PKC (IC50 = 0.6-1.3 μM)

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].

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).

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).

[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 that is perylo[1,12-def][1,3]dioxepine-6,11-dione substituted by hydroxy groups at positions 5 and 12, by methoxy groups at positions 7 and 10, and by 2-hydroxypropyl groups at positions 8 and 9 (the R,R-stereoisomer). It is a phytotoxin which was first isolated from the pathogenic soybean fungus, Cercospora kikuchii and later found in multiple members of the genus Cercospora. It has a role as a photosensitizing agent, a phytotoxin, a fungal metabolite and a reactive oxygen species generator. It is a polyphenol, a polyketide and an organic heterohexacyclic compound. It is a conjugate acid of a cercosporin(2-).
(+)-Cercosporin has been reported in Cercospora zeina with data available.

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Cercosporin is a naturally occurring perylenequinone. Although other perylenequinones have been extensively studied as photosensitizers in photodynamic therapy of cancer (PDT), cercosporin has been studied in this light only within the remits of phytopathology. Herein, we investigated the photocytotoxicity of cercosporin against two glioblastoma multiforme (T98G and U87) and one breast adenocarcinoma (MCF7) human cell lines. Cercosporin was found to be a potent singlet oxygen producer upon 532 nm excitation, while its cell loading was similar for MCF7 and U87, but approximately threefold higher for T98G cells. The subcellular localization of cercosporin was in all cases in both mitochondria and the endoplasmic reticulum. Light irradiation of cercosporin-incubated cells around 450 nm showed that T98G cells were more susceptible to cercosporin PDT, mainly due to their higher cercosporin uptake. Metabolic studies before and 1 h following cercosporin PDT showed that cercosporin PDT instigated a bioenergetic collapse in both the respiratory and glycolytic activities of all cell lines. In the dark, cercosporin exhibited a synergistic cytotoxicity with copper only in the most respiratory cell lines (MCF7 and T98G). Cercosporin is a potent photosensitizer, but with a short activation wavelength, mostly suitable for superficial PDT treatments, especially when it is necessary to avoid perforations.[1]

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The total syntheses of the PKC inhibitors (+)-calphostin D, (+)-phleichrome, cercosporin, and 10 novel perylenequinones are detailed. The highly convergent and flexible strategy developed employed an enantioselective oxidative biaryl coupling and a double cuprate epoxide opening, allowing the selective syntheses of all the possible stereoisomers in pure form. In addition, this strategy permitted rapid access to a broad range of analogues, including those not accessible from the natural products. These compounds provided a powerful means for evaluation of the perylenequinone structural features necessary to PKC activity. Simpler analogues were discovered with superior PKC inhibitory properties and superior photopotentiation in cancer cell lines relative to the more complex natural products.[2]

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.)