Celecoxib in combination with retinoid CD437 inhibits melanoma A375 cell in vitro

Abstract

This study aimed to investigate the effects of celecoxib, synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (CD437) and the combination of the two on cell proliferation, apoptosis, and cycle arrest of human malignant melanoma A375 cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazoliumbromide assay (MTT assay) was applied to determine the anti-proliferative effects of the drugs on human malignant melanoma A375 cells. Flow cytometry was performed to investigate the influence of the drugs on cell cycle and cell apoptosis. Both celecoxib and CD437 could inhibit the growth of human malignant melanoma A375 cells in a dose-dependent manner. Celecoxib at 80 μmol/L inhibited proliferation, induced apoptosis and G₂/M cell cycle arrest of human malignant melanoma A375 cells after treatment for 24 h [proliferation inhibiting rate: (50.2±2.51)%, apoptosis rate: (35.91±1.80)%]. CD437 at 10 μmol/L inhibited proliferation, induced apoptosis and G₀/G₁ cell cycle arrest of human malignant melanoma A375 cells after treatment for 24 h [proliferation inhibiting rate: (58.6±2.38)%, apoptosis rate: (28.03±0.77)%]. Celecoxib in combination with CD437 could significantly enhance the effects of inhibiting proliferation and inducing apoptosis of human malignant melanoma A375 cells 24 h after treatment compared with the drug alone [proliferation inhibiting rate: (68.92±1.72)%, apoptosis rate: (42.09±1.05)%, both P<0.05] and could decrease the proportion of the S phase in the cell cycle. Celecoxib could inhibit the growth of human malignant melanoma A375 cells by inducing apoptosis and G₂/M cycle arrest. CD437 could inhibit the growth of human malignant melanoma A375 cells by inducing apoptosis and G₀/G₁ cycle arrest. Celecoxib exhibited additive effects with CD437 on retarding the growth and inducing apoptosis of human malignant melanoma A375 cells. Celecoxib in combination with CD437 may become an effective method for prevention and treatment of human melanoma.

Keywords

celecoxib / CD437 / melanoma A375 cell / apoptosis / cycle arrest

Introduction

Melanoma is the most aggressive skin cancer, with rapid metastasis, high mortality and a poor prognosis. The incidence of melanoma in the world is rapidly rising, including in China. To date, no treatment has resulted in a clinically meaningful prolongation of overall survival. Thus, novel therapeutic strategies to treat melanoma have to be proposed. Celecoxib, which is a cyclooxygenase-2-specific inhibitor, can inhibit the proliferation of a large variety of tumors in vitro. Celecoxib may also be a potential anti-tumor therapeutic agent. Celecoxib inhibiting tumor growth and metastasis in animal experiments have been confirmed. Certain achievements have been made as to the preventive effect of celecoxib on colon cancer metastasis clinically. However, can celecoxib be applied to treat melanoma? Retinoids play an important role in cell proliferation and differentiation, and moreover, the anti-tumor effect of retinoids has been widely concerned. 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (CD437) is a novel synthetic retinoic acid derivative, which could induce apoptosis in a large variety of tumor cells in vitro, including some melanoma cell lines. Recently, some researchers found that celecoxib could enhance chemotherapy sensitivity to some tumors. In this study, the effects of celecoxib and synthetic retinoid CD437 on cell proliferation, apoptosis and cycle arrest of human malignant melanoma A375 cell were investgated.

Materials and methods

Materials

Fetal bovine serum (FBS), Dulbecco's modified Eagle media (DMEM) and Trypsin were purchased from GIBCO Invitrogen Corporation (USA). CD437, PI, 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazoliumbromide (MTT), RNase and DMSO were obtained from Sigma-Aldrich (USA). An AnnexinV-FITC kit was offered by JingMei Biotech Co., Ltd (Shanghai, China). Celecoxib was purchased from DeBioChem (Nanjing, China).

Cell lines and culture conditions

Human melanoma A375 cells (Chinese Type Culture Collection) were routinely cultured in DMEM supplemented with antibiotics and 10% heat-inactivated FBS at 37°C in a humidified atmosphere with 5% carbon dioxide.

MTT assay

The cells were seeded at densities ranging from 3 × 10³ to 6 × 10³ cells per well in 96-well tissue culture plates. After a 12 h incubation, cells were treated with different concentrations (0, 10, 20, 40, 80 μmol/L, respectively) of celecoxib and cells were treated with different concentrations (0, 1, 5, 10 μmol/L, respectively) of CD437, and then cells were treated with 80 μmol/L celecoxib in combination with 10 μmol/L CD437. After treating for 12, 24 and 48 h, the absorbance value at 490 nm was measured by an enzyme-linked immunoabsorbent assay reader (DG3022A). The percentage of growth inhibition was calculated with the equation: proliferation inhibition ratio= (1-A_t/A_c) ×100%. A_t and A_c represent the absorbance value of the treated and control cultures, respectively. IC50 (the drug concentration causing a 50% cell growth inhibition) was determined by interpolation from the dose-response curve.

Apoptosis and cycle arrest

The cells were seeded at densities ranging from 0.5×10⁵ to 1×10⁵ cells in a 25 cm² culture flask. After a 12 h incubation, the cells were treated with 80 μmol/L celecoxib, 10 μmol/L CD437, and 80 μmol/L celecoxib in combination with 10 μmol/L CD437, respectively, according to the above-mentioned MTT assay results. After a 24 h treatment, they were harvested by trypsinization and washed twice with phosphate buffered solution (selection of drug concentration and time point was done according to MTT assay results). The medium and phosphate buffered solution (PBS) were discarded and the cells were left in centrifuge tubes. On one hand, apoptosis cells were counted according to the AnnexinV-FITC kit instruction. On the other hand, the cells were fixed in ice-cold 70% ethanol for 18 h. After being washed twice with PBS, the dispersed cells were stained with propidium iodide (PI) and cell cycle analysis was performed. At least 10⁶ cells were analyzed each time. Flow cytometric analysis was conducted with a Becton Dickinson FACSCalibur and the accompanying CellQuest software.

Analysis

Data were expressed as \overline{x}\pm s. Independent-samples T test was used for statistical analysis using SPSS 12.0. A P <0.05 was considered statistically significant.

Results

Growth inhibition

Celecoxib could inhibit the growth of human melanoma A375 cells in a dose-dependent manner (Fig. 1). The concertration of celecoxib at which the celecoxib could show an effect on the A375 cells was higher than 20 μmol/L. Growth inhibition by celecoxib of A375 cells achieved its peak at the 24th hour of treatment. Proliferation inhibiting ratio (PIR) of 80 μmol/L celecoxib on A375 cells at the 24th hour of treatment was (50.2±2.51)%, and IC50 of celecoxib was 85.63 μmol/L. CD437 could also inhibit the growth of human melanoma A375 cells in a dose-dependent manner (Fig. 2). The PIR of 10 μmol/L CD437 on A375 cells was (58.6±2.38)% at the 24th hour of treatment, (61.74±1.54)% at the 48th hour of treatment, and the IC50_CD437 was 6.23 μmol/L. PIR of 80 μmol/L celecoxib in combination with 10 μmol/L CD437 on A375 cells achieved its peak at the 24th hour of treatment, and the PIR was (68.92±1.72)% (Fig. 3). Celecoxib exhibited additive effects with CD437 on retarding the growth of melanoma A375 cells [PIR_combination = (68.92±1.72)% vs PIR_CD437= (58.6±2.38)%, t = 8.61, P <0.05, ν = 10, n = 6, two-tailed test].

Apoptosis

Compared with the control group [apoptosis ratio: (12.07±0.3)%], both 80 μmol/L celecoxib and 10 μmol/L CD437 could significantly induce A375 apoptosis at the 24th hour of treatment [apoptosis ratio: (35.91±1.80)% and (28.03±0.77)%, respectively, Fig. 4). Celecoxib at 80 μmol/L in combination with 10 μmol/L CD437 could enhance the induced apoptosis of human melanoma A375 cells, and the apoptosis ratio was (42.09±1.05)% [apoptosis ratio_combination= (42.09±1.05)% vs apoptosis ratio_celecoxib = (35.91±1.80)%, t= 5.14, P <0.05, ν = 4, n = 3, two-tailed test, Fig. 4].

Cycle arrest

Compared with the control group, 80 μmol/L celecoxib obviously promoted G₂/M phase arrest in human melanoma A375 cells at the 24th h of treatment. Compared with the control group, the proportion of the G₂/M phase in the cell cycle increased from (6.49±0.16)% to (23.50±0.58)% and the proportion of the S phase in the cell cycle decreased from (32.43±0.81)% to (24.78±0.62)% by 80 μmol/L celecoxib. Compared with the control group, 10 μmol/L CD437 obviously promoted G₀/G₁ phase arrest in human melanoma A375 cells at the 24th h of treatment. Compared with the control group, the proportion of the G₀/G₁ phase in the cell cycle increased from (61.23 ± 1.11)% to (77.79 ± 1.99)% and the proportion of the S phase in the cell cycle decreased from (32.43 ± 0.81)% to (22.21 ± 0.56)% by 10 μmol/L CD437. Compared with the control group, the proportion of S phase in the cell cycle decreased further from (32.43±0.81)% to (16.24±0.41)% by 80 μmol/L celecoxib in combination with 10 μmol/L CD437, with the proportion of G₀/G₁ in the cell cycle being (77.85±1.94)%, and the proportion of G₂/M phase in cell cycle being (5.91±0.15)% (Fig. 5).

Discussion

Cyclooxygenase-2 (COX-2) is a highly inducible cyclooxygenase isoenzyme. Over expression of COX-2 not only participates in tumorigenesis, but is also involved in the regulation of invasion and metastasis of multifarious tumors [1-3]. Celecoxib, which is a cyclooxygenase-2-specific inhibitor, can inhibit the proliferation of a large variety of tumors, down-regulate the expression of vascular endothelial growth factor and matrix metalloproteinases, and prevent the invasion and metastasis of a large variety of tumors [4,5]. Recently, some researchers found that celecoxib could enhance chemotherapy sensitivity and radiosensitivity to some tumors [6,7]. Celecoxib plays its anticarcinogenic role either in a COX-2-dependent or COX-2-independent manner [8]. In this study, celecoxib could inhibit the growth of human melanoma A375 cell in a dose-dependent manner. The least concentration at which celecoxib could have an effect on A375 cell growth retardation was 20 μmol/L. Growth inhibition of celecoxib on A375 cells achieved its peak at the 24th h of treatment (IC50 of celecoxib: 85.63 μmol/L). Celecoxib could significantly induce A375 cell apoptosis and G₂/M cell cycle arrest. Celecoxib may be a potential optional drug for human melanoma.

Since all-trans-retinoic acid (ATRA) began to be applied in treating acute promyelocytic leukemia, the anti-tumor effect of retinoids has been widely researched. Recently, many researchers have been aiming at a class of adamantly-retinoids or retinoid-related molecules (RRMs). These compounds have a stronger apoptotic potential, lower level of toxicity and better pharmacokinetic profile than natural ligands. CD437 is the prototype of the promising compounds, which was originally developed as an RAR-γ–selective agonist [9,10]. As a representative of these compounds, CD437 has raised remarkable enthusiasm because it is endowed with selective apoptotic activity on a large variety of tumor cells including human melanoma cell lines WM1341, MeWo and S91 [11,12]. CD437 was reported to have stimulated the mitochondrial apoptosis pathway and activated apoptosis-regulative proteins including P53, P21, Bax/bcl-2, PTPC, Nur77/TR3 etc [13-17]. CD437 induces the apoptosis of ovarian adenocarcinoma cells via endoplasmic reticulum (ER) stress signaling [18]. CD437 also activates NF-κB and thereby induces death receptor 4 (DR4) expression and subsequent apoptosis of DU145 cells (prostatic carcinoma cell line) [19]. CD437 is a retinoic acid receptor (RAR) agonist, but surprisingly, when a RAR antagonist blocked RAR, CD437 still could induce apoptosis of prostate cancer cells, ovarian cancer cells and human lung cancer cells in vitro, which meant that CD437 could induce apoptosis by non-receptor-dependent mechanisms in vitro [19-21]. It was reported that the regulation of differentiation-associated cell-type-specific genes was related to an RAR-γ-dependent pathway, and apoptosis induction relied on a non-receptor-dependent pathway [22]. In this study, CD437 could also inhibit the growth of human melanoma A375 cells in a dose-dependent manner (IC50 of CD437: 6.23 μmol/L). CD437 could significantly induce A375 cell apoptosis and G₀/G₁ cell cycle arrest. Celecoxib exhibited additive effects with CD437 on retarding the growth of human malignant melanoma A375 cells. Celecoxib at 80 μmol/L in combination with 10 μmol/L CD437 could enhance the induction of apoptosis of human melanoma A375 cells. The S phase was decreased further by 32.43% to 16.24% by 80 μmol/L celecoxib in combination with 10 μmol/L CD437. Thus, celecoxib in combination with CD437 could enhance the proliferation-inhibiting effects and the apoptosis-inducing effects of human melanoma A375 cells. Celecoxib in combination with CD437 may become an effective method for prevention and treatment of human melanoma. Furthermore, animal experiments need to be perfomed to obtain more information.

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