Levobupivacaine inhibits proliferation and promotes apoptosis of breast cancer cells through PI3K/Akt/mTOR signalling pathway.

Objective This study aimed to test the hypothesis that levobupivacaine has anti-tumour effects on breast cancer cells Results Colony formation and transwell assay were used to determine breast cancer cells proliferation, whereas ow Cytometry (annexin V and PI staining) was used to investigate breast cancer cells apoptosis. The effects of levobupivacaine on cellular signalling and molecular response were studied with Quantitative Polymerase Chain Reaction (qPCR) and western blot. Induction of apoptosis was conrmed by cell viability, morphological changes showed cell shrinkage, rounding, and detachments from plates. The results of the western blot and qPCR indicated activation of Caspase-3 and inhibition of FOXO1. The results of the ow Cytometry conrmed that levobupivacaine inhibited breast cancer cell proliferation and enhanced apoptosis of breast cancer cells. Quantitative Polymerase Chain Reaction and Western blot analysis showed increased p21 and decreased cyclin D. However, the qPCR and western blot analysis showed that levobupivacaine signicantly increased Bax expression, accompanied by a signicant decreased Bcl (cid:0) expression and inhibition of PI3K/Akt/mTOR signalling pathway. These ndings suggested that levobupivacaine inhibits proliferation and promotes breast cancer cells apoptosis in vitro.


Introduction
Breast cancer is one of the most recorded cancer illness among women [1,2]. More than 40,000 patients die from breast related-cancer illness yearly despite the advances in chemotherapy and targeted treatments [3].
Molecular signalling pathways that are involved in breast malignant transformation have become evident as promising therapeutic targets [4]. Reports indicate that inhibiting this signalling pathway could inhibit cancer cells proliferation and also stimulate them toward cell death [5,6,7,8,9,10].
Growing evidence of local anaesthetics inhibiting cancer cell growth seems promising yet limited [11]. At the tissue level, administration of a certain amount of local anaesthetics either topical or local has shown to have a direct inhibitory effect on the action of epidermal growth factor receptor (EGFR), which is a potential target for anti-proliferation in cancer cells [12,13]. Evidence also shows that ropivacaine and lidocaine impede cancer cells growth, invasion, migration and increase apoptosis of lung cancer cells [14][15][16][17][18]. The effect of levobupivacaine on breast cancer cells is yet to be determined. The present study aimed to investigate the anti-tumour effects of levobupivacaine on breast cancer cells.

Ethics statement
The ethical committee of the Dalian Medical University First A liated Hospital approved for this study to be carried out.

Cell culture
We acquired MCF-7 and MDA-MB231 breast cancer cells from the ATCC (Beijing Zhongyuan limited, China). We regularly maintained the MCF-7 and MDA-MB-231 cells with high-glucose DMEM or DMEM/F12 (Gibco, USA) medium. Also, 10% fetal bovine serum (FBS) (Gibco, USA), penicillin 100 units/ml and streptomycin 100 µg/ml (TransGen Biotech, China) were used to supplement the medium to maintain the cells. The MCF-7 and MDA-MB231 cells were kept in an incubator at 37 • C humidi ed air with 5% CO2 atmospheric condition. The cells were repeatedly subcultured afterwards.

Western blot
At the log phase of treated MCF-7 and MDA-MB 231 cells growth, we harvested the cells and then washed them twice with ice-cold PBS. The procedure used for the western blot was the same as described elsewhere [19].

Colony formation assay
The procedure used for the colony formation assay was the same as previously described [19].

Transwell assay
The MCF-7 and MDA-MBA-231 cells (5 × 104) that were pre-treated with different dose of Levobupivacaine (0, 1, 2 mM) for 24 h and resuspended in culture medium with the same concentrations of levobupivacaine were seeded onto the coated membrane in the upper chamber of the transwell (24well millicell cell culture insert, 12 mm diameter, 8 µm pores; Merck KGaA, #P18P01250, China). The procedure used for the Transwell assay was the same as previously describe [19].

Data analysis
Values were expressed as the mean ± SD. Statistical analysis was performed with GraphPad Prism version 5.01(GraphPad Software, La Jolla, CA, US). One-way ANOVA was used to measure signi cance (p < 0.05). Dunnett's post hoc tests were used to test the difference between groups.

Levobupivacaine inhibits breast cancer cell viability
The MCF-7 and MDA-MBA-231 cell viability decreased as the concentration of levobupivacaine (0, 1, 2 and 3 mM) increased. For the MDA-MB-231 cells, more than 40% resulted in a cytotoxic effect, whereas the MCF-7 cells showed a similar responded cytotoxic effect of about 50% (Fig. 1A). Under a uorescence microscope, cells treated with Levobupivacaine showed morphological changes after 24 h exposure. The features of the treated breast cancer cells showed morphological changes including cell rounding, cell shrinkage, and almost detachment from the plates (Fig. 1B). The change of cell viability was found to be signi cant as the viability of breast cancer cells decreased with an increase in dosage of levobupivacaine treatment for 24 h.
Levobupivacaine inhibits proliferation in breast cancer cells Transwell assay indicated a signi cant decreased in the invasion ability of MCF-7 and MDA-MB-231 cells as the concentration of the levobupivacaine increased compared with the control (Fig. 2A). The results showed a signi cant decreased in the number of clones of the treated cells compared with the control (Fig. 2B). The data showed that the mRNA level of p21 signi cantly increased following levobupivacaine treatment (Fig. 2C). Western blot analysis showed a similar increased in p21 and decreased in FOXO1 and cyclin D1 expressions (Fig. 2D, E).

Levobupivacaine promote apoptosis in breast cancer cells
Levobupivacaine signi cantly reduced the number of cells showing nuclear staining compared with untreated cells (Fig. 3A). The qPCR data showed a decreased in BCL and an increased in Bax expressions in MCF-7 and MDA-MB-231 cells compared with the control (Fig. 3B, C). Consistently, Western blot analysis showed a similar decreased in BCL and increased expressions of Caspase 3 and Bax as the concentration of the levobupivacaine increased compared with control ( Fig. 3D, E).

Levobupivacaine inhibits proliferation and promotes apoptosis in breast cancer through PI3K/Akt/mTOR signalling pathway
The results showed a signi cant decreased in the expression of the nuclear localization of p-PI3K, p-Akt, and p-MTOR compared with the control using western blot ( Supplementary Fig. 1A, B).

Discussion
Several retrospective studies have demonstrated that regional anaesthesia is associated with a decreased risk of recurrence or metastasis of multiple carcinomas, including breast, prostate and cervical cancers [20][21][22]. Recent growing evidence demonstrates that local anaesthetics have an antitumour effect and may suppress the motility of cellular function and invasiveness more likely via voltage-gated sodium channel inhibition. The cellular modi cation of treated cells is likely dependent on the duration of exposure and the dose of local anaesthetic [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]. In this study, we used MCF-7 and MDA-MB-231 cells as models and found that levobupivacaine could effectively inhibit breast cancer cell proliferation and promotes apoptosis in vitro. The anti-proliferation and apoptosis effects observed in this study suggested that levobupivacaine may have the potential therapeutic effects against breast cancer.
PI3K/Akt/mTOR signalling pathway may play a vital role in cell proliferation, survival, development, metabolism, motility and also, regulation of the immune response. Breast cancer cell resistance to therapies can result from the activation of PI3K/Akt/mTOR signalling pathway [38][39][40][41]. Hence, making it an important object of study for comprehending the development and progression of breast cancer.
Consequently, in patients with breast cancer, PI3K/Akt/mTOR signalling pathway can be a target for diagnostic, treatment, and also for a prognostic purpose [2,[42][43][44][45][46][47]. In this study, the role of levobupivacaine on the expression of PI3K, Akt, and mTOR was investigated to illustrate the potential molecular mechanism. We observed a signi cantly decreased expression of p-Akt, p-PI3K, p-mTOR and subsequent decreased expression of FOXO, Cyclin D1 and Bcl-2 following levobupivacaine treatment which correlated with decreased breast cancer cells proliferation and increased apoptosis. The ndings of this study suggested that levobupivacaine inhibits proliferation and promotes apoptosis through the PI3K/Akt/mTOR signalling pathway which demonstrated possible antitumour effects of levobupivacaine on breast cancer. Conclusion levobupivacaine has the potency of reducing breast cancer cell viability, proliferation and causes cell death through the PI3K/Akt/mTOR signalling pathway. These pieces of evidence could lead to clinical studies which will seek to examine the anti-cancer effects of levobupivacaine which may increase the bene ts in cancer patient as well as improve patient care.

Limitations
Numerous studies have reported on the antitumour effects of local anaesthetics on various cancer cells [48][49][50]. However, our work is not without limitations.