Evaluating the Anti-Melanoma Effects and Toxicity of Cinnamaldehyde Analogues

Cinnamaldehyde (CA) showed potent activity against melanoma in our previous study, and the structure of unsaturated aldehydes is envisaged to play a role. Nevertheless, its limited drug availability restricts its clinical application. Therefore, a series of CA analogues were synthesized to evaluate their anti-melanoma activities across various melanoma cell lines. These compounds were also tested for their toxicity against the different normal cell lines. The compound with the most potential, CAD-14, exhibited potent activity against the A375, A875 and SK-MEL-1 cells, with IC50 values of 0.58, 0.65, and 0.82 µM, respectively. A preliminary molecular mechanism study of CAD-14 indicated that it could inhibit the p38 pathway to induce apoptosis, and suppress tumor growth by inhibiting the expression of ENO1. Furthermore, an acute toxicity study depicted that CAD-14 has better safety and tolerability than CA in vivo. These findings indicate that CAD-14 might be a lead compound for exploring effective anti-melanoma drugs.


Introduction
Recently, covalent inhibitors have attracted increasing attention as an important complement and alternative to non-covalent counterparts.Natural products have emerged as a fundamental resource for the development of covalent drugs [1].As the potential targets of nucleophilic residues, activated cysteine and serine residues are expected to be the most-attacked binding site by Michael acceptors, aziridines, epoxides, and β-lactams [2][3][4].While early covalent inhibitors relied heavily natural products, the design of contemporary targeted covalent inhibitors has also necessitated an element of serendipity in their development [5].
Melanoma originates in melanin cells in the skin, mucous membrane, and uveitis.Specifically, three types of genes are involved in melanoma genetic risk: those controlling telomerase, those controlling cell cycling and proliferation, and those controlling tumorimmunity interactions [6].The disease's high degree of malignancy, high metastasis, and poor prognosis are the major causes of skin cancer-related deaths [7].Malignant melanoma can regulate the local environment and body homeostasis through various mechanisms [8].Based on epidemiological data statistics, the global incidence rate of malignant melanoma is experiencing an annual increase of 3-5%, and 130,000 new cases of melanoma and 50,000 melanoma-related deaths are diagnosed yearly, making it one of the fastest-growing malignant tumors worldwide [9].The traditional treatment means are surgery, radiotherapy, and chemotherapy, but the five-year survival rate for patients with metastatic melanoma stands at a mere 27% due to the prevalence of adverse drug reactions and significant drug resistance [10].There has been growing acknowledgment of the widespread presence of v-rafmurine sarcoma viral oncogene homolog B (BRAF) mutational heterogeneity in melanoma within academic circles.Vemurafenib was the first to show efficacy in BRAF-mutant melanoma followed by dabrafenib.Although immunotherapy and targeted therapy for BRAF have significantly improved the overall survival and quality of life of patients with advanced melanoma, some patients still do not respond to them [11].Consequently, the development of novel drugs and the identification of their underlying mechanisms are of paramount importance.
Cinnamaldehyde (CA), a prominent natural product, serves as the main component of the volatile oil derived from cinnamon and various Cinnamomum species [12].Recent studies have depicted its enormous potential as a novel resource for research and development in the creation of anti-tumor medications.Our previous works have demonstrated that CA improves this anti-melanoma effect, and the structure of unsaturated aldehydes is envisaged to play a role [13].However, due to its limited drug availability, it is unsuitable for clinical use.This study aims to design and synthesize a series of CA analogues while retaining the α, β-unsaturated aldehyde moiety.We achieved the best activity from CAD-14 analogues during preliminary screening.Additionally, a preliminary molecular mechanism study of CAD-14 indicated that it could inhibit the p38 pathway to induce apoptosis.Meanwhile, CAD-14 inhibited tumor growth by inhibiting ENO1 in vivo.Furthermore, an acute toxicity study demonstrated that CAD-14 has superior safety and tolerability compared with CA.This study will establish a new theoretical foundation for CAD-14 as a potential anti-tumor drug.

Structure Elucidation of CA Analogues
Using CA as a basis, we retained its enal structure, then constructed a molecular skeleton and added selective groups using a drug molecular heterozygous design strategy.The electrical modification and steric hindrance substitution of a CA benzene ring were performed first, following the principle of least modification; this approach can enhance the activity and selectivity of the drug, but it may also significantly change the drug activity.In addition, the structure of the parent nucleus may also affect the drug's pharmacodynamic activity.Accordingly, we modified the benzene ring of the parent nucleus to introduce lipophilic furan ring and thiophene ring compounds.Analogues with biphenyl, naphthalene, and 1,4-dioxane were prepared as substitutes for the benzene ring of CA, to explore the impact of different parent structures on the compound's pharmacological properties.The chemical structures are illustrated in Scheme 1 and Figure 1.
Molecules 2023, 28, x FOR PEER REVIEW 2 of 17 reactions and significant drug resistance [10].There has been growing acknowledgment of the widespread presence of v-rafmurine sarcoma viral oncogene homolog B (BRAF) mutational heterogeneity in melanoma within academic circles.Vemurafenib was the first to show efficacy in BRAF-mutant melanoma followed by dabrafenib.Although immunotherapy and targeted therapy for BRAF have significantly improved the overall survival and quality of life of patients with advanced melanoma, some patients still do not respond to them [11].Consequently, the development of novel drugs and the identification of their underlying mechanisms are of paramount importance.Cinnamaldehyde (CA), a prominent natural product, serves as the main component of the volatile oil derived from cinnamon and various Cinnamomum species [12].Recent studies have depicted its enormous potential as a novel resource for research and development in the creation of anti-tumor medications.Our previous works have demonstrated that CA improves this anti-melanoma effect, and the structure of unsaturated aldehydes is envisaged to play a role [13].However, due to its limited drug availability, it is unsuitable for clinical use.This study aims to design and synthesize a series of CA analogues while retaining the α, β-unsaturated aldehyde moiety.We achieved the best activity from CAD-14 analogues during preliminary screening.Additionally, a preliminary molecular mechanism study of CAD-14 indicated that it could inhibit the p38 pathway to induce apoptosis.Meanwhile, CAD-14 inhibited tumor growth by inhibiting ENO1 in vivo.Furthermore, an acute toxicity study demonstrated that CAD-14 has superior safety and tolerability compared with CA.This study will establish a new theoretical foundation for CAD-14 as a potential anti-tumor drug.

Structure Elucidation of CA Analogues
Using CA as a basis, we retained its enal structure, then constructed a molecular skeleton and added selective groups using a drug molecular heterozygous design strategy.The electrical modification and steric hindrance substitution of a CA benzene ring were performed first, following the principle of least modification; this approach can enhance the activity and selectivity of the drug, but it may also significantly change the drug activity.In addition, the structure of the parent nucleus may also affect the drug's pharmacodynamic activity.Accordingly, we modified the benzene ring of the parent nucleus to introduce lipophilic furan ring and thiophene ring compounds.Analogues with biphenyl, naphthalene, and 1,4-dioxane were prepared as substitutes for the benzene ring of CA, to explore the impact of different parent structures on the compound's pharmacological properties.The chemical structures are illustrated in Scheme 1 and Figure 1.

Evaluating the Anti-Melanoma Activity of Synthesized Analogues
The initial screening of the synthesized analogues against various phenotypes' melanoma cell lines (A375, A875, SK-MEL-1) revealed that analogues CAD-2, CAD-7, CAD-10, CAD-12, CAD-14, CAD-19, and CAD-20 had strong activity (Figure 2A-C).The IC50 values of CAD-14 were 0.58, 0.65, and 0.82 µM in A375, A875, and SK-MEL-1 cell lines, respectively.These findings display that CAD-14 had the best effect, which was 80 times stronger than that of CA.Compared with the positive drug DTIC, the effect was nearly 15 times stronger (Figure 2D).These results suggest that CAD-14 may have stronger antimelanoma activity compared to CA.

Evaluating the Anti-Melanoma Activity of Synthesized Analogues
The initial screening of the synthesized analogues against various phenotypes' melanoma cell lines (A375, A875, SK-MEL-1) revealed that analogues CAD-2, CAD-7, CAD-10, CAD-12, CAD-14, CAD-19, and CAD-20 had strong activity (Figure 2A-C).The IC 50 values of CAD-14 were 0.58, 0.65, and 0.82 µM in A375, A875, and SK-MEL-1 cell lines, respectively.These findings display that CAD-14 had the best effect, which was 80 times stronger than that of CA.Compared with the positive drug DTIC, the effect was nearly 15 times stronger (Figure 2D).These results suggest that CAD-14 may have stronger anti-melanoma activity compared to CA.

CAD-14 Inhibited the Growth of A375 Cells and Arrested Cell Cycle
To evaluate the specificity of CAD-14 towards various cell types, multiple types of cancer cells and normal cells were tested.CAD-14 inhibited A375 cells more effectively than other cancer cells and was less sensitive to normal cell lines (Figure 3A).A375 cells were chosen for the follow-up experiments.A colony-forming assay was used to establish that CAD-14 exhibits proliferation inhibitory activity.Compared to the "Ctrl" group, CAD-14 significantly reduced the number of colonies (Figure 3B).To determine whether the cytotoxic effect of CAD-14 was associated with apoptosis, we tested the apoptosis-related protein via Western blotting.As depicted in Figure 3C,D and Supplement Figure S1, the expression of the pro-apoptotic protein Bak was strongly increased, while the expression of the anti-apoptotic proteins Bcl-2 and survivin was substantially diminished after treatment with CAD-14 in a dose-dependent manner.In order to investigate whether cell cycle arrest contributed to CAD-14-induced cell growth inhibition, the cell cycle distribution was analyzed using a flow cytometer.As displayed in Figure 3E,F, CAD-14 markedly induced S phase arrest in A375 cells in a concentration-dependent manner.These findings suggest that CAD-14 might substantially inhibit the proliferation of A375 cells and arrest the S phase.

CAD-14 Inhibited the Growth of A375 Cells and Arrested Cell Cycle
To evaluate the specificity of CAD-14 towards various cell types, multiple types of cancer cells and normal cells were tested.CAD-14 inhibited A375 cells more effectively than other cancer cells and was less sensitive to normal cell lines (Figure 3A).A375 cells were chosen for the follow-up experiments.A colony-forming assay was used to establish that CAD-14 exhibits proliferation inhibitory activity.Compared to the "Ctrl" group, CAD-14 significantly reduced the number of colonies (Figure 3B).To determine whether the cytotoxic effect of CAD-14 was associated with apoptosis, we tested the apoptosisrelated protein via Western blo ing.As depicted in Figure 3C,D and Supplement Figure S1, the expression of the pro-apoptotic protein Bak was strongly increased, while the expression of the anti-apoptotic proteins Bcl-2 and survivin was substantially diminished after treatment with CAD-14 in a dose-dependent manner.In order to investigate whether cell cycle arrest contributed to CAD-14-induced cell growth inhibition, the cell cycle distribution was analyzed using a flow cytometer.As displayed in Figure 3E,F, CAD-14 markedly induced S phase arrest in A375 cells in a concentration-dependent manner.These findings suggest that CAD-14 might substantially inhibit the proliferation of A375 cells and arrest the S phase.

CAD-14 Induced Apoptosis and Inhibited the P38 Pathway
To evaluate the effect of CAD-14 on ENO1, Western blot and a CETSA assay were then performed.The results indicated that CAD-14 changed the stability of ENO1 protein in a temperature-and dose-responsive manner (Figure 4A,B and Supplementary Figures S2 and S3).To predict the potential position of CAD-14 binding on ENO1, multiple

CAD-14 Induced Apoptosis and Inhibited the P38 Pathway
To evaluate the effect of CAD-14 on ENO1, Western blot and a CETSA assay were then performed.The results indicated that CAD-14 changed the stability of ENO1 protein in a temperature-and dose-responsive manner (Figure 4A,B and Supplementary Figures S2  and S3).To predict the potential position of CAD-14 binding on ENO1, multiple molecular dynamics simulations of ENO1 (PDB ID: 3B97) complexed with CAD-14 and other analogues were conducted.Molecular docking analysis revealed that CAD-14 might covalently bind to ENO1 at Cys356 via a Michael addition reaction (Figure 4C and Supplementary Table S1).Research has found that the p38 signaling pathway plays a crucial role in the occurrence/development of melanoma and has become one of the important targets for its clinical treatment [14].Therefore, we detected the expression of p38/P-p38.The results depict that CAD-14 suppressed the phosphorylation of p38 but did not affect its total protein levels (Figure 4D and Supplementary Figure S4).Accordingly, we propose that CAD-14 inhibited the p38 pathway to induce cell apoptosis.

CAD-14 Inhibited Tumor Growth by Inhibiting ENO1 In Vivo
To further evaluated whether ENO1 is a target protein of CAD-14, we next knockout the ENO1 gene in A375 cells; Figure 5A-C and Supplementary Figure S5 showed the efficiencies of three different recombinant ENO1 retroviral vectors.Based on these results, we finally selected "siENO1-2" for next animal experiments.The results showed that both "siENO1" and "CAD-14" groups can inhibit the tumor growth in the xenograft model compared with the "PBS" group (Figure 5D-F), and the effect was be er than dacarbazine.Besides this, we performed immunofluorescence staining on Ki67 (one proliferationrelated marker).There was obvious decrease in Ki67-positive cells in "CA" groups (Figure 5G).Meanwhile, we detected the expression of ENO1 and found that there were fewer ENO1 accumulated in the "siENO1" and "CAD-14" groups (Figure 5H).Taken together, these findings suggest that CAD-14 substantially inhibits the tumor growth via knock-out ENO1 in vivo.

CAD-14 Inhibited Tumor Growth by Inhibiting ENO1 In Vivo
To further evaluated whether ENO1 is a target protein of CAD-14, we next knock-out the ENO1 gene in A375 cells; Figure 5A-C and Supplementary Figure S5 showed the efficiencies of three different recombinant ENO1 retroviral vectors.Based on these results, we finally selected "siENO1-2" for next animal experiments.The results showed that both "siENO1" and "CAD-14" groups can inhibit the tumor growth in the xenograft model compared with the "PBS" group (Figure 5D-F), and the effect was better than dacarbazine.Besides this, we performed immunofluorescence staining on Ki67 (one proliferation-related marker).There was obvious decrease in Ki67-positive cells in "CA" groups (Figure 5G).Meanwhile, we detected the expression of ENO1 and found that there were fewer ENO1 accumulated in the "siENO1" and "CAD-14" groups (Figure 5H).Taken together, these findings suggest that CAD-14 substantially inhibits the tumor growth via knock-out ENO1 in vivo.

Acute Toxicity Study of CAD-14 In Vivo
To evaluate the toxicity of CAD-14 during embryonic development, an acute toxicity assay was performed using zebrafish embryos.As displayed in Figure 6, Supplementary Figure S12 and Table 1, death and malformation began to occur simultaneously after CAD-14 (128 µM) exposure for 96 h, with mortality rates of 90.00%.Furthermore, the median death concentration (IC50) was 83.68 µM.These results indicate that CAD-14 might have toxic and teratogenic effects after long-term treatment with high concentrations.

Acute Toxicity Study of CAD-14 In Vivo
To evaluate the toxicity of CAD-14 during embryonic development, an acute toxicity assay was performed using zebrafish embryos.As displayed in Figure 6, Supplementary Figure S12 and Table 1, death and malformation began to occur simultaneously after CAD-14 (128 µM) exposure for 96 h, with mortality rates of 90.00%.Furthermore, the median death concentration (IC 50 ) was 83.68 µM.These results indicate that CAD-14 might have toxic and teratogenic effects after long-term treatment with high concentrations.
To evaluate the toxicity of CAD-14 during embryonic development, an acute toxicity assay was performed using zebrafish embryos.As displayed in Figure 6, Supplementary Figure S12 and Table 1, death and malformation began to occur simultaneously after CAD-14 (128 µM) exposure for 96 h, with mortality rates of 90.00%.Furthermore, the median death concentration (IC50) was 83.68 µM.These results indicate that CAD-14 might have toxic and teratogenic effects after long-term treatment with high concentrations.

Discussion
α-β-unsaturated carbonyl groups are the most common and potentially active substructures in natural products.One sixth of all known natural products contain this chemical signature.However, there is controversy as to whether these compounds can be used as leads for drug design, because their potential Michael receptor function may lead to side effects such as cell damage or cytotoxicity [14].Simultaneously, α, β unsaturated carbonyl groups possess the capability to function as free-radical scavengers, facilitating the covalent binding of ligands to specific proteins, while also exhibiting antioxidant properties.Through α, β unsaturated carbonyl groups can interact directly with enzymes to activate certain enzymes, thereby preventing carcinogenesis.For example, quinone reductases and glutathione S-transferase activities can be activated, and subsequently inhibit the electrophilic reaction of carcinogens [15].These effects can be attributed to the properties of the Michael receptor.
Essentially, CA exhibits two distinct reaction pathways: one is the Michael addition reaction, wherein the thiol group of cysteine reacts with the double bond of α-β-unsaturated aldehyde-ketone; the other is the formation of imines, facilitated by an abnormally electrophilic aldehyde group [16].The literature has reported that CA can be covalently modified with the reactive cysteine of TRPA1, resulting in a Michael addition reaction, which activates signal transduction pathways and rapidly transmits potential tissue damage signals through the pain pathway [17].Most of the molecular targets of CA causing cellular effects are unknown.
ENO1 is expressed on the cell surface, can promote cancer invasion, and is subject to a series of specific post-translational modifications, such as acetylation, methylation, and phosphorylation [18].In tumor tissues, overexpressed ENO1 may be released into the peripheral blood through apoptosis of tumor cells or other pathways, thereby leading to up-regulation of ENO1 protein expression in the peripheral blood of tumor patients [19].In addition, ENO1 expression also affects the cell cycle.Studies have demonstrated that when the ENO1 gene is silenced by siRNA, the proliferation of HCC cells is inhibited, the S phase of the cell cycle is shortened, and the G2/M phase is prolonged [20].In this study, we designed a series of CA analogues through further structural optimization of CA.Among them, CAD-14 has strong anti-tumor effects because it is added the electronwithdrawing groups, increasing the electrophilicity of the aldehyde.Besides this, CAD-14 may participate in the Michael addition reaction with ENO1 through α and β-unsaturated aldehydes and ketones.This covalent binding changes the stability of ENO1, leads to S cell cycle arrest, and triggers apoptosis by inhibiting the p38 pathway.Meanwhile, CAD-14 may inhibit tumor growth by inhibiting ENO1 in vivo.This provides a more powerful basis for the study of melanoma treatment.Furthermore, this study also demonstrates that α-β-unsaturated aldehydes play a key role in anti-melanoma effects.
Recent pharmacokinetic and safety studies have revealed that in the acute poisoning of rats after intravenous injection of CA, the lethal dose (LD 50 ) value was 74.8 mg/kg [21].In addition, when the dose of CA reached a certain degree, the micronucleus rate in hepatocytes was increased (rats: 1100 mg/kg, mice: 850-1700 mg/kg).These results indicate that high doses of CA can cause genetic changes in hepatocytes [22].Zebrafish embryos are commonly used as animal models for testing drug toxicity.This study indicated that CAD-14 is safe at normal concentrations and could be used as a potential anti-cancer drug.

Western Blotting Assay
The A375 cells were treated with CAD-14 (0-0.8 µM), alone or with indicated agents, for 24 h.Whole-cell extracts were loaded for SDS-PAGE gels depending on the target protein sizes, and were explored with protein-specific antibodies followed by HRP-conjugated secondary antibodies.Bands corresponding to the antibodies were detected via ECL chemiluminescence.

Cell Cycle Analysis
The A375 cells were incubated with CAD-14 (0-0.4 µM) for 24 h.The cells were collected, washed in PBS, and fixed in ice-cold 75% (v/v) ethanol overnight at -20 • C. The cells were re-suspended, and the DNA content was tested using flow cytometry.

Cellular Thermal Shift Assay (CETSA)
In A375 cell lysates, CAD-14 (0.4 µM) was added for 24 h at different temperatures.Western blotting was used to quantify the effects on ENO1 protein levels.

Molecular Dynamics (MD) Assay
The three-dimensional structure of ENO1 (PDB ID: 3B97) was obtained from the PDB database.Molecular docking was performed using Schrödinge software (Schrödinge_2018, Schrödinge Inc., New York, NY, USA).All crystalline water molecules were removed and the structure of the ENO1 protein was minimized.Multiple ligand structures were generated using different ionization states, tautomerism and stereoisomers at pH 6.8-7.2.All cysteine residues in the protein were involved in the reaction, and the reaction type was a Michael addition.The binding site was analyzed using Pymol software (Schrödinge_2018, Schrödinge Inc., New York, NY, USA).

Building the Low ENO1-Expressing A375 Cells
The recombinant lentiviral vectors (1348084) were purchased from Genechem (Shanghai, China).The formal transfection was conducted with HitransG P (a virus infection enhancer, MOI = 50), and subsequent experiments were performed after 72 h.The transfected cells were cultured with complete medium containing 1 µg/mL puromycin (01100205, Labgic Technology, Beijing, China) to maintain the transfection efficiency.

Immunohistochemical Staining and Immunofluorescent Staining
Tumor tissues were isolated from the mice after sacrifice.Then, the tissues were fixed in 4% paraformaldehyde and embedded in paraffin.For immunohistochemistry staining, tissues were stained with Ki67 (GB111141, Servicebio, Wuhan, China).For immunofluorescent staining, tissues were stained with ENO1 (bs-3978R, Bioss, Beijing, China).

Developmental Toxicity Test of Zebrafish Embryos
The AB-wild-type adult zebrafish (three months old) were purchased from the Institute of Aquatic Biology, Chinese Academy of Sciences (Wuhan, China).During breeding, male and female fish were placed in separate spawning boxes at a 1:1 ratio.Then, early in the morning, they were mixed and laid eggs.During the exposure period, 96-well plates containing normal embryos were randomly selected and viewed daily under a light microscope to assess the developmental toxicity.

Statistical Analysis
The results are expressed herein as mean values ± SD, where significant differences between two groups were analyzed via a t-test, and the analysis of multiple groups was conducted with an analysis of variance (ANOVA) test.Differences of p < 0.05 were considered statistically significant.

Conclusions
This study demonstrated that CAD-14 could arrest the cell cycle and induce cell apoptosis, possibly via regulating the p38 pathway.CAD-14 bound to ENO1 and inhibited its stability.Meanwhile, CAD-14 could inhibit tumor growth by inhibiting ENO1 in vivo.Furthermore, an acute toxicity study demonstrated that CAD-14 has superior safety and tolerability compared with CA in vivo.These findings provide new understanding and indicate the potential of CAD-14 for the melanoma treatment.

Figure 1 .
Figure 1.The structures of CA analogues.

Figure 1 .
Figure 1.The structures of CA analogues.

Figure 2 .
Figure 2. Effect of CA analogues in melanoma cells.(A-D) The different melanoma cell lines were treated with 0-50 µM analogues for 72 h, and then IC50 value was assayed (A dashed blue line: 50% inhibition rate; red box: analogues with high activity).

Figure 2 .
Figure 2. Effect of CA analogues in melanoma cells.(A-D) The different melanoma cell lines were treated with 0-50 µM analogues for 72 h, and then IC 50 value was assayed (A dashed blue line: 50% inhibition rate; red box: analogues with high activity).Molecules 2023, 28, x FOR PEER REVIEW 5 of 17

Figure 3 .
Figure 3. CAD-14 inhibited the proliferation of A375 cells.(A) The different tumor and normal cell lines were treated with 0-200 µM CAD-14 for 72 h, and then IC50 value was assayed.(B) Cells were treated with 0-0.02 µM CAD-14 for 14 days, and Giemsa staining was used to conduct colony formation assays; the histogram depicts the number of colonies.(C,D) Cells were treated with 0-0.8 µM CAD-14 for 24 h; then, the related proteins were tested via Western blo ing.Histograms depict the intensity ratio.(E,F) Cells were treated with 0-0.4 µM CAD-14 for 24 h, then the DNA content of the cells was determined with a flow cytometry system.Histograms show the percentage of cells in G0/G1, G2/M, and S phase.Values represent the means ± SD. * p < 0.05, ** p < 0.01 versus "Ctrl" group.

Figure 3 .
Figure 3. CAD-14 inhibited the proliferation of A375 cells.(A) The different tumor and normal cell lines were treated with 0-200 µM CAD-14 for 72 h, and then IC 50 value was assayed.(B) Cells were treated with 0-0.02 µM CAD-14 for 14 days, and Giemsa staining was used to conduct colony formation assays; the histogram depicts the number of colonies.(C,D) Cells were treated with 0-0.8 µM CAD-14 for 24 h; then, the related proteins were tested via Western blotting.Histograms depict the intensity ratio.(E,F) Cells were treated with 0-0.4 µM CAD-14 for 24 h, then the DNA content of the cells was determined with a flow cytometry system.Histograms show the percentage of cells in G0/G1, G2/M, and S phase.Values represent the means ± SD. * p < 0.05, ** p < 0.01 versus "Ctrl" group.

17 Figure 5 .
Figure 5. CAD-14 inhibited the growth of A375 cells in vivo.(A-C) The A375 cells were transfected with three different recombinant ENO1 retroviral vectors and an NC virus for 72 h; the effect of recombinant retrovirus on ENO1 expression were evaluated via fluorescence photography and Western blo ing.Histograms show the intensity ratio.(n = 3).Values represent the means ± SD. ** p < 0.01 versus "NC" group.(D-F) Cells were injected subcutaneously into nude mice.Then, they were treated with Dacarbazine (10 mg/kg/day, as a positive control) or CAD-14 (5 mg/kg/day) for two weeks (the red arrow at day 7 represents the first day of treatment).The tumor volume was calculated (n = 5).(G,H) Immunofluorescent staining of Ki67 and ENO1 to detect proliferation ability and expression of ENO1 in tumor tissues, respectively.Values represent the means ± SD. * p < 0.05 versus "PBS" group.

Figure 5 .
Figure 5. CAD-14 inhibited the growth of A375 cells in vivo.(A-C) The A375 cells were transfected with three different recombinant ENO1 retroviral vectors and an NC virus for 72 h; the effect of recombinant retrovirus on ENO1 expression were evaluated via fluorescence photography and Western blotting.Histograms show the intensity ratio.(n = 3).Values represent the means ± SD. ** p < 0.01 versus "NC" group.(D-F) Cells were injected subcutaneously into nude mice.Then, they were treated with Dacarbazine (10 mg/kg/day, as a positive control) or CAD-14 (5 mg/kg/day) for two weeks (the red arrow at day 7 represents the first day of treatment).The tumor volume was calculated (n = 5).(G,H) Immunofluorescent staining of Ki67 and ENO1 to detect proliferation ability and expression of ENO1 in tumor tissues, respectively.Values represent the means ± SD. * p < 0.05 versus "PBS" group.

Figure 6 .
Figure 6.Developmental effects of CAD-14 on embryos.(A) The embryo morphology after CAD-14 treatment for 0-96 h.(B) Survival statistics in different time and concentration.

Table 1 .
Mortalities and malformation rates after treating CAD-14 for 96 h.