Cancer cells and tissues express VGSCs, and VGSCs activity increases the lateral motility and invasion of tumor cells in vitro (Fraser et al., 2005). VGSCs play an important role in the occurrence and development of tumors (Pedersen and Stock, 2013; Hofschröer et al., 2020), and functionally expressed in many types of tumor cells (epithelial carcinoma), including breast, cervical, ovarian, prostate, colon, skin, and lung cancers (Fraser et al., 2014). The overexpression of these channels enhances the metastasis cascade and tumor cell metastasis. LAs inhibit VGSCs function, and prevent VGSCs activity during and after surgery, which reduces the ability of cancer cells to escape and metastasize from the perioperative range of surgery. These effects reduce cell proliferation and indirectly increase patient survival.

Ropivacaine inhibited the invasion of SW620 colon cancer cells in a concentration range of 10–100 uM, which was similar to the current effect on the Nav1.5 mutant channel of the neonatal isoform, and this range is related to LAs blockade of sodium channels (Baptista-Hon et al., 2014). Shilpa Dutta et al. found that Nav1.5 was overexpressed in the highly invasive human breast cancer cell line MDA-MB-231. 1uM Nav1.5 blocker inhibited the invasion of MDA-MB-231 cells, and the rate of invasion inhibition was 30.3 ± 4.5%, and fortunately, cell viability was not affected (Dutta et al., 2018). LAs can block the VGSCs which blocks channels in resting, open, and inactivated states (inactivated states have the highest binding affinity) (Grandhi and Perona, 2020). Amide LAS, especially lidocaine, have more systemic anti-inflammatory benefits and effects on immune cells than other LAS agents (Van Der Wal et al., 2015).

The expression of the Nav1.5 can be blocked by Lidocaine in highly metastatic human breast cancer MDA-MB-231 cells (Fraser et al., 2014). Tetrodotoxin (TTX), which is a blocker of VGSCs, was used as a local anesthetic for the treatment of pain in cancer patients in clinical trials, and it showed significant anticancer effects in vivo and in vitro (Makarova et al., 2019).

Rho and RAC GTP enzymes regulate all types of cell migration (Zheng et al., 2020), and one of the activators of the Ras homolog gene family member A (RhoA) pathway is neuroepithelial cell gene 1 (NET1) (Pang et al., 2021). Rho-associated protein kinase (Rock) is a downstream effector of the RhoA pathway. Rock activates myosin phosphatase targeting subunit 1 (MYPT1), and it phosphorylates myosin on myosin light chain 1 (MLC1) (Dan et al., 2018). Low concentrations of bupivacaine (10–50 mM) reduced the migration of gastric cancer cells via the RhoA and MLC1 pathways but had no significant effect on tumor growth or survival (Dan et al., 2018). Bupivacaine decreased the phosphorylation of MYPT1 and MLC1, which reduced the migration of gastric cancer cells. Research also suggests that bupivacaine inhibits the migration of cancer cells by stimulating NET1 (Pang et al., 2021). NET1 is significantly up-regulated in gastric and breast cancers, which suggests that bupivacaine is involved in tumor migration via this pathway to inhibit tumor migration. Levobupivacaine and ropivacaine reduced tumor cell invasion and migration by reducing RhoA protein levels (Castelli et al., 2020).

Some studies showed that sodium channel blockade contributed to the anticancer activity of LAs (Fraser et al., 2014), but these studies also demonstrated that bupivacaine inhibited the migration of gastric cancer cells via the sodium-independent channel blocker RhoA and NET1 inhibition (Dan et al., 2018; Zheng et al., 2020; Pang et al., 2021). These results suggest that LAs are associated with the RhoA and NET1 pathways in addition to sodium channel blockade.

Cell cycle progression is a hallmark of cancer, primarily because Cyclins D1, E, and B2 are key regulators of the cell cycle, and are dysregulated in different cancers, including breast, esophageal, bladder, skin, and lung cancers (Icard et al., 2019). Treatment of breast cancer and melanoma cells with bupivacaine and lidocaine significantly decreased cell cycle proteins, which promoted cell cycle arrest (Castelli et al., 2020). Ropivacaine arrested liver cancer cells in the G2 phase (Le Gac et al., 2017). P53 is a cancer marker that induces cell cycle arrest at G2/M or G0/G1 and activates the expression of the cyclin-dependent kinase inhibitor p21. P27 is a tumor suppressor that regulates the G0 to S phase, and increased P53 phosphorylation (active form) and P27 were observed after treatment with LAs, which indicated impaired cell proliferation.

Golgi apparatus transporter 1A (GOLT1A) is significantly elevated in patients with lung adenocarcinoma, and it was associated with prognosis and pathological staging (Yang et al., 2018a). The down-regulation of GOLT1A inhibits cell proliferation and induces cell cycle arrest. After 2 mmol/L lidocaine for 24 h, the expression of GOLT1A, Cyclin D1, and Cyclin E1 was decreased, which prevented the cells from transitioning from the G1 phase to the S phase (Zhang et al., 2017). Notably, GOLT1A also modulates the sensitivity of breast cancer cells to tamoxifen and improved prognosis (Zhang et al., 2017).

Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that regulates cell proliferation and differentiation of epithelial cells and tumors, including head and neck, breast, colorectal, lung, and pancreatic cancers (Mitchell et al., 2018). EGFR may be incorrectly activated by a variety of mechanisms: Ligand-dependent dimerization, point mutation, partial deletion, or overexpression. EGFR is expressed in tumor and non-tumor cells in the tumor microenvironment (TME). EGFR plays a role in the stimulation of vascular endothelial growth factor (VEGF), fibroblast growth factor and interleukin-8 (IL-8), which suggests that it supports tumor cell proliferation, angiogenesis, and metastasis (Grapa et al., 2019).

EGFR is activated by specific ligands, such as pro-epidermal growth factor and transforming growth factor-beta (TGF-β). Once activated, EGFR dimers stimulate intrinsic protein tyrosine kinase activity in cells, which results in the automatic phosphorylation of several tyrosine residues within EGFR-expressing cells. Therefore, several signal transduction pathways, including mitogen-activated protein kinase (MAPK), protein kinase B (Akt) and c-jun N-terminal kinase (JNK), are initiated, which leads to DNA synthesis and cell proliferation (Sakaguchi et al., 2006). Lidocaine inhibited the proliferation of human tongue cancer cells (CAL27 strain). 400 microM Lidocaine inhibited serum- and EGF-induced CAL27 proliferation via inhibition of the auto-phosphorylation of EGFR tyrosine residues without cytotoxicity. With the increase of clinical concentration, 4,000 microM lidocaine inhibited the proliferation of CAL27 cells by inhibiting the activity of EGFR (Sakaguchi et al., 2006).

LAS preferentially induced the EGFR pathway in breast cancer cells (MCF-7) via exogenous and endogenous caspase-dependent apoptosis. The activation of EGFR leads to an increase in the downstream activity of caspase 8 and 9, which leads to the apoptosis of breast cancer cells (Li et al., 2018). Treatment of human hepatoma cells (HEP G2 cells) with 1 mM or 5 mM lidocaine showed a continuous increase in caspase 3 concentrations, which reached a maximum level after 24 h (Xing et al., 2017).

The increased activity of TME ion channels leads to an increase in intracellular calcium concentration. An increased level of Ca²⁺ in the cytosol promoted the formation of podosomes/invadopodia, which facilitated the invasion of cancer cells (Hantute-Ghesquier et al., 2018). Calcium is also a key regulator of cell invasion. Lidocaine inhibits chemokine-induced tumor cell migration via the direct inhibition of CXCR4 activity. Calcium signaling controls the progression and apoptosis of cancer cells via the transient receptor potential subfamily member 6 (TRVP6) channel of the transient receptor potential channel V subfamily (Xuan et al., 2016; Liu et al., 2020a). TRVP6 mRNA and protein were detected in ovarian, breast, prostate, thyroid cancer and colon cancers. These results suggest that TRVP6 plays an important role in tumorigenesis, progression and prognosis. After treatment with 100 μm lidocaine for 12 h, the expression of TRPV6 was reduced 50–80%, and the survival rate, migration and cell division of MDA-MB-231 cells were decreased (Xuan et al., 2016). TRPM7 also affects the activity of cancer cells, and it is involved in Ca²⁺ and Mg²⁺ steady-state ion channels (Liu et al., 2020a). Lidocaine inhibited glioma cell proliferation and metastasis via the blockade of TRPM7 channels, which prevented the cell cycle and induced protective autophagy (Leng et al., 2017).

MicroRNAs (miRNAs) play important roles in gene silencing and post-transcriptional regulation (Bartel, 2004). Several cancer-related miRNAs were sensitive to LAs in vitro, including miR-21, miR-145, miR-520a-3p, and miR-539 (Xuan et al., 2016; Yang et al., 2018b; Yang et al., 2018c; Yang et al., 2019). Lidocaine enhanced the toxicity of cisplatin in lung cancer via miR-21 regulation (Xuan et al., 2016), and inhibited the growth, migration and invasion of gastric cancer cells via the up-regulation of miR-145 (Sui et al., 2019). Adenosine triphosphate (ATP) levels are strongly associated with tumor cell growth and survival. Mitochondrial metabolism limits tumor proliferation and metastasis by altering ATP levels. Ropivacaine inhibited breast tumor proliferation and metastasis by destroying mitochondrial complexes I and II but not III or IV (Gong et al., 2018). Bupivacaine inhibited mitochondrial complexes I and III to inhibit thyroid tumor proliferation (Chang et al., 2014). Bupivacaine (1 mm and 5 mm) inhibited mitochondrial complexes I and II to induce a decrease in ATP. There was no similar decline in ATP levels or activity after bupivacaine administration in mutant gastric cancer cells without mitochondria (Gong et al., 2018). Bupivacaine inhibits the growth of tumor cells by reducing the level of ATP in mitochondria.

Hyperthermia is a non-invasive, localized cancer treatment option that induces targeted cancer cell death. Local hyperthermia induces cell damage in the tumor area with minimal damage to the surrounding tissue (usually 40–44°C) (Markowitz and Bressler, 2021). Under high temperature alone, human histiocytic lymphoma (U937) cells showed a certain degree of DNA fragmentation and nuclear fragmentation, but the degree of nuclear fragmentation was enhanced in a dose-dependent manner when amides were used in combination. LAS with higher liposolubility had a greater promotion effect of heat-induced apoptosis. Intracellular Ca²⁺ concentrations are elevated during high-temperature-induced apoptosis, and the Ca²⁺-chelating agents (BAPTAAM) inhibit DNA fragmentation, which suggests that calcium-dependent pathways are involved in hyperthermia-induced apoptosis (Arai et al., 2002). Moderate temperature (42°C) combined with a low concentration of lidocaine (0.2%) significantly increased skin cancer cell death (Raff et al., 2019). Previous studies also showed that cells in the intermediate and advanced phase of S are more sensitive to high temperatures, with an increased proportion of cells in S phase compared to five types of cancer cells (fibroblasts, keratocytes, melanoma, cervical cancer, basal cell carcinoma) were more sensitive to combination therapy (Raff et al., 2019). The causes of LAS combined with high temperature-induced cancer cell death include the formation of superoxides, a decrease in mitochondrial membrane potential, the activation of caspase 3 and an increase in intracellular Ca²⁺ (Raff et al., 2019).

Transient receptor potential melastatin 7 channels (TRPM7) participate in Ca²⁺ and Mg²⁺ steady-state ion balance, which affects cell viability. TRPM 7 is not regulated in many cancers, including head and neck, breast, thyroid, lung, and pancreatic cancers (Grandhi and Perona, 2020). TRPM7 overexpression in bladder cancer cells promoted the proliferation of cancer cells. Regulation of Ca²⁺ homeostasis is associated with cancer development (Gao et al., 2017). Some studies showed the migration of pancreatic cancer cells by TRPM7 via the regulation of Mg²⁺-dependent mechanisms (Rybarczyk et al., 2012). Another in vitro study showed that TRPM7 was associated with the growth of the human breast cancer cell line MCF-7 (Guilbert et al., 2009). Lidocaine inhibited the proliferation and metastasis of breast cancer cells by inhibiting the function of TRPM7 channels in breast cancer cell lines (Liu et al., 2021). Lidocaine inhibited TRPM7 channels in a concentration-dependent manner. The inhibition rate was 20% at 1 mM and 50% at 3 mM (Leng et al., 2017), but the dose of lidocaine in this study was higher than the clinical dose. Lidocaine prevented the cell cycle and induced protective autophagy in glioma cells by blocking TRPM7 channels (Leng et al., 2017). Another calcium channel, TRPV6, is also expressed in MDA-MB-231 human breast cancer cells, PC-3 prostate cancer cells and ES-2 ovarian cancer cells (Jiang et al., 2016). Lidocaine inhibited the migration and invasion of MDA-MB-231 cells by inhibiting this channel (Jiang et al., 2016).