Research ArticleExperimental Studies
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Acyclic Retinoid Inhibits the EGFR/AKT Signaling Pathway and Cancels Cisplatin-resistant Cell Characteristics

MAKITO MOTOYAMA, RYOTA SHIGEFUKU, NORIYOSHI TANAKA, MITSUAKI NISHIZAWA, KEIGO OSHIO, YOSHITOMO SUHARA and ICHIRO YAJIMA
Anticancer Research February 2025, 45 (2) 433-443; DOI: https://doi.org/10.21873/anticanres.17432

Abstract

Background/Aim: Lung cancer is among the most prevalent and lethal malignancies worldwide, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Overactivation of the EGFR/AKT signaling pathway contributes significantly to NSCLC progression and metastasis. Cisplatin, a widely used chemotherapeutic agent, faces limitations due to severe side effects and the emergence of resistant cancer cells. Acyclic retinoid (ACR), a synthetic derivative of vitamin A, has shown antitumor effects in hepatocellular carcinoma, but its efficacy against NSCLC and cisplatin-resistant cells remains unclear. This study aimed to investigate whether ACR could inhibit EGFR/AKT signaling and enhance therapeutic efficacy against NSCLC and cisplatin-resistant cells. Materials and Methods: Human NSCLC A549 cells, cisplatin-resistant A549 (A549CR) cells, and normal lung epithelial BEAS-2B cells were treated with ACR, alone or in combination with cisplatin. Cell viability, apoptosis, and changes in expression/phosphorylation of EGFR, AKT, and cell cycle regulators were assessed using cell viability assay, immunostaining, and immunoblotting. Results: ACR selectively reduced viability of A549 cells with less toxicity to BEAS-2B cells and induced apoptosis via cleaved Caspase-3 activation. ACR inhibited EGFR/AKT signaling and up-regulated p27KIP1 in A549 cells. The combination of ACR and cisplatin synergistically reduced cell viability and suppressed AKT phosphorylation. Notably, ACR also inhibited EGFR/AKT signaling in A549CR cells, restoring sensitivity to cisplatin and reversing EMT-like characteristics. Conclusion: ACR effectively inhibits EGFR/AKT signaling and enhances cisplatin sensitivity in NSCLC and cisplatin-resistant cells, suggesting its potential as a promising therapeutic strategy for lung cancer.

Key Words:

Lung cancer is one of the most common and deadly cancers worldwide, with an estimated 2 million new cases and 1.76 million deaths each year. Lung cancer is classified into two subtypes: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC accounts for approximately 85% of all lung cancers, and includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma (1, 2). Various molecules are involved in the development lung cancer, including epidermal growth factor receptor (EGFR), a receptor-type tyrosine kinase that plays a particularly important role. Over-expression or hyperactivation of EGFR has been reported in several types of malignant tumors, including NSCLC (3-9), while activation of the phosphatidylinositol-3 kinase/AKT (PI3K/AKT) pathway, an EGFR-dependent signaling pathway, is involved in the growth and metastasis of NSCLC (10).

Cisplatin (CDDP) is a chemotherapeutic agent widely used to treat lung cancer, particularly non-small cell lung cancer (NSCLC), which accounts for approximately 85% of all lung cancer cases (11). Platinum-based agents, such as cisplatin, carboplatin, and oxaliplatin, are regularly prescribed for the treatment of cancer. Cisplatin induces DNA damage and activates the apoptotic pathway by activating the MEK/ERK signaling pathway while inducing cell cycle arrest through the over-expression of p27KIP1, a cell cycle inhibitor (12-14). Although platinum-based drugs, including cisplatin, are often used to treat many carcinomas, their use is limited by severe and dose-limiting side effects. There are approximately 40 different side effects associated with these drugs. For example, a known dose-limiting side effect of cisplatin is nephrotoxicity (15). Other common side effects include anaphylaxis, cytopenia, hepatotoxicity, ototoxicity, cardiotoxicity, nausea and vomiting, diarrhea, mucositis, stomatitis, pain, alopecia, anorexia, cachexia, and asthenia. Due to the risk of these side effects, patients may require a reduction of 25% to 100% in their dose of platinum (15). Patients also require extensive monitoring of their biochemistry, renal function, and liver function, as well as hearing tests. Patients are typically prescribed additional non-chemotherapy-based medications to treat these side effects. These drugs include antiemetics, antibiotics, bone marrow growth factors, mannitol, propafenone, saline hydration, magnesium supplementation, monoclonal antibody cytokine blockers, and antioxidants (15). In addition to these side effects, a significant proportion of patients often experience recurrence due to drug resistance and toxicity in multiple organs, including the liver, kidneys, gastrointestinal tract, cardiovascular system, and nervous system.

In recent years, new approaches for cancer treatment have been developed, including combination therapy with cisplatin and natural products, which has been reported to not only enhance the therapeutic activity of cisplatin but also attenuate chemotherapy-induced toxicity (16). However, the use of cisplatin as an anticancer agent increases the risk of an individual acquiring resistant cells. Resistance mechanisms in tumor cells include decreased drug accumulation, increased detoxification activity, promotion of DNA repair capacity, and inactivation of cell death signals (17). The tumor microenvironment also plays an important role in the development of cisplatin resistance (18). The PI3K/AKT pathway is a critical link between extracellular stimuli and basic cellular processes, and plays a key role in tumor cell growth, apoptosis, and survival (19-21). Accumulating evidence has further confirmed that aberrant activation of PI3K and AKT contributes to drug resistance in many types of cancer, including lung cancer (22-26). One of the properties of cisplatin-resistant cells is epithelial-mesenchymal transition (EMT), which is known to be caused by the over-expression of N-cadherin and decreased expression of E-cadherin (27, 28). As the development of cisplatin-resistant cells can have a significant negative impact on patient prognosis, efforts are underway to inhibit the acquisition of resistance and to develop novel anticancer agents that can act effectively against resistant cells; however, fundamental solutions have not yet been achieved.

Acyclic retinoids (ACRs) are synthetic compounds derived from vitamin A1 that have been found to have important antitumor effects, particularly in the prevention of hepatocellular carcinoma (HCC) recurrence (29). The antitumor effect of ACR is reported to involve a pathway targeting the retinoid X receptor (RXR) on HCC cells (30-32). RXR dysfunction in patients with HCC is caused by activation of the Ras-mitogen-activated protein kinase (MAPK) signaling pathway, which is closely associated with liver carcinogenesis (30). ACR prevents the recurrence of HCC by inhibiting Ras-MAPK activation and subsequent phosphorylation of RXRα (30). Furthermore, ACR and angiotensin II receptor blockers (ARBs) have been shown to exert a combined protective effect against diethylnitrosamine-induced hepatocarcinogenesis in diabetic rats, with the combination of ACR and ARBs enhancing tumor suppression and improving intrahepatic angiogenesis, lipid peroxidation, and inflammatory status (33). These studies suggest that ACR is effective in preventing recurrence of HCC and may be used as a chemopreventive agent in high-risk patients with HCC (30). In this study, we exposed A549, a human NSCLC cell line, to ACR and studied the antitumor effects of ACR alone and in combination with cisplatin. We also established a cisplatin-resistant lung cancer cell line (A549CR) and studied its antitumor effects in these cisplatin-resistant cells.

Materials and Methods

Reagents. Acyclic retinoid was purchased from SIGMA (St. Louis, MO, USA), and cisplatin and DAPI were purchased from FUJIFILM Wako Chemicals (Wako, Osaka, Japan). Rabbit polyclonal antibodies against cleaved Caspase-3, EGFR, phosphor-EGFR (Tyr1068), phospho-AKT, and p27KIP1 (purchased from Cell Signaling, Danvers, MA, USA), and mouse monoclonal antibodies against alpha-TUBULIN (purchased from SIGMA), AKT (purchased from Cell Signaling), and E- and N-Cadherin (purchased from BD Bioscience, Franklin Lakes, NJ, USA) were used as primary antibodies for immunoblotting and immunohistochemistry.

Cell culture. A normal human lung epithelial cell line (BEAS-2B) and a human NSCLC cell line (A549) were supplied by the Riken BioResource Research Center (Wako) and the American Type Culture Collection (ATCC) (Manassas, VA, USA), respectively, and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (FUJIFILM Wako Chemicals) at 37°C in 5% CO2. All cell lines were tested for mycoplasma contamination and authenticated by short tandem repeat (STR) analysis.

Cell viability assay. The cell viability assay was performed using the crystal violet assay method, as described previously (34). Cells were fixed with 4% paraformaldehyde (PFA) and stained with 0.1% crystal violet. The absorbance at 595 nm in the stained cells solubilized with 0.1% sodium dodecyl sulfate (SDS) was measured using a microplate reader.

Immunohistochemistry. Cultured cells were fixed with 4% PFA in PBS for 20 min at 4°C. After rinsing off the fixative solution, the cells were incubated with Blocking One Histo (Nacalai Tesque, Kyoto, Japan) at room temperature for 10 min for blocking. After blocking, the primary antibody and goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 568 conjugate (Invitrogen, Waltham, MA, USA), were used to detect cleaved Caspase-3 with DAPI used for counterstaining.

Immunoblotting. Cells were washed twice with ice-cold PBS and lysed in 100 μl of lysis buffer [20 mM HEPES (pH 7.4), 150 mM NaCl, 12.5 mM b-glycerophosphate, 1.5 mM MgCl2, 2 mM EGTA, 10 mM NaF, 2 mM DTT, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 20 mM aprotinin, and 0.5% Triton X-100]. Whole cell lysates were resolved by SDS-PAGE and transferred to a ClearTrans® PVDF Membrane (FUJIFILM Wako Chemicals). The membranes were immunoblotted with various antibodies and the bound antibodies were visualized with horseradish peroxidase-conjugated antibodies against rabbit or mouse IgG (Calbiochem, St. Louis, MO, USA) using ImmunoStar® Zeta or ImmunoStar® LD (FUJIFILM Wako Chemicals).

Establishment of cisplatin-resistant A549 cells. A549 cells were cultured in 10-cm culture dishes. When 10-20% confluency was reached, the cells were exposed to cisplatin for 48 h, and then cultured and passaged in cisplatin-depleted medium until 90% confluency was achieved. Thereafter, the cisplatin concentration to which the A549 cells were exposed was increased by 0.66 μM with each passage and until they were finally exposed to 10 μM. Established resistant cells were maintained at 3.3 μM cisplatin and exposed to 10 μM cisplatin every 3 weeks.

Statistical analysis. All statistical analyses (Student’s t-test and Tukey–Kramer test) were performed using JMP Pro (version 16.0.0; SAS Institute Inc., Cary, NC, USA).

Results

To clarify the effect of ACR on lung cancer, we exposed the normal human lung epithelial cell line (BEAS-2B) and the human NSCLC cell line (A549) to ACR and measured the changes in their viability 48 and 72 h after ACR exposure (Figure 1). The results showed that only A549 cells exhibited a decrease in cell viability after being exposed to 3 and 10 μM ACR, whereas no change was observed in BEAS-2B cells. When BEAS-2B cells were exposed to 30 μM ACR, survival decreased to about 70%, but survival tended to be 7- to 14-fold higher than when A549 cells were exposed to 30 μM ACR (approximately 5 to 10% for A549 cells). These results indicate that ACR not only has an antitumor effect on human NSCLC cells but is also more toxic to lung cancer cells than to normal lung cells. To clarify whether apoptosis is involved in the reduction in viability of lung cancer cells following exposure to ACR, we used immunostaining to measure the expression level of cleaved Caspase-3, an apoptotic marker, using immunostaining (Figure 2). In A549 cells exposed to 10 μM ACR for 48 h, approximately 25% of the cells expressed cleaved Caspase-3, suggesting that ACR exposure induced apoptosis in lung cancer cells.

Figure 1.
Figure 1.

Effect of acyclic retinoid (ACR) on viability of human lung epithelial and NSCLC cell lines. Viability of BEAS-2B and A549 cells treated with ACR for 48 h (A) or 72 h (B). Data are shown as the mean±SD (n=5). **p<0.001 vs. A549 control cells, ##p<0.001 vs. BEAS-2B control cells, $p<0.05, $$p<0.01 vs. BEAS-2B cells treated with same concentrations of ACR by Tukey–Kramer test.

Figure 2.
Figure 2.
Figure 2.

Induction of apoptosis in A549 cells upon acyclic retinoid (ACR) exposure. A) Microscopic appearances of A549 cells treated with ACR. Cleaved Caspase-3-positive (red) cells by immunostaining DAPI was used to counterstain nuclei. Overlays of cleaved caspase-3 and DAPI stainings are also shown. B) The number of cleaved caspase-3-positive (apoptotic) cells (mean±SD; n=4) is presented. Significantly different (*p<0.05) by Student’s t-test.

To elucidate the mechanism via which ACR induces apoptosis in lung cancer cells, we investigated the effect of ACR on the EGFR/PI3K/AKT pathway (Figure 3); this pathway plays an important role in the development of lung cancer (4, 10). ACR exposure was found to significantly reduce the activation of the EGFR/PI3K/AKT pathway, i.e., the phosphorylation levels of EGFR and AKT in A549 cells in a concentration-dependent manner. This result suggests that ACR treatment of lung cancer cells may trigger apoptosis by targeting the EGFR/PI3K/AKT pathway. As p27KIP1 exerts its antitumor effect by inhibiting the cell cycle through cyclins and cyclin-dependent kinases, it has been reported that decreased p27KIP1 expression in lung cancer is a factor correlated with tumor aggressiveness and poor prognosis (35, 36). Measurement of p27KIP1 expression indicated that exposure of A549 cells to ACR significantly increased the expression level of p27KIP1 protein (Figure 3). However, treatment of normal lung epithelial cells with cisplatin and ACR had no effect on the phosphorylation levels of EGFR and AKT and the expression level of P27KIP1 (Supplementary Figure 1). These results suggest that ACR may exert its antitumor effects through the tumor-specific EGFR/AKT signaling pathway.

Figure 3.
Figure 3.

Acyclic retinoid (ACR)-induced modification of tumorigenesis-related signaling activities in A549 cells. A) Phosphorylation levels of EGFR and AKT, and protein level of p27KIP1 in A549 cells untreated or treated with ACR for 48 h. B. The intensities of the bands are presented as percentages (mean±SD; n=4) relative to those of untreated A549 cells. * and ** significantly different (*p<0.05; **p<0.01) by Tukey–Kramer test.

While the chemotherapeutic agent cisplatin is a widely used for the treatment of NSCLC, it is known to have serious side effects, including nephrotoxicity, anaphylaxis, and cytopenia (15, 16, 37). Recently, combination therapy comprising cisplatin and natural products has been evaluated as a new therapeutic strategy, and it has been suggested that natural products may enhance the therapeutic activity of cisplatin and reduce chemotherapy-induced toxicity (16). To investigate the potential of ACR in combination with cisplatin to reduce the various side effects of cisplatin, we simultaneously exposed A549 cells to ACR and cisplatin and measured their effects (Figure 4A). Exposure to Cisplatin (CDDP) was found to reduce the viability of A549 cells to approximately 40%, while exposure to cisplatin in combination with ACR enhanced the antitumor effect of cisplatin in a concentration-dependent manner.

Figure 4.
Figure 4.
Figure 4.

Effect of cisplatin in combination with acyclic retinoid (ACR) on A549 cells. A) Viability of A549 cells treated with cisplatin and/or acyclic retinoid (ACR) for 48 h. B) Phosphorylation level of AKT and protein level of p27KIP1 in A549 cells untreated or treated with cisplatin and/or ACR for 48 h. C) The intensities of the bands are presented as percentages (mean±SD; n=4) relative to untreated A549 cells. * and ** significantly different (*p<0.05; **p<0.01) by Tukey–Kramer test.

In lung cancer cells, cisplatin induces apoptosis by inducing the expression of p27KIP1 protein, a cell cycle inhibitor, leading to cell cycle arrest (12, 13). The exposure of A549 cells to cisplatin resulted in the concomitant up-regulation of p27KIP1 protein expression, whereas exposure to ACR induced greater p27KIP1 protein expression. Thus, our results showed that combined exposure to cisplatin and ACR up-regulated p27KIP1 protein expression (Figure 4B and C). Exposing cancer cells to cisplatin inhibits cell proliferation, but also induces the activation of AKT, a molecule that activates cell growth and survival, resulting in the induction of resistant cells (38). Exposing A549 cells to cisplatin greatly increased the level of phosphorylation of AKT. In contrast, ACR exposure not only reduced AKT phosphorylation but also inhibited the cisplatin-induced increase in AKT phosphorylation (Figure 4B and C).

To investigate the antitumor effect of ACR on cisplatin-resistant lung cancer cells, we established a cisplatin-resistant lung cancer cell line (A549CR) by the subjecting A549 cells to long-term exposure to cisplatin (Figure 5). Although the parental cells (A549) were small and irregularly shaped, the resistant cells (A549CR) showed a more elongated shape, similar to that previously reported for cisplatin-resistant cells (38, 39). Exposure to 3.3 μM cisplatin resulted in a significant decrease in the viability of parental cells, whereas that of resistant cells was unchanged; exposure to 19.8 μM cisplatin decreased the viability of parental cells to less than 10%, whereas the viability of the resistant cells was more than 40% (Figure 5B). The IC50 of cisplatin in the parental cells was 3.04 μM, whereas that in the resistant cells was 22.2 μM, an approximately seven-fold difference (IC50 of resistant cells/IC50 of parental cells). We also investigated the phosphorylation levels of AKT and the expression levels of E-cadherin and N-cadherin, which were previously reported to be markers of cisplatin-resistant cells. The phosphorylation level of AKT showed a more than three-fold increase in resistant cells compared with parental cells, while the level of E-cadherin expression decreased to less than half, and the level of N-cadherin expression increased more than seven-fold, suggesting that the cells established in this study exhibited the basic characteristics of cisplatin-resistant cells.

Figure 5.
Figure 5.

Establishment of a cisplatin-resistant A549 cell line (A549CR). A) Parental cell line (A549) and cisplatin-resistant A549 cells (A549CR) are presented. B) Viability of A549 and A549CR cells treated with cisplatin for 48 h. C) AKT phosphorylation and E-cadherin and N-cadherin protein levels in A549 and A549CR cells. D) Intensities of bands are presented as percentages (mean±SD; n=4) relative to A549 cells. Scale bar: 20μm. * and ** significantly different (*p<0.05; **p<0.01) by Tukey–Kramer test.

Next, we examined the antitumor effects of ACR on the established cisplatin-resistant cells (Figure 6). Exposure of A549CR cells to 20 μM ACR significantly decreased their viability in a concentration-dependent manner. The antitumor effect of ACR on resistant cells was comparable to that on parental A549 cells (Figure 6A), indicating that ACR has the same level of antitumor effect on resistant cells as it does on lung cancer cells. We also examined the effects of exposure to cisplatin combined with ACR on cisplatin-resistant cells. The viability of resistant cells was significantly reduced when they were exposed to a combination of 3.3 μM cisplatin and 20 μM ACR compared with exposure to 20 μM ACR alone, suggesting that certain concentrations of ACR in combination with cisplatin may increase the antitumor effect on resistant cells (Figure 6B).

Figure 6.
Figure 6.

Antitumor effects of acyclic retinoid (ACR) on cisplatin-resistant cells. A) Viability of parental cells (A549) and cisplatin-resistant cells (A549CR) treated with ACR for 48 h. B) Viability of A549CR cells treated with only ACR or both ACR and cisplatin (CDDP). * and ** significantly different (*p<0.05; **p<0.01) by Tukey–Kramer test.

Finally, we examined the mechanism of ACR antitumor effects on resistant cells (Figure 7). In parental cells, before acquiring resistance, ACR decreased the phosphorylation levels of EGFR and AKT and increased the expression level of p27KIP1 (Figure 3), whereas in cisplatin-resistant cells, although the phosphorylation levels of EGFR and AKT were decreased, no significant difference in the expression level of p27KIP1 was observed (Figure 7A and B). These results suggest that ACR inhibits EGFR/AKT signaling in the same way as in the parental cells, but its downstream effect may be different. It is known that the acquisition of resistance by cisplatin exposure is accompanied by increased phosphorylation of AKT, decreased E-cadherin levels and increased N-cadherin levels (27, 28), which agrees with our results (see Figure 5). Exposure of resistant cells to ACR significantly decreased the phosphorylation level of AKT and significantly increased the expression level of E-cadherin and significantly decreased the expression level of N-cadherin (Figure 7). These results suggest that ACR may have the ability to abrogate the resistant phenotype of cisplatin-resistant cells.

Figure 7.
Figure 7.

Effects of ACR on cancer-related molecules in cisplatin-resistant cell lines. A) Phosphorylation levels of EGFR and AKT, and protein levels of p27KIP1, E-cadherin and N-cadherin in A549CR cells treated with cisplatin and/or ACR for 48 h. B) Intensities of bands are presented as percentages (mean±SD; n=4) relative to untreated A549CR cells.

Discussion

In this study, we showed that ACR has a strong antitumor effect on a human lung cancer cell line (A549) and induces apoptosis via elevated expression of p27KIP1 protein and inhibition of the EGFR/AKT signaling pathway. We also found that a combination of cisplatin and ACR had a stronger antitumor effect than ACR alone and that this effect was induced by the strong inhibition of AKT activation by cisplatin and ACR. We then established cisplatin-resistant lung cancer cells and demonstrated that ACR had a strong antitumor effect on cisplatin-resistant cells via the suppression of AKT signaling. Previous studies on the effects of ACR on liver cancer have reported that ACR has a stronger inhibitory effect on the viability of liver cancer cells compared with its effect on the viability of normal liver cells (40). We found that ACR had a stronger inhibitory effect on the viability of lung cancer cells (A549) than on the viability of normal lung epithelial cells (BEAS-2B), indicating that ACR could be a promising component of therapies to treat lung cancer with fewer side effects.

EGFR is a receptor-type tyrosine kinase that is not only involved in the proliferation and survival of normal epithelial cells but has also been found to be abnormally activated in many malignancies, including NSCLC (3, 4). EGFR signaling activates various downstream signals, such as the JAK/STAT, MEK/ERK, PI3K/AKT, and PKC/NF-kB pathways; activation of the PI3K/AKT pathway in particular is involved in NSCLC growth and metastasis (10). In the present study, we demonstrated that ACR suppresses AKT signaling by strongly inhibiting EGFR activity, indicating that ACR could be highly effective in the treatment of lung cancer. EGFR tyrosine kinase inhibitors (TKIs), including gefitinib, erlotinib, and afatinib, are used as first-line agents to treat patients with NSCLC with EGFR-activating mutations (41). The inhibition of EGFR activation by ACR in our study indicates that it could contribute to the treatment of NSCLC with EGFR-activating mutations and have fewer side effects.

Although cisplatin is used to treat a variety of cancers, including NSCLC, it exhibits a variety of serious side effects (15), making dose control essential and, in some cases, forcing treatment interruption. In the present study, we showed that exposure to combination of cisplatin and ACR reduced the viability of lung cancer cells more effectively than exposure to cisplatin alone. These results could contribute to the development of a new treatment strategy in which ACR, which has fewer side effects, is used in combination with lower concentrations of cisplatin to achieve the same level of therapeutic efficacy. Cisplatin induces DNA damage and cell cycle arrest through the over-expression of p27KIP1 (12, 13). In this study, exposure of A549 lung cancer cells to a combination of cisplatin and ACR additively decreased further the viability of lung cancer cells by inducing increased expression of p27KIP1 indicating that the combined exposure may more effectively induce death of lung cancer cells. We also established a new cisplatin-resistant cell line by subjecting A549 lung cancer cells to long-term exposure to cisplatin. The IC50 of cisplatin in the cisplatin resistant cell line (A549CR) was 22.2 μM, which was approximately seven-fold higher than the IC50 of cisplatin in the parental cells, 3.04 μM. In addition, the A549CR cells showed similar characteristics to those of previously reported cisplatin-resistant lung cancer cells, with increased levels of phosphorylated AKT, decreased levels of E-cadherin expression, and increased levels of N-cadherin expression compared with the parental cells. Based on these results, newly established resistant cells are expected to contribute to solving problems related to cisplatin resistance.

Finally, we tested the effect of ACR on cisplatin-resistant cells and found that the antitumor effect of ACR on cisplatin-resistant cells was comparable to that of the parental cells. We also found that exposure to a combination of cisplatin and ACR, albeit at a specific concentration of 20 μM ACR, had a greater antitumor effect on cisplatin-resistant cells than on the parental cells. Furthermore, ACR exposure not only strongly inhibited the EGFR/AKT signaling pathway in cisplatin-resistant cells, but also strongly increased expression of E-cadherin and decreased expression of N-cadherin thus significantly contributing to the suppression of the development of resistant cells, which is a major problem in lung cancer therapy.

Conclusion

Our results suggest that ACR may be useful as a novel therapeutic agent to address the problems associated with cisplatin-based treatment strategies for lung cancer. Further detailed studies at the in vitro and in vivo levels are needed to develop therapeutic strategies including ACR for lung cancer cells and cisplatin-resistant lung cancer cells.

Footnotes

  • Authors’ Contributions

    IY and YS were responsible for the conception and design of the study. RS, MM, KO and NT conducted the experiments. RS, MM, KO and IY wrote the manuscript. IY, YS edited the manuscript. All Authors read and approved the final version of the manuscript.

  • Supplementary Material

    Supplementary Figure 1 is available at: DOI: 10.6084/m9.figshare.28160048.

  • Conflicts of Interest

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

  • Funding

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

  • Received December 12, 2024.
  • Revision received January 8, 2025.
  • Accepted January 10, 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).

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Acyclic Retinoid Inhibits the EGFR/AKT Signaling Pathway and Cancels Cisplatin-resistant Cell Characteristics
MAKITO MOTOYAMA, RYOTA SHIGEFUKU, NORIYOSHI TANAKA, MITSUAKI NISHIZAWA, KEIGO OSHIO, YOSHITOMO SUHARA, ICHIRO YAJIMA
Anticancer Research Feb 2025, 45 (2) 433-443; DOI: 10.21873/anticanres.17432
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