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Introduction

Two-dimensional (2D) monolayer cultures are traditionally used as models in cancer studies, as they are convenient and stable (1). However, 2D cultures are flat and elongated on plates, are grown in a medium with the same amount of nutrients and growth factors and lack cell-cell interactions (2). Therefore, despite their important roles in cancer research, 2D cultures have numerous limitations as in vivo cancer models. In vitro 3D tumor models, such as spheroids or organoids, have been successfully developed from different human cancers, including lung, prostate, breast and colon cancers. Unlike traditional 2D cell culture models, 3D cancer spheroid models provide an environment highly similar to the in vivo cancer microenvironment through cell-cell and cell-extracellular matrix interactions (1). Therefore, they are more relevant for drug discovery than 2D monolayer cultures (3).

Bladder cancer is the ninth most common cancer type worldwide and its incidence is four times higher in males than in females (4,5). At diagnosis, ~30 and 5% of bladder cancer cases present as the muscle-invasive and metastatic type, respectively. Although bladder cancer is chemosensitive, the recurrence rates after cisplatin-based chemotherapy in patients with advanced or metastatic bladder cancer are 30–40 and 100%, respectively (6).

Histone deacetylases (HDACs) are enzymes involved in cancer formation. They may regulate cancer cellular processes, including cell proliferation, apoptosis and migration, and overexpression of HDACs is associated with poor prognosis. HDAC knockdown inhibits urothelial carcinoma cell growth and induces apoptosis (7–9). In a previous study, it was reported that the HDAC inhibitor suberoylanilide hydroxamic acid re-sensitized cisplatin-resistant bladder cancer cells by inducing apoptosis and cell cycle arrest (10). Phosphoinositide 3-kinase (PI3K)/AKT signaling is also an important regulator of diverse cellular processes and is highly activated in bladder cancer (11,12).

CUDC-907 (Fimepinostat) is an orally available small-molecule drug that suppresses PI3K and HADCs. It exerts anticancer activities by inducing apoptosis and inhibiting the growth and metastases of various types of tumor, including prostate, lung and breast cancers (13–15); however, its anticancer effects on bladder cancer have not been previously reported, to the best of our knowledge. Thus, the present study aimed to investigate the anticancer effects of CUDC-907 on a 3D spheroid model of bladder cancer.

Materials and methods

Cell lines and reagents

T24 bladder cancer cells were obtained from the American Type Culture Collection. T24R2 cells resistant to 2 µg/ml cisplatin were generated via serial desensitization (16). The cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.). CUDC-907 (cat. no. S2759; Fig. 1) was obtained from Selleck Chemicals.

Figure 1. Chemical structure of CUDC-907 (Fimepinostat).

Spheroid formation

For spheroid formation, bladder cancer cells (2,500, 5,000 and 10,000 cells) were seeded in 96-well spheroid microplates (cat. no. CLS4515; Corning, Inc.) and then cultured in complete medium at 37°C with 5% CO2 for 4 days. Bladder cancer spheroids of ~230–280 µm were formed and treated with CUDC-907 at 37°C for 24 h (Fig. 2). The size and area of the formed spheroids were measured under a microscope (Olympus CKX53, ×40 magnification) using iSolution Lite ×64 software (IMT i-Solution, Inc.).

Figure 2. Schematic depicting the processes of the drug assay of bladder cancer spheroids.

Live/dead staining

The LIVE/DEAD Viability/Cytotoxicity Kit (cat. no. L3224; Invitrogen; Thermo Fisher Scientific, Inc.) was used to determine spheroid viability. The cell nuclei were stained with Hoechst 33342 solution (cat. no. 62249; Thermo Fisher Scientific, Inc.) and a live/dead stock solution was prepared by adding 1 µl of calcein-AM stain for marking live cells and 4 µl of ethidium homodimer-1 stain for marking dead cells into 1 ml of PBS. The stock solutions were applied to the cells at 37°C for 50 min. Subsequently, the cells were washed with PBS and observed using an inverted fluorescence microscope (LSM 710; Zeiss AG).

Cell counting Kit (CCK)-8 assay

The cell viabilities of the monolayers and spheroids were assessed using the CCK-8 assay. The monolayers and spheroids were treated with CUDC-907 at various concentrations (0, 0.1, 0.05, 1, 5, 10, 25 and 50 µM) for 24 h. To each well, 10 µl of CCK-8 reagent (cat. no. CK04; Dojindo Molecular Technologies) was added, followed by incubation at 37°C for 4 h. The absorbance was determined using a spectrophotometer (Molecular Devices, LLC) at 450 nm.

Matrigel migration assay

For the migration assay (17), the upper chambers of a Transwell insert (cat. no.3422; 6.5-mm insert; 8.0-µm pore size; Costar; Corning, Inc.) was pre-coated with 25 µl Matrigel® matrix (cat. no. 356234; 1 mg/ml; BD Biosciences) prior to adding FBS-free RPMI-1640 containing the spheroids. The lower chamber was loaded with 700 µl culture media containing 10% FBS and CUDC-907 at various concentrations (0, 5 and 10 µM). After 24 h of incubation, the migrated cells remaining on upper side were stained without wiping using the LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen; Thermo Fisher Scientific, Inc.) and Diff Quik (cat. no. 38721; Sysmex). Images of migrated cells were obtained using Leica LAS X Core 3.7.4 software under a microscope (magnification, 100×; Thunder Imager; Leica Microsystems GmbH).

Collagen invasion assay

For the 3D invasion assay, the spheroids were embedded in Cultrex 3D culture matrix rat collagen type I gel (cat. no. 3447-020-01; R&D Systems Inc.) and then incubated at 37°C for 30 min. Medium containing 10% FBS and CUDC-907 (0, 5 and 10 µM) was added to the collagen gel. After incubation at 37°C for 24 h, the invaded cells were stained using the LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen; Thermo Fisher Scientific, Inc.). Images of the invaded cells were acquired under a microscope. Image J software (National Institutes of Health) was used to measure the invaded area.

Reverse transcription-quantitative PCR

The total RNA (500 ng) of the spheroids was obtained using the RNeasy Mini-Kit (cat. no. 74104; Qiagen GmbH). The quality and quantity of RNA were measured using the Nanodrop ND 1000 (Thermo Fisher Scientific, Inc.). cDNA was synthesized using the iScript cDNA Synthesis Kit (cat. no. 1708891; Bio-Rad Laboratories, Inc.) in accordance with the manufacturer's instructions. For amplification, each PCR system included 2 µl cDNA, 2 µl primer (forward and reverse, 1:1), 10 µl Power SYBR Green PCR Master Mix (cat. no. 4367659; Applied Biosystems; Thermo Fisher Scientific, Inc.) and 6 µl H2O (final volume, 20 µl). Real-time PCR cycles consisted of 2 min at 50°C, a hold for 10 min at 95°C, and 40 cycles of 15 sec at 95°C and 1 min at 60°C. The expression of target genes was normalized to the level of GAPDH. The PCR primers are listed in Table I.

Table I. Real-time quantitative PCR primers used in the present study.

Table I.

Real-time quantitative PCR primers used in the present study.

Gene (direction)	Primer sequence 5′→3′
E-cadherin (F)	CAG CAC GTA CAC AGC CCT AA
E-cadherin (R)	ACC TGA GGC TTT GGA TTC CT
Vimentin (F)	GTT TCC AAG CCT GAC CTC AC
Vimentin (R)	GCT TCA ACG GCA AAG TTC TC
MMP-2 (F)	TTG ACG GTA AGG ACG GAC TC
MMP-2 (R)	ACT TGC AGT ACT CCC CAT CG
MMP-9 (F)	TTG ACA GCG ACA AGA AGT GG
MMP-9 (R)	CCC TCA GTG AAG CGG TAC AT
Bax (F)	AGA CAG GGG CCT TTT TGC TA
Bax (R)	AAT TCG CCG GAG ACA CTC G
Bcl-2 (F)	CTT TGA GTT CGG TGG GGT CA
Bcl-2 (R)	AGT TCC ACA AAG GCA TCC CA
Caspase-3 (F)	TGT TTG TGT GCT TCT GAG CC
Caspase-3 (R)	CAC GCC ATG TCA TCA TCA AC
Caspase-8 (F)	TCT GGA GCA TCT GCT GTC TG
Caspase-8 (R)	CCT GCC TGG TGT CTG AAG TT
Caspase-9 (F)	GGC TGT CTA CGG CAC AGA TGG A
Caspase-9 (R)	CTG GCT CGG GGT TAC TGC CAG
GAPDH (F)	CCA CTC CTC CAC CTT TGA CG
GAPDH (R)	CCA CCA CCC TGT TGC TGT AG

[i] F, forward; R, reverse.

Statistical analysis

All experiments were performed using the SPSS statistical software package (version 20.0; IBM Corporation). Values are expressed as the mean ± standard deviation. Differences were considered statistically significant at alpha=0.05 (P<0.05) according to one-way ANOVA. For multiple comparisons, Tukey's range test was performed.

Results

Treatment with CUDC-907 inhibits the survival of bladder cancer spheroids

The capacities of T24 and T24R2 cells to form spheroids were examined with an initial 1,000, 2,500 and 5,000 cells seeded per spheroid. At the end of the incubation period, images of the spheroids in each well were acquired (Fig. 3). The diameter of the spheroids increased as the number of seeded cells was increased. On day 3, the 2,500 cells of T24 and T24R2 formed compact, regular spheroids with clear boundaries and the diameters of the T24 and T24R2 spheroids were 258.2±5.12 and 241.6±1.82 µm, respectively. The viabilities of the spheroids were examined using live/dead staining. Analysis indicated that CUDC-907 treatment increased the number of dead cells (red-stained cells) of spheroids in a concentration-dependent manner (Fig. 4). The fluorescence intensity produced by EthD-1 (dead cell staining), which was measured using Image J software, also increased in a manner dependent on the dose of CUDC-907. After 24 h of culture, the volumes of the T24 and T24R2 spheroids treated with 10 µM CUDC-907 had decreased by 18.19 and 59.87%, respectively, compared with those of the untreated spheroids. In addition, the viabilities of the T24 and T24R2 monolayers and spheroids were evaluated using a CCK-8 assay. The T24 and T24R2 monolayers and spheroids exhibited differences in cell growth at 24 h. CUDC-907 treatment significantly reduced the viabilities of the T24 and T24R2 monolayers (Fig. 5A). The viabilities of the T24 and T24R2 spheroids were higher than those of their monolayers (Fig. 5B). The IC50 values of CUDC-907 were 3.5 and 2.8 times higher in the T24 and T24R2 spheroids, respectively, than in the monolayers (Table II). These data indicated that CUDC-907 inhibited the growth of bladder cancer spheroids.

Figure 3. 3D spheroid formation of bladder cancer cells. (A) T24 and (B) T24R2 (magnification, 40×) (C) T24 and (D) T24R2 (magnification, 200×). Images were obtained under an inverted fluorescence microscope. Calcein-AM stains live cells (green), EthD-1 stains dead cells (red) and Hoechst 33258 stains cell nuclei (blue). Values are expressed as the mean ± standard deviation of three independent experiments. Different letters indicate a statistically significant difference between columns with the same number of cells (P<0.05).

Figure 4. Live/dead viability of bladder cancer cell spheroids. (A) T24 spheroids and (B) T24R2 spheroids (scale bar, 100 µm). Images were acquired under an inverted fluorescence microscope. Calcein-AM stains live cells (green), EthD-1 stains dead cells (red) and Hoechst 33258 stains cell nuclei (blue). *P<0.05 vs. control.

Figure 5. Viability of bladder cancer spheroids by CCK-8 assay. (A) 2D cell culture and (B) 3D spheroids. Cell viability was detected using the CCK-8 assay. Values are expressed as the mean ± standard deviation of three independent experiments. *P<0.05 vs. control. CCK-8, Cell Counting Kit-8.

Table II. IC50 values of CUDC-907 in bladder cancer cells (µM).

Table II.

IC50 values of CUDC-907 in bladder cancer cells (µM).

Model	T24	T24R2
2D cells	7.74	15.84
3D spheroids	27.08	43.89

Treatment with CUDC-907 suppresses the migration of bladder cancer spheroids

To determine the effects of CUDC-907 on spheroid cell migration, spheroids were placed on the Matrigel-coated membrane in upper chamber and culture media containing CUDC-907 was added to the lower chamber. As presented in Fig. 6A and B, the migration distance of the bladder cancer spheroids in the control group was large. However, the bladder cancer spheroids treated with 10 µM CUDC-907 only exhibited slight movements through the matrix compared with the untreated spheroids. The Diff Quik staining results also indicated that the number of cells that migrated was markedly reduced in the presence of CUDC-907 (Fig. 6C). These data suggested that CUDC-907 treatment suppressed the migration of bladder cancer spheroids.

Figure 6. Effect of CUDC-907 on migration of bladder cancer spheroids. (A) Live/dead staining image of migrated cells in (A) T24 and (B) T24R2 spheroids (scale bar, 100 µm). (C) Diff Quik staining image of migrated cells in T24 and T24R2 3D spheroid (magnification, 100×). Images were obtained under an inverted fluorescence microscope. Calcein-AM stains live cells (green), EthD-1 stains dead cells (red) and Hoechst 33258 stains cell nuclei (blue).

Treatment with CUDC-907 suppresses the invasion of bladder cancer spheroids

Bladder cancer spheroids were embedded in collagen matrix and the invaded area was measured using ImageJ. The untreated bladder cancer spheroids displayed a considerable invasion capacity and the average invasion distance was large (Fig. 7A and B). Conversely, the spheroids treated with 10 µM CUDC-907 did not exhibit any significant changes in the collagen matrix. The invaded area of the spheroids also obviously decreased in a CUDC-907 concentration-dependent manner (Fig. 7C and D). In particular, the invaded areas of T24 and T24R2 spheroids treated with 10 µM CUDC-907 were reduced by 99.9 and 94.4%, respectively, compared to untreated spheroids. These results suggested that CUDC-907 inhibited the invasion of bladder cancer spheroids in a concentration-dependent manner.

Figure 7. Effect of CUDC-907 on invasion of bladder cancer spheroids. Live/dead staining image of invaded cells in (A) T24 and (B) T24R2 spheroids (magnification, ×100; scale bar, 100 µm). (C) Images of invading T24 and T24R2 spheroids (yellow; magnification, 200×; scale bar, 50 µm). (D) Area invaded by the T24 and T24R2 spheroids. Calcein-AM stains live cells (green), EthD-1 stains dead cells (red) and Hoechst 33258 stains cell nuclei (blue). The area of invasion was calculated using ImageJ software. *P<0.05 vs. control.

Treatment with CUDC-907 affects epithelial-mesenchymal transition (EMT), apoptosis and invasion of bladder cancer spheroids

Considering that Bax and caspases are pro-apoptotic genes (18), the influence of CUDC-907 on the mRNA expression of Bax and caspases was examined. As illustrated in Fig. 8, CUDC-907 treatment significantly increased the mRNA levels of Bax, caspase-3, caspase-8 and caspase-9 in T24 and T24R2 cells. Subsequently, the influence of CUDC-907 on EMT was assessed. CUDC-907 treatment significantly upregulated E-cadherin expression and downregulated vimentin expression in the T24 and T24R2 bladder cancer spheroids. In addition, the mRNA expression of MMP-2, which has a key role in the EMT for metastasis and invasion (19), was markedly reduced by CUDC-907 treatment in the T24 spheroids. These results demonstrated that CUDC-907 induced apoptosis and inhibited the EMT of the bladder cancer spheroids.

Figure 8. Effect of CUDC-907 on mRNA expression levels of EMT- and apoptosis-related genes in bladder cancer spheroids. Values are expressed as the mean ± standard deviation of three independent experiments. *P<0.05 vs. untreated control. EMT, epithelial-mesenchymal transition.

Discussion

When cancer patient-derived cells are cultured in 3D, cell-cell and cell-extracellular matrix interactions may change not only the type of cells but also the type and expression of key genes, and the actual microenvironment may be accurately established (2,20,21). Therefore, 3D cultured cells reflect cellular responses in vivo better than 2D cultured cells (21). It has previously been suggested that a 3D spheroid model may also be used as a tool for a drug screening assay instead of the in vivo model (22). Cells cultured in 2D cannot maintain a normal shape and are more susceptible to drugs than those cultured in 3D, owing to their differences in physical and physiological characteristics. In addition, 2D cultures are more sensitive to drugs than 3D cultures due to their different surface receptor organizations (2,20). Kim et al (23) reported that the viability of 5637 and T24 bladder cancer cells treated with rapamycin and BCG is higher in 3D cultures than in 2D cultures. The present results also showed that the resistance to CUDC-907 was greater in the spheroids than in the monolayers.

EMT promotes cancer progression processes, such as invasion and metastasis. During EMT, cancer cells detach from the primary tumor and acquire migratory capacity and invasiveness (24,25). In advanced bladder cancer, E-cadherin expression is lost, indicating increased aggressiveness and invasiveness of the disease (26). Loss of E-cadherin expression leads to increased invasion of bladder, lung and breast epithelial cancers (27). Vimentin is also a mesenchymal marker that induces EMT and is involved in bladder tumorigenesis (28). McConkey et al (29) reported a strong inverse relationship between E-cadherin and vimentin expression levels in bladder cancer. Zhang et al (30) found that CUDC-907 significantly inhibits migration and invasion by decreasing vimentin, a protein that regulates the migration and invasion of lung cancer cells. Similar to the above study, the present study demonstrated that CUDC-907 markedly upregulates E-cadherin mRNA expression and downregulates vimentin mRNA expression in bladder cancer spheroids. These data suggest that CUDC-907 inhibits cell migration and invasion by affecting EMT markers.

Apoptosis is a process of programmed cell death that efficiently clears damaged cells and is an intrinsic or extrinsic pathway mediated by caspases (18,31). The activities of pro-apoptotic caspase (aspartate-specific proteases), including the extracellular initiator caspase-8, intracellular initiator caspase-9 and effector caspase-3, are essential for apoptosis; thus, cancer cells evade apoptosis by blocking caspase activities (18,32,33). In the present study, CUDC-907 treatment upregulated the expression of caspase-8 and caspase-9, which promoted caspase-3 mRNA expression and induced apoptosis. In addition, the mRNA expression of Bax, a pro-apoptotic protein, was clearly increased by CUDC-907 treatment. Ishikawa and Mori (34) reported that CUDC-907 induces apoptosis by activating caspase-3, caspase-8 and caspase-9 and upregulating Bax in adult T-cell leukemia. Taken together, these results suggest that CUDC-907 exerts anticancer effects on bladder cancer spheroids by inhibiting EMT and promoting apoptosis.

In the present study, a 3D spheroid model of bladder cancer was established to screen CUDC-907 as an anticancer drug for bladder cancer. The results obtained using the spheroid model demonstrated that CUDC-907 exhibited anticancer effects by reducing cell viability, migration and invasion. Thus, the anticancer effects of CUDC-907 were achieved, at least in part, by inducing apoptosis and inhibiting EMT of bladder cancer spheroids. The established 3D spheroid model may be used for drug screening assays and CUDC-907 may act as a potential agent for bladder cancer treatment. However, since the present study used an in vitro model implemented in 3D and the patient-specific microenvironment was not considered, it is limited in its application to clinical practice. As for further studies, it will be necessary to confirm changes in biomarker expression or modification in bladder cancer resulting from treatment with CUDC-907.

Acknowledgements

Not applicable.

Funding

This work was supported by the SNUBH Research Fund (grant nos. 13-2020-0003 and 13-2022-0007).

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.

Authors' contributions

SL was involved in the study conception and design. JNH and DHK performed the experiments and drafted the paper. JSJ and HR analyzed and interpreted the data. JNH and HR corrected the manuscript. SL, JNH and JSL confirm the authenticity of all the raw data. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References