PKC (IC50 = 0.6-1.3 μM)
Cercosporin targets multiple cellular components through the generation of reactive oxygen species upon light activation. As a photosensitizer, the compound absorbs light energy and transfers it to molecular oxygen to produce singlet oxygen and other reactive oxygen species. These ROS cause oxidative damage to lipids, proteins, and DNA, leading to cell death. The compound also contains perylenequinone structural features necessary for PKC activity, with an IC₅₀ of 0.6-1.3 μM. Cercosporin's photodynamic therapy effect is mediated through the induction of oxidative stress, which triggers apoptotic or necrotic cell death depending on the cell type and treatment conditions. The compound's ability to generate ROS upon light activation makes it a valuable tool for studying photodynamic therapy mechanisms and the role of oxidative stress in cell death.

T98G cells exhibit a stronger response to cercosporin (0.8-8.0 μM; 30 s, 60 s, 90 s, 120 s) photodynamic therapy (PDT) compared to U87 or MCF7 cells. Additionally, the LD50 value of T98G cells (0.14 J cm2) is significantly lower than that of MCF-7 and U87 cell lines (0.26 and 0.24 J cm2, respectively) [1]. When copper and cerocosporin interact (0–3 μM; 24 hours), MCF7 and T98G cells experience synergistic cytotoxicity (S(CuSO4 + Cerco) ≥ S(CuSO4) x S(Cerco)) in U87 cells [1].
In vitro studies have demonstrated that Cercosporin exhibits potent photocytotoxic effects on various human tumor cell lines. The compound (0.8-8.0 μM) shows photodynamic therapy effects that are stronger in T98G glioblastoma cells than in U87 glioblastoma or MCF7 breast cancer cells. The LD₅₀ value for T98G cells (0.14 J/cm²) is much lower than the LD₅₀ values for MCF-7 and U87 cell lines (0.26 and 0.24 J/cm², respectively), indicating greater sensitivity of T98G cells to cercosporin-mediated PDT. Cercosporin (0-3 μM; 24 hours) interacts with copper to produce synergistic cytotoxicity in MCF7 and T98G cells, while showing barely additive effects in U87 cells. The compound's photodynamic therapy effect is dependent on light activation, with short activation wavelengths being most effective. Cercosporin also inhibits PKC activity with an IC₅₀ of 0.6-1.3 μM.

In vivo studies on Cercosporin have focused on its potential applications in photodynamic therapy for cancer treatment. The compound's short activation wavelength makes it particularly suitable for superficial PDT treatments, especially when it is necessary to avoid tissue perforation. The compound's efficacy and safety in animal models have been investigated, with studies showing that cercosporin accumulates in tumor tissues and, upon light activation, generates ROS that destroy cancer cells. However, the compound's phototoxic effects can also affect normal tissues, and careful dosing and light delivery are required to minimize off-target effects. Further in vivo studies are needed to fully characterize the compound's therapeutic potential and safety profile.

General Procedure for PKC Assay. [2]
Protein kinase C activity was measured following the literature protocol reported by Lown. 12 The stock solutions of histone (2 mg/mL) and OOPS-Na (1 mg/mL) in water were vortexed for 20 s to generate homogeneous solutions. The stock solution of DiC8 (1 mg/250 mL) in water was sonicated for 30 min. Inhibitor (e.g. Cercosporin (CGP049090)) stock solutions were prepared in 50% DMSO/water. Before assay, all stock solutions were vortexed for ~ 20 s to ensure complete homogeneity. The final volume of each reaction vial was 100 µL and 5% DMSO was present. The assay solution contained 20 mM Tris-HCl, pH 7.4, 350 µM CaCl2, 10 mM Mg(NO3) 2, 100 µg/mL OOPS-Na, 10 µL of 4 µg/mL DiC8, 10 µM ATP, 309 µM NaCl, 3 mM DTT, 1.55 mM EGTA, 0.03% glycerol and 0.0065 units PKC enzyme. Prior to S40 enzyme addition, the reaction mixture was vortexed for 10 s. Then, PKC was added and the solution was mixed gently (vortexing of the enzyme substantially reduced activity). After inhibitor addition (10 µL) and mixing, the reaction was initiated by addition of Mg(NO3)2 and ATP/ATP* followed by gentle mixing. Reactions were run at rt under overhead fluorescent light. The results were analyzed at 40 min and 80 min to generate duplicate points for each reaction vial. For analysis, 20 µL of reaction mixture was spotted on a P81 Whatmann chromatographic paper. The paper was air dried and washed with 0.75% phosphoric acid (4X) and acetone (1X). The paper was air dried and the amount of 32P-incorporated histone H1 was measured by scintillation counting (4 mL scintillation cocktail). For each assay, blanks consisting of no enzyme (background radiation) and enzyme with no inhibitor (100% activity) were run in parallel.

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PKC Catalytic Domain Assay. [2]
In comparison to our PKC general procedure, DiC8 and OOPS-Na were not used. Apart from this exception, all other materials were used in the same concentrations. A stock solution of 16 (500 µM) was prepared in 50% DMSO/water. Before assay, all stock solutions were vortexed for ~ 20 s to ensure complete homogeneity. The final volume of each reaction vial was 100 µL and 5% DMSO was present. The assay solution contained 20 mM Tris-HCl, pH 7.4, 350 µM CaCl2, 10 mM Mg(NO3)2, 10 µM ATP, 0.5 mM NaCl, 0.8 mM DTT, 5 µM EDTA, 5 µM EGTA, 0.05% glycerol and 0.0037 units PKC catalytic subunit. Prior to enzyme addition, the reaction mixture was vortexed for 10 s. Then, the PKC catalytic subunit was added and the solution was mixed gently. Next, the inhibitor (e.g. Cercosporin (CGP049090)) was added (10 µL; final concentration of inhibitor is 50 µM). After inhibitor addition and mixing, the S41 reaction was initiated by addition of Mg(NO3)2 and ATP/ATP* followed by gentle mixing. Three reactions: (1) PKC catalytic subunit and no inhibitor (100 % activity); (2) PKC catalytic subunit and 16 (50 µM); (3) no PKC catalytic subunit and no inhibitor (background), were run in parallel and in duplicate. The reactions were run at rt under overhead fluorescent light for 5 h. For analysis, 20 µL of reaction mixture was spotted on a P81 Whatmann chromatographic paper. The paper was air dried and washed with 0.75% phosphoric acid (4x) and acetone (1x). The paper was air dried and the amount of 32P-incorporated histone H1 was measured by scintillation counting (4 mL scintillation cocktail).
For non-cellular enzyme/receptor binding assays, Cercosporin can be evaluated for its photophysical properties, including absorption and emission spectra. The generation of singlet oxygen and other reactive oxygen species can be measured using specific probes such as singlet oxygen sensor green or electron paramagnetic resonance (EPR) spectroscopy. The compound's PKC inhibitory activity can be assessed using standard kinase activity assays with purified PKC enzyme and appropriate substrates. The compound's ability to interact with copper and other metal ions can be studied using spectroscopic techniques. The compound's photodynamic activity can be assessed by measuring the production of ROS upon light activation in cell-free systems.

Cell Viability Assay [1]
Cell Types: Human GBM cell line, T98G and U87; Breast cancer cell line, MCF-7
Tested Concentrations: 0 μM, 1 μM, 2 μM, 3 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Only in most cases demonstrated synergistic cytotoxicity with copper in respiratory cell lines (MCF-7 and T98G).
For in vitro cell-based assays, various cancer cell lines are cultured and treated with Cercosporin. Cells are typically treated with the compound at concentrations ranging from 0.8 to 8.0 μM, followed by exposure to light for specific durations (30 s, 60 s, 90 s, 120 s). Cell viability is assessed using MTT or similar assays to determine LD₅₀ values. ROS production is measured using fluorescent probes such as DCFH-DA. Apoptosis is evaluated by flow cytometry using Annexin V/PI staining or by assessing caspase activity. The synergistic effects of cercosporin with copper can be studied by co-treatment with copper sulfate.

For in vivo animal studies, Cercosporin can be administered to animal models of cancer for photodynamic therapy studies. The compound is typically administered intravenously or intratumorally, followed by light application to the tumor site at specific time intervals. Tumor growth, survival, and histopathological changes are assessed. The compound's photosensitizing effects on normal tissues are also evaluated. Dosing regimens, light delivery parameters, and treatment schedules depend on the specific experimental model and need to be optimized for each application.

The pharmacokinetic properties of Cercosporin include a molecular weight of 534.51 and a molecular formula of C₂₉H₂₆O₁₀. The compound has a relative density of 1.59 g/cm³. For in vitro studies, the compound can be dissolved in appropriate solvents such as DMSO. For in vivo administration, the compound can be formulated using various solvent systems, such as 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline. The powder should be stored at -20°C for up to 3 years, and in solvent at -80°C for up to 1 year. The compound is shipped with blue ice or at ambient temperature.

The toxicological profile of Cercosporin is primarily related to its phototoxic effects. As a photosensitizer, the compound can cause oxidative damage to normal tissues upon light exposure, which is the basis for both its therapeutic and toxic effects. The compound's short activation wavelength makes it suitable for superficial PDT treatments where it is necessary to avoid perforation. The compound's toxicity is light-dependent, and careful control of light exposure is required to minimize off-target effects. The compound is intended for research use only and is not for human use.

[1]. Cytotoxic and Photocytotoxic Effects of Cercosporin on Human Tumor Cell Lines. Photochem Photobiol. 2019 Jan;95(1):387-396.

[2]. Design, synthesis, and investigation of protein kinase C inhibitors: total syntheses of (+)-calphostin D, (+)-phleichrome, cercosporin, and new photoactive perylenequinones. J Am Chem Soc. 2009 Jul 8;131(26):9413-25.

Cercosporin is an organic heterohexacyclic compound with the structure perylene[1,12-def][1,3]dioxane-6,11-dione, substituted with hydroxyl groups at positions 5 and 12, methoxy groups at positions 7 and 10, and 2-hydroxypropyl groups at positions 8 and 9 (R,R-stereoisomers). It is a phytotoxin, initially isolated from the soybean pathogenic fungus Cercosporin kikuchii, and later found in various fungi of the Cercosporin genus. Cercosporin possesses multiple functions, including photosensitizer, phytotoxin, fungal metabolite, and reactive oxygen species (ROS) generator. It is a polyphenol, polyketide, and organic heterohexacyclic compound. It is the conjugate acid of Cercosporin (2-). (+)-Cercosporin has been reported in Cercosporin zeina, and relevant data are available. Cercosporin is a naturally occurring perylene quinone. While other perylene quinone compounds have been extensively studied as photosensitizers in photodynamic therapy (PDT), research on cercarin in this area has been limited to plant pathology. This article investigates the photocytotoxicity of cercarin on two glioblastoma (T98G and U87) and one breast cancer (MCF7) human cell lines. The study found that cercarin generates a large amount of singlet oxygen upon excitation at 532 nm, with similar loadings in MCF7 and U87 cells, but approximately three times the loading in T98G cells. In all cases, the subcellular localization of cercarin was located in the mitochondria and endoplasmic reticulum. Irradiation of cercarin-incubated cells with light at approximately 450 nm showed that T98G cells were more sensitive to cercarin photodynamic therapy (PDT), primarily due to their higher cercarin uptake. Metabolic studies conducted 1 hour before and after cerosporin PDT showed that cerosporin PDT caused bioenergy collapse in both respiratory and glycolytic activities in all cell lines. Under dark conditions, cerosporin showed synergistic cytotoxicity with copper only in the cell lines with the strongest respiration (MCF7 and T98G). Cerosporin is a potent photosensitizer, but its activation wavelength is short, making it primarily suitable for superficial photodynamic therapy (PDT), especially in cases where skin perforation needs to be avoided. [1]

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This article details the total synthesis of protein kinase C (PKC) inhibitors (+)-calphostin D, (+)-phleichrome, cerosporin, and 10 novel perylenequinone compounds. The highly convergent and flexible synthetic strategy developed employs enantioselective oxidative biaryl coupling and biscopper epoxide ring-opening reactions, enabling the selective synthesis of all possible pure stereoisomers. In addition, the strategy can rapidly synthesize a variety of analogs, including those that cannot be synthesized from natural products. These compounds provide a powerful means of characterizing the structure of perylenequinones required to assess PKC activity. Compared with more complex natural products, we have found some simpler analogs that exhibit superior PKC inhibitory activity and better photosynthetic effects in cancer cell lines. [2]
Cercosporin is a naturally occurring perylenequinone toxin produced by Cercospora fungi and the elsinochromes. It is a potent photosensitizer with a short activation wavelength, making it suitable for superficial photodynamic therapy applications. The compound contains perylenequinone structural features essential for PKC activity (IC₅₀: 0.6-1.3 μM). Cercosporin's photodynamic therapy effect is stronger in T98G cells than in U87 or MCF7 cells. The compound interacts with copper to produce synergistic cytotoxicity in MCF7 and T98G cells. Cercosporin has been studied for its potential applications in photodynamic therapy for cancer treatment. It is not currently approved for clinical use and is available only for research purposes.

C29H26O10

534.51074

534.153

C, 65.17; H, 4.90; O, 29.93

35082-49-6

91617

Brown to reddish brown solid powder

1.59g/cm3

886.9ºC at 760mmHg

299ºC

1.754

3.564

4

10

6

39

988

2

C[C@H](CC1=C2C3=C(C(=C(C4=C3C5=C6C2=C(C(=O)C=C6OCOC5=CC4=O)C(=C1OC)O)O)OC)C[C@@H](C)O)O

MXLWQNCWIIZUQT-GHMZBOCLSA-N

InChI=1S/C29H26O10/c1-10(30)5-12-18-19-13(6-11(2)31)29(37-4)27(35)21-15(33)8-17-23(25(19)21)22-16(38-9-39-17)7-14(32)20(24(18)22)26(34)28(12)36-3/h7-8,10-11,30-31,34-35H,5-6,9H2,1-4H3/t10-,11-/m1/s1

7,19-dihydroxy-5,21-bis[(2R)-2-hydroxypropyl]-6,20-dimethoxy-12,14-dioxahexacyclo[13.8.0.02,11.03,8.04,22.018,23]tricosa-1,3(8),4,6,10,15,18(23),19,21-nonaene-9,17-dione

CERCOSPORIN; Cercosporin from Cercospora hayii; 35082-49-6; 40501-77-7; NSC234486; 7,19-dihydroxy-5,21-bis(2-hydroxypropyl)-6,20-dimethoxy-12,14-dioxahexacyclo[13.8.0.02,11.03,8.04,22.018,23]tricosa-1,3(8),4,6,10,15,18(23),19,21-nonaene-9,17-dione; Isocercosporin; iso-Cercosporin;

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 : ≥ 10 mg/mL (~18.71 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.)