Paclitaxel-Induced Epidermal Alterations: An In Vitro Preclinical Assessment in Primary Keratinocytes and in a 3D Epidermis Model

Paclitaxel is a microtubule-stabilizing chemotherapeutic agent approved for the treatment of ovarian, non-small cell lung, head, neck, and breast cancers. Despite its beneficial effects on cancer and widespread use, paclitaxel also damages healthy tissues, including the skin. However, the mechanisms that drive these skin adverse events are not clearly understood. In the present study, we demonstrated, by using both primary epidermal keratinocytes (NHEK) and a 3D epidermis model, that paclitaxel impairs different cellular processes: paclitaxel increased the release of IL-1α, IL-6, and IL-8 inflammatory cytokines, produced reactive oxygen species (ROS) release and apoptosis, and reduced the endothelial tube formation in the dermal microvascular endothelial cells (HDMEC). Some of the mechanisms driving these adverse skin events in vitro are mediated by the activation of toll-like receptor 4 (TLR-4), which phosphorylate transcription of nuclear factor kappa B (NF-κb). This is the first study analyzing paclitaxel effects on healthy human epidermal cells with an epidermis 3D model, and will help in understanding paclitaxel’s effects on the skin.


Introduction
Paclitaxel is an anticancer drug, extracted from the bark of the Pacific yew tree Taxus brevifolia [1]. It was first approved in 1992 by the US Food and Drug Administration (FDA) for the treatment of advanced ovarian cancer and since then, it has been used in several cancers such as breast cancer, endometrial cancer, non-small-cell lung cancer, bladder cancer, cervical carcinoma, and AIDS-related Kaposi sarcoma [2]. Paclitaxel exerts its anticancer activity through its microtubule stabilization properties, which impair the dissociation of microtubules during mitosis. Therefore, paclitaxel disrupts mitosis and blocks cell cycle progression, leading to cell death [1][2][3]. The mechanisms by which paclitaxel leads to apoptosis are not clearly understood. However, several signaling pathways have been

Non-Cytotoxic Doses of Paclitaxel Induce Inflammation in a 3D Epidermis Model
The effects of paclitaxel on cell viability were examined in NHEK cells. Incubation with the positive control SLS demonstrated that both assays were sensitive to changes in viability and cytotoxicity. Treatment with increasing doses of paclitaxel (0.3-30 µM) for 24 h was safe for NHEK keratinocytes as it did not induce significant viability reduction and LDH release. The mean viability percentages were 86.53 ± 3.5%, 94.46 ± 0.8%, and 97.35 ± 1.085% at concentrations 30 µM, 3 µM, and 0.3 µM, respectively ( Figure 1A). The LDH release was lower than 2.5% in all doses examined and was not significant compared to the control ( Figure 1B). The stratification of the 3D epidermis cell model was confirmed by the hematoxylin-eosin staining as shown in Figure 2A. Keratinocytes were distributed into the principal epidermis layers. The basal, spinous, and granular layers are present in the reconstructed model and its terminal differentiation resulted in the presence of the stratum corneum, analogously to the epidermal in vivo structure of healthy skin. Since IL-1α, IL-6, and IL-8 are known as skin inflammation molecular markers [33][34][35], and these markers have been found to be upregulated by paclitaxel in some cancer cell lines [7,11,36], we analyzed whether paclitaxel could mediate an inflammatory response in the 3D epidermis model through the induction of such cytokines. Incubation of the 3D model with paclitaxel induced a significant dose-dependent release of IL-1α, IL-6, and IL-8 ( Figure 2B-D).
Int. J. Mol. Sci. 2022, 23,1142 3 of 18 effects of paclitaxel (0.3, 3 and 30 μM) in keratinocytes, lead us to investigate its molecular effects on primary human keratinocytes and in a 3D epidermis model.

Non-Cytotoxic Doses of Paclitaxel Induce Inflammation in a 3D Epidermis Model
The effects of paclitaxel on cell viability were examined in NHEK cells. Incubation with the positive control SLS demonstrated that both assays were sensitive to changes in viability and cytotoxicity. Treatment with increasing doses of paclitaxel (0.3-30 μM) for 24 h was safe for NHEK keratinocytes as it did not induce significant viability reduction and LDH release. The mean viability percentages were 86.53 ± 3.5%, 94.46 ± 0.8%, and 97.35 ± 1.085% at concentrations 30 μM, 3 μM, and 0.3 μM, respectively ( Figure 1A). The LDH release was lower than 2.5% in all doses examined and was not significant compared to the control ( Figure  1B). The stratification of the 3D epidermis cell model was confirmed by the hematoxylineosin staining as shown in Figure 2A. Keratinocytes were distributed into the principal epidermis layers. The basal, spinous, and granular layers are present in the reconstructed model and its terminal differentiation resulted in the presence of the stratum corneum, analogously to the epidermal in vivo structure of healthy skin. Since IL-1α, IL-6, and IL-8 are known as skin inflammation molecular markers [33][34][35], and these markers have been found to be upregulated by paclitaxel in some cancer cell lines [7,11,36], we analyzed whether paclitaxel could mediate an inflammatory response in the 3D epidermis model through the induction of such cytokines. Incubation of the 3D model with paclitaxel induced a significant dose-dependent release of IL-1α, IL-6, and IL-8 ( Figure 2B-D).

Paclitaxel-Induced Oxidative Stress Response
As it has been proposed that the apoptotic effects of paclitaxel may be mediated by its capacity to induce the release of reactive oxygen species (ROS) [25,37,38], the effect of paclitaxel on intracellular ROS levels was analyzed in NHEK cells and in a 3D epidermis model. As shown in Figure 3A, exposure to paclitaxel doses of 0.3, 3, and 30 µM for 4 h, caused a significant increase in ROS production. To assess which molecules might be taking part in the oxidant induction of paclitaxel, gene, and protein expression of nuclear factor erythroid-2-related factor 2 (Nrf2), superoxide dismutase (SOD1), and NADPH oxidase 4 (NOX4) were analyzed in the 3D epidermal model. Nrf2 is a transcription factor that regulates the endogenous antioxidant defense, SOD1 is a ROS scavenging gene and NOX4 is one of the primary enzymatic sources of ROS. Treatment of this model with paclitaxel for 24 h induced a decrease in the mRNA expression of SOD1 and Nrf2 in a dose-dependent manner ( Figure 3B). The same incubation time induced the upregulation of NOX4 in all paclitaxel doses ( Figure 3B). Figure 3C shows that incubating the 3D epidermis model for 24 h with paclitaxel produced the same response in the protein expression as in the mRNA

Paclitaxel-Induced Oxidative Stress Response
As it has been proposed that the apoptotic effects of paclitaxel may be mediated its capacity to induce the release of reactive oxygen species (ROS) [25,37,38], the effect paclitaxel on intracellular ROS levels was analyzed in NHEK cells and in a 3D epiderm model. As shown in Figure 3A, exposure to paclitaxel doses of 0.3, 3, and 30 μM for 4 caused a significant increase in ROS production. To assess which molecules might be t ing part in the oxidant induction of paclitaxel, gene, and protein expression of nucl factor erythroid-2-related factor 2 (Nrf2), superoxide dismutase (SOD1), and NADPH idase 4 (NOX4) were analyzed in the 3D epidermal model. Nrf2 is a transcription fac that regulates the endogenous antioxidant defense, SOD1 is a ROS scavenging gene a NOX4 is one of the primary enzymatic sources of ROS. Treatment of this model w paclitaxel for 24 h induced a decrease in the mRNA expression of SOD1 and Nrf2 in dose-dependent manner ( Figure 3B). The same incubation time induced the upregulati of NOX4 in all paclitaxel doses ( Figure 3B). Figure 3C shows that incubating the 3D e dermis model for 24 h with paclitaxel produced the same response in the protein expr

Paclitaxel-Induced Apoptosis
Detection of annexin V-FITC by flow cytometry was used to analyze the apoptosis percentage induced by paclitaxel in NHEK cells ( Figure 4A,B). Keratinocytes incubation with paclitaxel for 24 h induced an increase in cellular apoptosis. Representative propidium iodide versus annexin V-FITC plots are shown in Figure 4A for each condition. Higher doses of paclitaxel induced significantly higher apoptosis rates, reaching up to 25.8 ± 2.9% apoptosis at the highest dose 30 µM ( Figure 4B). To analyze the apoptosis molecular markers in the 3D epidermis model, gene and protein expression of p53, p21, and BCL2 were measured ( Figure 4C-E). 24 h of paclitaxel incubation reduced BCL2 mRNA expression while p53 and p21 were upregulated. All markers showed a dose-dependent modulation and statistically significant variations. Protein levels of BCL2 and p53 were also analyzed by Western blot and showed a dose-dependent increase in the case of p53, while protein levels of BCL2 decreased significantly ( Figure 4F).

Paclitaxel-Induced Apoptosis
Detection of annexin V-FITC by flow cytometry was used to analyze the apoptosis percentage induced by paclitaxel in NHEK cells (Figure 4 A,B). Keratinocytes incubation with paclitaxel for 24 h induced an increase in cellular apoptosis. Representative propidium iodide versus annexin V-FITC plots are shown in Figure 4A for each condition. Higher doses of paclitaxel induced significantly higher apoptosis rates, reaching up to 25.8 ± 2.9% apoptosis at the highest dose 30 μM ( Figure 4B). To analyze the apoptosis molecular markers in the 3D epidermis model, gene and protein expression of p53, p21, and BCL2 were measured ( Figure 4C-E). 24 h of paclitaxel incubation reduced BCL2 mRNA expression while p53 and p21 were upregulated. All markers showed a dose-dependent modulation and statistically significant variations. Protein levels of BCL2 and p53 were also analyzed by Western blot and showed a dose-dependent increase in the case of p53, while protein levels of BCL2 decreased significantly ( Figure 4F).

Paclitaxel-Targeted Angiogenesis
Paclitaxel has a strong anti-angiogenic activity in cancer cells through the suppression of the vascular endothelial growth factor (VEGF) expression, which plays a main role in the growth of new blood vessels, by activating the endothelial nitric oxide synthase (eNOS) [39][40][41]. To evaluate these events in the skin, the effect of paclitaxel on endothelial tube formation was examined in human dermal microvascular cells. Representative images for each condition are shown in Figure 5A. HDMECs in the control condition formed capillary-like structures. However, incubation with paclitaxel for 16 h showed an impairment of angiogenesis in all doses. The analysis was performed by measuring the significant decrease of the tube length, total branches and total loops created by HDMECs in the gel matrix after paclitaxel incubation ( Figure 5B). The molecular markers of angiogenesis, VEGF and eNOS, were also evaluated in the 3D epidermis model. After paclitaxel treatment, VEGF and eNOS mRNA expression were significantly reduced in a dose-dependent manner ( Figure 5C). The same decrease was induced by paclitaxel in VEGF protein levels ( Figure 5D).

NF-κB Transcription Factor Activation by Paclitaxel
The effect of paclitaxel on NF-κB activation was evaluated in the 3D epidermis model. The 3D model was incubated for 1 h with paclitaxel and both NF-κB unphosphorylated ( Figure 6A) and phosphorylated ( Figure 6B) forms were analyzed by Western blot. While the unphosphorylated form of the protein remained stable after incubation with all paclitaxel concentrations, NF-κB phosphorylation increased at all doses.

TLR4 Mediates Paclitaxel Effects on Human Keratinocytes
Previous evidence indicates that paclitaxel is a ligand to TLR4, which is expressed on innate immune cells, including macrophages [5,42]. However, there is no evidence in human skin. In this work, NHEK cells were transiently transfected with siRNA (-) control and siRNA-TLR4 to reduce TLR4 expression. The stimulation of NHEK cells with paclitaxel 3 µM on pro-inflammatory IL-1α, IL-6, and IL-8 cytokine release, and ROS production including SOD1, NOX4, and Nrf2 was significantly inhibited in cells transfected with siRNA-TLR4 ( Figure 7A-G). The effects of paclitaxel reducing the anti-apoptotic protein BCL2 were reduced in siRNA-TLR4 treated cells ( Figure 7H). The siRNA-TLR4 transfection also abrogated the effects of paclitaxel on eNOS and VEGF expression ( Figure 7I,J) and reduced the phosphorylation of NF-κB in NHEK cells ( Figure 7K).

NF-κB Transcription Factor Activation by Paclitaxel
The effect of paclitaxel on NF-κB activation was evaluated in the 3D epidermis model. The 3D model was incubated for 1 h with paclitaxel and both NF-κB unphosphorylated ( Figure 6A) and phosphorylated ( Figure 6B) forms were analyzed by Western blot. While the unphosphorylated form of the protein remained stable after incubation with all paclitaxel concentrations, NF-κB phosphorylation increased at all doses.

TLR4 Mediates Paclitaxel Effects on Human Keratinocytes
Previous evidence indicates that paclitaxel is a ligand to TLR4, which is expressed on innate immune cells, including macrophages [5,42]. However, there is no evidence in human skin. In this work, NHEK cells were transiently transfected with siRNA (-) control and siRNA-TLR4 to reduce TLR4 expression. The stimulation of NHEK cells with paclitaxel 3 μM on pro-inflammatory IL-1α, IL-6, and IL-8 cytokine release, and ROS production including SOD1, NOX4, and Nrf2 was significantly inhibited in cells transfected with siRNA-TLR4 ( Figure 7A-G). The effects of paclitaxel reducing the anti-apoptotic protein BCL2 were reduced in siRNA-TLR4 treated cells ( Figure 7H). The siRNA-TLR4 transfection also abrogated the effects of paclitaxel on eNOS and VEGF expression ( Figure 7I, J) and reduced the phosphorylation of NF-κB in NHEK cells ( Figure 7K).

Discussion
Paclitaxel's skin adverse effects have been described from a clinical point of view and the mechanisms underlying these events have not been described thoroughly. The effects of paclitaxel on the cellular mechanisms triggering its toxicity have been studied predominantly on malignant cells [43]. To date, few studies have examined these effects in normal cells. Therefore, we wanted to investigate in this study paclitaxel in primary keratinocytes and in a 3D epidermal model, to further comprehend the mechanisms triggering its skin alterations. This is the first study analyzing paclitaxel effects on both models, showing that paclitaxel induced inflammation, oxidative stress generation, apoptosis, and angiogenesis inhibition through the activation of TLR4 and NF-κB pathways. There is a limitation of the study to be mentioned. The feeder layer that supports the epidermal growth and differentiation of the keratinocytes in the 3D model is made by BALB/3T3 fibroblasts. There is a minor possibility that the presence of fibroblasts could affect the measurements performed in this study. However, due to the substantial difference in cell number between keratinocytes and fibroblasts, and the fact that the feeder layer is irradiated and therefore, growth-arrested, we considered that the contribution of fibroblasts to the results are minor and will not significantly bias our data.
We first examined the cytotoxic effects of paclitaxel within the clinically achievable plasma concentrations, ranging from 0.3 to 30 µM, on NHEK cells. None of the concentrations reduced cell viability significantly, nor induced LDH release. These concentrations were innocuous to keratinocytes and therefore were selected for further experiments. To validate the reconstruction of the 3D epidermis model, the hematoxylin-eosin staining demonstrated the development of a fully differentiated epidermis. The 3D epidermis model was used as a mimicker of a healthy epidermis to evaluate the molecular modulation induced after treatment with the clinically achievable plasma concentrations of paclitaxel.
As paclitaxel-induced inflammation may be a trigger in the development of skin adverse effects, we analyzed the expression of cytokines IL-1α, IL-6, and IL-8 in the 3D epidermis model. IL-1 is constitutively produced by keratinocytes in the stratum corneum [33] and released as a primary response to various stimuli. It also induces the release of secondary mediators, such as IL-6 and IL-8 [44]. IL-6 stimulates keratinocyte proliferation and is studied in diseases associated with epidermal hyperplasia and in wound healing. IL-8 promotes dendritic cell migration and the recruitment of monocytes and neutrophils after external stimuli, as key steps in the initiation phase of skin inflammation. Additionally, keratinocyte production of IL-8 has been observed in autoimmune-mediated diseases [35,45]. The levels of IL-1α, IL-6, and IL-8 released by the 3D epidermis model were increased after paclitaxel treatment. While there is a lack of information that relates IL-1α to paclitaxel, previous works have described that paclitaxel induces the upregulation of IL-6 in ovarian cancer cells through the TLR4-NF-κB cascade [7]. Further, plasma levels of IL-6 and IL-8 were increased in patients with breast cancer after paclitaxel administration [46]. Paclitaxel has been shown to activate IL-8 transcriptionally in ovarian carcinoma cells [47] and increases IL-8 synthesis in a subset of human lung carcinoma cell lines through an NF-κB-dependent mechanism [36]. Other authors have related paclitaxel to the upregulation of IL-8 through the kinase JKN in a human ovarian cancer cell line [11]. These findings show that paclitaxel can upregulate different cytokines in patients and cancer cell lines, in agreement with the results obtained in this report. However, this is the first study reporting paclitaxel-induced increase of IL-1α, IL-6, and IL-8 in a 3D epidermis model.
Regarding apoptosis, paclitaxel induces the activation of the kinase Raf1, which is responsible for the apoptotic control through the suppression of BCL2, an antiapoptotic protein [41,48]. In the absence of Raf1 activity, it can induce apoptosis under the influence of the proteins, p53 and p21 [4]. The tumor suppressor protein p53 regulates proliferation and apoptosis. In normal cells, DNA damage increases levels of p53, which then triggers a cell cycle arrest mediated by the p21 protein, to promote DNA damage repair mechanisms or apoptosis [4,49]. As expected, we observed an increase in apoptosis in NHEK keratinocytes after treatment with paclitaxel. In agreement with these results, paclitaxel has been shown to cause apoptosis in hair matrix keratinocytes [20,21]. Additionally, paclitaxel modulates the expression of BCL2 and increases p53 expression levels [50,51]. Here, we showed that treatment with paclitaxel in the 3D epidermis model reduced the BCL2 gene and protein levels and increased gene and protein expression of p21 and p53. These results show that the effects derived from the antineoplastic action of paclitaxel also occur in epidermal cells and its apoptotic effects in the skin might be driven by different signaling pathways, as shown by the modulation of BCL2, p53 and p21.
It has been proposed that the apoptotic effects of paclitaxel may be mediated by its capacity to induce the release of ROS [25,37,38]. However, the relationship of oxidative stress to the overall mechanism of paclitaxel is not well established. Therefore, we analyzed the oxidative stress response induced by paclitaxel in keratinocytes and the 3D epidermis model. Paclitaxel has been shown to induce ROS in multiple cell lines, such as the human breast cancer line [37,52,53], A549 cells [53], stromal fibroblasts [54], and endothelial cells [38]. Similarly, our results showed that exposing NHEK cells to increasing doses of paclitaxel increased ROS production. We then examined, in the 3D epidermis model, the gene and protein expression of SOD1, Nrf2, and NOX4, proteins involved in the regulation of ROS, and used as markers in the study of cellular oxidation. The SOD1 enzyme acts by dispersing oxygen superoxide and hydrogen peroxide in the cell and the transcription factor Nrf2 plays an important role in regulating the transcription of antioxidant proteins. On the other hand, NOX4 is a key factor in the intracellular homeostasis of oxidation reactions and is recognized as one of the main ROS producers [50,[55][56][57]. Treatment with paclitaxel in the 3D epidermis model led to a reduction in SOD1 and Nrf2 gene and protein expression, which implies that paclitaxel impairs its antioxidant response, as well as in cancer-associated fibroblasts [58]. Contrarily, NOX4 was upregulated by paclitaxel in the 3D epidermal model, consistently with the increase in ROS production seen on NHEK cells. Of note, NOX4 upregulation associated with an ROS increase has also been described by other authors in human breast cancer lines [37,52,53].
Paclitaxel accumulates in endothelial cells [59] and exhibits a strong anti-angiogenic activity in cancer cells through the suppression of VEGF expression [39,40]. VEGF takes part in the angiogenesis signaling pathway by activating the endothelial nitric oxide synthase eNOS [60], which has reduced expression after paclitaxel treatment in endothelial cells [41,51]. Physiological levels of nitric oxide (NO) are required to maintain the normal functioning of cells, including keratinocytes. NO is vital as a signaling molecule regulating multiple epidermal functions, including keratinocyte proliferation and differentiation, apoptosis, migration, and oxidative stress, as well as cytokine production [61]. NO is produced by eNOS, that is expressed in human keratinocytes in a similar way that VEGF [61,62]. In this work, we examined if the anti-angiogenic activity of paclitaxel could happen as well in the skin, by analyzing endothelial tube formation in primary human dermal microvascular endothelial cells (HDMEC). Treatment with paclitaxel in HDMEC produced a decrease in the generation of the endothelial tube. It affected the number of branches and loops formed, and the total length of the tube network. In the 3D epidermis model, VEGF and eNOS expression was reduced as well as the protein expression of VEGF. From these results, we speculated that reduced levels of eNOS imply low levels of physiological NO. This could affect the proper epidermis permeability and wound healing, and induce an imbalance of pro-inflammatory cytokines [61], as we have seen in our results. As some authors propose that paclitaxel anti-angiogenic properties may be regulated by ROS production in endothelial cells [38], we can infer that elevated levels of oxidative stress in skin keratinocytes might also drive the anti-angiogenic properties in HDMEC. Then, our results obtained in skin cells regarding the modulation of angiogenesis by paclitaxel, correlate with those seen on cancer cell lines and show that, since angiogenesis is a process closely related to skin alterations [63], it may be taking part in paclitaxel's skin adverse effects.
After paclitaxel treatment, activation of NF-κB transcription factor has been shown to play an important role in the regulation of inflammation, apoptosis, and cell cycle progression [64]. This transcription factor has also been associated with paclitaxel-induced ROS production and angiogenesis [38]. Given that, we decided to examine the activation of the transcription factor in a 3D epidermal model. Treatment of this model with paclitaxel induced the phosphorylation of the factor NF-κB, which indicated its activation, and its possible implication in driving the cellular processes investigated in this report. Between the different activators of NF-κB, TLR4 plays an important role in the innate immune response. The activation of TLR4 triggers different molecular pathways including JNK, P38 and NF-κB [65]. Previous reports have shown that paclitaxel can activate TLR4 in macrophages and dendritic cells, mimicking the effects of lipopolysaccharide secreting inflammatory cytokines [5,66]. TLR4 is expressed in human keratinocytes and its activation has been related to inflammatory, oxidative, and anti-proliferative effects [67], showing antineoplastic effects in cutaneous squamous cell carcinoma [68]. In this work, we showed novel evidence on the effects of paclitaxel activating TLR4 and promoting NF-κB phosphorylation to induce keratinocyte inflammation, oxidative stress, apoptosis, and dermal antiangiogenic activity. The reduction of TLR4 expression by siRNA-TLR4 partially abrogated the cellular effects induced by paclitaxel in keratinocytes. Currently, the dermatological adverse effects of paclitaxel have been described from a clinical perspective, but the knowledge about their cellular and molecular mechanisms is lagging. There is limited literature in which the effects of paclitaxel in healthy keratinocytes are explained. However, our results present novel evidence of the effects of paclitaxel on skin. This report shows that paclitaxel activates TLR-4 and promotes NF-κB phosphorylation, which results in the increase of oxidative stress, inflammation, and apoptosis, and the reduction of angiogenesis. These events could explain the direct skin side effects of paclitaxel in healthy skin, although the interplay between the different cellular processes and the associated signaling pathways are yet to be discovered.
3D epidermis cell models were reconstructed using the BALB/3T3 feeder-layer technique adapted from Mak et al. [69] and Arnette et al. [28]. In brief, 10 6 BALB/3T3 fibroblasts (Lonza, Basel, Switzerland) were seeded on collagen-coated Millicell inserts (Millicell-CM 12 mm, transparent Biophore Membrane; Millipore CORP., Bedford, UK) and placed into 6well plates (Corning Incorporated; Corning, NY, USA). Fibroblasts were cultured for 2 days in 1 mL Dulbecco's Modified Eagle Medium (DMEM, high glucose) (Gibco®, Life Technologies Corporation, Madrid, Spain) supplemented with 10% fetal calf serum (FCS) (Gibco®, Life Technologies Corporation, Madrid, Spain) and added to the apical and dorsal side of the insert. When fibroblasts reached 60-70% confluence, the monolayer was irradiated with UV light at 0.048 mW for 1 h with UVACUBE 400 (Honle UV Technology, Gilching, Ger-many) to establish the feeder layer. Then, primary adult epidermal keratinocytes (192627, Lonza, Basel, Switzerland) were seeded at a density of 0.5 × 10 6 cells/cm 2 . Cultures were grown at 37 • C and 95% air/5% CO 2 until approximately 60% confluency and then were switched to KGM-2, supplemented with SupplementMix and CaCl 2 60 µM (Promocell, Heidelberg, Germany) until confluent. Confluent cultures were raised to the air-liquid interface and cultured for 21 days until epidermal stratification was achieved. To validate the stratification, histological analysis was performed after 21 days. The reconstructed epidermis tissues were fixed with 10% formalin solution, dehydrated, and embedded in paraffin. Six-micrometer-thick sections were cut and stained with hematoxylin-eosin. Random photographs were taken of each sample with a Leica DM6000B microscope (Leica Biosystems; Wetzlar, Germany).

Cell Viability and Cell Death Assay
NHEK cells were cultured in a 96-well plate (Corning Incorporated, New York, NY, USA) until reaching 60% confluence. Then, cells were incubated for 24 h with different concentrations of paclitaxel (30, 3, 0.3, and 0.03 µM). Paclitaxel was dissolved in dimethyl sulfoxide (DMSO) and then, dilutions were performed in cell culture medium. The final concentration of DMSO in the culture did not exceed 0.001%. Cell viability 24 h after removal of the treatment was determined using the MTT assay. 1 mg/mL MTT solution was added to the treated cells and incubated for 3 h at 37 • C. After incubation, cells were washed with phosphate-buffered saline (PBS), and DMSO was added for 10 min to dissolve the formazan precipitate. Absorbance was measured at 572 nm using the plate reader Infinite M200 (Tecan Group Ltd., Männedorf, Switzerland). Data were normalized to control values. 24 h after treatment, the cell death assay was performed by measuring lactate dehydrogenase (LDH) release in the medium using the commercially available LDH cytotoxicity assay kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer's instructions. Absorbance was measured at 490 nm using the plate reader Infinite M200 (Tecan Group Ltd., Männedorf, Switzerland). LDH contents were normalized to the maximum LDH release. Sodium lauryl sulfate (SLS) was used in both experiments as a positive control at 80 µg/mL.

Cytokine Determination by ELISA
3D epidermal model tissues were incubated for 24 h with different paclitaxel concentrations (0.3, 3, and 30 µM). After incubation, the culture medium was collected for each condition. IL-8, IL-6, and IL-1α cytokine levels were analyzed using commercially available Quantikine ® ELISA kits (R&D Systems, Madrid, Spain) according to the manufacturer's protocol.

Real-Time RT-PCR and siRNA Experiments
3D epidermal model tissues were incubated for 24 h with different paclitaxel concentrations (0.3, 3, and 30 µM). After incubation, total RNA was extracted using TRIzol ® Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. Reverse transcription was performed in 500 ng of total RNA with a TaqMan reverse transcription reagents kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). cDNA was amplified with specific primers and probes predesigned by Applied Biosystems for SOD1 (Hs00533490_m1), Nrf2 (Hs00975961_g1), NOX4 (Hs01379108_m1), p21 (Hs01040810_m1), p53 (Hs01034249_m1), VEGF (Hs00900055_m1), BCL2 (Hs04986394_s1) and eNOS (Hs01574665_m1) in a QuantStudio™ 5 Real-Time PCR System, using a universal master mix (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). β-actin (Hs01060665_g1) was used as an endogenous control. The mean value of the replicates for each sample was calculated and expressed as the cycle threshold (Ct). The level of gene expression was then calculated as the difference (∆Ct) between the Ct value of the target gene and the Ct value of β-actin. The fold changes in the target gene mRNA levels were designated 2 −∆Ct .

Apoptosis
NHEK cells were seeded on 96-well plates and incubated for 24 h with different paclitaxel concentrations (0.3, 3 and 30 µM). Apoptosis was measured using a commercially available Annexin V-FITC apoptosis detection kit (ab14085, Abcam, Cambridge, UK). Cells were detached and collected along with the supernatant and incubated with annexin V-FITC in a final concentration of 3 µg/mL for 15 min. Then, annexin V binding buffer was added and prior to flow cytometric analysis, propidium iodide was added at 5 µg/mL. Flow cytometric analysis was performed by a BD LSRFortessa™ X-20 flow cytometer (BD Biosciences; San Jose, CA, USA). A minimum of 10,000 cells per sample were analyzed with Flow-Jo standard software (TreeStar Inc., Ashland, OR, USA).

Angiogenesis
Determination of changes in the angiogenesis process was analyzed using the commercial endothelial tube formation assay kit (Cell Biolabs, San Diego, CA, USA). HDMEC cells were seeded on the plates coated with the matrigel provided by the kit. Incubation with paclitaxel at different concentrations (0.3, 3, and 30 µM) was performed for 16 h, following the manufacturer's instructions, to allow the endothelial tube formation. After incubation, cells were stained with a calcein-acetoxymethyl-based staining solution (calcein-AM) for 30 min. Images were captured by fluorescence microscopy (Spectral Leica TCS SP2 microscope, Leica Biosystems; Wetzlar, Germany). The morphological features were quantitatively measured to characterize the capillary-like tube structure using the software WimTube™(Onimagin Technologies SCA, Córdoba, Spain). The software analysis provides the tube length, branching points, loops, and cell-covered area. Besides that, detailed overlay images are provided in which all branching points, tubes, and cells are noticeable.

Statistical Analyses
Results from cellular in vitro experiments were expressed as mean ± standard error (SE) of n experiments. Normal distribution for each data set was confirmed by the Kolmogorov-Smirnov test. Statistical analysis was carried out by multiple comparisons analysis of variance (ANOVA) followed by Bonferroni post hoc test. p < 0.05 was considered statistically significant.