Natural Product Regulates Autophagy in Cancer.

Anti-cancer effect of natural products has been widely known. As a sort of multi-target anti-cancer agents, natural compound's regulation on autophagy in cancer cells has been studied as a promising research to reveal the mechanism in oncogenesis, as well as a potential short way to anti-cancer drug discovery. In this chapter, we reviewed the cancer-autophagic-related studies on several natural product compounds. It was concluded that natural product compounds directly or indirectly regulated most of the target proteins on the autophagic signal pathways. Considering we have not seen the whole clear atlas of autophagy in oncogenesis yet, it is hard to raise up any conclusion that autophagy is always playing a positive role in oncogenesis and cancer progression.

The novel small molecular BH3 mimetics SM3 and its regulation of cell apoptosis and autophagy.

Bcl-2 family proteins play an important role in regulation of the cell survival and death. The inhibition of the anti-apoptotic proteins of Bcl-2 family leads to the apoptosis of cancer. BH3 mimetics have been developed targeting anti-apoptotic proteins of Bcl-2 family as small molecular drugs. It has been proved that BH3 mimetics has effect on apoptosis and proliferation in leukemia and some of them has been used in phase one or two clinical trials. Besides, with the development of the research on autophagic cell death, the antagonism and the synergism of autophagy and apoptosis is significant in cell death. As a hub of these two pathways of cell death, Bcl-2 protein is a potential target in basic research and clinical applications. In our studies, we found 32 potential BH3 mimetics compounds from 140,000 small molecular compounds via pharmacophore-based virtual screening. Furthermore, we demonstrated SM3, one of the 32 potential BH3 mimetics, induced autophagy and apoptosis simultaneously in dose-time dependence in A549 cell. SM3 induced apoptosis by intrinsic apoptosis pathway and induced autophagy by weakening the interaction between Beclin-1 and Bcl-2 complex. We wish to provide evidences and clues for the structural optimizing and further study of new compounds in the future.

Inhibition of phosphodiesterase 2 reverses gp91phox oxidase-mediated depression- and anxiety-like behavior.

Phosphodiesterase 2 (PDE2) plays an important role in treatment of stress-related depression through regulation of antioxidant defense and neuroprotective mechanisms. However, the causal relationship between PDE2 and the prevalence of depression and anxiety upon exposure to oxidative stress has not been investigated. The present study examined whether the effects of PDE2 inhibition on oxidative stress were directly involved in reduced ROS by regulating NADPH subunits gp91phox oxidase. The results suggested that the PDE2 inhibitor Bay 60-7550 reversed oxidative stress-induced behavioral signature, i.e. depression and anxiety. Pretreatment with the oxidizing agent DTNB completely blocked, while the reducing agent DTT and the NADPH oxidase inhibitor apocynin potentiated the effects of Bay 60-7550 on behavioral abnormalities, demonstrating the relationship between PDE2 and oxidative stress. Consistently, an in vitro test revealed the positive correlation between ROS and PDE2 levels. Moreover, Bay 60-7550 decreased corticosterone-induced gp91phox expression, which is the source of ROS. The subsequent study suggested that Bay 60-7550 induced decrease in ROS and increase in cAMP/cGMP, pVASP, pCREB, and the neurotrophic factor BDNF levels, which were completely blocked by CRISPR/Cas9-mediated gp91phox overexpression and potentiated by gp91phox siRNA-based antioxidant strategies. The in vivo test in stressed mice further suggested that gp91phox overexpression completely blocked the antidepressant- and anxiolytic-like effects of Bay 60-7550, while gp91phox knockdown enhanced such effects. These results provide solid evidence that the antidepressant- and anxiolytic-like effects of Bay 60-7550 against stress are causally related to down-regulation of gp91phox and activation of the cAMP/cGMP-pVASP-CREB-BDNF signaling pathway.

Inhibition of PDE2 reverses beta amyloid induced memory impairment through regulation of PKA/PKG-dependent neuro-inflammatory and apoptotic pathways.

Beta amyloid peptides (Aß) are known risk factors involved in cognitive impairment, neuroinflammatory and apoptotic processes in Alzheimer's disease (AD). Phosphodiesterase 2 (PDE2) inhibitors increase the intracellular cAMP and/or cGMP activities, which may ameliorate cognitive deficits associated with AD. However, it remains unclear whether PDE2 mediated neuroapoptotic and neuroinflammatory events, as well as cognitive performance in AD are related to cAMP/cGMP-dependent pathways. The present study investigated how the selective PDE2 inhibitor BAY60-7550 (BAY) affected Aß-induced learning and memory impairment in two classic rodent models. IL-22 and IL-17, Bax and Bcl-2, PKA/PKG and the brain derived neurotropic factor (BDNF) levels in hippocampus and cortex were detected with immunoblotting assay. The results showed that BAY reversed Aß-induced cognitive impairment as shown in the water maze test and step-down test. Moreover, BAY treatment reversed the Aß-induced changes in IL-22 and IL-17 and the ratio of Bax/Bcl-2. Changes in cAMP/cGMP levels, PKA/PKG and BDNF expression were also prevented by BAY. These effects of BAY on memory performance and related neurochemical changes were partially blocked by the PKG inhibitor KT 5823. These findings indicated that the protective effects of BAY against Aß-induced memory deficits might involve the regulation of neuroinflammation and neuronal apoptotic events.

Direct interaction between caffeic acid phenethyl ester and human neutrophil elastase inhibits the growth and migration of PANC-1 cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant tumors of the digestive system, but the mechanisms of its development and progression are unclear. Inflammation is thought to be fundamental to pancreatic cancer development and caffeic acid phenethyl ester (CAPE) is an active component of honey bee resin or propolis with anti-inflammatory and anticancer activities. We investigated the inhibitory effects of CAPE on cell growth and migration induced by human neutrophil elastase (HNE) and report that HNE induced cancer cell migration at low doses and growth at higher doses. In contrast, lower CAPE doses inhibited migration and higher doses of CAPE inhibited the growth induced by HNE. HNE activity was significantly inhibited by CAPE (7.5-120 µM). Using quantitative real-time PCR and western blotting, we observed that CAPE (18-60 µM) did not affect transcription and translation of α1-antitrypsin (α1-AT), an endogenous HNE inhibitor. However, in an in silico drug target docking model, we found that CAPE directly bound to the binding pocket of HNE (25.66 kcal/mol) according to CDOCKER, and the residue of the catalytic site stabilized the interaction between CAPE and HNE as evidenced by molecular dynamic simulation. Response unit (RU) values of surface plasmon resonance (SPR) significantly increased with incremental CAPE doses (7.5-120 µM), indicating that CAPE could directly bind to HNE in a concentration-dependent manner. Thus, CAPE is an effective inhibitor of HNE via direct interaction whereby it inhibits the migration and growth of PANC-1 cells in a dose-dependent manner.

Nordihydroguaiaretic acid impairs prostate cancer cell migration and tumor metastasis by suppressing neuropilin 1.

Tumor metastasis is a major cause leading to the deaths of cancer patients. Nordihydroguaiaretic acid (NDGA) is a natural product that has been demonstrated to show therapeutic values in multiple diseases. In this study, we report that NDGA can inhibit cell migration and tumor metastasis via a novel mechanism. NDGA suppresses NRP1 function by downregulating its expression, which leads to attenuated cell motility, cell adhesion to ECM and FAK signaling in cancer cells. Moreover, due to its cross-cell type activity on NRP1 suppression, NDGA also impairs angiogenesis function of endothelial cells and fibronectin assembly by fibroblasts, both of which are critical to promote metastasis. Based on these comprehensive effects, NDGA effectively suppresses tumor metastasis in nude mice model. Our findings reveal a novel mechanism underlying the anti-metastasis function of NDGA and indicate the potential value of NDGA in NRP1 targeting therapy for selected subtypes of cancer.

Bisdemethoxycurcumin exerts pro-apoptotic effects in human pancreatic adenocarcinoma cells through mitochondrial dysfunction and a GRP78-dependent pathway.

Pancreatic cancer is a highly aggressive malignancy, which is intrinsically resistant to current chemotherapies. Herein, we investigate whether bisdemethoxycurcumin (BDMC), a derivative of curcumin, potentiates gemcitabine in human pancreatic cancer cells. The result suggests that BDMC sensitizes gemcitabine by inducing mitochondrial dysfunctions and apoptosis in PANC-1 and MiaPaCa-2 pancreatic cancer cells. Utilizing two-dimensional gel electrophoresis and mass spectrometry, we identify 13 essential proteins with significantly altered expressions in response to gemcitabine alone or combined with BDMC. Protein-protein interaction network analysis pinpoints glucose-regulated protein 78 (GRP78) as the key hub activated by BDMC. We then reveal that BDMC upregulates GRP78 and facilitates apoptosis through eIF2α/CHOP pathway. Moreover, DJ-1 and prohibitin, two identified markers of chemoresistance, are increased by gemcitabine in PANC-1 cells. This could be meaningfully reversed by BDMC, suggesting that BDMC partially offsets the chemoresistance induced by gemcitabine. In summary, these findings show that BDMC promotes apoptosis through a GRP78-dependent pathway and mitochondrial dysfunctions, and potentiates the antitumor effect of gemcitabine in human pancreatic cancer cells.

Neuroprotective Effects of a Standardized Flavonoid Extract from Safflower against a Rotenone-Induced Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is a major age-related neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra par compacta (SNpc). Rotenone is a neurotoxin that is routinely used to model PD to aid in understanding the mechanisms of neuronal death. Safflower (Carthamus tinctorius. L.) has long been used to treat cerebrovascular diseases in China. This plant contains flavonoids, which have been reported to be effective in models of neurodegenerative disease. We previously reported that kaempferol derivatives from safflower could bind DJ-1, a protein associated with PD, and that a flavonoid extract from safflower exhibited neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of PD. In this study, a standardized safflower flavonoid extract (SAFE) was isolated from safflower and found to primarily contain flavonoids. The aim of the current study was to confirm the neuroprotective effects of SAFE in rotenone-induced Parkinson rats. The results showed that SAFE treatment increased body weight and improved rearing behavior and grip strength. SAFE (35 or 70 mg/kg/day) treatment reversed the decreased protein expression of tyrosine hydroxylase, dopamine transporter and DJ-1 and increased the levels of dopamine and its metabolite. In contrast, acetylcholine levels were decreased. SAFE treatment also led to partial inhibition of PD-associated changes in extracellular space diffusion parameters. These changes were detected using a magnetic resonance imaging (MRI) tracer-based method, which provides novel information regarding neuronal loss and astrocyte activation. Thus, our results indicate that SAFE represents a potential therapeutic herbal treatment for PD.

EGCG reverses human neutrophil elastase-induced migration in A549 cells by directly binding to HNE and by regulating α1-AT.

Lung carcinogenesis is a complex process that occurs in unregulated inflammatory environment. EGCG has been extensively investigated as a multi-targeting anti-tumor and anti-inflammatory compound. In this study, we demonstrated a novel mechanism by which EGCG reverses the neutrophil elastase-induced migration of A549 cells. We found that neutrophil elastase directly triggered human adenocarcinoma A549 cell migration and that EGCG suppressed the elevation of tumor cell migration induced by neutrophil elastase. We observed that EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity based on the CDOCKER algorithm, MD stimulation by GROMACS, SPR assay and elastase enzymatic activity assay. As the natural inhibitor of neutrophil elastase, α1-antitrypsin is synthesized in tumor cells. We further demonstrated that the expression of α1-antitrypsin was up-regulated after EGCG treatment in neutrophil elastase-treated A549 cells. We preliminarily discovered that the EGCG-mediated induction of α1-antitrypsin expression might be correlated with the regulatory effect of EGCG on the PI3K/Akt pathway. Overall, our results suggest that EGCG ameliorates the neutrophil elastase-induced migration of A549 cells. The mechanism underlying this effect may include two processes: EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity; EGCG enhances the expression of α1-antitrypsin by regulating the PI3K/AKT pathway.

Enoxaparin sensitizes human non-small-cell lung carcinomas to gefitinib by inhibiting DOCK1 expression, vimentin phosphorylation, and Akt activation.

Gefitinib is widely used for the treatment of lung cancer in patients with sensitizing epidermal growth factor receptor mutations, but patients tend to develop resistance after an average of 10 months. Low molecular weight heparins, such as enoxaparin, potently inhibit experimental metastasis. This study aimed to determine the potential of combined enoxaparin and gefitinib (enoxaparin + gefitinib) treatment to inhibit tumor resistance to gefitinib both in vitro and in vivo. A549 and H1975 cell migration was analyzed in wound closure and Transwell assays. Akt and extracellular signal-related kinase 1/2 signaling pathways were identified, and a proteomics analysis was conducted using SDS-PAGE/liquid chromatography-tandem mass spectrometry analysis. Molecular interaction networks were visualized using the Cytoscape bioinformatics platform. Protein expression of dedicator of cytokinesis 1 (DOCK1) and cytoskeleton intermediate filament vimentin were identified using an enzyme-linked immunosorbent assay, Western blot, and small interfering RNA transfection of A549 cells. In xenograft A549-luc-C8 tumors in nude mice, enoxaparin + gefitinib inhibited tumor growth and reduced lung colony formation compared with gefitinib alone. Furthermore, the combination had stronger inhibitory effects on cell migration than either agent used individually. Additional enoxaparin administration resulted in better effective inhibition of Akt activity compared with gefitinib alone. Proteomics and network analysis implicated DOCK1 as the key node molecule. Western blot verified the effective inhibition of the expression of DOCK1 and vimentin phosphorylation by enoxaparin + gefitinib compared with gefitinib alone. DOCK1 knockdown confirmed its role in cell migration, Akt expression, and vimentin phosphorylation. Our data indicate that enoxaparin sensitizes gefitinib antitumor and antimigration activity in lung cancer by suppressing DOCK1 expression, Akt activity, and vimentin phosphorylation.

Hypoxia-inducible factor-1α dependent pathways mediate the renoprotective role of acetazolamide against renal ischemia-reperfusion injury.

BACKGROUND/AIMS: Acute kidney injury (AKI) is a major complication of kidney transplantation, resulting in early graft dysfunction. Since diuretic acetazolamide (AZA) has been shown to improve contrast induced AKI, we hypothesized that AZA also protected against ischemia-reperfusion (I/R) caused AKI. METHODS: An in vivo mouse renal I/R injury model and an in vitro H2O2 stimulated HK-2 cell injury model were utilized to examine the renoprotective effect of AZA. Renal injury and blood flow were measured. Nitric oxide synthase (eNOS)/Nitric oxide (NO), cell apoptosis and hypoxia-inducible factor-1α (HIF-1α) changes were analyzed. RESULTS: AZA reduced kidney injury scores and improved renal function by decreasing serum creatinine and BUN levels after I/R. Impaired renal blood flow was restored by increasing eNOS activities and NO production, as indicated by Laser Doppler imaging. TUNEL staining presented that AZA reduced apoptotic cells due to attenuated caspase activation and increased Bcl-2/Bax ratio. Furthermore, HIF-1α induction by AZA was demonstrated. AZA also enhanced in vitro NO production, reduced cell apoptosis and increased HIF-1α expression. Knockdown of HIF-1α by RNAi confirmed that AZA exerted its protective role depending on HIF-1α. AZA's effects were significantly reduced by Akt inhibitor LY294002. CONCLUSIONS: The present study demonstrated that AZA exerted a renoprotective role against I/R induced AKI through activating HIF-1α and downstream pathways.

Genistein accelerates refractory wound healing by suppressing superoxide and FoxO1/iNOS pathway in type 1 diabetes.

Refractory wounds in diabetic patients constitute a serious complication that often leads to amputation with limited treatment regimens. The present study was designed to determine the protective effect of the soy isoflavone genistein on diabetic wound healing and investigate underlying mechanisms. Streptozotocin (STZ)-induced type 1 diabetic mice with full-thickness excisional wounds received 0.2, 1 or 5mg/kg/day of genistein via subcutaneous injection. Genistein dose-dependently rescued the delay of wound closure in diabetic mice. A dose of 5 mg/kg/day of genistein treatment significantly increased the mean perfusion rate, and in vitro treatment with genistein protected against high glucose-induced impairment of capillary tube formation in cultured endothelial cells. Diabetic conditions significantly increased superoxide anion (O(2)·(-)) production and nitrotyrosine formation, and decreased nitrite levels in wound tissues. Genistein treatment at all doses normalized the elevated O(2)·(-) production and nitrotyrosine formation, and reversed the attenuated nitrite level. In diabetic wound tissues, the inducible nitric oxide synthase (iNOS) was activated, and genistein administration prevented increased iNOS activity. Moreover, genistein attenuated diabetic cutaneous silent information regulator 1 and forkhead box O transcription factor 1 (FoxO1) levels and potentiated ac-FoxO1 in a dose-dependent manner. Genistein rescued the delayed wound healing and improved wound angiogenesis in STZ-induced type 1 diabetes in mice, at least in part, by suppression of FoxO1, iNOS activity and oxidative stress.

Roles of vimentin and 14-3-3 zeta/delta in the inhibitory effects of heparin on PC-3M cell proliferation and B16-F10-luc-G5 cells metastasis.

AIM: To investigate the inhibitory effects of heparin on PC-3M cells proliferation in vitro and B16-F10-luc-G5 cells metastasis in Balb/c nude mice and identify the protein expression patterns to elucidate the action mechanism of heparin. METHODS: Human prostate cancer PC-3M cells were incubated with heparin 0.5 to 125 µg/mL for 24 h. The proliferation of PC-3M cells was assessed by MTS assay. BrdU incoporation and Ki67 expression were detected using a high content screening (HCS) assay. The cell cycle and apoptosis of PC-3M cells were tested by flow cytometry. B16-F10-luc-G5 cardinoma cells were injected into the lateral tail vein of 6-week old male Balb/c nude mice and heparin 30 mg/kg was administered iv 30 min before and 24 h after injection. The metasis of B16-F10-luc-G5 cells was detected by bioluminescence assay. Activated partial thromboplastin time (APTT) and hemorheological parameters were measured on d 14 after injection of B16-F10-luc-G5 carcinoma cells in Balb/c mice. The global protein changes in PC-3M cells and frozen lung tissues from mice burdened with B16-F10-luc-G5 cells were determined by 2-dimensional gel electrophoresis and image analysis. The protein expression of vimentin and 14-3-3 zeta/delta was measured by Western blot. The mRNA transcription of vimentin, transforming growth factor (TGF)-ß, E-cadherin, and α(v)-integrin was measured by RT-PCR. RESULTS: Heparin 25 and 125 µg/mL significantly inhibited the proliferation, arrested the cells in G(1) phase, and suppressed BrdU incorporation and Ki67 expression in PC-3M cells compared with the model group. But it had no significant effect on apoptosis of PC-3M cells. Heparin 30 mg/kg markedly inhibits the metastasis of B16-F10-luc-G5 cells on day 8. Additionally, heparin administration maintained relatively normal red blood hematocrit but had no influence on APTT in nude mice burdened with B16-F10-luc-G5 cells. Thirty of down-regulated protein spots were identified after heparin treatment, many of which are related to tumor development, extracellular signaling, energy metabolism, and cellular proliferation. Vimentin and 14-3-3 zeta/delta were identified in common in PC-3M cells and the lungs of mice bearing B16-F10-luc-G5 carcinoma cells. Heparin 25 and 125 µg/mL decreased the protein expression of vimentin and 14-3-3 zeta/delta and the mRNA expression of α(v)-integrin. Heparin 125 µg/mL decreased vimentin and E-cadherin mRNA transcription while increased TGF-ß mRNA transcription in the PC-3M cells, but the differences were not significant. Transfection of vimentin-targeted siRNA for 48 h significantly decreased the BrdU incoporation and Ki67 expression in PC-3M cells. CONCLUSION: Heparin inhibited PC-3M cell proliferation in vitro and B16-F10-luc-G5 cells metastasis in nude mice by inhibition of vimentin, 14-3-3 zeta/delta, and α(v)-integrin expression.

Ginsenoside Rg3 attenuates cell migration via inhibition of aquaporin 1 expression in PC-3M prostate cancer cells.

Ginsenoside Rg3 (Rg3), one of the bioactive extracts found in ginseng root, was reported to have anti-cancer activity in various cancer models. The anti-proliferation effect of Rg3 on prostate cancer cells has been well reported. To test whether Rg3 has an anti-metastatic effect on prostate cancer, we treated a highly metastatic PC-3M prostate cancer cell line with Rg3. We found that Rg3 (10µM) led to remarkable inhibition of PC-3M cell migration. Simultaneously, exposure to Rg3 suppressed expression of the aquaporin 1 (AQP1) water channel protein, which has previously been reported to be involved in cell migration. Overexpression of AQP1 attenuated Rg3-induced inhibition of cell migration, and introduction of a shRNA targeting AQP1 abrogated the inhibitory effect of Rg3, although the basal level of cell migration was decreased by RNA interference. In mechanism study, estrogen receptor- and glucocorticoid receptor-dependent pathways are proved uninvolved in the AQP1 regulation by Rg3. However, Rg3 treatment triggered the activation of p38 MAPK; and SB202190, a specific inhibitor of p38 MAPK, antagonized the Rg3-induced regulation of AQP1 and cell migration, suggesting a crucial role for p38 in the regulation process. Deletion analysis of the promoter region of AQP1 was also conducted using dual-luciferase assay, which indicated that the -1000 bp to -200 bp promoter region was involved in the AQP1 regulation by Rg3. In all, we conclude that Rg3 effectively suppresses migration of PC-3M cells by down-regulating AQP1 expression through p38 MAPK pathway and some transcription factors acting on the AQP1 promoter.

Inhibitory effect of epigallocatechin gallate on the proliferation of Lewis Lung Cancer mice and A549 and Calu-3 lung cancer cells / 中国药学杂志

OBJECTIVE: To determine the inhibitory effect of EGCG on the Lewis Lung Cancer Model, to compare and preliminary to discuss the mechanism of the inhibitory effect of EGCG on the proliferation of A549 cell line and Calu-3 cell line. METHODS: Observed the inhibitory effect of EGCG on Lewis Lung Cancer in vivo. Compared the inhibitory effect of different dosages of EGCG on Calu-3 and A549 cell lines with WST-8 assay. Observed the opoptosis promotional effect of EGCG on Calu-3 cells. Compared the inhibitory effect of different dosage of EGCG on Calu-3 and A549 cell lines in PI cell number counting assay. RESULTS: EGCG inhibited the proliferation of Lewis Lung Cancer in vivo. Results of WST-8 assay showed that EGCG inhibited the proliferation of Calu-3 cell line in dose-dependent manner. However, A549 cell line showed sign of drug-resistance to EGCG. The inhibitory effect of EGCG on the proliferation of Calu-3 cell line through increasing apoptosis was observed with TUNEL assay. Cell number of Calu-3 was obviously decreased by treating of EGCG, but was not changed in A549 cell line. CONCLUSION: EGCG inhibited proliferation of Lewis Lung Cacer in vivo, and inhibited proliferation of Calu-3 cell line but didn't play the inhibitory role on A549 cell line in vitro. Copyright 2012 by the Chinese Pharmaceutical Association.

[Molecular mechanisms of diabetic wound healing].

Refractory wound is a severe complication that leads to limb amputation in diabetes, and is also a leading cause of hospitalizations in diabetic patients with limited treatment regimens. However, the underlying mechanisms remain to be fully elucidated. Various factors contribute to delayed diabetic wound healing, such as growth factor, nitric oxide (NO), reactive oxygen species (ROS), matrix metalloproteinases (MMPs), microRNA, endothelial progenitor cells (EPCs), etc. In this review, we will discuss the recent findings on the molecular mechanisms underlying impaired diabetic wound healing.