LRP6 (inhibits phosphorylation), DVL2 (inhibits phosphorylation), GSK3β (inhibits Ser9 phosphorylation, thereby increasing its activity) [1]

Prodigiosin treatment (25–500 nM; 24 hours) decreased the viability of breast cancer cells, with an IC50 value of 261.2 in MDA-MB-468 cells and 62.52 nM in MDA-MB-231 cells at 48 hours. nM[1]. Prodigiosin treatment (25-500 nM; 24 hours) markedly decreased the levels of total β-catenin, active β-catenin, and phosphorylated LRP6 and DVL2. In HEK293T cells, prodigiosin strongly suppresses GSK3β Ser9 phosphorylation, indicating increased GSK3β activity [1]. Breast cancer cells can undergo apoptosis and be inhibited from proliferating when exposed to prodigiosine [1]. In transfected HEK293T cells, prodigiosin treatment (25–500 nM; 24 hours) dose-dependently inhibits Wnt signaling triggered by Wnt1, Wnt3, Wnt1/LRP6, Wnt3/LRP6, and Disheveled 2 (DVL2). The Wnt3A-CM-induced transcription was dose-dependently suppressed by prodigiosin administration. When Wnt transfection or Wnt3A therapy is applied, the SuperTopFlash reporter gene's transcription is inhibited by prodigiosin [1]. Applying this substance to cultures of Batrachochytrium dendrobatidis and B. Prodigiosin and salamandrivorans both significantly restrict growth, having MIC values of 10 μM and 50 μM, respectively [2].

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In HEK293T cells, Prodigiosin (25-500 nM) dose-dependently inhibited SuperTopFlash reporter activity induced by Wnt1, Wnt3, Wnt1/LRP6, Wnt3/LRP6, DVL2, and Wnt3A-conditioned medium (Wnt3A-CM), but did not suppress β-catenin-induced reporter activity. It also did not influence AP-1 or NFAT reporter gene luciferase activity [1].
In HEK293T cells transfected with Wnt1, Prodigiosin treatment (25-500 nM) for 24 h significantly reduced the levels of phosphorylated LRP6, phosphorylated DVL2 (slower-migrating band), active β-catenin, and total β-catenin [1].
In HEK293T cells, Prodigiosin treatment (25-500 nM) for 24 h noticeably inhibited the phosphorylation of GSK3β at Ser9, indicative of increased GSK3β activity [1].
In breast cancer MDA-MB-231 and MDA-MB-468 cells, Prodigiosin (25-500 nM) for 24 h decreased the levels of phosphorylated LRP6, total LRP6, phosphorylated and unphosphorylated DVL2, Ser9 phosphorylated GSK3β, active β-catenin, and total β-catenin [1].
In MDA-MB-231 and MDA-MB-468 cells, Prodigiosin at concentrations as low as 25 nM decreased the mRNA expression of cyclin D1, LEF1, and fibronectin (Wnt target genes), but had no effect on FZD5 expression. It also significantly suppressed the protein levels of cyclin D1 and LEF1 [1].
Prodigiosin reduced the viability of breast cancer cells with IC50 values at 48 h of 62.52 nM in MDA-MB-231 cells and 261.2 nM in MDA-MB-468 cells. It also resulted in a dose-dependent decrease in BrdU incorporation in these cells and induced apoptosis as indicated by the detection of cleaved caspase-3 [1].
Prodigiosin suppressed the migration of MDA-MB-231 and MDA-MB-468 cells in a dose-dependent manner in in vitro scratch and transwell assays, and significantly reduced the number of invaded cells in Matrigel-coated transwell assays [1].
Prodigiosin (10-50 µM) caused significant growth inhibition of Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). The minimum inhibitory concentrations (MIC) were 10 µM for Bd and 50 µM for Bsal. The IC50 was 3.8 µM for Bd and 27.3 µM for Bsal [2].
Prodigiosin did not show aerosol activity (non-volatile) against Bd or Bsal in a divided petri plate assay [2].

Tumor growth was markedly reduced by prodigiosin (5 mg/kg; intraperitoneal injection; twice weekly; for 3 weeks). Ki-67, a marker of proliferation, can be expressed less frequently in tumor cells when treated with prodigiosin [1].

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In a MDA-MB-231 breast cancer xenograft mouse model, intraperitoneal (i.p.) injection of Prodigiosin at 5 mg/kg twice weekly for 3 weeks significantly inhibited tumor growth, decreased tumor cell density (H&E staining), and reduced the expression of the proliferation marker Ki-67. Prodigiosin treatment also decreased the expression of active and total β-catenin (immunohistochemical staining), and significantly reduced protein levels of phosphorylated LRP6, phosphorylated and unphosphorylated DVL2, Ser9 phosphorylated GSK3β, active and total β-catenin, LEF1, and cyclin D1. Real-time PCR analyses showed a marked reduction in the mRNA expression of cyclin D1, LEF1, and fibronectin in prodigiosin-treated tumors [1].
In MMTV-Wnt1 transgenic mice, i.p. injection of Prodigiosin at 5 mg/kg twice weekly for 3 weeks caused tumor regression, reduced tumor cell density and proliferation (Ki-67 staining). Prodigiosin-treated tumors showed reduced expression of active and total β-catenin (immunohistochemical staining), and decreased levels of phosphorylated LRP6, phosphorylated and unphosphorylated DVL2, Ser9 phosphorylated GSK3β, and active and total β-catenin. A treatment-induced decrease in mRNA expression of cyclin D1, LEF1, and fibronectin was also observed [1].

Cell proliferation assay[1]
Cell Types: MDA-MB-231 and MDA-MB-468 Cell
Tested Concentrations: 10 nM, 25 nM, 50 nM, 100 nM, 250 nM, 500 nM, 1000 nM, 2500 nM, 5000 nM
Incubation Duration: 24 hrs (hours), 48 hrs (hours).
Experimental Results: Reduce the viability of breast cancer cells. The IC50 value of MDA-MB-231 cells at 48 hrs (hours) was 62.52 nM, and the IC50 value of MDA-MB-468 cells was 261.2 nM.

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Western Blot Analysis[1]
Cell Types: HEK293T Cell
Tested Concentrations: 50 nM, 100 nM, 250 nM, 500 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: Dramatically diminished levels of phosphorylated LRP6 and DVL2, active β-catenin and total β- Catenin.

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For SuperTopFlash reporter assays, HEK293T cells were transfected with the SuperTopFlash reporter plasmid together with various expression plasmids (Wnt1, Wnt3, Wnt1/LRP6, Wnt3/LRP6, DVL2, or β-catenin) or treated with Wnt3A-conditioned medium. Transfected or treated cells were then incubated with vehicle or Prodigiosin (25-500 nM) for 24 h. Luciferase values were normalized to β-galactosidase (β-gal) activities [1].
For Western blot analysis, HEK293T cells or breast cancer cells (MDA-MB-231, MDA-MB-468) were treated with indicated concentrations of Prodigiosin for 24 h. Cell extracts were then subjected to SDS/PAGE and immunoblotting using specific antibodies against phosphorylated LRP6, total LRP6, DVL2, active β-catenin, total β-catenin, Ser9 phosphorylated GSK3β, total GSK3β, cyclin D1, LEF1, and cleaved caspase-3 [1].
For quantitative PCR, total RNA was extracted from treated cells, reverse-transcribed into cDNA, and then subjected to quantitative PCR analysis to detect the mRNA expression of cyclin D1, fibronectin, LEF1, and FZD5 [1].
For cell viability assay, breast cancer cells were treated with Prodigiosin for 48 h, and viability was assessed. IC50 values were calculated [1].
For proliferation assay, BrdU incorporation assay was performed on breast cancer cells treated with Prodigiosin [1].
For migration and invasion assays, scratch assay and transwell assays (with or without Matrigel coating) were used. MDA-MB-231 and MDA-MB-468 cells were treated with indicated concentrations of Prodigiosin, and migrated or invaded cells were stained and counted [1].
For antifungal activity, 96-well growth inhibition assays were performed. Zoospores of Bd or Bsal (2x10^6 zoospores/mL) were incubated with various concentrations of Prodigiosin (1-50 µM). Growth was measured as optical density at 490 nm over several days. Minimum inhibitory concentrations (MIC) and IC50 values were determined [2].

Animal/Disease Models: Female BALB/c nude mice were injected with MDA-MB-231 cells [1].
Doses: 5 mg/kg.
Route of Administration: intraperitoneal (ip) injection; twice a week; for 3 consecutive weeks.
Experimental Results: Dramatically inhibited mouse tumor growth.

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For the MDA-MB-231 xenograft model, nude mice were injected subcutaneously with MDA-MB-231 cells. When tumor volumes reached approximately 50 mm³, mice were treated by intraperitoneal (i.p.) injection with vehicle or Prodigiosin at 5 mg/kg twice weekly for 3 weeks. After treatment, mice were killed, and tumor volumes and weights were measured. Tumors were collected for histological analysis (H&E and Ki-67 staining), immunohistochemical staining (active and total β-catenin), immunoblot analysis, and real-time PCR [1].
For the MMTV-Wnt1 transgenic mouse model, female mice that developed mammary tumors (approximately 50 mm³) were treated with i.p. injection of either vehicle or Prodigiosin (5 mg/kg) twice weekly. After a 3-week treatment regimen, mice were killed and tumors were removed for immunohistochemical staining, immunoblot analyses, and real-time PCR [1].

In the MDA-MB-231 xenograft mouse model, Prodigiosin treatment (5 mg/kg, i.p., twice weekly for 3 weeks) did not affect the body weight of the mice, suggesting low systemic toxicity at this dose and schedule [1].

[1]. Prodigiosin inhibits Wnt/β-catenin signaling and exerts anticancer activity in breast cancer cells. Proc Natl Acad Sci U S A. 2016 Nov 15;113(46):13150-13155.

[2]. Prodigiosin, Violacein, and Volatile Organic Compounds Produced by Widespread Cutaneous Bacteria of Amphibians Can Inhibit Two Batrachochytrium Fungal Pathogens. Microb Ecol. 2018 May;75(4):1049-1062.

Vibrio rubigin is a tripyrrole compound, a red pigment with antibacterial properties produced by Serratia marcescens. It functions as an antibacterial agent, biopigment, bacterial metabolite, apoptosis inducer, and antitumor agent. It is a tripyrrole, aromatic ether, and cyclic compound. Vibrio rubigin has been reported to be present in Serratia marcescens and Vibrio aerogenes, and relevant data are available. 4-Methoxy-5-((5-methyl-4-pentyl-2H-pyrrole-2-ylidene)methyl)-2,2'-bi-1H-pyrrole. A toxic, bright red tripyrrole pigment from Serratia marcescens and other bacteria. It possesses antibacterial, anticoccal, antimalarial, and antifungal activities, but is primarily used as a biochemical tool. See also: Vibrio rubigin (note moved to).

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Prodigiosin is a natural red pigment produced by numerous bacterial species, with antibacterial, antifungal, antiprotozoal, antimalarial, immunosuppressive, and anticancer properties. It has shown potent cytotoxic activity against human cancer cell lines with multidrug resistance phenotypes or defects in apoptotic pathways, with much less toxicity to normal cells [1].
Prodigiosin can chelate copper and zinc, alter cellular pH, and its mechanism may involve inhibition of vacuolar H+-ATPase (V-ATPase), interfering with vesicle acidification necessary for endocytosis and activation of the FZD/LRP6 complex [1].
The Prodigiosin analog Obatoclax is currently in clinical trials for treating hematologic malignancies. Similar to prodigiosin, obatoclax inhibited Wnt signaling activated by Wnt1, Wnt3, Wnt1/LRP6, Wnt3/LRP6, and DVL2, and suppressed Wnt3A-CM-induced transcriptional activity of the SuperTopFlash reporter [1].
Prodigiosin has been detected in Serratia plymuthica and Serratia marcescens isolates from amphibian skin. Its production is influenced by growth medium and temperature, with greater production on TGhL medium compared to LB medium [2].

C20H25N3O

323.44

323.2

82-89-3

Prodigiosin hydrochloride;56144-17-3

135455579

Brown to red solid powder

1.12g/cm3

542.4ºC at 760 mmHg

281.8ºC

1.591

4.776

2

2

7

24

523

0

CCCCCC1=C(NC(=C1)/C=C\2/C(=CC(=N2)C3=CC=CN3)OC)C

SHUNBVWMKSXXOM-UNOMPAQXSA-N

InChI=1S/C20H25N3O/c1-4-5-6-8-15-11-16(22-14(15)2)12-19-20(24-3)13-18(23-19)17-9-7-10-21-17/h7,9-13,21-22H,4-6,8H2,1-3H3/b19-12-

(2Z,5Z)-4-methoxy-5-((5-methyl-4-pentyl-1H-pyrrol-2-yl)methylene)-1,5-dihydro-2,2'-bipyrrolylidene

Prodigiosine BRN 4526727 NSC 47147

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 : ~25 mg/mL (~77.30 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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