Research ArticleExperimental Studies
Open Access

Acyclic Retinoid Overcomes Vemurafenib Resistance in Melanoma Cells via Dual Inhibition of MAPK and PI3K/AKT/mTOR Pathways

YUI SUZUKI, SOICHIRO USUKI, MITSUAKI NISHIZAWA, NORIYOSHI TANAKA, YOSHITOMO SUHARA and ICHIRO YAJIMA
Anticancer Research June 2025, 45 (6) 2265-2278; DOI: https://doi.org/10.21873/anticanres.17601

Abstract

Background/Aim: To investigate the effects of acyclic retinoid (ACR) on v-raf murine sarcoma viral oncogene homolog BV600E (BRAFV600E)-mutant melanoma cells and its potential to overcome vemurafenib resistance by targeting the mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase 1 (AKT)/mammalian target of rapamycin (mTOR) pathways.

Materials and Methods: The BRAFV600E-mutant melanoma cell lines, A375 and SK-Mel28, were treated with ACR alone or in combination with low-dose vemurafenib. Cell viability was measured and vemurafenib-resistant A375 cells (A375VR) were developed through prolonged exposure to vemurafenib. Western blotting was used to analyze the phosphorylation of extracellular-regulated kinase 1 and 2 (ERK1/2), AKT, phospho-p70 S6 kinase (p70S6K), and Eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) as well as the expression of cell cycle- and apoptosis-related proteins.

Results: ACR reduced the viability of A375 and SK-Mel28 cells by inhibiting ERK1/2 phosphorylation and increasing cleavage of caspase-3. Combined treatment with ACR and low-dose vemurafenib enhanced the effects on melanoma cells. In A375VR cells, ACR reduced cell viability by inhibiting both the MAPK and PI3K/AKT/mTOR pathways, as evidenced by the reduced phosphorylation of ERK1/2, AKT, p70S6K, and 4EBP1. ACR also reduced cyclin D1 and BCL2 levels while increasing expression of cyclin-dependent kinase inhibitory protein 1 (p27KIP1).

Conclusion: ACR exhibited potent anticancer effects on BRAFV600E-mutant and vemurafenib-resistant melanoma cells by dual-targeting of MAPK and PI3K/AKT/mTOR pathways, indicating its potential as a novel therapeutic agent for melanoma treatment.

Keywords:

Introduction

Malignant melanoma is the primary cause of skin cancer mortality, with more than 60,000 deaths reported globally by 2022 (1). The incidence of melanoma has continuously increased in recent decades (2), further emphasizing the critical need to develop more effective treatments. Despite advances in early detection and surgical intervention, the prognosis of advanced melanoma remains poor (3), necessitating the exploration of new therapeutic strategies. Malignant melanoma is characterized by a series of mutations in genes that regulate proliferation [v-raf murine sarcoma viral oncogene homolog B (BRAF), neuroblastoma RAS viral oncogene homolog (NRAS), and neurofibromatosis type 1 (NF1)], survival, cell-cycle control, and sensitivity to apoptosis [cyclin-dependent kinase inhibitor 2A (CDKN2A) and tumor protein p53 (TP53)]. All these factors contribute to cancer development, progression, and invasion (4, 5).

RAF proteins are serine/threonine-specific kinases that initiate signaling pathways downstream of the membrane-bound small G protein RAS and activate the downstream mitogen-activated protein kinase kinase (MEK)–mitogen-activated protein kinase 1 and 3 (ERK1/2) pathway. One of the kinase family members, BRAF, has been implicated in melanoma (66%) (6), colorectal cancer (10%) (7), non-small cell lung cancer (10%) (8), hairy-cell leukemia (100%) (7). BRAF mutations are observed in approximately 50-70% of melanomas (6), with the most common being a substitution of valine to glutamic acid at codon 600 (V600E). This mutation results in constant BRAF kinase activity and abnormal activation of the downstream MEK-ERK1/2 signaling pathway. This aberrant activation is required for proliferation, invasion, and survival of BRAF-mutant melanoma cells (9-11).

In addition to the RAS/RAF/MEK/ERK signaling pathway, various other pathways are involved in melanoma. The phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase 1 (AKT)/mammalian target of rapamycin (mTOR) pathway is one of the most important pathways involved in melanoma growth, survival, and invasion (12). Both the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways regulate cancer cell growth and survival by modulating cell-cycle regulatory molecules, including cyclin D1 and cyclin-dependent kinase inhibitory protein 1 (p27KIP1) proteins, and cell survival molecules, including BCL2 apoptosis regulator (BCL2) (13-17). Crosstalk between these pathways may contribute to therapeutic resistance (18), highlighting the biological complexity of melanoma.

Identification of these molecular mechanisms in melanoma has led to the development of new anticancer agents that target these molecules. Vemurafenib (PLX4032) was the first drug approved for the treatment of BRAFV600E-mutant melanoma (19) and demonstrated improved response rate, progression-free survival, and overall survival in clinical trials (20). Unfortunately, most patients experience relapse within 6 to 7 months (21).

Mechanisms of resistance to BRAF inhibitors include secondary mutations in NRAS and MEK, activation of alternative survival pathways, and upregulation of platelet-derived growth factor receptor beta (PDGFRβ) [reviewed in (22)]. Despite advancements in targeting the BRAFV600E mutation, resistance to treatments, such as vemurafenib, presents a significant challenge, underscoring the need for novel therapeutic strategies. The molecular mechanisms underlying resistance to MAPK pathway inhibition include BRAF amplification, BRAF splicing, NRAS mutations, MEK mutations, loss-of-function mutations in neurofibromatosis type 1 gene, and activation of alternative signaling pathways (e.g., PI3K–AKT–mTOR) (23, 24). Therefore, there is a need for new and innovative therapies to treat BRAF-driven cancer.

Retinoids, derivatives of vitamin A, have long been studied for their antiproliferative and differentiation-inducing effects on various cancer types [reviewed in (25)]. Initially developed as an orally administered vitamin A derivative, acyclic retinoid (ACR: all-trans-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid; also known as peretinoin and NIK-333) functions as an agonist of both the nuclear retinoic acid receptor (RAR) and retinoid X receptor (RXR) (26, 27) and regulates various intracellular signaling pathways by inhibiting the phosphorylation of ERK1/2 (28, 29). Several pharmacological studies have also shown that ACR inhibited the recurrence of hepatocellular carcinoma by suppressing the carcinogenesis of precancerous lesions in the liver and inhibiting the growth of potential tumors (30, 31). Furthermore, retinoids have been reported to modulate immune responses and enhance the efficacy of immunotherapies (32). Considering the emerging role of immunotherapy in melanoma treatment (33), combining retinoids with existing therapies may result in synergistic effects.

A series of recent studies have reported that ACR inhibited tumor growth through mechanisms such as induction of apoptosis and cell-cycle arrest in several cancer types other than hepatocellular carcinoma, including colonic, esophageal, and pancreatic cancer (34-36). Despite these findings, the antitumor efficacy of ACR in melanoma remains largely unexplored, and its potential therapeutic effects and underlying molecular mechanisms in this cancer type have yet to be elucidated.

While combination therapies in the treatment of melanoma have the potential to provide therapeutic effects that cannot be achieved with single agents of molecular targeted therapy or immunotherapy, there are inherent challenges, such as increased risk of side effects, acquisition of resistance, and treatment costs. The combination of multiple drugs increases the risk of systemic adverse events and immune-related side-effects owing to the overlap of the side-effects of each drug. Serious immune-related side-effects have been frequently reported to occur with immune checkpoint inhibitor combination therapy. Therefore, new therapeutic agents are required to address these issues (37).

In this study, we investigated the antitumor effects of ACR on human melanoma cell lines (A375 and SK-Mel28) harboring the BRAFV600E mutation, alone and in combination with vemurafenib, a typical BRAFV600E inhibitor. We also analyzed the antitumor effects of ACR on our established vemurafenib-resistant cell line (A375VR) and changes in the expression and activity of various relevant molecules. Our findings may provide a basis for the development of novel therapeutic strategies targeting BRAFV600E-mutant melanoma and overcoming resistance to current treatments.

Materials and Methods

Reagents and materials. Acyclic retinoid was purchased from Sigma (St. Louis, MO, USA), and vemurafenib was purchased from FUJIFILM Wako Chemicals (Osaka, Japan). Rabbit polyclonal antibodies against ERK1/2, cleaved caspase-3, p70 S6 kinase (p70S6K; Thr389), phospho-p70S6K, phospho- eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), epidermal growth factor receptor (EGFR), phospho-EGFR (Tyr1068), phospho-AKT, and p27KIP1, and BCL2 were purchased from Cell Signaling Technology (Danvers, MA, USA); mouse monoclonal antibodies against phospho-ERK1/2 and alpha-tubulin were from Sigma; and antibodies to AKT, 4EBP1, and cyclin D1 purchased from Cell Signaling Technology were used as primary antibodies for immunoblotting and immunohistochemistry.

Cell culture. The human melanoma cell lines SK-Mel28 and A375 were supplied by the Riken BioResource Research Center (Tsukuba, Japan) and the American Type Culture Collection (Manassas, VA, USA), respectively, and cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (FUJIFILM Wako Chemicals) at 37°C in 5% CO2. The cells were routinely subcultured every 2-3 days to maintain exponential growth. Mycoplasma contamination was tested using a MycoAler Mycoplasma Detection Kit (Lonza, Basel, Switzerland), and was found to be negative. For experimental assays, cells were seeded at appropriate densities to ensure logarithmic growth during the treatment period.

Establishment of vemurafenib-resistant A375VR cells. A375 cells were cultured in 10-cm culture dishes at 10-20% confluency, exposed to 1 μM vemurafenib for 48 h, and then cultured and passaged until 90% confluency was achieved. The cells were then reseeded, and vemurafenib-resistant cells were established by repeating the above steps for 8 weeks. Established resistant cells were maintained with 1 μM vemurafenib (Figure 1).

Figure 1.
Figure 1.

Flowchart of the establishment of vemurafenib-resistant cells.

Cell viability assay. Cell viability was assessed using the resazurin method [ready-to-use solution; Tokyo Chemical Industry (Tokyo, Japan)]. Briefly, cells were seeded in 96-well plates at a density of 5×103 cells per well and allowed to adhere overnight. Cells were treated with 0 to 60 μM acyclic retinoid with/without 0.03 to 0.1 μM vemurafenib for 48 to 72 h; cells treated with an equivalent volume of dimethyl sulfoxide (vehicle) served as the negative control. After treatment, ready-to-use resazurin solution was then added at a volume equal to 10% of the volume of cell culture medium and the cells were incubated for 2 h. Finally, the absorbance of each well was measured at 570 nm. Cell viability was expressed as a percentage of the optical density of the control wells treated with dimethyl sulfoxide (set as 100%). ACR and vemurafenib concentrations were chosen to achieve approximately 50% viability in the melanoma cell lines used.

Immunoblotting. Cells treated with/without agents were washed twice with ice-cold phosphate-buffered saline and lysed in 100 μl of lysis buffer [20 mM HEPES (pH 7.4), 150 mM NaCl, 12.5 mM β-glycerophosphate, 1.5 mM MgCl2, 2 mM ethylene glycol bis(beta-aminoethylether)-N,N,N,N-tetraacetic acid, 10 mM NaF, 2 mM dithiothreitol, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 0.5% Triton X-100]. Whole cell lysates were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a ClearTrans PVDF Membrane (FUJIFILM Wako Chemicals). The membranes were immunoblotted with the antibodies described earlier and the bound antibodies were visualized with horseradish peroxidase-conjugated antibodies against rabbit or mouse IgG (Calbiochem, Darmstadt, Germany) using ImmunoStar Zeta or ImmunoStar LD (FUJIFILM Wako Chemicals). Images were captured using Lumicube (Liponics, Tokyo, Japan). Bands were quantified using Just TLC software (Liponics). Protein expression was normalized to that of alpha-tubulin.

Statistical analysis. All experiments were performed independently at least thrice. Data are presented as mean±standard deviation. Statistical analyses were conducted using JMP Pro (version 16.0.0; SAS Institute Inc., Cary, NC, USA). Prior to the analyses, the normality of data distribution was assessed using the Shapiro–Wilk test, and the homogeneity of variance was tested using Levene’s test. Multiple group comparisons were conducted using one-way analysis of variance followed by the Tukey–Kramer post-hoc test. Statistical significance was set at p<0.05.

Results

To clarify the effect of ACR on melanoma, two human melanoma cell lines (A375 and SK-Mel28) were exposed to different concentrations of ACR (0, 3, 10, 30 and 60 μM), and their viability was measured after 48 and 72 h using the resazurin assay (Figure 2). Both cell lines exhibited a significant dose-dependent decrease in cell viability after ACR treatment. At 30 μM ACR, the cell viability was reduced to approximately 20% in A375 cells and 10% in SK-Mel28 cells after 72 h of exposure. These results indicated that ACR exerts potent cytotoxic effects on melanoma cells.

Figure 2.
Figure 2.

Exposure to acyclic retinoid (ACR) reduced viability of human melanoma cells. Viability of A375 cells (A) and SK-Mel28 cells (B) treated with ACR for 48 and 72 h are presented. Data are shown as the mean±SD (n=5). Significantly different from the control or between groups connected by horizontal bars at: *p<0.05 or **p<0.01 (Tukey–Kramer test).

To further elucidate the mechanisms underlying the antitumor effects of ACR in melanoma cells, we investigated its effect on the key signaling pathways involved in cell proliferation and apoptosis. Specifically, we measured the phosphorylation level of ERK1/2, a critical component of the MAPK pathway, which is frequently dysregulated in melanoma; the expression of p27KIP1, a cyclin-dependent kinase inhibitor involved in cell-cycle regulation, and the expression of cleaved caspase-3, a hallmark of apoptosis (Figure 3). Western blot analysis revealed that ACR treatment significantly reduced the phosphorylation of ERK1/2 in A375 cells (Figure 3B and C), indicating inhibition of the MAPK signaling pathway. Interestingly, ACR treatment significantly increased the protein expression of p27KIP1 (Figure 3D), suggesting alterations in cell-cycle regulation. Furthermore, ACR treatment significantly increased the expression of cleaved caspase-3 (Figure 3E), confirming the induction of the apoptotic pathway in melanoma cells. Collectively, these results suggest that ACR inhibits key signaling pathways in melanoma, affects cell-cycle dynamics, and induces apoptosis.

Figure 3.
Figure 3.

Acyclic retinoid (ACR) inhibits mitogen-activated protein kinase 1 and 3 (ERK1/2) phosphorylation and induces cleavage of caspase-3 in A375 melanoma cells. (A) Western blotting of phosphorylated (p) and unphosphorylated ERK1/2, p27KIP1 and cleaved/uncleaved caspase 3 proteins in A375 cells untreated or treated with ACR for 48 h. Quantification of band intensities of pERK1 (B), pERK2 (C), p27KIP1 (D) and cleaved caspase 3 (E). Data are presented as percentages (mean±SD; n=4) relative to those of untreated A375 cells. Significantly different from the control or between groups connected by horizontal bars at: *p<0.05 or **p<0.01 (Tukey–Kramer test).

Vemurafenib causes serious side-effects during melanoma treatment, including squamous cell carcinoma, cutaneous squamous cell carcinoma, liver failure, and liver dysfunction (38, 39). We hypothesized that the combination of ACR and vemurafenib might create a new treatment modality that would reduce the dose of vemurafenib required without reducing its antitumor effect. To test this hypothesis, we measured cell viability of A375 melanoma cells treated with low concentrations of vemurafenib (0.01 and 0.1 μM) both alone and in combination with ACR (Figure 4). Treatment with 0.1 μM vemurafenib alone reduced cell viability to approximately 80% of the control level. In contrast, the combination treatment with 0.1 μM vemurafenib and ACR significantly reduced cell viability to approximately 20%, which was significantly lower than that of both treatments alone. This suggests that simultaneous treatment with ACR can provide effective antitumor activity at lower vemurafenib concentrations. Therefore, combination treatment may provide sufficient antitumor efficacy while potentially reducing the incidence of side-effects caused by vemurafenib. Additionally, combination therapy may delay the development of drug resistance in melanoma cells (18).

Figure 4.
Figure 4.

Antitumor effects of acyclic retinoid (ACR) in combination with vemurafenib in A375 melanoma cells. The viability of A375 cells treated with ACR and/or vemurafenib for 48 h is shown. Data are shown as the mean±SD (n=5). Significantly different from the control or between groups connected by horizontal bars at: *p<0.05 or **p<0.01 (Tukey–Kramer test).

Another significant problem with vemurafenib therapy is the development of resistant cells after prolonged administration (21). To investigate the antitumor effects of ACR on resistant cells, we established vemurafenib-resistant melanoma cell lines (A375VR-A, -B, and -C) (Figure 5). All three clones exhibited reduced sensitivity to vemurafenib compared with the parental cells, as demonstrated by the higher cell viability under vemurafenib exposure (Figure 5A). Specifically, the resistant cell lines showed a minimal decrease in viability when exposed to 0.1 μM vemurafenib, indicating the successful establishment of resistance. We further investigated the phosphorylation levels of ERK1/2 downstream of BRAFV600E, the target of vemurafenib. In parental A375 cells, 0.1 μM vemurafenib inhibited ERK1/2 and its phosphorylation; however, in all three resistant cell lines, this effect was noticeably reduced (Figure 5B and C). This suggests that the MAPK pathway was reactivated in the resistant cells, despite the presence of vemurafenib. In studies of drug resistance in melanoma, an increase in the half-maximal inhibitory concentration (IC50) value by about 10-fold compared to the parental strain is indicative of acquired resistance (40, 41). IC50 values calculated from cell viability assays showed the resistant cell lines had approximately 9-fold (A375VR-A) and 10-fold (A375VR-B and A375VR-C) higher IC50 values compared to the parental strain. These results confirmed that the resistant cells established in this study, especially A375VR-B and A375VR-C, acquired significant drug resistance to vemurafenib. All subsequent experiments were conducted using A375VR-C as the vemurafenib-resistant cell line.

Figure 5.
Figure 5.

Establishment of vemurafenib-resistant melanoma cell lines. (A) Cell viability of parental A375 cells and three vemurafenib-resistant clones (A375VR-A, -B, and -C) treated with vemurafenib (Vem) for 48 h are presented. Data are shown as the mean±SD (n=5). Significantly different from the parental A375 control at: *p<0.05, or **p<0.01 (Tukey–Kramer test). (B) Western blotting of phosphorylated (p) and unphosphorylated mitogen-activated protein kinase 1 and 2 (ERK1/2) in A375 cells treated with vemurafenib for 48 h. (C) Quantification of band intensities of pERK1 and pERK2. The intensities of the bands are presented as percentages (mean±SD; n=5) relative to those of untreated A375 cells. ***Significantly different at p<0.001 (Tukey–Kramer test).

To determine the effect of ACR on vemurafenib-resistant melanoma cells, we exposed A375VR-C cells to ACR and measured cell viability and phosphorylation levels of MEK and ERK1/2 downstream of BRAF signaling (Figure 6). Exposure of vemurafenib-resistant cells to vemurafenib increased cell viability, whereas ACR exposure significantly reduced the viability of resistant cells (Figure 6A). To elucidate the mechanism underlying this effect, we investigated the activity of the MEK/ERK signaling pathway in resistant cells. Levels of phosphorylation of MEK proteins were not significantly altered by ACR treatment (Figure 6B). However, phosphorylation of ERK1/2 was significantly reduced by ACR (Figure 6B). These results suggest that ACR may reduce cell viability by inhibiting ERK1/2 activity downstream of MEK, potentially bypassing the resistance mechanisms affecting the upstream components of the pathway.

Figure 6.
Figure 6.

Acyclic retinoid (ACR) reduced cell viability and mitogen-activated protein kinase 1 and 3 (ERK1/2) phosphorylation in vemurafenib-resistant melanoma cells. (A) Viability of vemurafenib-resistant A375VR cells treated with ACR with/without vemurafenib for 48 h is presented. (B) Western blotting of phosphorylated mitogen-activated protein kinase kinase (MEK) and ERK1/2 in A375VR cells treated with ACR with/without vemurafenib for 48 h are shown. (C) Quantification of band intensities of MEK and ERK1/2. The intensities of the bands are presented as percentages (mean±SD; n=5) relative to those of the untreated A375VR cells. Significantly different from the untreated control or between groups connected by horizontal bars at: *p<0.05, **p<0.01, or ***p<0.001 (Tukey–Kramer test).

In this study, we found that ACR affected a part of the MEK/ERK signaling pathway. To examine its effects on other pathways implicated in melanoma progression and drug resistance, we investigated the phosphorylation of AKT, p70S6K, and 4EBP1, which are key molecules involved in the PI3K/AKT/mTOR pathway (Figure 7); this pathway contributes to melanoma cell survival and resistance to targeted therapies (12). Exposure of vemurafenib-resistant cells to vemurafenib did not affect the phosphorylation of these proteins, indicating that the PI3K/AKT/mTOR pathway remained active in resistant cells despite vemurafenib treatment. In contrast, exposure to ACR significantly reduced the phosphorylation of AKT, p70S6K, and 4EBP1 in vemurafenib-resistant cells (Figure 7B). These results suggest that ACR exerts antitumor effects by simultaneously targeting the MEK/ERK and PI3K/AKT/mTOR signaling pathways in vemurafenib-resistant melanoma cells. The dual inhibition of these pathways may enhance the suppression of cell proliferation and survival, providing a potential strategy for overcoming drug resistance (18).

Figure 7.
Figure 7.

Effects of acyclic retinoid (ACR) on phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase 1 (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, and cell-cycle-, and survival-related proteins in vemurafenib-resistant cells. (A) Western blotting of phosphorylated (p) and unphosphorylated AKT, p70 S6 kinase (p70S6K), eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), cyclin-dependent kinase inhibitory protein 1 (p27KIP1), BCL2 apoptosis regulator (BCL2) and cyclin D1, in A375VR cells treated with ACR with/without vemurafenib for 48 h. (B) Quantification of band intensities of are presented as percentages (mean±SD; n=3) relative to those of untreated A375VR cells. Significantly different from the control or between groups connected by horizontal bars at: *p<0.05 or **p<0.01 (Tukey–Kramer test).

The PI3K/AKT/mTOR signaling pathway is involved in cell proliferation and survival through the regulation of cyclin D1, p27KIP1, and BCL2 (13-17). We investigated the effect of ACR on the expression of these molecules in the vemurafenib-resistant cells (Figure 7). Vemurafenib exposure did not affect the expression of these proteins in resistant cells, which was consistent with the observed resistance. However, ACR exposure reduced the expression of cyclin D1 and BCL2 in vemurafenib-resistant cells (Figure 7B), which are associated with cell-cycle progression and anti-apoptotic activity, respectively. Additionally, ACR treatment increased the expression of p27KIP1 protein (Figure 7B), a cyclin-dependent kinase inhibitor that induces cell-cycle arrest. These results suggest that ACR inhibits cell proliferation via the suppression of PI3K/AKT/mTOR signaling and promotes apoptosis by downregulating survival proteins and upregulating cell-cycle inhibitors. Collectively, these findings indicated that ACR may overcome vemurafenib resistance by targeting multiple pathways involved in melanoma cell survival and proliferation.

Discussion

The findings of this study demonstrate that ACR, a non-cyclic derivative of vitamin A, exerts significant antitumor effects on BRAFV600E-mutant, specifically A375 and SK-Mel28 melanoma cells. This suggests that ACR holds promise for the treatment of melanoma, thereby broadening its therapeutic applicability (25). Furthermore, the results indicated that ACR exhibited a remarkable ability to circumvent melanoma cell resistance to vemurafenib, which is a significant clinical challenge in the treatment of advanced melanoma. This suggests that ACR may be useful for enhancing current therapeutic strategies when used in combination with existing treatments or as a standalone therapy, particularly in cases in which resistance to BRAF inhibitors limits treatment efficacy.

ACR demonstrated a pronounced dose-dependent reduction in cell viability in both A375 and SK-Mel28 melanoma cell lines, indicating its broad and potent antiproliferative activity against melanoma cells harboring the BRAFV600E mutation. This finding extends the previously reported antitumor effects of ACR in hepatocellular carcinoma (30, 31), highlighting its potential utility in treating cancers beyond hepatocellular carcinoma. The ability of ACR to inhibit ERK1/2 phosphorylation and modulate the expression of key regulatory proteins, such as p27KIP1 and increasing cleaved caspase-3, suggests that ACR mediates its antitumor effects by interfering with the MAPK pathway and inducing apoptosis of melanoma cells.

Moreover, the combination of ACR and vemurafenib significantly reduced melanoma cell viability compared to these agents alone, suggesting that ACR might potentially enhance the efficacy of vemurafenib while possibly reducing the required dosage and associated side-effects. Given that vemurafenib is associated with significant adverse events, including cutaneous squamous cell carcinoma and hepatotoxicity (38, 39), lowering its dosage without compromising its therapeutic efficacy may be highly beneficial for patients. This combination therapeutic approach may also help delay or prevent the onset of drug resistance in melanoma cells (18).

A major challenge in melanoma treatment is the development of resistance to BRAF inhibitors such as vemurafenib, which often leads to disease progression after an initial period of response (21). In this study, we successfully established vemurafenib-resistant melanoma cell lines (A375VR) that exhibited increased ERK1/2 phosphorylation and elevated IC50 values compared to parental cells, consistent with previous reports of acquired resistance in melanoma (40, 41). Importantly, ACR effectively reduced the viability of these resistant cells, likely through inhibition of ERK1/2 phosphorylation, indicating that ACR can bypass or overcome the mechanisms responsible for resistance to vemurafenib. Activation of the PI3K/AKT/mTOR pathway in resistant melanoma cells is a well-known mechanism that contributes to continued cell survival and proliferation despite BRAF inhibition (24). The ability of ACR to reduce the phosphorylation of AKT, p70S6K, and 4EBP1 suggests that it can target and inhibit the PI3K/AKT/mTOR pathway, thereby suppressing this alternative survival pathway that is often upregulated in resistant cells. This dual inhibition of the MAPK and PI3K/AKT/mTOR pathways by ACR may enhance its antitumor efficacy and represents a promising strategy for overcoming drug resistance in melanoma (18). Furthermore, recent studies have emphasized the importance of targeting multiple pathways and stem-like cell populations in melanoma therapy. Kanai et al. demonstrated that resveratrol, when used in combination with all-trans retinoic acid (ATRA), enhanced the differentiation of melanoma stem-like cells, suggesting a potential strategy for reducing tumor resistance and recurrence through differentiation therapy (42). Although our study utilized ACR, which differs structurally from ATRA, this finding underscores the broader therapeutic relevance of retinoids in melanoma management, particularly in modulating cellular plasticity. Additionally, it has been reported that a novel imidazothiazole-based BRAF V600E inhibitor enabled resistance to be overcome by inhibiting RAF dimerization, highlighting the importance of targeting upstream RAF signaling dynamics (43). While our study focused on downstream signaling pathways, such as ERK and AKT, these findings collectively support the need for multi-faceted therapeutic approaches. In support of our dual-pathway inhibition hypothesis, Lin et al. reported that regulator of G protein signaling 2 (RGS2) suppresses melanoma growth by simultaneously inhibiting the MAPK and PI3K/AKT pathways (44). This our results align closely with this, further validating the therapeutic potential of agents such as ACR, which can target both signaling axes to overcome drug resistance.

The potential variability among the cell lines may affect the reproducibility and generalizability of our findings. Although we used well-established cell lines in this study, inherent differences in their genetic and phenotypic profiles could have contributed to the variability in treatment responses. Future studies that incorporate a broader range of cell lines, including patient-derived models, are recommended to validate these results.

Although ACR inhibited ERK1/2 and reduced AKT phosphorylation in melanoma cells, it did not significantly affect MEK phosphorylation. This suggests that ACR may suppress ERK1/2 activity through mechanisms that bypass the classical MEK–ERK pathway, potentially involving alternative signaling cascades. One potential mechanism involves the sphingosine kinase 1 (SPHK1)–sphingosine-1-phosphate (S1P) signaling pathway, which has been implicated in ERK1/2 activation independently of MEK (45). ACR inhibition of AKT phosphorylation may attenuate SPHK1 activity, leading to the reduced production of S1P, a known regulator of ERK1/2 activation. This indirect suppression of ERK1/2 phosphorylation via the AKT–SPHK1–S1P axis presents a novel mechanism by which ACR exerts its antitumor effects, particularly in vemurafenib-resistant melanoma cells, in which the PI3K/AKT pathway is frequently hyperactivated (see Figure 8). Further studies are required to clarify the precise molecular interactions between ACR and AKT, SPHK1, or ERK1/2 in melanoma cells. The role of retinoids in modulating immune responses has been increasingly recognized because they can influence the differentiation and function of various immune cells (32). Retinoids enhance the efficacy of immunotherapies by modulating T-cell responses and reducing immunosuppression in the tumor microenvironment. Considering the success of immune checkpoint inhibitors in melanoma treatment (33), ACR may synergize with immunotherapy to further improve patient outcomes. The potential combination of ACR and immunotherapeutic agents warrants further investigation in preclinical and clinical settings.

Figure 8.
Figure 8.

Hypothetical mechanism for suppression of melanoma-related signaling pathways by acyclic retinoid (ACR). The white arrows indicate the effects of ACR on each molecule analyzed in this study: the sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) axis is activated by AKT serine/threonine kinase 1 (AKT), which in turn activates mitogen-activated protein kinases 1 and 3 (ERK), suggesting that the SPHK1/S1P axis is involved in the antitumor effects of ACR. 4EBP1: Eukaryotic translation initiation factor 4E-binding protein 1; BRAFV600E: v-raf murine sarcoma viral oncogene homolog BV600E; MEK: mitogen-activated protein kinase kinase; mTOR: mammalian target of rapamycin; NRAS: neuroblastoma RAS viral oncogene homolog; p70S6K: p70 S6 kinase; PI3K: phosphoinositide 3-kinase; S1PRs: sphingosine 1-phosphate receptors.

Finally, ACR-mediated modulation of cell-cycle and survival-related proteins, such as the reduction of cyclin D1 and BCL2 expression, and upregulation of p27KIP1, supports its role in inhibiting cell proliferation and promoting apoptosis of melanoma cells. The PI3K/AKT/mTOR pathway is known to regulate these proteins, and their altered expression following ACR treatment further highlights the impact of ACR on mechanisms of melanoma cell survival (15, 16). Additionally, the downregulation of BCL2, an anti-apoptotic protein, may sensitize melanoma cells to apoptotic stimuli, potentially enhancing the effectiveness of pro-apoptotic agents in combination with ACR (46). In hepatocellular carcinoma, where clinical trials have already been conducted, ACR was well tolerated and none of the side-effects identified in patients treated with ACR, including urinary albumin, hypertension, and headache, were serious (47). Therefore, similar side-effects are likely to occur when ACR is used to treat melanoma but are not expected to be significant.

Conclusion

This study demonstrated that ACR exerts potent anticancer effects in vitro, suggesting its potential as a novel therapeutic agent for melanoma treatment. Our results indicate that ACR can inhibit cell proliferation and may induce apoptosis through dual targeting of the MAPK and PI3K/AKT/mTOR pathways, with its ability to modulate key regulatory proteins and potentially enhance immune responses, thereby supporting its feasibility for further exploration in oncology. However, the conclusions drawn from our study are limited by the absence of in vivo data. Validation using appropriate animal models is essential to better understand the pharmacokinetics, biodistribution, and toxicity profile of ACR under physiologically relevant conditions. Such studies would confirm the therapeutic potential observed in vitro and help to establish the safety margin necessary for clinical application. Despite these limitations, our findings represent a significant first step toward establishing ACR as a promising anticancer compound. Future in vivo and clinical studies are needed to clarify the full range of therapeutic efficacy and validate its utility in established or emerging cancer treatments.

Footnotes

  • Authors’ Contributions

    IY conceived, designed, and supervised the study. YS designed and performed all experiments with the assistance of MN, NT, SU, and YS. MN and SU performed the cell culture and western blot analyses, respectively. NT and YS supported the ACR-related experiments. IY and YS wrote the manuscript. I-Y is responsible for the overall content.

  • Conflicts of Interest

    The Authors declare that they have no conflicts of interest related to this study.

  • Funding

    This work was supported in part by the Grants-in-Aid for Scientific Research (C) (grant number 22K12393).

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine-learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received April 3, 2025.
  • Revision received April 19, 2025.
  • Accepted April 24, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

References

  1. ↵
    1. International Agency for Research on Cancer
    : Skin cancer: World Health Organization; 2022. Available at: https://www.iarc.who.int/cancer-type/skin-cancer [Last accessed on April 20, 2025]
  2. ↵
    1. Urban K,
    2. Mehrmal S,
    3. Uppal P,
    4. Giesey RL,
    5. Delost GR
    : The global burden of skin cancer: A longitudinal analysis from the Global Burden of Disease Study, 1990-2017. JAAD Int 2: 98-108, 2021. DOI: 10.1016/j.jdin.2020.10.013
    OpenUrlCrossRefPubMed
  3. ↵
    1. Lee KA,
    2. Nathan P
    : Cutaneous melanoma – a review of systemic therapies. Acta Derm Venereol 100(11): adv00141, 2020. DOI: 10.2340/00015555-3496
    OpenUrlCrossRefPubMed
  4. ↵
    1. Cancer Genome Atlas Network
    : Genomic classification of cutaneous melanoma. Cell 161(7): 1681-1696, 2015. DOI: 10.1016/j.cell.2015.05.044
    OpenUrlCrossRefPubMed
  5. ↵
    1. Shain AH,
    2. Bastian BC
    : From melanocytes to melanomas. Nat Rev Cancer 16(6): 345-358, 2016. DOI: 10.1038/nrc.2016.37
    OpenUrlCrossRefPubMed
  6. ↵
    1. Davies H,
    2. Bignell GR,
    3. Cox C,
    4. Stephens P,
    5. Edkins S,
    6. Clegg S,
    7. Teague J,
    8. Woffendin H,
    9. Garnett MJ,
    10. Bottomley W,
    11. Davis N,
    12. Dicks E,
    13. Ewing R,
    14. Floyd Y,
    15. Gray K,
    16. Hall S,
    17. Hawes R,
    18. Hughes J,
    19. Kosmidou V,
    20. Menzies A,
    21. Mould C,
    22. Parker A,
    23. Stevens C,
    24. Watt S,
    25. Hooper S,
    26. Wilson R,
    27. Jayatilake H,
    28. Gusterson BA,
    29. Cooper C,
    30. Shipley J,
    31. Hargrave D,
    32. Pritchard-Jones K,
    33. Maitland N,
    34. Chenevix-Trench G,
    35. Riggins GJ,
    36. Bigner DD,
    37. Palmieri G,
    38. Cossu A,
    39. Flanagan A,
    40. Nicholson A,
    41. Ho JW,
    42. Leung SY,
    43. Yuen ST,
    44. Weber BL,
    45. Seigler HF,
    46. Darrow TL,
    47. Paterson H,
    48. Marais R,
    49. Marshall CJ,
    50. Wooster R,
    51. Stratton MR,
    52. Futreal PA
    : Mutations of the BRAF gene in human cancer. Nature 417(6892): 949-954, 2002. DOI: 10.1038/nature00766
    OpenUrlCrossRefPubMed
  7. ↵
    1. Barras D
    : BRAF mutation in colorectal cancer: an update. Biomark Cancer 7(Suppl 1): 9-12, 2015. DOI: 10.4137/BIC.S25248
    OpenUrlCrossRefPubMed
  8. ↵
    1. Nguyen-Ngoc T,
    2. Bouchaab H,
    3. Adjei AA,
    4. Peters S
    : BRAF alterations as therapeutic targets in non-small-cell lung cancer. J Thorac Oncol 10(10): 1396-1403, 2015. DOI: 10.1097/JTO.0000000000000644
    OpenUrlCrossRefPubMed
  9. ↵
    1. Hingorani SR,
    2. Jacobetz MA,
    3. Robertson GP,
    4. Herlyn M,
    5. Tuveson DA
    : Suppression of BRAF(v599e) in human melanoma abrogates transformation. Cancer Res 63(17): 5198-5202, 2003.
    OpenUrlAbstract/FREE Full Text
    1. Karasarides M,
    2. Chiloeches A,
    3. Hayward R,
    4. Niculescu-Duvaz D,
    5. Scanlon I,
    6. Friedlos F,
    7. Ogilvie L,
    8. Hedley D,
    9. Martin J,
    10. Marshall CJ,
    11. Springer CJ,
    12. Marais R
    : B-RAF is a therapeutic target in melanoma. Oncogene 23(37): 6292-6298, 2004. DOI: 10.1038/sj.onc.1207785
    OpenUrlCrossRefPubMed
  11. ↵
    1. Bhatt KV,
    2. Spofford LS,
    3. Aram G,
    4. McMullen M,
    5. Pumiglia K,
    6. Aplin AE
    : Adhesion control of cyclin D1 and p27Kip1 levels is deregulated in melanoma cells through BRAF-MEK-ERK signaling. Oncogene 24(21): 3459-3471, 2005. DOI: 10.1038/sj.onc.1208544
    OpenUrlCrossRefPubMed
  12. ↵
    1. Chamcheu JC,
    2. Roy T,
    3. Uddin MB,
    4. Banang-Mbeumi S,
    5. Chamcheu RN,
    6. Walker AL,
    7. Liu YY,
    8. Huang S
    : Role and therapeutic targeting of the PI3K/Akt/mTOR signaling pathway in skin cancer: a review of current status and future trends on natural and synthetic agents therapy. Cells 8(8): 803, 2019. DOI: 10.3390/cells8080803
    OpenUrlCrossRef
  13. ↵
    1. Schloesser RJ,
    2. Huang J,
    3. Klein PS,
    4. Manji HK
    : Cellular plasticity cascades in the pathophysiology and treatment of bipolar disorder. Neuropsychopharmacology 33(1): 110-133, 2008. DOI: 10.1038/sj.npp.1301575
    OpenUrlCrossRefPubMed
    1. Markman B,
    2. Dienstmann R,
    3. Tabernero J
    : Targeting the PI3K/Akt/mTOR pathway – beyond rapalogs. Oncotarget 1(7): 530-543, 2010. DOI: 10.18632/oncotarget.188
    OpenUrlCrossRefPubMed
  15. ↵
    1. Vu T,
    2. Claret FX
    : Trastuzumab: updated mechanisms of action and resistance in breast cancer. Front Oncol 2: 62, 2012. DOI: 10.3389/fonc.2012.00062
    OpenUrlCrossRefPubMed
  16. ↵
    1. González-Ruiz L,
    2. González-Moles MÁ,
    3. González-Ruiz I,
    4. Ruiz-Ávila I,
    5. Ayén Á,
    6. Ramos-García P
    : An update on the implications of cyclin D1 in melanomas. Pigment Cell Melanoma Res 33(6): 788-805, 2020. DOI: 10.1111/pcmr.12874
    OpenUrlCrossRefPubMed
  17. ↵
    1. He Y,
    2. Sun MM,
    3. Zhang GG,
    4. Yang J,
    5. Chen KS,
    6. Xu WW,
    7. Li B
    : Targeting PI3K/Akt signal transduction for cancer therapy. Signal Transduct Target Ther 6(1): 425, 2021. DOI: 10.1038/s41392-021-00828-5
    OpenUrlCrossRef
  18. ↵
    1. Griffin M,
    2. Scotto D,
    3. Josephs DH,
    4. Mele S,
    5. Crescioli S,
    6. Bax HJ,
    7. Pellizzari G,
    8. Wynne MD,
    9. Nakamura M,
    10. Hoffmann RM,
    11. Ilieva KM,
    12. Cheung A,
    13. Spicer JF,
    14. Papa S,
    15. Lacy KE,
    16. Karagiannis SN
    : BRAF inhibitors: resistance and the promise of combination treatments for melanoma. Oncotarget 8(44): 78174-78192, 2017. DOI: 10.18632/oncotarget.19836
    OpenUrlCrossRefPubMed
  19. ↵
    1. Bollag G,
    2. Tsai J,
    3. Zhang J,
    4. Zhang C,
    5. Ibrahim P,
    6. Nolop K,
    7. Hirth P
    : Vemurafenib: the first drug approved for BRAF-mutant cancer. Nat Rev Drug Discov 11(11): 873-886, 2012. DOI: 10.1038/nrd3847
    OpenUrlCrossRefPubMed
  20. ↵
    1. Flaherty KT,
    2. Puzanov I,
    3. Kim KB,
    4. Ribas A,
    5. McArthur GA,
    6. Sosman JA,
    7. O’Dwyer PJ,
    8. Lee RJ,
    9. Grippo JF,
    10. Nolop K,
    11. Chapman PB
    : Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 363(9): 809-819, 2010. DOI: 10.1056/NEJMoa1002011
    OpenUrlCrossRefPubMed
  21. ↵
    1. Shi H,
    2. Hugo W,
    3. Kong X,
    4. Hong A,
    5. Koya RC,
    6. Moriceau G,
    7. Chodon T,
    8. Guo R,
    9. Johnson DB,
    10. Dahlman KB,
    11. Kelley MC,
    12. Kefford RF,
    13. Chmielowski B,
    14. Glaspy JA,
    15. Sosman JA,
    16. van Baren N,
    17. Long GV,
    18. Ribas A,
    19. Lo RS
    : Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy. Cancer Discov 4(1): 80-93, 2014. DOI: 10.1158/2159-8290.CD-13-0642
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Proietti I,
    2. Skroza N,
    3. Bernardini N,
    4. Tolino E,
    5. Balduzzi V,
    6. Marchesiello A,
    7. Michelini S,
    8. Volpe S,
    9. Mambrin A,
    10. Mangino G,
    11. Romeo G,
    12. Maddalena P,
    13. Rees C,
    14. Potenza C
    : Mechanisms of acquired BRAF inhibitor resistance in melanoma: a systematic review. Cancers (Basel) 12(10): 2801, 2020. DOI: 10.3390/cancers12102801
    OpenUrlCrossRef
  23. ↵
    1. Welsh SJ,
    2. Rizos H,
    3. Scolyer RA,
    4. Long GV
    : Resistance to combination BRAF and MEK inhibition in metastatic melanoma: Where to next? Eur J Cancer 62: 76-85, 2016. DOI: 10.1016/j.ejca.2016.04.005
    OpenUrlCrossRefPubMed
  24. ↵
    1. Manzano JL,
    2. Layos L,
    3. Bugés C,
    4. de Los Llanos Gil M,
    5. Vila L,
    6. Martínez-Balibrea E,
    7. Martínez-Cardús A
    : Resistant mechanisms to BRAF inhibitors in melanoma. Ann Transl Med 4(12): 237, 2016. DOI: 10.21037/atm.2016.06.07
    OpenUrlCrossRefPubMed
  25. ↵
    1. Jin Y,
    2. Teh SS,
    3. Lau HLN,
    4. Xiao J,
    5. Mah SH
    : Retinoids as anti-cancer agents and their mechanisms of action. Am J Cancer Res 12(3): 938-960, 2022.
    OpenUrlPubMed
  26. ↵
    1. Yamada Y,
    2. Shidoji Y,
    3. Fukutomi Y,
    4. Ishikawa T,
    5. Kaneko T,
    6. Nakagama H,
    7. Imawari M,
    8. Moriwaki H,
    9. Muto Y
    : Positive and negative regulations of albumin gene expression by retinoids in human hepatoma cell lines. Mol Carcinog 10(3): 151-158, 1994. DOI: 10.1002/mc.2940100306
    OpenUrlCrossRefPubMed
  27. ↵
    1. Araki H,
    2. Shidoji Y,
    3. Yamada Y,
    4. Moriwaki H,
    5. Muto Y
    : Retinoid agonist activities of synthetic geranyl geranoic acid derivatives. Biochem Biophys Res Commun 209(1): 66-72, 1995. DOI: 10.1006/bbrc.1995.1471
    OpenUrlCrossRefPubMed
  28. ↵
    1. Matsushima-Nishiwaki R,
    2. Okuno M,
    3. Takano Y,
    4. Kojima S,
    5. Friedman SL,
    6. Moriwaki H
    : Molecular mechanism for growth suppression of human hepatocellular carcinoma cells by acyclic retinoid. Carcinogenesis 24(8): 1353-1359, 2003. DOI: 10.1093/carcin/bgg090
    OpenUrlCrossRefPubMed
  29. ↵
    1. Komi Y,
    2. Sogabe Y,
    3. Ishibashi N,
    4. Sato Y,
    5. Moriwaki H,
    6. Shimokado K,
    7. Kojima S
    : Acyclic retinoid inhibits angiogenesis by suppressing the MAPK pathway. Lab Invest 90(1): 52-60, 2010. DOI: 10.1038/labinvest.2009.110
    OpenUrlCrossRefPubMed
  30. ↵
    1. Okada H,
    2. Honda M,
    3. Campbell JS,
    4. Sakai Y,
    5. Yamashita T,
    6. Takebuchi Y,
    7. Hada K,
    8. Shirasaki T,
    9. Takabatake R,
    10. Nakamura M,
    11. Sunagozaka H,
    12. Tanaka T,
    13. Fausto N,
    14. Kaneko S
    : Acyclic retinoid targets platelet-derived growth factor signaling in the prevention of hepatic fibrosis and hepatocellular carcinoma development. Cancer Res 72(17): 4459-4471, 2012. DOI: 10.1158/0008-5472.CAN-12-0028
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Honda M,
    2. Yamashita T,
    3. Yamashita T,
    4. Arai K,
    5. Sakai Y,
    6. Sakai A,
    7. Nakamura M,
    8. Mizukoshi E,
    9. Kaneko S
    : Peretinoin, an acyclic retinoid, improves the hepatic gene signature of chronic hepatitis C following curative therapy of hepatocellular carcinoma. BMC Cancer 13: 191, 2013. DOI: 10.1186/1471-2407-13-191
    OpenUrlCrossRefPubMed
  32. ↵
    1. Pino-Lagos K,
    2. Benson MJ,
    3. Noelle RJ
    : Retinoic acid in the immune system. Ann N Y Acad Sci 1143: 170-187, 2008. DOI: 10.1196/annals.1443.017
    OpenUrlCrossRefPubMed
  33. ↵
    1. Dany M,
    2. Nganga R,
    3. Chidiac A,
    4. Hanna E,
    5. Matar S,
    6. Elston D
    : Advances in immunotherapy for melanoma management. Hum Vaccin Immunother 12(10): 2501-2511, 2016. DOI: 10.1080/21645515.2016.1190889
    OpenUrlCrossRefPubMed
  34. ↵
    1. Suzui M,
    2. Sunagawa N,
    3. Chiba I,
    4. Moriwaki H,
    5. Yoshimi N
    : Acyclic retinoid, a novel synthetic retinoid, induces growth inhibition, apoptosis, and changes in mRNA expression of cell cycle- and differentiation-related molecules in human colon carcinoma cells. Int J Oncol 28(5): 1193-1199, 2006. DOI: 10.3892/ijo.28.5.1193
    OpenUrlCrossRefPubMed
    1. Shimizu M,
    2. Suzui M,
    3. Deguchi A,
    4. Lim JTE,
    5. Weinstein IB
    : Effects of acyclic retinoid on growth, cell cycle control, epidermal growth factor receptor signaling, and gene expression in human squamous cell carcinoma cells. Clin Cancer Res 10(3): 1130-1140, 2004. DOI: 10.1158/1078-0432.ccr-0714-3
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Nakagawa T,
    2. Shimizu M,
    3. Shirakami Y,
    4. Tatebe H,
    5. Yasuda I,
    6. Tsurumi H,
    7. Moriwaki H
    : Synergistic effects of acyclic retinoid and gemcitabine on growth inhibition in pancreatic cancer cells. Cancer Lett 273(2): 250-256, 2009. DOI: 10.1016/j.canlet.2008.08.004
    OpenUrlCrossRefPubMed
  37. ↵
    1. Fujimura T,
    2. Yoshino K,
    3. Kato H,
    4. Fukushima S,
    5. Ishizuki S,
    6. Otsuka A,
    7. Matsushita S,
    8. Amagai R,
    9. Muto Y,
    10. Yamazaki E,
    11. Kambayashi Y,
    12. Yahata T,
    13. Miyata T,
    14. Fujisawa Y,
    15. Asano Y
    : A phase II multicentre study of plasminogen activator inhibitor-1 inhibitor (TM5614) plus nivolumab for treating anti-programmed cell death 1 antibody-refractory malignant melanoma: TM5614-MM trial. Br J Dermatol 191(5): 691-697, 2024. DOI: 10.1093/bjd/ljae231
    OpenUrlCrossRefPubMed
  38. ↵
    1. Hagen B,
    2. Trinh VA
    : Managing side effects of vemurafenib therapy for advanced melanoma. J Adv Pract Oncol 5(6): 400-410, 2014.
    OpenUrlPubMed
  39. ↵
    1. Kim A,
    2. Cohen MS
    : The discovery of vemurafenib for the treatment of BRAF-mutated metastatic melanoma. Expert Opin Drug Discov 11(9): 907-916, 2016. DOI: 10.1080/17460441.2016.1201057
    OpenUrlCrossRefPubMed
  40. ↵
    1. Cordaro FG,
    2. De Presbiteris AL,
    3. Camerlingo R,
    4. Mozzillo N,
    5. Pirozzi G,
    6. Cavalcanti E,
    7. Manca A,
    8. Palmieri G,
    9. Cossu A,
    10. Ciliberto G,
    11. Ascierto PA,
    12. Travali S,
    13. Patriarca EJ,
    14. Caputo E
    : Phenotype characterization of human melanoma cells resistant to dabrafenib. Oncol Rep 38(5): 2741-2751, 2017. DOI: 10.3892/or.2017.5963
    OpenUrlCrossRefPubMed
  41. ↵
    1. Strub T,
    2. Ghiraldini FG,
    3. Carcamo S,
    4. Li M,
    5. Wroblewska A,
    6. Singh R,
    7. Goldberg MS,
    8. Hasson D,
    9. Wang Z,
    10. Gallagher SJ,
    11. Hersey P,
    12. Ma’ayan A,
    13. Long GV,
    14. Scolyer RA,
    15. Brown B,
    16. Zheng B,
    17. Bernstein E
    : SIRT6 haploinsufficiency induces BRAF(V600E) melanoma cell resistance to MAPK inhibitors via IGF signalling. Nat Commun 9(1): 3440, 2018. DOI: 10.1038/s41467-018-05966-z
    OpenUrlCrossRefPubMed
  42. ↵
    1. Kanai M,
    2. Shinagawa A,
    3. Ota M,
    4. Virgona N,
    5. Yano T
    : Resveratrol can differentiate human melanoma stem-like cells from spheroids treated with all-trans retinoic acid. Anticancer Res 44(12): 5283-5292, 2024. DOI: 10.21873/anticanres.17356
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Poudel M,
    2. Kim G,
    3. Bhattarai PY,
    4. Shin S,
    5. Zaraei S,
    6. Oh CH,
    7. Choi HS
    : Potent imidazothiazole-based inhibitor of BRAF V600E overcomes acquired resistance via inhibition of RAF dimerization in PLX4032-resistant melanoma. Anticancer Res 42(6): 2911-2921, 2022. DOI: 10.21873/anticanres.15773
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Lin SJ,
    2. Huang YC,
    3. Chen HY,
    4. Fang JY,
    5. Hsu SY,
    6. Shih HY,
    7. Liu YC,
    8. Cheng YC
    : RGS2 suppresses melanoma growth via inhibiting MAPK and AKT signaling pathways. Anticancer Res 41(12): 6135-6145, 2021. DOI: 10.21873/anticanres.15433
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. El Hed A,
    2. Khaitan A,
    3. Kozhaya L,
    4. Manel N,
    5. Daskalakis D,
    6. Borkowsky W,
    7. Valentine F,
    8. Littman DR,
    9. Unutmaz D
    : Susceptibility of human Th17 cells to human immunodeficiency virus and their perturbation during infection. J Infect Dis 201(6): 843-854, 2010. DOI: 10.1086/651021
    OpenUrlCrossRefPubMed
  46. ↵
    1. Kale J,
    2. Osterlund EJ,
    3. Andrews DW
    : BCL-2 family proteins: changing partners in the dance towards death. Cell Death Differ 25(1): 65-80, 2018. DOI: 10.1038/cdd.2017.186
    OpenUrlCrossRefPubMed
  47. ↵
    1. Muto Y,
    2. Moriwaki H,
    3. Ninomiya M,
    4. Adachi S,
    5. Saito A,
    6. Takasaki KT,
    7. Tanaka T,
    8. Tsurumi K,
    9. Okuno M,
    10. Tomita E,
    11. Nakamura T,
    12. Kojima T
    : Prevention of second primary tumors by an acyclic retinoid, polyprenoic acid, in patients with hepatocellular carcinoma. N Engl J Med 334(24): 1561-1568, 1996. DOI: 10.1056/NEJM199606133342402
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
Acyclic Retinoid Overcomes Vemurafenib Resistance in Melanoma Cells via Dual Inhibition of MAPK and PI3K/AKT/mTOR Pathways
YUI SUZUKI, SOICHIRO USUKI, MITSUAKI NISHIZAWA, NORIYOSHI TANAKA, YOSHITOMO SUHARA, ICHIRO YAJIMA
Anticancer Research Jun 2025, 45 (6) 2265-2278; DOI: 10.21873/anticanres.17601
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords