One of the telltale symptoms of oral submucous fibrosis (OSF) is a burning sensation in the mouth when eating spicy foods. Vesicular stomatitis/blister formation following the intake of foods laced with chilies (Capsicum annum and Capsicum frutescence) has led to the hypothesis that supports the etiological role of chilies in the development of OSF. This was reinforced by Sirsat and Khanolkar in 1960 (Sirsat and Khanolkar, 1960a; Sirsat and Khanolkar, 1960b), who were able to produce a mucosal connective tissue response similar to OSF by topical application of capsaicin (8-methyl-N-vanillyl-6-nonenamide), the active ingredient in chilies, in protein depleted and vitamin B deficient Wistar rats (Sirsat and Khanolkar, 1960a). Histologically, the epithelial changes and sub-epithelial hyalinization were reminiscent of OSF, but no changes were noted in the deeper connective tissue characteristic of fibrosis (Sirsat and Khanolkar, 1960a; Sirsat and Khanolkar, 1960b). Since these alterations were non-specific and indicative of local reaction to irritants, the authors were guarded in proposing chilies as the causative factor in OSF (Sirsat and Khanolkar, 1960a; Murti et al., 1995). Thus, they reported that ‘conditioned mucosa’ owing to vitamin and protein deficiency might make the mucosa susceptible to OSF. However, it has been observed that the dietary state may not be the primary factor as patients with advanced OSF have difficulty having a normal diet. Also, there are no interventional studies that support the protective effects of nutritional supplements against OSF (Cox and Walker, 1996).

Several investigators have supported the chili hypothesis (Sirsat and Khanolkar, 1960a; Sirsat and Pindborg, 1967; Shiau and Kwan, 1979) with some suggesting an allergic reaction to chilies (Sirsat and Khanolkar, 1960a; Pindborg et al., 1964; Pindborg and Singh, 1964; Pindborg and Sirsat, 1966; Sirsat and Pindborg, 1967; Pindborg et al., 1968; Shiau and Kwan, 1979). Hypersensitivity to chilies and a susceptible mucosa result in increased eosinophilia in tissue and serum suggesting that OSF arises as a result of an allergic reaction to chilies (Desa, 1957; Sirsat and Khanolkar, 1960a; Rao, 1962; Pindborg et al., 1964; Pindborg and Singh, 1964; Pindborg and Sirsat, 1966; Wahi et al., 1966; Sirsat and Pindborg, 1967; Pindborg et al., 1968; Hamner et al., 1971; Shiau and Kwan, 1979). In this paper, we provide an explanation for the continuous burning pain in OSF and attempt to correct the myth that chili is an etiologic factor in the development of OSF. We explain the molecular mechanisms of capsaicin-mediated antifibrotic and anticarcinogenic effects and draw attention to the therapeutic use of capsaicin in reversing fibrosis and preventing the malignant transformation of OSF.

The role of chilies in the development of OSF has been widely discussed in the literature. While the formation of vesicles and secondary eosinophilia has been considered to be the manifestation of an allergic response to chili (Pindborg and Singh, 1964; Pindborg and Sirsat, 1966), several investigators have refuted this association (Desa, 1957; Rao, 1962; Wahi et al., 1966; Hamner et al., 1971; Shiau and Kwan, 1979; Gupta et al., 1980). Even though the formation of vesicles occurred within 3 hours of consumption of food strongly laced with chili (Pindborg and Sirsat, 1966), only 30% of OSF patients showed above normal absolute eosinophil counts (Wahi et al., 1966).

In a case series analysis by Desa (1957) only 6/64 patients with OSF (9.4%) reported excessive use of chilies (Desa, 1957). Contesting the chili hypothesis, Wahi et al. (1966) showed that neither the amount nor the duration of chili consumption contributed to OSF progression (Wahi et al., 1966). Likewise, Hamner et al. (1971) failed to produce OSF in hamster cheek cells with topical application of 2% capsaicin dissolved in mineral oil or freshly ground chili powder 3 times per week for 2-years (Hamner et al., 1971). Furthermore, OSF is not reported in Mexico and South America, where the dietary intake of chilies equals or even exceeds that in India (Cox and Walker, 1996). Shiau and Kwan (1979) in their report on 35 OSF patients in Taiwan did not see any causal association between chili consumption and OSF (Shiau and Kwan, 1979). Gupta et al. (1980) in their 10-years follow-up of Indian villagers could not correlate the disease process to any other dietary factors except the use of areca nut (AN) (Gupta et al., 1980). Seedat and van Wyk (1988) could not find any association between OSF and the use of tobacco, lime, and chili among those who chewed AN (Seedat and van Wyk, 1988).

While the role of capsaicin in fibrosis has been challenged, several studies have shown that capsaicin has proven antifibrotic properties (Wang Q et al., 2014; Perumal et al., 2015; Choi et al., 2017; Mendivil et al., 2019; Sheng et al., 2020). Capsaicin activation of TRPV-1 increases intracellular calcium ([Ca²⁺]_i), upregulates AMP-activated protein kinase (AMPK), and SIRT-1, which then upregulates endothelial nitric oxide synthase (eNOS) (McCarty et al., 2015), thus improving the endothelium-dependent vasodilation (EDV). Improved EDV could lead to alleviation of hypoxia and inhibition of fibrosis (Figure 2A).

Increased [Ca²⁺]_i also upregulates endothelial-specific transcription factor Krüppel-like Factor 2 (KLF2) through the activation of Ca²⁺/calmodulin-dependent protein kinase kinase-β (CaMKK-β) and downstream phosphorylation of AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase 2 (ERK2), histone deacetylase-5 (HDAC-5), and myocyte enhancer factor-2 (MEF2) (McCarty et al., 2015). KLF2 also upregulates eNOS, antioxidant enzymes responsive to nuclear factor erythroid 2-related factor 2 (NRF2), and thrombomodulin. The upregulation of these antioxidant enzymes downregulates inflammatory genes like nuclear factor-kappa B (NF-κB), IL-6, TNF-α, and Monocyte Chemoattractant Protein-1 (MCP-1) (McCarty et al., 2015; Choi et al., 2017). TRPV-1 upregulates uncoupling protein 2 (UCP2) through peroxisome proliferator-activated receptor delta (PPAR-δ). Augmented UCP2 has proven to be important in promoting EDV in mice (McCarty et al., 2015). Besides that, peroxisome proliferator-activated receptor gamma (PPAR-γ) is upregulated (McCarty et al., 2015), which mediates its antifibrotic effects by inhibiting the TGF-β1/Smad pathway through SMAD-7 (Choi et al., 2017). Inhibition of the TGF-β1/Smad pathway decreases collagen I (Col-I), collagen III, a-SMA, and fibronectin (Fn) deposits in the tissue (Wang Q et al., 2014; Choi et al., 2017) (Figure 2A).

In the liver, capsaicin has been shown to attenuate fibrosis through the inhibition of notch signaling, inhibiting M1-polarization of macrophages, and reducing TNF-α secretion (Sheng et al., 2020). Capsaicin along with sulforaphane synergistically inhibited fibrosis of the liver by upregulating PPAR-γ and Nrf2. This, in turn, upregulated catalase and downregulated proinflammatory cytokines like TGF-β1, TNF-α, IL-6, and Type I collagen (Mendivil et al., 2019). Nrf2 interacts with the antioxidant response element of the catalase gene to enhance its production (Mendivil et al., 2019).

Similarly, in benzo(a)pyrene-induced lung cancer the levels of ECM components like collagen, elastin, uronic acid, and glycosaminoglycans such as hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate were elevated, but upon treatment with capsaicin, they were significantly reduced (Cho et al., 2017). TRPV-1 activation protects against renal and cardiac fibrosis through inhibition of the TGF-β/Smad2/3 signaling pathway (Wang Q. et al., 2014; Yu et al., 2018). In the peritoneal macrophages, the anti-inflammatory activity of capsaicin is facilitated by IkB-α degradation (Kim et al., 2003). In the retina, capsaicin renders a protective effect on ischemia-induced injury (Wang et al., 2017). On the contrary, conjunctival epithelial cells possessing TRPV1 channels seem to be profibrotic and proinflammatory (Yang et al., 2013; Khajavi et al., 2014). This is attributable to corneal epithelial cells containing the highest density of capsaicin-sensitive TRPV1 channels in the human body (Muller et al., 2003; Marfurt et al., 2010).

In oral mucosa TRPV1 channels functions as an environment sensing protein (Takahashi et al., 2020). However, the effect of capsaicin is dose-dependent with lower doses being antifibrotic, and higher doses being profibrotic (Yu et al., 2018). The antifibrotic mechanism of capsaicin and the pathways involved have been discussed in Table 1.

The effect of capsaicin on the properties of collagen was studied using a combination of biophysical and computational tools. It was found that capsaicin strongly suppressed collagen fibril formation, increased the stability of collagen fibers in tendons, and had no effect on the stability of collagen. While capsaicin did not promote the disassembly of collagen fibrils it moderately protected the collagen fibrils from enzymatic degradation. However, computational studies have revealed the functions of the aromatic and amide regions of capsaicin in the capsaicin-collagen interaction. Capsaicin is bound to the “GFOGER”, “EKG”, and “GLO” triple-helical conformational sequences within Collagen-I to inhibit collagen fibril formation (Perumal et al., 2015) (Figure 2B).

The pro-carcinogenic effect of capsaicin in higher doses has been previously described (Murti et al., 1995) (Friedman et al., 2019). However, several studies have also demonstrated the potential anticancer effects of capsaicin (Ip et al., 2012; Gonzales et al., 2014; Clark and Lee, 2016; Cho et al., 2017; Dai et al., 2018; Friedman et al., 2018; Friedman et al., 2019; Mosqueda-Solis et al., 2021) and its non-genotoxic properties (Cho et al., 2017). Capsaicin was shown to reduce the incidence of oral epithelial dysplasia (OED) in a 4-nitroquinoline 1-oxide (4-NQO) induced tongue cancer model in male rats (Tanaka et al., 2002). Capsaicin prevents the transformation of OED to oral squamous cell carcinoma (OSCC) in mice through the reduction of anti-apoptotic marker B-cell lymphoma 2 (BCL2) (Mohamed and AlQarni, 2019). In a recent systematic review by Mosqueda-Solís et al., on capsaicin intake and oral carcinogenesis, a definitive protective effect of capsaicin on the development of oral cancer in mice was demonstrated (Mosqueda-Solis et al., 2021). Additional evidence of capsaicin being an anticarcinogenic agent comes from the fact that stomach cancer is less common in the Indian subcontinent where the consumption of spicy food is not unusual (Barad et al., 2014). This is particularly beneficial among cisplatin-resistant stomach cancer cells whereby cisplatin inducible Aurora-A kinase is degraded by capsaicin to induce apoptosis (Huh et al., 2011).

Capsaicin restores the wild-type p53 (p53_Wt) and degrades mutant p53 (p53_Mut) (Cho et al., 2017), and promotes apoptosis. Capsaicin decreases the BCL2/BAX ratio which in turn increases the permeability of the outer mitochondrial membrane (OMM) causing the release of cytochrome-c (Cyt-C) and its cytosolization. The cytosolic Cyt-C activates Caspase 9 (Cas-9) and Caspase 3 (Cas-3) which then cause apoptosis of the cell (Cho et al., 2017) (Figure 3).

Gonzales et al., in 2014 reported the anticancer effect of capsaicin on oral cancer cells through enhanced apoptosis, independent of the TRPV-1 receptor (Gonzales et al., 2014). Capsaicin has been shown to induce apoptosis in human tongue cancer cell lines SCC4, SCC25, and HSC3 via mitochondria-dependent and independent pathways (Ip et al., 2012; Gonzales et al., 2014). The apoptosis was mediated by p53 through enhanced phosphorylation at serine residues 15, 20, 392 and increased acetylation (through the inhibition of SIRT1) (Clark and Lee, 2016).

Another mechanism supporting the anticancer activity of capsaicin is via glycogen synthase kinase-3β (GSK3β)- and protein kinase C-δ (PKC-δ)-dependent phosphorylation of CCAAT enhancer-binding proteinβ (C/EBPβ). Phosphorylation of C/EBPβ augments the binding affinity of C/EBPβ onto non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) promoter, which activates the transcription of NAG-1 genes. Activating transcription factor 3 (ATF3) enhances the recruitment of C/EBPβ to the NAG-1 promoter by acting either as a bridge protein or by helping in the formation of a supramolecular complex along with other transcription factors. The activation of NAG-1 genes then triggers apoptosis (Lee et al., 2010; Clark and Lee, 2016) (Figure 3).

Additionally, capsaicin-induced a G0/G1 phase arrest in oral cancer cells through the reduction of cyclin-D (CCND), cyclin-dependent kinase 2 (CDK2), and cyclin-dependent kinase 6 (CDK6), and an increase in the levels of p21 and p16 (Ip et al., 2012). Furthermore, the anti-cancer effects of capsaicin have been demonstrated by the inhibition of Epidermal Growth Factor Receptor (EGFR) and PI3K/Akt/mTOR signaling pathways (Dai et al., 2018), and through IL-6/Signal Transducer and Activator of Transcription- 3 (STAT-3)/Cyclin D1 (CCND1) and survivin pathways (Cho et al., 2017) (Figure 3).

Capsaicin inhibits angiogenesis both in vitro and in vivo. It has been shown to cause the degradation of HIF-1α through the scaffold/matrix-associated region-binding protein 1 (SMAR1) and by the stabilization of p53 (Clark and Lee, 2016; Cho et al., 2017). SMAR1 upregulation leads to downregulation of Cox-2 and PGE-2 and reduced transcription of HIF-1α (Cho et al., 2017). Reduction in HIF-1α leads to reduced transcription of vascular endothelial growth factor (VEGF) and suppression of angiogenesis (Clark and Lee, 2016; Cho et al., 2017). Capsaicin inhibits VEGF-induced vessel sprouting and angiogenesis by the suppression of p38 MAPK, protein kinase B (PKB), and FAK pathways (Clark and Lee, 2016) (Figure 3).

The expression of the growth modulator, TRPV-1, was investigated in the pre-malignant and malignant lesions of the tongue. TRPV-1 expression was dramatically increased in all grades of oral squamous cell carcinoma (OSCC). An increase in TRPV-1 was also noted in the OSCC cell lines (Marincsak et al., 2009). This was, however, not due to the pro-carcinogenic effects of capsaicin as Ca⁺⁺ channels were triggered minimally (evidence for the activation of the receptor) upon engagement of the receptor (Gonzales et al., 2014). Additionally, TRPV1 channels were inactive in primary tongue cancer-derived cell lines SCC4, SCC25, and HSC3 (Gonzales et al., 2014). Capsaicin seemingly had an anti-cancer effect in already transformed oral cells through a mechanism independent of TRPV1 (Gonzales et al., 2014), by acting as an antagonist of coenzyme-Q (Co-Q), inhibiting the electron transport system (ETS), thereby generating ROS, and inducing apoptosis (Figure 3). On the contrary, the immortalized keratinocyte cell line OKF6-TERT2 did demonstrate cytotoxicity to capsaicin via TRPV1 channels (Gonzales et al., 2014). Therefore, capsaicin has anticancer effects in the background of OSF via a TRPV1-dependent mechanism.

Hou et al. showed that the T/C + C/C genotypes and C allelic type for miR-499a produced reduced amounts of miR-499a when compared to the TT genotype or T allelic type (Hou et al., 2015). miR-499a negatively regulated the proto-oncogene ETS-1, which in turn upregulated the connective tissue growth factor (CTGF), a well-known orchestrator of fibrosis (Hou et al., 2015). The T/C + C/C genotypes and C allelic type for miR-499a had a greater risk for BQ-induced OSF than the TT genotype or T allelic type (Hou et al., 2015). The higher risk of fibrosis in T/C + C/C genotypes and C allelic type was due to decreased expression of miR-499a, which in turn caused the activation of ETS-1 and the upregulation of CTGF. The reduction in miR-499 expression was correlated with advanced stage and larger tumor size (Hou et al., 2015). Incidentally, mir-499a levels were restored by capsaicin (Zheng et al., 2015), which in turn caused a significant reduction in ETS-1 and CTGF. Thus, the restoration of miR-499 expression through capsaicin may avert the malignancy arising in OSF through downregulation of the ETS-1/CTGF pathway and degradation of androgen receptor (Wu et al., 2015; Zheng et al., 2015) (Figure 2C).

A reduction in the dendritic cell (DC) population in pre-malignant and malignant oral lesions with higher grades (Pellicioli et al., 2017), may promote malignancy through immune evasion. Several studies have shown the downregulation of immune response among those who chew areca nut (Wang et al., 2011; Wang et al., 2012; Chang et al., 2014; Lee et al., 2014). Areca nut extract (ANE) inhibits the differentiation and function of DCs (Wang et al., 2012; Lee et al., 2014). Furthermore, augmentation of myeloid-derived suppressor cells (MDSC) via continued areca nut use fosters malignancy through auxiliary downregulation of the immune response (Dolcetti et al., 2010; Wang et al., 2011). Notably, several studies have shown that capsaicin can restore the function of the immune system and stimulate the DCs (Gilardini Montani et al., 2015; Lee et al., 2016). Intertumoral depletion of CD8+T-cells/cytotoxic T cells (CTLs) and upregulation of regulatory T cells (Tregs) are also responsible for the immune escape of tumors (Cho et al., 2017). Capsaicin could counter the immune escape of tumors by upregulating the CTLs and downregulating Tregs. Moreover, capsaicin could also lead to enhanced sensitization of stromal macrophages to tumors and increase tumor antigen presentation thereby enhancing the destruction of tumors by T cells (Cho et al., 2017) (Figure 3).

The debate over whether capsaicin is pro-carcinogenic or anti-carcinogenic as well as if it exerts pro-fibrotic or anti-fibrotic effects continue. There is overwhelming evidence confirming that capsaicin, when used in its purest form and at an appropriate dose, is anti-fibrotic and anti-carcinogenic. Repeated application of capsaicin causes the depletion of substance-P (a tumor promoter), thereby reducing inflammation. It has been established that capsaicin activates PPARγ through TRPV-independent mechanisms by activating the liver X receptors (LXR), which inhibit the activation of the NF-κB pathway via LXR response elements (LXREs). Blocking NF-κB activation suppresses the production of inflammatory cytokines like IL-1β, IL-6, and TNFα (Cho et al., 2017).

Capsaicin is anti-carcinogenic and thereby inhibits microsomal phase-I xenobiotic-metabolizing enzymes like CYP-1A1, CYP1A2, CYP3A4/5, CYP2B6, CYP2C19, CYP2D6, and CYP2E1, leading to reduced activation of carcinogens. Capsaicin inhibits the mutagenicity of tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by blocking its metabolic activation by microsomal enzymes (Cho et al., 2017). Reduced lipid peroxidation through the restoration of antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PD), and glutathione reductase (GR) represent some other novel anti-cancer mechanisms mediated by capsaicin (Cho et al., 2017). Additionally, capsaicin and evodiamine restore chemosensitivity by inhibiting the P-Glycoprotein (P-gp) and ATP-binding cassette transporter G2 (ABCG2), which is an important drug efflux pump mediating chemoresistance (Cho et al., 2017; Friedman et al., 2018). The treatment of HSC-3 and SAS oral cancer cell lines expressing a mutant p53 gene with capsaicin, augmented chemosensitivity of chemotherapeutic agents like 5-FU, cisplatin, docetaxel and doxorubicin through increased autophagy and deceased ribophorin-II. This resulted in increased necroptosis of these cells as evidenced by elevated levels of necroptotic markers, the phospho-Mixed Lineage Kinase domain-like (MLKL) and phospho-Receptor interacting protein kinase 3 (RIP3) (Huang et al., 2021). The anticancer potential of capsaicin and its analogs is discussed in detail in Table 2 and their chemical structure have been shown in Figure 4. Structurally capsaicin has been divided into three regions aromatic, amide and hydrophobic regions which are designated region A, region B and region C, respectively. Capsaicin has been shown to act as a classic agonist of the TRPV-1 channel, while evodiamine is a potent selective agonist and differs from capsaicin in region C. Capsiate and Dihydrocapsiate differ from capsaicin in region B. The occurrence of a carbon-carbon double bond in region C in capsaicin and the lack of it dihydrocapsaicin, chemically differentiate from each other (Friedman et al., 2018) (Figure 4).

The role of chilies or spicy food in the genesis of OSF has long been debated. Capsaicin has a protective role in attenuating fibrosis and is potentially therapeutic in reversing conditions linked to collagen accumulation. The continuous burning pain observed in the mucosa of OSF patients is due to the activation of mast cells and P2X receptors. This pain further gets aggravated upon the activation of TRPV-1 channels by capsaicin. Higher doses of capsaicin, however, would result in the deactivation of TRPV-1 channels. An appropriate dosage of capsaicin may ameliorate fibrosis and prevent the malignant progression of OSF. Judicious use of capsaicin and its delivery as a topical agent may have therapeutic benefits in reducing the sensation of pain through receptor desensitization, by optimizing anti-fibrotic function, thus preventing the malignant transformation of OSF.