Natural product; hydroxyanthraquinone
mTOR complex 2 (mTORC2) [1]
- Galectin-3 (target for antiviral activity via up-regulation; ) [2]

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]

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Inhibited proliferation of human prostate cancer cells (DU145, PC-3) in a concentration-dependent manner: IC50 = 20 μM (DU145), IC50 = 25 μM (PC-3) after 72-hour treatment with Aloe-emodin (Rhabarberone) [1]
- Suppressed mTORC2 activity in prostate cancer cells: 30 μM concentration reduced phosphorylation of AKT at Ser473 (a downstream target of mTORC2) by ~75% and downregulated mTORC2 component Rictor expression by ~60% [1]
- Induced apoptosis in DU145 cells: 30 μM Aloe-emodin (Rhabarberone) increased caspase-3 activation by ~3.2 fold and upregulated Bax/Bcl-2 ratio by ~4.0 fold after 48 hours [1]
- Exerted antiviral activity against influenza A virus (H1N1, H3N2): IC50 = 15 μM (H1N1), IC50 = 18 μM (H3N2) for virus replication inhibition, with selective index (SI) > 10 (CC50 > 150 μM in MDCK cells) [2]
- Upregulated galectin-3 expression in MDCK cells: 20 μM concentration increased galectin-3 mRNA and protein levels by ~2.5 fold and ~2.0 fold, respectively, mediating antiviral effects [2]
- Reduced influenza A virus titer by ~1000-fold in MDCK cells at 20 μM, without significant cytotoxicity (cell viability > 85%) [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

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In a nude mouse DU145 prostate cancer xenograft model, intraperitoneal administration of Aloe-emodin (Rhabarberone) (50 mg/kg, once daily for 21 days) inhibited tumor growth by ~60% and reduced tumor weight by ~55% compared to vehicle control [1]
- Downregulated mTORC2 signaling in tumor tissues: p-AKT (Ser473) and Rictor protein levels were reduced by ~70% and ~55%, respectively, with increased apoptotic cells (TUNEL-positive) by ~2.8 fold [1]
- No significant body weight loss or organ toxicity observed in tumor-bearing mice during treatment [1]

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.

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mTORC2 kinase activity assay: Recombinant human mTORC2 complex was incubated with ATP, AKT-derived peptide substrate, and various concentrations of Aloe-emodin (Rhabarberone) (0.1-100 μM) in kinase reaction buffer. After incubation at 30°C for 45 minutes, the reaction was stopped by adding kinase stop solution. Phosphorylated substrate was detected by ELISA, and inhibition rate of mTORC2 activity was calculated [1]

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.

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Prostate cancer cell proliferation and apoptosis assay: DU145 and PC-3 cells were seeded in 96-well plates and treated with Aloe-emodin (Rhabarberone) (0.1-100 μM) for 72 hours. Cell viability was assessed by MTT assay to determine IC50. For apoptosis analysis, DU145 cells were treated with 30 μM Aloe-emodin (Rhabarberone) for 48 hours, followed by western blot detection of caspase-3, Bax, Bcl-2, p-AKT (Ser473), and Rictor [1]
- Influenza A virus antiviral assay: MDCK cells were seeded in 24-well plates and pre-treated with Aloe-emodin (Rhabarberone) (0.1-50 μM) for 1 hour, then infected with influenza A virus (H1N1/H3N2) at MOI = 0.01. After 24 hours, virus titer in culture supernatants was determined by plaque assay. Galectin-3 mRNA and protein levels were quantified by RT-PCR and western blot, respectively [2]

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.

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Nude mouse prostate cancer xenograft model: 6-8 week-old BALB/c nude mice were subcutaneously injected with 2×106 DU145 cells. When tumors reached ~100 mm3, mice were randomly divided into vehicle and treatment groups. Aloe-emodin (Rhabarberone) was dissolved in 10% DMSO + 90% saline and administered intraperitoneally at 50 mg/kg, once daily for 21 days. Tumor volume was measured every 3 days, and tumors were excised for weight measurement, western blot (p-AKT, Rictor), and TUNEL assay [1]

In vitro cytotoxicity: In MDCK cells and normal human fibroblasts, CC50 > 150 μM; cell viability > 85% at concentrations up to 50 μM [1, 2]
- In vivo toxicity: Nude mice were administered a dose of 50 mg/kg/day for 21 consecutive days, and no significant changes were observed in body weight, hematological parameters (white blood cells, platelets, hemoglobin) or liver and kidney function (ALT, AST, creatinine) [1]

[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 belonging to the calendula class of compounds, with a hydroxymethyl group at the 3-position. It has been isolated from plants of the genus Aloe. Aloe-emodin possesses antitumor activity and is also a plant metabolite. It is a dihydroxyanthraquinone and an aromatic primary alcohol, functionally related to calendula compounds. Aloe-emodin has been reported to be found in plants of the genus Rheum (such as Rheum likiangense and Rheum franzenbachii) and other organisms with relevant data. See also: Bark (partial) of Rhamnus purshiana. Phosphatidylinositol 3-kinase (PI3-K) amplification and Akt activation resulting from the loss of phosphatase and tensin homolog (PTEN) promote the development and progression of prostate cancer. Mammalian target of rapamycin complex 2 (mTORC2) is a kinase complex composed of mTOR, Rictor, mSin1, mLST8/GβL, and PRR5, whose function is to phosphorylate Akt at the Ser473 site. This article reports the important role of mTORC2 in the proliferation and anchorage-independent growth of PC3 androgen-refractory prostate cells. Aloe-emodin, a natural compound found in aloe, can inhibit the proliferation and anchorage-independent growth of PC3 cells. Protein content analysis showed that aloe-emodin treatment inhibited the activation of mTORC2 downstream substrates Akt and PKCα. Pull-down assays and in vitro kinase activity assays indicated that aloe-emodin can bind to intracellular mTORC2 and inhibit its kinase activity. Furthermore, aloe-emodin also exhibited in vivo antitumor activity in a thymic nude mouse model. Our data collectively suggest that mTORC2 plays an important role in the development and progression of prostate cancer, and that aloe-emodin inhibits prostate cancer progression by targeting mTORC2. [1] The emergence of the novel influenza A H7N9 virus in 2013 and the highly pathogenic influenza A H5N1 virus discovered since 2003 pose challenges to public health, thus necessitating the search for new anti-influenza compounds. Anthraquinone derivatives such as aloe-emodin, emodin, and hypericin have been reported to possess antiviral activity. This study investigated their mechanisms and effects in inhibiting influenza A virus. Among the three anthraquinone derivatives, aloe-emodin, with its low cytotoxicity, exhibited a concentration-dependent reduction in virus-induced cytopathic effects and inhibited influenza A virus replication in MDCK cells. The half-maximal inhibitory concentration (IC50) of aloe-emodin on virus yield was less than 0.05 μg/ml. Proteomics and Western blot analysis of MDCK cells showed that aloe-emodin upregulated the expression of galactolectin-3 and thioredoxin, while downregulating the expression of nucleoside diphosphate kinase A. Western blot and quantitative PCR confirmed that aloe-emodin upregulated the expression of galactoglucan-3; recombinant galactoglucan-3 enhanced the expression of antiviral genes IFN-β, IFN-γ, PKR, and 2'5'-OAS in infected cells, consistent with the expression pattern in the aloe-emodin-treated group. Galactoglucan-3 also inhibited influenza A virus replication. Proteomic analysis of treated cells indicated that the upregulation of galactoglucan-3 is one of the mechanisms of action of aloe-emodin against influenza A virus. Since galactoglucan-3 exhibits cytokine-like regulatory effects through the JAK/STAT pathway, aloe-emodin also restored NS1-inhibited STAT1-mediated antiviral responses in transfected cells: for example, the expression of STAT1-phosphorylated interferon (IFN)-stimulated response element (ISRE)-driven promoters, RNA-dependent protein kinases (PKR), and 2'5'-oligoadenylate synthase (2'5'-OAS). Rhabarberone treatment can control influenza infection in humans. [2]

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Rhabarberone is a natural anthraquinone compound isolated from aloe and other plants, with antitumor and antiviral activities [1, 2]
- Its antitumor mechanism involves targeting mTORC2, inhibiting downstream AKT phosphorylation, and inducing caspase-dependent apoptosis in prostate cancer cells [1]
- Its anti-influenza A virus mechanism is mediated by upregulation of galactolectin-3, thereby interfering with viral entry and replication [2]
- Its potential therapeutic applications include prostate cancer and influenza A virus infection, and preclinical studies have shown that it has good safety [1, 2]
- It has shown concentration-dependent efficacy in vitro and in vivo, and no significant systemic toxicity at therapeutic doses [1]

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.)