PKC ( EC50 = 11.7 nM ); NF-κB; SphK; protein kinase C (PKC)

PMA induces Thy-1 up-regulation, raises Thy-1 mRNA and protein levels in endothelial cells, and prevents the formation of capillary-like tubes and endothelial cell migration.[1]

Induction of Ear Edema Model (PMA-induced Method)
Pathogenic Mechanism:
This model activates the protein kinase C (PKC) signaling pathway through phorbol 12-myristate 13-acetate (PMA), leading to:
• Stimulation of phospholipase A2 (PLA2) activation
• Promotion of inflammatory mediator release (e.g., prostaglandins)
• Local vasodilation and increased vascular permeability
• Inflammatory cell infiltration and tissue edema

Standard Modeling Protocol:
Experimental Animals: Female Swiss mice
Weight Range: 25-30 g
Experimental Groups: Must include a solvent control group

Administration Parameters:
• ‌Drug Concentration:‌ 100 μg/mL PMA (dissolved in appropriate solvent)
• ‌Dosing Volume:‌ 20 μL
• ‌Administration Site:‌ Single ear (left or right)
• ‌Administration Method:‌ Topical application (evenly spread on inner and outer ear surfaces)
• ‌Observation Timeframe:‌ Peak effect typically occurs 4-6 hours post-administration

Model Validation Criteria:

‌3.1 Primary Evaluation Criteria:‌
• ‌Ear Thickness Difference:‌ Measured with a caliper; edema ear should show ≥50% increase compared to control
• ‌Vascular Permeability:‌ Significant increase in Evans blue extravasation (quantitative detection)
‌3.2 Secondary Evaluation Criteria (Optional):‌
• ‌Histological Examination:‌ Degree of inflammatory cell infiltration
• ‌Inflammatory Cytokine Detection:‌ Levels of pro-inflammatory factors (e.g., TNF-α, IL-1β)
• ‌Myeloperoxidase (MPO) Activity Assay‌

Precautions:
• Strictly control environmental conditions (temperature: 22±2°C; humidity)
• Conduct experiments at fixed time intervals to minimize circadian rhythm effects
• Solvent control group should receive the same volume of PMA-dissolving solvent
• Animals should acclimate for at least 3 days prior to experiments
• Avoid mechanical stimulation that may affect ear condition

Model Characteristics:
• ‌Success Rate:‌ >90%
• ‌Peak Inflammation Time:‌ 4-6 hours post-administration
• ‌Duration:‌ Significant inflammatory response observable within 24-48 hours
• ‌Applications:‌ Anti-inflammatory drug screening, inflammation mechanism research, etc.
Note: Specific experimental parameters may be adjusted based on research objectives, but modifications must be clearly justified in the literature. A preliminary pilot study is recommended for first-time model establishment to optimize conditions.

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Research Protocol for PMA-induced Foot Edema Model
Pathogenic Mechanism
PMA activates the protein kinase C (PKC) signaling pathway, stimulating phospholipase A2 (PLA2) activation, promoting the release of inflammatory mediators such as prostaglandins, leading to local vasodilation and increased vascular permeability, ultimately resulting in inflammatory cell infiltration and tissue edema. This mechanism shares similarities with edema formation in pathological conditions like nephrotic syndrome.
Experimental Animal Selection
• Rat model: Male adult Wistar rats, 200-220g
• Mouse model: Male Swiss albino mice, 25-30g

Modeling Method
1. Administration parameters:
o Drug concentration: 2.5μg PMA in 20μL vehicle
o Administration site: Topical application to unilateral ear
o Administration method: Single dose

2. Key experimental design points:
o Must include solvent control group
o Other inhibitory products should be administered 4 hours before sacrifice
Model Validation Criteria
Primary Evaluation Criteria
• Visual monitoring: Significant increase in mass difference between ears (measured with caliper)
• Biochemical indicators: Stimulation of superoxide anion production in macrophages (quantitative detection)

Secondary Evaluation Criteria (Optional)
• Changes in vascular permeability (Evans blue extravasation method)
• Inflammatory factor level detection (TNF-α, IL-1β, etc.)
• Myeloperoxidase (MPO) activity assay

Model Characteristics
• Peak inflammation time: 4-6 hours post-administration
• Duration: Significant inflammatory response observable within 24-48 hours
• Applications: Anti-inflammatory drug screening, inflammation mechanism research, etc.

Precautions
3. Strictly control environmental temperature and humidity (22±2°C)
4. Animals require at least 3 days of acclimation
5. Avoid mechanical stimulation affecting ear condition during operation
6. Solvent control group should receive equal volume of PMA-dissolving vehicle
Note: Specific experimental parameters may be adjusted according to research purposes, but modifications must be clearly justified in the literature. Preliminary experiments are recommended for initial model establishment to determine optimal conditions.

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PMA first increases endothelial cell migration in the zebrafish model, then it activates the PKC-δ/Syk/NF-κB-mediated pathway to up-regulate Thy-1, which in turn prevents endothelial cell migration.[1]

Researchers previously showed that overexpression of Thy-1 inhibited and knock-down of Thy-1 enhanced endothelial cell migration. Here, Researchers used phorbol-12-myristate-13-acetate (PMA) as an inducer for Thy-1 expression to investigate molecular mechanisms underlying Thy-1 up-regulation. The data showed that increased levels of Thy-1 mRNA and protein in endothelial cells were observed at 14-18 hours and 20-28 hours after PMA treatment, respectively. Treatment with PMA for 32 hours induced Thy-1 up-regulation and inhibited capillary-like tube formation and endothelial cell migration. These effects were abolished by Röttlerin (a PKC-δ inhibitor), but not Gö6976 (a PKC-α/β inhibitor). Moreover, pre-treatment with Bay 61-3606 (a Syk inhibitor) or Bay 11-7082 (a NF-κB inhibitor) abolished the PMA-induced Thy-1 up-regulation and migration inhibition in endothelial cells. Using the zebrafish model, Researchers showed that PMA up-regulated Thy-1 and inhibited angiogenesis through the PKC-δ-mediated pathway. Surprisingly, they found that short-term (8-10 hours) PMA treatment enhanced endothelial cell migration. However, this effect was not observed in PMA-treated Thy-1-overexpressed endothelial cells. Taken together, our results suggest that PMA initially enhanced endothelial cell migration, subsequently activating the PKC-δ/Syk/NF-κB-mediated pathway to up-regulate Thy-1, which in turn inhibited endothelial cell migration. Our results also suggest that Thy-1 might play a role in termination of angiogenesis.[7]

Monolayer cultured αT3-1 and LβT-2 cells are grown in DMEM in a humidified incubator with 5% CO2 at 37°C. Serum starvation lasts 16 hours when 0.1% FCS is added to the same medium. Then, for the duration specified, GnRH and PMA are added. αT3-1 cells can be transfected temporarily using either jetPRIME or ExGen 500, whereas LβT2 cells can only be transfected using the jetPRIME transfection reagent. In experiments involving dominant-negative (DN) PKCs, 1.5 μg of p38α-GFP or 3 μg of the DN-PKCs constructs are transfected into αT3-1 cells (in 6 cm plates) in combination with pCDNA3, the control vector. Transfections of LβT2 cells are carried out (in 10 cm plates) using 4 μg of p38α-GFP in combination with 9 μg of either the DN-PKCs constructs or the control vector, pCDNA3. The cells are serum starved (0.1% FCS) for 16 hours approximately 30 hours after transfection. They are then stimulated with GnRH or PMA, twice washed with ice-cold PBS, treated with the lysis buffer, and then subjected to one freeze-thaw cycle. After harvesting the cells, the supernatants are taken for immunoprecipitation experiments and centrifuged at 15,000 x g for 15 minutes at 4°C.[7]

Rats: Male Wistar rats weighing between 250 and 280 grams are used in all experiments. Seven groups of fifteen thirty-five Wistar rats are randomly assigned. (1) A 0.9% normal saline injection is administered to rats in the sham group (n = 21); (2) A 0.9% normal saline injection is administered to rats in the IR group (n = 21) 30 minutes prior to middle cerebral artery occlusion (MCAO); (3) A lateral cerebral ventricle injection of CBX (5 μg/mL×10 μL) is administered to rats in the Carbenoxolone (CBX) group (n = 21) 30 minutes prior to MCAO; (4) Rats in the Sch-6783 group (n = 21) receive a lateral cerebral ventricle injection of DZX (2 mM×30 μL) 30 minutes before MCAO; (5) Rats in the 5-HD group (n = 21) receive a lateral cerebral ventricle injection of 5-HD (100 mM×10 μL); (6) The rats in the DZX + Ro group (n = 15) receive a lateral cerebral ventricle injection of DZX, and after 10 min, Ro-31-8425 (400 μg/kg) is injected 15 minutes before MCAO; (7) Rats in the 5-HD+PMA group (n = 15) receive an intraperitoneal injection of PMA (200 μg/kg) following the injection of 5-HD and DZX.

Absorption, Distribution and Excretion
…Mouse skin localization assays…determined that 3–6 hours after application of tritium-labeled PMA to the skin, the stratum corneum above basal cells was highly labeled, while sebaceous glands and hair follicles were moderately labeled. After 48 hours, some labeling remained in sebaceous glands and hair follicles. …The half-life of the promoter was approximately 24 hours. Metabolism/Metabolites …The main metabolic pathway of TPA is the hydrolysis of two ester groups…In rodent skin models, all hydrolysates lacked pro-tumor activity, which is the main toxicological effect of TPA. Metabolic hydrolysis requires esterase activity, which varies by tissue and species. Both ester groups of TPA can be hydrolyzed in mouse skin and cultured cells to produce the monoesters 12-tetradecanoylphorbolol and phorbolol-13-acetate, as well as the complete hydrolysate phorbolol. In mouse skin, the reduction of the ketone group at the C-3 position was identified as another metabolic pathway. Notably, no other metabolites were detected during microsomal incubation, indicating that cytochrome 450-mediated oxidative metabolism is not involved in TPA metabolism. Ester hydrolysis was also the only metabolic reaction observed in various cultured cells. TPA hydrolysis paralleled the loss of ornithine decarboxylase (ODC)-induced activity. Since ODC is a marker of tumor promotion, these findings suggest that none of the three hydrolytic metabolites of TPA (two monoesters and phorbol) possess pro-tumorigenic activity. The hydrolysis rates of TPA and its structural analogue phorbol-12,13-didecanoate (PDD) differed significantly in cultured fibroblasts from different animal species, suggesting that the hydrolytic metabolism of phorbol diester depends on cell type and the diester's chemical structure…
…In vivo studies of the metabolism of radiolabeled TPA were conducted in mouse dorsal skin. Several novel lipophilic metabolites were detected in addition to the hydrolytic metabolites and identified as esterification of TPA with long-chain fatty acids at the C-20 hydroxyl group. These TPA-20 acylates did not appear to have pro-tumorigenic activity, but were partially hydrolyzed back to TPA in mouse skin… A few in vitro studies involving human cells have shown that many cultured human cell lines do not metabolize TPA to a high degree… Biological half-life… After application of tritium-labeled PMA to mouse skin… the promoter half-life was approximately 24 hours. The calculated terminal half-life was 11 ± 3.9 hours (from five infusions in four patients)…

Toxicity Summary
Identification and Uses: TPA is a colorless powder. It is used in cancer research to study tumor-promoting mechanisms, screen potential inhibitors, and serve as a positive control for tumor promoters. It has been used as an experimental drug to treat leukemia, lymphoma, and other types of cancer. Human Exposure and Toxicity: TPA is a human platelet aggregator. TPA has been used in clinical trials in patients with relapsed malignancies, particularly hematologic malignancies, including severe leukemia. The aim of this trial was to use TPA as an inducer to induce apoptosis and differentiation at low doses. The application of TPA was based on existing cell inhibitor regimens, with 35 patients receiving low-dose, constant-rate infusions over a specific time period. Following treatment, some patients experienced serious side effects such as transient fatigue, anemia, neutropenia and thrombocytopenia, mild dyspnea, nausea, fever, chills, and cardiovascular reactions including syncope and hypotension. However, only one patient experienced a tumor response, manifested as tumor shrinkage. TPA is routinely used in in vitro human cell studies. Animal studies: TPA has been shown to promote tumorigenesis in mouse skin bioassays, mouse forestomach assays, and in vitro cell proliferation assays. However, there is no evidence that TPA possesses tumor-initiating properties. This study investigated tumor initiation and promotion in mouse forestomach epithelium. Mice were administered a single dose of 7,12-dimethylbenzo[a]anthracene (DMBA) (50 mg/kg body weight) via intragastric instillation, followed by repeated administration at a dose of 10 mg/kg body weight (twice weekly) for 35 weeks. Of the 50 mice treated, 45 developed forestomach tumors (papillary tumors). No forestomach tumors were observed in the untreated control group or mice treated with TPA alone, while papillomas were observed in 10 mice treated with DMBA alone. TPA was not shown to have genotoxicity. TPA has shown effects of inducing chromosome breakage, mutagenesis, and sister chromatid exchange in some experimental systems, but these effects are mediated by secondary products (possibly derived from arachidonic acid) produced by cells in response to tumor promoters under culture conditions with low levels of antioxidants in the culture medium and serum. In whole rat embryonic cultures, TPA exposure led to forebrain shrinkage, growth retardation, and incomplete body axial rotation. Post-culture, embryonic E-cadherin mRNA levels were found to be abundant. TPA is commonly used in in vitro animal cell studies.

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Interactions
To determine the time interval between initiation and promotion, and the effect of mouse age on the two-stage carcinogenicity, 20 μg of 7,12-dimethylbenzo[a]anthracene (the initiator) was applied once to the dorsal skin of five groups of female ICR/ha Swiss mice, and 2.5 μg of PMA was applied three times a week. Mice in groups 1, 2, 3, 4, and 5 were 6, 44, 56, 6, and 6 weeks old at the initial treatment (initiator), respectively; the intervals between the initial and subsequent treatments (promoter) were 2, 2, 2, 36, and 56 weeks, respectively; the number of mice in each group was 120, 20, 50, 35, and 35, respectively; the incidence of papilloma in each group was 100%, 100%, 56%, 90%, and 57%, respectively; and the incidence of squamous cell carcinoma in each group was 50%, 30%, 6%, 25%, and 11%, respectively. The corresponding control groups received only one drug at varying intervals. The results showed that skin cancer could be induced regardless of whether the interval between initiation and promotion was 2, 36, or 56 weeks. In groups 3 and 5, the cancer incidence was significantly reduced; these two groups of mice began secondary treatment at 58 and 62 weeks of age, respectively. /Excerpt from Table/
Because endogenous proteases may play a role in the mechanism of action of tumor promoters, the inhibitory effects of three known protease inhibitors on the two-stage carcinogenesis process were tested. The protease inhibitors…toluenesulfonyl chloride methyl ketone, toluenesulfonylphenylalanine chloride methyl ketone, and toluenesulfonylarginine methyl ester… were treated in the mouse ear after a single dose of 7,12-dimethylbenzanthracene induction, followed by…PMA promotion. The inhibitors were administered three times weekly immediately after the promoter treatment. The protease inhibitors delayed the onset of the first-stage tumor, altered the overall rate pattern of tumor development, and resulted in a decrease in tumor incidence.
Low doses of sulfur mustard, i.e., bis(β-chloroethyl) sulfide, completely inhibited the two-stage carcinogenesis process in mouse skin. Thirty female ICR/ha mice in each group received either a control treatment or different combinations of the inducer, promoter, and inhibitor test compounds for 400 days. 7,12-Dimethylbenzo[a]anthracene (DMBA), 20 μg/0.1 mL acetone, used once, as an initiator. PMA, as a promoter, was used three times a week at a concentration of 2.5 μg/0.1 mL acetone. BCS, bis(β-chloroethyl) sulfide, was used 14 days after the initiator. DMBA + PMA + BCS (twice a week) induced papilloma in 1 mouse, with the first tumor appearing on day 90; DMBA + PMA + BCS (three times a week) induced papilloma in 2 mice, with tumors appearing on day 209; DMBA + PMA alone induced papilloma in 27 mice and squamous cell carcinoma in 16 mice, with tumors appearing on day 40; PMA alone induced papilloma in 4 mice, with tumors appearing on day 218. DMBA + BCS (twice weekly) induced papilloma in one mouse at 385 days; DMBA + BCS (three times weekly) did not induced papilloma in mice; BCS alone (three times weekly) induced papilloma in one mouse at 323 days; DMBA + acetone induced papilloma in one mouse only at 219 days; no papilloma was observed in the control group (acetone only) or the group that did not receive any of the test compounds. /Excerpt from table/
This study aimed to determine the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and diethyl dithiocarbamate (DDTC), alone or in combination, on human pancreatic cancer cells cultured in vitro and forming xenograft tumors in nude mice. Pancreatic cancer cells were treated with DDTC or TPA alone or in combination, and the number of viable cells was determined by trypan blue staining exclusion method, while the number of apoptotic cells was determined by morphological observation under a fluorescence microscope after propidium iodide staining. The results showed that DDTC or TPA treatment alone could inhibit the growth of pancreatic cancer cells and promote their apoptosis in a concentration-dependent manner. In both monolayer and three-dimensional (3D) cultured PANC-1 cells, the combined treatment with DDTC and TPA was significantly more effective than either treatment alone. NF-κB-dependent reporter gene expression analysis and Western blot analysis indicated that the significant effect of the combined treatment on PANC-1 cells was associated with the inhibition of DDTC-induced nuclear factor-κB (NF-κB) activation and the reduction of Bcl-2 expression. Furthermore, treatment with DDTC + TPA in nude mice significantly inhibited the growth of PANC-1 xenograft tumors. These results suggest that the combined use of TPA and DDTC may be an effective strategy for inhibiting pancreatic cancer growth.
For more complete data on interactions of 12-O-tetradecanoylphorbol-13-acetate (16 in total), please visit the HSDB record page.

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Human toxicity: The intravenous LD50 in mice was 309 μg/kg. Adverse Reactions: Reproductive toxicity—Chemicals that are toxic to the reproductive system, including defects in offspring and impairment of male or female reproductive function. Reproductive toxicity includes developmental effects. Please refer to the Reproductive Toxicity Risk Assessment Guidelines.

[1]. Autocrine and paracrine roles of sphingosine-1-phosphate. TRENDS in Endocrinology and Metabolism. Volume 18, Issue 8, October 2007, Pages 300-307. https://doi.org/10.1016/j.tem.2007.07.005

[2]. MPTP-Induced Depletion in Basolateral Amygdala via Decrease of D2R Activation Suppresses GABAA Receptors Expression and LTD Induction Leading to Anxiety-Like Behaviors. Front Mol Neurosci. 2017 Aug 7;10:247.

[3]. Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3. J Leukoc Biol. 1996;59(4):555-561.

[4]. Differential roles of PKC isoforms (PKCs) and Ca2+ in GnRH and phorbol 12-myristate 13-acetate (PMA) stimulation of p38MAPK phosphorylation in immortalized gonadotrope cells. Mol Cell Endocrinol. 2017 Jan 5;439:141-154.

[5]. The phorbol 12-myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium. PLoS One. 2018;13(3):e0193601.

[6]. Mechanism of Mitochondrial Connexin43's Protection of the Neurovascular Unit under Acute Cerebral Ischemia-Reperfusion Injury. Int J Mol Sci. 2016 May 5;17(5). pii: E679.

[7]. PMA inhibits endothelial cell migration through activating the PKC-δ/Syk/NF-κB-mediated up-regulation of Thy-1. Sci Rep. 2018 Nov 2;8(1):16247.

12-O-Tetradecanoylphorbol-13-acetate is a white crystalline solid. (NTP, 1992)
Phrabolol 13-acetate 12-myristate is a phorbol ester whose structure is such that the hydroxyl groups at the phorbolol cyclopropane linker (position 13) and the adjacent carbon atom (position 12) are converted into the corresponding acetate and myristate, respectively. It is the main active ingredient in Croton tiglium seed oil. It has been used as a tumor promoter in rodent skin cancer and has been associated with increased proliferation of malignant cells. However, its function is controversial because reduced cell proliferation has also been observed in several cancer cell types. It has multiple functions, including as a protein kinase C agonist, antitumor agent, reactive oxygen species generator, plant metabolite, mitogen, carcinogen, and apoptosis inducer. It is an acetate, tetradecanoate, diester, tertiary α-hydroxy ketone, and phorbol ester.
Phosporine 12-Myristate 13-acetate diester is an inducer of extracellular traps (NETs) in neutrophils.
Phosporine 12-Myristate 13-acetate has been reported in Iris tectorum, Phormidium tenue, and other organisms with relevant data.
Tetradecanoylphorbol acetate (TPA) is a phorbol ester with potential antitumor activity. TPA can induce the maturation and differentiation of hematopoietic cell lines, including leukemia cells. This substance may induce gene expression and protein kinase C (PKC) activity. In addition to its potential antitumor activity, TPA may also have protumogenetic activity. (NCI04)
Phosporine esters are polycyclic compounds isolated from croton oil, in which two hydroxyl groups on adjacent carbon atoms are esterified with fatty acids. The most common of these derivatives is phorbol myristoyl acetate (PMA). These are potent co-carcinogens or tumor promoters, diacylglycerol analogs that irreversibly activate protein kinase C. A phorbol ester found in croton oil possesses highly potent tumor-promoting activity. It stimulates DNA and RNA synthesis. Mechanism of Action The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) plays different roles in cell cycle regulation in various tumor cell types. The mechanism between TPA and the breast cancer cell cycle is not fully elucidated. Therefore, we investigated the mechanism by which TPA regulates the breast cancer cell cycle. Our results show that TPA increases p21 expression levels in wild-type p53 MCF-7 cells and mutant p53 MDA-MB-231 cells in a dose-dependent manner. Conversely, TPA decreases p53 expression in MCF-7 cells but has no effect on MDA-MB-231 cells. Next, we investigated the regulatory mechanisms of TPA on p21 and p53 expression. The results showed that TPA-induced upregulation of p21 and downregulation of p53 could be reversed by the MEK1/2 inhibitor UO126, but not by the JNK inhibitor SP600125 or the p38 inhibitor SB203580, even though TPA increased the phosphorylation levels of ERK and JNK in MCF-7 cells. Furthermore, UO126 treatment also restored TPA-induced G2/M phase arrest. To verify p21 expression via the MEK/ERK pathway, we transfected cells with constitutively active (CA)-MEK adenovirus. The results showed that CA-MEK overexpression significantly upregulated p21 expression. In conclusion, we believe that TPA mutually regulates the expression levels of p21 and p53 through a MEK/ERK-dependent pathway. In breast cancer cells, TPA-induced upregulation of p21 is mediated through a p53-independent mechanism.

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Therapeutic Use
/Clinical Trials/ ClinicalTrials.gov is a registry and results database that catalogs human clinical studies funded by public and private sources worldwide. The website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov provides summary information on the study protocol, including: the disease or condition; the intervention (e.g., the medical product, behavior, or procedure under investigation); the study title, description, and design; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information from other health websites, such as the NLM's MedlinePlus (for patient health information) and PubMed (for citations and abstracts of academic articles in the medical field). The database contains 12-O-tetradecanoylphorbol-13-acetate. Phorbol ester activates protein kinase C and regulates multiple downstream cellular signaling pathways. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a phorbol ester that can induce differentiation or apoptosis in various cell lines at low concentrations. A phase I dose-escalation trial of TPA in patients with relapsed or refractory malignancies was conducted. The starting dose was 0.063 mg/m², and most patients received intravenous TPA infusions on days 1–5 and 8–12, followed by a 2-week rest period before re-treatment. A total of 35 patients were treated. Post-treatment, TPA-like activity levels in the blood were monitored using bioassays. Serious adverse events included gross hematuria, grand mal seizures, syncope, and hypotension in a few patients. Many patients experienced transient fatigue, mild dyspnea, fever, chills, and muscle aches shortly after infusion. Dose-limiting toxicities included syncope and hypotension at a dose of 0.188 mg/m². Only one patient experienced a tumor response. These studies determined that 0.125 mg/m² is the maximum tolerated dose when TPA is used according to this dosing regimen. We investigated the roles of PKC and Ca2+ in p38MAPK phosphorylation in gonadotropin-releasing hormone (GnRH)-stimulated gonadotropic cell lines αT3-1 and LβT2. GnRH induced a slow and a rapid increase in p38MAPK phosphorylation in αT3-1 and LβT2 cells, respectively, while PMA elicited a slow response. Using PKC dominant-negative inhibitors and activated protein C kinase receptor (RACK) peptide inhibitors, we revealed that PKCα, PKCβII, PKCδ, and PKCε play different roles in p38MAPK phosphorylation in a ligand- and cell environment-dependent manner. This seemingly contradictory finding—that GnRH- and PMA-activated PKC play different roles in p38MAPK phosphorylation—may stem from differences in PKC localization. In αT3-1 cells, basal, GnRH, and PMA-induced p38MAPK phosphorylation was caused by voltage-gated Ca2+ channel-mediated Ca2+ influx and Ca2+ mobilization; while in differentiated LβT2 gonadotropic cells, it was caused only by Ca2+ mobilization. p38MAPK is located on the cell membrane and is induced by GnRH (approximately 5 minutes) to translocate to the nucleus. Thus, we have identified PKC and Ca2+ pools involved in GnRH-stimulated p38MAPK phosphorylation. [4]

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THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) have been widely used as a model for studying human macrophage function and biology. However, the conditions used for differentiation, particularly the concentration of PMA and the duration of treatment, vary considerably. This paper compares several differentiation conditions and the ability of THP-1 macrophages to interact with the facultative intracellular pathogen Salmonella typhimurium. The results showed that THP-1 macrophages differentiated under high concentrations of PMA died rapidly after infection, while THP-1 macrophages differentiated under low concentrations of PMA survived and were able to control intracellular bacteria like primary human macrophages. [5]

C36H56O8

616.8251

616.397

C, 70.10; H, 9.15; O, 20.75

16561-29-8

27924

White to off-white solid powder

1.2±0.1 g/cm3

698.1±55.0 °C at 760 mmHg

162 °F (NTP, 1992)
50-70 °C (melting pt-freezing pt)

208.1±25.0 °C

0.0±5.0 mmHg at 25°C

1.553

7.71

3

8

17

44

1150

8

O(C(C([H])([H])[H])=O)[C@@]12[C@@]([H])([C@@]([H])(C([H])([H])[H])[C@@]3([C@]4([H])C([H])=C(C([H])([H])[H])C([C@]4(C([H])([H])C(C([H])([H])O[H])=C([H])[C@@]3([H])[C@]1([H])C2(C([H])([H])[H])C([H])([H])[H])O[H])=O)O[H])OC(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])=O

PHEDXBVPIONUQT-RGYGYFBISA-N

InChI=1S/C36H56O8/c1-7-8-9-10-11-12-13-14-15-16-17-18-29(39)43-32-24(3)35(42)27(30-33(5,6)36(30,32)44-25(4)38)20-26(22-37)21-34(41)28(35)19-23(2)31(34)40/h19-20,24,27-28,30,32,37,41-42H,7-18,21-22H2,1-6H3/t24-,27+,28-,30-,32-,34-,35-,36-/m1/s1

[(1S,2S,6R,10S,11R,13S,14R,15R)-13-acetyloxy-1,6-dihydroxy-8-(hydroxymethyl)-4,12,12,15-tetramethyl-5-oxo-14-tetracyclo[8.5.0.02,6.011,13]pentadeca-3,8-dienyl] tetradecanoate

TPA; NSC262244; PD616; PMA; Phorbol myristate acetate; NSC 262244; Phorbol 12-myristate 13-acetate; 16561-29-8; phorbol-12-myristate-13-acetate; 12-O-Tetradecanoylphorbol-13-acetate; 12-O-Tetradecanoylphorbol 13-acetate; Tetradecanoylphorbol acetate; Phorbol ester; Factor A1; NSC-262244; PD-616; RP-323; PD 616; RP 323; PMA; RP323

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~162.1 mM)
Ethanol: ~100 mg/mL (~162.1 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.05 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 25.0 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.

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Solubility in Formulation 2: 2.5 mg/mL (4.05 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 ultrasonication.
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 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: ≥ 2.5 mg/mL (4.05 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.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (4.05 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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.

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Solubility in Formulation 5: 2.5 mg/mL (4.05 mM) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH 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 6: ≥ 2.5 mg/mL (4.05 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 7: 5%DMSO + Corn oil: 5.0mg/ml (8.11mM)

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