Apoptosis

QBS is a potent cytotoxic compound that induces cell cycle arrest and apoptosis. Treatment of Jurkat T cells with QBS increased the levels of cyclin B1 as well as phosphorylated-cdc2, which was accompanied by reduced activity of cdc2 kinase, suggesting that QBS may induce cell cycle arrest at G2 phase. Structural analogues of QBS also exhibited similar effects on cell cycle progression and cell viability. Long-term treatment with QBS resulted in DNA fragmentation, cytochrome C release, and PARP cleavage, and an increase in the number of subdiploidy cells, indicative of cellular apoptosis. Moreover, QBS-induced apoptosis was blocked by z-VAD-fmk, a pan-caspase inhibitor. These results suggest that QBS is a novel and potent compound that induces G2 arrest and subsequent apoptosis, implicating it as a putative candidate for chemotherapy.[1]

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QBS induces cell death in Jurkat T cells [1]
To identify novel compounds that have cytotoxic activities in leukemia cell lines, we screened a chemical library containing 11,000 compounds, using Jurkat T cells. In the initial screening, 24 compounds that reduced viability more than 50% (EC50 < 2 μM) were selected based on the MTT conversion assay (data not shown). 2-Amino-N-quinoline-8-yl-benzenesulfonamide (Fig. 1A) was identified as the most potent chemical that decreased cell viability among the 24 selected chemicals (EC50 = 0.77 ± 0.05 μM). As shown in Fig. 1B, decrease of cell viability was apparent at a concentration of 0.5 μM and reached approximately 70% at 1 μM. The cytotoxic effect of QBS was also observed in other lymphoma cell lines, such as Molt-4, Raji and Ramos cells (EC50 = 0.87 ± 0.04, 1.26 ± 0.09, and 0.96 ± 0.04 μM, respectively). These results suggest that QBS might be an effective cytotoxic agent in leukemia cell lines.

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QBS triggers caspase-dependent apoptosis [1]
To elucidate the pathway affected by QBS that reduces cell viability, we firstly examined whether QBS induced apoptosis. From Hoechst 33342 staining and DNA-ploidy assay, it was revealed that QBS induced nuclear condensation, DNA fragmentation (Fig. 2A, upper panel) and increased the sub-G1 peak (Fig. 2A lower panel), which are hallmarks of apoptosis. After 48 h of QBS treatment, the sub-G1 population reached 32%, whereas it was only 2.8% in untreated cells. In addition, oligonucleosomal cleavage of genomic DNA was increased by QBS treatment in a concentration-dependent manner (Fig. 2C). Moreover, typical nuclear morphology of apoptotic cells by QBS treatment was confirmed by electron microscopy (Fig. 2B). Next, we investigated whether cytochrome C was released to the cytosol from mitochondria by immunofluorescent staining using monoclonal antibody against cytochrome C. As shown in Fig. 2D, cytochrome C released into the cytoplasm was observed only in apoptotic cells which showed nuclear condensation and DNA fragmentation. Furthermore, 116 kDa PARP was obviously cleaved into its characteristic 85 kDa fragment after 36 h of QBS treatment (Fig. 2E). Taken together, these findings indicate that QBS triggers apoptosis in Jurkat cells.

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QBS induces G2 phase arrest by DNA damage in Jurkat T cells [1]
From DNA-ploidy analysis, a significant inhibitory effect of QBS on cell cycle progression was observed at 18 h after QBS treatment (Fig. 4A). QBS treatment resulted in an accumulation of cells at the G2/M phase (21%, 40%, and 58% at 0, 18, and 24 h after QBS treatment, respectively) with a corresponding reduction in the number of cells at the G1 phase. Similar results were obtained when other leukemia cell lines such as MOLT-4, Raji, and Ramos were treated with QBS (data not shown).

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QBS analogues arrest Jurkat T cells in the G2/M phase of the cell cycle [1]
To investigate the structure–activity relationship (SAR) of QBS, we examined the effect of four QBS derivatives which possess quinoline and benzenesulfonamide groups. QBS derivatives we tested were N-8-quinolinyl-benzenesulfonamide, N-phenyl-4-quinolinesulfonamide, N,4-dimethyl-N-8-quinolinylbenzenesulfonamide, and 4-methyl-N-4-quinolinylbenzenesulfonamide, which hereafter are called QBS-1, PQS, QBS-2 and QBS-3, respectively (Fig. 6). The structure of QBS-1, which is a sulfonamide derivative, is similar to that of QBS except for the absence of amine group. In PQS, the phenyl and quinoline groups are connected by the nitrogen and the sulfur atom of sulfonamide group, in a reverse way to that of QBS-1. The chemical structure of QBS-2 is analogous to QBS-1. However, the difference between QBS-2 and other compounds used in this work originates from the tertiary amine group having different basicity compared to the secondary one (Fig. 6). The structure of QBS-3 is similar to that of QBS-1 except for its methyl group substitution. Among the four tested QBS analogues, QBS-1 and PQS dramatically increased the G2/M cell population compared with the DMSO-treated control (52.6 ± 2.1%, 49.2 ± 2.4% and 22.3 ± 3.4% in QBS-1, PQS and DMSO-treated cells, respectively) (Fig. 6). PQS inhibited the cdc2-kinase activity as well as inducing G2 phase arrest (Fig. 5C). However, QBS-2 and QBS-3 did not have any significant effects on cell cycle arrest (24.3 ± 3.6%, 25.6 ± 2.8% in QBS-2 and QBS-3-treated cells, respectively) (Fig. 6).

Cell viability measurement [1]
To determine the effect of various chemicals on cell viability, cells (2 × 104 cells/ml) were seeded in 96-well plates and treated with the chemicals in RPMI1640 medium containing 2% FBS, at the concentrations as indicated. For inhibition of apoptosis, cells were pretreated with a pan-caspase inhibitor, z-VAD-fmk (30 μM) 30 min prior to QBS treatment. DMSO (0.1%, final concentration) was treated as a vehicle control. After treatment with the chemicals, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 0.5 mg/ml) was added, and cells were incubated at 37 °C for 2 h in a CO2 incubator. After centrifugation at 2000 rpm for 10 min, supernatant was carefully removed and DMSO was added. Absorbance was measured at 570 nm in a microplate reader.

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DNA ploidy analysis [1]
Cells were suspended in phosphate-buffered saline (PBS) containing 5 mM EDTA and fixed in 100% ethanol. RNase A (2 μg/ml) was added to the suspended cells, and the cells were incubated at room temperature for 30 min. Then, propidium iodide (50 μg/ml) was added before reading. DNA contents of the cells were analyzed on a FACScan flow cytometer, which was also used to determine the percentage of cells in the different phases of the cell cycle.

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Morphological analysis of apoptotic cells [1]
Morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with 2 μg/ml Hoechst 33342 fluorochrome, followed by examination under a fluorescence microscope.

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Caspase-3 activity assay [1]
Jurkat cells were harvested and sonicated. Following centrifugation at 15,000 rpm for 10 min, 20 μg protein of supernatant in buffer containing 100 mM Hepes, 10% sucrose, 5 mM dithiothreitol, 10-6% NP-40, and 0.1% CHAPS (pH 7.25) was added to each well of a 96-well plate with 50 μM DEVD-aminomethylcoumarin (AMC). After incubation at 37 °C for 1 h, the cleaved free AMC (excitation of 355 nm, emission of 460 nm) was detected using a fluorometer.

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Immunoblotting [1]
Cells were lysed in lysis buffer (50 mM Tris–Cl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP-40, 1 mM PMSF). Equal amount of protein in each sample were separated by SDS–polyacrylamide gel electrophoresis and transferred to Hybond ECL nitrocellulose membranes. The membrane was blocked with 5% skim milk, and sequentially incubated with primary and secondary antibodies.

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Cyclin B1-associated cdc2 kinase activity assay [1]
Cells were washed in ice-cold PBS and lysed on ice in lysis buffer (40 mM Tris–Cl, pH 7.5, 120 mM NaCl, 0.1% NP-40, 1 mM PMSF, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM Na3VO4, 10 mM NaF). Lysates were incubated with a monoclonal mouse anti-cyclin B1 antibody for 2 h at 4 °C and protein G-Sepharose slurry was added. The mixture was incubated for 2 h at 4 °C. For kinase assay, the immunecomplexes were washed three times in lysis buffer and then twice in the assay reaction buffer, containing 25 mM Tris–HCl, pH 7.5, and 10 mM MgCl2. The immunoprecipitates were incubated with 2 μg of histone H1 in 20 μl of reaction buffer and 2 μCi of [32P] ATP for 30 min at 37 °C. The reaction was terminated by addition of SDS loading buffer. The mixture obtained was boiled for 5 min, loaded onto a 10% SDS polyacrylamide gel and autoradiographed.

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Immunofluorescence staining [1]
Jurkat cells were fixed in 4% paraformaldehyde for 30 min at room temperature. Fixed cells were permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate for 3 min at 4 °C and then sequentially incubated with mouse anti-cytochrome C antibody, biotinylated anti-mouse IgG, streptavidin-FITC and 3 μg/ml of Hoechst 33342 (Molecular Probe). Stained cells were examined under a fluorescence microscope. In staining of cyclin B1, the nuclei of cells was stained with propidium iodide (2 μg/ml) after treatment with RNase A (2 μg/ml) for 30 min. Stained cells were observed under a confocal microscopy.

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Comet assay [1]
The comet assay was performed under alkaline conditions based on the procedure reported by Singh et al. Briefly, 85 μl of 0.8% of normal agarose (NA) was added on a microscope slide pre-layered with 1.5% of NA. Cell suspension (3 × 104 cells) was mixed with 75 μl of 0.5% of low melting point agarose (LMPA) kept at 37 °C and added onto the microscope slide. After the top layer of agarose was solidified, the slides were immersed in lysis solution(2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, to which 1% Triton X-100 and 10% DMSO, pH 10) for 1 h at 4 °C in the dark. The slides were then covered with fresh alkaline buffer (1 mM EDTA and 300 mM NaOH, pH 13) in a horizontal electrophoresis unit. The cells were then exposed to the alkaline conditions for 20 min to allow DNA unwinding, and exposure of single-strand breaks and alkali-labile sites. Next, the DNA was electrophoresed at 25 V/300 mA (0.7 V/cm) for 20 min. All steps were conducted under subdued lighting to prevent additional DNA damage. After electrophoresis, the slides were neutralized with 0.4 M Tris (pH 7.5) and air-dried and stored at room temperature until DNA migration was checked. DNA was stained with propidium iodide (20 μg/ml distilled water, 25 μg per slide) right before observation.

[1]. G2 arrest and apoptosis by 2-amino-N-quinoline-8-yl-benzenesulfonamide (QBS), a novel cytotoxic compound. Biochem Pharmacol. 2005 May 1;69(9):1333-41.

We screened a library of 11,000 small molecular weight chemicals, looking for compounds that affect cell viability. We have identified 2-amino-N-quinoline-8-yl-benzenesulfonamide (QBS) as a potent cytotoxic compound that induces cell cycle arrest and apoptosis. Treatment of Jurkat T cells with QBS increased the levels of cyclin B1 as well as phosphorylated-cdc2, which was accompanied by reduced activity of cdc2 kinase, suggesting that QBS may induce cell cycle arrest at G2 phase. Structural analogues of QBS also exhibited similar effects on cell cycle progression and cell viability. Long-term treatment with QBS resulted in DNA fragmentation, cytochrome C release, and PARP cleavage, and an increase in the number of subdiploidy cells, indicative of cellular apoptosis. Moreover, QBS-induced apoptosis was blocked by z-VAD-fmk, a pan-caspase inhibitor. These results suggest that QBS is a novel and potent compound that induces G2 arrest and subsequent apoptosis, implicating it as a putative candidate for chemotherapy. [1]

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Interestingly, our SAR study with QBS analogues leads to the conclusion that the activity of QBS requires not only the presence, but also their correct steric configuration of both quinoline and sulfonamide groups. As described in Section 3, the constitutional structure of QBS-1 is the same as that of QBS, except for the presence of an amine group. The unsubstituted phenyl and quinoline groups in PQS are bounded to the nitrogen and the sulfur atom of the sulfonamide group, respectively, in the opposite way of QBS-1. Among the four analogues tested, two structural isomers of QBS, QBS-1 and PQS were able to induce G2 phase arrest, implying that the relative position of the nitrogen atom in the quinoline with sulfonamide group is not critical for the activity of elicited by these compounds. QBS-2 and QBS-3, however, containing p-methyl substituted phenyl and quinoline moieties failed to induce cell cycle arrest. The methyl group substituted at the phenyl ring endows structural differences of QBS-2 and QBS-3 versus QBS-1 and PQS. The effects of these structural analogues on cell cycle arrest corresponded to the results of cell viability assay. As expected, QBS-1 and PQS, but not QBS-2 and QBS-3, were capable of inducing cell death (date not shown). Collectively, these results indicate that even though these compounds have structural similarities, i.e., a sulfonamide group, a phenyl and a quinoline unit, the changes in basicity by tertiary amine and/or methyl group substitution can make important differences with respect to their abilities to cause DNA damage and G2 phase arrest. [1]
In conclusion, we suggest QBS as a novel, potent inducer of DNA damage and G2 arrest, followed by apoptosis. Moreover, QBS was found to affect cell cycle progression in other several leukemic cell lines, as well as in Jurkat T cells. Importantly, the SAR study revealed that the effect of QBS and its derivatives on cell cycle arrest and cell death are related to the spatial configuration of quinoline and sulfonamide groups, which can be useful information for the development of novel chemotherapeutic agents. We believe that further investigation into the mechanism responsible for QBS-induced apoptosis would provide chemotherapeutic strategies in leukemia therapy based on novel insights.[1]

C15H13N3O2S

299.34762

299.073

C, 60.19; H, 4.38; N, 14.04; O, 10.69; S, 10.71

16082-64-7

16082-64-7

1070159

Solid powder

1.437g/cm3

540.7ºC at 760 mmHg

280.8ºC

9.36E-12mmHg at 25°C

1.727

4.352

2

5

3

21

448

0

NC1=CC=CC=C1S(NC1=CC=CC2=CC=CN=C12)(=O)=O

NIOOKXAMJQVDGB-UHFFFAOYSA-N

InChI=1S/C15H13N3O2S/c16-12-7-1-2-9-14(12)21(19,20)18-13-8-3-5-11-6-4-10-17-15(11)13/h1-10,18H,16H2

2-amino-N-quinolin-8-ylbenzenesulfonamide

CU-242; CU242; CU 242; 16082-64-7; 2-Amino-N-quinolin-8-yl-benzenesulfonamide; DTXSID90360049; DTXCID80311101; 633-904-4; Benzenesulfonamide, 2-amino-N-8-quinolinyl-; NUN82647; 2-amino-N-(quinolin-8-yl)benzenesulfonamide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)