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Prostratin

Cat No.:V5545 Purity: ≥98%
Prostratin is a naturally occurring diterpenoid that is an activator of PKC with Ki of 12.5 nM and can inhibit the activity of HIV-1.
Prostratin
Prostratin Chemical Structure CAS No.: 60857-08-1
Product category: New15
This product is for research use only, not for human use. We do not sell to patients.
Size Price Stock Qty
1mg
5mg
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Product Description
Prostratin is a naturally occurring diterpenoid that is an activator of PKC with Ki of 12.5 nM and can inhibit the activity of HIV-1.
Biological Activity I Assay Protocols (From Reference)
ln Vitro
Prostratin has a Ki of 210 nM and transcribes [3H]PDBu binding to CEM cells [1]. Transcriptional acute myeloid leukemia (AML) cell lines (HL-60, NB4, and U937 cells) are not able to proliferate at concentrations of prostratin (125-1000 nM). In HL-60 cells, prostatretin (125–100 nM) influences cell cycle-related molecules (pRb phosphorylation, CDK, and p21) and causes G1 arrest in AML cells. Prostratin also activates PKC, which causes AML cell lines to become active. Moreover, humidity produced by prostaglandin is necessary for PKC-driven activation of MEK/ERK/MAP signal load [2]. For HIV-1-induced activation, prostatin induction necessitates the active form of PKD3. Moreover, prostratein activates PKD3 via PKCε of the new PKC subfamily [2].
Cell Assay
Cell viability assay [2]
Cell Types: HL-60, NB4 and U937 Cell
Tested Concentrations: 125nM, 250nM, 500nM, 1000nM
Incubation Duration: 24 hrs (hours), 48 hrs (hours), 72 hrs (hours)
Experimental Results: dose-dependent inhibition 3]. Growth of acute myeloid leukemia (AML) cell lines.

Cell cycle analysis[2]
Cell Types: HL-60, NB4 and U937 Cell
Tested Concentrations: 125 nM, 250 nM, 500 nM, 1000 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced G0/G1 phase accumulation in a concentration-dependent manner.

Western Blot Analysis[2]
Cell Types: HL-60 Cell
Tested Concentrations: 125 nM, 250 nM, 500 nM, 1000 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: Effects of cell cycle related molecules (pRb phosphorylation, CDK and p21) in HL- in 60 cells.
References

[1]. A nonpromoting phorbol from the samoan medicinal plant Homalanthus nutans inhibits cell killing by HIV-1. J Med Chem. 1992 May 29;35(11):1978-86.

[2]. The protein kinase C agonist prostratin induces differentiation of human myeloid leukemia cells and enhances cellular differentiation by chemotherapeutic agents. Cancer Lett. 2015 Jan 28;356(2 Pt B):686-96.

[3]. Protein kinase D3 is essential for prostratin-activated transcription of integrated HIV-1 provirus promoter via NF-κB signaling pathway. Biomed Res Int. 2014;2014:968027.

Additional Infomation
Prostratin is a phorbol ester. It has a role as a metabolite.
Prostratin has been reported in Euphorbia triangularis, Euphorbia fischeriana, and other organisms with data available.
These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C22H30O6
Molecular Weight
390.4700
Exact Mass
390.204
CAS #
60857-08-1
PubChem CID
454217
Appearance
White to off-white solid powder
Density
1.3±0.1 g/cm3
Boiling Point
550.5±50.0 °C at 760 mmHg
Melting Point
216-219℃
Flash Point
188.7±23.6 °C
Vapour Pressure
0.0±3.4 mmHg at 25°C
Index of Refraction
1.600
LogP
1.84
Hydrogen Bond Donor Count
3
Hydrogen Bond Acceptor Count
6
Rotatable Bond Count
3
Heavy Atom Count
28
Complexity
825
Defined Atom Stereocenter Count
7
SMILES
C[C@@H]1C[C@@]2([C@@H](C2(C)C)[C@H]3[C@]1([C@@H]4C=C(C(=O)[C@]4(CC(=C3)CO)O)C)O)OC(=O)C
InChi Key
BOJKFRKNLSCGHY-HXGSDTCMSA-N
InChi Code
InChI=1S/C22H30O6/c1-11-6-16-20(26,18(11)25)9-14(10-23)7-15-17-19(4,5)21(17,28-13(3)24)8-12(2)22(15,16)27/h6-7,12,15-17,23,26-27H,8-10H2,1-5H3/t12-,15+,16-,17-,20-,21+,22-/m1/s1
Chemical Name
[(1R,2S,6R,10S,11R,13S,15R)-1,6-dihydroxy-8-(hydroxymethyl)-4,12,12,15-tetramethyl-5-oxo-13-tetracyclo[8.5.0.02,6.011,13]pentadeca-3,8-dienyl] acetate
HS Tariff Code
2934.99.9001
Storage

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.
Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
DMSO : ~50 mg/mL (~128.05 mM)
Solubility (In Vivo)
Solubility in Formulation 1: ≥ 1.25 mg/mL (3.20 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 12.5 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.

Solubility in Formulation 2: ≥ 1.25 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 12.5 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: ≥ 1.25 mg/mL (3.20 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.


 (Please use freshly prepared in vivo formulations for optimal results.)
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 2.5610 mL 12.8051 mL 25.6102 mL
5 mM 0.5122 mL 2.5610 mL 5.1220 mL
10 mM 0.2561 mL 1.2805 mL 2.5610 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.

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What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?
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What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:
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  • The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box
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Note: Chemical formula is case sensitive: C12H18N3O4  c12h18n3o4
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In vivo Formulation Calculator (Clear solution)
Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)
Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)
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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.

Biological Data
  • The active form of PKD3 is required for prostratin-induced HIV-1 transcription activation. (a) Expression profile of 3 PKDs in HeLa and Jurkat cells. RNA isolated from HeLa or Jurkat cells was analyzed by qRT-PCR for the expression levels of PKD1, PKD2, or PKD3, respectively. Data from 3 independent experiments were averaged and plotted. (b) Effect of shRNA knockdown on prostratin-activated HIV-1 expression in HeLa cells. The cells were cotransfected with HIV-LTR-Luc reporter construct and indicated shRNA for 48 hrs, followed by a 6 hr treatment of 2 μM prostratin as indicated. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (c) Effect of shRNA knockdown on prostratin-activated expression of latent HIV-1 provirus in J-Lat 2D10 cells. The cells were infected with indicated shRNA for 48 hrs, followed by a 16 hr treatment of 0.5 μM prostratin as indicated. The GFP-positive cells were detected by flow cytometry and plotted based on 3 independent experiments as in Figure 1(b). (d) Prostratin activates PKD3 by inducing the phosphorylation at Ser731/735 of its activation loop. HeLa cells transfected with GFP-PKD3 cDNA were treated with 2 μM of prostratin for indicated time. The phosphorylation levels of Ser731/735 of GFP-PKD3 in cell lysates were measured by Western blotting, with the levels of bulk GFP-PKD3 shown below.[3]. Wang H, et al. Protein kinase D3 is essential for prostratin-activated transcription of integrated HIV-1 provirus promoter via NF-κB signaling pathway. Biomed Res Int. 2014;2014:968027.
  • Prostratin activates PKD3 via PKCε of novel PKC subfamily. (a) Effect of PKC/PKD inhibitor on prostratin-stimulated HIV-1 expression in HeLa cells. HeLa HIV-LTR-Luc cells were pretreated with indicated inhibitor for 1 hr, followed by 2 μM prostratin treatment for 6 hrs. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated sample set to 1.0. (b) Effect of PKC/PKD inhibitor on prostratin-stimulated expression of latent HIV-1 provirus in 2D10 Jurkat cells. 2D10 cells were cotreated with indicated inhibitor plus 0.5 μM prostratin for 16 hrs, followed by flow cytometry assay. The percentage of GFP-positive cells was plotted based on 3 independent experiments. (c) Effect of PKC inhibitors on prostratin-induced PKD3 activation. HeLa cells transfected with GFP-PKD3 were pretreated with indicated kinase inhibitor for 1 hr, followed by 2 μM prostratin treatment for 2 hrs. The phosphorylation levels of Ser731/735 of expressed GFP-PKD3 in cell lysates were analyzed by Western blotting, with the levels of bulk GFP-PKD3 shown at the bottom. (d) Effect of nPKC knockdown on prostratin-induced HIV-1 expression. HeLa cells were cotransfected HIV-LTR-luciferase construct with shRNA for PKCθ or PKCε for 48 hrs, followed by 2 μM prostratin treatment for 6 hrs. The levels of luciferase activity were plotted based on 3 independent experiments, with the level of untreated sample set to 1.0. [3]. Wang H, et al. Protein kinase D3 is essential for prostratin-activated transcription of integrated HIV-1 provirus promoter via NF-κB signaling pathway. Biomed Res Int. 2014;2014:968027.
  • Prostratin-induced HIV-1 transcription depends on NF-κB. (a) Schematics of HIV-1 promoter deletion mutations. dSp1 (without Sp1 binding sites), dEnh (without NF-κB enhancer element), dTAR (without TAR RNA sequences), or dSp1/dEnh (without Sp1 or NF-κB enhancer element). (b) Effect of promoter mutation on prostratin-induced HIV-1 transcription. HeLa cells were transfected with HIV-LTR-Luc reporter constructs containing the indicated promoter deletions, followed by prostratin treatment. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0. (c) Effect of prostratin treatment on RelA and Sp1 recruitment to promoter. HIV-LTR-Luc cells were treated with prostratin for 1 hr and subjected to chromatin immunoprecipitation (ChIP) analysis with indicated antibody. The levels of DNA isolated by ChIP were analyzed by quantitative PCR (qPCR) with the primers targeting promoter region of HIV-LTR as indicated on the left and plotted based on 2 independent experiments, with the level of untreated cells set to 1.0. (d) Effect of inhibiting NF-κB signaling on prostratin-stimulated HIV-1 transcription. HIV-LTR-Luc cells were pretreated with inhibitor BAY, followed by prostratin treatment. The luciferase activities were plotted based on 3 independent experiments, with the level of untreated cells set to 1.0.[3]. Wang H, et al. Protein kinase D3 is essential for prostratin-activated transcription of integrated HIV-1 provirus promoter via NF-κB signaling pathway. Biomed Res Int. 2014;2014:968027.
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