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Corresponding author: s.bimonte@istitutotumori.na.it The effects of the combination of (-)-Epigallocatechin-3-gallate and Tapentadol on the growth of human triple negative breast cancer MDA.MB231 cells Bimonte S.1; Cascella M. 1; Del Vecchio V.2 ; Barbieri A.2; Falco M.2; Schiavone V.3; Arra C.2; Cuomo A.1 1Division of Anesthesia and Pain Medicine - IRCCS “ Fondazione G. Pascale ”, Napoli, Italia; 2 S.S.D. Sperimentazione Animale- IRCCS “ Fondazione G. Pascale”, Napoli, Italia. 3Division of Anesthesia and Intensive Care, Hospital “Pineta Grande”, Castel Volturno, Caserta, Italia . Corresponding author: s.bimonte@istitutotumori.na.it Introduction Background: Breast cancer is characterized by a higher rate of mortality and is considered one of the deadliest type of cancer. Recently it has been demonstrated that (-)-epigallocatechin-3-gallate (EGCG), a principal catechin of green tea, is able to inhibit the growth of MDA.MB231 breast cancer cells by influencing different signaling pathways, including the apoptosis. Furthermore, EGCG is also used in the treatment of bone cancer pain. Tapentadol, the opioid drug acting at the level of noradrenaline (norepinephrine) reuptake inhibition (NRI) and μ-opioid receptor (MOR), is able to modulate bone cancer pain and to influence the cancer cell viability by regulating the apoptosis. Material and methods: In this study, we reported results from the combination of EGCG and tapentadol on breast cancer cell growth. Results:. In vitro results showed that the cell proliferation, the viability and the apoptosis of MDA.MB231 cells were impaired by the combination of EGCG and tapentadol. Specifically, our data show that EGCG and tapentadol are able to inhibit the proliferation of MDA.MB231 cells by enhancing the apoptosis. Conclusions: These findings suggest that the combination of these substances could represent a new strategy for the treatment of patients with triple-negative breast cancer. Figure 2. EGCG and tapentadol inhibit the proliferation of MDA.MB231 cells. MTT assay shows a dose-dependent inhibition on the viability of cells treated with EGCG and TAP (tapentadol). A stronger effect is observed in the combined group of treatment. Data presented as a mean ± standard deviation. Significance: *p< 0.05, **p< 0.01 and ***p< 0.001 by analysis of variance, compared with controls. Figure 1. Effects of EGCG and tapentadol on MDA.MB231 cells migration. MDA.MB231 cells were incubated in medium alone (Control), B) EGCG 40 μM, C) TAP 20 μg/ml (tapentadol), D) EGCG 40 μM + TAP 20 μg/ml. At 48 h (D) the inhibitory effect of EGCG and TAP, was clearly evident (*P value < 0.05). Figure 3. The effects of EGCG and TAP on MDA.MB231 apoptosis. In vitro apoptosis assay by flow cytometry showed the effect of the substances on MDA.MB231 cell apoptosis. A) EGCG; B) TAP; C) EGCG plus Tap. An enhancement of the apoptosis level on cells was observed with the combination of both compounds. Data presented as a mean ± standard deviation. Significance: **p< 0.01 and ***p< 0.001 by analysis of variance, compared with control. Figure 4. Effect of EGCG and tapentadol on the apoptosis in MDA.MB231 cells. A) Western blot analysis shows that EGCG and TAP enhance p53’s expression in MDA.MB231 cells treated with two substances (EGCG: 40 μM plus TAP: 20 μg/ml) respect to controls (CTR) and to single treatments (EGCG: 40 μM; TAP: 20 μg/ml) EGCG 40; β-actin was used as loading control. Conclusions In vitro results showed that the cell proliferation, the viability and the apoptosis of MDA.MB231 cells were impaired by the combination of EGCG and tapentadol. Specifically, our data show that EGCG and tapentadol are able to inhibit the proliferation of MDA.MB231 cells by enhancing the apoptosis. These findings suggest that the combination of these substances could represent a new strategy for the treatment of patients with triple-negative breast cancer.