Licochalcone A inhibits proliferation and promotes apoptosis of colon cancer cell by targeting programmed cell death-ligand 1 via the NF-κB and Ras/Raf/MEK pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhiza glabra L., a traditional medicinal, has a history of thousands of years. It is widely used in clinic and has been listed in Chinese Pharmacopoeia. Licochalcone A is a phenolic chalcone compound and a characteristic chalcone of Glycyrrhiza glabra L. It has many pharmacological activities, such as anti-cancer, anti-inflammatory, anti-viral and anti-angiogenic activities. AIM OF THE STUDY: In this study, we explored the anti-tumor activity and potential mechanism of licochalcone A in vitro and in vivo. MATERIALS AND METHODS: In vitro, the mechanism of licochalcone A at inhibiting PD-L1 expression was investigated by molecular docking, western blotting, RT-PCR, flow cytometry, immunofluorescence and immunoprecipitation assays. The co-culture model of T cells and tumor cells was used to detect the activity of cytotoxic T lymphocytes. Colony formation, EdU labelling and apoptosis assays were used to detect changes in cellular proliferation and apoptosis. In vivo, anti-tumor activity of licochalcone A was assessed in a xenograft model of HCT116 cells. RESULTS: In the present study, we found that licochalcone A suppressed the expression of programmed cell death ligand-1 (PD-L1), which plays a key role in regulating the immune response. In addition, licochalcone A inhibited the expressions of p65 and Ras. Immunoprecipitation experiment showed that licochalcone A suppressed the expression of PD-L1 by blocking the interaction between p65 and Ras. In the co-culture model of T cells and tumor cells, licochalcone A pretreatment enhanced the activity of cytotoxic T lymphocytes and restored the ability to kill tumor cells. In addition, we showed that licochalcone A inhibited cell proliferation and promoted cell apoptosis by targeting PD-L1. In vivo xenograft assay confirmed that licochalcone A inhibited the growth of tumor xenografts. CONCLUSION: In general, these results reveal the previously unknown properties of licochalcone A and provide new insights into the anticancer mechanism of this compound.

Dihydrosanguinarine suppresses pancreatic cancer cells via regulation of mut-p53/WT-p53 and the Ras/Raf/Mek/Erk pathway.

BACKGROUND: There have been some reports implicating the pharmacologic action of Dihydrosanguinarine (DHSA), but little research including the effects of it on cancer cells. PANC-1 cells have mutations in K-Ras and TP53, which respectively express mutant K-Ras and p53 protein, and the mutations in Ras/p53 have been believed with closely relationship to the occurrence of various tumors. PURPOSE: To reveal the inhibition of Dihydrosanguinarine on pancreatic cancer cells (PANC-1 and SW1990) proliferation by inducing G0/G1 and G2/M phase arrest via the downregulation of mut-p53 protein, inducing apoptosis and inhibiting invasiveness through the Ras/Mek/Erk signaling pathway. METHODS: Human pancreatic cancer cell lines were cultured with cisplatin and DHSA. Then, cell proliferation, the cell cycle and apoptosis were measured by CCK-8 and flow cytometry. The migratory and invasive abilities of pancreatic cancer cells were evaluated by transwell assay. The expression levels of mRNA and protein were measured by RT-PCR and western blotting. RESULTS: The results showed that DHSA treatment inhibited cell proliferation, migration and invasion in a time- and dose-dependent manner and led to induction of cell cycle arrest and apoptosis. G0/G1 and G2/M phase arrest inhibited the viability of PANC-1 cells by downregulating the expression of mut-p53 protein. Decreased levels of C-Raf and Erk phosphorylation in DHSA-treated PANC-1 and SW1990 cells were observed in a time- and dose-dependent manner. However, the total expression of p53 and Ras proteins had a different change in PANC-1 and SW1990 cells. CONCLUSIONS: Our findings offer the novel perspective that DHSA inhibits pancreatic cancer cells through a bidirectional regulation between mut-p53/-Ras and WT-p53/-Ras to restore the dynamic balance by Ras and p53 proteins.

Mutations of RAS/RAF Proto-oncogenes Impair Survival After Cytoreductive Surgery and HIPEC for Peritoneal Metastasis of Colorectal Origin.

BACKGROUND: Adequate selection of patients with peritoneal metastasis (PM) for cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) remains critical for successful long-term outcomes. Factors reflecting tumor biology are currently poorly represented in the selection process. The prognostic relevance of RAS/RAF mutations in patients with PM remains unclear. METHODS: Survival data of patients with colorectal PM operated in 6 European tertiary centers were retrospectively collected and predictive factors for survival identified by Cox regression analyses. A simple point-based risk score was developed to allow patient selection and outcome prediction. RESULTS: Data of 524 patients with a median age of 59 years and a median peritoneal cancer index of 7 (interquartile range: 3-12) were collected. A complete resection was possible in 505 patients; overall morbidity and 90-day mortality were 50.9% and 2.1%, respectively. PCI [hazard ratio (HR): 1.08], N1 stage (HR: 2.15), N2 stage (HR: 2.57), G3 stage (HR: 1.80) as well as KRAS (HR: 1.46) and BRAF (HR: 3.97) mutations were found to significantly impair survival after CRS/HIPEC on multivariate analyses. Mutations of RAS/RAF impaired survival independently of targeted treatment against EGFR. Consequently, a simple point-based risk score termed BIOSCOPE (BIOlogical Score of COlorectal PEritoneal metastasis) based on PCI, N-, G-, and RAS/RAF status was developed, which showed good discrimination [development area under the curve (AUC) = 0.72, validation AUC = 0.70], calibration (P = 0.401) and allowed categorization of patients into 4 groups with strongly divergent survival outcomes. CONCLUSION: RAS/RAF mutations impair survival after CRS/HIPEC. The novel BIOSCOPE score reflects tumor biology, adequately stratifies long-term outcomes, and improves patient assessment and selection.

Electroacupuncture serum inhibits TNF­α­mediated chondrocyte inflammation via the Ras­Raf­MEK1/2­ERK1/2 signaling pathway.

The Ras­Raf­mitogen­activated protein kinase kinase (MEK)1/2­extracellular signal­regulated kinase (ERK)1/2 signaling pathway contributes to the release of chondral matrix­degrading enzymes and accelerates the degradation of articular cartilage. Electroacupuncture (EA) treatment has been widely used for the treatment of osteoarthritis (OA); however, the mechanism underlying the effects of EA on OA remains unclear. Therefore, the present study evaluated the anti­inflammatory effects and potential underlying mechanisms of EA serum (EAS) on tumor necrosis factor (TNF)­α­mediated chondrocyte inflammation. A total of 30 Sprague Dawley rats were randomly divided into three groups: The blank group; experimental group I, which received 15 min of EA treatment; and experimental group II, which received 30 min of EA treatment. Subsequently, serum samples were obtained. Chondrocytes were isolated from the knee cartilage of Sprague Dawley rats, and were identified using collagen type II immunohistochemistry. TNF­α­treated chondrocytes were used as a cell model, and subsequently the cells were treated with EAS from each group for various durations. The results demonstrated that EAS treatment significantly promoted the viability and inhibited the apoptosis of TNF­α­treated chondrocytes. In addition, interleukin (IL)­1ß concentration was significantly increased in the model group compared with in the control group, whereas EAS significantly reduced IL­1ß concentration in TNF­α­treated chondrocytes. Furthermore, the protein expression levels of Ras, Raf and MEK1/2 were reduced in the EAS groups compared with in the model group. EAS also significantly inhibited the phosphorylation of ERK1/2, and the expression of downstream regulators matrix metalloproteinase (MMP)­3 and MMP­13. In conclusion, these results indicated that EAS may inhibit TNF­α­mediated chondrocyte inflammation via the Ras­Raf­MEK1/2­ERK1/2 signaling pathway in vitro, thus suggesting that EAS may be considered a potential therapeutic strategy for the treatment of OA.

Griffipavixanthone, a dimeric xanthone extracted from edible plants, inhibits tumor metastasis and proliferation via downregulation of the RAF pathway in esophageal cancer.

Metastasis causes a large number of deaths among esophageal cancer patients. The activation of RAF family proteins elevates tumor metastasis and proliferation. In screen targeting the RAF protein, we identified that Griffipavixanthone (GPX), a dimeric xanthone isolated from Garcinia esculenta, is a B-RAF and C-RAF inhibitor against esophageal cancer cells. Using wound healing, transwell migration and matrigel invasion assays, we confirmed that GPX significantly inhibited cell migration and invasion. Furthermore, exposure to GPX rendered cell proliferation and induced G2/M cell cycle arrest. Our mechanistic study showed that GPX suppressed cancer metastasis and proliferation through downregulation of RAF-MEK-ERK cascades proteins as well as RAF mRNA levels. In a pulmonary metastasis model, the intraperitoneal injection of GPX significantly suppressed esophageal tumor metastasis and ERK protein level in vivo. In conclusion, our present study suggested that GPX could inhibit tumor migration, invasion and proliferation in vitro and in vivo, which indicated the potential of GPX for preventing and treating esophageal cancer.

Effect of Chaihushugan San on expression of the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in the hippocampi of perimenopausal rats induced by immobilization stress.

OBJECTIVE: We wished to study the impact of Chaihushugan San (CSS) on the behavior of perimenopausal rats with liver-Qi stagnation (LQS) and to investigate the effect of CSS on signal transduction of the Raf/mitogen-activated protein kinase (MEK)/ extracellular signal-regulated kinase (ERK) cascade in the hippocampi of rats induced by immobilization. METHODS: Twenty 52-week-old female rats were divided into two groups by the random number table method: model control group (MCG) and CSS group (CSSG), with 10 rats in each group. Ten-week-old female rats were used as the normal control group (NCG). CSS effects were assessed using rats exposed to immobilization stress by measuring body weight and sucrose consumption, serum hormone levels, and observing performance in the open field test (OFT). Molecular mechanisms were examined by measuring the effect of CSS on expression of Raf1, MEK1/2 and ERK1/2 mRNA in hippocampi using quantitative real-time polymerase chain reaction and by measuring levels of these proteins and related phospho-proteins using Western blotting. RESULTS: Perimenopausal rats with LQS had decreased locomotor activity; reduced sucrose consumption; and increased serum levels of corticotropin releasing hormone (CRH) and corticosterone (CORT). Activation of hippocampal Raf/MEK/ERK cascade was suppressed significantly in the MCG, and activation was increased after 21 days of CSS treatment. CONCLUSION: CSS has significant effects upon relief of the symptoms of LQS in immobilization-induced rats. The mechanism underlying this action might (at least in part) be mediated by reversal of disruption of the Raf/MEK/ERK pathway.

Astragaloside IV Attenuates Glutamate-Induced Neurotoxicity in PC12 Cells through Raf-MEK-ERK Pathway.

Astragaloside IV (AGS-IV) is a main active ingredient of Astragalus membranaceus Bunge, a medicinal herb prescribed as an immunostimulant, hepatoprotective, antiperspirant, a diuretic or a tonic as documented in Chinese Materia Medica. In the present study, we employed a high-throughput comparative proteomic approach based on 2D-nano-LC-MS/MS to investigate the possible mechanism of action involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. Differential proteins were identified, among which 13 proteins survived the stringent filter criteria and were further included for functional discussion. Two proteins (vimentin and Gap43) were randomly selected, and their expression levels were further confirmed by western blots analysis. The results matched well with those of proteomics. Furthermore, network analysis of protein-protein interactions (PPI) and pathways enrichment with AGS-IV associated proteins were carried out to illustrate its underlying molecular mechanism. Proteins associated with signal transduction, immune system, signaling molecules and interaction, and energy metabolism play important roles in neuroprotective effect of AGS-IV and Raf-MEK-ERK pathway was involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. This study demonstrates that comparative proteomics based on shotgun approach is a valuable tool for molecular mechanism studies, since it allows the simultaneously evaluate the global proteins alterations.

[In vitro studies of Raf-CREB, Akt-CREB, and CaMK II -CREB signal transduction pathway regulated by ginsenosides Rb1, Rg1 and Re].

OBJECTIVE: Effects of ginsenoside Rb1, Rg1 and Re on neurotrophic factor signal transduction pathway using liposome-mediated transfection of eukaryotic cells approach. METHOD: The injury model was established by treating SH-SY5Y cells with 0.6 mmol x L(-1) of corticosterone (CORT) by 24 h. SH-SY5Y cell were pretreated with CORT for 30 min followed by co-treated with 120,60 and 20 micromol x L(-1) of Rb1, 120, 80 and 40 micromol x L(-1) of Rg1 and 120, 80 and 40 micromol x L(-1) of Re for 24 h. Cells viability was determined by Cell Counting Kit (CCK) assay. CREB expressing Luciferase reporter gene was constructed and transfected with plasmid containing hRaf, hcAMP, hAkt, hCaMK gene into human embryonic kidney (HEK293) cells using liposornal transfection reagent lipofection 2000. The expression of CREB before and after it addion of Rb1, Rg1 and Re was examined by Luc assay system and Western blotting. RESULT: Compared with normal control group, CORT significantly decreased the viability of SH-SY5Y cells to 67.21% (P < 0.01). CCK results show that Rb1 (60 micromol x L(-1)), Rg1 (80 micromol x L(-1)) and Re (80 micromol x L(-1)) on SH-SY5Y cells have significant protective effect (P < 0.01). Lucassay and Western blotting results show that the gene and protein levels of CREB increased significantly through the pathway of Raf and Akt with Rb1 and Rg1 (P < 0.01), Re can increase significantly the gene and protein levels of CREB through the pathway of Raf and CaMK II. CONCLUSION: Rb1, Rg1 and Re protects SH-SY5Y cells from CORT-induced damage and the neuroprotective mechanism may be associated with the Raf-CREB, Akt-CREB and CaMK II -CREB pathways.

VEGFR, RET, and RAF/MEK/ERK pathway take part in the inhibition of osteosarcoma MG63 cells with sorafenib treatment.

Osteosarcoma (OS) is the leading primary malignant bone tumor in children and young adults. It is response for a high mortality rate. Nowadays, few researches have been performed on sorafenib against OS and no tools are available to guide the use of sorafenib in the OS treatment. In this study, we aim to investigate the effect of sorafenib on OS cell MG63 and figure the potential effective molecular pathway of its function. In the present study, we performed assays of cell proliferation, RT-PCR, and western blot to investigate the effect of sorafenib on OS MG63 cells and to elucidate the molecular actions of sorafenib against RTKs VEGFR2 and RET, as well as MEK/ERK signaling pathway. The present study confirmed that sorafenib could inhibit the proliferation of OS MG63 cells and caused a series of biomolecule effects, including the change of VEGFR2 and ERK gene expression, and the phosphorylation alteration of VEGFR2, RET, and MEK1 proteins. VEGFR2, RET, and MEK/ERK signaling pathway are involved in the pharmacological mechanism of sorafenib. They are potential candidate targets for OS treatment.

Inhibition of Raf-MEK-ERK and hypoxia pathways by Phyllanthus prevents metastasis in human lung (A549) cancer cell line.

BACKGROUND: Lung cancer constitutes one of the malignancies with the greatest incidence and mortality rates with 1.6 million new cases and 1.4 million deaths each year. Prognosis remains poor due to deleterious development of multidrug resistance resulting in less than 15% lung cancer patients reaching five years survival. We have previously shown that Phyllanthus induced apoptosis in conjunction with its antimetastastic action. In the current study, we aimed to determine the signaling pathways utilized by Phyllanthus to exert its antimetastatic activities. METHODS: Cancer 10-pathway reporter array was performed to screen the pathways affected by Phyllanthus in lung carcinoma cell line (A549) to exert its antimetastatic effects. Results from this array were then confirmed with western blotting, cell cycle analysis, zymography technique, and cell based ELISA assay for human total iNOS. Two-dimensional gel electrophoresis was subsequently carried out to study the differential protein expressions in A549 after treatment with Phyllanthus. RESULTS: Phyllanthus was observed to cause antimetastatic activities by inhibiting ERK1/2 pathway via suppression of Raf protein. Inhibition of this pathway resulted in the suppression of MMP2, MMP7, and MMP9 expression to stop A549 metastasis. Phyllanthus also inhibits hypoxia pathway via inhibition of HIF-1α that led to reduced VEGF and iNOS expressions. Proteomic analysis revealed a number of proteins downregulated by Phyllanthus that were involved in metastatic processes, including invasion and mobility proteins (cytoskeletal proteins), transcriptional proteins (proliferating cell nuclear antigen; zinc finger protein), antiapoptotic protein (Bcl2) and various glycolytic enzymes. Among the four Phyllanthus species tested, P. urinaria showed the greatest antimetastatic activity. CONCLUSIONS: Phyllanthus inhibits A549 metastasis by suppressing ERK1/2 and hypoxia pathways that led to suppression of various critical proteins for A549 invasion and migration.

Wogonin induces cross-regulation between autophagy and apoptosis via a variety of Akt pathway in human nasopharyngeal carcinoma cells.

Autophagy as well as apoptosis is an emerging target for cancer therapy. Wogonin, a flavonoid compound derived from the traditional Chinese medicine of Huang-Qin, has anticancer activity in many cancer cells including human nasopharyngeal carcinoma (NPC). However, the involvement of autophagy in the wogonin-induced apoptosis of NPC cells was still uninvestigated. In this study, we found wogonin-induced autophagy had interference on the process of apoptosis. Wogonin-induced autophagy formation evidenced by LC3 I/II cleavage, acridine orange (AO)-stained vacuoles and the autophagosome/autolysosome images of TEM analysis. Activation of autophagy with rapamycin resulted in increased wogonin-mediated autophagy via inhibition of mTOR/P70S6K pathway. The functional relevance of autophagy in the antitumor activity was investigated by annexin V-positive stained cells and PARP cleavage. Induction of autophagy by rapamycin ameliorated the wogonin-mediated apoptosis, whereas inhibition of autophagy by 3-methyladenine (3-MA) or bafilomycin A1 increased the apoptotic effect. Interestingly, this study also found, in addition the mTOR/P70S6K pathway, wogonin also inhibited Raf/ERK pathway, a variety of Akt pathways. Inactivation of PI(3) K/Akt by their inhibitors significantly induced apoptosis and markedly sensitized the NPC cells to wogonin-induced apoptosis. This anticancer effect of Akt was further confirmed by SH6, a specific inhibitor of Akt. Importantly, inactivation of its downstream molecule ERK by PD98059, a MEK inhibitor, also induced apoptosis. This study indicated wogonin-induced both autophagy and apoptosis through a variety of Akt pathways and suggested modulation of autophagy might provide profoundly the potential therapeutic effect.

Raf: a strategic target for therapeutic development against cancer.

The mitogen-activated protein kinase (MAPK) signaling pathway plays a critical role in transmitting proliferative signals generated by cell surface receptors and cytoplasmic signaling elements to the nucleus. Several important signaling elements of the MAPK pathway, particularly Ras and Raf, are encoded by oncogenes, and as such, their structures and functions can be modified, rendering them constitutively active. Because the MAPK pathway is dysregulated in a notable proportion of human malignancies, many of its aberrant and critical components represent strategic targets for therapeutic development against cancer. Raf, which is an essential serine/threonine kinase constituent of the MAPK pathway and a downstream effector of the central signal transduction mediator Ras, is activated in a wide range of human malignancies by aberrant signaling upstream of the protein (eg, growth factor receptors and mutant Ras) and activating mutations of the protein itself, both of which confer a proliferative advantage. Three isoforms of Raf have been identified, and therapeutics targeting Raf, including small-molecule inhibitors and antisense oligodeoxyribonucleotides (ASON), are undergoing clinical evaluation. The outcomes of these investigations may have far-reaching implications in the management of many types of human cancer. This review outlines the structure and diverse functions of Raf, the rationale for targeting Raf as a therapeutic strategy against cancer, and the present status of various therapeutic approaches including ASONs and small molecules, particularly sorafenib (BAY 43-9006).