Introduction
In medicinal chemistry, many compounds with high biological activities derive from scaffolds, and their unique bioactivity may depend on substituent group (1). In addition, designing and transforming the substituent group of small molecule compounds have become an effective way for drug discovery. Benzothiazole is a heterocyclic scaffold with a benzene ring fused with a five-membered thiazole ring, and it is recognized as an important basis of the nucleus for drug synthesis (2,3). Benzothiazole derivatives have a wide range of biological activities (4), such as anticancer (5,6), antimicrobial (7) and immunosuppressive activity (8), benzothiazole analogues are also biologically active compounds in the central nervous system (9). In addition, benzothiazole derivatives as commercial drugs are extensively used in the clinical treatment of numerous diseases (10,11).
Our research team first reported a novel benzothiazole derivative BD750 [2-(2-benzothiazoleyl)-4,5,6,7-tetrahydro-2H-indazol-3-ol, C14H13N3OS, MW: 271.3]. BD750 has significantly shown immunosuppressive activity by inhibiting T cell proliferation (8). However, its poor solubility in water is unsatisfactory. In addition, the poor water solubility may make it difficult in clinical application. Thus, our research team was committed to design, synthesize, screen and biological evaluate of novel water-soluble benzothiazole derivatives based on BD750. Then we find BD926, a new water-soluble benzothiazole derivative in which the H-2 of benzothiazol is replaced by 4,5,6,7-tetrahydro-2H-indazol-3-olate group and it has shown good water solubility of approximately 25 mg/ml in water (12).
In the present study, we investigated the antitumor biological activity of BD926 in the human Ramos B-lymphoma cell line, and explored its potential mechanisms. Our findings aim to clarify that BD926 may be a potential antitumor drug.
Materials and methods
Drug and reagents
BD926, Sodium 2-(2-benzothiazoleyl)-4,5,6,7-tetrahydro-2H-indazol-3-olate (Fig. 1A), was synthesized previously by our team and the structure was confirmed by 1H-NMR, 13C-NMR and HRMS (ESI). Purity (85%) was measured by HPLC analysis (12). BD926 was dissolved in ultrapure water at a stock concentration of 10 mM and stored at −20°V.
The antibodies against GAPDH, PARP, Bcl-xl, Bcl-2, Bid, caspase-3, caspase-8, caspase-9 were purchased from Cell Signaling Technology. The caspase-12 antibody was purchased from Abcam. Primers were synthesized by the Shanghai Biological Engineering Company of China. All other chemicals were of analytical grade.
Cell culture
B lymphoma cell lines Ramos, Raji, HTB-60 and CA46, T lymphoma cell lines CEM, HL-60, Jurkat and K562, myeloma cell lines MM.1S, MM.1R, U266 and PRMI-8226 were saved by our laboratory. The cells were propagated under humidified conditions with 5% CO2 at 37°C in RPMI-1640 medium and supplemented with 10% fetal bovine serum (FBS; both from Gibco, Gaithersburg, MD, USA), 100 units/ml penicillin and streptomycin. To ensure that there was no mycoplasma contamination present, antibiotic treatment was performed regularly.
Human peripheral blood mononuclear cells (PBMC) were isolated from three healthy donors by density-gradient centrifugation using lymphocyte separation liquid (Nycomed AS, Oslo, Norway) and cultivated in the complete medium of RPMI-1640 containing 10% FBS. The blood donors were healthy individuals who provided informed consent.
Cell proliferation assay
Cell viability was performed by the CCK-8 assay (Dojindo Laboratories, Kumamoto, Japan). Briefly, the exponentially growing cells (1E4 cells/well) were seeded in a 96-well flat bottom microtiter plate and treated with BD926 for 24 or 48 h. Then, 20 μl of CCK-8 solution was added to each well and incubated for another 4 h at 37°C. The absorbance of each well was measured with Spectra microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA) at 450 nm wavelength and the median inhibitory concentration (IC50) was calculated with the GraphPad Prism software. Three replicate wells were used for each analysis. The results were obtained from three separate experiments.
Cell cycle analysis
After treated with BD926 for 12 h, the cells were harvested and washed briefly in cold PBS, then fixed in 75% ice-cold ethanol overnight. The samples were concentrated after removal of ethanol. Propidium iodide (PI) staining solution (1% Triton X-100, 0.01% RNase, 0.05% PI) (Sigma-Aldrich, St. Louis, MO, USA) was added to the samples to stain cellular DNA at 4°C for 30 min in dark. The cell cycle distribution was measured and analyzed by flow cytometry (FCM) (BD FACS Accuri C6; BD Biosciences, San Jose, CA USA).
Morphological observation under phase contrast microscope
Ramos cells were seeded in 12-well plates and treated with BD926 for 24 h. Then, the morphology of Ramos cells were observed under a phase contrast microscope (Olympus, Tokyo, Japan).
Apoptosis analysis by flow cytometry
Annexin V-FITC/PI apoptosis detection kit (Roche Diagnostics, Indianapolis, IN, USA) was carried out to detect the apoptotic cells. Briefly, after treated with different concentrations of BD926 for 12 h, Ramos cells were harvested and washed with cold PBS. The cells were then stained with Annexin V-FITC/PI then were analyzed by FCM.
Mitochondrial membrane potential testing
JC-1 is a fluorescent probe for the detection of mitochondrial membrane potential (MMP). After treated with BD926 for 12 h, changes in mitochondrial transmembrane potential (ΔΨm) were evaluated by staining cells with JC-1 (Roche Diagnostics). Cell culture and drug treatment were done as described above. The harvested Ramos cells were washed with cold PBS, incubated with JC-1 (5 mg/ml) at 37°C for 30 min in the dark, then measured by FCM.
Cytochrome c detecting
Ramos cells were treated with BD926 for 12 h, washed with PBS, fixed (BD™ Phosflow Fix Buffer) and permeabilized (BD Phosflow™ Perm Buffer), then stained with anti-cytochrome c/FITC (BD Biosciences) antibody. The resistance type with FITC labeled rat lgG0/G1 antibodies were used for comparison. FCM was used to detect cytochrome c content of the cytoplasm in Ramos cells.
Western blot analysis
After treated with BD926 for 24 h, Ramos cells were lysed in RIPA buffer (Bioteke Corp., Beijing, China) and the lysate was centrifuged at 13,000 × g at 4°C for 15 min. The supernatant was harvested and the protein concentration was measured by BCA method (Bioteke). Equal amounts of total proteins were subjected to 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA, USA). After electrophoresis, the membranes were blocked at room temperature for 1.5 h and incubated in the respective primary antibodies at 4°C overnight, then the bound antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA). The immunostaining signal was visualized by enhanced chemiluminescence (Millipore).
Real-time quantitative RT-PCR
Total RNA was extracted from the treatment cells using the acid guanidinium thiocyanate-phenol-chloroform method (TRIzol; Takara Bio, Dalian, China), and cDNAs were synthesized with the PrimeScript™ RT reagent kit (Takara Bio). Quantitative PCR was performed using the Bio-Rad CFX96 Real-Time PCR detection system with the TaqMan probe method (Roche Diagnostics). The primers are 5′-cagatgaaaatgggggtaccta-3′ (F) and 5′-tcaagagt ggtgaagatttttgat-3′ (R) for CHOP; 5′-agctgtagcgtatggtgctg-3′ (F) and 5′-aaggggacatacatcaagcagt-3′ (R) for GRP78.
The mRNA expression levels were calculated with the 2−ΔΔCT method and expressed in relative quantification units. A control without cDNA was run in parallel with each assay. Each reaction was amplified in triplicate, and relative mRNA levels were normalized to GAPDH.
ROS measurement
To assess the generation of ROS, BD926-treated cells (1×105) were incubated with 10 μM 2,7-dichlorofluorescein diacetate (DCFH-DA). Within the cells, DCFH-DA is converted to DCFH, which can be oxidized to the fluorescent compound DCF in the presence of ROS. Cell culture and drug treatment were done as described above, and Ramos cells were washed with PBS after incubated with DCFH-DA (Bi Yun Tian Corp., Kuaidamao, China) at 25°C for 30 min in the dark. Then the expression of cell fluorescence signal was detected by FCM.
Statistical analysis
The results of the statistical analysis used GraphPad Prism 5 software with one-way ANOVA and Dunnett's test, analysis, comparison between group differences. Descriptive statistics were calculated with mean ± standard deviation, and P<0.05 showed a statistically significant difference.
Results
BD926 inhibits the proliferation of tumor cells
In order to confirm the anticancer activity of BD926 in cell models, we examined the growth inhibition effect of BD926 on tumor cell lines including B lymphoma cells, T lymphoma cells and myeloma cells. Cells were treated with BD926 for 24 h and then the cell viability was assayed by CCK-8. The results showed that BD926 significantly inhibited the cell proliferation with IC50 from 6.0 to 42.2 μM depending on different tumor cell lines (Fig. 1B–D).
The present study aimed to clarify the anticancer effect and mechanism of BD926 based on Ramos cells. The result showed, BD926 exhibited a significant cell proliferation inhibition with IC50 ~3 μM for 48 h and 10.9 μM for 24 h in Ramos cells (Fig. 1E). This indicated that BD926 treatment decreased the viability of Ramos cells in a time- and dose-dependent manner. The 50% inhibition concentration of cytotoxicity (CC50) of BD926 in resting human PBMC was as high as 200 μM for 48 h (Fig. 1F). This result suggested that BD926 had almost no cytotoxic effect on resting human PBMC under the effective inhibition concentration of tumor cells, which indicated that BD926 could effectively inhibit tumor proliferation and exhibited low toxicity in PBMC.
BD926 induces Ramos cell cycle arrest at G0/G1 phase
Because the proliferation of Ramos cells was suppressed by BD926, we determined how BD926 blocks the cell cycle progression by FCM analysis. The Ramos cells were treated by BD926 for 12 h, the cellular DNA was stained with PI and analysed via FCM. The results are shown in Fig. 2. A dose-dependent increase in the cell population of G0/G1 phase was observed. BD926 treatment increased the percentage of G0/G1 cells from 34.0% in non-treated group to 52.4% in 20 μM BD926-treated group in Ramos cells. The results indicated that BD926 induced Ramos cell cycle arrest at G0/G1 phase.
BD926 induces apoptosis of Ramos cells
To obtain BD926-induced apoptosis information of Ramos cells, cell morphological changes were observed by phase contrast microscopy. BD926 induced significant apoptotic morphological changes in Ramos cells. Cells treated with BD926 became deformed, smaller, cell membrane integrity and foaming phenomenon was seen along with apoptotic bodies (Fig. 3A).
To confirm this cell death, we also used Annexin V-FITC and PI fluorescence staining to detect the apoptosis of Ramos cells by FCM. A dose-dependent increase in the population of apoptotic cells was observed, and the percentage of apoptotic cells reached 27.9% at the concentration of 20 μM BD926 compared with 4.6% in the negative control (Fig. 3). To explore whether BD926-induced apoptosis was specifically associated with caspase activation, we examined whether Z-VAD-FMK (a general caspase inhibitor) could affect apoptosis. As shown in Fig. 3D, treatment with 20 μM BD926 combined with 20 mM Z-VAD-FMK decreased the percentage of apoptotic cells compared with BD926 treatment alone.
BD926 induces the Ramos cell apoptosis via mitochondrial pathway
Effect of BD926 on mitochondrial membrane potential (MMP)
Mitochondria are involved in the regulation of apoptotic cell death (13), and the change of mitochondrial membrane potential (ΔΨm) is known to be one of the important factors for mitochondrial dysfunction. Therefore, we examined ΔΨm with JC-1 staining by FCM, on account of the dissipated MMP preventing the accumulation of JC-1 in the mitochondria, and made a shift from red (JC-1 aggregates) to green fluorescence (JC-1 monomers). As shown in Fig. 4A, BD926 depolarized the MMP in a dose-dependent manner. In addition, BD926 at a concentration of 20 μM induced 21.0% decrease of ΔΨm in Ramos cells compared with 2.3% in the negative control.
Effect of BD926 on cytochrome c
A limited step in the intrinsic apoptotic pathway is related to the release of cytochrome c from mitochondria into the cytosol. To observe the change in Ramos cells, anti-cytochrome c/FITC antibody was used. As shown in Fig. 4C, BD926 induced the release of cytochrome c in dose-dependent manner. BD926 at a concentration of 20 μM induced the release of cytochrome c reaching 31.6% in Ramos cells compared with 0.9% in the negative control.
Apoptotic protein detection
To evaluate the contribution of active caspases in BD926 induced-apoptosis, we characterized the activation of caspase-9/-8/-3 and PARP by western blot analysis. As shown in Fig. 4E, a decrease in pro-caspases-9/-8/-3 and an increase in the levels of their cleaved forms were observed in Ramos cells. The activation of Bid and PARP protein were also observed, but the change of Bcl-2 and Bcl-xl were not significant.
Effect of BD926 on the endoplasmic reticulum apoptosis pathway
Endoplasmic reticulum (ER) stress have shown to be involved in the cell apoptosis (14,15). To confirm whether BD926 induced apoptosis by ER stress signaling, we examined the mRNA transcription of the markers in ER apoptosis pathway using real-time reverse transcription PCR. As shown in Fig. 5A, BD926-treated cells significantly increased the mRNA expression levels of ER stress-related molecules, including glucose regulated protein 78 (Grp78) which is a major protective player of the unfolded protein response (UPR) and pro-apoptotic transcriptional regulator C/EBP homologous protein GADD153/CHOP.
Caspase-12, an ER resident caspase, is localized on the ER and activated during apoptosis induced by ER stress (16). Different from caspase-7/-8/-9, caspase-12 is a specific medium which associated with ER stress-induced apoptosis (17). As the results showed, BD926 induced the activation of caspase-12 and the cleaved caspase-12 expression levels were significantly increased by BD926 (Fig. 5B). Moreover, the poly(ADP-ribose) polymerase (PARP) cleavage was also increased significantly. These results indicated that BD926 induced ER stress-associated apoptosis.
Reactive oxygen species (ROS) plays a crucial role in BD926-induced apoptosis
ROS is a mediator of intracellular signaling cascades. The excessive generation of ROS can induce oxidative stress, loss of cell functioning and apoptosis (18). To confirm whether BD926-induced apoptosis was triggered by ROS accumulation, the intracellular ROS level was measured using the ROS-detecting fluorescence dye DCFH-DA. As shown in Fig. 6A, according to the ratio of DCF-positive cells we judged that the level of ROS was significantly increased in the cells treated with BD926. In addition, BD926 at a concentration of 20 μM induced 20.7% DCF-positive cells in Ramos cells compared with 1.4% in the negative control. We also found ROS inhibitor NAC attenuated BD926-induced apoptosis (Fig. 6C). This finding suggested that ROS mediated BD926-induced apoptosis.
Discussion
The chemotherapeutic agents enhancing oxidative stress are toxic to the cancer cells because they are involved in the biological processes including cell cycle arrest, DNA repair and apoptosis. ROS is a well known mediator of intracellular signaling of cascades. The excessive generation of ROS can induce oxidative stress, loss of cell functioning and apoptosis (14). It is also reported that ROS accumulation could lead to mitochondrial dysfunction via depolarizing the mitochondrial membrane potential (18). Our findings indicate that BD926 induces apoptosis in human Ramos B-lymphoma cells, accompanying with ROS generation. ROS inhibitor NAC significantly reduces the BD926-induced ROS production and attenuates the apoptosis in Ramos cells, suggesting that ROS may play a key role in BD926-induced the apoptosis of Ramos cells.
The results showed that BD926 enhanced the disruption of MMP, induced the release of cytochrome c, then increased the expression of cleaved-Bid, cleaved caspase-9/-3 and its downstream target cleaved-PARP leading to activation of the mitochondrial apoptotic pathway. The increased mRNA expression of CHOP and the activated caspase-12 are also observed, which indicated that BD926 induced ER-associated apoptosis. Moreover, BD926 also resulted in cell cycle arrest at the G0/G1 stage. In brief, we propose that BD926 activates apoptosis by increasing ROS production, which triggers the ER stress and the mitochondrial membrane dysfunction in Ramos cells, accompanied by DNA damage and cell cycle arrest (Fig. 6D).
BD926, a new water-soluble benzothiazole derivative in which the H-2 of benzothiazol was replaced by 4,5,6,7-tetra-hydro-2H-indazol-3-olate group. In this study, we found that BD926 showed a growth inhibition effect against human tumor cell lines including B lymphoma cells, T lymphoma cells and myeloma cells. BD926 induced apoptosis of human Ramos B-lymphoma cells in a dose- and time-dependent manner. Importantly, BD926 had almost no cytotoxic effect on resting human PBMC under the effective inhibition concentration on tumor cells, which indicated that BD926 was able to effectively inhibit tumor proliferation and maintain low toxicity, a prerequisite for a lead compound.
The present study provides important insights into molecular mechanisms of the anticancer biological activities of BD926 in cell models, and the potential value of BD926 as a novel candidate antitumor drug. H-2 of benzothiazol was replaced by 4,5,6,7-tetrahydro-2H-indazol-3-olate group contributing to improve the biological activity and water solubility, which will promote the clinical application process of the benzothiazole derivatives BD926. Our findings suggest that BD926 may be a promising lead compound, design and development of new benzothiazole derivatives based on BD926 have wide reaching prospects in cancer chemotherapy.