CORR Insights®: Bupivacaine and Lidocaine Induce Apoptosis in Osteosarcoma Tumor Cells

Where Are We Now?

Cell death, known as apoptosis, generally consists of intrinsic and extrinsic biochemical pathways involving the protease caspase enzymes. The intrinsic pathway involves a local anesthetic to activate the mitochondria stress-induced pathway, which releases caspase-activating proteins into the cytosol, thereby triggering apoptosis. Local anesthetics are known to bind and induce structural changes some place on the cell membrane. In cardiac cells, for example, local anesthetics are generally sodium channel blockers. The pores of the voltage-gated sodium channels within membranes become affected by local anesthetics. One study [4] proposed that the drug’s positively charged amine produces an electrostatic barrier to ion permeation and inhibits the normal action potential [4]. Without membranes containing associated pores or channels, cells and the mitochondria could not survive. Binding of molecules via proposed mechanisms cause malfunction and even apoptosis.

The extrinsic pathway relies on caspase activation through a signaling mechanism at the plasma cell membrane. This pathway involves ligand binding (such as a local anesthetic or other protein molecule) then transduction of a signal through various receptors and domains, causing caspase activity within the cell, leading to apoptosis. Membrane-associated signals, in turn, induce apoptosis via activation of the “death proteins” within the cell. Ultimately, multiple caspases are involved.

Cross-talk can also occur between the intrinsic and extrinsic pathway [7]. In studying various tumor cell lines, cytotoxicity, or cell death, includes but is not limited to evaluation and measurement after exposure to the agent (local anesthetics) of cell viability and colony formation, DNA fragmentation, reactive oxygen species, and Western blot (measurement of protein expression).

In human thyroid cells, local anesthetics activate the mitochondrial apoptotic pathway [2], causing permeability and release of cytochrome c by activation of Bax. This forms an oligomeric pore, and permeabilization of the mitochondrial outer membrane occurs. Treatment with lidocaine and bupivacaine can lead to a reduction in the Bcl-2 levels with a concomitant increase in Bax levels. In this pathway, caspase-9 is predominant. In human breast cancer cells, local anesthetics could induce multiple caspase enzymes found in both the intrinsics and extrinsic pathways [3]. Measured caspases included 3, 8, and 9.

The current study by Mirshahidi and colleagues [5] describes a proposed mechanism by which lidocaine and bupivacaine induce in vitro apoptosis in human and rat osteosarcoma cell lines in both a time- and dose-dependent manner. The results here correlated with the conclusion that the intrinsic pathway (caspase-9) is involved, specifically, decreased expression of Bcl-2 and increased expression of Bax. The change in ratio of Bcl-2/Bax facilitates the opening of the mitochondrial permeability transition pore and the release of cytochome c, apoptosis inducing factor and proaptotic proteins. This sustains caspase in the active state. However, survivin, which antagonizes the apoptotic pathway in both the intrinsic and extrinsic pathways, was reduced. This implies that the extrinsic pathway (caspase-8 and the TNF family receptors such as Fas) [7] could not be ruled out here.

Where Do We Need To Go?

We know that cell death occurs specifically due to the action of proteases (caspases), which affect membranes of both the mitochondria and cell itself. Identifying a common mechanism of action of binding of local anesthetics to other molecules or to the membrane in various cell types/lines can help explain similar mechanisms of apoptosis in osteosarcoma. While similarities exist for some tumor cell lines and the caspase activation, the actual structure of molecular and chemical binding of the local anesthetics to the osteosarcoma cell to direct apoptosis has not been proposed, and identifying where and how binding of the local anesthetics occurs is still needed.

Regarding osteosarcoma, the authors of the current study note that “the increased cellular levels of reactive oxygen species cause channels on the mitochondrial membrane to open, which release apoptosis-promoting factors into the cytosol and lead to activation of apoptosis and direct damage to nuclear and mitochondrial DNA” [5]. How this is specifically structurally programmed with resultant committed cell death is unknown. Where and how the local anesthetics bind and then signal membrane pore opening/disruptions is an interesting focus for future exploration.

The basic science of orthopaedics [1] includes structure and function of molecules. In the current study [5], the actual mechanism of binding of a local anesthetic and facilitating the opening of the mitochondrial pore allowing the release of cytochrome c is unknown. The results of research on structure and binding of local anesthetics direct future investigation to the cell and mitochondrial membrane. Characteristics of the local anesthetics include a positively charged amine group. Future research should focus on how the binding of drugs such as local anesthetics may result in conformational changes of any membrane bound or matrix molecules due to electrostatic changes resulting from ion formation.

Mirshahidi and colleagues [5] offered a topic for future studies: membranes and pores. The mechanism whereby local anesthetics bind, and how caspase is activated and subsequently metabolized, lies in the chemical structure of local anesthetics. But what causes the membranes to transduce signals? Complex pathways, such as the caspase cascade, involves multiple membrane components of the cell. Initially, in vitro and in vivo studies may have to rely on a static proposed mechanism (molecular modeling) of specific biochemical binding of molecules such as local anesthetics to predict the definitive mechanism for which apoptosis can be turned on and off on tumor cells like osteosarcoma. To date, it has also been found that the amide-linked local anesthetics like lidocaine inhibit inflammatory Src signaling involved in migration of adenocarcinoma cells. This study indicated that the amide-, but not ester-linked local anesthetics may provide beneficial antimetastatic effects. This effect by the amine local anesthetic is known to be independent of their known role as sodium-channel blockers [6], but the actual mechanism of action (exact binding) is unclear.

How Do We Get There?

Future studies will involve molecular modeling. One methodology that has been used is homology modeling and ligand docking [8]. Voltage-gated channels have found a common pharmacophore and atomic mechanism of action for various drugs in the eukaryotic sodium channel by using the radiographic structure of a prokaryotic channel. Applied computer modeling applies various ligands such as lidocaine. In tumor cells, the drug’s (lidocaine) positively charged amine may produce an electrostatic barrier to ion permeation, may bind other negatively charged molecules within the pore or membrane channel, or may even bind to the receptor itself on the membrane. Structural changes to the membranes of the cell in apoptosis are not fully known. Molecular modeling may explain how association of a negatively charged amine may produce the complex cascade of apoptosis.

Future studies on tumor suppression and apoptosis will benefit from utilizing known crystallographic structures of molecules, enzymes, and receptors and then applying this homology modeling based on known chemical structure and binding of local anesthetics to cardiac and other cells. Complex as this topic is, this is a start.