2‐Methoxyestradiol inhibits the proliferation level in keloid fibroblasts through p38 in the MAPK/Erk signaling pathway

The MAPK/Erk signaling pathway is a classic pathway in cell proliferation. Our former study showed that keloid tissue revealed a higher proliferation level than physiological scars and normal skin. As a natural metabolite of estradiol, 2‐methoxyestradiol (2ME2) showed an inhibition proliferation effect on tumor cells.

We drew the conclusion that a keloid's continuous growth characteristics may be caused by an imbalance state between a high proliferation level and a relatively low apoptosis level. 3,4In this case, an effective drug for keloids should possess a double effect of proapoptosis and antiproliferation.
2-Methoxyestradiol (2ME2), considered to be a new promising chemotherapy drug, showed satisfying antitumor activity by inhibiting cell proliferation and inducing cell apoptosis in a variety of malignant cell lines. 5,6In our previous study, we explored the effect of 2ME2 toward keloid fibroblasts and drew the conclusion that 2ME2 improved the apoptosis level in keloid fibroblasts due to caspase-dependent mechanisms. 7To further study the effect of 2ME2 toward the keloid fibroblast proliferation level and explore its potential mechanisms, we conducted the following study.

| Patients, sample management, cell culture, and treatment
The Bioethical Committee of our Hospital approved this study protocol, and all patients signed informed consents.From June 2021 to December 2021, 12 patients (ages ranging from 18 to 47 years, median age: 32 years, Table 1) were randomly selected from the Department of Plastic Surgery at our Hospital: six keloid patients (gender ratio 1:1, median age: 32 years) and six non-keloid patients (gender ratio 1:1, median age: 31 years).The keloids were caused by trauma and finally diagnosed using a pathological examination.All patients reported no history of systemic disorders, recent drug taking, or receiving other treatment that might influence the study results.The keloid and nonkeloid patients showed no significant differences in age, gender, or sample sites.

| Fibroblast cell activity assessment
The CCK-8 assay (Dojindo) was used to detect the fibroblast cell activity, and the protocols were conducted based on the kit instructions.Fibroblasts were seeded at 5000 cells/well in 96-well plates for 4 days.A microplate reader was used to measure the optical density at 450 nm.

| Immunofluorescence observation
Immunofluorescence staining procedures were conducted based on our previous study. 7The primary antibody information was anti-p38

| Statistical analysis
Study data are presented as mean ± standard deviation (SD).A oneway analysis of variance (ANOVA) followed by a least significant difference (LSD) t-test were used for the statistical analyses using SPSS 24.0 software (SPSS Inc.).p < 0.05 was considered to be statistically significant.

| Cell activity assessment
The keloid fibroblast cell activity and the normal skin fibroblast cell activity in 4 days are shown in Figure 1 and Table 2.The results showed that both the keloid and normal skin fibroblast cell activities increased with time.During the first 2 days, no significant differences were found between the keloid and non-keloid groups.On the third day, a significant difference was found between the N group and the K/K+DMSO group.On the fourth day, both 2ME2 and doramapimod significantly inhibited keloid fibroblast cell activity compared with the K group.However, keloid fibroblast cell activity was similar between the K+2ME2 group and the K+IN group.

| Immunofluorescence studies
The immunofluorescence staining images are presented in Figure 2.
In this portion, the protein expression of p38, PCNA, and HIF-1α was observed.All of the protein expressions mentioned above were similar, and the strongest expression intensity was between the K group and the K+DMSO group.The N group showed the lowest expression intensity of all the proteins.Compared with the K group, 2ME2 and doramapimod inhibited protein expression intensities in the K+2ME2 group and the K+IN group.

| Protein expression of the proliferation factors
A quantitative analysis of the protein expression levels of the MAPK/ Erk pathway factors and the related proliferation proteins, HIF-1α and PCNA, was conducted using western blot and is shown in Figure 3 and Table 3.In these results, 2ME2 and doramapimod significantly decreased most of the key factor (p38, c-Myc, ATF2, and c-Jun) expressions of the MAPK/Erk pathway and PCNA expression.In addition, 2ME2 remarkably decreased HIF-1α expression; however, doramapimod did not show an inhibition effect.On the contrary, the N group showed the highest expression of STAT1, and the lowest expression was found in the K and K+DMSO groups.2ME2 and doramapimod increased STAT1 expression in both groups.

| DISCUSS ION
0][11] The imbalance state between cell proliferation and apoptosis may lead to keloid continuous growth characteristics. 7 this case, effective drugs for keloid treatment may not only promote the cell apoptosis level but also inhibit the cell proliferation level.
2ME2 is an investigational drug for chemotherapy that is generated by the sequential hydroxylation of estradiol through the enzyme cytochrome P450 isoform 1A1 to produce 2-hydroxyestradiol.This is then followed by a conjugation reaction of the enzyme catechol-O-methyltransferase generating 2ME2 from 2-hydroxyestradiol. 124][15] In recent studies, researchers have found other antitumor mechanisms of 2ME2.Lu's study demonstrated that 2ME2 could inhibit the expression of HIF-1α and confer radiosensitivity in ECA-109 cells. 16Wu's study concluded that 2ME2 inhibits nasopharyngeal carcinoma CNE-2 F I G U R E 1 Cell activity in all the groups at different time points.

TA B L E 2
Cell activity in all groups.
stem-like cell (NPCSC) proliferation and migration and reduces the radioresistance of NPCSCs through NF-κB/HIF-1 pathway inactivation and epithelial-mesenchymal transition reversal. 17 and keloids.In our previous study, we primarily concluded that a keloid's continuous growth characteristic may be caused by an imbalance state between a high proliferation level and a relatively low apoptosis level. 3,4It seems that 2ME2's effect may ameliorate the imbalance state at the same time.We found that 2ME2 could improve the keloid fibroblast cell apoptosis level through a caspase-dependent mechanism. 7We conducted this study to explore 2ME2's effect toward the proliferation of keloid fibroblasts and its potential mechanism.
In this study, we choose the p38 universal inhibitor "doramapimod" in the mechanism study.The keloid fibroblast cell activity was measured via the CCK-8 kit with time.The results showed that both 2ME2 and doramapimod effectively inhibited keloid fibroblast cell activity from 96 h after intervention.In addition, the K+2ME2+IN group showed the best effect for inhibiting keloid fibroblast cell activity from the third day on.
The MAPK/Erk signaling pathway is the most classic pathway in the proliferation process. 191][22][23] In addition, PCNA and HIF-1α participate in the proliferation process. 22,24The immunofluorescence results showed the qualitative expressions of p38, PCNA, and HIF-1α.
Compared with the K group, 2ME2 and doramapimod decreased the expression of p38, PCNA, and HIF-1α.In the western blot results, 2ME2 decreased the p38, c-Myc, ATF2, c-Jun, PCNA, and HIF-1α expressions.Interestingly, in our former work, we found no significant difference in the STAT1 expression among normal skin, physiological scars, and keloids at the tissue level.However, normal skin fibroblasts showed the highest STAT1 expression among all the groups, and 2ME2 increased the STAT1 expression of keloid fibroblasts.We inferred that it might have been the tissue microenvironment that caused the STAT1 expression differences.In addition, doramapimod and 2ME2 showed a synergistic effect in inhibiting the activation of the MAPK/Erk pathway.
Based on the results mentioned above, we inferred that 2ME2 may inhibit the keloid fibroblast proliferation level by targeting p38 in the MAPK/Erk pathway.
The concentration-effect curves of 5-FU, TA, and 2ME2 are shown in Figure 4.In this case, 2ME2 showed satisfying treating effect toward keloid fibroblasts with low drug concentration, which may also fibrosis. 25Tang also concluded that 2ME2 showed satisfying treatment effect for psoriasis in vivo and in vitro. 26Clinical trials have also focused on 2ME2, such as prostate cancer, 27 breast cancer, 28 and multiple myeloma. 29Also, 2ME2 emerges as a promising new candidate for the treatment of endometriosis. 30Clinical use of 2ME2 may be limited by its poor water solubility, which may cause 2ME2 plasma concentrations lower than the effective dose.
However, topical use of 2ME2 may become a practical way to assure its effective concentration.F I G U R E 4 Concentration-effect curves of 5-FU, TA, and 2-methoxyestradiol for 12, 24, and 48 h.
Long 18    first introduced 2ME2 in keloid research.His study indicated that 2ME2 increased radiation-induced apoptosis of keloid fibroblasts by targeting HIF-1α.From then, we continued the work with 2ME2

F I G U R E 2 F I G U R E 3
Immunofluorescence staining images (400×) of p38, PCNA, and HIF-1α.The green area represents cells with positive expression of the target protein.The blue area represents the DNA area.2-Methoxyestradiol and doramapimod decreased the expression levels of p38, PCNA, and HIF-1α (n = 6 in each group).reduce side effects.2ME2 may become a new agent for treating keloid in clinical practice.One question may appear as to why we preferred 2ME2 over doramapimod.We analyzed this question according to the following aspects: (1) 2ME2 is a new chemotherapeutic drug of low concentration and high effectiveness.In this study, doramapimod (20 μM) showed its effect at nearly three times the concentration of 2ME2 (6.975 μM).It is well known that the drug concentration is proportional to the toxicity.A drug of low concentration and high effectiveness can not only assure the treatment success but also provide patient safety.(2)2ME2 is much cheaper than doramapimod.As of the time of purchasing the reagents for this study, 2ME2 (10 mM) was 579.28 RMB; however, doramapimod (10 mM) was 2403.35RMB.In this case, choosing 2ME2 can decrease patient economic costs.In addition, 2ME2 may also show therapeutic effect in dermatology.Kim's study showed that 2ME2 may be a potent therapeutic agent for radiation-induced skin injury by reducing radiation-induced inflammation, skin thickness, and vascular Relative protein amounts for all of the target proteins.The expression levels of all factors (except STAT1) were significantly decreased by the 2-methoxyestradiol treatment.2ME2 increased the expression of STAT1 of the keloid fibroblasts.Values are shown as mean ± SD (n = 6 in each group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. the N group; #p < 0.05, ###p < 0.001).