. 2022 Feb 8;23(3):1894. doi: 10.3390/ijms23031894

Table 2.

The effect of lidocaine, morphine, ropivacaine and levobupivacaine on BC cell lines and its influence in other treatment regimens.

Authors Year	Type of Study	Drug Concentration	Outcome	Observations
Lidocaine
Liu et al. [28] 2021	In vitro MDA-MB-231 AU565 T47D MDA-MB-468 MCF-7 BT474 BT-20	0.3–3 mM	↓	Viability, migration and TRPM7 function on BC cell lines were tested. Lidocaine at 1 and 3 mM (24 h) significantly suppresses the viability of BC cell lines with exception of MCF-7, and with greater effects on AU565, T47D and BT-20 cell lines. Lidocaine at 0.3 mM (24 h) only inhibited the viability of AU565 cell line. Lidocaine at 1 and 3 mM (24 h) suppressed cell migration in all the cell lines but at 0.3 mM the migration is only supressed on MDA-MB-231, AU565, and BT474. TRPM7 plays a role in mediating lidocaine’s effects on viability and migration of MDA-MB-231, AU565, T47D and MDA-MB-468.
Lin et al. [29] 2021	In vitro MCF-7	0.01–0.2 mmol/L	↓	Lidocaine inhibited proliferation, migration, and invasion of BC cell line MCF-7 by modulating the MicroRNA-495-3p/Fibroblast Growth Factor 9 axis. The overexpression of FGF9 inhibited the inhibitory effect of lidocaine on the proliferation, migration, and invasion of breast cancer MCF-7 cells.
Freeman et al. [26] 2019	In vivo Female BALB/c with 4T1 tumour cells	1.5–2 mg/kg/h	↓	Perioperative administration of lidocaine in a BC murine model of surgery, during sevoflurane anaesthesia, reduced the metastatic burden of lung tissue but not the hepatic colonies. No statistical differences were found in serum VEGF and IL-6 concentrations between groups 4 weeks after perioperative administration. Lidocaine infusions were stopped before the postoperative period, presenting a study limitation.
Chamaraux-Tran et al. [30] 2018	In vitro MCF-10A MCF-7 MDA-MB-231 SKBr3 HER²⁺	0.1–10 mM	↓	The viability of MCF-7 and SkBr3 HER²⁺ cell lines reduced significantly at 1 mM lidocaine and more (4 h). The MDA-MB-231 and MCF10A cell lines were more sensitive to lidocaine 4 h treatment, showing a significant viability reduction starting at 0.5 mM. Exposure to lidocaine at 0.1 mM (24 h) resulted in a marked inhibition of the migration of both MDA-MB-231 and SkBr3 HER²⁺ cell lines compared to MCF10A normal cells. MCF-7 and MCF-10A cell lines did not shown any significant migratory ability after lidocaine treatment. Lidocaine (0.1 mM) compromised the anchorage-independent growth of the MDA-MB-231 cell line.
Chamaraux-Tran et al. [30] 2018	In vivo SCID female mice inoculated with MDA-MB-231 cells	100 mg/kg	↓	Intraperitoneal lidocaine improved survival of mice model of MDA-MB-231 peritoneal carcinomatosis and reduced tumour growth.
Li et al. [31] 2018	In vitro MCF-10A	10–100 μM	=	Lidocaine decreased viability and induced significant cellular toxicity only on tumour cells (MDA-MB-231 and MCF-7 cells) exclusively with concentrations range of 0.3 to 10 mM (48 h). Also, a significant apoptotic response was observed, in this range, for MDA-MD-231 cells. At plasma concentrations (10 μM), lidocaine (24 h) promoted cell cycle arrest from phase S to phase G2/M in MDA-MB-231 cell line. Interestingly, at 10× plasma concentration (100 μM) the shift from G0/1 to S phase was already seen after 6 h. A selective effect was shown considering that the viability of non-cancer human breast epithelial MCF10A cells was not affected.
	In vitro MCF-10A	0.3–10 mM	=
	MCF-7	10–100 μM	=
	MCF-7	0.3–10 mM	↓
	MDA-MB-231	10–100 μM	=
	MDA-MB-231	0.3–10 mM	↓
Agostino et al. [32] 2018	In vitro MDA-MB-231	0.001–100 μM	↓	Lidocaine at concentrations of 10 or 100 μM (24 h) inhibited CXCR4 and mediated migration of MDA-MB-231 cell line, involving changes in intracellular calcium release and cytoskeleton remodelling.
Jiang et al. [33] 2016	In vitro MDA-MB-231	0.01–0.1 μM	=	Inhibitory effect on cell invasion was enhanced with increasing concentrations of lidocaine. With 10 μM, 100 μM and 1 mM of lidocaine (24 h) cell migration of the MDA-MB-231 cells was remarkably inhibited. Lidocaine effects on migration and invasion could occur partly as a result of the downregulation of TRPV6 expression. Lidocaine was able to significantly decrease cell viability in a concentration-dependent manner from 1 to 10 mM (4 h). However, lower concentrations (≤1 mM) of lidocaine exhibited no marked cytotoxicity.
Jiang et al. [33] 2016	In vitro MDA-MB-231	1–10 mM	↓
Chang et al. [34] 2014	In vitro MCF-10A	2–32 mM	=	Lidocaine decreased MCF-7 cells viability, increasing cell death by apoptosis, in a dose- and time-dependent manner (24 h). In vitro apoptotic effects of lidocaine are reproducible in vivo.
	MCF-7	1–16 mM	↓
	In vivo Female BALB/c nude mice inoculated with MCF-7 cells	21.3 mM	↓
Lirk et al. [23] 2014	In vitro BT-20 MCF-7	10–100 μM	↓	Authors incubated BC cell lines with lidocaine to assess demethylating properties. Lidocaine (10 or 100 μM) and the chemotherapeutic 5-aza-2′-deoxycytidine (DAC) at 0.1 or 0.5 μM demonstrated to have additive demethylating effects in BT-20 cell line after 72 h treatment. In MCF-7 cells, only the combined treatment with 0.5 μM DAC and 10 μM lidocaine revealed a stronger demethylation. The concentrations used were insufficient to cause direct cytotoxicity. Methylation bases between the cell lines shown to have different properties. Biological heterogeneity may have had a role in different outcomes of anaesthetics interventions.
Lirk et al. [35] 2012	In vitro BT-20 MCF-7	0.01–0.1 mM	=	Treatment with 1 mM lidocaine (72 and 96 h) resulted in significant reductions in cell number, while lower concentrations of local anaesthetics had no effect. There was an increase in the apoptosis rate upon lidocaine (1, 0.1, and 0.01 mM) 72 and 96 h treatment. At clinically relevant concentrations (1 mM), lidocaine demethylated DNA of MCF-7 cells after 72 h. Whereas treatment with 0.1 and 0.01 mM lidocaine revealed a significant demethylation after 72 and 96 h. In BT-20 cell line, was observed a dose-dependent decrease in DNA methylation in response to lidocaine (1, 0.1, and 0.01 mM) after 72 and 96 h. Demethylating tumour-suppressive effects may only be detectable in specific types of cancer due to differential methylation profiles. The cell lines used may have had genotypic and phenotypic derivations since their validation.
Lirk et al. [35] 2012	In vitro BT-20 MCF-7	1 mM	↓
Ropivacaine
Zhao et al. [36] 2021	In vitro MDA-MB-231 MDA-MB-468 MCF-10A SKBr3 HER²⁺ MCF7 BT474	1 mmol/L	↓	Ropivacaine inhibited proliferation, decreased migration and invasion and induced apoptosis of breast cancer cells MDA-MB-231 and MCF-7. This LA might inhibit the progression of all the BC cell lines tested by modulating the miR-27b-3p /YAP axis.
Zhao et al. [36] 2021	In vivo Balb/c nude mice injected with MDA-MB-231 cells	40 μmol/Kg	↓	Treatment with ropivacaine repressed the cell growth of MDA-MB-231 cells in vivo, while miR-27b-3p inhibitor could reverse this effect. Thus, confirming the results obtained in vitro.
Castelli et al. [37] 2019	In vitro MDA-MB-231	5–1000 μM	↓	Ropivacaine at 5 μM and more decreased significantly cell viability after 48 h of treatment. This LA (50 μM) resulted in 50% mortality of MDA-MB-231 cell line after 24 h treatment. Ropivacaine promoted apoptosis paralleled by the inactivation of survival pathways, such as PI3K/Akt/GS3K/β-catenin. This LA was able decrease cell proliferation by inactivating Wnt/GSK3β/β-catenin pathway. Ropivacaine was able to decrease RhoA and the active form of FAK protein level, indicating a reduction in cell invasion and migration.
Li et al. [31] 2018	In vitro MCF-10A	3.5–35 μM	=	Ropivacaine decreased viability, inhibited migration, and induced significant cellular toxicity of MDA-MB-231 only in concentrations of 0.3 to 10 mM (48 h). At plasma concentrations (3.5 μM), ropivacaine (24 h) promoted cell cycle arrest from phase S to phase G2/M in MDA-MB-231 cell line. Curiously, 24 h treatment with this LA blocked cell cycle before mitosis of MDA-MB-231 cells treated at 10× plasma concentrations (35 μM). Ropivacaine did not affect viability or cellular toxicity of non-tumorigenic human breast epithelial MCF10A cells.
	In vitro MCF-10A	0.3–10 mM	=
	MCF-7	3.5–35 μM	=
	MCF-7	0.3–10 mM	↓
	MDA-MB-231	3.5–35 μM	↓
	MDA-MB-231	0.3–10 mM	↓
Gong et al. [25] 2018	In vitro MDA-MB-468 SKBr3 HER²⁺	0.1–1 mM	↓	After 72 h of treatment, ropivacaine at concentrations of 0.5 and 1 mM significantly inhibited proliferation and induced apoptosis in a concentration-dependent manner. SKBr3 HER²⁺ cells appear to be more sensitive to ropivacaine than MDA- MB-468 cells. Ropivacaine significantly inhibited growth, survival, and anchorage-independent colony formation (72 h). Interestingly, ropivacaine at 0.5 mM significantly inhibits colony formation but does not affect growth and survival. Ropivacaine inhibited mitochondrial respiration by suppressing mitochondrial respiratory complex I and II activities, leading to energy depletion, oxidative stress, and damage. It was demonstrated a synergism between ropivacaine and 5-FU, likely by suppressing Akt/mTOR signalling pathway.
Lirk et al. [23] 2014	In vitro BT-20 MCF-7	3–30 μM	=	Ropivacaine showed no cytotoxic effect in either BC cell line. Ropivacaine after a 72 h treatment decreased methylation in BT-20 cells. Ropivacaine plus DAC revealed no increased demethylating effect in BT-20 or MCF-7 cells. Methylation bases between the cell lines shown to have different properties. Biological heterogeneity may have had a role in different outcomes of anaesthetic interventions.
Levobupivacaine
Kwakye et al. [38] 2020	In vitro MCF-7 MDA-MB-231	1–3 mM	↓	Levobupivacaine inhibited proliferation and promoted apoptosis in BC cells. Levobupivacaine after a 24 h treatment significantly decreased in the invasion ability of MCF-7 and MDA-MB-231 cells in a dose-dependent manner. Findings demonstrated a significantly increase of BAX expression and were associated with a decreased of BCL-2 expression and inhibition of PI3K/Akt/mTOR signalling pathway.
Castelli et al. [37] 2019	In vitro MDA-MB-231	5–1000 μM	↓	Levobupivacaine at 10 μM and more decreased significantly cell viability after 24 h of treatment. This LA (50 μM) resulted in 50% mortality of MDA-MB-231 cell line after 24 h treatment. Levobupivacaine promoted the inactivation of survival pathways such as PI3K/Akt/GS3K/β-catenin, contributing to cell death by apoptosis. Levobupivacaine was able decrease cell proliferation by inactivating Wnt/GSK3β/β-catenin pathway. This LA was able to decrease RhoA and the active form of FAK protein level, indicating a reduction in cell invasion and migration.
Li et al. [31] 2018	In vitro MCF-10A	2.5–25 μM	=	Levobupivacaine decreased viability, significantly inhibited migration, and induced significant cellular toxicity of MDA-MB-231 and MCF-7 cells only in concentrations of 0.3 to 10 mM (48 h). Levobupivacaine at plasma concentrations (2.5 μM) promoted a cell cycle arrest from phase S to phase G2/M in MDA-MB-231 cell line (24 h). Interestingly, at 10× plasma concentration (25 μM) the shift from G0/1 to S phase was already seen after 6 h. Levobupivacaine did not affect viability or cellular toxicity of non-tumorigenic human breast epithelial MCF10A cells.
	In vitro MCF-10A	0.3–10 mM	=
	MCF-7	2.5–25 μM	=
	MCF-7	0.3–10 mM	↓
	MDA-MB-231	2.5–25 μM	=
	MDA-MB-231	0.3–10 mM	↓
Morphine
Cheng et al. [39] 2019	In vitro MDA-MB-231	10 μmol/mL	↑	Morphine promoted lung metastasis 3 weeks after BC surgery in animal models. Morphine promoted postoperative recurrence, tumour proliferation and angiogenesis and reduced tumour cell apoptosis. PI3K-c-Myc signalling pathway may be related to angiogenesis promoted by morphine. Authors did not describe how morphine promoted the unexpected increased expression of TSP-1.
Cheng et al. [39] 2019	In vivo BALB/c-nu specific-pathogen-free mice with MDA-MB-231 cells	10 mg/kg	↑
Chen et al. [40] 2017	In vitro MCF-7	0.01–10 μM	↓	Morphine inhibited cell growth by blocking the cell cycle and promoted apoptosis in MCF-7 cells. Naloxone could not reverse morphine effects, which indicated that the inhibition of cell growth and proliferation by morphine could be an independent effect, not associated with opioid receptors.
Bimonte et al. [41] 2015	In vitro MCF-7 MDA-MB-231	1–100 μM	↑	Morphine enhanced proliferation, migration, and inhibited apoptosis of BC cell lines at 48 h in a dose-dependent manner.
Bimonte et al. [41] 2015	In vivo Foxn1^nu/nu mice with MDA-MB-231	0.714–1.43 mg/kg/day	↑	Morphine promoted tumour growth and angiogenesis.
Doornebal et al. [42] 2015	In vivo Female wild-type syngeneic FVB/N mice	10 mg/kg/12 h	=	Morphine in the presence or in the absence of surgery-induced tissue damage and pain, neither facilitated de novo metastatic dissemination nor promoted outgrowth of minimal residual disease after surgery. It did not exclude the possibility that anaesthetic techniques may influence the progression of the disease due to the intrinsic properties of the drugs.
Doornebal et al. [42] 2015	Female MMTV-NeuT mice (BALB/c background)	20 mg/kg/12 h	=
Niu et al. [43] 2015	In vitro MCF-7 BT549 MCF-10A	1–10 μM	↑	Morphine contributed to chemoresistance via expanding the population of cancer stem cells and promoted tumour growth in vitro. Compared with the normal saline group, morphine group showed a larger tumour volume after 21 days. Morphine enhanced the tumorigenicity of BC cells in vivo, however, this effect could be blocked by nalmefene.
Niu et al. [43] 2015	In vivo NOD/SCID mouse model inoculated with BT549 cells	5–15 mg/kg	↑
Nguyen et al. [44] 2014	In vivo C3TAG mice henceforth	0.5–1.5 mg/kg/day	↑	Morphine did not affect the onset of tumour development, but it promoted growth of existing tumours, and reduced overall survival in mice. Mast cell activation by morphine might have contributed to increased cytokine and substance P levels, leading to cancer progression and refractory pain.
Ge et al. [24] 2014	In vitro MCF-7	50–1250 μM	↓	Morphine at 250 μM and 1250 μM (48 h) significantly inhibited proliferation and induced apoptosis in MCF-7 cells. In combination with 500 μM of the chemotherapeutic agent 5-Fluorouracil (5-FU) there was an inhibition of proliferation and apoptotic promotion in MCF-7 cells.
Ecimovic et al. [45] 2011	In vitro MDA-MB-231 MCF-7	10–100 ng/mL	↑	Morphine increased both expression of NET1 and cell migration, but not when NET1 was silenced, suggesting that NET1 contributes to directly mediating the effect of morphine on BC cell migration.
Ustun et al. [46] 2010	In vivo BALB/c bearing Ehrlich carcinoma	0.714 mg/kg/day	↑	Morphine was able to induce angiogenesis. The study was not designed to study the underlying mechanism.

↑: enhance cancer; ↓: inhibit cancer grow or metastasis; =: no effect on cancer.