Effect of anticancer activity of aripiprazole main metabolite OPC-14857 on malignant glioblastoma
Shuhei Nakao, Yuki Uemichi, Shuji Nagano, Miyuki Mabuchi, Shiho Ohmori, Daichi Enomoto, Takehiko Ueyama, Tadashi Shimizu, Mária Deli, Mária Deli, Mária Deli, Mária Deli, Mária Deli
TL;DR
This study explores how OPC-14857, a metabolite of aripiprazole, may have anticancer effects against glioblastoma, a deadly brain tumor, potentially offering new treatment options.
Contribution
The study reveals that OPC-14857, a metabolite of aripiprazole, shows promising anticancer activity against glioblastoma, possibly as a repurposed drug.
Findings
OPC-14857 showed stronger anticancer effects than temozolomide in glioblastoma cell lines.
OPC-14857 induced G2/M phase cell cycle arrest and suppressed cell migration.
Both OPC-14857 and aripiprazole increased doxorubicin efficacy via P-glycoprotein inhibition.
Abstract
Glioblastoma is an incurable and highly malignant brain tumor that poses challenges in surgical and chemotherapeutic treatments. Aripiprazole (ARP), an antipsychotic drug, exerts cytotoxic effects against various cancers. In the present study, we compared the inhibitory effect of ARP on cell proliferation with that of its main metabolite, OPC-14857 (OPC), using glioblastoma cell lines (U251, T98G, and U87 cells) to explore their potential for repurposing against brain tumors. Both demonstrated more potent anticancer activity than temozolomide, the current standard clinical therapy for malignant glioblastoma. Additionally, we assessed their effects on the cell cycle, cytoskeleton, cell migration, and protein expression. The anti-proliferative and anti-migratory activities of OPC were similar to those of ARP. Moreover, there were no differences in the effects of cell death inhibitors on…
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Fig 8- —http://dx.doi.org/10.13039/100019568Biosignal Research Center, Kobe University
- —http://dx.doi.org/10.13039/501100001691Japan Society for the Promotion of Science
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Taxonomy
TopicsCancer, Stress, Anesthesia, and Immune Response · Cell death mechanisms and regulation · Drug Transport and Resistance Mechanisms
1. Introduction
Glioblastoma is a highly malignant brain tumor and one of the deadliest of all carcinomas. The 5-year survival rate for affected patients is 6.9%, and even in the group with the highest survival rate (15–39 years old), the survival rate is only 26.6% [1]. At the time of diagnosis, it has typically diffusely infiltrated the brain, rendering complete surgical resection rarely curative. Moreover, therapeutic resistance arises from multiple, interacting factors, including cancer stem cells (CSCs), an immunosuppressive and heterogeneous tumor microenvironment intratumoral heterogeneity, and adaptive/compensatory signaling—rather than from a single attribute. Accordingly, effective treatment may require a multi-target, multi-step strategy instead of a single-target approach [2,3]. Additionally, most chemotherapeutic agents poorly penetrate the blood-brain barrier (BBB), limiting their application [4].
Although temozolomide (TMZ) partially crosses the BBB, its concentration in cerebrospinal fluid is approximately 20% of that in plasma [5,6]. In 2005, a phase III trial comparing radiotherapy alone to radiotherapy combined with TMZ showed that the combination treatment prolonged survival. Although the median survival with the combination was only 2.5 months longer, increasing from 12.1 months to 14.6 months [7], resistance to TMZ often reduces the efficacy of chemotherapy for glioblastoma. One of the major mechanisms of this resistance is the expression of O-6-methylguanine-DNA methyltransferase (MGMT) [5,8]. Therefore, the discovery of new anticancer drugs with different pharmacological mechanisms and greater activity than TMZ is desired.
Drug repurposing is the process of discovering new pharmacological effects of currently market-approved drugs or developing discontinued compounds and applying these new effects to treat a disease different from its original therapeutic purpose [9]. Recently, a drug repurposing strategy against glioblastoma has been reported for BBB-penetrable anti-epileptic drugs, antidepressants, and antipsychotic drugs [10–14]. Several studies have investigated the treatment of glioblastoma using combinations of existing drugs (e.g., the AVRO regimen, [15]). Aripiprazole (ARP, Fig 1), a D2 receptor modulator, is an atypical antipsychotic drug widely used to treat schizophrenia and bipolar disorder. Anticancer activity of aripiprazole has been reported across multiple tumor types, for example glioblastoma, breast cancer, gastric adenosquamous carcinoma, and colon carcinoma cells [16–19].
Structures of Aripiprazole and OPC-14857.
Most drugs are metabolized in vivo, and the effects of their metabolites are often different from those of the parent compounds. In certain cases, metabolites may retain, diminish, or even acquire distinct pharmacological activities. For successful drug repurposing, it is necessary to focus not only on the drug but also on the activity of its metabolites. We previously reported that the major metabolite (HUHS190) of naftopidil showed stronger anticancer activity than naftopidil in bladder cancer treatment in vitro and was effective in vivo [20].
In the present study, we focused on exploring the use of OPC-14857 (OPC, Fig 1), a major ARP metabolite formed by CYP2D6 and CYP3A4 [21,22], as a drug-repurposing strategy for employing pharmaceutical metabolites against glioblastoma. We aimed to compare the anticancer effects of ARP and OPC on malignant glioblastoma cells. We also examined the relationship between the anticancer effects and factors such as cell death, cell cycle progression, and cytoskeleton formation.
2. Materials and methods
2.1. Cell culture
Human glioblastoma cell lines (U251, T98G, and U87) were obtained from Dr. T. Sasayama (Kobe University, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium (Fujifilm Wako, 043−30085, Osaka-shi, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Biosera, FB-1360/500, Cholet, France). The cells were grown in an incubator at 37°C under 5% CO_2_ and subcultured at 80% confluence using trypsin-ethylenediaminetetraacetic acid (Nacalai tesque, 35553−74, Kyoto-shi, Kyoto, Japan). HEK293 cells were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB9068, Ibaraki-shi, Osaka, Japan). The cells were cultured in Eagle’s minimal essential medium (Fujifilm Wako, 051−07615, Osaka-shi, Osaka, Japan) supplemented with 10% horse serum (HS; Gibco, Thermo Fisher Scientific, 16050−122, Waltham, MA, USA), 1% GlutaMAX^TM^ (Gibco, Thermo Fisher Scientific, 35050−061, Waltham, MA, USA) and 1% penicillin/streptomycin (Nacalai tesque, 09367−34, Kyoto-shi, Kyoto, Japan) using the same conditions as those for U251 cells.
2.2. Chemicals
ARP (Tokyo Chemical Industry (TCI), A2496, Chuo-ku, Tokyo, Japan), Necrostatin-1 (Cayman Chemical, 11658, Ann Arbor, Michigan, USA), Z-VAD-fmk (Peptide institute, 3188-v, Ibaraki-shi, Osaka, Japan), and Ferrostatin-1 (TCI, F1302, Chuo-ku, Tokyo, Japan) were purchased from commercial sources. IM-54 was synthesized according to the reported literature [23]. OPC-14857 (OPC) was synthesized from ARP with 2,3-dichloro-5,6-dicyano-p-benzoquinone (TCI, D1070, Chuo-ku, Tokyo, Japan). To a solution of ARP (112 mg, 0.25 mmol) in dichloromethane (1.0 mL), 2,3-dichloro-5,6-dicyano-p-benzoquinone was added (170 mg, 0.75 mmol) at room temperature. The reaction mixture was stirred for 3 h, after which H_2_O and 1 M aq. were added. The mixture was extracted using chloroform. The organic layer was then dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified using column chromatography (CHCl_3_: MeOH = 100:1–10:1) to obtain OPC as a white solid (77.7 mg, 70%). ^1^H NMR (400 MHz, CDCl_3_): δ 7.74 (d, J = 9.6 Hz, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.16–7.11 (m, 2H), 6.95 (dd, J = 6.8, 3.2 Hz, 1H), 6.88 (d, J = 2.4 Hz, 1H), 6.81 (dd, J = 8.8, 2.4 Hz, 1H), 6.56 (d, J = 9.2 Hz, 1H), 4.11 (t, J = 6.2 Hz, 2H), 3.08 (br s, 4H), 2.67 (br s. 4H), 2.51 (t, J = 7.4 Hz, 2H), 2.29 (br s, 1H), 1.88 (m, 2H), 1.74 (m, 2H).
2.3. Cell viability assay
U251, U87, and T98G cells were seeded at a density of 3,000 cells/well in a 96-well plate (100 μL) and incubated at 37°C for 24 h. Subsequently, the cells were treated with the test compound (10 μL ARP or OPC in dimethylsulfoxide (DMSO), the final concentration was 0, 10, 20, 30, 40, and 50 μM, 0.1% DMSO) and incubated at 37°C for 24 h. HEK293 cells were seeded at a density of 3,250 cells/well in 96 well plate (100 μL), and treated with the same method (final concentration was 0, 1, 5, 20 50 µM). Cell viability was determined using Cell Counting Kit-8 (Dojindo, CK04, Kamimashiki-gun, Kumamoto, Japan), according to the instructions provided by the manufacturer. Briefly, after 2 h incubation, absorbance was measured at 450 nm using VersaMax (Molecular Devices, San Jose, California, USA). Cell viability was calculated with the absorbance of each treated group referred to that of the untreated control, which was set at 100%.
2.4. Recovery activity assay of cell death inhibitors against ARP or OPC
U251 cells were seeded at a density of 3,000 cells/well in 96-well plates in a 100 μL culture medium and incubated at 37°C for 24 h in a humidified atmosphere containing 5% CO_2_. The cells were subsequently treated with each compound (final concentration: IM-54 = 1 µM, Necrostatin-1 = 10 µM, Z-VAD = 20 µM, and Ferrostatin-1 = 1 µM, 0.2% DMSO), with or without ARP or OPC (30 μM, 0.2% DMSO) in a 100 μL culture medium for 24 h. After the addition of 10 µL CCK-8, the plates were incubated at 37°C for about 2 h and absorbance was measured at 450 nm using VersaMax (Molecular Devices, San Jose, California, USA). The cell viability was determined using Cell Counting Kit-8.
2.5. Wound healing assay
U251 cells were seeded at a density of 80,000 cells in a 35 mm dish and incubated at 37°C for 48 h to 90%–100% confluence. The cells were scratched with a 200 μL tip (Watson, 1272−703CS, Arakawa-ku, Tokyo, Japan) and subsequently treated with ARP or OPC (final concentration: 0 or 30 μM) at 37°C for 24 h. The cells were observed under a fluorescence microscope (BZ-X800; Keyence, Higashiyodogawa-ku, Osaka, Japan).
2.6. Fluorescence imaging
U251 cells were seeded at a density of 80,000 cells/well in a 35 mm glass base dish (AGC Techno glass, 3971−035, Haibara-gun, Shizuoka, Japan) and incubated at 37°C for 24 h. They were treated with ARP or OPC (final concentration was 0, 30 μM, 0.1% DMSO) and incubated at 37°C for 24 h. Subsequently, the cytoskeleton was dyed with Deoxyribonuclease I, Alexa Fluor ^TM^ 488 Conjugate (Thermo Fisher Scientific, D12371, Waltham, Massachusetts, USA), and Phalloidin, Rhodamine X conjugated (Fujifilm Wako, 165−21641, Osaka-shi, Osaka, Japan) according to the instructions provided by the manufacturer. Briefly, the medium was removed and the cells were washed with phosphate-buffered saline (PBS) (-) (Fujifilm Wako, 166−23555, Osaka-shi, Osaka, Japan). Subsequently, 4% formaldehyde/PBS (-) was added to the cells at ambient temperature for 30 min and washed with PBS (-). The cells were incubated with 0.3% Triton X-100/PBS (-) for 10 min and washed with PBS (-). Fluorescent compounds were added to the cells and the dishes were stored at 4°C overnight. They were subsequently observed under a fluorescence microscope (BZ-X800; excitation/emission: 495 nm/519 nm or 556 nm/574 nm). The fluorescence intensity was quantified using a BZ-X800 Analyzer (Keyence, Higashiyodogawa-ku, Osaka, Japan). The ratio (total intensity of red (F-actin)/total intensity of green (G-actin)) of the total values was calculated and compared.
2.7. Cell cycle analysis using flow cytometry
U251 cells were seeded at a density of 1.5 × 10^6^ cells/dish and incubated at 37°C for 24 h. Subsequently, the cells were treated with the test compound (ARP or OPC, final concentration: 30 μM, 0.1% DMSO) and incubated at 37°C for 24 h. They were washed with PBS (-) and collected with a cell scraper. After centrifugation (480 G, 5 min, ambient temperature), the supernatant was removed, and the precipitate was suspended in PBS (-) and transferred to 1.5 mL tubes. The tubes were centrifuged (520 G, 5 min, 4°C) again to remove the supernatant and resuspended in ice-cold 300 µL PBS (-). Additionally, 700 µL of ethanol, previously stored at −20°C, was added and cells were fixed at −32°C overnight. Samples were thawed and centrifuged (520 G, 5 min, 4°C) to remove the supernatant, and the residue was washed three times with 1% bovine serum albumin (BSA; Merck, A2153-50G, Darmstadt, Germany)/PBS (-). 1% BSA/PBS (-) was added to the residue and stored at 4°C for 24 h, and 10 mg/mL RNase (Merck, R4875, Darmstadt, Germany) solution was added to the sample (25 µL, final concentration was 500 µg/mL). The samples were placed in a water bath at 37°C for 20 min, centrifuged (520 G, 5 min, 4°C), and washed three times with 1% BSA/PBS (-). The precipitate was suspended in 1% BSA/PBS (-) (500 µL) and 1 mg/mL of propidium iodide (Fujifilm Wako, 163–26284, Osaka-shi, Osaka, Japan) (1 µL) was added. The fluorescence intensity of individual cells was determined using a FACSAria III flow cytometer (Becton Biosciences, Franklin Lakes, New Jersey, USA).
2.8. Effect of ARP or OPC in combination with doxorubicin (DOX)
The test compounds were dissolved in DMSO and subsequently diluted to 1/1000 in a culture medium to achieve final concentrations of 30 μM for ARP and OPC, or 75 nM for DOX. U251 cells were plated at a density of 3,000 cells/well in a 96-well plate and incubated at 37°C in a humidified atmosphere containing 5% CO_2_ for 24 h. Following medium removal, the cells were treated with the test compounds at various concentrations in a 100 μL medium containing 0.2% DMSO and cultured for an additional 24 h. To quantitatively assess synergistic effects, Bliss analysis was performed according to a previously reported method [24]. Briefly, the cell viability of U251 cells was measured following treatment with either ARP or DOX alone, and the expected combined effect was calculated as the product of the individual viabilities. This expected value was then compared to the experimentally observed viability following co-treatment with ARP and DOX. A synergistic effect was defined when the observed viability was lower than the calculated value. The same approach was applied to OPC.
2.9. Immunoblot analyses
U251 cells were exposed to ARP (20 µM) or OPC (20 µM) for 24 h. The cells were extracted with Radio-Immunoprecipitation assay (RIPA) buffer containing 50 mM Tris-HCl, 150 mM NaCl, 0.1% aodium dodecyl sulfate (SDS), 1% Triton X-100, 0.5% sodium deoxycholate, 1.0 mM ethylenediaminetetraacetic acid (EDTA). Proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore). The membrane was immunoblotted with anti-phosphorylated (p-)Akt, anti-Akt, anti-survivin, anti-Src, anti-p53, anti-caspase-3 (Cell Signaling Technology, 4691, 4060, 2808, 2123, 30313, 9662) and anti-GAPDH (cytosolic marker, Proteintech) antibody using immunoreaction enhancer solution (Can Get Signal, Toyobo). An ECL system was used for detection. The protein expression level was quantified by ImageJ software (National Institute of Health).
2.10. Statistical analysis
The results are expressed as mean ± standard error of the mean. Dunnett's multiple comparisons test was used to assess the significance of differences between the control group and the groups treated with the compounds, using JMP Pro^®^ 16 software (SAS Institute, Cary, NC, USA). Statistical significance was determined using a p-value < 0.05.
3. Results
3.1. Activity of ARP and OPC against various malignant glioblastoma cells and non-cancerous cells
The anticancer activities of ARP and OPC were investigated in U251, T98G, and U87 cell lines. Both compounds inhibited glioblastoma cell growth in a dose-dependent manner, and almost completely at 50 µM (Fig 2A, 2B, 2C). The IC₅₀values of ARP for U251, T98G, and U87 cell growth were 17.6 µM, 26.9 µM and 20.0 µM, respectively (Fig 2D). The IC₅₀ values of OPC for U251, T98G, and U87 cell growth were 18.6 µM, 22.7 µM and 18.3 µM, respectively (Fig 2D). On the other hand, temozolomide had no effect on glioblastoma cell viability even at 300 μM (S1 Fig). The cell viability of HEK293 with ARP and OPC was evaluated, as shown in Fig 2D. The IC₅₀ values of ARP and OPC against HEK293 were 18.1, 25.6 µM respectively. However, cell viability at 50 µM was higher in HEK293 compared to glioblastoma cells; for ARP, viability was 1.1% in U251, 1.5% in T98G, 1.5% in U87, and 36% in HEK293 cells; for OPC, viability was 7.7% in U251, 3.5% in T98G, 6.8% in U87, and 38% in HEK293 cells.
Effects of Aripiprazole (ARP) and OPC-14857 (OPC) on glioblastoma cells.Inhibitory effects of cell proliferation on U251 (A), T98G (B), U87 (C) and HEK293 (D) cells. IC₅₀ values of ARP and OPC in each cell line (E). The cells were seeded in a 96-well plate and incubated at 37°C for 24 h. Subsequently, the cells were treated with the test compound and incubated at 37°C for 24 h. Cell viability was determined using Cell Counting Kit-8, according to the instructions provided by the manufacturer. The symbol * (p < 0.05) indicates significant differences between ARP and OPC by one-way analysis of variance and student t-test. Data are shown as the mean ± SEM (n = 3).
3.2. Effects of ARP and OPC on cell migration
The effect of ARP and OPC on glioblastoma cell migration was investigated using a wound-healing assay. U251 cells with ARP or OPC (30 µM) were treated for 24 h and both compounds significantly reduced cell migration compared to the control group, as shown in Fig 3.
Effect of ARP (30 µM) or OPC (30 µM) on cell migration in U251 cells.U251 cells were seeded at a density of 80,000 cells in a 35 mm dish and incubated at 37°C for 48 h to 90%–100% confluence. The cells were treated with ARP (30 µM) or OPC (30 µM). The cells were scratched on a petri dish using a tip and observed under a microscope (0 h and 24 h).
3.3. Recovery activity of cell death inhibitors against ARP or OPC
The anti-cancer effects of ARP and OPC on U251 cells were compared in terms of cell viability in the presence and absence of cell death inhibitors. Recovery activity was not observed in the presence of inhibitors of caspase (Z-VAD-fmk), necroptosis (Nec-1), ferroptosis (Fer-1), or peroxide-derived necrosis-like cell death (IM-54) (Fig 4A and 4B). In contrast, IM-54 slightly enhanced the growth-inhibitory effects of ARP on U251 cells (Fig 4A).
Recovery activity of cell death inhibitors against ARP (A, 30 μM) or OPC (B, 30 μM) and other compounds in U251 cells.U251 cells were treated with ARP (A) or OPC (B) and several cell death inhibitors (Z-VAD, Nec-1, Fer-1, and IM-54) for 24 h. Cell viability was determined using a water-soluble tetrazolium assay. Each error bar represents the mean ± SEM (n = 4). Statistical analysis was performed using one-way analysis of variance and subsequent Dunnett’s test in each compound with or without ARP or OPC. The symbol * (p < 0.05) indicates significant differences between non-inhibitor and each cell death inhibitor treatment by one-way analysis of variance and subsequent Dunnett’s test. The symbol ns indicates not significant compared with the non-inhibitor. The 100% viable cells correspond to untreated cells (0.1% DMSO).
3.4. Effects of ARP and OPC on the cytoskeleton
To investigate the effects of ARP and OPC on the filamentous actin cytoskeleton, immunofluorescence staining using rhodamine X-conjugated phalloidin (which specifically binds to F-actin) and Alexa Fluor 488-conjugated deoxyribonuclease I (which specifically binds to G-actin) was performed. In the ARP-treated group, the F-actin/G-actin (F/G) ratio significantly decreased, whereas it remained unchanged in the OPC-treated group (Fig 5B). In addition, exposure to ARP and OPC reduced the accumulation of F-actin at the membrane periphery, leading to changes in the distribution of F-actin (Fig 5A, white arrowheads).
Effects of ARP or OPC on the formation and distribution of actin filaments.A) Red shows F-actin stained by phalloidin-rhodamine conjugated and green shows G-actin stained by Alexa Fluor™ 488 DNase I. The right column shows the merged staining of F-actin and G-actin. Cells with membrane-localized F-actin are indicated by arrows. B) The fluorescence intensity ratio of ARP and OPC in F-actin and G-actin staining. The F-actin to G-actin (F/G) ratio was calculated using approximately 5 images. Statistical analyses were performed using Dunnett’s test. The Symbol *** (p < 0.001, n = 5) indicates significant differences between DMSO and ARP or OPC (30 µM).
3.5. Effects of ARP and OPC on the cell cycle
We investigated the effects of ARP (30 µM) and OPC (30 µM) on cell cycle progression. In U251 cells, 24 h after the addition of ARP, there was no change in the G1 and G2/M phases, and fewer cells were in the S phase compared to those in the control group (Fig 6A, 6B, and 6D). In contrast, the addition of OPC decreased the number of cells in the G1 phase and increased the number of cells in the S and G2/M phases compared to those in the control group (Fig 6A, 6C, 6D).
Effects of ARP and OPC on the cell cycle in U251 cells.After treatment with DMSO (A), ARP (B), or OPC (C), the number of cells in each cell cycle phase was measured using flow cytometry with propidium iodide staining. A-C) Cell cycle distribution was analyzed using flow cytometry. D) Histograms represent the percentage of cells distributed in each cell cycle when the total cell count was 100%. Error bars represent mean ± SEM (n = 3). The symbols *** (p < 0.001), ** (p < 0.01), and * (p < 0.05) indicate significant differences between each drug and DMSO treatment by one-way analysis of variance followed by Dunnett’s test.
3.6. Combined effect of DOX and ARP or OPC
We then examined the combined effects of DOX, ARP, and OPC. In the presence of DOX (75 nM) alone, cell viability was reduced to 33% compared to the control group, while cell viability in the presence of ARP or OPC (5 µM) alone was almost the same as control. In contrast, the combination of DOX and ARP or OPC significantly reduced cell viability compared to DOX alone (Fig 7, DOX plus ARP: 23.9%, DOX plus OPC: 24.7%). Synergistic effects were quantitatively evaluated using Bliss analysis. As shown in Fig 7, the cell viability of U251 cells following treatment with either ARP or OPC (5 µM) alone was 101% and 100%, respectively, relative to the DMSO control (set as 100%). In contrast, treatment with DOX (75 nM) alone resulted in a cell viability of 33%.
Combined experiment of DOX (75 nM) and ARP or OPC (5 μM) in U251 cells.U251 cells were treated with DOX in the presence or absence of ARP or OPC for 48 h. Cell viability was determined using a water-soluble tetrazolium assay. Error bars represent mean ± SEM (n = 4). Statistical analyses were performed using Dunnett’s test. ** (p < 0.01) indicates significant differences between DOX alone and DOX plus ARP or OPC.
3.7. Effect of ARP and OPC on protein expression
To investigate the effects of ARP and OPC (20 µM) on protein expression, Western blotting was performed. The expression levels of survivin (an anti-apoptotic protein), p53 (a tumor suppressor protein), Akt and its activated form p-Akt (serine/threonine kinases involved in survival pathways), caspase-3 (a pro-apoptotic effector), and Src (a tumor-promoting protein) were analyzed following treatment with ARP or OPC. Survivin expression was significantly decreased upon treatment with both compounds. p53 expression was significantly reduced by OPC, while Src expression was significantly increased under the same condition. On the other hand, the expression levels of Akt, p-Akt, and caspase-3 remained unchanged.
4. Discussion
In the present study, we investigated the inhibitory effects of ARP and its metabolite, OPC, on human glioblastoma cell growth and examined the details of this inhibition. Although an inhibitory effect of ARP on glioblastoma cell proliferation has been reported [16,17], none of the studies mentioned its metabolites. Therefore, we investigated the inhibitory effects of OPC and ARP on the growth of U251, T98G, and U87 cells. To investigate this effect in detail, we focused on U251 cells, a representative glioblastoma cell line, and examined the competition with various cell death inhibitors. Subsequently, we examined the effects on cell migration, cell cycle, cytoskeleton formation, the combination effects with DOX, and the expression of key signaling proteins involved in cell survival and apoptosis by western blotting.
The antiproliferative and anti-migratory activities of OPC and ARP were similar (Fig 2A-2C and Fig 3). Both compounds exhibited more potent anticancer activities than TMZ, which is the current standard clinical therapy for malignant glioblastoma (Fig 2A, 2B, and S1). TMZ remained above 50% cell viability even at the highest tested concentration (300 µM), so IC₅₀ value of TMZ could not be precisely determined in our assay system. Moreover, no difference in the effects of cell death inhibitors on the anticancer activities of ARP and OPC existed (Fig 4A and 4B). In pharmacokinetic studies of ARP, OPC accounted for approximately 55% of the AUC of ARP in the plasma [25].
The C_max_ values of ARP and OPC were approximately 69 nM and 6.5 nM, respectively [25]. In our study, the IC₅₀ of ARP and OPC was higher than the C_max_. The in vitro IC₅₀ values obtained in this study for both ARP and OPC were approximately 20 µM against U251, T98G, and U87 cells. Therefore, there is a substantial discrepancy between these values and the reported maximum plasma concentrations, indicating that immediate clinical application may be challenging. However, the findings of this study could serve as a basis for further development of drug discovery seeds, with the goal of reducing the gap between in vitro efficacy and clinically achievable concentrations. Notably, ARP has a high oral bioavailability of 87% and has been clearly shown to cross the blood–brain barrier [25]. In addition, its metabolite OPC retains biological activity, suggesting that ARP has favorable pharmacokinetic properties as a seed compound. Compared with temozolomide (TMZ), a DNA-alkylating agent currently used in glioblastoma treatment, ARP and OPC exhibit markedly different in vitro activity profiles. Given their chemical structures, they are unlikely to act as alkylating agents, and thus would offer a differentiated mechanism of action.
Although the IC₅₀ values of ARP and OPC in HEK293 cells were similar to those observed in glioblastoma cells, a clear difference in viability was noted at 50 μM: less than 10% in glioblastoma cells versus over 35% in HEK293 cells. This may indicate partial selectivity in cellular responses at higher concentrations. A previous study reported that ARP exhibited no cytotoxicity in HEK293 cells at concentrations up to 100 µM [17]. In contrast, our data showed a measurable reduction in viability with an IC₅₀ around 20 µM. This discrepancy may be attributed to several experimental differences, such as assay type, exposure duration, or cell line subclones. For example, the previous study employed a 72 h MTT assay, whereas we used a 24 h exposure with a CCK-8 assay. Differences in assay sensitivity, metabolic activity, or proliferation rate could partially account for the observed variation. To better evaluate the therapeutic window and potential off-target effects, further studies using primary astrocytes may provide more physiologically relevant insights.
The anticancer effect of ARP against glioblastoma cells has been reported to be related to apoptosis rather than necrosis [17]. However, in the present study, neither ARP nor OPC were recovered by cell death inhibitors, including caspase inhibitors (Fig 4A and 4B). Our study was performed under conditions of 20%−30% survival by ARP and OPC (30 µM), whereas the previous study using ARP was carried out under conditions of 70% survival [17]. Therefore, although apoptosis may be partly responsible for the antiproliferative effect, our results suggest that the main anticancer effects of ARP and OPC are antiproliferative rather than cell death-inducing.
Our present study showed that ARP significantly decreased the F/G ratio compared to the control group (Fig 5), suggesting that ARP may inhibit actin polymerization. On the other hand, it has been reported that ARP does not affect the formation of F-actin [17]. The difference from the present study is that only F-actin was observed in that study, without considering G-actin, and the F/G-actin ratio was not calculated. Differences in the conditions of the two studies may have led to varying results. The F/G-actin ratio after OPC exposure was not significantly different from that in the control, suggesting that OPC exposure does not inhibit actin polymerization.
In the present study, the changes in the cell cycle associated with the inhibition of cell proliferation and migration by OPC or ARP were examined. OPC exposure significantly decreased the G1 phase and increased the G2/M phase. In contrast, no significant changes were observed after the exposure to ARP, with only a slight decrease in the number of cells in the S phase (Fig 6). According to a study on the anticancer effect of lycorine, a natural product, an increase in the G2/M phase did not affect the expression of F-actin but affected actin turnover, resulting in morphological changes and suppression of cancer cell growth [26]. Our results suggest that OPC causes G2/M phase arrest and affects actin, thereby altering its localization to inhibit cell proliferation and migration.
Although single treatment with ARP or OPC showed no anticancer effect at 5 μM, their addition to DOX enhanced its anticancer effect (Fig 7). In our preliminary experiments, we tested combination treatments using 30 µM of ARP and OPC; however, under this condition, the antiproliferative effects of the single agents were already strong, making it difficult to detect any additive or synergistic effects. Therefore, we focused on the 5 µM condition in the present study. Clinically, it is important to expand the applicability of existing anticancer drugs to glioblastomas because the choice of standard drugs is limited. DOX has low permeability across the BBB and is not currently indicated [27]. However, advances in drug delivery technology have suggested that DOX can be applied to glioblastomas [28–30]. ARP has been reported to inhibit P-glycoprotein (P-gp) [31]. Our findings suggest that ARP or OPC inhibit P-gp and show a combined effect with DOX on glioblastoma. According to Bliss analysis, synergy is inferred when the experimentally observed viability following combination treatment is lower than the expected value calculated as the product of the viabilities of each single-agent treatment. The observed cell viabilities under combination treatment were 24% (ARP 5 µM plus DOX 75 nM) and 25% (OPC 5 µM plus DOX 75 nM), both of which were lower than the expected values based on individual treatments. These results suggest that both ARP and OPC exert synergistic effects when combined with DOX.
To further elucidate the mechanism underlying the growth-inhibitory effects of ARP and OPC, we examined the expression of key signaling proteins involved in cell survival and apoptosis by Western blotting (Fig 8). Treatment with either compound significantly reduced the expression of survivin, an anti-apoptotic protein, suggesting an increase in apoptotic susceptibility. This is consistent with previous reports demonstrating that survivin downregulation contributes to the anticancer effects of ARP and related compounds in cancer stem cells [16]. In this present study, OPC induced a more pronounced reduction in survivin levels compared to ARP, indicating that the metabolite may serve as a more potent suppressor of survivin-mediated signaling. In addition, OPC treatment resulted in significant decrease in p53 expression along with an increase in Src levels. These changes may reflect a cellular stress [32] response or compensatory signaling activation [33]. No significant changes were observed in the expression levels of Akt, phosphorylated Akt (p-Akt), or caspase-3, indicating that the observed growth inhibition may occur via pathways independent of canonical PI3K/Akt signaling and caspase-dependent apoptosis. Collectively, these findings suggest that ARP and OPC exert their antiproliferative effects, at least in part, by downregulating survivin expression, while OPC may additionally modulate other tumor-associated signaling pathways such as p53 and Src.
Comparison of the expression levels of signaling proteins related to cell survival and apoptosis following treatment with ARP or OPC.U251 cells were exposed to ARP (20 µM) or OPC (20 µM) for 24 h, after which the cells were detached, disrupted by ultrasonication, and the proteins were extracted for Western blot analysis. The results of western blotting were shown in A) survivin and p53, D) Akt, p-Akt and caspase-3, H) Src. Each protein expression was expressed relative to the DMSO control (set to 1) and normalized to GAPDH as a loading control, B) survivin, C) p53, E) p-Akt, F) Akt, G) Caspase-3, I) Src. In this experiment, proteins were transferred from the gel to the membrane, after which the membrane was sectioned and individually probed with antibodies. The images shown in this figure are unprocessed. Statistical analyses were performed using Dunnett’s test. Error bars represent mean ± SEM (n = 3). The symbols * (p < 0.05) and ** (p < 0.01) indicate significant differences between DMSO and ARP or OPC. The symbol ns indicates not significant compared with the non-inhibitor.
Several D2 receptor–modulating drugs beyond ARP have been reported to suppress glioblastoma cell growth [15]. For example, the D2 antagonist perphenazine has been shown to potentiate the cytotoxic effect of temozolomide (TMZ) in glioblastoma models [34]. A common feature across these agents is their ability to modulate D2 receptor signaling. It has been reported that higher levels of D2 receptor expression are associated with poorer prognosis in glioblastoma patients and are inversely correlated with temozolomide sensitivity in glioblastoma cells [35]. Moreover, sustained activation of D2 receptor enhances the sphere-forming capacity of glioblastoma cells and increases their tumorigenic potential in orthotopic xenograft models [36]. Thus, it is difficult to exclude the possibility that changes in intracellular signaling downstream of D2 modulation contribute—at least to some extent—to the viability effects observed in glioblastoma cells, including those reported here. Although prior studies on ARP [16–18] have not comprehensively delineated D2-dependent versus D2-independent mechanisms in this context, there are reports indicating that aripiprazole’s cytotoxicity at higher concentrations can occur independently of its D2 receptor activity (e.g., in hepatocytes) [37]. Because we did not directly interrogate D2 signaling in the present study, the extent to which ARP and OPC influence glioblastoma cell viability through D2 receptor modulation remains unresolved and warrants further investigation.
While future in vivo studies are warranted, several important considerations must be taken into account when using xenograft models. First, human cancer cell lines used in such models have limited genetic backgrounds and often fail to capture the full heterogeneity of tumors, thereby limiting their ability to reflect actual drug responses in patients [38]. Second, transplanted tumor cells can be gradually replaced by host cells over time [38]. Third, although the immune system plays a crucial role in real tumors, xenograft models are typically established in severely immunodeficient animals, raising concerns about whether these models accurately recapitulate the tumor microenvironment [39]. Therefore, further investigations are necessary to translate the findings of this study into in vivo settings.
In conclusion, our observations revealed no difference in cell proliferation and chemotactic inhibition between ARP and OPC, but ARP had stronger inhibitory activity than the clinical drug, TMZ. In contrast, ARP and OPC affected actin filament formation and cell cycle progression differently, which may be attributed to structural differences such as the presence of a conjugated amide moiety in OPC. Western blot analysis further revealed that both compounds suppressed survivin expression, with OPC inducing a more pronounced downregulation. OPC also uniquely modulated the expression of p53 and Src, suggesting a distinct mechanism of action from ARP. Additionally, OPC and ARP enhance the efficacy of DOX by inhibiting P-gp. Although our observations were limited to in vitro studies, our results suggest that OPC may have sustained anticancer effects, even when ARP is metabolized in humans. These insights underscore the importance of evaluating active metabolites when considering drug repositioning strategies for glioblastoma.
Supporting information
S1 FigEffect of Temozolomide on U251 cells. Inhibitory effect of cell proliferation on U251 cells.(TIF)
S1 FileRaw data.(XLSX)
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