Skin cancer is the most common malignancy worldwide and is rapidly rising in incidence, representing a significant public health challenge. The β-blocker, carvedilol, has shown promising effects in preventing skin cancer. However, as a potent β-blocker, repurposing carvedilol to an anticancer agent is limited by cardiovascular effects. Carvedilol is a racemic mixture consisting of equimolar S- and R-carvedilol, whereas the R-carvedilol enantiomer does not possess β-blocking activity. Because previous studies suggest that carvedilol's cancer preventive activity is independent of β-blockade, we examined the skin cancer preventive activity of R-carvedilol compared with S-carvedilol and the racemic carvedilol. R- and S-carvedilol were equally effective in preventing EGF-induced neoplastic transformation of the mouse epidermal JB6 Cl 41-5a (JB6 P+) cells and displayed similar attenuation of EGF-induced ELK-1 activity. R-carvedilol appeared slightly better than S-carvedilol against UV-induced intracellular oxidative stress and release of prostaglandin E2 from the JB6 P+ cells. In an acute UV-induced skin damage and inflammation mouse model using a single irradiation of 300 mJ/cm2 UV, topical treatment with R-carvedilol dose dependently attenuated skin edema and reduced epidermal thickening, Ki-67 staining, COX-2 protein, and IL6 and IL1β mRNA levels similar to carvedilol. In a chronic UV (50–150 mJ/cm2) induced skin carcinogenesis model in mice with pretreatment of test agents, topical treatment with R-carvedilol, but not racemic carvedilol, significantly delayed and reduced skin squamous cell carcinoma development. Therefore, as an enantiomer present in an FDA-approved agent, R-carvedilol may be a better option for developing a safer and more effective preventive agent for skin carcinogenesis.

Prevention Relevance:

In this study, we demonstrated the skin cancer preventive activity of R-carvedilol, the non-β-blocking enantiomer present in the racemic β-blocker, carvedilol. As R-carvedilol does not have β-blocking activity, such a preventive treatment would not lead to common cardiovascular side effects of β-blockers.

Basal and squamous cell carcinoma (SCC), collectively named nonmelanoma skin cancer (NMSC), is the most common cancer type in the United States and worldwide (1). The primary etiologic factor for NMSC is the solar UV radiation, mainly consisting of UVA (320–400 nm) and UVB (290–320 nm; ref. 2). In contrast to most other tumor types, skin cancer incidence is increasing at an alarming rate in the United States (3, 4). Although most NMSCs are not fatal, the tumor can destroy facial sensory organs, such as the nose, ear, and lips (5), and, therefore, have an enormous impact on health care costs. There is a strong need to develop preventive agents that inhibit and reverse UV-induced biochemical changes leading to skin carcinogenesis.

Approved by the FDA in 1995, carvedilol is a third-generation receptor subtype nonselective β-blocker with α-adrenergic receptor (AR) blocking and antioxidant properties (6–8). Recent data suggest that carvedilol has preventive properties against malignant transformation and carcinogenesis induced by EGF, chemical carcinogens, and UV radiation (9–12). However, as a highly potent β-blocker (IC50 value of ∼1 nmol/L; ref. 13), repurposing carvedilol to an anticancer agent faces obstacles because by blocking the β-ARs it reduces cardiac output and thus, may cause undesirable cardiovascular side effects, such as bradycardia, hypotension, and reduced exercise capacity. Although systemic effects may be ameliorated through topical administration, systemic absorption through the skin can still result in cardiovascular disturbances. Such potential side effects associated with carvedilol become a significant concern for repurposing this agent for cancer prevention.

Notably, not all β-blockers have preventive activities against EGF-induced epidermal cell transformation (9, 11, 14). Furthermore, previous studies demonstrated that pharmacologic inhibition of α1- and β2-ARs and genetic knockdown of β2-ARs with short hairpin RNA failed to abrogate carvedilol-mediated inhibition of EGF-induced JB6 Cl 41-5a (JB6 P+) cell transformation (14). These results indicate that the preventive effects of carvedilol against skin carcinogenesis are independent of β-blockade.

Carvedilol is a racemic mixture of equal amounts of S-carvedilol and R-carvedilol (Fig. 1A). The difference in stereochemistry influences the enantiomers' optical activity and, subsequently, their pharmacologic effects. For example, the enantiomers exhibit different AR binding affinities: while both bind to the α-AR, only S-carvedilol exhibits affinity to the β-AR (15). Therefore, only S-carvedilol is responsible for the β-AR blocking activity associated with the cardiovascular properties that the racemic carvedilol exhibits. Indeed, previous studies confirmed that R-carvedilol does not significantly alters heart rate and blood pressure in mice (16, 17). Thus, R-carvedilol represents an attractive cancer preventative agent if the cancer preventive properties of racemic carvedilol are not stereoselective.

Figure 1.

Comparison of the effects of S- and R-carvedilol (CAR) on EGF-mediated effects. A, Chemical structures of carvedilol enantiomers: the β-locking S-carvedilol and non-β-blocking R-carvedilol. B, Normalized JB6 P+ cell soft agar colony formation assay and SRB assay. JB6 P+ cells were seeded in agar containing 10 ng/mL EGF combined with various S- or R-carvedilol concentrations, and colonies with 10+ cells were counted after a 10-day incubation period. Data are normalized to colonies obtained by EGF treatment only (n = 8). SRB assay data for JB6 P+ cells treated with S- or R-carvedilol at various concentrations for 72 hours are normalized to the negative control (n = 3). C, Effects of S- or R-carvedilol on EGF-induced activity of ELK-1 promoter–driven luciferase activity in HEK-293 cells normalized via Renilla luciferase coexpression (n = 3). Black stars represent the group is statistically different from 10 ng/mL EGF treatment (P < 0.05), and gray stars indicate a statistical difference between the group and control according to an ANOVA with Tukey–Kramer multiple comparison post hoc test.

Figure 1.

Comparison of the effects of S- and R-carvedilol (CAR) on EGF-mediated effects. A, Chemical structures of carvedilol enantiomers: the β-locking S-carvedilol and non-β-blocking R-carvedilol. B, Normalized JB6 P+ cell soft agar colony formation assay and SRB assay. JB6 P+ cells were seeded in agar containing 10 ng/mL EGF combined with various S- or R-carvedilol concentrations, and colonies with 10+ cells were counted after a 10-day incubation period. Data are normalized to colonies obtained by EGF treatment only (n = 8). SRB assay data for JB6 P+ cells treated with S- or R-carvedilol at various concentrations for 72 hours are normalized to the negative control (n = 3). C, Effects of S- or R-carvedilol on EGF-induced activity of ELK-1 promoter–driven luciferase activity in HEK-293 cells normalized via Renilla luciferase coexpression (n = 3). Black stars represent the group is statistically different from 10 ng/mL EGF treatment (P < 0.05), and gray stars indicate a statistical difference between the group and control according to an ANOVA with Tukey–Kramer multiple comparison post hoc test.

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In this study, we provide preclinical evidence that R-carvedilol has a similar preventive effect as the racemic carvedilol and S-carvedilol against EGF-induced JB6 P+ cell transformation, as well as UV-induced oxidative stress and prostaglandin E2 (PGE2) production. R-carvedilol displayed an equal or better inhibitory effect in hairless mice than carvedilol against a single-dose UV-induced acute skin damage and inflammation. In a chronic UV exposure model that generates tumors as early as 17 weeks, pretreatment with R-carvedilol significantly delayed tumor formation compared with pretreatment with carvedilol. Because R-carvedilol does not lower heart rate or blood pressure, R-carvedilol warrants further cancer preventative studies and is a strong candidate as an anticancer agent.

Compounds

Carvedilol was purchased from Tocris Bioscience for cell culture work or from TCI American for animal work. S- and R-carvedilol for cell culture work and for short-term UV mouse study were purchased from Toronto Research Chemicals. For long-term UV mouse study, the optically pure R-carvedilol was synthesized at Chem-Impex International, Inc. and verified by HPLC analysis. These compounds were reconstituted in DMSO as a stock solution at 10 mmol/L and stored at −20°C. Carvedilol and R-carvedilol were diluted from the DMSO stock in acetone immediately before in vivo studies. EGF was purchased from PeproTech and was dissolved in sterile deionized water at a 100 μg/mL stock and stored at −20°C.

Cell culture

The mouse epidermal cell line JB6 P+, a subline sensitive to promotion by growth factors and environmental stressors, such as UV, was purchased from the ATCC in 2011. The cells were cultured in Eagle minimum essential medium (ATCC) containing 4% (volume/volume) heat-inactivated FBS and 1% penicillin/streptomycin. The cells were incubated in a humidified atmosphere of 37°C and 5% CO2/95% air. HEK-293 cells were grown in DMEM (Genesee Scientific) containing 10% FBS and 1% penicillin/streptomycin at 37°C and 5% CO2/95% air. Authentication for these cell lines has not been done by the authors.

UV light source

UV lamps emitting UVB (280–320 nm; 54% of total energy), UVA (320–400 nm; 37% of total energy), UVC (100–280 nm; 2% of total energy), and visible light (400–450 nm; 7% of total energy; catalog nos., #95-0042-08 and #95-0043-13, UVP) were used to irradiate in vitro and in vivo experiments. Stable power output (mW/cm2) was measured using an UVX Radiometer (#97-0015-02, UVP) coupled with a sensor with a calibration point of 310 nm (UVX-31, #97-0016-04, UVP), and exposure time was calculated using the following formula: dose (mJ/cm2) = exposure time (s) × output intensity (mW/cm2). Quality control of the lamps and exposure time was calculated and monitored before using each of the lamps to account for power output changes.

Anchorage-independent growth assay in soft agar

In a 96-well tissue culture plate, 2 × 103 JB6 P+ cells were mixed with 0.33% agar and suspended on top of a solidified bottom layer containing 0.5% agar. Nobel Agar (Sigma-Aldrich) was prepared in PBS, autoclaved, and stored at 4°C. EGF (10 ng/mL) was used to promote and stimulate the anchorage-independent growth of JB6 P+ cells. The test compounds, carvedilol, R-, or S-carvedilol, were mixed with EGF and added to both agar layers. Plates were incubated at 37°C with 5% CO2/95% air for 10–14 days. Colonies larger than 10 cells were counted manually under a microscope.

Sulforhodamine B cytotoxicity assay

Cell viability for drug's cytotoxicity was determined using the sulforhodamine B (SRB) assay. A total of 3,000 cells per well were seeded in a 96-well plate and allowed to attach overnight. The cells were then treated with a serial dilution of R- or S-carvedilol. The compounds were incubated for 72 hours before the cells were fixed overnight with 10% trichloroacetic acid and stained with SRB Sodium Salt (Sigma-Aldrich). The excess staining solution was removed with 1% acetic acid and allowed to dry. The dye was then dissolved with a 10% tris base solution and read using a μQuant Microplate Reader (BioTek Instruments).

Cellular reactive oxygen species detection

UV-induced production of reactive oxygen species (ROS) was detected via the H2DCFDA ROS Indicator (Invitrogen). A total of 10,000 JB6 P+ cells were seeded on a 96-well plate and allowed to attach overnight. The media were washed and replaced with 50 μL of PBS before being exposed to 25 mJ/cm2 UVB. The PBS was replaced with culture media with or without test drugs and allowed to incubate for 24 hours; afterward, the media were washed and replaced with a 10 μmol/L H2DCFDA PBS solution for 30 minutes. The dye was then washed and replaced with 100 μL of PBS, and the plate was read using the Clariostar Microplate Reader (BMG Labtech).

Luciferase reporter gene assay

HEK-293 cells were transfected with pRL-TK-Luc Renilla luciferase (Promega) and pELK1-luc (Signosis) plasmids at a ratio of 1:4 using FuGENE HD Transfection Reagent (Roche Applied Science). Twenty-four hours later, the cells were serum starved in DMEM supplemented with 0.1% FBS for 16 hours. Cells were pretreated with drugs for 30 minutes, then cotreated with EGF (10 ng/mL) for 6 hours. Cell lysates were analyzed by the Dual-luciferase Reporter Gene Assay (Promega) with Renilla luciferase serving as a normalization factor.

ELISA for PGE2 secreted from cell culture

PGE2 activity was determined by culturing JB6 P+ cells in 12-well plates. The cells were pretreated with or without carvedilol, S-, or R-carvedilol for 2 hours, and were then exposed to 60 mJ/cm2 UV. After 24 hours of incubation, the culture media were removed from the plates, and PGE2 concentrations were determined via competitive ELISA according to the manufacturer's protocol (Cayman Chemical Co.).

Acute UV exposure in vivo study

All animal studies were performed with the approval of the Western University of Health Sciences' Institutional Animal Care and Use Committee (Pomona, CA). Mice had access to water and food ad libitum and housed on a 12-hour light/dark cycle with 35% humidity. A total of 36 female SKH-1 mice (Charles River) were randomly divided into six groups; n = 6 per group: (i) vehicle-treated control, (ii) UV exposed, followed by vehicle treatment, (iii) UV exposed, followed by 10 μmol/L carvedilol treatment, and (iv–ix) UV exposed, followed by 0.1, 1 or 10 μmol/L R-carvedilol treatment. The volume for topical treatment was 200 μL, with acetone as the vehicle. The UV dose was 300 mJ/cm2. The mice were euthanized 6 hours after the UV exposure, and dorsal skin samples were excised and snap frozen for further analysis.

DNA isolation and cyclobutane pyrimidine dimers (CPD) dot blot analysis

Genomic DNA was isolated from dorsal skin samples using a QIAamp DNA Mini Kit (Qiagen). The DNA samples (100 ng) were vacuum transferred to a Nitrocellulose Membrane (0.45 μm, Thermo Fisher Scientific) using a Bio-Dot SF Microfiltration Apparatus (Bio-Rad). CPDs were detected using an anti-CPD mAb (Kamiya). Following antibody detection, total DNA amounts were visualized by SYBR-Gold (Invitrogen) staining; total DNA in each sample was used to normalize the CPD values.

RNA isolation and qPCR analysis

Total RNA was isolated from whole-skin tissue using the RNeasy Mini Kit (Qiagen). cDNA was obtained with the High Capacity cDNA Reverse Transcriptase Kit (Thermo Fisher Scientific). cDNA and PerfeCTa SYBR Green Supermix (Quanta Biosciences, Inc.) were combined with primers for mouse genes and β-actin (the primer sequences are available upon request). qPCR was performed on a CFX96 Real-time Thermal Cycler Detection System (Bio-Rad) and analyzed with the 2–ΔΔCt method with β-actin as the normalization control.

Western blot analysis

Protein was extracted from skin tissue by grinding liquid nitrogen–frozen skin into a fine powder with a prechilled mortar and pestle before adding RIPA Lysis Buffer (Santa Cruz Biotechnology) containing PMSF, Na3VO4, and protease inhibitors. The tissues were further homogenized using an OMNI tissue master 123 handheld homogenizer. Protein (30 μg) was resolved by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Anti-COX-2 (1:1,000, Cayman Chemicals) and anti-β-actin (Cell Signaling Technology) antibodies in 5% nonfat milk and 5% sodium azide were applied to the membrane overnight, washed six times, and then exposed to goat anti-rabbit IgG-horseradish peroxidase (Cell Signaling Technology) diluted 1:20,000 in 5% nonfat milk at room temperature for 60 minutes. Membranes were visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

Histology and IHC analysis

The skin tissues were fixed in formalin, processed, and embedded in paraffin blocks. Five-millimeter-thick prepared sections of the paraffin-embedded skin tissues were placed onto positively charged glass slides. The prepared sections on glass slides were then deparaffinized three times (5 minutes) in xylene, followed by dehydration in graded ethanol, and finally rehydrated in running tap water. For antigen retrieval, sections were boiled in 10 mmol/L citrate buffer (pH 6) for 10 minutes. The prepared sections were incubated with hydrogen peroxide for 10 minutes to minimize nonspecific staining, and then rinsed three times (5 minutes each) with PBST (0.05% Tween-20). Blocking solution was applied for 1 hour, and then sections were incubated with diluted rabbit mAbs, namely anti-COX-2 (1:600, Cell Signaling Technology, catalog no., 12282) and Ki-67 (1:400, Cell Signaling Technology, catalog no., 12202), overnight at 4°C in a humid chamber. Further processing was done according to the instructions of the detection system. The slides were imaged with a microscope at 20×–40× magnification (Leica DM750). Scoring of the images from IHC analysis was conducted using a semiquantitative scoring system. Ki-67 was scored by counting the positively stained cells in five fields at various locations along the skin section to obtain the average for each mouse skin (n = 3 mice for short-term UV study and n = 3–5 mice for long-term UV study). For the extent of staining of COX-2, the following system was used: 0, no staining; 1, <25% of cells positive; 2, >25% and ≤50% of cells positive; and 3, >50% of cells positive. For staining intensity, the following system was used: 0, no staining; 1, faint staining; 2, moderate staining; and 3, strong staining. To quantitate COX-2 staining, an expression index was calculated by extent of staining multiplied by the intensity of staining.

UV-induced murine skin tumorigenesis

Six- to 8-week-old female SKH-1 mice (Charles River Laboratories) were randomly divided into four groups. The groups were (i) non-UV exposure vehicle treated (n = 5), (ii) UV-exposed vehicle treated (n = 9), (iii) UV-exposed carvedilol treated (n = 9), and (iv) UV-exposed R-carvedilol treated (n = 9). Mice were topically treated with 200 μL acetone (vehicle), 10 μmol/L carvedilol, or R-carvedilol dissolved in 200 μL acetone three times per week for 2 weeks before UVB exposure. The mice were irradiated with gradually increasing UV levels three times a week for 27 weeks with an initial dose of 50 mJ/cm2 that was increased each week by 25 mJ/cm2 to 150 mJ/cm2, which continued for the duration of the experiment. The treatment regimen was applied 30 minutes before UV irradiation and continued throughout the experiment. During the UV exposure, mice roamed freely in acrylic cages on a rotating platform with rotational placement ensuring consistent and equal dorsal distribution of UV irradiation. Tumors of at least 1 mm in diameter were counted and measured with a caliper weekly. The tumor volume was calculated according to the formula: (width)2 × length/2. After 27 weeks, the mice were sacrificed and nontumorous skin and tumor tissues were harvested and processed for histologic analysis.

Statistical analysis

All data in the text are described as mean ± SD, data in histograms are expressed as individual data points with a line representing the group mean, and line graphs as mean ± SD or ± SEM; data presentation and error quantification are described in the figure legends. All data were graphed, and curves were generated using beta versions of GraphPad Prism 9.0.0. The Grubs test identified outliers in the raw data within GraphPad Prism. NCSS 2019 was used to analyze the data after removing any outliers via ANOVA; Tukey–Kramer post hoc tests were used for parametric data, and Kruskal–Wallis post hoc tests were used for nonparametric data. Tumor formation was graphed using a Kaplan–Meier survival curve showing the incidence of tumor formation in GraphPad Prism and analyzed using a Mantel–Cox log-rank test in NCSS 2019. For all statistical analyses, groups were considered statistically different when P < 0.05, and denotation of statistical difference is described in the figure legends.

Effects of R- and S-carvedilol on EGF-mediated neoplastic transformation of JB6 P+ cells

Previous studies indicate that racemic carvedilol inhibits EGF-induced transformation of JB6 P+ cells with a 95% confidence that the IC50 value is between 175 and 342 nmol/L (14). Similar experiments were conducted to determine the inhibitory effect of optically pure R- and S-carvedilol. R- and S-enantiomers produced nearly identical inhibitory effects on EGF-induced JB6 P+ cell transformation (Fig. 1B); the 95% confidence interval of the IC50 values was 222–372 nmol/L for S-carvedilol and 231–376 nmol/L for R-carvedilol. The IC50 values were not different from previous data. Intriguingly, the hill slope for both concentration–response curves was significantly negative, with an average 95% confidence interval of −0.63 to −0.49. Because colony formation is affected by cell viability, the SRB colorimetric assay in the monolayer culture of JB6 P+ cells was used to determine the cytotoxicity for the test agents in parallel with the colony formation assays. The SRB data indicate that, like racemic carvedilol (14), R- and S-carvedilol begin to show toxic effects at 10 μmol/L and are toxic toward JB6 P+ cells at 100 μmol/L (Fig. 1B). Because the concentrations that caused cytotoxicity were much higher than the concentrations required to inhibit colony formation, the antitransformation activity of R- and S-carvedilol is not due to a cytotoxic effect.

Effects of R- and S-carvedilol on EGF-mediated ELK-1 activation in HEK-293 cells

Previous data demonstrated that racemic carvedilol blocked EGF-induced activation of ELK-1 promoter activity (11). Like carvedilol, R- and S-carvedilol inhibited EGF-mediated ELK-1 promoter activity in a dose-dependent fashion without displaying any statistically different inhibitory effects on basal ELK-1 promoter activity (Fig. 1C). These data further support the notion that R- and S-carvedilol similarly affect the oncogenic signaling mediated by EGF in HEK-293 cells.

Effects of R- and S-carvedilol against UV-induced ROS formation in JB6 P+ cells

A primary effect of UV-induced damage is ROS generation; therefore, the antioxidant properties of R- and S-carvedilol were examined. JB6 P+ cells were irradiated with 25 mJ/cm2 UV, and then immediately incubated with varying concentrations of either R- or S-carvedilol for 24 hours. The amount of ROS was determined using the fluorescence probe, DCFH2DA. UV exposure significantly increased the production of ROS by 2.45 ± 0.71 fold. Post-UV exposure drug treatment with R-carvedilol resulted in a dose–response relationship that brought ROS production down to basal levels; whereas, S-carvedilol also returned the ROS production levels to basal, but did not follow a classic dose response (Fig. 2A). These results demonstrated that both R- and S-carvedilol in the optically pure form have antioxidant properties like the racemic mixture.

Figure 2.

Effects of S- and R-carvedilol (CAR) on UV-induced effects in JB6 P+ cells. A, The level of intracellular ROS induced by 25 mJ/cm2 UV, followed by a 24-hour incubation with varying doses of R- or S-carvedilol was detected using the cell permeant dye, DCFH2DA (DCF). Data are normalized to the control and represented as mean ± SEM, n = 8. B, PGE2 levels were determined by ELISA in culture media from untreated or UV-irradiated JB6 culture treated with DMSO (the vehicle), racemic carvedilol, S-, or R-carvedilol (10 μmol/L), n = 3. A two-factor ANOVA followed by a Tukey–Kramer multiple comparison post hoc test was used to assess statistical differences at P < 0.05, and differences are denoted by different Greek letters.

Figure 2.

Effects of S- and R-carvedilol (CAR) on UV-induced effects in JB6 P+ cells. A, The level of intracellular ROS induced by 25 mJ/cm2 UV, followed by a 24-hour incubation with varying doses of R- or S-carvedilol was detected using the cell permeant dye, DCFH2DA (DCF). Data are normalized to the control and represented as mean ± SEM, n = 8. B, PGE2 levels were determined by ELISA in culture media from untreated or UV-irradiated JB6 culture treated with DMSO (the vehicle), racemic carvedilol, S-, or R-carvedilol (10 μmol/L), n = 3. A two-factor ANOVA followed by a Tukey–Kramer multiple comparison post hoc test was used to assess statistical differences at P < 0.05, and differences are denoted by different Greek letters.

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Effects of R-carvedilol on UV-induced epidermal PGE2 secretion

Because UV-induced epidermal production of PGE2 is involved in skin carcinogenesis and agents inhibiting PGE2 production are chemopreventive (18), R- and S-carvedilol's effect on UV-induced PGE2 release from JB6 P+ was examined. Exposure of JB6 P+ cells to 60 mJ/cm2 UV statistically increased PGE2 secretion into the culture media by 3.93-fold (Fig. 2B). Individual treatment with 10 μmol/L racemic carvedilol and R-carvedilol, but not S-carvedilol, for 2 hours before and 24 hours after UV exposure statistically reduced PGE2 secretion into the culture media. However, none of the drug treatment was able to bring the PGE2 level to that of negative control.

Effects of R-carvedilol on single-dose UV-induced skin damage in SKH-1 hairless mice

We further sought to evaluate the effects of R-carvedilol on acute UV-induced skin damage in mice. A single dose of 300 mJ/cm2 UV was utilized to examine R-carvedilol's proximal effects in SKH-1 hairless mice. The mice were treated topically with acetone (vehicle control), 10 μmol/L racemic carvedilol as a positive control (10), or varying doses of R-carvedilol (0.1, 1, and 10 μmol/L) immediately after UV irradiation. The mice were sacrificed 6 hours after irradiation, and skin samples were taken for further analysis. Hematoxylin and eosin (H&E) staining (Fig. 3A) was utilized to visualize the epidermis, for which the thickness was measured (Fig. 3B). UV treatment significantly increased epidermal thickness from the negative control (no UV, vehicle), and topical treatment of 10 μmol/L carvedilol and R-carvedilol statistically ablated UV-induced epidermal thickening. Analogous to the H&E data, IHC analysis indicated an increased expression of Ki-67 in the basal epidermal layers of the mouse skin after UV radiation (Fig. 3A and C). Carvedilol and R-carvedilol treatment similarly reduced the number of Ki-67–positive cells to levels between the negative control mice and UV-treated mice (Fig. 3C).

Figure 3.

Effects of carvedilol (CAR) and R-carvedilol on short-term UV radiation–mediated skin changes of SKH-1 mice. Mice were treated with UV (300 mJ/cm2), with or without topical 10 μmol/L racemic carvedilol or various R-carvedilol doses, for 6 hours. A, Representative microphotographs of mouse skin stained with H&E and IHC of Ki-67 treated with vehicle, 10 μmol/L racemic carvedilol, or 10 μmol/L R-carvedilol. B, Measurement of epidermal thickness in H&E-stained slides of mice treated with vehicle, 10 μmol/L racemic carvedilol, or 10 μmol/L R-carvedilol. The thickness was measured 10 times at various locations along the epidermis and averaged to obtain a single-skin sample data (n = 3). C, Quantification of the number of Ki-67–positive cells treated with vehicle, 10 μmol/L racemic carvedilol, or 10 μmol/L R-carvedilol; n = 3. D, Genomic DNA was isolated from the skin tissues and probed for CPD adducts via slot blot and reprobed for total DNA via SYBR gold staining. The data are presented as CPD/DNA; n = 6. IL1β (E), IL6 (F), and PCNA (G) mRNA expression in the skin was examined via qPCR, expressed as 2–ΔΔCt, n = 6. An ANOVA followed by a Tukey–Kramer multiple comparison post hoc test was used to assess statistical differences at P < 0.05, and differences are denoted by different Greek letters.

Figure 3.

Effects of carvedilol (CAR) and R-carvedilol on short-term UV radiation–mediated skin changes of SKH-1 mice. Mice were treated with UV (300 mJ/cm2), with or without topical 10 μmol/L racemic carvedilol or various R-carvedilol doses, for 6 hours. A, Representative microphotographs of mouse skin stained with H&E and IHC of Ki-67 treated with vehicle, 10 μmol/L racemic carvedilol, or 10 μmol/L R-carvedilol. B, Measurement of epidermal thickness in H&E-stained slides of mice treated with vehicle, 10 μmol/L racemic carvedilol, or 10 μmol/L R-carvedilol. The thickness was measured 10 times at various locations along the epidermis and averaged to obtain a single-skin sample data (n = 3). C, Quantification of the number of Ki-67–positive cells treated with vehicle, 10 μmol/L racemic carvedilol, or 10 μmol/L R-carvedilol; n = 3. D, Genomic DNA was isolated from the skin tissues and probed for CPD adducts via slot blot and reprobed for total DNA via SYBR gold staining. The data are presented as CPD/DNA; n = 6. IL1β (E), IL6 (F), and PCNA (G) mRNA expression in the skin was examined via qPCR, expressed as 2–ΔΔCt, n = 6. An ANOVA followed by a Tukey–Kramer multiple comparison post hoc test was used to assess statistical differences at P < 0.05, and differences are denoted by different Greek letters.

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The skin DNA damage level was quantified by detecting cyclobutane pyrimidine dimers (CPD; Fig. 3D). UV irradiation induced statistically greater CPD formation (Fig. 3D); however, the considerable variation prevented any observable statistical differences induced by carvedilol and R-carvedilol. Nevertheless, 10 μmol/L R-carvedilol resulted in an intermediate response that was not statistically differentiable from the control and UV exposure, suggesting a potential role of R-carvedilol in preventing DNA damage. As a second measure of the DNA damage pathway, PCNA mRNA levels were quantified via qRT-PCR. Because of high variability in the control and UV samples, the coefficient of variation was 57% and 60% for the negative control and UV-irradiated mice, respectively, as well as because of the relatively low increase in UV-induced PCNA expression, 2.7 ± 1.6 fold over basal, no statistical differences were observed (Fig. 3G).

COX-2 is a known molecular target upregulated by UV irradiation; therefore, carvedilol and R-carvedilol effects on UV-induced COX-2 mRNA and protein expression were examined (Fig. 4). UV irradiation statistically increased COX-2 mRNA levels in the epidermis by an average of 11.7 ± 9.5 fold greater than controls; however, there was a great deal of variation among the mice (Fig. 4A). Like the CPD assay, racemic carvedilol and R-carvedilol reduced the mean COX-2 mRNA expression to an intermediate response that was not statistically differentiable from the control and UV exposure. Protein expression of COX-2, on the other hand, was not statistically increased by UV (Fig. 4B), but treatment with 10 μmol/L R-carvedilol resulted in statistically lower COX-2 protein levels after UV exposure. The changes in epidermal thickness and expression of COX-2 were visualized via histology and IHC (Fig. 4C). The images suggested that most of the UV-induced COX-2 expression was localized to the epidermis, with fainter staining in the dermis. Moreover, the dermis appeared to have less staining when treated with R-carvedilol and racemic carvedilol. The expression index analysis of COX-2 showed statistically significant attenuation of UV-induced COX-2 by both carvedilol and R-carvedilol (Fig. 4C).

Figure 4.

Effects of carvedilol (CAR) and R-carvedilol on UV-mediated expression of COX-2 in skin tissues of SKH-1 mice. Mice were treated with UV (300 mJ/cm2), with or without topical 10 μmol/L racemic carvedilol or various R-carvedilol doses, for 6 hours. COX-2 mRNA (A) and protein (B) expression in the skin was examined via qPCR, expressed as 2–ΔΔCt, and Western blotting, respectively; n = 6. C, Representative microphotographs of mouse skin stained for COX-2 and treated with vehicle, 10 μmol/L racemic carvedilol, or 10 μmol/L R-carvedilol. The expression index was used to quantify the extent and intensity of COX-2 expression in each sample (n = 3). An ANOVA followed by a Tukey–Kramer multiple comparison post hoc test was used to assess statistical differences at P < 0.05, and differences are denoted by different Greek letters.

Figure 4.

Effects of carvedilol (CAR) and R-carvedilol on UV-mediated expression of COX-2 in skin tissues of SKH-1 mice. Mice were treated with UV (300 mJ/cm2), with or without topical 10 μmol/L racemic carvedilol or various R-carvedilol doses, for 6 hours. COX-2 mRNA (A) and protein (B) expression in the skin was examined via qPCR, expressed as 2–ΔΔCt, and Western blotting, respectively; n = 6. C, Representative microphotographs of mouse skin stained for COX-2 and treated with vehicle, 10 μmol/L racemic carvedilol, or 10 μmol/L R-carvedilol. The expression index was used to quantify the extent and intensity of COX-2 expression in each sample (n = 3). An ANOVA followed by a Tukey–Kramer multiple comparison post hoc test was used to assess statistical differences at P < 0.05, and differences are denoted by different Greek letters.

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Acute UV exposure can cause inflammatory reaction to UV. Because previous study showed an upregulation of IL1β and IL6 mRNA expression 6 hours after single-dose UV radiation (10), qRT-PCR evaluation of IL1β (gene symbol Il1b) and IL6 (gene symbol Il6; Fig. 3E and F) was conducted to examine further whether carvedilol effects are stereoselective. UV irradiation of the skin markedly and uniformly increased IL1β message by 4.11 ± 0.09 fold over the control (Fig. 3E). Notably, the control had considerable variation with a normalized mean of 1.00 ± 0.89. Carvedilol did not statistically reduce IL1β expression, but decreased the mean while mimicking the variance of the control. R-carvedilol did not demonstrate a concentration—response relationship and was not statistically different from carvedilol treatment, while at 0.1 and 10 μmol/L, R-carvedilol significantly attenuated UV-induced upregulation of IL1β (Fig. 3E). Like IL1β, IL6 levels were increased, but not uniformly, by 39.9 ± 17.7 fold, and all treatments statistically reduced IL6 similarly (Fig. 3F).

Effects of topically applied carvedilol or R-carvedilol on chronic UV-induced skin tumorigenesis in mice

We further investigated R-carvedilol's chemopreventive activity in a murine skin carcinogenesis model compared with the racemic carvedilol. SKH-1 hairless mice were topically treated for 2 weeks with 200 μL vehicle control (acetone), carvedilol (10 μmol/L in acetone), or R-carvedilol (10 μmol/L in acetone). The mice were next irradiated by gradually increasing levels of UV radiation from 50 to 150 mJ/cm2 three times a week for 27 weeks to induce skin tumors, as documented previously (19). The drug treatment was applied 30 minutes before each UV exposure. At week 17, tumors became visible on the vehicle control group, while carvedilol- and R-carvedilol–treated animals showed a 3- and 5-week delay in tumor formation, respectively, and the control animals never exposed to UV did not develop any tumor (Fig. 5A). Expectedly, UV induces a significant risk of developing tumors (P = 0.0047). Although pretreatment with carvedilol delayed tumor formation, carvedilol failed to protect against UV tumor formation (P = 0.3564) and was statistically different from the naïve control group (P = 0.0417). R-carvedilol displayed a protective effect against UV-induced tumor formation (P = 0.0102) and was not statistically different from the naïve controls (P = 0.0566). However, R-carvedilol did not differ from carvedilol (P = 0.2019). The tumor multiplicity (number of tumors/mouse) data in Fig. 5B consistently show that R-carvedilol significantly reduced tumor numbers (R-carvedilol vs. UV, P = 0.0401, χ2 analysis), while carvedilol treatment was not significant (carvedilol vs. UV, P = 0.157724). Tumor multiplicity in the R-carvedilol treatment group was not statistically differentiable from the negative control because of 37% of the animals that never developed any tumors (Fig. 5B). The tumor burden (total tumor volume/mouse) data showed a trend of attenuated tumor burden in carvedilol and R-carvedilol treatment groups without statistical significance possibly due to the large variations (Fig. 5C). Visually, R-carvedilol treatment improved the skin damage and tumor formation of the mice in comparison with UV-only controls (Fig. 5D).

Figure 5.

Effects of carvedilol (CAR) and R-carvedilol on the development of UV-induced skin tumors in SKH-1 mice. Mice were pretreated with 10 μmol/L carvedilol, R-carvedilol, or acetone for 2 weeks. The mice were then exposed to gradual doses of UV up to 150 mJ/cm2 three times per week, and drug treatments were given 30 minutes before each irradiation. A, Tumor incidence is graphed as a Kaplan–Meier curve and analyzed via a Mantel–Cox log-rank test. The number of tumors per mouse (B) and average tumor volume per mouse (C) are plotted and analyzed via a χ2 analysis to assess statistical differences at P < 0.05 (*). n = 5 for control; n = 9 for other groups. D, Representative photographs of mice from the UV-treated and UV-treated plus R-carvedilol groups panel.

Figure 5.

Effects of carvedilol (CAR) and R-carvedilol on the development of UV-induced skin tumors in SKH-1 mice. Mice were pretreated with 10 μmol/L carvedilol, R-carvedilol, or acetone for 2 weeks. The mice were then exposed to gradual doses of UV up to 150 mJ/cm2 three times per week, and drug treatments were given 30 minutes before each irradiation. A, Tumor incidence is graphed as a Kaplan–Meier curve and analyzed via a Mantel–Cox log-rank test. The number of tumors per mouse (B) and average tumor volume per mouse (C) are plotted and analyzed via a χ2 analysis to assess statistical differences at P < 0.05 (*). n = 5 for control; n = 9 for other groups. D, Representative photographs of mice from the UV-treated and UV-treated plus R-carvedilol groups panel.

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For each mouse in different treatment groups, one tumor with largest volume was dissected for histologic analysis. Among the nine skin tumors in the UV-only control group, eight were diagnosed as papillomas, one was SCC. In carvedilol-treated group, all five were papillomas. However, in R-carvedilol–treated group, there was no SCC, two of five were diagnosed as papillomas, and three were diagnosed as epidermal dysplasia. The representative H&E images are shown in Fig. 6A. To confirm the treatment effects of carvedilol and R-carvedilol on chronic UV-induced skin inflammation, we examined the normal looking skin dissected from the mice that were exposed to long-term UV treatment. As shown in Fig. 6B and C, chronic exposure of mouse skin to UV substantially induced epidermal hyperplasia (H&E images), higher number of Ki-67–positive cells (IHC), and higher expression index of COX-2 (IHC). These changes were significantly reduced by carvedilol treatment, and R-carvedilol showed the same degree of treatment effects. Thus, the long-term UV carcinogenesis studies confirm that R-carvedilol is indeed chemopreventive.

Figure 6.

Effects of carvedilol (CAR) and R-carvedilol on UV-induced skin tumors and inflammation in the skin surrounding the tumors in SKH-1 mice. Mice were pretreated with 10 μmol/L carvedilol, R-carvedilol, or acetone for 2 weeks. The mice were then exposed to gradual doses of UV up to 150 mJ/cm2 three times per week, and drug treatments were given 30 minutes before each irradiation. A, Histology of tumor tissues showing SCC, papilloma, and epidermal dysplasia in control and R-carvedilol treatment groups. B, Representative microphotographs of mouse skin stained with H&E and IHC of Ki-67 and COX-2 treated with vehicle, racemic carvedilol, or R-carvedilol. C, Measurement of epidermal thickness in H&E-stained slides of mice treated with vehicle, 10 μmol/L racemic carvedilol, or 10 μmol/L R-carvedilol. The thickness was measured five times at various locations along the epidermis and averaged to obtain a single-skin sample data (n = 5). D, Quantification of the number of Ki-67–positive cells treated with vehicle, 10 μmol/L racemic carvedilol, or 10 μmol/L R-carvedilol (n = 5). E, The expression index was used to quantify the extent and intensity of COX-2 expression in each sample (n = 5). An ANOVA followed by a Tukey–Kramer multiple comparison post hoc test was used to assess statistical differences at P < 0.05, and differences are denoted by different Greek letters.

Figure 6.

Effects of carvedilol (CAR) and R-carvedilol on UV-induced skin tumors and inflammation in the skin surrounding the tumors in SKH-1 mice. Mice were pretreated with 10 μmol/L carvedilol, R-carvedilol, or acetone for 2 weeks. The mice were then exposed to gradual doses of UV up to 150 mJ/cm2 three times per week, and drug treatments were given 30 minutes before each irradiation. A, Histology of tumor tissues showing SCC, papilloma, and epidermal dysplasia in control and R-carvedilol treatment groups. B, Representative microphotographs of mouse skin stained with H&E and IHC of Ki-67 and COX-2 treated with vehicle, racemic carvedilol, or R-carvedilol. C, Measurement of epidermal thickness in H&E-stained slides of mice treated with vehicle, 10 μmol/L racemic carvedilol, or 10 μmol/L R-carvedilol. The thickness was measured five times at various locations along the epidermis and averaged to obtain a single-skin sample data (n = 5). D, Quantification of the number of Ki-67–positive cells treated with vehicle, 10 μmol/L racemic carvedilol, or 10 μmol/L R-carvedilol (n = 5). E, The expression index was used to quantify the extent and intensity of COX-2 expression in each sample (n = 5). An ANOVA followed by a Tukey–Kramer multiple comparison post hoc test was used to assess statistical differences at P < 0.05, and differences are denoted by different Greek letters.

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The β-blocker, carvedilol, has been reported to have skin cancer preventive effects (9, 10). However, not all β-blockers prevent EGF-induced JB6 P+ colony formation in soft agar, which is the gold standard in vitro transformation assay (14). Carvedilol is unique among β-blockers; clinical studies demonstrate that carvedilol has particular survival advantages in heart failure over other β-blockers (20). Carvedilol is one of the most effective β-blockers in preventing ventricular tachyarrhythmias and reducing heart failure mortality (21). Interestingly, in a large population-based cohort study, long-term carvedilol use was related to a significant cancer risk reduction across all cancer types (22).

Although carvedilol is a safe drug for long-term use for patients with cardiovascular diseases, its effect on the heart rate, cardiac output, and blood pressure hinders its development into a cancer preventive agent. Not all β-blockers exhibit cancer preventive activity and the cancer preventative activity of carvedilol is independent of β-blockade (14). Clinically available carvedilol is a racemic mixture of equal amounts of S- and R-carvedilol. Notably, S-carvedilol is a β-blocker, while R-carvedilol does not possess β-blocking activity (16). In this study, the non-β-blocking enantiomer R-carvedilol was examined to determine whether it retains the cancer preventive properties of the racemic carvedilol. As a constituent of racemic carvedilol, R-carvedilol has been prescribed for decades, suggesting that R-carvedilol is a safe pharmaceutical agent. Dosing healthy human volunteers with R-carvedilol displayed no significant adverse effects (16), and R-carvedilol did not lower heart rate or blood pressure in mice (16, 17). Preclinical and clinical application of R-carvedilol as an anticancer agent has never been investigated; thus, this is the first study demonstrating the anticancer properties of R-carvedilol.

The results demonstrated that R- and S-carvedilol display similar protective effects in EGF-treated JB6 P+ cells and HEK-293 cells (Fig. 1), as well as UV-irradiated JB6 P+ cells (Fig. 2). The data obtained from R- and S-carvedilol are within the 95% confidence values of previous studies with racemic carvedilol (11, 14). Because previous studies indicate that the cancer preventative effects of carvedilol are independent of β-ARs, R-carvedilol is not a β-blocker and yet produces similar pharmacologic effects as carvedilol, and R- and S-carvedilol were nearly indistinguishable in the presented data; the cancer preventative properties of carvedilol are independent of β-ARs, and the pharmacologic effect is not sensitive to the chirality of carvedilol. This study has compared S- and R-carvedilol in vitro and the racemic carvedilol was used for comparison with R-carvedilol in vivo, because the racemic carvedilol is an FDA-approved agent, and all cancer prevention studies in published work have used the racemic carvedilol (9, 10, 11).

Treating mice acutely with 300 mJ/cm2 UV induces skin damage and allows for initial UV-induced signal transduction to be evaluated. UV-induced epidermal thickening occurs within 6 hours (Fig. 3), which is primarily due to edema and inflammation, as well as cellular proliferation, at this early timepoint. Racemic carvedilol and R-carvedilol inhibited epidermal thickening to maintain skin thickness identical to naïve control mice (Fig. 3B). Ablation of skin thickening suggests that the pharmacologic effect may be preventing UV-induced edema and inflammation. Supporting this conclusion, the UV-induced cell proliferation marker Ki-67 was reduced by carvedilol and R-carvedilol (Fig. 3C), and DNA damage was also attenuated (Fig. 3D). PCNA mRNA expression, which is involved in cell proliferation and DNA repair (23), showed the same trend that UV-induced upregulation was attenuated by carvedilol and R-carvedilol (Fig. 3G).

Further investigation into the immediate increase in inflammatory molecules proved interesting as R-carvedilol showed slightly increased efficacy compared with racemic carvedilol. COX-2 expression was examined via qRT-PCR, Western blotting, and IHC (Fig. 4). Although UV-induced transcription of COX-2, racemic, and R-carvedilol had modest effect on mRNA upregulation (Fig. 4A). However, R-carvedilol dose dependently reduced COX-2 protein expression to levels below the naïve control mice, suggesting that the mechanism of inhibiting COX-2 protein expression is independent of UV irradiation (Fig. 4B). Histologic examination of where COX-2 was expressed after UV irradiation demonstrated that the bulk of the expression occurred in the epidermis, which was strongly attenuated by both carvedilol and R-carvedilol (Fig. 4C). Dermal COX-2 expression occurred across all samples, suggesting that the mRNA and protein levels in the naïve control mice were primarily from the dermal layer. Further studies should use laser capture microdissection to determine whether there is a spatial component to R-carvedilol–mediated reduction in COX-2 expression. Understanding the tissue and cellular location of R-carvedilol–mediated effects may illuminate the mechanism underlying R-carvedilol chemopreventive effects. The results obtained from these COX-2 expression studies confirm that carvedilol and R-carvedilol prevent skin cancer via inhibiting the activation of COX-2, which is essential for tumor promotion/progression (24).

Six hours after 300 mJ/cm2, UV irradiation induces transcription of IL1β and IL6 (Fig. 3); however, racemic carvedilol and R-carvedilol only consistently inhibit IL6 mRNA expression (Fig. 3F). UVB increases IL1α, which subsequently increases IL6 with a peak at 6 hours (25); therefore, carvedilol could be inhibiting IL1α or the IL1α signaling cascade resulting in IL6 transcription. Further studies are required to determine the effects of R-carvedilol on IL1α expression, and assays directly examining IL1α-mediated signaling can be carried out in cultured keratinocytes to identify the yet undescribed mechanism of action underlying carvedilol-mediated skin cancer prevention.

Treating mice chronically with low doses of UV (50–150 mJ/cm2) three times a week mimics environmental exposure to UV as the mice never presented with erythema (sunburn), but begin to develop skin tumors at 17 weeks (Fig. 5A). Unlike previous studies, where test compounds were administered after UV exposure, racemic carvedilol and R-carvedilol were administered 30 minutes before UV exposure in this study because the sunscreen effects of carvedilol does not play a role in its cancer preventive effects (10). Although pretreatment with racemic carvedilol delayed the first incidence of tumors by 3 weeks, there was no statistically significant protection from developing tumors (Fig. 5A and B). Previous data, where carvedilol was administered after UV exposure, resulted in a similar 3-week delay in tumor formation and statistically significant protection from developing tumors (10). Therefore, the carvedilol application timing appears to alter the cancer preventative effects, suggesting that pharmacokinetics must be examined to optimize the cancer preventative properties of carvedilol, and by extension, R-carvedilol. However, R-carvedilol delayed tumor formation for a longer duration, 5 weeks, and proved to be statistically different from UV treatment, but not from racemic carvedilol treatment (Fig. 5A). R-carvedilol's effects are predominately dependent on reducing and preventing the onset and multiplicity of tumors in this chronic carcinogen exposure model because the average tumor volume at the end of experiments was not statistically different from UV-treated animals (Fig. 5C). As the model drives tumor progression in a relatively short time compared with environmental exposure in humans, a delay of 3–5 weeks and prevention of 44% of the subjects from developing tumors could have dramatic clinical effects, as seen in the Taiwanese retrospective study (22).

The purpose of this study was to determine whether the non-β-blocking R-carvedilol can be explored as a cancer preventative agent. The rationale underlying the study was to identify an agent that can be used without cardiovascular effects. However, a secondary goal was to identify potential mechanisms of action to understand how carvedilol prevents UV-induced tumorigenesis. Identifying the unknown targets for carvedilol could have significant implications for understanding carcinogenesis and chemoprevention of cancer, as well as the development of new cancer preventive strategies. Thus far, studies examining the mechanism of action of carvedilol eliminated β-blockade (14) and likely eliminated scavenging ROS (26) as chemopreventive mechanisms. In this study, the R-carvedilol concentration–response curves generated from the soft agar colony formation assay suggest that there are either multiple binding sites displaying negative cooperation or multiple targets involved with varying affinities. R-carvedilol's interaction with multiple targets, known as polypharmacology, is an intriguing possibility as scavenging ROS plus interaction with an unknown secondary target may be required for racemic carvedilol and R-carvedilol to prevent tumorigenesis. Polypharmacology creates challenges in deciphering the unknown mechanism of actions of a drug as all reasonable targets must be identified and manipulated in various combinations. Toward identifying novel targets for R-carvedilol, a second hint to the mechanism of action comes from the short-term UV exposure studies where UV-induced increase in thickening and inflammation of the epidermis was ablated (Figs. 3 and 4). The molecular target that approaches the similar degree of inhibition seen in Fig. 3 is the IL6 PCR data (Fig. 3F) and the COX-2 data in Fig. 4. Inflammation within the skin is an essential process in skin cancer development and progression (27), and carvedilol may be inhibiting signal transduction or cellular infiltration that results in IL6 and COX-2 production. The reduction of epidermal thickening of the epidermis in carvedilol- and UV-treated mice may be mediated, in part, by reducing IL6 (28) and/or by inhibiting COX-2 (24). However, it is also possible that a lack of damage in carvedilol-treated skin, as measured by the skin thickness in this study, abrogated the need for IL6- and COX-2–mediated reparative processes (29). Further studies are necessary to decipher potential targets of R-carvedilol. Carvedilol and R-carvedilol demonstrated comparable effects against UV-induced epidermal thickening, Ki-67 and COX-2 expression, and inflammation markers. However, R-carvedilol showed increased activity in reducing tumor incidence and multiplicity compared with carvedilol. The exact reason of why the racemic carvedilol is less effective in suppressing tumor development is unknown because carvedilol and R-carvedilol similarly attenuated epidermal proliferation and inflammation. Future studies are needed to identify the unique anticancer mechanisms for R-carvedilol, by the use of head-to-head comparison of R- and S-carvedilol.

In conclusion, this study has revealed that the skin cancer preventative properties of carvedilol are not dictated by chirality. Specifically, the non-β-blocking enantiomer, R-carvedilol, exhibits the same degree of skin cancer preventive activity as racemic carvedilol and S-carvedilol. Because R-carvedilol does not have β-blocking activity, using R-carvedilol as a preventive treatment will not lead to predictable β-blocker cardiovascular effects that are undesirable for a cancer preventative treatment. Thus, R-carvedilol should be an effective and safe skin cancer prevention treatment for long-term clinical use.

S. Liang reports grants from NIH during the conduct of the study. Y. Huang reports a patent for 63/151,711 issued. No disclosures were reported by the other authors.

S. Liang: Data curation, investigation, methodology, writing–original draft. M.A. Shamim: Investigation, methodology. A. Shahid: Formal analysis, investigation, visualization, methodology. M. Chen: Investigation, methodology. K.H. Cleveland: Investigation, methodology. C. Parsa: Resources, methodology. R. Orlando: Resources, project administration. B.T. Andresen: Conceptualization, data curation, formal analysis, writing–review and editing. Y. Huang: Conceptualization, resources, supervision, funding acquisition, writing–original draft, writing–review and editing.

The research reported was partly supported by the NCI of the NIH under award number R15CA227946 (to Y. Huang). This work was also supported by Western University of Health Sciences Intramural Student Funds as part of the Graduate Program (to M.A. Shamim, M. Chen, and K.H. Cleveland). We thank Lily Kong Lim, A.S.C.P. (H.T.), at Beverly Hospital for preparing formalin-fixed, paraffin-embedded tissues and H&E staining.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data