VEGF-R2

In HUVEC, (Z)-GugguLsterone (10, 20 μM; 24 or 48 hours) lowers the levels of VEGF-R2 protein [1]. Through FXR-mediated ACE2 modulation, (Z)-Guggulsterone (10 μM; 24) decreases primary airways, disturbs ACE2 and SHP levels in organoids, and lessens SARS-CoV-2 infection in many cell types [2].

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The z-guggulsterone treatment inhibited capillary-like tube formation (in vitro neovascularization) by human umbilical vein endothelial cells (HUVEC) and migration by HUVEC and DU145 human prostate cancer cells in a concentration- and time-dependent manner. The z- and E-isomers of guggulsterone seemed equipotent as inhibitors of HUVEC tube formation[1].

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Our previous studies have shown that z-guggulsterone, a constituent of Indian Ayurvedic medicinal plant Commiphora mukul, inhibits the growth of human prostate cancer cells by causing apoptosis. We now report a novel response to z-guggulsterone involving the inhibition of angiogenesis in vitro and in vivo. The z-guggulsterone treatment inhibited capillary-like tube formation (in vitro neovascularization) by human umbilical vein endothelial cells (HUVEC) and migration by HUVEC and DU145 human prostate cancer cells in a concentration- and time-dependent manner. The z- and E-isomers of guggulsterone seemed equipotent as inhibitors of HUVEC tube formation. The z-guggulsterone-mediated inhibition of angiogenesis in vitro correlated with the suppression of secretion of proangiogenic growth factors [e.g., vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor], down-regulation of VEGF receptor 2 (VEGF-R2) protein level, and inactivation of Akt. The z-guggulsterone-mediated suppression of DU145 cell migration was increased by knockdown of VEGF-R2 protein level. Ectopic expression of constitutively active Akt in DU145 cells conferred protection against z-guggulsterone-mediated inhibition of cell migration [1].

(Z)-Guggulsterone (silica; 1 mg; 5 x weekly) dramatically lowers wet weight and tumor volume [1].
Oral gavage of 1 mg z-guggulsterone/d (five times/wk) to male nude mice inhibited in vivo angiogenesis in DU145-Matrigel plug assay as evidenced by a statistically significant decrease in tumor burden, microvessel area (staining for angiogenic markers factor VIII and CD31), and VEGF-R2 protein expression. In conclusion, the present study reveals that z-guggulsterone inhibits angiogenesis by suppressing the VEGF-VEGF-R2-Akt signaling axis. Together, our results provide compelling rationale for further preclinical and clinical investigation of z-guggulsterone for its efficacy against prostate cancer[1].

The z-guggulsterone-mediated inhibition of angiogenesis in vitro correlated with the suppression of secretion of proangiogenic growth factors [e.g., vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor], down-regulation of VEGF receptor 2 (VEGF-R2) protein level, and inactivation of Akt. The z-guggulsterone-mediated suppression of DU145 cell migration was increased by knockdown of VEGF-R2 protein level. Ectopic expression of constitutively active Akt in DU145 cells conferred protection against z-guggulsterone-mediated inhibition of cell migration[3].

Western Blot Analysis [1]
Cell Types: Vascular Endothelial Growth Factor (VEGF)
Tested Concentrations: 10, 20 μM
Incubation Duration: 24 or 48 hrs (hours)
Experimental Results: Caused a decrease in VEGF-R2 protein levels in HUVEC.

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Cell Culture and Cell Viability Assay [1]
HUVEC were purchased from Clonetics and maintained in endothelial cell growth medium-2 (EGM2 MV SingleQuots) supplemented with 5% fetal bovine serum. Monolayer cultures of DU145 cells were maintained as we have previously described. Stock solutions of each isomer of guggulsterone were prepared in DMSO and diluted with complete medium. An equal volume of DMSO (final concentration <0.2%) was added to the controls. The effects of z- and E-guggulsterone treatments on HUVEC viability was determined by sulforhodamine B assay as we have previously described.

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In vitro Capillary-Like Tube Structure Formation and Migration Assays [1]
The effects of z- and E-guggulsterone treatments on in vitro angiogenesis were determined by tube formation assay as we have previously reported. The HUVEC seeded on Matrigel differentiate and form capillary-like tube structures. In some tube formation experiments, the HUVEC were exposed to 20 μmol/L of z-guggulsterone for 24 h in the absence or presence of 1 μmol/L of the Akt-1/2 inhibitor. The effect of z-guggulsterone treatment on in vitro migration by HUVEC or DU145 cells was determined using a Transwell Boyden Chamber containing a polycarbonate filter (pore size 8 μm) as we have previously described. In some migration assays, HUVEC or DU145 cells were treated with 20 μmol/L of z-guggulsterone for 24 h in the absence or presence of 1 μmol/L of Akt-1/2 inhibitor.

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Immunoblotting [1]
The immunoblotting of total Akt, phosphorylated Akt, and VEGF-R2 was done as we have previously described. Briefly, HUVEC or DU145 cells were treated with desired concentrations of z-guggulsterone for specified time periods, and both floating and attached cells were collected. The cell lysates were prepared as we have previously described. The lysate proteins were resolved by 6% to 10% SDS-PAGE and transferred onto polyvinylidene fluoride membrane. After treatment with the desired primary and secondary antibodies, the immunoreactive bands were visualized using an enhanced chemiluminescence method. The blots were stripped and reprobed with antiactin antibody to correct for differences in protein loading. Changes in protein levels were determined by densitometric scanning of the immunoreactive bands. The immunoblotting for each protein was done at least twice using independently prepared lysates.

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Analyses of Growth Factors, Interleukins, and MMPs [1]
HUVEC or DU145 cells (2 × 105) were seeded in 24-well plates and allowed to attach by overnight incubation. Cells were treated with the desired concentrations of z-guggulsterone or DMSO (control) for 24 and 48 h. Subsequently, the culture medium was collected and used to determine the secretion of VEGF, EGF, G-CSF, FGF, IL-12, IL-17, MMP-2, and MMP-9 using commercially available ELISA kits as we have previously described.

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RNA Interference of VEGF-R2 [1]
RNA interference of VEGF-R2 was done using a VEGF-R2–targeted short interfering RNA (siRNA). A nonspecific control siRNA was purchased from Qiagen. For transfection, DU145 cells (5 × 104) were seeded in six-well plates and allowed to attach overnight. Cells were transfected with 200 nmol/L of control nonspecific siRNA or VEGF-R2–targeted siRNA using OligofectAMINE according to the manufacturer's recommendations. Twenty-four hours after transfection, the cells were treated with DMSO (control) or 20 μmol/L of z-guggulsterone for 24 h. The cells were collected and processed for analysis of migration and immunoblotting as described above.

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Ectopic Expression of Constitutively Active Akt [1]
DU145 cells were transiently transfected with pCMV6 vector encoding constitutively active Akt-1 (Myr-Akt1-HA) or empty vector using Fugene 6 transfection regent. Briefly, DU145 cells were seeded in six-well plates at a density of 2 × 105 cells/mL and allowed to attach by overnight incubation. Cells were transfected with the expression vector encoding constitutively active Akt or empty vector. Twenty-four hours after transfection, the cells were treated with 20 μmol/L of z-guggulsterone or DMSO (control) for 24 h and processed for immunoblotting of total or phosphorylated Akt levels and migration assay.

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ChIP [2]
Approximately 6 × 106 cells were used for each ChIP, and cells were incubated with fresh medium with 100 μM of CDCA, UDCA/Ursodeoxycholic acid or z-guggulsterone/ZGG 2 h before collection. ChIP was performed using the True Micro ChiP kit according to the manufacturer’s instructions. In brief, following pre-clearing, the lysate was incubated overnight with the FXR antibody (Supplementary Table 1) or non-immune IgG. ChIP was completed and immunoprecipitated DNA was purified using MicroChip DiaPure columns. Samples were analysed by qPCR using the ΔΔCt approach as previously described51 (see Supplementary Table 3 for primer sequences). Primers flanking the FXRE on the well-known FXR target gene OSTα (also known as SLC51A; ref. 54) were used as a positive control, whereas primers flanking a site distant from the FXRE on the ACE2 promoter were used as a negative control. The results were normalized to the enrichment observed with non-immune IgG ChIP controls.

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Luciferase reporter [2]
Two different fragments containing the FXRE IR-1 in the ACE2 gene and in the SHP gene (also known as NR0B2) were amplified using human genomic DNA as a template and inserted onto a pGL3-promoter luciferase vector. The ACE2 and SHP IR-1 mutants were generated using a site-directed mutagenesis approach. Sequences of primers used are reported in Supplementary Table 4. These gene reporter constructs were co-transfected with a commercially available FXR expression plasmid into HEK293 cells using TransIT-293 Transfection Reagent. Twenty-four hours after transfection, cells were treated with 50 μM of CDCA, UDCA/Ursodeoxycholic acid and z-guggulsterone/ZGG in fresh medium for 8 h. Luciferase activity was determined with the GLO-Luciferase Reporter Assay System and values were normalized to the empty pGL3 vector.

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Cytotoxicity and viability [2]
Primary organoids were treated with 0.1 μM–100 μM of CDCA, UDCA/Ursodeoxycholic acid or z-guggulsterone/ZGG and the percentage of viable cells was counted using trypan blue and a Countess II cell counter. Cellular viability in primary organoids treated with 10 μM of CDCA, UDCA or ZGG was measured using the resazurin-based assay PrestoBlue using SoftMax Pro 5.4.4 on a SpectraMax M2.

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Luciferase reporter for SARS-CoV-2 replication [2]
A luciferase reporter for SARS-CoV-2 protease activity during viral replication was generated as previously described28 In brief, HEK293T reporter cells stably expressing ACE2, renilla luciferase (Rluc) and SARS-CoV-2 papain-like protease-activatable circularly permuted firefly luciferase (FFluc) were seeded in flat-bottomed 96-well plates. The following morning, cells were treated with the indicated doses of CDCA, UDCA/Ursodeoxycholic acid and z-guggulsterone/ZGG, and infected with SARS-CoV-2 at a MOI of 0.01. The SARS-CoV-2 RdRp inhibitor remdesivir and a neutralizing antibody cocktail blocking the interaction between SARS-CoV-2 spike and ACE2 (REGN-COV2) were included as positive controls. After 24 h, cells were lysed in Dual-Glo Luciferase Buffer diluted 1:1 with PBS and 1% NP-40. Lysates were then transferred to opaque 96-well plates, and viral replication quantified as the ratio of FFluc/Rluc activity measured using the Dual-Glo kit according to the manufacturer’s instructions. FFluc/Rluc ratios were expressed as a fraction of the maximum, then analysed using the Sigmoidal, 4PL, X is log(concentration) function in GraphPad Prism.

Animal/Disease Models: Male nude mice (5-6 weeks old) were subcutaneously (sc) (sc) implanted with Matrigel plugs containing DU145 cells.
Doses: 1 mg.
Route of Administration: po (po (oral gavage)) 5 times a week.
Experimental Results: Resulting in statistically significant tumor volume and wet tumor weight. reduce.

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In vivo Matrigel Plug Assay [1]
The effect of z-guggulsterone administration on in vivo angiogenesis was determined by DU145-Matrigel plug assay. Male nude mice (5–6 weeks old) were purchased from Taconic and randomized into two groups of five mice per group. The mice were orally gavaged with 0.1 mL of vehicle (PBS) or 1 mg of z-guggulsterone/mouse in 0.1 mL of PBS (corresponding to ∼40 mg z-guggulsterone/kg body weight) five times per week for 2 weeks prior to Matrigel plug implantation. The Matrigel plugs containing 3 × 106 DU145 cells were implanted s.c. into the flank of each mouse. The z-guggulsterone and vehicle administration was continued for two more weeks. Tumor volume was determined by using a caliper as we have previously described. Body weights of the vehicle-treated control and z-guggulsterone–treated mice were recorded weekly. Mice from each group were also monitored for other symptoms of side effects, including food and water withdrawal and impaired posture or movement. Animals were sacrificed 14 days after Matrigel plug implantation. At the termination of the experiment, the Matrigel plugs from control and z-guggulsterone–treated mice were removed and fixed in 10% neutral-buffered formalin. The fixed Matrigel plugs from control and z-guggulsterone administered mice were dehydrated, embedded in paraffin, and sectioned at 4 μm of thickness. Sections from control and z-guggulsterone administered mice were used for immunohistochemical analysis of CD31, factor VIII, and VEGF-R2. Quantitative image analysis of the microvessel area based on CD31 and factor VIII immunostaining was done using Image Analysis software.

[1]. z-Guggulsterone, a constituent of Ayurvedic medicinal plant Commiphora mukul, inhibits angiogenesis in vitro and in vivo. Mol Cancer Ther. 2008 Jan;7(1):171-80.

[2]. FXR inhibition may protect from SARS-CoV-2 infection by reducing ACE2. Nature. 2023 Mar;615(7950):134-142.

Guggulsterone is a 3-hydroxy steroid. It has a role as an androgen.
Guggulsterone has been reported in Commiphora mukul and Commiphora wightii with data available.

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Our previous studies have shown that z-guggulsterone, a constituent of Indian Ayurvedic medicinal plant Commiphora mukul, inhibits the growth of human prostate cancer cells by causing apoptosis. We now report a novel response to z-guggulsterone involving the inhibition of angiogenesis in vitro and in vivo. The z-guggulsterone treatment inhibited capillary-like tube formation (in vitro neovascularization) by human umbilical vein endothelial cells (HUVEC) and migration by HUVEC and DU145 human prostate cancer cells in a concentration- and time-dependent manner. The z- and E-isomers of guggulsterone seemed equipotent as inhibitors of HUVEC tube formation. The z-guggulsterone-mediated inhibition of angiogenesis in vitro correlated with the suppression of secretion of proangiogenic growth factors [e.g., vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor], down-regulation of VEGF receptor 2 (VEGF-R2) protein level, and inactivation of Akt. The z-guggulsterone-mediated suppression of DU145 cell migration was increased by knockdown of VEGF-R2 protein level. Ectopic expression of constitutively active Akt in DU145 cells conferred protection against z-guggulsterone-mediated inhibition of cell migration. Oral gavage of 1 mg z-guggulsterone/d (five times/wk) to male nude mice inhibited in vivo angiogenesis in DU145-Matrigel plug assay as evidenced by a statistically significant decrease in tumor burden, microvessel area (staining for angiogenic markers factor VIII and CD31), and VEGF-R2 protein expression. In conclusion, the present study reveals that z-guggulsterone inhibits angiogenesis by suppressing the VEGF-VEGF-R2-Akt signaling axis. Together, our results provide compelling rationale for further preclinical and clinical investigation of z-guggulsterone for its efficacy against prostate cancer.[1]

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Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.[2]

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Tumor angiogenesis (neovascularization) is a highly complex process that is regulated by multiple proangiogenic growth factors and their corresponding receptors. Based on the results of the present study, it seems reasonable to conclude that inhibition of the VEGF–VEGF-R2–Akt signaling axis may be an important mechanism in the antiangiogenic effects of z-guggulsterone. This conclusion is supported by the following observations: (a) z-guggulsterone–mediated inhibition of tube formation and migration correlates with the suppression of secretion of VEGF, which provides prosurvival signals to normal and tumor-derived endothelial cells mediated by receptor tyrosine kinase VEGF-R2; (b) z-guggulsterone treatment down-regulates the protein levels of VEGF-R2; (c) z-guggulsterone–mediated suppression of DU145 cell migration is intensified by the knockdown of VEGF-R2 protein levels; (d) z-guggulsterone inhibits Akt in HUVEC and DU145 cells and inhibition of HUVEC tube formation by this agent is intensified by pharmacologic inhibition of Akt. However, the precise mechanism by which z-guggulsterone reduces the secretion of VEGF or down-regulates VEGF-R2 protein level is not clear and requires further investigation. In conclusion, the present study reveals that z-guggulsterone inhibits angiogenesis in vitro and in vivo. The z-guggulsterone–mediated inhibition of angiogenesis is associated with the inactivation of Akt, suppression of growth factor (VEGF and G-CSF), IL-17 and MMP-2 secretion, and down-regulation of VEGF-R2 protein expression.[1]

C21H28O2

312.45

312.208

C, 80.73; H, 9.03; O, 10.24

39025-23-5

39025-23-5

6450278

White to off-white solid powder

1.1±0.1 g/cm3

463.3±45.0 °C at 760 mmHg

188-190°

172.3±25.7 °C

0.0±1.1 mmHg at 25°C

1.557

3.65

0

2

0

23

640

5

C/C=C/1\C(=O)C[C@H]2[C@@H]3CCC4=CC(=O)CC[C@]4(C)[C@H]3CC[C@]12C

WDXRGPWQVHZTQJ-OSJVMJFVSA-N

InChI=1S/C21H28O2/c1-4-16-19(23)12-18-15-6-5-13-11-14(22)7-9-20(13,2)17(15)8-10-21(16,18)3/h4,11,15,17-18H,5-10,12H2,1-3H3/b16-4+/t15-,17+,18+,20+,21-/m1/s1

(8R,9S,10R,13S,14S,17Z)-17-ethylidene-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15-decahydrocyclopenta[a]phenanthrene-3,16-dione

Z-Guggulsterone; (Z)-Guggulsterone; Z-Guggulsterone; Guggulsterone; 39025-23-5; 95975-55-6; Guggulsterones Z; Cis-Guggulsterone; Guggulsterone E&Z; (Z)-Guggulsterone

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)

DMSO: 5~10 mg/mL (16.0~32.0 mM)
Ethanol: ~2 mg/mL (~6.4 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (3.20 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 10.0 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.

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Solubility in Formulation 2: 10 mg/mL (32.01 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.

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