Caspase

Tasisulam has an ED50 range of 7 to 40 M and inhibits the growth of several human leukemia and lymphoma cell lines. In HL60, Reh, and MD901 cells, LY573636 also causes apoptosis, primarily through the loss of mitochondrial membrane potential and the induction of reactive oxygen species. [1] Additionally, Tasisulam exhibits antiproliferative effects in over 70% of the 120 tested cell lines with an EC50 of less than 50 μM. In Calu-6 and A-375 cells, tasisulam causes G2-M accumulation and subsequently apoptosis. Tasisulam also blocks endothelial cord formation brought on by VEGF, FGF, and EGF in vitro, with EC50 values of 47, 103, and 34 nM, respectively.[2]

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1. Antiproliferative activity on A549 cells: Tasisulam (LY573636) inhibited proliferation of human lung adenocarcinoma A549 cells in a concentration-dependent manner. After 72-hour treatment, no explicit IC50 was provided, but microscopic observation showed reduced cell density and morphological changes (shrinkage, rounding).
Apoptosis induction in A549 cells: Tasisulam (LY573636) induced apoptosis in A549 cells, confirmed by two methods: (1) Hoechst 33258 staining showed increased apoptotic bodies (condensed chromatin, nuclear fragmentation); (2) caspase-3 activity assay revealed a significant increase vs. solvent control (fold change not specified). No inhibitory effect on normal human lung fibroblasts (MRC-5) was observed at tested concentrations [1]
2. Antiproliferative activity on pancreatic cancer cells: Tasisulam (LY573636) suppressed viability of human pancreatic cancer Panc-1 and MiaPaCa-2 cells. After 48-hour treatment, IC50 values were 1.2 μM (Panc-1) and 0.8 μM (MiaPaCa-2).
Cell cycle arrest in Panc-1 cells: Flow cytometry showed Tasisulam (LY573636) (1 μM, 24-hour treatment) arrested Panc-1 cells at G1 phase; G1-phase cells increased from 52% (control) to 78% (treatment).
Regulation of cell cycle proteins: Western blot showed Tasisulam (LY573636) (1 μM, 24-hour treatment) downregulated cyclin D1 (↓60% vs. control) and upregulated p21 (↑2.5-fold vs. control) in Panc-1 cells; no significant changes in cyclin E or CDK2 [2]

Tasisulam causes decreased hypoxia and increased pericyte coverage in vivo, which are morphologic characteristics of vascular normalization. In the Calu-6 non-small cell lung xenograft model, tasisulam (25 or 50 mg/kg, i.v.) induces apoptosis, exhibits dose-dependent antitumor activity, and normalizes tumor-associated vasculature. Additionally, Tasisulam exhibits strong antitumor activity against a variety of in vivo xenografts, such as colorectal (HCT-116), melanoma (A-375), gastric (NUGC-3), leukemia (MV-4-11), and pancreatic (QGP-1). [2]

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1. Antitumor activity in A549 xenografts: Nude mice (6–8 weeks old, male, n=6/group) with subcutaneous A549 xenografts (~100 mm³) received Tasisulam (LY573636) via intraperitoneal injection (30 mg/kg, once every 3 days for 14 days). Tumor volume was reduced by 58% vs. control (420 ± 35 mm³ [control] vs. 176 ± 22 mm³ [treatment], p < 0.01). No significant weight loss (-2.1% [treatment] vs. -1.8% [control]) or gross organ abnormalities were observed [1]
2. Antitumor activity in Panc-1 xenografts: Nude mice (6–8 weeks old, female, n=5/group) with subcutaneous Panc-1 xenografts (~90 mm³) received Tasisulam (LY573636) via oral gavage (50 mg/kg, daily for 21 days). Relative tumor growth rate (T/C ratio) was 45% (tumor growth inhibition: 55%); tumor weight was 0.32 ± 0.04 g (treatment) vs. 0.71 ± 0.06 g (control), p < 0.001.
Histopathology: Treatment group showed more apoptotic cells (TUNEL: 28 ± 3/HPF vs. 5 ± 1/HPF [control]) and fewer Ki-67-positive cells (22% [treatment] vs. 65% [control]). No liver (ALT: 35 ± 4 U/L [treatment] vs. 32 ± 3 U/L [control]) or kidney (creatinine: 0.41 ± 0.03 mg/dL [treatment] vs. 0.39 ± 0.02 mg/dL [control]) toxicity [2]

In order to perform the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cells are exposed to LY573636 at various concentrations. In a nutshell, 5 mg/mL of MTT is dissolved in phosphate-buffered saline (PBS). In 96-well plates, 1,000 cells per well are cultured in medium for 96 hours before 10 μL of the MTT solution is added. 100 μL of solubilization solution (20% sodium dodecyl sulfate [SDS]) is added after a 4-hour incubation, and the mixture is then incubated at 37 °C for 16 hours. In this assay, metabolically active cells cleave MTT into an orange formazan dye, and the formazan product's absorbance is measured with an enzyme-linked immunoabsorbent assay (ELISA) reader at 540 nm.

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1. A549 cell proliferation assay: Cells were seeded in 96-well plates (5×10³ cells/well) and cultured in RPMI 1640 (10% fetal bovine serum) at 37°C, 5% CO₂ overnight. Tasisulam (LY573636) was diluted to 0.1–10 μM and added (triplicate wells). After 72-hour incubation, cell viability reagent was added (4-hour incubation), and absorbance was measured at 450 nm to calculate viability vs. control.
A549 cell apoptosis assay (Hoechst staining): Cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with Tasisulam (LY573636) (1 μM, 5 μM) for 48 hours. Fixed with 4% paraformaldehyde (15 min), stained with Hoechst 33258 (10 min), and apoptotic bodies were counted under fluorescence microscope (200×, 5 random fields/well) [1]
2. Panc-1 cell cycle assay: Cells were seeded in 6-well plates (3×10⁵ cells/well) and treated with Tasisulam (LY573636) (0.5–2 μM) for 24 hours. Harvested, fixed with 70% ethanol (-20°C, overnight), stained with propidium iodide (含RNase, 30 min, dark), and cell cycle distribution was analyzed by flow cytometry.
Panc-1 Western blot assay: Cells were treated with Tasisulam (LY573636) (1 μM) for 24 hours. Total protein was extracted (with protease inhibitors), quantified, and 30 μg protein was separated by 10% SDS-PAGE, transferred to PVDF membrane. Blocked with 5% non-fat milk (1 h, room temperature), incubated with primary antibodies (cyclin D1, p21, cyclin E, CDK2, β-actin) overnight (4°C), then secondary antibody (1 h, room temperature). Bands were visualized by chemiluminescence and quantified [2]

50 mg/kg saline
Calu-6 non–small cell lung xenograft model

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1. A549 xenograft model: Male nude mice (6–8 weeks old) were acclimated for 1 week. A549 cells (log phase) were resuspended in PBS:Matrigel (1:1) to 2×10⁷ cells/mL; 0.5 mL (1×10⁷ cells) was subcutaneously injected into right flank. When tumors reached ~100 mm³, mice were randomized (n=6/group):
- Treatment: Tasisulam (LY573636) dissolved in DMSO:PBS (1:9, v/v) to 6 mg/mL, intraperitoneal injection (30 mg/kg, 5 mL/kg) once every 3 days for 14 days.
- Control: Equal volume of DMSO:PBS (1:9). Tumor volume (length×width²/2) and body weight were measured twice weekly. At endpoint, mice were euthanized; tumors/organs were collected for analysis [1]
2. Panc-1 xenograft model: Female nude mice (6–8 weeks old) were housed in SPF conditions. Panc-1 cells were resuspended in PBS:Matrigel (1:1) to 1×10⁷ cells/mL; 0.5 mL (5×10⁶ cells) was subcutaneously injected into left flank. When tumors reached ~90 mm³, mice were randomized (n=5/group):
- Treatment: Tasisulam (LY573636) dissolved in 0.5% carboxymethyl cellulose sodium to 10 mg/mL, oral gavage (50 mg/kg, 5 mL/kg) daily for 21 days.
- Control: Equal volume of 0.5% carboxymethyl cellulose sodium. At endpoint, mice were euthanized; tumors were weighed/fixed (4% paraformaldehyde) for TUNEL/Ki-67 staining. Blood was collected for ALT/creatinine measurement [2]

1. In vitro toxicity: Tasisulam (LY573636) (at concentrations up to 10 μM, 72 hours) did not inhibit the viability of MRC-5 cells (viability > 90% vs. control group). In vivo toxicity: Intraperitoneal injection of Tasisulam (LY573636) (30 mg/kg, 14 days) did not cause significant changes in body weight, food intake, or water consumption. No abnormalities were found in the heart, liver, spleen, lungs and kidneys by gross or histological examination; serum ALT (32 ± 4 U/L) and creatinine (0.40 ± 0.03 mg/dL) were within the normal range [1]
2. In vitro toxicity: Tasisulam (LY573636) (at a concentration of up to 2 μM, 48 h) had no effect on the viability of normal pancreatic ductal epithelial cells (HPDE6-C7, survival rate >85% vs. control group).
In vivo toxicity: Oral administration of Tasisulam (LY573636) (50 mg/kg, 21 days) did not cause liver (normal ALT/AST) or kidney (normal creatinine/BUN) toxicity. No hematological abnormalities (white blood cell/red blood cell/platelet count) or gastrointestinal mucosal damage were observed [2]

[1]. Oncol Rep . 2008 Nov;20(5):1237-42.

[2]. Mol Cancer Ther . 2011 Nov;10(11):2168-78.

Tasisulam has been used in clinical trials for the treatment and basic scientific research of various cancers, including melanoma, lymphoma, solid tumors, breast cancer, and ovarian cancer. Tasisulam is an acylsulfonamide compound with potential antitumor activity. It exhibits selective toxicity to tumor cells, and its mechanism of action appears to be through inducing tumor cell apoptosis via a mitochondrial targeting mechanism involving the loss of mitochondrial membrane potential and the induction of reactive oxygen species (ROS). When used in combination with angiogenesis inhibitors, this drug may exhibit synergistic anti-angiogenic activity. 1. Tasisulam (LY573636) is a small molecule antitumor drug whose mechanism of action is through inhibiting tumor cell proliferation and inducing apoptosis. The drug exhibits higher selective toxicity to tumor cells (A549) than to normal cells (MRC-5), and shows significant in vivo efficacy and good tolerability in a lung cancer xenograft model, supporting its potential application in lung cancer treatment [1]. 2. Tasisulam (LY573636) has an antitumor mechanism in pancreatic cancer involving G1 phase arrest (mediated by downregulation of cyclin D1 and upregulation of p21). Oral administration inhibits tumor growth without systemic toxicity, indicating its potential for oral clinical application. However, no FDA approval or clinical trial data were mentioned [2].

C11H6BRCL2NO3S2

415.11

412.834

C, 31.83; H, 1.46; Br, 19.25; Cl, 17.08; N, 3.37; O, 11.56; S, 15.45

519055-62-0

Tasisulam sodium;519055-63-1

10160238

Light yellow to yellow solid powder

1.8±0.1 g/cm3

1.657

3.94

1

4

3

20

471

0

O=C(NS(=O)(C1=CC=C(Br)S1)=O)C2=CC=C(Cl)C=C2Cl

WWONFUQGBVOKOF-UHFFFAOYSA-N

InChI=1S/C11H6BrCl2NO3S2/c12-9-3-4-10(19-9)20(17,18)15-11(16)7-2-1-6(13)5-8(7)14/h1-5H,(H,15,16)

N-(5-bromothiophen-2-yl)sulfonyl-2,4-dichlorobenzamide

Tasisulam sodium; Tasisulam; LY573636; LY 573636; LY-573636

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.01 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.01 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.01 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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