Polycyclic aromatic hydrocarbons in bone homeostasis.

Prolonged exposure to polycyclic aromatic hydrocarbons (PAHs) may result in autoimmune diseases, such as rheumatoid arthritis (RA) and osteoporosis (OP), which are based on an imbalance in bone homeostasis. These diseases are characterized by bone erosion and even a disruption in homeostasis, including in osteoblasts and osteoclasts. Current evidence indicates that multiple factors affect the progression of bone homeostasis, such as genetic susceptibility and epigenetic modifications. However, environmental factors, especially PAHs from various sources, have been shown to play an increasingly prominent role in the progression of bone homeostasis. Hence, it is essential to investigate the effects and pathogenesis of PAHs in bone homeostasis. In this review, recent progress is summarized concerning the effects and mechanisms of PAHs and their ligands and receptors in bone homeostasis. Moreover, strategies based on the effects and mechanisms of PAHs in the regulation of the bone balance and alleviation of bone destruction are also reviewed. We further discuss the future challenges and perspectives regarding the roles of PAHs in autoimmune diseases based on bone homeostasis.

Wild-Type KRAS Allele Effects on Druggable Targets in KRAS Mutant Lung Adenocarcinomas.

KRAS mutations are one of the most common oncogenic drivers in non-small cell lung cancer (NSCLC) and in lung adenocarcinomas in particular. Development of therapeutics targeting KRAS has been incredibly challenging, prompting indirect inhibition of downstream targets such as MEK and ERK. Such inhibitors, unfortunately, come with limited clinical efficacy, and therefore the demand for developing novel therapeutic strategies remains an urgent need for these patients. Exploring the influence of wild-type (WT) KRAS on druggable targets can uncover new vulnerabilities for the treatment of KRAS mutant lung adenocarcinomas. Using commercially available KRAS mutant lung adenocarcinoma cell lines, we explored the influence of WT KRAS on signaling networks and druggable targets. Expression and/or activation of 183 signaling proteins, most of which are targets of FDA-approved drugs, were captured by reverse-phase protein microarray (RPPA). Selected findings were validated on a cohort of 23 surgical biospecimens using the RPPA. Kinase-driven signatures associated with the presence of the KRAS WT allele were detected along the MAPK and AKT/mTOR signaling pathway and alterations of cell cycle regulators. FoxM1 emerged as a potential vulnerability of tumors retaining the KRAS WT allele both in cell lines and in the clinical samples. Our findings suggest that loss of WT KRAS impacts on signaling events and druggable targets in KRAS mutant lung adenocarcinomas.

The impact of the phytotherapeutic agent quercetin on expression of genes and activity of signaling pathways.

Quercetin is a flavonoid existing in different herbs, fruits, seeds, nuts and tea. It has beneficial effects on human health through mediating antioxidant activities, immune-modulatory impacts and regulating metabolic pathways. These effects are most probably induced through modulation of activity of signaling pathways and expression of genes. Several in vitro studies have verified anti-proliferative effects of quercetin and its effect on expression of apoptotic genes and cell cycle-related genes. Moreover, through modulation of a number of proteins such as NF-kB, PARP, STAT3, Bax, Bcl-2, COX2, and cytokines, quercetin has beneficial effects in neurodegenerative disorders, liver diseases and diabetes. PI3K/AKT is the mostly linked pathway with beneficial effects of quercetin. In the current manuscript, we explain the impact of quercetin on expression of genes and function of cellular signaling cascades in different contexts.

Dimethyl fumarate abridged tauo-/amyloidopathy in a D-Galactose/ovariectomy-induced Alzheimer's-like disease: Modulation of AMPK/SIRT-1, AKT/CREB/BDNF, AKT/GSK-3ß, adiponectin/Adipo1R, and NF-κB/IL-1ß/ROS trajectories.

Since the role of estrogen in postmenauposal-associated dementia is still debatable, this issue urges the search for other medications. Dimethyl fumarate (DMF) is a drug used for the treatment of multiple sclerosis and has shown a neuroprotective effect against other neurodegenerative diseases. Accordingly, the present study aimed to evaluate the effect of DMF on an experimental model of Alzheimer disease (AD) using D-galactose (D-Gal) administered to ovariectomized (OVX) rats, resembling a postmenopausal dementia paradigm. Adult 18-month old female Wistar rats were allocated into sham-operated and OVX/D-Gal groups that were either left untreated or treated with DMF for 56 days starting three weeks after sham-operation or ovariectomy. DMF succeeded to ameliorate cognitive (learning/short- and long-term memory) deficits and to enhance the dampened overall activity (NOR, Barnes-/Y-maze tests). These behavioral upturns were associated with increased intact neurons (Nissl stain) and a reduction in OVX/D-Gal-mediated hippocampal CA1 neurodegeneration and astrocyte activation assessed as GFAP immunoreactivity. Mechanistically, DMF suppressed the hippocampal contents of AD-surrogate markers; viz., apolipoprotein (APO)-E1, BACE1, Aß42, and hyperphosphorylated Tau. Additionally, DMF has augmented the neuroprotective parameters p-AKT, its downstream target CREB and BDNF. Besides, it activated AMPK, and enhanced SIRT-1, as well as antioxidant defenses (SOD, GSH). On the other hand, DMF inhibited the transcription factor NF-κB, IL-1ß, adiponectin/adiponectin receptor type (AdipoR)1, GSK-3ß, and MDA. Accordingly, in this postmenopausal AD model, DMF treatment by pursuing the adiponectin/AdipoR1, AMPK/SIRT-1, AKT/CREB/BDNF, AKT/GSK-3ß, and APO-E1 quartet hampered the associated tauo-/amyloidopathy and NF-κB-mediated oxidative/inflammatory responses to advance insights into its anti-amnesic effect.

Shenmai injection improves doxorubicin cardiotoxicity via miR-30a/Beclin 1.

BACKGROUND: Shenmai Injection (SMI) has been widely used in the treatment of cardiovascular diseases and can reduce side effects when combined with chemotherapy drugs. However, the potential protective mechanism of SMI on the cardiotoxicity caused by anthracyclines has not been clear. METHODS: We used network pharmacology methods to collect the compound components in SMI and myocardial injury targets, constructed a 'drug-disease' target interaction network relationship diagram, and screened the core targets to predict the potential mechanism of SMI in treating cardiotoxicity of anthracyclines. In addition, the rat model of doxorubicin cardiotoxicity was induced by injecting doxorubicin through the tail vein. The rats were randomized in the model group, miR-30a agomir group, SMI low-dose group, SMI high-dose group，and the control group. The cardiac ultrasound was used to evaluate the structure and function of the rat heart. HE staining was used to observe the pathological changes of the rat myocardium. Transmission electron microscopy was used to observe myocardial autophagosomes. The expression of miR-30a and Beclin 1 mRNA in the rat myocardium was detected by RT-qPCR. Western Blot detected the expression of LC3-II/LC3-I and p62 protein. RESULTS: The network pharmacological analysis found that SMI could act synergistically through multiple targets and multiple pathways, which might exert a myocardial protective effect through PI3K-Akt signaling pathways and cancer microRNAs. In vivo, compared with the control group, the treatment group could improve the cardiac structure and function, and reduce myocardial pathological damage and the number of autophagosomes. The expression of miR-30a in the myocardium of rats in miR-30a agomir group and SMI group increased (P < 0.01)，Beclin 1 mRNA was decreased (P < 0.01)，LC3-Ⅱ/LC3-I protein was decreased (P < 0.01 or P < 0.05)，and p62 protein was increased (P < 0.01 or P < 0.05). CONCLUSIONS: SMI has the characteristics of multi-component, multi-target, and multi-pathway. It can inhibit myocardial excessive autophagy by regulating the expression of miR-30a/Beclin 1 and alleviate the myocardial injury induced by doxorubicin.

Up-regulation of nPKC contributes to proliferation of mice pulmonary artery smooth muscle cells in hypoxia-induced pulmonary hypertension.

This study is designed to investigate the role of novel protein kinases C (nPKC) in mediating pulmonary artery smooth muscle cells (PASMCs) proliferation in pulmonary hypertension (PH) and the underlying mechanisms. Mouse PASMCs was isolated using magnetic separation technology. The PASMCs were divided into 24 h group, 48 h group and 72 h group according to different hypoxia treatment time, then detected cell proliferation rate and nPKC expression level in each group. We treated PASMCs with agonists or inhibitors of PKCdelta (PKCδ) and PKCepsilon (PKCε) and exposed them to hypoxia or normoxia for 72 h, then measured the proliferation of PASMCs. We also constructed a lentiviral vector containing siRNA fragments for inhibiting PKCδ and PKCε to transfected PASMCs, then examined their proliferation. PASMCs isolated successfully by magnetic separation method and were in good condition. Hypoxia promoted the proliferation of PASMCs, and the treatment for 72 h had the most significant effect. Hypoxia upregulated the expression of PKCδ and PKCε in mouse PASMCs, leading to PASMCs proliferation. Moreover, Our study demonstrated that hypoxia induced upregulation of PKCδ and PKCε expression resulting to the proliferation of PASMCs via up-regulating the phosphorylation of AKT and ERK. Our study provides clear evidence that increased nPKC expression contributes to PASMCs proliferation and uncovers the correlation between AKT and ERK pathways and nPKC-mediated proliferation of PASMCs. These findings may provide novel targets for molecular therapy of pulmonary hypertension.

Puerarin suppresses the hepatic gluconeogenesis via activation of PI3K/Akt signaling pathway in diabetic rats and HepG2 cells.

Pueraria, a Chinese herbal medicine, plays an important role in many classic prescriptions for the treatment of diabetes. Puerarin is the main component of pueraria. The current in vivo and in vitro research mainly focus on exploring the potential mechanism of puerarin in inhibiting hepatic gluconeogenesis. The type 2 diabetic rats were established by a combination of small dosage of streptozotocin (STZ) injection with high-fat diet. After the administration of puerarin 4 weeks, the parameters of the glucose and lipid metabolism were determined. HepG2 cells were treated by palmitic acid (PA) to induce the insulin resistance in vitro model. After the treatment of puerarin, the glucose consumption and cell viability were examined. Then, the protein expression of PI3K, Akt, pAkt, pFOXO1, FOXO1, PEPCK and G6pase in liver tissue and HepG2 cells were evaluated by western blot. RT-PCR was used to measure the content of PEPCK, G6pase mRNA in liver tissue. The results showed that puerarin administration significantly decrease the level of FBG, HbA1C and triglycerides in diabetic rats. Mechanistic research showed that puerarin activating PI3K/Akt is puerarin-mediated beneficial effects and can be reversed by inhibitor of PI3K or Akt. In conclusion, puerarin inhibits hepatic gluconeogenesis by activating PI3K/Akt signaling pathway.

Oxyberberine, an absorbed metabolite of berberine, possess superior hypoglycemic effect via regulating the PI3K/Akt and Nrf2 signaling pathways.

Berberine (BBR) is a promising anti-diabetic isoquinoline alkaloid from Rhizoma coptidis, while its bioavailability was extremely low. Here, the existing form and pharmacokinetics of BBR were comparatively characterized in conventional and antibiotic-induced pseudo germ-free (PGF) rats. Furthermore, we comparatively investigated the antidiabetic effect and potential mechanism of BBR and its intestinal oxidative metabolite oxyberberine (OBB) in STZ-induced diabetic rats. Results showed that BBR and OBB existed mainly as protein-bound form in blood, while protein-bound OBB was significantly depleted in PGF rats. Treatment with OBB and BBR effectively decreased clinical symptoms of diabetic rats, reduced blood glucose level, ameliorated the pancreatic damage, and mitigated oxidative stress and inflammatory markers. However, the anti-diabetes effect of BBR was obviously compromised by antibiotics. In addition, OBB exerted superior anti-diabetes effect to BBR of the same dose, significantly up-regulated the mRNA expression of Nrf2 signaling pathway and substantially promoted the pancreatic levels of PI3K/Akt signaling pathway. In conclusion, BBR and its absorbed oxidative metabolite OBB were mainly presented and transported in the protein-bound form in vivo. The gut microbiota may play an important role in the anti-diabetes effect of BBR through transforming itself into the superior hypoglycemic metabolite OBB. OBB possessed favorable hypoglycemic and pancreatic ß-cells protective effects, which may stand a huge potential to be further developed into a promising anti-diabetes candidate.

Qingjie Fuzheng Granule suppresses lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway.

SCOPE: To investigate the effect of Qingjie Fuzheng Granule (QFG) on lymphangiogenesis and lymphatic metastasis in colorectal cancer. METHODS: The effects of QFG on the expression and secretion of vascular endothelial growth factor-C (VEGF-C) in HCT-116 cells were investigated both in vitro and in vivo. HCT-116 cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. The VEGF-C expression level was determined using RT-qPCR and western blotting, and the VEGF-C concentration in supernatant was measured by ELISA. Tumor xenograft models of HCT-116 cells were generated using BALB/c nude mice, and the mice were randomly divided into a control group (gavaged with normal saline) and QFG group (gavaged with 2 g/kg QFG). The effect of QFG on tumor growth was evaluated by comparing the volume and weight of tumors between two groups. Immunohistochemistry (IHC) and RT-qPCR were performed to detect the expression levels of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR-3), and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1). ELISA was performed to measure the concentration of serum VEGF-C. TMT proteomics technology and Reactome pathway analysis were used to explore the mechanism of QFG inhibiting lymphangiogenesis in tumor. The VEGF-C (5 ng/mL)-stimulated human lymphatic endothelial cell (HLEC) model was conducted to evaluate the effect of QFG on lymphangiogenesis in vitro. The model cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. Cell viability was then determined using an MTT assay. The cell migration, invasion, and tube-formation ability were analyzed using transwell migration, matrigel invasion and tube formation assays, respectively. The underlying mechanism was uncovered, the levels of VEGFR-3, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), p-PI3K/PI3K, p-AKT/AKT and p-mTOR/ mTOR were detected using western blotting. RESULTS: QFG significantly reduced VEGF-C expression and secretion in HCT-116 cells. QFG evidently suppressed in vivo tumor growth and the expression of VEGF-C, VEGFR-3, and LYVE-1. The serum VEGF-C level was also reduced by QFG. Moreover, TMT proteomics technology and Reactome pathway analysis identified 95 differentially expressed protein and multiple enriched pathway about matrix metalloproteinase and extracellular matrix, which is direct associate with lymphangiogenesis. In vitro experiment, QFG inhibited the viability, migration, invasion and tube formation of HLECs. Additionally, QFG reduced the VEGFR-3, MMP-2, MMP-9 expression levels, and the p-PI3K/PI3K, p-AKT/AKT, p-mTOR/ mTOR ratios. CONCLUSION: QFG can exert its effect on both tumor cells and HLECs, exhibiting ani- lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway pathway.

Effect of casein kinase 1α inhibition on autophagy flux and the AKT/phospho-ß-catenin (S552) axis in HCT116, a RAS-mutated colorectal cancer cell line.

The Wnt/ß-catenin pathway, which interferes with cell proliferation, differentiation, and autophagy, is commonly dysregulated in colorectal cancer (CRC). Mutation of the RAS oncogene is the most prevalent genetic alteration in CRC and has been linked to activation of protein kinase B (AKT) signaling. Phosphorylation of ß-catenin at Ser 552 by AKT contributes to ß-catenin stability, transcriptional activity, and increase of cell proliferation. Casein kinase 1 alpha (CK1α) is an enzyme that simultaneously regulates Wnt/ß-catenin and AKT. The link of the AKT and Wnt pathway to autophagy in RAS-mutated CRC cells has not well identified. Therefore, we investigated how pharmacological CK1α inhibition (D4476) is involved in regulation of autophagy, Wnt/ß-catenin, and AKT pathways in RAS-mutated CRC cell lines. qRT-PCR and immunoblotting experiments revealed that phospho-AKT (S473) and phospho-ß-catenin (S552) are constitutively increased in RAS-mutated CRC cell lines, in parallel with augmented CK1α expression. The results also showed that D4476 significantly reduced the AKT/phospho-ß-catenin (S552) axis concomitantly with autophagy flux inhibition in RAS-mutated CRC cells. Furthermore, D4476 significantly induced apoptosis in RAS-mutated CRC cells. In conclusion, our results indicate that CK1α inhibition reduces autophagy flux and promotes apoptosis by interfering with the AKT/phospho-ß-catenin (S552) axis in RAS-mutated CRC cells.

Integrative analysis of epigenomics, transcriptomics, and proteomics to identify key targets and pathways of Weining granule for gastric cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Weining granule (WNG) is a "Qi-Enriching and Kidney-Tonifying, Spleen-Reinforcing and Stasis-Removing" formula for gastric cancer (GC). Past research we noted WNG inhibited cell growth and raised apoptosis in GC. However, the underlying mechanism of WNG for GC have yet to be systematically clarified. AIM OF THE STUDY: We sought to characterize the molecular landscape of GC cells in vitro after WNG treated, to identify the molecular targets and pathways that were associated with WNG for inducing the apoptosis of GC cells, and further to clarify underlying molecular mechanism of WNG for GC. MATERIALS AND METHODS: We performed the techniques of RNA sequencing, tandem mass tags (TMT) based quantitative proteomics, and reduced representation bisulfite sequencing (RRBS) in WNG-treated/or untreated SGC-7901 GC cells to gain a comprehensive molecular portrait of WNG treatment. Then we integrated methylomics, transcriptomics, and proteomics data to carry out the bioinformatics analysis, and constructed the protein-protein interaction (PPI) network to identify molecular targets, and to discover the underlying signaling pathways associated with WNG for GC by network analysis. Besides, we verified the candidate target genes by Kaplan-Meier plotter database. RESULTS: We identified 1249 significant differentially expressed genes (DEGs) from RNA expression datasets, 191 significant differentially abunabundant proteins (DAPs) from proteomics datasets, and 8293 significant differentially methylated regions (DMRs) from DNA methylation datasets. GO and KEGG analysis showed DEGs, DAPs, and DMRs enriched in the cancer-related biological processes of calcium signaling pathway, pathways in cancer, metabolic pathways, MAPK signaling pathway, PI3K-Akt signaling pathway, and transcriptional misregulation in cancer. We integrated three profile datasets and performed network analysis to distinguish the hub genes, and finally the genes of SOD2, HMOX1, MMP1, SRXN1, NOTCH1, MAPK14, TXNIP, VEGFA, POLR2F, and HSPA9 were identified. The Kaplan-Meier plotter confirmed that SOD2, MMP1, SRXN1, NOTCH1, MAPK14, TXNIP, VEGFA, and HSPA9 were significantly correlated with OS in GC patients (P < 0.01), while HMOX1 and POLR2F expression were not significantly relevant to survival of GC patients (P > 0.01). CONCLUSIONS: SOD2, MMP1, SRXN1, NOTCH1, MAPK14, TXNIP, VEGFA, and HSPA9 were the predictive pharmaceutical targets of WNG for GC. The anticancer function of WNG was significantly associated with the pathways of focal adhesion pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, and Wnt signaling pathway.

l-Theanine Prevents Long-Term Affective and Cognitive Side Effects of Adolescent Δ-9-Tetrahydrocannabinol Exposure and Blocks Associated Molecular and Neuronal Abnormalities in the Mesocorticolimbic Circuitry.

Chronic adolescent exposure to Δ-9-tetrahydrocannabinol (THC) is linked to elevated neuropsychiatric risk and induces neuronal, molecular and behavioral abnormalities resembling neuropsychiatric endophenotypes. Previous evidence has revealed that the mesocorticolimbic circuitry, including the prefrontal cortex (PFC) and mesolimbic dopamine (DA) pathway are particularly susceptible to THC-induced pathologic alterations, including dysregulation of DAergic activity states, loss of PFC GABAergic inhibitory control and affective and cognitive abnormalities. There are currently limited pharmacological intervention strategies capable of preventing THC-induced neuropathological adaptations. l-Theanine is an amino acid analog of l-glutamate and l-glutamine derived from various plant sources, including green tea leaves. l-Theanine has previously been shown to modulate levels of GABA, DA, and glutamate in various neural regions and to possess neuroprotective properties. Using a preclinical model of adolescent THC exposure in male rats, we report that l-theanine pretreatment before adolescent THC exposure is capable of preventing long-term, THC-induced dysregulation of both PFC and VTA DAergic activity states, a neuroprotective effect that persists into adulthood. In addition, pretreatment with l-theanine blocked THC-induced downregulation of local GSK-3 (glycogen synthase kinase 3) and Akt signaling pathways directly in the PFC, two biomarkers previously associated with cannabis-related psychiatric risk and subcortical DAergic dysregulation. Finally, l-theanine powerfully blocked the development of both affective and cognitive abnormalities commonly associated with adolescent THC exposure, further demonstrating functional and long-term neuroprotective effects of l-theanine in the mesocorticolimbic system.SIGNIFICANCE STATEMENT With the increasing trend of cannabis legalization and consumption during adolescence, it is essential to expand knowledge on the potential effects of adolescent cannabis exposure on brain development and identify potential pharmacological strategies to minimize Δ-9-tetrahydrocannabinol (THC)-induced neuropathology. Previous evidence demonstrates that adolescent THC exposure induces long-lasting affective and cognitive abnormalities, mesocorticolimbic dysregulation, and schizophrenia-like molecular biomarkers that persist into adulthood. We demonstrate for the first time that l-theanine, an amino acid analog of l-glutamate and l-glutamine, is capable of preventing long-term THC side effects. l-Theanine prevented the development of THC-induced behavioral aberrations, blocked cortical downregulation of local GSK-3 (glycogen synthase kinase 3) and Akt signaling pathways, and normalized dysregulation of both PFC and VTA DAergic activity, demonstrating powerful and functional neuroprotective effects against THC-induced developmental neuropathology.

Pinellia ternata attenuates carotid artery intimal hyperplasia and increases endothelial progenitor cell activity via the PI3K/Akt signalling pathway in wire-injured rats.

CONTEXT: Clinically, Pinellia ternata (Thunb.) Breit. (Araceae) (PT) has been widely used in the treatment of atherosclerosis and hyperlipidaemia, but the underlying mechanisms are still not clearly understood. OBJECTIVE: This research was conducted to confirm the mechanism by which PT affects carotid artery intimal hyperplasia. MATERIALS AND METHODS: An intestinal hyperplasia Sprague-Dawley rat model was established by carotid artery injury. The rats were randomly divided into five groups (n = 8): sham, model, PT (with daily intragastric administration of 10 g/mL/kg PT tubers water extract), PT+LY294002 (with intraperitoneal injection of 50 mg/kg LY294002 + 10 g/mL/kg PT) and endothelial progenitor cells (EPCs) (with injection of 5 × 105/cells), and treated for 4 or 8 weeks. RESULTS: HE staining showed that PT attenuated intimal hyperplasia. RT-PCR, Western blotting and immunohistochemistry showed that PT increased the expression of vascular endothelial growth factor (VEGF) and eNOS in the atherosclerotic carotid artery. PT increased the Dil-acLDL+/FITC-UEA-1+ population (from 0.41 ± 0.085% to 0.60 ± 0.092%) in the blood, decreased TCHO, TG, LDL-C, IL-6 and TNF-α levels, and increased HDL-C and IL-10 levels in the blood. However, these changes were reversed by the PI3K/Akt pathway inhibitor LY294002. DISCUSSION AND CONCLUSIONS: PT can be developed as an atherosclerosis and carotid intimal hyperplasia treatment drug. Therefore, further study will focus on the effects of PT on intimal hyperplasia in wire-injured atherosclerosis patients and explore in depth some other relevant molecular mechanisms.

The Impact of Icariside II on Human Prostate Cancer Cell Proliferation, Mobility, and Autophagy via PI3K-AKT-mTOR Signaling Pathway.

INTRODUCTION: The flavonol glycoside icariside II (ICA II) has been shown to exhibit a range of anti-tumor properties. Herein, we evaluated the impact of ICA II on human prostate cancer cell proliferation, motility, and autophagy, and we further evaluated the molecular mechanisms underlying these effects. METHODS: We treated DU145 human prostate cancer cells with a range of ICA II doses and then assessed their proliferation via CCK-8 assay, while flow cytometry was used to monitor apoptosis and cell cycle progression. We further utilized wound healing and transwell assays to probe the impact of ICA II on migration and invasion, and assessed autophagy via laser confocal fluorescence microscopy. Western blotting was further utilized to measure LC3-II/I, Beclin-1, P70S6K, PI3K, AKT, mTOR, phospho-AKT, phospho-mTOR, and phospho-P70S6K levels, with qRT-PCR being used to evaluate the expression of specific genes at the mRNA level. RESULTS: We found that ICA II was capable of mediating the dose- and time-dependent suppression of DU145 cell proliferation, causing these cells to enter a state of cell cycle arrest and apoptosis. We further determined that ICA II treatment was associated with significant impairment of prostate cancer cell migration and invasion, whereas autophagy was enhanced in treated cells relative to untreated controls. CONCLUSION: Our results indicate that ICA II treatment is capable of suppressing human prostate tumor cell proliferation and migration while enhancing autophagy via modulating the PI3K-AKT-mTOR signaling pathway. As such, ICA II may be an ideal candidate drug for the treatment of prostate cancer.

ISL Induces Apoptosis and Autophagy in Hepatocellular Carcinoma via Downregulation of PI3K/AKT/mTOR Pathway in vivo and in vitro.

AIMS: Isoliquiritigenin (ISL), a flavonoid from Glycyrrhiza glabra, has previously been reported to have anti-tumor effects in vivo and in vitro. However, the mechanisms whereby ISL exerts its anticancer effects remain poorly understood in hepatocellular carcinoma (HCC). PURPOSE: In the present study, we investigated the anticancer efficacy and associated mechanisms of ISL in HCC MHCC97-H and SMMC7721 cells. RESULTS: We found that ISL inhibited cell viability and proliferation and induced apoptosis in a dose- and time-dependent manner in liver cancer lines. Furthermore, ISL could activate autophagy in HCC cells, and the autophagy inhibitor HCQ enhances ISL-induced apoptosis in HCC cells. Additionally, ISL induced apoptosis and autophagy through inhibition of the PI3K/Akt/mTOR pathway. Most importantly, in a xenograft tumor model in nude mice, data showed that the administration of ISL decreased tumor growth and concurrently promoted the expression of LC3-II and cleaved-caspase-3. Interestingly, we found that ISL inhibits mTOR by docking onto the ATP-binding pocket of mTOR (ie, it competes with ATP). We thus suggest that mTOR is a potential target for ISL inhibition of hepatocellular carcinoma development, which could be of interest for future investigations. CONCLUSION: Taken together, the results reveal that ISL effectively inhibited proliferation and induced apoptosis in HCC through autophagy induction in vivo and in vitro, probably via the PI3K/Akt/mTOR pathway. ISL may be a potential therapeutic agent for hepatocellular carcinoma.

Piperlonguminine and Piperine Analogues as TrxR Inhibitors that Promote ROS and Autophagy and Regulate p38 and Akt/mTOR Signaling.

The natural products piperlongumine and piperine have been shown to inhibit cancer cell proliferation through elevation of reactive oxidative species (ROS) and eventually cell death, but only have modest cytotoxic potencies. A series of 14 novel phenylallylidenecyclohexenone analogues based on piperlongumine and piperine therefore were designed and synthesized, and their pharmacological properties were evaluated. Most of the compounds produced antiproliferative activities against five human cancer cells with IC50 values lower than those of piperlongumine and piperine. Among these, compound 9m exerted the most potent antiproliferative activity against drug-resistant Bel-7402/5-FU human liver cancer 5-FU resistant cells (IC50 = 0.8 µM), which was approximately 10-fold lower than piperlongumine (IC50 = 8.4 µM). Further, 9m showed considerably lower cytotoxicity against LO2 human normal liver epithelial cells compared to Bel-7402/5-FU. Mechanistically, compound 9m inhibited thioredoxin reductase (TrxR) activity, increased ROS levels, reduced mitochondrial transmembrane potential (MTP), and induced autophagy in Bel-7402/5-FU cells via regulation of autophagy-related proteins LC3, p62, and beclin-1. Finally, 9m activated significantly the p38 signaling pathways and suppressed the Akt/mTOR signaling pathways. In conclusion, 9m could be a promising candidate for the treatment of drug-resistant cancer cells and, as such, warrants further investigation.

Cedrol, a Sesquiterpene Alcohol, Enhances the Anticancer Efficacy of Temozolomide in Attenuating Drug Resistance via Regulation of the DNA Damage Response and MGMT Expression.

Glioblastoma (GBM) is a common and aggressive brain tumor with a median survival of 12-15 months. Temozolomide (TMZ) is a first-line chemotherapeutic agent used in GBM therapy, but the occurrence of drug resistance limits its antitumor activity. The natural compound cedrol has remarkable antitumor activity and is derived from Cedrus atlantica. In this study, we investigated the combined effect of TMZ and cedrol in GBM cells in vitro and in vivo. The TMZ and cedrol combination treatment resulted in consistently higher suppression of cell proliferation via regulation of the AKT and MAPK signaling pathways in GBM cells. The combination treatment induced cell cycle arrest, cell apoptosis, and DNA damage better than either drug alone. Furthermore, cedrol reduced the expression of proteins associated with drug resistance, including O6-methlyguanine-DNA-methyltransferase (MGMT), multidrug resistance protein 1 (MDR1), and CD133 in TMZ-treated GBM cells. In the animal study, the combination treatment significantly suppressed tumor growth through the induction of cell apoptosis and decreased TMZ drug resistance. Moreover, cedrol-treated mice exhibited no significant differences in body weight and improved TMZ-induced liver damage. These results imply that cedrol may be a potential novel agent for combination treatment with TMZ for GBM therapy that deserves further investigation.

PI3K/AKT/mTOR pathway-related long non-coding RNAs: roles and mechanisms in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide with high prevalence and lethality. The oncogenic phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway is a classic dysregulated pathway involved in the pathogenesis of HCC. However, the underlying mechanism for how PI3K/AKT/mTOR pathway aberrantly activates HCC has not been entirely elucidated. The recognition of the functional roles of long non-coding RNAs (lncRNAs) in PI3K/AKT/mTOR signaling axis sheds light on a new dimension to our understanding of hepatocarcinogenesis. In this review, we comprehensively summarize 67 dysregulated PI3K/AKT/mTOR pathway-related lncRNAs in HCC. Many studies have indicated that the 67 dysregulated lncRNAs show oncogenic or anti-oncogenic effects in HCC by regulation on epigenetic, transcriptional and post-transcriptional levels and they play pivotal roles in the initiation of HCC in diverse biological processes like proliferation, metastasis, drug resistance, radio-resistance, energy metabolism, autophagy and so on. Besides, many of these lncRNAs are associated with clinicopathological features and clinical prognosis in HCC, which may provide a potential future application in the diagnosis and therapy of HCC.

Aloe-emodin induces autophagy and apoptotic cell death in non-small cell lung cancer cells via Akt/mTOR and MAPK signaling.

Lung cancer has a relatively poor prognosis, and the clinical efficacy of targeted drugs remains unsatisfactory. Therefore, the search for safe and efficient novel antitumor drugs has become an urgent problem in the treatment of lung cancer. Aloe-emodin (AE), a medicinal herb, has been demonstrated to exhibit many pharmacological effects on tumor cells, such as lung cancer cells. However, the anticancer properties of AE have not been fully exploited by modern medicine, as their mechanisms of action are not yet known. In this study, the bioassay results demonstrated that AE reduced the viability of the non-small cell lung cancer cell line A549 and NCI-H1299 in a dose- and time-dependent manner. Moreover, AE induced caspase-dependent apoptosis and autophagy. AE induced autophagy through activation of MAPK signaling and inhibition of the Akt/mTOR pathway. We also found that AE-induced autophagy was attenuated by the reactive oxygen species scavenger N-acetylcysteine, indicating that reactive oxygen species played a key role in AE-mediated autophagy in A549 and NCI-H1299 cells. Furthermore, AE induced reactive oxygen species-dependent autophagy in A549 and NCI-H1299 cells, which triggered apoptosis. Additionally, AE showed synergistic cytotoxic effects with the antitumor drug gemcitabine in A549 and NCI-H1299 cells. In brief, these results showed that AE might be useful for developing a therapeutic candidate for lung cancer complications.

Puerarin attenuates the endothelial-mesenchymal transition induced by oxidative stress in human coronary artery endothelial cells through PI3K/AKT pathway.

Endothelial-mesenchymal transition (EndMT) is a process in which endothelial cells lose their specific morphology/markers and undergo a dramatic remodeling of the cytoskeleton. It has been implicated in the progression of cardiovascular diseases such as cardiac fibrosis and cardiac dysfunction. Recent study indicated that puerarin could inhibit EndMT against cardiac fibrosis. However, the precise role of puerarin in EndMT and the underlying molecular mechanisms remain unclear. EndMT was induced by H2O2 (150 µM) in human coronary artery endothelial cells (HCAECs). HCAECs were exposed to H2O2 for six days with or without puerarin pretreated 2 h. The protein changes of EndMT markers (CD31, VE-cadherin, FSP1 and α-SMA) in HCAECs were detected. The levels of phosphoinositide-3-kinase (PI3K) and protein kinase B (AKT) proteins were analyzed by Western Blot. Wound healing and transwell assay were carried out to examine cell chemotaxis. Puerarin mitigated H2O2-induced EndMT as indicated by alleviating the reduced expression of CD31 and VE-cadherin and inhibiting the upregulation of α-SMA and FSP1. Furthermore, the mechanisms study showed that puerarin activated the PI3K/AKT pathway by inhibiting reactive oxygen species and further attenuated EndMT. On the other hand, PI3K inhibitor LY294002 reversed this effect imposed by puerarin. Puerarin alleviated the migration of mesenchymal-like cells through reducing MMPs protein expression. These results implicated that puerarin exhibited cytoprotective effects against H2O2-induced EndMT in HCAECs through alleviating oxidative stress, activating the PI3K/AKT pathway and limiting cell migration.