Capsaicin effects on ion channel function have been tested at high capsaicin concentrations and in TRPV1-free cell systems, ensuring that capsaicin actions are TRPV1-independent. Under these conditions, it has been also reported that capsaicin modifies the function of some voltage-dependent sodium channels (VDSCs), such as the Nav1.5, a mechanosensitive channel highly expressed in cardiac tissue that drives the excitability of cardiomyocytes (19). High capsaicin concentration (30 to 100 μM) negatively regulates the Nav1.5 channel activation, an effect produced by decreasing the lipid bilayer’s stiffness and disjoining force which stabilizes the inactive state of this channel (13, 17). Remarkably, capsaicin also reduces the Nav1.5 peak current produced by pressure and shear stress without affecting the channel’s voltage-dependence gating, suggesting that the mechanism implied changes on the membrane properties (13, 20, 21).

Capsaicin also inhibits inward (Na⁺ and Ca²⁺) and outward (K⁺) currents from rabbit ventricular myocytes, impacting the cardiac excitability (21). Remarkably, the capsaicin effect over these channels is gradual, reaching steady-state inhibition at 3 to 6 min after capsaicin application, and partially reversed. The co-application of capsaicin and capsazepine (a TRPV1 antagonist) preserved capsaicin’s inhibitory effect on these channels, pointing a TRPV1-independent mechanism. Thus, the Na⁺, K⁺ and Ca²⁺ currents inhibition by capsaicin are through the modification of membrane properties. These results prompted us to question the physiological role of capsaicin in cardiac tissues. Considering that cardiac excitability needs the coordination of depolarization and repolarization events to propagate the electrical activity, the accurate ion channel function is strictly necessary. The computer modeling of rabbit ventricular action potential with the rectifier K⁺ currents inhibited at low capsaicin concentration emulate marked prolongation of action potential duration at 50% of the amplitude (APD50), while the inhibition of voltage-gated Na⁺ and Ca²⁺ and inward rectifier K⁺ currents at high capsaicin concentrations leads to a shortening of APD50 (21); prolongation and shortening of APD50 may cause cardiac arrhythmia (22). Thus, capsaicin insertion into the membrane of cardiomyocytes affects their excitability, having harmful effects.

In recent years, the number of reports about capsaicin’s role in different kinds of cancer has increased. Interestingly, a recent report demonstrated that capsaicin interacts with the transmembrane isoforms hCA IX and hCA XII, these are tumor-associated human Carbonic Anhydrases (hCAs) (with a K_I of 0.28 and 0.064 μM, respectively) (23). These metalloenzymes catalyze the reversible hydration of CO₂ to bicarbonate and protons, maintaining the suitable acid–base balance in the tissues (24). Capsaicin interacts with the active site of these transmembrane isoforms and encloses its alkyl chain to the hydrophobic surface of the active site. The capsaicin amide group establishes electrostatic interactions with zinc ion and H-bond between its carbonyl group and the backbone of T200 and T198 from hCA IX and hCA XII, respectively, Figure 1D upper. The biological relevance of the capsaicin inhibitory effect over hCA IX and hCA XII was probed in the A549 cell line (adenocarcinoma human alveolar basal epithelial cells) which overexpress these enzymes, the capsaicin treatment decreases the high cell migration profile; furthermore, hCA IX upregulates the MMP9 expression and capsaicin treatment reverted it (23). Other reports show that capsaicin also inhibits the overactivation of the cytosolic isoenzymes I and II (hCA I and hCA II, respectively) (25, 26).

The capsaicin anticancer effects have also been related to the inhibition of the tumor-associate NADH oxidase (tNOX), which displays specific and lot of expression in the cell surface of several types of cancer (27). These effects have been reported in malignant melanoma, bladder carcinoma, and some gastric cancer cells, among others (28–30). The treatment of these cells with capsaicin produces cell cycle arrest and apoptosis. Furthermore, direct interaction between capsaicin and tNOX was demonstrated using cellular thermal shift assay (CETSA). This molecular association triggers tNOX to degradation, affecting SIRT1 and consequently producing apoptosis (28–30).

Capsaicin also produces cytotoxic effects in cancer cells through the inhibition of heat shock proteins 90 and 70 (Hsp90 and Hsp70) overexpressed in certain tumors (31). These proteins form a co-chaperone helper system that folds mutant proteins, resulting in a favorable tumor phenotype. Remarkably, capsaicin directly interacts with the ATP-binding pocket located at the N-terminus of Hsp90 (32). This interaction blocks the ATPase activity of Hsp90 producing the Hsp70 uncoupling and its lysosomal degradation. Furthermore, cancer cell lines co-treated with capsaicin and a well-characterized Hsp90 inhibitor, 17-AAG, showed improved anti-tumor effects (32).

The capsaicin role in cancer also extends to the regulation of enzymes implied in the histone methylation. Specifically, capsaicin acts as an inhibitor of the Lysine Specific Demethylase 1A (KDM1A/LSD1) (33), which is overexpressed in different kinds of cancer (34). Capsaicin reversibly and specifically inhibits LSD1 with an IC50 = 0.6 ± 0.04 μM. Docking analysis suggested that capsaicin interacts with the FAD binding cavity (33). Interestingly, LSD1 inhibition by capsaicin decreases the proliferation, migration and invasion of a gastric cancer cell line (BGC-823) (33), suggesting that this vanilloid compound could have protective roles against this type of cancer. Additionally, some metabolites produced during microbial intestinal transformation of capsaicin and dihydrocapsaicin also display inhibitory effects toward LSD1 (35, 36).

Capsaicin is also linked to the regulation of enzymes that modify DNA methylation. For instance, there are clues that capsaicin may inhibit DNA methyltransferase 1 (DNMT1) (37). Molecular docking simulations showed capsaicin interactions with DNMT1 through hydrogen bonding with G35, Q40, T467 and Q572 residues and establish hydrophobic interactions with L71, W465, Y564 and A568. DNMT1 produces hypermethylation of the genes’ promoter regions coding for the cell adhesion molecule 1 (CADM1) and suppressor of cytokine signaling (SOCS1), which are suppressors of malignant tumor development (38, 39). The HeLa cells (an adenocarcinoma cervical cancer cell line) display DNMT1 overactivity, resulting in the hypermethylation of these genes and downregulation of their expression. Interestingly, HeLa cells exposed to 20 μM capsaicin for a long time showed reversed hypermethylation of CADM1 and SOCS1, restoring the expression of these tumor suppressors. Moreover, high capsaicin treatment produces a decrease in the HeLa cell viability (37). These results suggest that capsaicin could be an anti-methylation agent with the potential for use in the co-treatment of cervical cancer.

The capsaicin’s potential role as an anticancer compound has also been explored in human small-cell lung cancer (40). The long-term treatment of these cells with high capsaicin concentration induces apoptosis through calpain activation; interestingly, this effect dependents on the TRPV6 expression, an abundant receptor expressed in several types of cancer. Similarly, capsaicin produces apoptosis in gastric cancer cells in a TRPV6-dependent manner (41). Since this channel is not directly activated by capsaicin (42, 43), the molecular mechanism of this effect is still unresolved.

The above information briefly exemplifies the capsaicin effects on some cancer cell lines where this compound produces its effects through direct interaction with specific molecular targets. Besides the large number of reports about the antitumoral capsaicin effects, it has also been reported some contrary effects, for example, long-term exposure to low and high capsaicin concentration on some colon and bladder cancer cells (44, 45), positively regulate the epithelial-mesenchymal transition, enhancing their metastatic features, highlighting that we carefully must analyze the benefits and risks of use of capsaicin as a coadjutant in anticancer treatments (30). Therefore, the potential anticancer effects of capsaicin require evaluation for each type of tumor and an appropriate assessment of the optimal concentration and exposure times to obtain greater specificity in the putative anticancer response.

Initial studies demonstrated that capsaicin (10 to 50 μM) inhibits the release of prostaglandin E2 from LPS-stimulated peritoneal macrophages. This action is through inhibiting the Cyclooxygenase-2 (COX-2) activity without modifying its expression (46).

Recently, it has shown that capsaicin binds to the COX-2 (47), Pyruvate kinase isoenzyme type M2 (PKM2) and Lactate Dehydrogenase A (LDHA) (48). The report showed that capsaicin inhibited lipopolysaccharide-mediated TNF-𝛼, IL-1𝛽, IL-6 and iNOS expression in the RAW264.7 macrophage cell line, suggesting that capsaicin has anti-inflammatory activity in a TRPV1-independent way since this cell line does not express TRPV1 (48). Interestingly, in a sepsis mouse model treated with capsaicin improved the rate of survival, and upon further exploration with a chemical proteomics approach was discovered that capsaicin exerts its effects through direct interaction with PKM2, LDHA and COX-2 (48). The interaction of capsaicin with these proteins requires key cysteines (C424 of PKM2 and C84 of LDHA), which was confirmed through site-directed mutagenesis studies and molecular modeling (48).

Furthermore, through enzymatic kinetic studies it has also been reported that capsaicin binds to and inhibits human secretory Phospholipase A2 (sPLA2), a key enzyme involved in the hydrolysis of phospholipids to release proinflammatory mediators (47). Molecular modeling showed that capsaicin interaction with sPLA2 is carried out by hydrogen bonds formed between the NH and keto groups of capsaicin with the enzyme residues H47 and G29, respectively, Figure 1D lower. Additionally, the evaluation of the catalytic activity of PLA2 in presence of its substrate and capsaicin showed that the V_max was not modified, however, the K_m of the enzyme increased in the presence of capsaicin (from 0.14 to 0.25 mM), suggesting that capsaicin competes with the substrate for the binding site in PLA2. Thus, the interaction between capsaicin and PLA2 may prevent the production of inflammatory mediators (47).

The above evidence exemplifies capsaicin’s anti-inflammatory actions; however, it is crucial to consider that capsaicin also has pro-inflammatory effects through TRPV1 activation, this releases Substance P and Calcitonin Gene-Related Peptide, which feeds back the inflammatory response (neurogenic inflammation) (49, 50). Moreover, it has been reported that capsaicin activates human dendritic cells through its classical receptor, TRPV1, having a pivotal role in the immune response (51). These are essential clues to consider when capsaicin is proposed as an anti-inflammatory compound since the immediate effect will always be through TRPV1 activation due to its high affinity, followed by actions on its non-classical targets. Interestingly, the capsaicin long-term exposition of TRPV1 produces its desensitization (52); thus, prolonged use of capsaicin could lead to the loss of its pro-inflammatory effects, with the prevalence of anti-inflammatory effects through non-classical targets.

The effects of capsaicin on the airways have also been explored. It was described that capsaicin potentiates the activity of cystic fibrosis transmembrane conductance regulator (CFTR), an anionic channel regulated by cAMP (53). Electrophysiological experiments carried out in CFTR-expressing cells without TRPV1 expression (NIH-3T3 or CHO cells) showed that capsaicin enhanced cAMP-stimulated CFTR currents. The capsaicin concentration to produce the half-maximal potentiation was calculated as ~48 μM. Interestingly, single-channel recording showed that capsaicin increases the open probability (Po) of CFTR produced by forskolin (53). Furthermore, experiments carried out with genistein, a potentiator of CFTR activity, and high concentration of capsaicin decrease the genistein potentiation of cAMP-stimulated CFTR currents; otherwise, when capsaicin was applied together with low genistein concentration, the CFTR currents were potentiated, suggesting that both compounds act through a common binding site, probably intracellularly located (53).

Capsaicin potentiator effect on CFTR activity has also been reported in the human airway epithelial Calu-3 cells (54). Experiments to measure the apical Cl-currents (aI_Cl) showed that capsaicin increases the forskolin-induced aI_Cl, effect reduced by the presence of CFTR inhibitors. Interestingly, capsaicin has opposite effects over several HCO₃⁻/CI^-uptake transporters located on the basolateral membrane, indicating that capsaicin strongly impacts the physiology of the airways, and it could be considered for the treatment of CFTR-related inflammatory diseases.

Recently, the beneficial effects of capsaicin in gastrointestinal physiology have also been reported, (55). The reports show that capsaicin modifies the short-circuit current (I_sc), which is related to intestinal chloride secretion. Studies achieved in intestinal samples isolated from WT mice showed that I_sc stimulated by carbachol and caffeine were attenuated by capsaicin (30 μM) (55). The capsaicin effect was preserved in intestinal samples from TRPV1 knockout mice; however, this effect was lost in the intestine from TRPV4 knockout mice, suggesting that capsaicin attenuates intestinal I_sc in a TRPV4-dependent manner. Capsaicin’s inhibitory effects on TRPV4 activity were evaluated in intestinal epithelial cells (IEC-6) and fluorometric assays revealed that capsaicin inhibits TRPV4 activation through its specific agonists (55), indicating that capsaicin inhibits the intestinal CI^-secretion affecting the TRPV4 function. Interestingly, the jejunal Na⁺ absorption is improved by capsaicin through its inhibitory actions over TRPV4, since reduced Na⁺ absorption is related to diarrhea, capsaicin could act as an antidiarrheal agent (55).

Gastrointestinal function is closer to the vascular system, ensuring adequate blood supply to absorb nutrients and distribute oxygen. Alteration in the vasculature is associated with some inflammatory conditions such as intestinal bowel syndrome (IBS). The endothelial cells play a pivotal role in ensuring vasorelaxation, where endothelium-dependent hyperpolarization (EDH) regulates the resistance vessel and the mesenteric circulation (56). TRPV4 expression is abundant in endothelial cells, and its activation is associated with increased intracellular Ca²⁺ and the concomitant activation of calcium-activated K⁺ channels producing EDH. Peculiarly, capsaicin induces endothelial hyperpolarization through TRPV4 activation, stimulating vasorelaxation. This effect improves blood supply in WT colitis-induce mice but have no effect on the TRPV4 knockout mice (57). These observations allow the use of capsaicin as a natural compound in combination with anti-colitis treatments to improve the health of patients with this illness.

Atypical capsaicin roles comprise antiviral effects. Studies performed in A549 cells infected with VSV, H1N1, or ECMV (vesicular stomatitis-, human influenza-, and encephalomyocarditis-virus, respectively) and treated with high capsaicin concentration exhibited marked inhibition of viral replication (58). In vivo experiments were also conducted using the VSV intraperitoneal injection in control and capsaicin pretreated mice. The results showed the attenuation of VSV replication in the liver, lung, and spleen of capsaicin-treated mice; capsaicin also reduced the expression of some inflammatory mediators such as TNFα, IlIB, and Il6. Similar experiments were carried out with capsaicin and AMG517, a selective TRPV1 antagonists, which failed to prevent the antiviral capsaicin effect, discarding the TRPV1 involvement.

The mechanism underlines that capsaicin’s antiviral action implicates STAT3, a transcriptional repressor of antiviral immunity. Interestingly, capsaicin induces STAT3 to lysosomal degradation. Apparently, capsaicin interacts in the SH2 domain of STAT3 forming hydrogen bonds with S636 and S623 residues of STAT3 (58). Furthermore, these data agree with a report that demonstrated that capsaicin negatively regulates STAT3 on primary cells of lymphoma and induces the exposition of Damage Associated Molecular Patterns (DAMPs) inducing immunogenic cell death (59); these results demonstrate the effects of capsaicin targeting STAT3 lysosomal degradation enhancing immunity response.

Similar antiviral capsaicin effects have been reported for the porcine enteric coronaviruses. Experiments using IPEC-J2 cells (intestinal porcine enterocytes) demonstrated that the replication of porcine virus required an increase of intracellular Ca²⁺ (60). These cells endogenously express the TRPV4 channel and capsaicin inhibits the calcium entry through this channel and consequently suppress the replication viral (60).

It has also been reported that capsaicin interacts with collagen (61). The reports found that increasing concentrations of capsaicin reduced the fibril formation (61). Surprisingly, once these collagen fibrils are assembled, capsaicin protects them from enzymatic degradation. Molecular modeling was carried out to infer the interaction of capsaicin with the triple helical conformation of collagen, suggesting that the phenol ring of capsaicin could potentially bind to the “GFOGER,” “EKG,” and “GLO” sequences in the collagen fibril (61).

Peculiarly, capsaicin also has the potential to bind directly to DNA. Using calf thymus DNA (ctDNA) and diverse biophysical tools demonstrated that H-bond and Van der Waals forces are the main interactions between capsaicin and ctDNA. Molecular docking showed that capsaicin bound to ctDNA in the minor groove, placing it in a GC-rich region. Capsaicin-DNA interactions are stabilized by two hydrogen bonds, each with guanine, and have the potential to form hydrophobic interactions with adenine rings (62). The biological effects of capsaicin-DNA interactions remain unresolved, opening a field to research the implications of this interaction and its possible impact on the function of DNA.

Capsaicin, used as a natural co-adjuvant to relieve some pathological conditions, should considered the proper use of this compound since high concentrations can accumulate in the plasma membrane and produce collateral harmful consequences, such as cardiovascular alterations.

Curiously, some of the unclassical capsaicin interactions are beyond transmembrane localization, suggesting that capsaicin would have to be able to achieve the nucleoplasm to interact with LSD1 and DNMT1 or be available in the cytoplasm to exert its actions over some enzymes as Hsp90. It will be possible through the capsaicin accumulation in the plasma membrane and its internalization through endocytic vesicles, where the capsaicin would be inserted in the “tail-up” and “head-down” configuration, leaving accessible the head and neck domains to establish H-bond with the amino acids of its intracellular molecular targets, which is a typical interaction between this capsaicinoid and the proteins before mentioned.

Another interesting perspective is detailing the molecular mechanism of capsaicin action over the function of other TRPV channels, impacting gastrointestinal and cancer cell physiology. The capsaicin insertion to membranes could be the mechanism to regulate the function of TRPV4 and TRPV6 since these channels also play a role as mechanosensors (63, 64), thus any modification on the membrane fluidity could affect their function; it will be interesting to assay the function of these channels inserted in artificial membranes applying low and high concentrations of capsaicin to determinate how the capsaicin insertion into membranes affects the function of these channels.

Finally, the main future perspective of using capsaicin is having an efficient delivery system, and the nano micelles could be a great option. Thus, further research is required to find the optimal use of capsaicin beyond an analgesic alternative.