Cassinello F.1, Moris L.2, López Pérez V.2, Olmedillas S.3, Villagrasa A.3, García Arranz M.3

1Hospital Universitario Nuestra Señora de Candelaria, Dept of Anaesthesiology, Santa Cruz de Tenerife, Spain, 2Fundación Jiménez Díaz, Dept of Anaesthesiology, Madrid, Spain,3Fundación Jiménez Díaz, New Therapies, Madrid, Spain

TITLE: 
EFFECT OF LIDOCAINE AND SUBSTANCE P ON MIGRATION AND PROLIFERATION "IN VITRO" OF HUMAN ADIPOSE DERIVED STEM CELLS (hASCs) AND BREAST CANCER CELLS (MDA-MB-231) 

BACKGROUND AND GOALS: 
Local anaesthetics inhibit proliferation and migration while Substance P (SP) has demonstrated the opposite effect (1,2,3). Local anaesthetics seem to inhibit both SP binding and the related increase in intracellular calcium (4). Our objectives were: 
To demonstrate migration and proliferation inhibition induced by lidocaine of hASCs and MDA-MB-231 breast cancer cells. And to demonstrate SP can reduce the effect of lidocaine on migration and proliferation of hASCs and MDA-MB-231 breast cancer cells.

MATERIALS AND METHODS: 
Cells were treated with different doses of lidocaine (4000, 10000 y 20000 µM) and/or SP (50 y 500 nM). Migration was evaluated with a wound healing scratch assay after incubation at 37oC and 5% CO2 for 24 hours. The wounded area was imaged at the beginning and at 12 hours and analyzed with Image J Software. The results are shown as a percentage of remaining denuded area.
Proliferation was evaluated with AlamarBlue reagent viability assay. After 24 hours, cells were treated with different doses of lidocaine (4000,10000 y 2000 µM) and/or SP (50 y 500 nM).
All assays were repeated  (x3) and the percentage difference between treated and control cells were calculated. Kruskall-Wallis test was used to compare mean values and p<0,05 considered significant. 

RESULTS AND DISCUSSION: 
Lidocaine inhibited significantly migration at 4000μM compared with control of hASCs cells (p=0,05) and also of MDA-MB-231 (p=0,0001). Substance P did not influence migration alone or with lidocaine (Figure 1).
Lidocaine inhibited proliferation at 20000 μM compared with control of hASCs cells (p=0,0001) and also of MDA-MB-231 (p=0,0002). Adding substance P (50 nM) reduced the inhibition effect of lidocaine on viability of hASCs cells (p=0,03) and also of MDA-MB-231 (p=0,006) (Table1).

CONCLUSIONS:
-	Lidocaine inhibited migration and proliferation. 
-	Substance P partially antagonizes the inhibition of proliferation induced by lidocaine, but does not alter the decrease on migration.  
References:
1.	Cassinello F, Prieto I, Del Olmo M, Rivas S, Strichartz G. Cancer Surgery: How may anesthesia influence outcome. Journal of Clinical Anesthesia 2015; 27: 262-272.
2.	Sakaguchi M, Kuroda Y, Hirose M. The antiproliferative effect of lidocaine on human tongue cancer cells with inhibition of the activity of epidermal growth factor receptor. Anesth Analg 2006; 102:1103-1107.
3.	Palma C. Tachykinins and their receptors in human malignancies. Curr Drug Targets 2006;7(8):1043-52.
4.	Li YM, Wingrove DE, Too HP, Marnerakis M, Stimson ER, Strichartz GR,et al. Local anesthetics inhibit substance P binding and evoked increases in intracellular Ca2+. Anesthesiology 1995; 82(1):166-73.

Table 1. Proliferation study

hASCs	MDA-MB-231
Control %	Lido 20000	Lido 20000 + SP 50	Lido 20000	Lido 20000 + SP 50
100	30	66	30	62
100	32	65	30	61
100	31	64	28	61
100	31	65 *	29	61*
LIDOCAINE (uM) + SP (nM) %VIABILITY 24H. All results are media of 3 assays (x3). Last line presents the average of all studies ± SD. (*p<0.05)

Figure 1. Migration Assay.

Effect of lidocaine and substance P on migration and proliferation &quot;in vitro&quot; of human adipose derived stem cells (HascS) and breast cancer cells (mda-mb-231)

Effect of lidocaine and substance P on migration and proliferation &quot;in vitro&quot; of human adipose derived stem cells (HascS) and breast cancer cells (mda-mb-231)

Effect of lidocaine and substance P on migration and proliferation "in vitro" of human adipose derived stem cells (HascS) and breast cancer cells (mda-mb-231)