In the US, bladder cancer is the second most frequent genitourinary malignant tumor, and is a fatal disease [1
]. Most human bladder cancers belong to the category of transitional cell carcinoma, which can be further divided into invasive and non-invasive bladder cancer. T2 and higher stages of muscle invasive bladder cancer have a poor prognosis and a high risk of metastasis [2
]. Non-muscle-invasive bladder cancer is usually treated with transurethral resection of the bladder tumor, followed by close monitoring or Bacillus Calmette–Guérin (BCG) instillation [5
]. However, the recurrence rate in patients with high-grade non-muscle-invasive bladder cancer after transurethral resection exceeds 75%, with a low survival rate [6
]. Since the recurrence rate of bladder cancer is still high, and many medications currently used for bladder cancer have strong side effects [3
], the development of new drugs for bladder cancer treatment remains an important issue.
Marine soft corals are rich in biologically-active substances, and have been shown to exert anti-inflammatory, anti-fungal, anti-viral, anti-cancer, and cytotoxic activities [7
]. Cembrane -type diterpene are common secondary metabolites of marine and terrestrial organisms, and cytotoxicity is one of the major characteristics of compounds of this type [11
]. Previous studies have shown that compounds extracted from soft corals, such as diterpenes, diterpenoids, and prostanoids can induce apoptosis in cancer cells, including colon cancer, oral squamous carcinoma, breast cancer, cervical cancer, hepatocellular carcinoma, bladder cancer, and melanoma cells [14
]. The apoptotic process includes intrinsic and extrinsic pathways [21
]. Studies have shown that many organelles in the cell may trigger the intrinsic pathway to induce apoptosis when stress occurs. Mitochondria and the endoplasmic reticulum (ER) are the two major organelles in which stress-induced apoptosis takes place [22
]. Mitochondria provide the chemical energy required for cellular activities, and are the main components in the cell in which oxidative phosphorylation and adenosine triphosphate (ATP) synthesis take place [25
Mitochondria are also involved in cell differentiation, cell signaling, and apoptosis, and have the ability to regulate cell growth and control the cell cycle [26
]. During apoptosis, Bax translocates from the cytoplasm to the outer membrane of the mitochondria, and Bax and Bak oligomers form pores in the outer membrane, triggering mitochondrial dysfunction [27
The functions of the ER include regulation of protein synthesis, protein folding, post-translational modification, and maintenance of intracellular calcium homeostasis [30
]. When ER stress occurs, in order to relieve the stress and promote survival, cells need to initiate specific signaling pathways to limit protein synthesis, increase protein folding ability, and degrade misfolded proteins. The unfolded protein response (UPR) is a series of processes by which the ER transmits the stress signal from its lumen into the cytosol and nucleus. UPR-related genes are induced by UPR sensors, activating transcription factor 6 (ATF6) and inositol requiring enzyme 1α (IRE1-α) in response to ER stress to promote correct protein folding. Another UPR sensory protein, PKR-like ER-associated kinase (PERK), detects an overload of biosynthetic protein folding in the ER, and initiates limitation of new protein synthesis by phosphorylation of eukaryotic initiation factor 2α (eIF2α) [23
]. Additionally, cells may prevent excessive accumulation of misfolded proteins in the ER through ER-associated degradation (ERAD). If the level of misfolded proteins does not reduce, cells will undergo apoptosis via pathways involving IRE1-α, caspase-12, and PERK/CHOP [32
]. Moreover, results by scholars have shown that apoptosis in human bladder cancer cells is associated with endoplasmic reticulum stress [33
Flacidoxide-13-acetate is a cembrane-type diterpene extracted from the cultured soft coral Sinularia gibberosa
. Our previous study revealed that flacidoxide-13-acetate reduces cancer cell migration and invasion in T24 and RT4 human bladder cancer cell lines [36
]. In this study, we investigated the mechanisms of its apoptotic and antiproliferative activities in human bladder cancer, and aimed to provide useful information to inform the development of flacidoxide-13-acetate as a new drug for bladder cancer treatment.
4. Material and Methods
Flaccidoxide-13-acetate was isolated from cultured-type soft coral Sinularia gibberosa by Dr. Jui-Hsin Su. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and phosphate-buffered saline (PBS), were purchased from Biowest (Nuaillé, France). Polyvinylidene difluoride (PVDF) membranes were purchased from Millipore (Bellerica, MA, USA). Protease inhibitor cocktail, dimethyl sulfoxide (DMSO), salubrinal, and goat anti-rabbit and horseradish peroxidase-conjugated immunoglobulin (Ig) G were obtained from Sigma (St. Louis, MO, USA). Cell extraction buffer was acquired from BioSource International (Camarillo, CA, USA). An annexin V-FITC/PI apoptosis detection kit was purchased from Pharmingen (San Diego, CA, USA). The enhanced chemiluminescence (ECL) Western blotting reagents were obtained from Pierce Biotechnology (Rockford, IL, USA). Cytochrome C releasing apoptosis assay kit was purchased form Biovision (Milpitas, CA, USA).
4.2. Cell Culture and Drug Treatment
Human bladder cancer RT4 and T24 cell lines were obtained from the Taiwan Food Industry Research and Development Institute (Hsinchu, Taiwan). The cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100µg/mL streptomycin, and 100 units/mL penicillin in a humidified 5% CO2 incubator at 37 °C. Cells were treated with various concentrations of flaccidoxide-13-acetate and harvested after 24 h of incubation. DMSO was added to the control group, and cultured for 24 h for subsequent studies. All experiments were performed three times to determine their reproducibility.
4.3. Cell Viability Assay
The effects of flaccidoxide-13-acetate on the T24 and RT4 cell lines were evaluated by MTT assay. Cells (1 × 105 cells/well) were seeded in 96 well plates. The cells were treated with various concentrations of flaccidoxide-13-acetate (5, 10, 15, 20, and 25µM). After 24 h of incubation, MTT solution (0.5 mg/mL in PBS) was added to each well. The plates were incubated for 4 h at 37 °C, after which the culture medium was removed and the cells were dissolved in 200 μL DMSO. The absorbance was measured at 595 nm using a microplate ELISA reader (Bio-Rad, Hercules, CA, USA) and DMSO was used as the control. Samples were analyzed and all experiments were repeated three times.
4.4. Flow Cytometric Assay
T24 and RT4 cells were seeded onto 5 cm petri dishes, treated with different concentrations of flaccidoxide-13-acetate for 24 h. The cells were then collected and fixed in 70% cold ethanol at 4 °C overnight. The cells were subsequently stained with 10 μg/mL Annexin V–FITC and 5 μg/mL propidium iodide (PI) for 30 minutes at 37 °C. Apoptosis processes induced by flaccidoxide-13-acetate were analyzed using a FACScalibur flow cytometer and Cell-Quest software (Becton-Dickinson, Mansfield, MA, USA).
4.5. Colony Formation Assay
T24 and RT4 cells were seeded in 24 well plates (2000 cells/well) and incubated for 24 h. The cells were treated with various concentrations (5, 10, 15, and 20 μM) of flaccidoxide-13-acetate in 2 mL of serum complete media and incubated for 10 days. The colonies were then washed with PBS and fixed with methanol for 15 min and stained with 0.15% crystal violet. The colonies were counted and scanned using a high-resolution scanner Scan Maker 9800XL (MiCROTEK, Hsinchu, Taiwan).
4.6. Antibody and Western Blot Assay
Rabbit anti-human ERK, p-ERK, JNK, p-JNK, GRP78, ATF4, and cleaved-ATF6 antibodies were purchased from ProteinTech Group (Chicago, IL, USA). Rabbit anti-human antibodies against AKT, p-AKT, PI3K, p-PI3K, Mcl-1, Bad, p-Bad, Bcl-xl, Bcl-2, Bax, p38, p-p38, PERK, p-PERK, elF2α, p-elF2α, pro-caspase 3, cleaved caspase 3, pro-caspase 9, cleaved caspase 9, cytochrome C, and CHOP were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-human β-actin antibodies were obtained from Sigma (St Louis, MO, USA). Cytosolic cytochrome C were separated using a cytochrome C releasing apoptosis assay kit (Biovision, Milpitas, CA, USA).
The flaccidoxide-13-acetate treated sample and DMSO treated control samples (total protein 25 μg) were separated by 12.5% SDS-PAGE, and the proteins on the gel were transferred to a PVDF membrane. The membrane containing transferred protein was blocked in PBS buffer and incubated with primary antibody at 4 °C overnight, followed by secondary antibodies (goat anti-rabbit or goat anti-mouse and horseradish peroxidase conjugate, 1:5000 dilution in 2% dehydrated skim milk) for 2 h at 4 °C. The signals were detected with an enhanced chemiluminescence detection kit.
4.7. Inhibitor Assessment
In order to further determine the effects of p38, ERK, and JNK on flaccidoxide-13-acetate-induced cell proliferation arrest, a total of 1 × 105 cells were seeded in a 24 well plate and pre-incubated for 2 h with specific inhibitors for p38 (SB2203580), JNK (SP600125), and ERK (PD98059) prior to flaccidoxide-13-acetate administration. Afterwards, the cell viability rate was determined by MTT assay.
4.8. Statistical Analysis
The results of the MTT assay and colony formation assay were subjected to Student’s test (Sigma-Stat2.0, San Rafael, CA, USA). Results with p < 0.05 were considered statistically significant.