in MCF-7 and HT-29 cell lines. This study found that combining cimetidine or famotidine with doxorubicin at a dose of 25 micromolar reduced the viability of cancer cells to below 50%. These findings suggest that H2-receptor antagonists may act as supportive agents that potentiate the cytotoxicity of conventional chemotherapeutics. In another study, H2 receptor antagonist cimetidine has been shown to improve the outcomes in cancer patients via inhibiting cancer proliferation as histamine activation is thought as a growth factor in cancer cells [17]. Our findings align with previous reports suggesting that H2-receptor antagonists may have anticancer properties beyond their traditional role in gastric acid suppression.
In this study, the potentially cytotoxic concentrations of famotidine were substantially higher than those used therapeutically. Therefore, the observed effects are likely attributable not only to the pharmacological action of famotidine but also to nonspecific high-dose–related toxicity. At these concentrations, the compound appears to exert physiologically cytotoxic effects, suggesting that the findings primarily reflect dose-dependent cellular stress rather than clinically relevant pharmacodynamic activity. Consequently, the translational implications of these results should be interpreted with caution.
Another observation from our study is the consistency between the MTT assay and the Live/Dead fluorescence results, which mutually reinforce the cytotoxic nature of famotidine at higher doses [18]. The dual-assay approach strengthens the validity of the findings by capturing both metabolic activity (via MTT) and membrane integrity (via fluorescence staining) [19].
A critical consideration is the clinical relevance of the concentrations required to produce cytotoxicity in vitro. Therapeutic plasma levels achieved with standard dosing regimens are considerably lower than the concentrations that produced substantial cytotoxic effects in our experiments. For instance, a typical 40 mg oral dose of famotidine results in peak plasma concentrations of only 75–100 ng/mL within a few hours [20, 21]. In another study, the cytotoxic effects of famotidine on peripheral blood mononuclear cells found that cytotoxic activity was enhanced at a concentration of 10 ng/mL, which is equivalent to the serum level achieved following a 20 mg intravenous dose of famotidine [22]. These values are substantially lower than the cytotoxic concentration observed in our study. However, these results may still hold relevance in the context of drug repurposing strategies, particularly if famotidine is found to enhance the efficacy of conventional chemotherapeutics or sensitize resistant cancer cells through synergistic mechanisms. Furthermore, advanced drug-delivery technologies may enable targeted delivery of higher local concentrations to tumor tissue, potentially achieving antineoplastic effects without exposing patients to toxic systemic levels [23].

Limitations
A limitation of this study is the use of commercially available tablet formulations instead of the pure compound. As a result, the potential influence of excipients on the observed outcomes cannot be entirely excluded

Conclusion
This study provides evidence supporting the physiologically toxic effect of famotidine in breast cancer cells.