Synergistic effects of next-generation Tumor-Treating Fields technology and cinnamaldehyde/cinnamon oil on apoptosis, cell cycle arrest and growth suppression in triple-negative breast cancer cells

Background: Triple-negative breast cancer (TNBC), one of the most aggressive subtypes of breast cancer, accounts for 10-20% of breast cancers. Tumor-Treating Fields (TTF), disrupt mitotic spindle formation and induce apoptosis in TNBC cells. Cinnamaldehyde (CA), the active component of cinnamon, inhibits TNBC proliferation and promotes apoptosis. This study evaluated whether CA or cinnamon oil (CO) acts in synergy with TTF to enhance growth suppression, apoptosis, and cell-cycle arrest in TNBC cells. Methods: MDA-MB-231 TNBC cells were seeded at 75,000 cells/mL and treated with CA (150 µM), CO (1:300 dilution), or vehicle controls. Frequency-modulated TTF (FM-TTFields) were applied continuously for 72 hours at 150 kHz (±10 kHz modulation using an 8.3 mHz triangle wave), 1.2 V RMS /cm. Cell proliferation was assessed by CyQUANT assay and Celigo imaging; apoptosis and cell-cycle distribution were analyzed by Annexin V/PI flow cytometry. Synergy was quantified using Bliss independence and Highest Single Agent (HSA) models. Results: Cell death synergy: Cell death from combined treatments TTF+CA (96.7%) and TTF+CO (96.3%) exceeded single agents CA (93.6%), CO (86.1%), and TTF (40.0%). Synergy analysis by HSA and Bliss models confirmed synergy, stronger for TTF+CO (HSA Δ = +10.3 ± 2.5 points (pts), p = 0.02; Bliss Δ = +4.7 ± 1.7 pts, p = 0.042) than TTF+CA (HSA Δ = +3.2 ± 1.4 pts, p = 0.06; Bliss Δ = +0.9 ± 1.0 pt, p = 0.27). Combining TTF with CA or CO yields synergistic benefits, with TTF+CO showing the greatest effect. Apoptosis synergy (Annexin V/PI): TTF+CO (49.9%) and TTF+CA (65.5%) greatly surpassed CA (20.6%), CO (13.9%), and TTF (1.4%). Combinations increased apoptosis ~36-fold (TTF+CO) and ~48-fold (TTF+CD). Synergy was highest for TTF+CA (HSA Δ = +44.3 pts; Bliss Δ = +43.2 pts) versus TTF+CO (HSA Δ = +35.1 pts; Bliss Δ = +33.9 pts). Combining TTF with CA or CO substantially enhances apoptosis, with TTF+CA most effective. Cell-cycle modulation: CA and CO enriched the S phase relative to DMSO (Δ%: CO +10 pts; CA +8 pts). TTF induced strong G2/M accumulation (+18 pts). Combining TTF with CO further increased G2/M (+12 pts) with minimal S-phase change (+1 pt), while CA+TTF produced only a small G2/M shift (+2 pts). These patterns suggest mechanistic complementarity: CA and CO primarily arrest cells in the S phase, whereas TTF induce mitotic arrest (M phase). Conclusions: Combining FM-TTFields with cinnamon oil or cinnamaldehyde enhances TNBC cell death and apoptosis through complementary mechanisms-S-phase arrest and mitotic disruption-resulting in significant synergy for FM-TTFields combined with cinnamon oil or cinnamaldehyde. These findings support a novel, combinatorial strategy for treating TNBC. Future studies should validate these results in vivo and explore clinical translation.