Natural product; hydroxyanthraquinone

In a dose-dependent manner, aloe emodin (0–15 μM; 24-96 hours) suppresses PC3 cell proliferation [1]. Aloe emodin (0–15 μM; 24 hours) prevents Akt and PKCα, two downstream substrates of mTORC2, from activating. Aloe emodin has the ability to attach itself to intracellular mTORC2 and stop kinase activation [1].

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Up-regulation of galectin-3 by aloe-emodin[2]; Inhibitory ability of galectin-3 on in vitro replication of influenza A virus[2]; Signaling induction of aloe-emodin in influenza A NS1-expressing cells. [2]

Through the inhibition of mTOR complex 2 activity, aloe emodin (10–50 mg/kg) suppresses the growth of tumors [1].
Based on our in vitro and ex vivo results, we next determined whether aloe-emodin could suppress tumor growth in vivo. The results show that the mean tumor weight was decreased in the aloe-emodin-treated group (Figure 6A, P < 0.05) and the mean tumor volume in the vehicle-treated group increased faster than that in the aloe-emodin-treated group (Figure 6B, P < 0.05). The body weights of vehicle-treated and aloe-emodin-treated mice (50 mg/kg) were not different (32.4 ± 0.70 g versus 31.8 ± 0.61 g, P ≥ 0.05). Tumor extracts from vehicle-treated and aloe-emodin-treated mice (i.e. euthanized on the same day of the experiment) were prepared and phosphorylation of Akt was analyzed. Western blotting analysis revealed that the aloe-emodin-treated tumor extracts exhibited substantially decreased Akt phosphorylation at Ser473 compared with vehicle-treated tumors

mTOR in vitro kinase assay[1]
The glutathione-S-transferase-tagged fusion Akt1 proteins were purified using glutathione-Sepharose 4B beads and eluted with 10 mM reduced glutathione in 50 mM Tris–HCl (pH 8.0) buffer. Active mTOR (1362-end, 250 ng) was incubated with dimethyl sulfoxide or aloe-emodin and then reacted with purified Akt1 fusion proteins (1 μg). Reactions were conducted in kinase buffer containing 50 μM unlabeled ATP with or without 10 μCi of [γ-32P]ATP at 30°C for 30 min. Reactions were terminated and proteins resolved by 8% SDS–polyacrylamide gel electrophoresis and visualized by autoradiography.

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mTORC2 in vitro kinase assay[1]
mTORC2 was pulled down with a Rictor antibody as described by Sarbassov et al. Purified Akt1 fusion proteins (1 μg) were used for an in vitro kinase assay. Reactions were conducted in kinase buffer containing 25 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 100 mM potassium acetate, 1 mM MgCl2 and 50 μM ATP at 30°C for 30 min. Reactions were terminated and proteins resolved by 8% or 6% SDS–polyacrylamide gel electrophoresis and visualized by Western blotting.

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PI3-K in vitro kinase assay[1]
The PI3-K in vitro kinase assay was performed as described. Briefly, 100 ng active PI3-K was incubated with dimethyl sulfoxide or aloe-emodin for 15 min and then reacted with phosphatidylinositol sodium salt. Reactions were performed in kinase buffer containing 50 μM unlabeled ATP with or without 10 μCi of [γ-32P]ATP at 30°C for 20 min. Reactions were terminated and resolved by thin layer chromatography and visualized by autoradiography.

Cell Viability Assay[1]
Cell Types: PC3 cells
Tested Concentrations: 0 μM, 2.5 μM, 5 μM, 10 μM or 15 μM
Incubation Duration: 24 h, 48 h, 72 h or 96 h
Experimental Results: Suppressed proliferation and anchorage-independent growth of PC3 cells.

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Western Blot Analysis[1]
Cell Types: PC3 cells
Tested Concentrations: 0 μM, 2.5 μM, 5 μM, 10 μM or 15 μM
Incubation Duration: 24 h
Experimental Results: Inhibited the activation of the downstream substrates of mTORC2, Akt and PKCα.

Animal/Disease Models: Athymic nude mice (BALB/c nude mouse, 6 weeks old)[1]
Doses: 10 mg/kg, 50 mg/kg (20% PEG400 in autoclaved PBS)
Route of Administration: intraperitoneal (ip)injection, five times per week; for 28 days
Experimental Results: demonstrated tumor suppression effects in vivo in an athymic nude mouse model.

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In vivo tumor growth assay[1]
Athymic nude mice (BALB/c nude mouse, 6 weeks old) were were maintained under ‘specific pathogen free’ conditions and all animal studies were conducted according to guidelines approved by the KRIBB-IACUC. Animals were acclimated for 2 weeks before the study and had free access to food and water. The animals were housed in climate-controlled quarters with a 12-h light/12-h dark cycle. Animals were randomly assigned to the following groups: vehicle group (n = 12); 10 mg/kg aloe-emodin group (n = 12); 50 mg/kg aloe-emodin group (n = 12) and 50 mg/kg aloe-emodin control group (n = 12). Each mouse was administered aloe-emodin (10 or 50 mg/kg body weight in 100 μl of 20% PEG400 in autoclaved phosphate-buffered saline as vehicle) or only vehicle five times per week by intraperitoneal injection. After 3 days of treatment, PC3 cells (1 × 106) were injected subcutaneously into the right flank of mice in the respective groups. Following injection, mice continued to be administered aloe-emodin or vehicle. Mice in the 50 mg/kg aloe-emodin control group were not injected with cells but maintained for comparison of body weight and tumor development. Mice were weighed and tumors measured by caliper three times per week. Tumor volume was calculated from measurements of two diameters of the individual tumor according to the following formula: tumor volume (mm3) = (length × width × width/2). Mice were monitored until day 28 and at that time, mice were euthanized and tumors extracted.

[1]. Aloe-emodin suppresses prostate cancer by targeting the mTOR complex 2. Carcinogenesis. 2012 Jul;33(7):1406-11.

[2]. Antiviral activity of aloe-emodin against influenza A virus via galectin-3 up-regulation. Eur J Pharmacol. 2014 Sep 5;738:125-32.

Aloe emodin is a dihydroxyanthraquinone that is chrysazin carrying a hydroxymethyl group at position 3. It has been isolated from plant species of the genus Aloe. It has a role as an antineoplastic agent and a plant metabolite. It is a dihydroxyanthraquinone and an aromatic primary alcohol. It is functionally related to a chrysazin.
Aloe-emodin has been reported in Rheum likiangense, Rheum franzenbachii, and other organisms with data available.
See also: Frangula purshiana Bark (part of).

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Phosphatidylinositol 3-kinase (PI3-K) amplification and phosphatase and tensin homolog (PTEN) deletion-caused Akt activation contribute to the development of prostate cancer. Mammalian target of rapamycin complex 2 (mTORC2) is a kinase complex comprised of mTOR, Rictor, mSin1, mLST8/GβL and PRR5 and functions in the phosphorylation of Akt at Ser473. Herein, we report that mTORC2 plays an important role in PC3 androgen refractory prostate cell proliferation and anchorage-independent growth. Aloe-emodin, a natural compound found in aloe, inhibited both proliferation and anchorage-independent growth of PC3 cells. Protein content analysis suggested that activation of the downstream substrates of mTORC2, Akt and PKCα, was inhibited by aloe-emodin treatment. Pull-down assay and in vitro kinase assay results indicated that aloe-emodin could bind with mTORC2 in cells and inhibit its kinase activity. Aloe-emodin also exhibited tumor suppression effects in vivo in an athymic nude mouse model. Collectively, our data suggest that mTORC2 plays an important role in prostate cancer development and aloe-emodin suppresses prostate cancer progression by targeting mTORC2.[1]

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Novel influenza A H7N9 virus, which emerged in 2013, and highly pathogenic H5N1 virus, identified since 2003, pose challenges to public health and necessitate quest for new anti-influenza compounds. Anthraquinone derivatives like aloe-emodin, emodin and chrysophanol, reportedly exhibit antiviral activity. This study probes their inhibitory mechanism and effect against influenza A virus. Of three anthraquinone derivatives, aloe-emodin, with a lower cytotoxicity showed concentration-dependently reducing virus-induced cytopathic effect and inhibiting replication of influenza A in MDCK cells. 50% inhibitory concentration value of aloe-emodin on virus yield was less than 0.05 μg/ml. Proteomics and Western blot of MDCK cells indicated aloe-emodin up-regulating galectin-3, and thioredoxin as well as down-regulating nucleoside diphosphate kinase A. Western blot and quantitative PCR confirmed aloe-emodin up-regulating galectin-3 expression; recombinant galectin-3 augmented expression of antiviral genes IFN-β, IFN-γ, PKR and 2'5',-OAS in infected cells, agreeing with expression pattern of those treated with aloe-emodin. Galectin-3 also inhibited influenza A virus replication. Proteomic analysis of treated cells indicated galectin-3 up-regulation as one anti-influenza A virus action by aloe-emodin. Since galectin-3 exhibited cytokine-like regulatory actions via JAK/STAT pathways, aloe-emodin also restored NS1-inhibited STAT1-mediated antiviral responses in transfected cells: e.g., STAT1 phosphorylation of interferon (IFN) stimulation response element (ISRE)-driven promoter, RNA-dependent protein kinase (PKR) and 2'5',-oligoadenylate synthetase (2'5',-OAS) expression. Treatment with aloe-emodin could control influenza infection in humans.[2]

C15H10O5

270.24

270.052

C, 66.67; H, 3.73; O, 29.60

481-72-1

10207

Typically exists as Brown to orange solids at room temperature

1.6±0.1 g/cm3

568.8±50.0 °C at 760 mmHg

223-224°C

311.9±26.6 °C

0.0±1.6 mmHg at 25°C

1.746

3.38

3

5

1

20

421

0

O([H])C1=C([H])C(C([H])([H])O[H])=C([H])C2C(C3C([H])=C([H])C([H])=C(C=3C(C=21)=O)O[H])=O

YDQWDHRMZQUTBA-UHFFFAOYSA-N

InChI=1S/C15H10O5/c16-6-7-4-9-13(11(18)5-7)15(20)12-8(14(9)19)2-1-3-10(12)17/h1-5,16-18H,6H2

1,8-dihydroxy-3-(hydroxymethyl)anthracene-9,10-dione

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)

Solubility in Formulation 1: 2.08 mg/mL (7.70 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: 2.08 mg/mL (7.70 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 20.8 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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 (Please use freshly prepared in vivo formulations for optimal results.)