PKC; PKCδ(IC50 = 3-6 μM); PKCα,β,γ (IC50 = 30-42 μM); PKCε,η,ζ(IC50 = 80-100 μM)
Rottlerin is a dose-dependent inhibitor of PKCθ (IC50 ≈ 1.25 μM) and PKCδ (IC50 ≥ 6.0 μM). At concentrations < 6.0 μM, it shows selective inhibition for PKCθ over PKCδ and PKCε/λ.

In primary HMVECs, rottlerin (20 μM, 2/6/24 hours) significantly and time-dependently lowers the levels of cyclin D-1 mRNA [2]. In HMVEC, rottlerin (20 μM) causes cell growth [2].

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Rottlerin, a natural product purified from Mallotus philippinensis, has a number of target molecules and biological effects. We recently found that Rottlerin caused growth arrest in MCF-7 breast cancer cells and human immortalized keratinocytes, through inhibition of NFκB and downregulation of cyclin D-1. To evaluate whether this effect could be generalized to primary cells, human microvascular endothelial cells were treated with Rottlerin. In this study, we demonstrated that Rottlerin prevents basal and TNFα-stimulated NFκB nuclear migration and DNA binding also in human microvascular endothelial cell, where NFκB inhibition was accompanied by the downregulation of NFκB target gene products, such as cyclin D-1 and endothelin-1, which are essential molecules for endothelial cell proliferation and survival. Rottlerin, indeed, inhibited human microvascular endothelial cells proliferation and tube formation on Matrigel. Rottlerin also increases cytoplasmic free calcium and nitric oxide levels and downregulates endothelin converting enzyme-1 expression, thus contributing to the drop in endothelin-1 and growth arrest. These results suggest that Rottlerin may prove useful in the development of therapeutic agents against angiogenesis[2].

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Rottlerin inhibited HIV-1 replication in T cells. In MT-2 cells, IC50 was 5.2 μM, resulting in more than 20-fold reduction. In Jurkat cells, IC50 was 2.2 μM, with more than 20-fold reduction. In anti-CD3/CD28-activated peripheral blood lymphocytes (PBLs), IC50 was 4.4 μM, leading to more than 4-fold reduction.
Rottlerin (3.0 μM) significantly inhibited HIV-1 proviral integration in MT-2 cells.
Rottlerin induced a dose-dependent inhibition of HIV-1 LTR transactivation in basal conditions and upon PMA activation in Jurkat cells. It also inhibited NF-κB-dependent luciferase expression and Tat-mediated HIV-1 transactivation.
Stable RNA interference of PKCθ mRNA in Jurkat and MT-2 cells (achieving 60-70% knockdown) led to more than 3-fold and 4-fold reduction in HIV-1 replication, respectively, compared to control cells.
Transient co-transfection of PBLs with plasmids for PKCθ shRNA and HIV-1 proviral clone resulted in more than 6-fold reduction in HIV-1 replication.
Rottlerin did not inhibit HIV-1 replication in PKCθ non-expressing U87.CD4.CXCR4 cells at concentrations within the low cytotoxicity range.
Rottlerin did not interfere with viral entry, as it inhibited VSV-pseudotyped HIV-1 infection (independent of CD4/CXCR4) and did not significantly modify cell surface expression of CD4, CXCR4, or CCR5.
Rottlerin (3.0 μM) did not modify the amount of early (R/U5) and late (LTR/gag) HIV-1 reverse transcription products in MT-2 cells.
Rottlerin (3.0 μM) reduced phosphorylation of PKCθ at Thr538, prevented its translocation to lipid rafts, and repressed the PKCθ pathway effector NF-κB by inhibiting IκBα phosphorylation/degradation and NF-κB nuclear binding activity.

In Balb C nude mice, Rottlerin (20 mg/kg, once daily, five days a week, for six weeks) inhibits the growth of AsPC-1 pancreatic tumors without causing toxicity [3]. Rottlerin activates caspase-3 and cleaves poly(ADP-ribose) polymerase (PARP) to induce apoptosis.

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Rottlerin-treated mice showed a significant inhibition in tumor growth which was associated with suppression of cell proliferation, activation of capase-3 and cleavage of PARP. Rottlerin inhibited the expression of Bcl-2, cyclin D1, CDK2 and CDK6, and induced the expression of Bax in tumor tissues compared to untreated control. Rottlerin inhibited the markers of angiogenesis (Cox-2, VEGF, VEGFR, and IL-8), and metastasis (MMP-2 and MMP-9), thus blocking production of tumorigenic mediators in tumor microenvironment. Rottlerin also inhibited epithelial-mesenchymal transition by up-regulating E-cadherin and inhibiting the expression of Slug and Snail. Furthermore, rottlerin treatment of xenografted tumors or pancreatic cancer cells isolated from Kras(G12D) mice showed a significant inhibition in Akt, Shh and Notch pathways compared to control groups. These data suggest that rottlerin can inhibit pancreatic cancer growth by suppressing multiple signaling pathways which are constitutively active in pancreatic cancer. Taken together, our data show that the rottlerin induces apoptosis and inhibits pancreatic cancer growth by targeting Akt, Notch and Shh signaling pathways, and provide a new therapeutic approach with translational potential for humans.[3]

Rottlerin, a compound from Mallotus philippinensis, is shown to inhibit protein kinases with some specificity for PKC. To some extent, the novel inhibitor is able to differentiate between PKC isoenzymes, with IC50 values for PKC delta of 3-6 microM, PKC alpha,beta,gamma of 30-42 microM and PKC epsilon,eta,zeta of 80-100 microM. Inhibition of PKC appears, at least in part, to be due to a competition between rottlerin and ATP. Among the protein kinases tested, only CaM-kinase III is suppressed by rottlerin as effectively as PKC delta. The chemical structure of rottlerin might serve as a basis for the development of novel inhibitors with improved selectivity for a distinct PKC isoenzyme, such as PKC delta, or for CaM-kinase III [1].

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PKCθ and PKCδ enzymatic activity was assayed using an immunoprecipitation kinase assay. Jurkat cells were incubated with inhibitors for 18 hours, lysed, and cytosolic extracts were immunoprecipitated with anti-PKCθ or anti-PKCδ antibodies. The immunoprecipitates were incubated with a PKCε peptide substrate, phosphatidylserine, diacylglycerol, and [γ-32P]ATP for 10 minutes at 30°C. The reaction was stopped by spotting onto phosphocellulose P81 paper, washed with phosphoric acid, and radioactivity was counted. Inhibition of enzymatic activity was analyzed, and IC50 was calculated.

Western blot analysis[2]
Cell Types: primary HMVEC (human microvascular endothelial cells).
Tested Concentrations: 20μM.
Incubation Duration: 2, 6, 24 hrs (hours).
Experimental Results: Cyclin D-1 mRNA levels were Dramatically diminished in a time-dependent manner. After 2 hrs (hours) of treatment, mRNA levels diminished to 50% of control, after 6 hrs (hours) to approximately 40%, and after 24 hrs (hours) to 20%. A similar trend was observed at the protein level, with a decrease of approximately 50% after 2 hrs (hours), an 80% decrease after 6 hrs (hours), and a decrease to almost undetectable levels after 24 hrs (hours).

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Cell proliferation assay [2]
Cell Types: primary HMVEC (human microvascular endothelial cells).
Tested Concentrations: 20μM.
Incubation Duration: 24/48 hrs (hours).
Experimental Results: demonstrated strong growth inhibition, with thymidine incorporation diminished by approximately 75% and 80%, respectively, relative to control cells (DMSO 0.1%).

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For HIV-1 infection assays, PBLs activated with anti-CD3/CD28 or PHA/IL-2 for 3 days, or MT-2 cells, were pretreated with Rottlerin for 30 minutes and then infected with HIV-1 NL4.3 strains for 2 hours. After washing, the compound was added again and left in culture for 2-7 days. Supernatants were collected for p24 antigen measurement, and cells were lysed for Renilla or luciferase activity quantification. IC50 was determined using a 96-well plate format with recombinant HIV-1 NL4.3-Renilla and increasing concentrations of Rottlerin, measuring Renilla activity 48 hours post-infection.
Cell viability was determined using a luminescent cell viability assay quantifying ATP levels in metabolically active cells after incubation with Rottlerin for 72 hours. Cell viability was also assessed by bright-field microscopy and flow cytometry with propidium iodide staining.
Cell proliferation was measured using a non-radioactive cell proliferation assay. PBLs were cultured with or without PHA and Rottlerin for 72 hours, followed by addition of a tetrazolium compound mixture. Absorbance was measured at 490 nm after 3 hours.
For analysis of HIV-1 reverse transcription, MT-2 cells infected with HIV-1 NL4.3-wt for 18 hours were used to extract total DNA. Semiquantitative PCR was performed to amplify short (R/U5) and long (LTR/gag) reverse transcriptase products.
For analysis of HIV-1 proviral integration, a semiquantitative nested Alu-PCR assay was performed on genomic DNA extracted from MT-2 cells infected with HIV-1 NL4.3-wt for 18 hours.
For transcriptional activity assays, Jurkat cells were transfected with LTR-LUC or 3κB-LUC reporter vectors, treated with Rottlerin and/or PMA, and luciferase activity was measured after 18 hours.
For DNA affinity immunoblotting, nuclear extracts from Jurkat cells were incubated with a biotin-labeled probe containing κB consensus motifs. Protein-DNA complexes were captured with streptavidin-agarose, separated by SDS-PAGE, and analyzed by immunoblotting for p65/RelA.
For generation of stable PKCθ knockdown cells, Jurkat and MT-2 cells were co-transfected with shRNA plasmids (pGeneClip-iPKCθ-1 and pGeneClip-iPKCθ-3) by electroporation and selected with puromycin.

Animal/Disease Models: Balb C nude mice (4-6 weeks old) were injected with AsPC-1 cells (2×106 cells mixed with Matrigel, 50:50 ratio) [3].
Doses: 0 or 20 mg/kg.
Route of Administration: Administer one time/day, 5 days a week, for 6 weeks.
Experimental Results: Inhibited the growth of AsPC-1 pancreatic tumors in Balb C nude mice and had no effect on the body weight of AsPC-1 tumor-bearing mice.

Rats were orally administered LDLo 750 mg/kg. Indian Journal of Physiology and Pharmacology, 3(168), 1959 [PMID:13841348]

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Cytotoxicity was assessed in resting PBL, Jurkat, and MT-2 cells treated with escalating doses of Rottlerin for 72 hours. Results showed that cytotoxicity was induced in PBL cells at concentrations ≥15 μM and in Jurkat cells at concentrations ≥10 μM. No significant cytotoxicity was observed in MT-2 cells even at a concentration of 100 μM. The half-maximal cytotoxic concentration (CC50) was calculated using an S-type dose-response formula. In HIV-1-infected MT-2 cells, long-term treatment (7 days) with Rottlerin did not significantly reduce cell viability; instead, it increased cell viability by more than twofold by protecting cells from HIV-induced cytopathic effects/syncytial formation.
Rottlerin inhibited PHA-induced T cell proliferation in a dose-dependent manner, maintaining T cell proliferation levels at resting cell levels at a concentration of 3.0 μM.

[1]. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun. 1994 Feb 28;199(1):93-8.

[2]. Rottlerin exhibits antiangiogenic effects in vitro. Chem Biol Drug Des. 2011 Jun;77(6):460-70.

[3]. Rottlerin suppresses growth of human pancreatic tumors in nude mice, and pancreatic cancer cells isolated from KrasG12D mice. Cancer Letters 353 (2014) 32-40.

[4]. Protein kinase Ctheta is a specific target for inhibition of the HIV type 1 replication in CD4+ T lymphocytes. J Biol Chem. 2011 Aug 5;286(31):27363-77.

[5]. Kinase inhibitors tyrphostin 9 and rottlerin block early steps of rabies virus cycle. Antiviral Res. 2019 Aug;168:51-60.

Rottlerin is a chromenolate compound with the structure 2,2-dimethyl-2H-chromene, substituted with hydroxyl groups at positions 5 and 7, substituted with 3-acetyl-2,4,6-trihydroxy-5-methylbenzyl at position 6, and substituted with (1E)-3-oxo-1-phenylprop-1-en-3-yl at position 8. It is a potassium channel opener, isolated from Mallotus philippensis. Rottlerin possesses various pharmacological activities, including antitumor activity, apoptosis induction, as a metabolite, a K-ATP channel agonist, antihypertensive activity, and anti-allergic activity. It is a ketone, chromenolate, glycerol, methyl ketone, and aromatic ketone. Rottlerin has been reported to exist in Mallotus philippensis, and relevant data exist. This study aims to investigate the molecular mechanism by which Rottlerin inhibits the growth of human pancreatic tumors in Balb C nude mice and the growth of pancreatic cancer cells isolated from Kras(G12D) mice. AsPC-1 cells were subcutaneously injected into Balb C nude mice, and tumor-bearing mice were treated with roteglin. Cell proliferation and apoptosis were detected by Ki67 and TUNEL staining, respectively. The expression of Akt, Notch, and Sonic Hedgehog (Shh) signaling pathway components was detected by immunohistochemistry, Western blot analysis, and/or qRT-PCR. The effects of roteglin on pancreatic cancer cells isolated from Kras (G12D) mice were also investigated. Tumor growth in roteglin-treated mice was significantly inhibited, which was associated with cell proliferation inhibition, caspase-3 activation, and PARP lysis. Compared with the untreated control group, roteglin inhibited the expression of Bcl-2, cyclin D1, CDK2, and CDK6 in tumor tissue and induced Bax expression. Roteglin inhibited angiogenesis markers (Cox-2, VEGF, VEGFR, and IL-8) and metastasis markers (MMP-2 and MMP-9), thereby blocking the production of tumorigenic mediators in the tumor microenvironment. Roterrin also inhibits epithelial-mesenchymal transition by upregulating E-cadherin and inhibiting the expression of Slug and Snail. In addition, compared with the control group, the Akt, Shh and Notch signaling pathways were significantly inhibited after treatment with Roterrin on xenograft tumors or pancreatic cancer cells isolated from Kras (G12D) mice. These data suggest that roderrin can inhibit the growth of pancreatic cancer by inhibiting multiple signaling pathways that are persistently activated in pancreatic cancer. In summary, our data suggest that roderrin can provide a novel therapeutic approach with transformation potential for humans by inducing apoptosis and inhibiting the growth of pancreatic cancer by targeting the Akt, Notch and Shh signaling pathways. [3] The integration of the HIV-1 genome into CD4(+) T cells produces a latent viral reservoir with a long half-life, thereby hindering the clearance of infection. Controlling viral replication is crucial to reducing the size of the latent viral reservoir, especially during primary infection of CD4(+) T cells with a large number of HIV-1 infections. During primary infection, the addition of immunosuppressants to highly active antiretroviral therapy can suppress HIV-1 replication by limiting T cell activation, but these drugs may pose a risk of inducing lymphoproliferative disorders. Selective inhibition of protein kinase C (PKC), which is crucial for T cell function, can limit T cell activation and HIV-1 replication without causing systemic immunosuppression, as PKC is primarily expressed in T cells. Therefore, we analyzed the effect of the dose-dependent PKC inhibitor rodttlerin on HIV-1 replication in T cells. Rottlerin reduced HIV-1 replication by more than 20-fold in MT-2 cells (IC50 = 5.2 μM) and Jurkat cells (IC50 = 2.2 μM), and by more than 4-fold in peripheral blood lymphocytes (IC50 = 4.4 μM). At concentrations below 6.0 μM, selective inhibition of PKC (but not PKCδ or -ζ) by Rottlerin was observed, reducing the phosphorylation level of Thr(538) residues on the activation loop of the kinase catalytic domain and preventing the translocation of PKC to lipid rafts. As a result, the major effector molecule at the end of the PKC pathway, NF-κB, was inhibited. Rottlerin also significantly inhibited HIV-1 integration. In recent years, several specific PKC inhibitors have been designed for the treatment of autoimmune diseases. Combining these inhibitors with highly potent antiretroviral therapy during primary infection may help prevent large-scale viral infection and replication of infected CD4(+) T cells, thereby reducing the size of the viral reservoir in the early stages of infection. [4] Rabies virus (RABV) is a neurotropic virus that causes fatal encephalitis in humans and animals and still causes up to 59,000 deaths worldwide each year. To date, only prophylactic or post-exposure vaccination can prevent the disease, but there is no effective treatment. After screening a library of compounds containing 80 kinase inhibitors, we found two effective RABV infection inhibitors: tyrphostin 9 and rodttlerin. Mechanism studies showed that both inhibitors interfere with early steps of the viral cycle and prevent viral replication. In the presence of tyrphostin 9, the process of viral entry into the cell via endocytosis is disrupted, preventing viral particles from entering the cytoplasm normally; while rodttlerin inhibits transcription, likely by reducing intracellular ATP concentration, thereby inhibiting viral genome replication. [5]

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Rottlerin (also known as marrotoxin) is a cell-permeable protein kinase C (PKC) inhibitor used as a research tool. Its inhibitory effect on HIV-1 replication persists from the onset of infection in long-term culture.
Rottlerin was used in this study to demonstrate the importance of PKCθ in HIV-1 replication in T cells, rather than to promote it as a clinical candidate drug. The compound has a narrow therapeutic window and is used only for research.
PKCθ is selectively expressed on T cells and is crucial for T cell receptor-mediated activation and the NF-κB signaling pathway. Specific inhibition of PKCθ can limit T cell activation and HIV-1 replication without causing systemic immunosuppression.
The authors suggest that specific PKCθ inhibitors designed for autoimmune diseases could be combined with highly active antiretroviral therapy (HAART) for primary HIV-1 infection to reduce viral replication and the establishment of a latent viral reservoir.

C30H28O8

516.53852

516.178

C, 69.76; H, 5.46; O, 24.78

82-08-6

5281847

Brown to reddish brown solid powder

1.4±0.1 g/cm3

800.4±65.0 °C at 760 mmHg

200 °C

266.0±27.8 °C

0.0±2.9 mmHg at 25°C

1.682

8.66

5

8

6

38

921

0

CC1=C(C(=C(C(=C1O)C(=O)C)O)CC2=C(C(=C3C(=C2O)C=CC(O3)(C)C)C(=O)/C=C/C4=CC=CC=C4)O)O

DEZFNHCVIZBHBI-ZHACJKMWSA-N

InChI=1S/C30H28O8/c1-15-24(33)19(27(36)22(16(2)31)25(15)34)14-20-26(35)18-12-13-30(3,4)38-29(18)23(28(20)37)21(32)11-10-17-8-6-5-7-9-17/h5-13,33-37H,14H2,1-4H3/b11-10+

(E)-1-(6-((3-Acetyl-2,4,6-trihydroxy-5-methylphenyl)methyl)-5,7-dihydroxy-2,2-dimethyl-2H-1-benzopyran-8-yl)-3-phenyl-2-propen-1-one

Mallotoxin; NSC 56346; rottlerin; Mallotoxin; 82-08-6; Kamalin; UNII-E29LP3ZMUH; E29LP3ZMUH; EINECS 201-395-4; NSC 94525; NSC56346; NSC94525; Kamalin; NSC-56346; NSC-94525.

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 : ~12.5 mg/mL (~24.20 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.42 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 12.5 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: ≥ 1.25 mg/mL (2.42 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 22 mg/mL (42.59 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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