Figure 6. Celastrol identified as a novel HSD3B7 inhibitor that impairs ccRCC cells growth in vitro and in vivo.

A, Reaction scheme for monitoring HSD3B7 activity via luminescence assay.

B, Percent of control (POC) for high throughput screening compounds towards HSD3B7 and coupling enzymes. Compounds with less than 20% HSD3B7 activity and greater than 75% counter screening enzyme activity are highlighted in red.

C, IC₅₀ Dose-response curves for isoginkgetin and celastrol on HSD3B7 activity (n=3).

D, Structure comparison between celastrol and 7α-hydroxycholesterol.

E, Annexin-V/PI staining and flow cytometry analysis performed on A498 cells after 24h of celastrol treatment (1, 2, 5μM).

F, Proliferation assay performed on A498 cells grown in media with 10% FBS and treated with celastrol (2μM), or vehicle control (DMSO).

G, Annexin-V/PI staining and flow cytometry analysis performed on RPTEC cells after 24h of celastrol treatment (2μM).

H, Liquid chromatography-tandem mass spectrometry (LC/MS) analysis assessing HSD3B7 upstream product, 7α-hydroxycholesterol in A498 cells treated with celastrol (2μM), or vehicle control (DMSO) for indicated time point.

I, Tumor growth curves from A498 cells subcutaneously implanted in NIH-III nude mice treated with vehicle (PEG400 (45%), 1X PBS (45%), and EtOH (10%)) or celastrol (0.2mg/kg) by intraperitoneal injection daily for 23 days after tumor volume reached ~100mm³. Tumor volume was assessed at the indicated timepoints using caliper measurements (n=9 mice for celastrol treated group and n=8 mice for vehicle treated group).

J, Tumor weight from A498 cells subcutaneously implanted in NIH-III nude mice treated with vehicle or celastrol (0.2mg/kg) by intraperitoneal injection daily for 23 days.

K, Representative photographs of A498 tumors grown in NIH-III nude mice treated with vehicle or celastrol (0.2mg/kg) by intraperitoneal injection daily for 23 days.

L, Tumor volume from A498 cells subcutaneously implanted in NIH-III nude mice treated with vehicle or celastrol (0.2mg/kg) by intraperitoneal injection daily for 23 days.

M, Liquid chromatography-tandem mass spectrometry (LC/MS) analysis assessing HSD3B7 upstream product, 7α-hydroxy-cholesterol in A498 tumors treated with celastrol (0.2 mg/kg) or vehicle control for 23 days.

N, Liquid chromatography-tandem mass spectrometry (LC/MS) analysis assessing cholesterol in A498 tumors treated with celastrol (0.2 mg/kg) or vehicle control for 23 days.

(All experiments were performed in at least triplicates and statistical analysis was applied with *=P<0.05, **=P<0.01, ***=<0.001, n.s=non-significant).