1. Introduction
Ultraviolet B (UVB) radiation (280–315 nm) is regarded as one of the major predisposing risk factors of skin cancer as it is known to induce DNA damage, oxidative stress, inflammation and cell cycle dysregulation, pathological events that can lead to malignant transformation in cells [
1,
2,
3]. Keratinocytes are the primary targets of UVB-induced toxic effects [
4,
5], as a result the most commonly occurring skin cancers develop in the epidermal layer through a multistage process involving initiation, promotion and progression [
6,
7,
8]. In the complex process of photocarcinogenesis, two distinct pathways regulating DNA damage repair and inflammation have been identified [
9,
10,
11]. The one pathway occurs upon DNA damage which involves cell cycle arrest, DNA misrepair genetic mutations, cell cycle dysregulation and apoptosis in cells. In the other pathway, UVB activates the production of various pro-inflammatory mediators including the primary (TNF-α and interleukin-1 (IL-1α and β)) and secondary (IL-6, IL-8, IL-10 and IL-4) cytokines that mediate inflammation and immunosuppresion [
9,
12]. A key signalling molecule responsible for the early inflammatory events in skin is IL-1α, a primary cytokine that is constitutively produced in keratinocytes as a biologically active precursor molecule that remains in the cytosol [
13,
14,
15]. Upon cell activation by inflammatory stimuli, cytosolic IL-1α translocates to the nucleus where it activates the production of pro-inflammatory cytokines and growth factors that are involved in the recruitment of immune cells producing reactive oxygen species (ROS) and cell proliferation [
16,
17].
In damaged cells, IL-1α is passively released into the extracellular environment where it mediates the inflammatory process via IL-1R-dependent mechanisms. The necrotic release is associated with epidermal hyperplasia and malignant transformation in skin [
18,
19,
20]. Due to its role in inflammation and cancer development, IL-1α has been used as a biomarker in various in vivo and in vitro models to evaluate the progression of the inflammatory process during cancer development [
20,
21,
22]. However, as the necrotic release of IL-1α is associated with an exacerbated inflammatory response, it has been suggested that extracellular IL-1α signalling may not be a suitable model for chemoprevention studies and mechanisms, focusing on the inactivation of intracellular IL-1α (icIL-1α) via apoptosis, have thus been suggested [
23]. Consequently, an in vitro UVB irradiation cell model was developed in which induction of icIL-1α could be inhibited either by transcription modulation or augmentation of apoptosis [
24]. Since, in vitro models, utilising primary or cultured keratinocytes have served as useful tools in mechanistic and drug development studies of inflammation [
25,
26,
27,
28], the UVB/HaCaT model was also recommended as a screening tool that could be used to determine the anti-inflammatory and chemopreventive efficacy of novel compounds in the inflammatory process.
Naturally occurring antioxidants such as polyphenolic plant constituents have been the focus of research studies concerned with the development of novel anti-inflammatory and chemopreventive agents in skin [
29,
30,
31,
32]. One of the most extensively studied natural agents in skin is green and black tea (
Camellia sinensis) and their polyphenolic constituents, which have been shown to prevent inflammation by inhibiting the synthesis of IL-1α in mouse skin. The latter is suggested to be one of the underlying mechanisms involved in the anti-tumour activity of both green and black tea extracts [
33,
34].
Rooibos (
Aspalathus linearis) and honeybush (
Cyclopia) species are South African herbal teas that have traditionally been used in the treatment of skin disorders and in skin care products due to their antioxidant, anti-inflammatory, immunomodulatory, anti-proliferative and anticancer activities [
35,
36,
37]. These biological properties, partly attributed to the dihydrochalcones of rooibos and the xanthones and flavanones of honeybush, have also been associated with the prevention of various chronic illnesses including cancer. The anti-carcinogenic properties of rooibos and honeybush (
C. intermedia) extracts have been demonstrated in mouse skin carcinogenesis models [
38,
39]. Modulation of inflammation by the polyphenolic constituents has been proposed as a key mode of action underlying the protective activity of the extracts. The anti-inflammatory properties of rooibos and honeybush against UVB and PMA-induced COX-2 expression and oedema have been demonstrated and implicated in the anti-tumour activity of the herbal tea extracts in skin [
39,
40]. However, the modulatory activity of the herbal teas against primary cytokines involved in the early stages of UVB-induced inflammation as well as the effect on cell growth parameters still needs to be elucidated.
The aim of the current study was therefore to determine the effect of methanol and aqueous extracts of rooibos and two honeybush species against UVB-induced icIL-1α accumulation in keratinocytes in relation to various cell growth indices. Green tea served as a benchmark. The extracts used in the current study were the same used previously, in the evaluation of the potential role of polyphenols in the modulation of skin cell viability by
Aspalathus linearis and
Cyclopia spp in vitro [
24]. Depending on the extract type, the herbal teas showed differential effects in the reduction of icIL-1α in UVB-exposed HaCaT keratinocytes and this was closely related to the reduction in cell viability and induction of apoptosis.
3. Discussion
One of the adverse biological effects in skin following UVB radiation involves the increased production and release of the primary cytokine, intracellular IL-1α (icIL-1α) by keratinocytes [
41]. The release leads to the subsequent activation of other epidermal and dermal cells resulting in the induction of an inflammatory response and cell proliferation [
16,
42]. Chronic release, as mediated by different external factors such as exposure to UVB or cancer tumour promoters, is associated with epidermal hyperplasia and skin carcinogenesis [
18,
25]. Since extracellular IL-1α is associated with an exacerbated inflammatory response and the pathogenesis of disease, mechanisms involving inactivation of icIL-1α via transcription modulation and/or programmed cell death have been identified and recommended as a target in chemoprevention [
16,
23,
43]. UVB radiation is also known to increase the level of ROS by disrupting mitochondrial function causing oxidative DNA damage [
4,
44].
The protective mechanism against DNA damage involves the induction of cell cycle arrest that allows for DNA repair while irreparable damage triggers apoptosis in a p53 dependent manner [
5]. Unrepaired DNA lesions can generate mutations that confer a selective growth advantage to pre-neoplastic cell populations as they become more resistant to apoptotic cell death eventually resulting in the development of cancer. Therefore, a key step proposed during chemoprevention of UVB-induced cell damage is the elimination of transformed cells by apoptotic cell death [
45].
Naturally occurring plant polyphenols have been targeted for use in cutaneous photo protection as they can modulate UVB-induced inflammatory and apoptotic signalling pathways associated with oxidative stress and DNA damage in skin [
30,
32,
45]. Rooibos and honeybush also possess antioxidant and anti-inflammatory properties that have been implicated in the photoprotective and anti-carcinogenic mechanisms in skin [
38,
39,
40]. The anti-inflammatory activity against cytokine production by quercetin, rutin and luteolin, also present in rooibos, has been shown to involve inhibition of pro-inflammatory cytokine synthesis of via suppression of transcription factors and MAP kinases [
31,
46]. Mangiferin and hesperidin, major polyphenolic constituents of honeybush, were shown to inhibit the inflammatory response by suppressing cytokines as well as other pro-inflammatory mediators such as adhesion molecules and prostaglandins in vitro and in vivo [
47,
48,
49]. However, the activity of these herbal teas against the adverse effects of UVB irradiation on cell integrity and the induction of primary cytokines still needs to be elucidated. Recently, an in vitro UVB model HaCaT cell model for chemoprevention has been developed for screening the anti-inflammatory efficacy of novel compounds against IL-1α accumulation [
43]. The anti-inflammatory activity exhibited against icIL-1α could either involve direct modulation of transcription machinery or pro-apoptotic effects via enhanced caspase-3 activity. Therefore the current study investigated the modulatory activity of methanol and aqueous extracts of rooibos and two selected
Cyclopia species against icIL-1α accumulation and different cell growth parameters in the UVB/HaCaT keratinocytes chemoprevention in vitro model. The activity of the herbal tea extracts was benchmarked against extracts of green tea, the protective properties of which have been studied extensively in skin [
50]. Green tea and rooibos extracts inhibited UVB-induced icIL-1α accumulation without increasing the levels of exIL-1α. The effect on icIL-1α was strongly correlated with reduction in cell viability suggesting that the inhibitory effect on icIL-1α production resulted from a reduction in cell viability and not direct modulation of its transcription machinery as previously demonstrated for dexamethasone and ibuprofen [
43]. Since the extracts did not induce the release of icIL-1α, reduction of cell viability could not be associated with necrotic cell death. A strong inverse correlation between icIL-1α inhibition and enhanced UVB-induced caspase-3 activity indicated that the anti-inflammatory activity of the extracts against icIL-1α is indirect and it involves the removal of cells with increased levels of icIL-1α via apoptosis. It is known that, in cells undergoing apoptosis, icIL-1α is retained in the nuclear chromatin fraction thus preventing its release into the extracellular environment [
23]. A similar mechanism is therefore proposed in the present study whereby rooibos and green tea extracts control sterile inflammation by promoting nuclear retention of UVB-induced icIL-1α in the keratinocytes via apoptosis.
The pro-apoptotic activity of the green tea extracts in the present study may involve the major compounds, EGCG and the alkaloid, caffeine, since the methanol extract, exhibiting stronger activity in skin cells, was previously shown to contain the highest levels of these compounds [
24]. These green tea constituents, specifically EGCG, have been shown to prevent tumour formation by enhancing apoptosis in UVB irradiated mouse skin in vivo [
51,
52]. In addition, the underlying mechanisms involved in the pro-apoptotic activity of caffeine have also been demonstrated in a HaCaT cell premalignant model known to have UVB typical p53 mutations [
53]. Caffeine was shown to specifically targets keratinocytes with unrepaired DNA damage by inhibiting AKT/protein kinase B involved in UVB-induced anti-apoptotic mechanisms. Since the pro-apoptotic activity of rooibos and green tea extracts in the HaCaT cell line was also demonstrated following UVB-irradiation in the present study, these extracts also seem to target cells with unrepaired DNA damage. This is corroborated by the previous study where the HaCaT keratinocytes was also the most sensitive cell line with respect to the reduction in cell viability by rooibos and green tea extracts when compared to normal and cancer skin cells [
24]. Although caffeine was significantly higher in the methanol extract of green tea, no appreciable difference in the alkaloid equivalents associated with the IC
50 level against cell viability was noticed between the methanol and aqueous extracts. Therefore, even though caffeine is likely to play a role in the pro-apoptotic activity of green tea it may not be the main active constituent. In rooibos, the polyphenolic equivalents (as TP and FLAVA), as well as the individual flavonoids associated with IC
50 values against cell viability, were also similar between the methanol and aqueous extracts. However, when considering apoptosis, the methanol extract, known to have high levels of the monomeric flavonoids and FLAVA constituents, was more active, further emphasising the role of pro-oxidant effects of these polyphenolic compounds in the promotion of UVB-induced apoptosis associated with oxidative stress [
24]. Of the rooibos polyphenols very little is known about the anti-inflammatory effects of aspalathin, a dihydrochalcone and major flavonoid present in unoxidized rooibos. Quercetin and rutin have been reported to effect apoptosis in cancer cells including UVB-irradiated HaCaT keratinocytes [
54,
55]. Iso-orientin and vitexin are also known to effect apoptosis in cancer cells by disrupting the mitochondrial signalling pathways [
56,
57].
When considering the effect of green tea and rooibos extracts against cell viability and cell proliferation, no significant differences between UVB irradiated and non-irradiated cells were noticed, indicating that these extracts do not offer any protective effect against UVB in cells. Since UVB is known to induce oxidative stress in cells [
4], this suggests that the extracts do not provide protection against ROS-induced cellular damage. This implies a synergistic pro-oxidative interaction between UVB and the polyphenolic constituent in the green tea and rooibos extracts. The underlying mechanism is likely to involve polyphenol/iron interactions and defects of the mitochondrial respiratory chain complexes that are related to iron depletion [
24,
58,
59]. In vitro studies have indicated that complex II defects in the mitochondrial electron transport chain can result in decline of ATP levels, cell cycle arrest and oxidative stress [
60,
61]. Complex II defects have also been shown to mediate UVA-induced ROS production in HaCaT epidermal keratinocytes [
44]. Therefore, it is likely that reduction of cell viability and cell proliferation exhibited by the green tea and rooibos extracts further enhance oxidative stress, either through pro-oxidant effects involving auto-oxidation and/or by inducing complex II defects. Oxidative stress resulting from complex II inhibition and auto-oxidation of green tea polyphenols has been associated with induction of apoptosis in different cell lines [
62,
63,
64]. In the present study, rooibos and green tea extracts displayed similar activity by enhancing the UVB-induced reduction in cell viability and this was associated with the induction of apoptosis in the keratinocytes. As pre-neoplastic or initiated cells are more prone to undergo apoptosis due to DNA damage [
65], it is likely that the pro-oxidant properties of these extracts, and the resultant oxidative stress, accelerate UVB damaged in the keratinocytes thereby facilitating apoptosis and subsequent removal of inflamed cells containing high levels of biologically active icIL-1α.
The honeybush species displayed different effects compared to green tea and rooibos. This could be related to the activity of their major polyphenolic constituents, xanthones and flavanones. UVB-irradiated cells exhibited resistance to reduction of cell viability and inhibition of cell proliferation by the methanol extracts of
C. intermedia and
C. subternata. This indicated that these honeybush extracts have a cytoprotective effect against UVB-induced damage related to the induction of oxidative stress. The cytoprotective effect was further emphasised by the reduction of UVB-induced caspase-3 activity associated with apoptosis that was exhibited by the methanol extracts of
C. intermedia and
C. subternata. Since the methanol extracts have high levels of xanthones and flavanones, the cytoprotective effect of honeybush species may be mediated by these major polyphenolic constituents, as suggested previously [
24]. Mangiferin has been shown to protect human skin keratinocytes against oxidative cell death [
66] while hesperidin has been found to exhibit protective effects against radiation-induced DNA damage and cell proliferation in bone marrow cells [
67]. Other phenolic constituents of honeybush, i.e., the flavanone, eriocitrin, was reported to be more effective at protecting lipid membrane against oxidative stress than hesperidin [
68], while the flavone, scolymoside is also known to exhibit a strong radical scavenging activity [
69]. Of interest is that a high level of scolymoside associated with the IC
50 against cell viability of the methanol extract of
C. subternata, which exhibited a protection against apoptosis. In addition, the protection against these cell survival parameters by the methanol extract of
C. subternata was also associated with high levels of phloretin-3′,5′-di-
C-glucoside and iriflophenone-3-
C-glucoside. Therefore, it is possible that the xanthones may be acting additively and/or synergistically with hesperidin and other polyphenolic constituents to protect against UVB-induced oxidative stress.
The cytoprotective effect of honeybush has also been reported to be dependent on a specific xanthone-to-flavanone ratio and/or the FLAVA constituents. A respective decrease in the xanthone-to flavanone ratio and increase in the FLAVA content seems to mediate the cytotoxic activity of the extracts [
24]. This also became apparent in the current study as an increased activity in the reduction of cell viability, inhibition of cell proliferation and an increased induction of apoptosis was noticed by the aqueous extracts of both honeybush species. Similar to green tea and rooibos, the cytotoxic effect of the honeybush species may be mediated by pro-oxidant polyphenol/iron interactions by these constituents. In this regard the pro-oxidant activity of the tannin-like polymeric compounds, including the proanthocyanidins that occur in higher levels in the aqueous extracts, could play a determining role. However, the role of these polymeric constituents in the pro-oxidant and pro-apoptotic activity of honeybush still needs to be elucidated. With respect to green tea far higher FLAVA levels were also associated with the IC
50 value for the reduction in cell viability and icIL-1α by the aqueous extract. This would imply that more hydrophilic constituents may exhibited a protective effect against cell death counteracting the effect of the catechins. In contrast, the two rooibos extracts display remarkably similar effects when comparing the levels of the polyphenolic constituents associated with their IC
50 values for reduction in cell viability and icIL-1α accumulation. In both cases the aqueous extract also exhibited a reduced effect on the induction of apoptosis. Therefore, the relative differences in the anti- and/or pro-oxidant potential are associated with variations in the type of polyphenolic constituents as well as their relative concentrations in comprising the methanol and aqueous extracts of green tea and the herbal teas.
Regarding the modulation of UVB-induced icIL-1α accumulation by the honeybush species, the aqueous extract of C. intermedia inhibited icIL-1α production in a dose dependent manner, but this effect was far weaker than that of green tea and rooibos extracts. The inhibitory effect was dose-dependent and correlated strongly with the reduction in cell viability and induction of caspase-3 activity, suggesting that the anti-inflammatory effect follows a similar mechanism involving apoptosis as has been described earlier for green tea and rooibos. On the other hand, the methanol extracts of both honeybush species enhanced icIL-1α accumulation, suggestive of a pro-inflammatory effect that was closely associated with a reduction in UVB-induced apoptosis without adversely affecting cell viability. The pro-inflammatory activity of the honeybush species is possibly mediated by two mechanisms. In the first mechanism, inflamed cells with high levels of biologically active icIL-1α are protected from entering into apoptosis through antioxidant mechanisms that involve polyphenol/iron interactions. In the second mechanism, known/unknown compounds in honeybush may stimulate icIL-1α production through mechanisms that involve transcription modulation, but this needs further investigation. Since the aqueous extract of C. subternata enhanced icIL-1α production by reducing cell viability as well as inducing apoptosis, could be related to the second proposed mechanism involving transcription regulation. It would appear that thresholds exist for the effective removal of UVB-induced icIL-1α in keratinocytes by the herbal teas which is governed by different mechanisms involving complex keratinocyte/polyphenolic interactions.
In summary, the current study indicated that rooibos extracts and the aqueous extract of
C. intermedia, similar to green tea extracts, exhibit indirect anti-inflammatory effects against UVB-induced damage in keratinocytes by removing damaged inflamed skin cells with high levels of icIL-1α via apoptosis. The underlying mechanisms in this indirect “anti-inflammatory” effect may involve pro-oxidative polyphenol/iron interactions that result from mitochondrial dysfunction and oxidative damage targeting damaged cells to undergo apoptosis. On the other hand, the methanol extracts of honeybush exhibited a pro-inflammatory activity mainly associated with cytoprotective effects, presumably mediated by antioxidant mechanisms that also involve polyphenol/iron protective interactions. However, there might also be a stimulatory role involving transcription modulation in the pro-inflammatory activity of the aqueous extract of
C. subternata. The indirect anti-inflammatory effect of green tea, rooibos and the aqueous extract of
C. intermedia or specific combination thereof may be useful in the prevention of UVB-induced chronic inflammation and photo-carcinogenesis. These extracts may be considered as an additive in skin care products directed for use following excessive sun exposure. Although methanol extracts of honeybush seem to protect against UVB-induced harmful effects, these extracts should rather be further characterised in a pre-exposure model for protection against UVB induced oxidative stress. In this regard, mangiferin has been shown to possess cytoprotective and anti-genotoxic activity in vitro [
70]. A recent study also reported on the antioxidant and anti-inflammatory potential of fermented
C. intermedia extract in a pre-treatment UVB-HaCaT model further supporting the proposed photo protective properties of honeybush in the skin [
71]. Future studies on rooibos and species-specific honeybush aqueous extracts should investigate the role of iron-related pro-oxidant mechanisms in effecting mitochondrial dysfunction, enhancement of oxidative stress and apoptosis as well as the subsequent removal of UVB damaged keratinocytes and the attenuation of inflammation.