New application of in silico methods in identifying mechanisms of action and key components of anti-cancer herbal formulation YIV-906 (PHY906).

YIV-906 (formally PHY906, KD018) is a four-herb formulation that is currently being developed to improve the therapeutic index and ameliorate the side effects of many chemotherapeutic drugs including sorafenib, irinotecan, and capecitabine. However, as a promising anti-cancer adjuvant, the molecular mechanism of action of YIV-906 remains unrevealed due to its multi-component and multi-target features. Since YIV-906 has been shown to induce apoptosis and autophagy in cancer cells through modulating the negative regulators of ERK1/2, namely DUSPs, it is of great interest to elucidate the key components that cause the therapeutic effect of YIV-906. In this work, we investigated the mechanism of YIV-906 inhibiting DUSPs, using a broad spectrum of molecular modelling techniques, including molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations. In total, MD simulations and binding free energy calculations were performed for 99 DUSP-ligand complexes. We found that some herbal components or their metabolites could inhibit DUSPs. Based on the docking scores and binding free energies, the sulfation and glucuronidation metabolites of the S ingredient in YIV-906 play a leading role in inhibiting DUSPs, although several original herbal chemicals with carboxyl groups from the P and Z ingredients also make contributions to this inhibitory effect. It is not a surprise that the electrostatic interaction plays the dominant role in the ligand binding process, given the fact that several charged residues reside in the binding pockets of DUSPs. Our MD simulation results demonstrate that the sulfate moieties and carboxyl moieties of the advantageous ligands from YIV-906 can occupy the enzymes' catalytic sites, mimicking the endogenous phosphate substrates of DUSPs. As such, the ligand binding can inhibit the association of DUSPs and ERK1/2, which in turn reduces the dephosphorylation of ERK1/2 and causes cell cycle arrest in the tumor. Our modelling study provides useful insights into the rational design of highly potent anti-cancer drugs targeting DUSPs. Finally, we have demonstrated that multi-scale molecular modelling techniques are able to elucidate molecular mechanisms involving complex molecular systems.

Isobavachalcone exerts anti­proliferative and pro­apoptotic effects on human liver cancer cells by targeting the ERKs/RSK2 signaling pathway.

Aberrant activation of the extracellular signal­regulated kinases (ERKs)/ribosomal S6 kinase 2 (RSK2) signaling pathway is frequently determined in various human tumor types, including liver cancer, and has been considered as a promising target for cancer chemoprevention and therapy. In the present study, using computer­aided virtual screening and molecular docking, isobavachalcone (IBC), a natural chalcone compound, was identified to be an ATP­competitive inhibitor targeting ERK1/2 and RSK2. Cell Counting Kit­8, EdU incorporation and colony formation assays were used to detect the effects of IBC on cell viability and proliferation, and the results demonstrated that IBC effectively inhibited the proliferation of liver cancer HepG2 and Hep3B cells, whereas it had no notable cytotoxic effect on immortal liver L02 cells. Flow cytometric analysis and western blotting further revealed that IBC caused significant levels of apoptosis on liver cancer cells via the caspase­dependent mitochondria pathway. The computer prediction was confirmed with pull­down and in vitro kinase assays, in which IBC directly bound with ERK1/2 and RSK2, and dose­dependently blocked RSK2 kinase activity in liver cancer cells. Treatment of HepG2 or Hep3B cells with IBC significantly attenuated epidermal growth factor­induced phosphorylation of RSK2 and resulted in the reduced activation of its downstream substrates including cAMP response element­binding protein, activating transcription factor 1, histone H3 and activating protein­1. Enforced RSK2 expression in L02 cells could increase the effect of IBC on suppressing cell growth. Conversely, knockdown of RSK2 reduced the inhibitory effect of IBC on HepG2 cell proliferation. Overall, the present data indicated that ERKs/RSK2 signaling serves a pivotal role in IBC­induced suppression of liver cancer cells and that IBC may be a potential therapeutic candidate for human cancer with elevated ERKs/RSK2 activity.

Altered behavior of adult obese rats by monosodium l-glutamate neonatal treatment is related to hypercorticosteronemia and activation of hypothalamic ERK1 and ERK2.

OBJECTIVES: Obesity is a metabolic and hormonal disorder with serious social and psychological impacts. There is a close relationship among obesity, neuroendocrine homeostasis and behavioral patterns. However, few data are available in the literature regarding this subject. This study assessed behavior and memory of adult obese rats by monosodium l-glutamate (MSG) neonatal treatment or highly palatable dietary treatment. METHODS: MSG obesity was induced by subcutaneous injections of MSG (4 mg/g) during the first 5 days of life (Ob-MSG); control group (C-MSG), received saline solution equimolar. Both groups were fed with commercial chow. To induce dietary obesity, 21-day-old rats were assigned to two experimental diets: highly palatable diet (Ob-Diet) and control diet (C-Diet) composed of commercial chow. Ninety-day-old animals were submitted to behavioral assessment by the open-field test and short- and long-term memory by the object recognition test. Biometric variables were obtained, the Lee index was calculated and mass of retroperitoneal and perigonadal fat pads was measured. Furthermore, an altered behavioral profile was investigated by quantification of plasmatic corticosterone, expression, and activity of hypothalamic extracellular signal-regulated kinase protein (ERK) 1 and 2. RESULTS: Increased Lee index and fat pads were observed in Ob-MSG and Ob-Diet groups. Ob-MSG presented a higher level of anxiety and impaired long-term memory compared to C-MSG, while there was no difference between Ob-Diet and C-Diet. The Ob-MSG group presented a higher level of plasmatic corticosterone and increased phosphorylation of hypothalamic ERK1 and 2. DISCUSSION: Both treatments induced obesity but only Ob-MSG showed altered behavioral parameters, which is related to increased concentration of corticosterone and hypothalamic ERK1 and 2 activation.

Scopoletin, an active principle of tree tobacco (Nicotiana glauca) inhibits human tumor vascularization in xenograft models and modulates ERK1, VEGF-A, and FGF-2 in computer model.

We recently reported the antineovascularization effect of scopoletin on rat aorta and identified its potential anti-angiogenic activity. Scopoletin could be useful as a systemic chemotherapeutic agent against angiogenesis-dependent malignancies if its antitumorigenic activity is investigated and scientifically proven using a suitable human tumor xenograft model. In the present study, bioassay-guided (anti-angiogenesis) phytochemical investigation was conducted on Nicotiana glauca extract which led to the isolation of scopoletin. Further, anti-angiogenic activity of scopoletin was characterized using ex vivo, in vivo and in silico angiogenesis models. Finally, the antitumorigenic efficacy of scopoletin was studied in human colorectal tumor xenograft model using athymic nude mice. For the first time, an in vivo anticancer activity of scopoletin was reported and characterized using xenograft models. Scopoletin caused significant suppression of sprouting of microvessels in rat aortic explants with IC50 (median inhibitory concentration) 0.06µM. Scopoletin (100 and 200mg/kg) strongly inhibited (59.72 and 89.4%, respectively) vascularization in matrigel plugs implanted in nude mice. In the tumor xenograft model, scopoletin showed remarkable inhibition on tumor growth (34.2 and 94.7% at 100 and 200mg/kg, respectively). Tumor histology revealed drastic reduction of the extent of vascularization. Further, immunostaining of CD31 and NG2 receptors in the histological sections confirmed the antivascular effect of scopoletin in tumor vasculature. In computer modeling, scopoletin showed strong ligand affinity and binding energies toward the following angiogenic factors: protein kinase (ERK1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2). These results suggest that the antitumor activity of scopoletin may be due to its strong anti-angiogenic effect, which may be mediated by its effective inhibition of ERK1, VEGF-A, and FGF-2.