Acquired resistance to vemurafenib develops through reactivation of RAF/MEK/ERK signaling or bypass mechanisms. Recent combination therapies such as a MEK inhibitor combined with vemurafenib show improvement in major clinical end points, but the percentage of patients with adverse toxic events is higher than with vemurafenib monotherapy and most patients ultimately relapse. Therefore, there is an urgent need to develop new antimelanoma drugs and/or adjuvant agents for vemurafenib therapy. In this study, we created a novel semiorganically modified derivative, DETD-35, from deoxyelephantopin (DET), a plant sesquiterpene lactone demonstrated as an anti-inflammatory and anti–mammary tumor agent. Our results show that DETD-35 inhibited proliferation of a panel of melanoma cell lines, including acquired vemurafenib resistance A375 cells (A375-R) established in this study, with superior activities to DET and no cytotoxicity to normal melanocytes. DETD-35 suppressed tumor growth and reduced tumor mass as effectively as vemurafenib in A375 xenograft study. Furthermore, DETD-35 also reduced tumor growth in both acquired (A375-R) and intrinsic (A2058) vemurafenib resistance xenograft models, where vemurafenib showed no antitumor activity. Notably, the combination of DETD-35 and vemurafenib exhibited the most significant effects in both in vitro and in vivo xenograft studies due to synergism of the compound and the drug. Mechanistic studies suggested that DETD-35 overcame acquired vemurafenib resistance at least in part through deregulating MEK-ERK, Akt, and STAT3 signaling pathways and promoting apoptosis of cancer cells. Overall, our results suggest that DETD-35 may be useful as a therapeutic or adjuvant agent against BRAFV600E mutant and acquired vemurafenib resistance melanoma. Mol Cancer Ther; 15(6); 1163–76. ©2016 AACR.

This article is featured in Highlights of This Issue, p. 1153

Melanoma is the most lethal form of skin cancer, and cases are increasing year by year. In the United States, the estimated number of new melanoma cases in 2001 was 51,400. This figure grew by 43.7% to 73,873 cases in 2015 (1, 2). Mutation in BRAF, V-raf murine sarcoma viral oncogene homolog B1 protein, BRAFV600E is found in approximately 50% of melanoma patients and drives RAF/MEK/ERK MAPK signaling to promote cancer progression (3). Therefore, a number of inhibitors targeting BRAF mutation or MAPK signaling have been developed. For example, vemurafenib (PLX4032), a small-molecule specifically targeting BRAFV600E mutation, was discovered by Plexxikon in 2008 (4) and received FDA approval for treatment of late-stage melanoma in 2011 due to its promising clinical efficacy. Unfortunately, patients developed drug resistance and eventually relapsed within an average of 7 months after vemurafenib therapy (5). Acquired resistance to PLX4032 in human melanoma was mainly through paradoxical activation of MAPK signaling or bypass mechanisms (6–11). The reactivation of MEK-ERK signaling is driven by either a RAF-independent pathway through overexpression of MAP kinase kinase kinase 8 (MAP3K8, or COT) or the activation of the receptor tyrosine kinases (RTK; refs. 12–14). RTK signaling is initially suppressed by the elevated state of ERK-dependent feedback in BRAFV600E mutant melanoma cells in which RAS activity is low and BRAFV600E functions as a monomer. Upon vemurafenib treatment, inhibition of ERK activity relieves ERK-dependent feedback and allows ligand-dependent signaling by growth factors, leading to a rebound in RAS activity that promotes the formation of RAF dimers and reactivation of MEK-ERK signaling despite the presence of vemurafenib (15). These findings led to clinical trials, such as a MEK inhibitor combined with vemurafenib or other BRAF inhibitors (16). Nonetheless, although recent combination therapy showed improvement in major clinical end points, the percentage of patients with adverse toxic events is higher than with vemurafenib monotherapy, and most patients ultimately relapse (16–18). Therefore, it is important to continue to develop new antimelanoma drugs and/or novel adjuvant approaches for vemurafenib therapy.

Elephantopus scaber L. is a traditional Chinese medicine that has been used for various inflammation-related syndromes. Our laboratory has isolated a bioactive compound deoxyelephantopin (DET) from E. scaber which shows anti-inflammation, hepatoprotective, and anti–breast cancer activities by modulating multiple mechanisms, such as activation of the JNK-mediated apoptotic pathways, deregulation of the IKK/NFκB pathway and the ubiquitin proteasome machinery, and impeding TS/A cell motility by inhibiting calpain-mediated adhesion dynamics and inducing reactive oxygen species (ROS) and aggresome formation (19–22). Based on a structural modeling analysis, we previously observed that the specific carbonyl group at C12, C15, and C16 of DET directly interacts with RelA subunit of NF-κB through interhydrogen bonding. This interaction between DET and RelA likely averts the RelA subunit from binding to its DNA target (20). Notably, DET is also observed to be superior to paclitaxel (PTX) in prolonging the lifespan/survival rate in TS/A mammary tumor isograft-bearing mice (19). In addition, DET has been reported to possess antilung adenocarcinoma (23), nasopharyngeal carcinoma (24) and human lymphocyte proliferation in vitro, and antiascites tumor in vivo (25). Together, we consider DET as a novel lead compound for further modification or optimization of its structure to create a more potent drug lead for cancer management. We have thus initiated semiorganic synthesis of DET derivatives (DETD) in order to obtain a potent DETD for melanoma treatment.

In this report, we provide a comprehensive evaluation of the preclinical activity profile of DETD-35, a derivative of DET, in BRAFV600E mutant and acquired vemurafenib resistance melanoma cells in vitro and in vivo. DET and vemurafenib were also evaluated in parallel. We showed that DETD-35 effectively inhibited BRAFV600E mutant and acquired vemurafenib resistance xenograft tumor growth in mice, with superior activities to parental compound DET. The synergism of DETD-35 and vemurafenib led to better in vitro and in vivo efficacy compared with treatment with either agent alone. This study demonstrates the novel therapeutic potential of DETD-35 against BRAFV600E mutant and acquired vemurafenib resistance in melanoma.

Preparation of DET and organic synthesis of DETDs

DET isolation followed the protocol published elsewhere (19). DETD-3 was derived from DET by translactonization via the hydrolysis of DET followed by acidic treatment. DETD-5, -6, -13, -17, -19, -24, -31, -33, -35, -39, and -43 were further synthesized from DETD-3 (26).

Chemicals and reagents

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), N-acetyl-L-cysteine (NAC), and crystal violet were purchased from Sigma-Aldrich. DMEM, FBS, and the antibiotic mixture (penicillin–streptomycin) were purchased from Invitrogen. PLX4032 and MEK inhibitor (PD0325901) were purchased from Selleck Chemical. All other solvents or chemicals were of reagent or high-performance liquid chromatography (HPLC) grade.

Cell lines and culture conditions

Normal human epithelial cell melanocytes (ATCC PCS-200-012) and a spectrum of wild-type or mutant melanoma cell lines, including murine B16-F10 (ATCC CRL-6475) and human cell lines MeWo (ATCC HTB-65), A375 (ATCC CRL-1619), A2058 (ATCC CRL-11147), and SK-MEL-2 (ATCC HTB-68) were obtained from the ATCC. All cell lines were purchased during 2010–2012 and authenticated originally by the ATCC using short tandem repeat analysis. Cells were expanded and frozen down at low passage within 1 month after the receipt of the original stocks. Cells were then thawed and used within 15 passages without further authentication for this study. Cells were cultured in manufacturers' suggested medium supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified 5% CO2 incubator.

Animals

Female NOD/SCID mice bred in the Laboratory Animal Core Facility (Agricultural Biotechnology Research Center) were given a standard laboratory diet and distilled H2O ad libitum and kept on a 12-hour light/dark cycle at 22 ± 2°C. All experimental protocols (No: 11-02-137) were approved by the Institutional Animal Care and Utilization Committee, Academia Sinica, Taiwan.

Cell viability assay

Cells were seeded in 96-well plates overnight and treated with compounds for 24 to 72 hours. Cell growth was determined by MTT-based colorimetric assays according to Scudiero and colleagues (27). Viability of the cells treated with vehicle-only (0.5% DMSO) was defined as 100% viability. Survival of cells after treatment with compound or PLX4032 was calculated using the following formula: viable cell number (%) = [OD570 (treated cell culture)/OD570 (vehicle control)] × 100.

Compound–drug combination assay

Concentrations of DETD-35 and PLX4032 for compound–drug combination study were selected based on their IC50 values from MTT assay. The Chou–Talalay method was used to determine the effects of a compound–drug combination on A375 cells (28). The interaction of DETD-35 and PLX4032 was determined by the combination index (CI), and the CI plot was generated using CompuSyn software. The combined effects of two compounds can be categorized as follows: CI < 1 indicates synergism; CI = 1 indicates an additive effect; and CI > 1 indicates antagonism between the two compounds tested (28).

Colony-formation assay

Colony-forming cell growth was obtained by growing A375 or A375-R cells in 24-well plates with indicated treatments for 21 days. The medium containing the compound or drug was refreshed every 3 days. Cell colonies were stained with 0.1% (w/v) crystal violet in PBS, dried, and photographed. Cell-retained crystal violet was extracted with 20% (v/v) acetic acid in water and quantitatively measured by absorbance at 595 nm. Relative colony-forming percentage in each treatment was compared with the control treatment.

Cell-cycle analysis

Cell-cycle analysis was performed as described by Shyur and colleagues (29). Cells were synchronized by incubation in medium containing 1% FBS for 12 hours, and the culture was then incubated with fresh medium/10% FBS-containing vehicle or compound for 24 hours. Both adherent and floating cells were collected, washed with PBS, and fixed with 1 mL of ice-cold 70% ethanol overnight at 4°C. Cells were stained with propidium iodide/RNase A solution for 30 minutes at room temperature in the dark and then analyzed by flow cytometry (Flow cytometer BD LSR II).

Apoptosis detection

Cells were seeded in 6-well plates and treated with vehicle or indicated compounds. After 72 hours of incubation, apoptotic cells were analyzed using the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen) according to the manufacturer's instructions and flow cytometry.

Western blot analysis

Total cellular proteins were prepared from tested cells as previously described (30). Protein concentration was determined by the Bradford method (Bio-Rad). Protein samples were resolved by 5% to 20% gradient SDS-PAGE and then underwent immunoblotting. Primary antibodies against ERK1/2 (Sc-94), Bax (Sc-20067), Akt (Sc-5298), JNK (Sc-571), c-Jun (Sc-1694), and STAT3 (Sc-482) were purchased from Santa Cruz Biotechnology; phospho-Src (GTX81151) was purchased from GeneTex; Src (2778-1) was purchased from Epitomics; phospho-Akt (#4058), phospho-STAT3 (#9131), phospho-MEK (#9121), MEK (#9122), phospho-ERK1/2 (#9106), p27 (#2552), cyclin D1 (#2978), BcL-xL (#2764), phospho-SAPK/JNK (#9251), phospho-c-Jun (#9261), caspase-7 (#9492), cleaved caspase-7 (#9491), PARP (#9542), and cleaved PARP (#5625) were purchased from Cell Signaling Technology; and actin (MAB1501) was purchased from Merck Millipore. Appropriate horseradish peroxidase–conjugated secondary antibodies were used. Protein bands reacting to specific antibodies were visualized by use of enhanced chemiluminescence (Amersham) with exposure to chemiluminescence light film (BioMax; Kodak Co.). The resulting images were quantified by densitometry using Bio-Profil (Bio-1D 97.04; Vilber-Lourmat).

Caspase-3 activity assay

Caspase-3 activity was detected using the NucView 488 Caspase-3 Assay Kit for live cells (Biotium; Catalog No. 30029). Briefly, A375 and A375-R cells were seeded in 10 cm plates, treated with vehicle or compound for 72 hours, and then the cells were trypsinized and resuspended at a density of 1 × 106 cells/mL. Cell suspension (0.2 mL) was pipetted into a test tube and incubated with a 5 μmol/L NucView 488 substrate concentration for 30 minutes at room temperature in the dark. After that, 300 μL of PBS was added into each test tube and analyzed by flow cytometry. The intensity of the fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 515 nm.

Measurement of ROS

ROS level in the cells was measured using the Total ROS Detection Kit (Enzo; Catalog No. 51011). Briefly, A375 and A375-R cells were seeded in 10-cm plates for 18 hours. Cells were pretreated with or without 5 mmol/L NAC for 1 hour before treating with vehicle or compounds for 2 hours. The cells were then trypsinized and resuspended in 500 μL of ROS detection solution which can be reacted to a wide range of reactive species, such as hydrogen peroxide, peroxynitrite, hydroxyl radicals, nitric oxide, and peroxy radical, yielding a green fluorescent product, and incubated for 30 minutes at 37°C. The generated ROS level was then analyzed by flow cytometry.

In vivo xenograft tumor models

A375, A375-R, or A2058 cells (3 × 106) were injected subcutaneously to the flanks of 6-week-old NOD/SCID mice. Once the tumor volume reached around 50 mm3, the mice were randomized into treatment groups (n = 8 per group), and the treatments were started. Body weight and tumor volume of the mice were recorded every 3 days using formula a × b2 × 0.5, where a and b represent the larger and smaller tumor diameters (31). Tumor growth inhibition (TGI) was calculated as 100 × [1 − (mean tumor volume final − mean tumor volume initial)treatment group/(mean tumor volume final − mean tumor volume initial)vehicle group] (31).

Immunohistochemistry analysis

The formalin-fixed and paraffin-embedded tumor sections (4-μm thick) were heat immobilized and deparaffinized with xylene and rehydrated in a graded series of ethanol with a final wash in distilled water. Antigen retrieval involved use of Target Retrieval Solution (DakoCytomation) in a Decloaking Chamber (Biocare Medical). The following antibodies were used for immunohistochemistry analysis: phospho-Src (GTX81151) from GeneTex; and phospho-Akt (#4060), phospho-MEK (#2338), phospho-ERK1/2 (#4370), Ki-67 (#9027), cleaved caspase-3 (#9661), and cleaved PARP (#5625) from Cell Signaling Technology.

Statistical analysis

Data are expressed as mean ± SEM. Statistical analyses were conducted using an SAS program (SAS Institute), and significant differences within treatments were determined by ANOVA. P values of less than 0.05 were considered statistically significant.

DET and DETDs inhibit melanoma cell proliferation in vitro

DET had an antiproliferative effect on human A375 or mouse B16-F10 melanoma cells at an IC50 value of 6.0 μmol/L; however, the re-lactonized derivative DETD-3 did not show antimelanoma cell proliferation activity within the same tested concentration up to 10 μmol/L (Table 1). Thus, the hydroxyl group at the C-6 position in DETD-3 was further modified to improve the activity and/or study the structure-activity relationship. Attaching an acyl group on the C-6 hydroxyl group can produce an ester, which may act not only as a prodrug, but also influence the interaction of the drug. An additional acyl group might provide an H-bond acceptor, of appropriate size to fill in the cavity space of the protein binding site, with decent hydrophobicity, and so on. The aliphatic groups of various sizes were connected to produce DETD-17, -5, -13, -19, and -24. An aromatic group offers a planarity and π-electron–rich space, which may play an important role in ligand–protein interaction. Therefore, benzoyl, phenyl acetoxyl, and cinnamoyl groups were also connected to the C-6 hydroxyl to produce DETD-31, -33, -43, -35, -6, and -39, respectively. These ester derivatives could have various distances between the aromatic ring and the terpene skeleton. Chemical structures of DET and DETDs are shown in Table 1. 

Table 1.

Structure of DET and its derivatives (DETDs) and their effects on proliferation of different melanoma cell lines and normal melanocytesa

Structure of DET and its derivatives (DETDs) and their effects on proliferation of different melanoma cell lines and normal melanocytesa
Structure of DET and its derivatives (DETDs) and their effects on proliferation of different melanoma cell lines and normal melanocytesa

All synthesized DETDs together with the parental phytocompound DET as a reference were evaluated for antimelanoma cell proliferation activity using wild-type as well as mutant melanoma cell lines B16-F10, MeWo, A375, A2058, SK-MEL-2, and normal melanocytes. DET only inhibited B16-F10 and A375 cell proliferation, but showed no detectable activities against other human melanoma cells at tested concentrations up to 10 μmol/L. Among DETDs with aliphatic ester groups, only DETD-5 showed comparable antiproliferative activities against the tested melanoma cells, except wild-type MeWo and normal melanocytes, with IC50 values of 4.1 to 7.8 μmol/L. Other aliphatic ester derivatives, such as DETD-17, -13, -19, and -24, did not show inhibitory effects on any of the tested human melanoma cells. Compared with aliphatic ester derivatives, interestingly, most DETDs with the aromatic ester group inhibited the melanoma cell growth without affecting normal cells. Aromatic ester derivatives, DETD-33, -35, -6, and -39, inhibited all tested melanoma cell proliferation with IC50 values of 3.9–9.9, 2.2–6.7, 2.5–6.0, and 1.6–3.5 μmol/L, respectively. Particularly, DETD-35 with 10π electronic naphthalene group and DETD-39 with 3-methoxycinnamate group demonstrated two times more potent antiproliferation against the tested melanoma cells than DET itself. These results indicate that the feature of the aromatic ring on the ester group is favored for the antimelanoma activity, and more electron-rich systems tend to be more potent. DETD-39 is slightly cytotoxic to normal human melanocytes with an IC50 of 9.0 μmol/L (Table 1), whereas DETD-35 was demonstrated to have little or no toxicity to normal melanocytes, with only 9.1% inhibition detected at 10 μmol/L concentration. Therefore, we chose DETD-35 for further investigation.

DETD-35 shows synergism with vemurafenib in inhibiting A375 cell proliferation

Compound–drug combination assay was carried out to evaluate the potential synergism between vemurafenib and DETD-35. The MTT-based antiproliferation assay showed that the IC50 of PLX4032 and DETD-35 on A375 cells were 0.07 and 0.85 μmol/L, respectively, when 2 × 103 cells were initially seeded and treated with the compound for 72 hours (Fig. 1A and B). A greater dose-dependent cytotoxicity of DETD-35 to A375 cells was observed at the tested compound concentrations up to 20 μmol/L in which less than 10% viable cells were detected (data not shown). MTT assay with a fixed concentration of PLX4032 (10 nmol/L) combined with increasing concentrations of DETD-35 (0.1 to 1 μmol/L) showed better inhibition compared with DETD-35 treatment alone at the same tested concentrations (Fig. 1C). The CI values calculated using CalcuSyn software for combination treatment of DETD-35 and vemurafenib were within 0.60 to 1.05, indicating either synergism or an additive effect of the compound–drug combination (Fig. 1D).

Figure 1.

DETD-35 shows synergism with vemurafenib in vitro. A–C, A375 melanoma cells were treated with the indicated concentrations of PLX4032, DETD-35, and PLX4032+DETD-35 for 72 hours, respectively. Cell viability was determined using MTT assay. D, compound–drug combination study using CalcuSyn software. CI < 1 indicates synergism; CI = 1 indicates additive effect; CI > 1 indicates antagonism between the two compounds tested. E, top, colony-formation assay showing long-term (21-day treatment) antiproliferative effects of PLX4032, DETD-35, and DET against A375 cells. Crystal violet–stained cells are shown. Bottom, quantification of the crystal violet absorbance at 595 nm after 21 days of treatment. Data are mean ± SEM, n = 4. Mean without a common letter differs, P < 0.05. F, cell-cycle analysis, cells were treated with the indicated concentrations of compounds for 24 hours. After fixation, cells were stained with propidium iodide and analyzed by flow cytometry. G, combination of DETD-35 or DET with PLX4032 increased levels of apoptosis in A375 melanoma cells. Cells were treated with the indicated concentrations of the compounds for 72 hours. Apoptosis status in cells was measured by Annexin V/FITC plus propidium iodide double staining and flow cytometry. Top, representative quadrant diagrams showing cell distribution in Q1 (early apoptosis), Q2 (apoptosis), Q3 (live), and Q4 (dead). Bottom, apoptotic fraction was calculated by adding distribution percentage in Q1, Q2, and Q4 together. Data are mean ± SEM, n = 3. Mean without a common letter differs, P < 0.05.

Figure 1.

DETD-35 shows synergism with vemurafenib in vitro. A–C, A375 melanoma cells were treated with the indicated concentrations of PLX4032, DETD-35, and PLX4032+DETD-35 for 72 hours, respectively. Cell viability was determined using MTT assay. D, compound–drug combination study using CalcuSyn software. CI < 1 indicates synergism; CI = 1 indicates additive effect; CI > 1 indicates antagonism between the two compounds tested. E, top, colony-formation assay showing long-term (21-day treatment) antiproliferative effects of PLX4032, DETD-35, and DET against A375 cells. Crystal violet–stained cells are shown. Bottom, quantification of the crystal violet absorbance at 595 nm after 21 days of treatment. Data are mean ± SEM, n = 4. Mean without a common letter differs, P < 0.05. F, cell-cycle analysis, cells were treated with the indicated concentrations of compounds for 24 hours. After fixation, cells were stained with propidium iodide and analyzed by flow cytometry. G, combination of DETD-35 or DET with PLX4032 increased levels of apoptosis in A375 melanoma cells. Cells were treated with the indicated concentrations of the compounds for 72 hours. Apoptosis status in cells was measured by Annexin V/FITC plus propidium iodide double staining and flow cytometry. Top, representative quadrant diagrams showing cell distribution in Q1 (early apoptosis), Q2 (apoptosis), Q3 (live), and Q4 (dead). Bottom, apoptotic fraction was calculated by adding distribution percentage in Q1, Q2, and Q4 together. Data are mean ± SEM, n = 3. Mean without a common letter differs, P < 0.05.

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We also evaluated the long-term antiproliferative effects of DETD-35 and PLX4032 (treatment for 21 days) using colony-formation assay. The parental compound DET was also evaluated in parallel. PLX4032 inhibited colony formation of A375 cells, with 55% inhibition at 0.1 μmol/L treatment. DETD-35 or DET alone also showed inhibition in a dose-dependent manner. Of note, cells treated with DETD-35 at 0.5 μmol/L, 1.25 μmol/L, and 2.5 μmol/L inhibited 42%, 71%, and 98% colony formation, respectively, and 1 μmol/L, 2.5 μmol/L, and 5 μmol/L of DET treatment inhibited 38%, 65%, and 91% colony formation, respectively, of A375 cells. Importantly, the combination treatment displayed better effects than the individual compound or drug treatment. At 0.1 μmol/L PLX4032 plus 1.25 μmol/L DETD-35 or 2.5 μmol/L DET, the inhibition reached 88% and 81%, respectively (Fig. 1E). The antiproliferative effect of PLX4032 was strongly associated with induction of G1-phase cell-cycle arrest (Fig. 1F). DETD-35 did not have any significant effect on A375 DNA distribution in the cell cycle, whereas DET at 5 μmol/L induced G2–M-phase arrest (Fig. 1F). Both DETD-35 and DET treatment induced high levels of A375 cell apoptosis, up to 85.2% and 67.8%, respectively; however, PLX4032 treatment only induced 10.4% apoptosis in the cells (Fig. 1G). We found that the profound effect of PLX4032 on inducing G1-phase cell-cycle arrest dominated the effect of compound (DETD-35 or DET)–drug combination in A375 cells, whereas DETD-35 or DET sensitized PLX4032 induced A375 cell apoptosis to 91% and 69%, respectively (Fig. 1F and G).

Figure 2.

DETD-35 suppresses MEK-ERK signaling and induces apoptosis in A375 melanoma cells. A, cells were treated with indicated concentrations of the compounds for 16 hours before lysis. Cell lysates were subjected to Western blotting against the specific antibodies indicated, and actin was used as an internal or loading control. Quantification of specific protein expression, which was normalized to actin first followed by comparison of the fold change within different treatments to vehicle treatment. B, cells were treated with indicated concentrations of the compounds for 72 hours, and caspase-3 activity was determined by measuring cleavage of a fluorogenic substrate using flow cytometry (RFU, relative fluorescence units). Data are mean ± SEM, n = 3. Mean without a common letter differ, P < 0.05.

Figure 2.

DETD-35 suppresses MEK-ERK signaling and induces apoptosis in A375 melanoma cells. A, cells were treated with indicated concentrations of the compounds for 16 hours before lysis. Cell lysates were subjected to Western blotting against the specific antibodies indicated, and actin was used as an internal or loading control. Quantification of specific protein expression, which was normalized to actin first followed by comparison of the fold change within different treatments to vehicle treatment. B, cells were treated with indicated concentrations of the compounds for 72 hours, and caspase-3 activity was determined by measuring cleavage of a fluorogenic substrate using flow cytometry (RFU, relative fluorescence units). Data are mean ± SEM, n = 3. Mean without a common letter differ, P < 0.05.

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DETD-35 deregulates MEK-ERK signaling and induces apoptotic hallmarks in A375 cells

Next, we investigated the underlying mechanisms by which DET, DETD-35, and PLX4032 inhibited A375 melanoma cell activity. We observed that PLX4032 inhibited MEK and ERK phosphorylation, increased p27 expression, and suppressed cyclin D1 expression as shown by Western blotting (Fig. 2A). DETD-35 or DET also inhibited MEK and ERK phosphorylation, but had no effect on p27 and cyclin D1 expression; however, both compounds induced the apoptotic hallmarks, the cleaved form of PARP accumulation, and caspase-3 activity in the cells (Fig. 2A and B). The compound–drug combination showed inhibition of pMEK, pERK, and cyclin D1 expression as well as induction of p27 and cleaved PARP protein levels (Fig. 2A), and caspase-3 activity (Fig. 2B), indicating combination treatment integrated the effects of PLX4032 and DET or DETD-35 in A375 cells. Other apoptotic markers Bax and BcL-xL were not responsive to PLX4032, DET, or DETD-35 treatment.

DETD-35 alone or in combination with vemurafenib suppresses tumor growth in an A375 orthotopic xenograft model

We used an A375 human melanoma xenograft model to further evaluate the in vivo bioefficacy of DET, DETD-35, PLX4032, and the compound–drug combination. Supplementary Fig. S1A displays the detailed experimental design for A375 xenograft study. Figure 3A shows representative tumor tissues from the tumor control and treatment groups of the A375 xenograft study. PLX4032 (20 mg/kg/day, 24 doses in total) was effective in suppressing tumor growth with 87.4% TGI and reduction of tumor mass by 71.9%. We observed that DETD-35 and DET (20 mg/kg/every 2 days, 12 doses in total) also suppressed tumor growth with 87.1% and 71.0% TGI and reduced tumor mass by 70.5% and 47.5%, respectively (Fig. 3B and C), indicating DETD-35 was as effective as vemurafenib and possessed better antimelanoma growth activity than DET. Importantly, the compound–drug combination, in which DETD-35 (20 mg/kg/every 4 days, 6 doses in total) and PLX4032 (20 mg/kg/every 2 days, 12 doses in total) were administered alternately and the treatment frequency was only half of the single compound administration frequency, showed similar antitumor efficacy as the single-agent treatment (91.0% TGI and 72.3% reduction in tumor mass), suggesting synergism of DETD-35 and PLX4032 in mice (Fig. 3B and C). In addition, there were no indications of toxicity such as body weight loss or changes in organ index in the treatment groups (Supplementary Fig. S1B and S1C). Immunohistochemical analysis showed PLX4032 significantly inhibited pMEK, pERK, and proliferation marker Ki-67 levels, and slightly induced the apoptotic markers, i.e., cleaved caspase-3 and cleaved PARP levels in the tumor tissues (Fig. 3D). We observed that DETD-35 and DET also significantly inhibited pMEK, pERK, and Ki-67 levels in which the inhibitory effect of DETD-35 was significantly better than DET (Fig. 3D). Moreover, both compounds showed stronger induction of cleaved caspase-3 and cleaved PARP levels compared with PLX4032 treatment. Importantly, we found that the compound–drug combination reduced levels of pMEK, pERK, and Ki-67 as did PLX4032 and induced levels of cleaved caspase-3 and cleaved PARP as did DETD-35 (Fig. 3D). These results were in good agreement with results observed in the in vitro studies.

Figure 3.

DETD-35 inhibits BRAFV600E mutant melanoma growth in vivo. NOD/SCID mice bearing A375 xenografts were treated with vehicle, PLX4032 (20 mg/kg/day), DETD-35 or DET (20 mg/kg/every 2 days), and a compound–drug combination with alternate administration of DETD-35 or DET (20 mg/kg/every 4 days) plus PLX4032 (20 mg/kg/every 2 days), when tumor volume reached around 50 mm3. A, representative tumor tissues isolated from each treatment group. B, tumor volume was measured every 3 days and plotted as mean ± SEM. C, the weight of the dissected tumors from each treatment group. Data are mean ± SEM, n = 8. Mean without a common letter differs, P < 0.05. D, top, representative immunohistochemistry images of tumor tissues with positive staining of phospho-MEK, phospho-ERK1/2, Ki-67, cleaved caspase-3 and cleaved PARP (brownish color), and nuclear (blue color) by hematoxylin staining are shown (scale bar, 50 μm). Bottom, quantification of phospho-MEK, phospho-ERK1/2, Ki-67, cleaved caspase-3, and cleaved PARP–positive stained cells. Data are mean ± SEM, n = 3. Mean without a common letter differs, P < 0.05.

Figure 3.

DETD-35 inhibits BRAFV600E mutant melanoma growth in vivo. NOD/SCID mice bearing A375 xenografts were treated with vehicle, PLX4032 (20 mg/kg/day), DETD-35 or DET (20 mg/kg/every 2 days), and a compound–drug combination with alternate administration of DETD-35 or DET (20 mg/kg/every 4 days) plus PLX4032 (20 mg/kg/every 2 days), when tumor volume reached around 50 mm3. A, representative tumor tissues isolated from each treatment group. B, tumor volume was measured every 3 days and plotted as mean ± SEM. C, the weight of the dissected tumors from each treatment group. Data are mean ± SEM, n = 8. Mean without a common letter differs, P < 0.05. D, top, representative immunohistochemistry images of tumor tissues with positive staining of phospho-MEK, phospho-ERK1/2, Ki-67, cleaved caspase-3 and cleaved PARP (brownish color), and nuclear (blue color) by hematoxylin staining are shown (scale bar, 50 μm). Bottom, quantification of phospho-MEK, phospho-ERK1/2, Ki-67, cleaved caspase-3, and cleaved PARP–positive stained cells. Data are mean ± SEM, n = 3. Mean without a common letter differs, P < 0.05.

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DETD-35 overcomes acquired vemurafenib resistance in vitro

To study whether or not DETD-35 could overcome acquired resistance to BRAF inhibitor in A375 cells, we first generated the A375 cell line which was resistant to PLX4032 (A375-R) as previously reported (10). Briefly, A375 melanoma cells were seeded in T75 plates and treated with increasing concentrations of PLX4032 (10, 20, 50, 100, 250, 500, 1,000, 2,000, 5,000 nmol/L) through approximately 2 months and 20 passages. We have successfully developed acquired resistance cell line A375-R which is 100-fold less sensitive to PLX4032 treatment compared with parental cell line (IC50: 7.34 vs. 0.07 μmol/L). Importantly, DETD-35 and DET were still effective against the resistance cell line (Fig. 4A–C). PLX4032 revealed no inhibitory effect on colony formation, whereas DETD-35 and DET inhibited colony formation of A375-R cells in a dose-dependent manner (Fig. 4D). Moreover, the compound–drug combination showed dramatic inhibition of A375-R cell colony formation, indicating DETD-35 or DET could sensitize the antiproliferative effect of PLX4032 (Fig. 4D). Consistent with the antiproliferation results, PLX4032 had no effect on cell-cycle distribution and apoptosis of A375-R cells. DETD-35 had no effect on cell-cycle distribution, whereas DET caused G2–M-phase cell-cycle arrest in A375-R cells. Furthermore, both DET and DETD-35 induced high levels of (86%–89%) apoptosis in A375-R cells (Supplementary Fig. S2A and S2B). For mechanistic studies, we included MEK inhibitor combined with PLX4032 as a positive control because MEKi combination with vemurafenib is used to treat BRAFV600E melanoma patients clinically (16). We found that PLX4032 inhibited pSrc, pMEK, pERK, pAkt, and pSTAT3 protein levels in A375 cells. In contrast, phosphoproteins were reactivated in PLX4032-treated A375-R cells. Moreover, PLX4032 could not induce p27 expression or reduce cyclin D1 level in A375-R cells, in the same as observed in PLX4032-treated A375 cells (Fig. 4E). DETD-35 and DET, however, could suppress the phosphorylated proteins, suggesting inhibition of their reactivation in resistant A375 cells. Both compounds were also observed to induce cleaved PARP expression and caspase-3 activity in A375-R cells (Fig. 4E and Supplementary Fig. S2C). Compound–drug combination with DETD-35 or DET plus PLX4032 also inhibited levels of pSrc, pMEK, pERK, pAkt, and pSTAT3 proteins, in the same way as the MEK inhibitor+PLX4032 (Fig. 4E). Interestingly, the compound–drug combination induced cleaved PARP expression, whereas MEK inhibitor+PLX4032 treatment induced p27 expression and reduced cyclin D1 expression (Fig. 4E).

Figure 4.

DETD-35 overcomes acquired vemurafenib resistance in vitro. A–C, A375-R cells were treated with indicated concentrations of PLX4032, DETD-35, and DET for 72 hours. Cell viability was determined using MTT assay. D, top, colony-formation assay showing long-term (21-day treatment) antiproliferative effects of PLX4032, DETD-35, and DET against A375-R cells. Crystal violet–stained cells are shown. Bottom, quantification of the crystal violet absorbance at 595 nm after 21 days of treatment. Data are mean ± SEM, n = 4. Mean without a common letter differs, P < 0.05. E, A375-R cells were treated with the indicated concentrations of compounds for 16 hours before lysis. Cell lysates were subjected to Western blotting using the specific antibodies indicated, and actin was used as an internal or loading control. Quantification of specific protein expression, which was normalized to actin first followed by comparison of the fold change within different treatments to vehicle treatment.

Figure 4.

DETD-35 overcomes acquired vemurafenib resistance in vitro. A–C, A375-R cells were treated with indicated concentrations of PLX4032, DETD-35, and DET for 72 hours. Cell viability was determined using MTT assay. D, top, colony-formation assay showing long-term (21-day treatment) antiproliferative effects of PLX4032, DETD-35, and DET against A375-R cells. Crystal violet–stained cells are shown. Bottom, quantification of the crystal violet absorbance at 595 nm after 21 days of treatment. Data are mean ± SEM, n = 4. Mean without a common letter differs, P < 0.05. E, A375-R cells were treated with the indicated concentrations of compounds for 16 hours before lysis. Cell lysates were subjected to Western blotting using the specific antibodies indicated, and actin was used as an internal or loading control. Quantification of specific protein expression, which was normalized to actin first followed by comparison of the fold change within different treatments to vehicle treatment.

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DETD-35 overcomes acquired and intrinsic vemurafenib resistance in vivo

We used A375-R (acquired vemurafenib resistance) and A2058 (intrinsic vemurafenib resistance due to lack of PTEN expression) xenograft mouse models to evaluate the in vivo bioefficacy of DETD-35, DET, PLX4032, and the compound–drug combination against vemurafenib resistance melanoma. Supplementary Fig. S3A displays the detailed experimental design for A375-R and A2058 xenograft study. Figure 5A shows representative tumor tissues from the A375-R xenograft study. PLX4032 treatment (75 mg/kg/day, 24 doses in total) showed no antitumor activity with similar tumor growth rate and sizes compared with the tumor control group in both A375-R (Fig. 5B and C) and A2058 (Fig. 5D and E) xenograft models, as expected. DETD-35 (20 mg/kg/every 2 days, 12 doses in total) effectively inhibited A375-R (Fig. 5B and C) and A2058 (Fig. 5D and E) tumor growth in mice with 47.1% and 73.7% TGI and a reduction in tumor mass of 46.3% and 69.0%, respectively. DET (20 mg/kg/every 2 days, 12 doses in total) only inhibited A375-R tumors with 31.2% TGI (Fig. 5B). Notably, the compound–drug combination in which alternate administration of DETD-35 (20 mg/kg/every 2 days, 12 doses in total) and PLX4032 (75 mg/kg/every 2 days, 12 doses in total) showed the best antitumor activity in both vemurafenib resistance xenografts A375-R (Fig. 5B and C) and A2058 (Fig. 5D and E), with 76.5% and 79.0% TGI and 65.2% and 75.3% reduction in tumor mass, respectively. There was no indication of toxicity such as body weight loss or organ index changes in the treatment groups (Supplementary Fig. S3B–S3E). Consistent with in vitro studies, immunohistochemical analyses showed that DETD-35 and DET treatment or the compound–drug combination significantly reduced pSrc, pMEK, pERK, pAKt, and Ki-67 levels, and increased cleaved caspase-3 and cleaved PARP levels in A375-R xenograft tissues, but PLX4032 alone did not have effect on the expression of these proteins (Fig. 5F). Notably, DETD-35 showed more activity than DET, a result which mirrored the observations in the A375 xenograft study.

Figure 5.

DETD-35 overcomes both intrinsic and acquired vemurafenib resistance in mice. NOD/SCID mice bearing A375-R and A2058 xenografts were treated with vehicle, PLX4032 (75 mg/kg/day), DETD-35 or DET (20 mg/kg/every 2 days), and a compound–drug combination with alternate administration of DETD-35 or DET (20 mg/kg/every 2 days) and PLX4032 (75 mg/kg/every 2 days), when tumor volume reached around 50 mm3. A, representative tumor tissues isolated from A375-R xenograft. B, tumor volume of A375-R xenograft was measured every 3 days and plotted as mean ± SEM. C, the weight of the dissected tumors from A375-R xenograft. D, tumor volume of A2058 xenograft was measured every 3 days and plotted as mean ± SEM. E, the weight of the dissected tumors from A2058 xenograft. Data are mean ± SEM, n = 8. Means without a common letter differ, P < 0.05. F, top, representative immunohistochemistry images of A375-R tumor for positive staining of phospho-Src, phospho-MEK, phospho-ERK1/2, phospho-Akt, Ki-67, cleaved caspase-3, and cleaved PARP (scale bar, 50 μm). Bottom, quantification of phospho-Src, phospho-MEK, phospho-ERK1/2, phospho-Akt, Ki-67, cleaved caspase-3, and cleaved PARP–positive stained cells from tumor tissue sections of A375-R xenograft study. Data are mean ± SEM, n = 3. Mean without a common letter differs, P < 0.05.

Figure 5.

DETD-35 overcomes both intrinsic and acquired vemurafenib resistance in mice. NOD/SCID mice bearing A375-R and A2058 xenografts were treated with vehicle, PLX4032 (75 mg/kg/day), DETD-35 or DET (20 mg/kg/every 2 days), and a compound–drug combination with alternate administration of DETD-35 or DET (20 mg/kg/every 2 days) and PLX4032 (75 mg/kg/every 2 days), when tumor volume reached around 50 mm3. A, representative tumor tissues isolated from A375-R xenograft. B, tumor volume of A375-R xenograft was measured every 3 days and plotted as mean ± SEM. C, the weight of the dissected tumors from A375-R xenograft. D, tumor volume of A2058 xenograft was measured every 3 days and plotted as mean ± SEM. E, the weight of the dissected tumors from A2058 xenograft. Data are mean ± SEM, n = 8. Means without a common letter differ, P < 0.05. F, top, representative immunohistochemistry images of A375-R tumor for positive staining of phospho-Src, phospho-MEK, phospho-ERK1/2, phospho-Akt, Ki-67, cleaved caspase-3, and cleaved PARP (scale bar, 50 μm). Bottom, quantification of phospho-Src, phospho-MEK, phospho-ERK1/2, phospho-Akt, Ki-67, cleaved caspase-3, and cleaved PARP–positive stained cells from tumor tissue sections of A375-R xenograft study. Data are mean ± SEM, n = 3. Mean without a common letter differs, P < 0.05.

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DETD-35 induces generation of ROS to trigger apoptotic cell death in A375 and A375-R cells

Previously, we demonstrated that DET treatment can induce oxidative stress to facilitate the activation of the c-Jun N-terminal kinase (JNK) signaling pathway toward apoptotic cell death in TS/A cells (19). We evaluated whether DETD-35 exerts the same mechanism by inducing ROS-mediated apoptosis in A375 and A375-R cells. We first performed the proliferation assay in the cells with or without pretreatment with the ROS scavenger NAC. As shown in Fig. 6, the 1 μmol/L DETD-35 and 2 μmol/L DET treatment inhibited 59% and 55% A375 cell viability and 69% and 65% A375-R cell viability, respectively, and 5 mmol/L NAC alone did not affect either type of cell viability (Fig. 6A and B). However, treatment with NAC for 1 hour before treating with either DET or DETD-35 restored cell viability in both A375 and A375-R cells (Fig. 6A and B), suggesting that the presence of ROS is correlated to the inhibitory effect of DET or DETD-35 on melanoma cell viability. We then examined the ROS levels in the cells treated with DET or DETD-35 using flow cytometry. Treatment with 2.5 μmol/L DETD-35 or 5 μmol/L DET for 2 hours significantly increased (more than 2-fold) ROS level in both A375 and A375-R cells, as indicated by the data in Fig. 6C; this effect could be significantly reversed by pretreating the cells with 5 mmol/L NAC for 1 hour (Fig. 6C). Because oxidative stress can activate JNK, which subsequently induces c-Jun activation and phosphorylation to trigger apoptosis in cancer cells (19), we thus further analyzed JNK signaling molecules in the cells with the same treatment by Western blotting. We observed that DETD-35 significantly activated p-c-Jun and c-Jun protein levels at early time points (2, 4, and 8 hours) in both A375 and A375-R cells, and less effect was observed for the DET treatment (Fig. 6D). Furthermore, cell apoptosis markers, cleaved forms of caspase-7 (cl-caspase-7) and PARP (cl-PARP), were strongly induced by DETD-35 and DET treatment at a later treatment time point (24 hours) in both A375 and A375-R cells (Fig. 6D). Notably, pretreatment with 5 mmol/L NAC for 1 hour significantly blockaded DETD-35–induced cascade protein expression or activation of p-c-Jun/c-Jun, cl-caspase-7, and cl-PARP in both cell lines. Interestingly, although DET was also observed to induce ROS production in both cell lines and the significant DET-induced apoptosis could be also reversed by NAC pretreatment, the activation of p-c-Jun/c-Jun was found much less significant compared with that of DETD-35. Together, our results indicate that DETD-35 or DET-induced apoptotic cell death in A375 and A375-R cells was in part through generation of ROS, yet different pathways might be involved in both compound effects.

Figure 6.

DETD-35 induces oxidative stress to trigger apoptosis in A375 and A375-R cells. A and B, A375 cells and A375-R cells, respectively, were pretreated with or without NAC for 1 hour followed by treatment with DETD-35 or DET at the indicated concentrations for 72 hours. Cell viability was determined using MTT assay. C, top, ROS generation in A375 cells and A375-R cells treated with DETD-35 and DET alone or pretreated with NAC for 1 hour followed by compound treatment for 2 hours. Flow cytometry and green fluorescent dye (ROS detection probe) were used in this experiment. Bottom, quantification of the generated ROS in the cells under the same treatment as top panel. Data are mean ± SEM, n = 4. Mean without a common letter differs, P < 0.05. D, cell lysates from A375 and A375-R cells pretreated with or without NAC for 1 hour followed by treatment with 2.5 μmol/L DETD-35 and 5 μmol/L DET at the indicated times were collected and subjected to Western blotting using the specific antibodies indicated, and actin was used as an internal or loading control.

Figure 6.

DETD-35 induces oxidative stress to trigger apoptosis in A375 and A375-R cells. A and B, A375 cells and A375-R cells, respectively, were pretreated with or without NAC for 1 hour followed by treatment with DETD-35 or DET at the indicated concentrations for 72 hours. Cell viability was determined using MTT assay. C, top, ROS generation in A375 cells and A375-R cells treated with DETD-35 and DET alone or pretreated with NAC for 1 hour followed by compound treatment for 2 hours. Flow cytometry and green fluorescent dye (ROS detection probe) were used in this experiment. Bottom, quantification of the generated ROS in the cells under the same treatment as top panel. Data are mean ± SEM, n = 4. Mean without a common letter differs, P < 0.05. D, cell lysates from A375 and A375-R cells pretreated with or without NAC for 1 hour followed by treatment with 2.5 μmol/L DETD-35 and 5 μmol/L DET at the indicated times were collected and subjected to Western blotting using the specific antibodies indicated, and actin was used as an internal or loading control.

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From the early 1980s to 2010, dacarbazine was the only FDA-approved chemotherapeutic drug for the treatment of metastatic melanoma with 15% to 20% overall response, 4 to 6 months of medium response duration, and less than 5% complete response (32). However, management of melanoma has improved dramatically in recent years. For example, the immunotherapeutic agent ipilimumab which acts against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and targeted therapeutics agents, such as vemurafenib and dabrafenib (BRAF inhibitor), show a remarkable tumor response rate and prolong patient survival leading to a new era of melanoma therapies (33). Unfortunately, melanoma tumors frequently relapse due to acquired drug resistance to both immunotherapeutics and targeted therapeutics agents; consequently, there is a growing and urgent need to develop novel or alternative therapeutic approaches for cutaneous melanoma. In this study, we created a phytocompound derivative DETD-35 and demonstrated that it possessed a better effect than the parental natural compound DET on inhibiting the proliferation of a panel of melanoma cells with different genetic backgrounds. In vitro mechanistic studies suggested that DETD-35 acted through inducing cell apoptosis while vemurafenib mainly caused G1-phase cell-cycle arrest in A375 cells. Notably, treatment with a combination of DETD-35 and vemurafenib not only induced apoptosis (increased in cleaved PARP expression and caspase-3 activity) but also caused G1-phase cell-cycle arrest (increased p27 expression and suppressed cyclin D1 expression) in A375 cells. These data suggest the synergistic or complementary effect of the compound–drug combination, resulting in better inhibitory effects on colony formation and induction of more significant apoptosis in melanoma cells compared with compound or drug treatment alone. When antitumor growth activity was evaluated in vivo in parallel with FDA-approved drug vemurafenib against BRAFV600E mutant melanoma, DETD-35 showed comparable bioefficacy. Furthermore, the compound–drug combination showed statistically similar in vivo bioefficacy as a single compound treatment in which the treatment frequency of the compound–drug combination was only half of the single compound administration frequency. These data suggested that DETD-35 possesses both therapeutic and adjuvant activity against BRAFV600E mutant melanoma.

Resistance to vemurafenib therapy is mainly due to reactivation of MEK-ERK signaling in cells. In addition, activated RTK-driven alternative mechanisms, such as the PI3K-Akt survival pathway, also contributed to resistance to vemurafenib (9). Furthermore, Girotti and colleagues recently showed that EGFR-SRC family kinase (SFK)-STAT3 signaling is upregulated in vemurafenib-resistant cells (34). Consistent with those previous studies, we showed the reactivation of phosphorylated forms of Src, MEK, ERK, Akt, and STAT-3 in our in-house established resistance cell line A375-R. DETD-35 inhibited the reactivation of the proteins in the resistance cells in the same way as observed with the positive control MEK inhibitor. Interestingly, we observed that MEKi overcame acquired vemurafenib resistance by restoring the expression of p27 and inhibiting cyclin D1 that led to G1-phase cell-cycle arrest in the cells. In contrast, DETD-35 displayed a different mechanism to the MEK inhibitor, sensitizing vemurafenib to inhibit proliferative capability and enhance apoptosis of A375-R cells in xenograft mice. Moreover, DETD-35 also sensitized vemurafenib to inhibit intrinsic resistance tumor (A2058 xenograft) growth in mice. These data indicated the novel effect of DETD-35 in circumvention of both acquired and intrinsic vemurafenib-resistant melanoma growth in mice.

Recent studies have shown multiple vemurafenib resistance mechanisms between patients, within patients, and even within the same tumor biopsy (35, 36). It is therefore anticipated that multiple pathway inhibition combined with vemurafenib will achieve durable clinical control of BRAFV600E melanoma (35, 36). In our study, we showed that DETD-35 could deregulate multiple signaling molecules, including MEK-ERK, Akt, and STAT3 in vemurafenib-resistant melanoma cells A375-R, as shown in the schematic model (Supplementary Fig. S4), which supports, at least in part, the novel pharmacologic activity of the new compound DETD-35. The complementary and synergistic activities of DETD-35 and vemurafenib against BRAFV600E mutant melanoma and acquired vemurafenib resistance melanoma growth in mice suggested a novel strategy for the management of late-stage BRAFV600E melanoma by using DETD-35 as a direct therapy or an adjuvant agent of vemurafenib.

No potential conflicts of interest were disclosed.

Conception and design: J.-H. Feng, K. Nakagawa-Goto, K.-H. Lee, L.-F. Shyur

Development of methodology: K.-H. Lee, L.-F. Shyur

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Nakagawa-Goto, L.-F. Shyur

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.-H. Feng, L.-F. Shyur

Writing, review, and/or revision of the manuscript: J.-H. Feng, K. Nakagawa-Goto, K.-H. Lee, L.-F. Shyur

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.-H. Feng, K. Nakagawa-Goto, L.-F. Shyur

Study supervision: L.-F. Shyur

The authors thank Dr. Yu-Chuan Liang and Laboratory Animal Core Facility of Agricultural Biotechnology Research Center, Academia Sinica, Taiwan, for the experimental NOD/SCID mice.

This work was supported by a grant from Ministry of Science and Technology (MOST 103-2320-B-001-008-MY3), Taiwan and institutional grant awarded to L.-F. Shyur. This study was also supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT KAKENHI, Japan) awarded to K. Nakagawa-Goto (grant numbers 25293024 and 25670054) and an NIH grant from the NCI awarded to K.-H. Lee (CA-177584).

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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