In this study we compared six commonly used local anesthetics at plasma concentrations and above, on breast cancer cell viability, migration, and cell division. This information on the potency and efficacy of local anesthetics may be used as a basis for selecting local anesthetics for study in animal models of cancer and in clinical trials comparing the effects of different types of anesthesia on cancer proliferation.
Previous studies have been limited mostly to lidocaine and bupivacaine, at millimolar concentrations. Here we screened five amide local anesthetics (lidocaine, mepivacaine, levobupivacaine, and ropivacaine) and one ester local anesthetic chloroprocaine. In one study, 4.5 mM lidocaine and 1.3 mM bupivacaine were found to inhibit the viability of MCF-7 cells by inducing apoptosis . A second study found that lidocaine at concentrations higher than 1 mM significantly impaired cell viability of MDA-MB-231 cells, prostatic cancer PC-3 cells, and ovarian cancer ES-2 cells . Another more recent study showed that 5 mM lidocaine or ropivacaine significantly inhibited the growth of human hepatocellular carcinoma through modulation of cell cycle-related genes . Here, we confirmed the direct toxic effects of all tested local anesthetics at millimolar concentrations (1 ~ 10 mM) on breast cancer cells as determined by MTT and LDH assays (Figs. 1 and 2). The clinical preparation of lidocaine for local injection ranges from 0.5% (18.5 mM) to 2% (74 mM). However, the tissue concentrations following local infiltration is difficult to measure. It depends on the speed of injection, the concentration and volume, time of measurement, and the tissue composition and blood supplies. Only a few studies analyzed the tissue concentration of lidocaine. In a recent study using rabbit, the concentration of lidocaine reached peak value in jaw bone (114 μg/g) and oral mucosa tissue (156 μg/g) after 0.5 mL of 2% lidocaine injection for 10 min, which is estimated to be 0.42 mM and 0.58 mM assuming a tissue density of 1 g/ml . This is probably an underestimate for molar concentration since tissues are composed of both “solid” and “soluble” compositions, or cellular and extracellular compartments. It is quite likely, the breast tissue concentrations after local infiltration of 0.5% (18.5 mM) lidocaine range from mini-molar, sub-millimolar and micromolar depending on the time and proximity of injection. Thus, the effects of both 10 × plasma and mini-molar concentrations are clinically relevant and might potentially be beneficial against postoperative metastasis. Currently, there is one ongoing clinical trial with an expected 1600 patient enrollment and an estimated completion date of 2021, testing the effects of local peritumor infiltration with 60 ml of 0.5% (18.5 mM) lidocaine in breast cancer patients (NCT01916317) [2, 16]. It will be interesting to compare the results of this trial with a trial evaluating the effect of intravenous lidocaine on postoperative outcome of patients with breast cancers (NCT01204242) .
Although local anesthetics may reach sub-millimolar concentrations at the site of injection, plasma concentrations following regional anesthesia are considerably lower. Among the local anesthetics used clinically, lidocaine is the only local anesthetic that can be administered intravenously at an anti-arrhythmic dose, that is, a plasma concentration of 5–20 μM [7, 8]. The plasma concentration after regional anesthesia with lidocaine is in a similar range . Lidocaine at this dose has been used in several “innovative” ways. For example, it has been used for neuroprotection in cardiac surgery patients [19, 20], for reduction of opiate usage in ambulatory surgery patients , and for reduction of postoperative ileus and pain following colon resection . It would be very attractive if this intravenous level of lidocaine could suppress the viability and motility of circulating cancer cells. However, we did not detect any significant effects of lidocaine (or any other local anesthetic) in this dose range. The plasma concentration of lidocaine effectively blocks neuronal voltage gated sodium channels , but this does not apply to cancer cells. However, with 3-day treatments at 10 times of plasma concentration, we found that some local anesthetics, particularly levobupivacaine and chloroprocaine, directly inhibited viability of both breast cancer cell lines MDA-MB-231 and MCF-7 (Fig. 3), but not the non-cancerous breast epithelial cell line MCF-10A. Although lidocaine is more widely studied among other local anesthetics, our results suggest that levobupivacaine induced a more potent reduction of cell viability than other local anesthetics on breast cancer cells. Similarly, Jose et al. has demonstrated a strong cytotoxic effect of levobupivacaine on cancer cell viability through inhibiting mitochondrial energy production . Moreover, we observed that triple negative breast cancer cells (MDA-MB-231) are more sensitive than estrogen receptor-positive breast cancer cells (MCF-7) in response to local anesthetics, which indicate a cell-type specific effect.
Inhibition of cell migration is another way in which local anesthetics might affect cancer cells. It has been reported that 1 mM lidocaine inhibited the invasion and migration of MDA-MB-231 cells, prostatic cancer PC-3 cells, and ovarian cancer ES-2 cells . We did not find any significant direct effects of lidocaine on breast cancer migration at the plasma concentration (10 μM). However, mepivacaine, levobupivacaine, ropivacaine, and chloroprocaine significantly inhibited the migration of MDA-MB-231 and/or MCF-7 at 10 times of plasma concentration (Fig. 4).
To further explore the effects of local anesthetics at plasma concentrations on breast cancer cell function, we looked for changes in the cell cycle in MDA-MB-231 cells. The cell cycle and cell growth are tightly regulated in normal cell, but genomic and epigenetic dysregulation lead to the uncontrolled proliferation of cancer cells. Few studies have investigated the effect of local anesthetics on the cell cycle. Le Gac and colleagues analyzed lidocaine and ropivacaine on human hepatocellular carcinoma cells. They found that 100 μM ropivacaine arrested the cell cycle at the G2 phase, whereas 100 μM lidocaine had little effect. They also observed that ropivacaine selectively modulated the expression of key cell cycle-related genes . In our study, 24-h treatment with any of the six local anesthetics at plasma concentration or 10 times of plasma concentration led to an increase in cells in S phase and a decrease in G0/G1 (Fig. 7a). This indicates an arrest in the cell cycle process from S (DNA replication) phase to G2/M phase, and may result in arresting mitosis and cellular apoptosis. Consistent with the above study of human hepatocellular carcinoma, ropivacaine at 35 μM increased the percentage of cells in the G2 phase, which may attribute to the blockage of cell cycle from G2 (preparation for cell division) to M (cell division). Further research is needed to examine the detail mechanism of cell cycle arrest in local anesthetic-treated breast cancer cells.
The potential beneficial effects of using local anesthesia during cancer surgery include attenuating surgical stress from neuroendocrine disturbance that promote the development of metastasis , reducing usage of systemic anesthesia and opiates , which inhibit cell-mediated immunity, and a direct effect on the cancer cells. Our results show that it is difficult to delineate a common mechanism to account for the direct inhibition of cancer cell growth by all the tested local anesthetics. We have shown that different local anesthetics may exert differential effects by various mechanisms in cancer cells. For example, levobupivacaine and chloroprocaine clearly exhibited anti-proliferation and anti-migration effect on breast cancer cells, while ropivacaine affects the cell cycle of breast cancer cells. Moreover, the two breast cancer cell lines we employed in this study displayed differential responses to local anesthetics. This indicates that heterogeneity of breast cancer may play an important role in determining the usefulness of local anesthetics on decreasing cancer recurrence. Therefore, future mechanistic studies must focus on specific breast cancer subtypes. Although, clinical trials point towards beneficial effects of local anesthetics on cancer metastasis; we found that plasma concentrations of a variety of local anesthetics had no significant effects on the viability and migration of two subtypes of breast cancer cells. It may be that local anesthetics affect cancer metastasis through modulation of the tumor microenvironment. This will be the direction of our future studies.