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Purity: ≥98%
RSL3 is a novel and potent inhibitor of glutathione peroxidase 4 (GPX4), which is able to reduce the expression of GPX4 protein, and induces ferroptotic death of head and neck cancer cell. RSL3 increases the expression of p62 and Nrf2 and inactivates Keap1 in HN3-rslR cells.
| Targets |
GPX4/glutathione peroxidase 4
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|---|---|
| ln Vitro |
With an IC50 of 5.8 μM in HN3-rslR cells and 0.48 μM in HN3 cells, RSL3 (0-8 μM, 72 hours) significantly lowers the viability of HN3 cells [1]. In HN3-rslR cells, RSL3 (0-8 μM, 24 hours) inactivates Keap1, raises the expression of p62 and Nrf2, and decreases the expression of GPX4 protein [1].
RSL3 or ML-162 induced the ferroptosis of HNC cells to varying degrees.[1] Resistance to ferroptosis was associated with p62 and Nrf2 expression in RSL3-treated HNC cells.[1] Inhibition of Nrf2 sensitized chemoresistant HNC cells to RSL3 treatment in vitro [1]. |
| ln Vivo |
Factor HN3R cell tumor growth can be markedly inhibited by combining trigonelline with RSL3 (100 mg/kg, intratumoral injection, twice weekly for 20 days) [1].
In mice xenograft models, all the mice survived well during and after cell implantation and treatment with vehicle, RSL3, trigonelline, or RSL3 plus trigonelline. They were euthanized 20 days after treatment. RSL3 or trigonelline alone did not significantly inhibit in vivo tumor growth compared with the vehicle control (P > 0.1) (Fig. 6A and B). However, tumor growth was significantly suppressed by RSL3 plus trigonelline (P < 0.01). Body weight and daily food intake did not change significantly in the control or treatment groups (P < 0.05) (Fig. 6C). The levels of ferrous iron, lipid ROS, and RPA measured in the in vivo tumors were significantly higher in the RSL3 plus trigonelline combination group than the control or other treatment groups (P < 0.01). A histological examination of vital organs did not reveal any significant differences between the groups [1]. |
| Enzyme Assay |
ROS production measurement[1]
Cellular ROS generation in the supernatant of HNC cell lysates treated for 24 h was measured by adding, for 30 min at 37 °C, either 10 µM 2′,7′-dichlorofluorescein diacetate for cytosolic ROS or 2 µM C11-BODIPY C11 for lipid peroxidation. ROS levels were analyzed using a FACSCalibur flow cytometer equipped with CellQuest Pro. Labile iron pool (LIP) and iron assays[1] Each sample was seeded in plates and treated with the indicated drugs. After 4 h, the supernatant was removed and the plates were washed twice with Hank's Balanced Salt Solution (HBSS). The cells were labeled by adding 8 μg/ML Calcein AM in HBSS and were incubated on plates for 30 min at 37 °C. After 30 min, the labeling solution was removed and the cells washed twice with PBS before trypsinization with Trypsin-EDTA. They were then neutralized by adding 4% FBS in HBSS and centrifuged for 3 min at 1500 rpm. The collected cells were washed once with HBSS while vortexing and were centrifuged for 3 min at 1500 rpm. They were then resuspended in 250 μL HBSS while vortexing, and go to FACS. Labile iron levels were analyzed using a FACSCalibur flow cytometer equipped with CellQuest Pro, and ferrous iron levels in the cells or tissue extracts were measured by using an iron assay kit. Nrf2 transcriptional activity assay[1] The transcriptional activity of Nrf2 was assayed using a Cignal Antioxidant Response Reporter kit, according to the manufacturer's instructions. |
| Cell Assay |
Cell viability assay[1]
Cell Types: HN3 cells, HN3-rslR Cell Tested Concentrations: 0-8 μM Incubation Duration: 72 hrs (hours) Experimental Results: IC50 values in HN3 and HN3-rslR cells were 0.48 µM and 5.8 µM respectively[1] . Western Blot Analysis [1] Cell Types: HN3-rslR Cell Tested Concentrations: 0-8 μM Incubation Duration: 24 hrs (hours) Experimental Results: Inhibited GPX4 expression, increased p62 and Nrf2 levels, and diminished Keap1 levels. |
| Animal Protocol |
Animal/Disease Models: Tenweeks old athymic BALB/c male nude mice (nu/nu) carrying HN3R cells [1]
Doses: 100 mg/kg combined with trigonelline (50 mg/kg): intratumoral injection, Experimental Results: Significant reduction in tumor volume in mice treated with trigonelline twice a week for 20 days. Tumor xenograft[1] Ten-week-old athymic BALB/c male nude mice (nu/nu) were used and HN3R cells were injected subcutaneously into the flank of each. As soon as gross nodules from the tumor implants were detected, the mice were subjected to one of four treatments: vehicle; RSL3 (100 mg/kg intratumorally twice per week); trigonelline (50 mg/kg daily via oral administration); or RSL3 plus trigonelline. Each group included 10 mice. Tumor size and body weight were measured twice a week, and the tumor volume was calculated as (length × width2)/2. After scarification, the tumors were isolated and cellular lipid ROS and ferrous iron levels were measured. |
| References |
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| Additional Infomation |
Glutathione peroxidase 4 (GPX4) is a regulator of ferroptosis (iron-dependent, non-apoptotic cell death); its inhibition can render therapy-resistant cancer cells susceptible to ferroptosis. However, some cancer cells develop mechanisms protective against ferroptosis; understanding these mechanisms could help overcome chemoresistance. In this study, we investigated the molecular mechanisms underlying resistance to ferroptosis induced by GPX4 inhibition in head and neck cancer (HNC). The effects of two GPX4 inhibitors, (1S, 3R)-RSL3 and ML-162, and of trigonelline were tested in HNC cell lines, including cisplatin-resistant (HN3R) and acquired RSL3-resistant (HN3-rslR) cells. The effects of the inhibitors and trigonelline, as well as of inhibition of the p62, Keap1, or Nrf2 genes, were assessed by cell viability, cell death, lipid ROS production, and protein expression, and in mouse tumor xenograft models. Treatment with RSL3 or ML-162 induced the ferroptosis of HNC cells to varying degrees. RSL3 or ML-162 treatment increased the expression of p62 and Nrf2 in chemoresistant HN3R and HN3-rslR cells, inactivated Keap1, and increased expression of the phospho-PERK-ATF4-SESN2 pathway. Transcriptional activation of Nrf2 was associated with resistance to ferroptosis. Overexpression of Nrf2 by inhibiting Keap1 or Nrf2 gene transfection rendered chemosensitive HN3 cells resistant to RSL3. However, Nrf2 inhibition or p62 silencing sensitized HN3R cells to RSL3. Trigonelline sensitized chemoresistant HNC cells to RSL3 treatment in a mouse model transplanted with HN3R. Thus, activation of the Nrf2-ARE pathway contributed to the resistance of HNC cells to GPX4 inhibition, and inhibition of this pathway reversed the resistance to ferroptosis in HNC.[1]
|
| Molecular Formula |
C23H21CLN2O5
|
|---|---|
| Molecular Weight |
440.8762
|
| Exact Mass |
440.113
|
| Elemental Analysis |
C, 62.66; H, 4.80; Cl, 8.04; N, 6.35; O, 18.14
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| CAS # |
1219810-16-8
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| PubChem CID |
1750826
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
641.3±55.0 °C at 760 mmHg
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| Flash Point |
341.6±31.5 °C
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| Vapour Pressure |
0.0±1.9 mmHg at 25°C
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| Index of Refraction |
1.636
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| LogP |
3.07
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
|
| Heavy Atom Count |
31
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| Complexity |
696
|
| Defined Atom Stereocenter Count |
2
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| SMILES |
COC(=O)[C@H]1CC2=C([C@@H](N1C(=O)CCl)C3=CC=C(C=C3)C(=O)OC)NC4=CC=CC=C24
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| InChi Key |
TXJZRSRTYPUYRW-NQIIRXRSSA-N
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| InChi Code |
InChI=1S/C23H21ClN2O5/c1-30-22(28)14-9-7-13(8-10-14)21-20-16(15-5-3-4-6-17(15)25-20)11-18(23(29)31-2)26(21)19(27)12-24/h3-10,18,21,25H,11-12H2,1-2H3/t18-,21+/m1/s1
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| Chemical Name |
methyl (1S,3R)-2-(2-chloroacetyl)-1-(4-methoxycarbonylphenyl)-1,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylate
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| Synonyms |
(1S,3R)-RSL3; 1S,3R-RSL3; RSL3 (1S,3R-); CHEMBL4747331; (1S,3R)-Methyl 2-(2-chloroacetyl)-1-(4-(methoxycarbonyl)phenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate; RSL3 1S,3R-;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~226.82 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 5 mg/mL (11.34 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.67 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.72 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. Solubility in Formulation 4: ≥ 0.56 mg/mL (1.27 mM) (saturation unknown) in 10% DMF 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 5: 20 mg/mL (45.36 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2682 mL | 11.3410 mL | 22.6819 mL | |
| 5 mM | 0.4536 mL | 2.2682 mL | 4.5364 mL | |
| 10 mM | 0.2268 mL | 1.1341 mL | 2.2682 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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