Riboflavin protects against pancreatic cancer metastasis by targeting TGF-ß receptor 1.

The inhibition of transforming growth factor-ß1 (TGF-ß1) signaling by targeting TGF-ß receptor 1 (TßR1) has been considered as an ideal approach for the prevention of pancreatic cancer metastasis. Utilizing a pharmacophore model for TßR1 inhibitors, candidate compounds with the potential TßR1 binding ability were screened from the U.S. Food and Drug Administration (FDA) database, and riboflavin (RF) with a highest fit value was chosen to investigate its binding ability to TßR1 and effect on TGF-ß1 signaling in pancreatic cancer cells. Molecular docking and cellular thermal shift assay (CETSA) proved that RF at pharmacological concentrations could directly bind to TßR1. Further studies showed that pharmacological concentrations of RF in vitro could block TGF-ß1 signaling, suppress the migration and invasion, and prevent epithelial-mesenchymal transition (EMT) process of pancreatic cancer cells in the absence or presence of TGF-ß1 stimulation, indicating that RF presented anti-metastatic effect in pancreatic cancer cells. Knockdown of TßR1 could significantly attenuate the effects of RF on the migration and EMT process in pancreatic cancer cells, further confirming that the anti-metastatic effect of RF was achieved by blocking TGF-ß1 signaling after binding to TßR1. Moreover, in a mouse model of pancreatic cancer metastasis, it was certified that RF administration could block lung and liver metastases, TGF-ß1 signaling and EMT process of pancreatic cancer in vivo. In summary, our findings showed that RF could block TGF-ß1 signaling by directly binding to TßR1, thereby suppressing the metastasis of pancreatic cancer cells by inhibiting EMT process both in vitro and in vivo.

Design and synthesis of dual cathepsin L and S inhibitors and antimetastatic activity evaluation in pancreatic cancer cells.

Currently, the migration and invasion of cancer cells remain the main factors of poor prognosis in the majority of cancer patients. Developing an effective antimetastatic agent is crucial for cancer therapy. Our recent research revealed that Cat L and S are expressed concurrently in metastatic pancreatic cancer cells. Asperphenamate analog ASPER-29, which exhibits dual Cat L and S inhibitory potency, showed a definite antimetastatic effect on pancreatic cancer BxPC-3 and PANC-1 cells. To further improve the antimetastatic ability of asperphenamate-type molecules, 24 derivatives were designed and synthesized by a scaffold-hopping strategy. The cathepsin inhibitory activity assay results showed that most of the derivatives exhibited dual inhibitory effects on Cat L and S. Among all derivatives, Compound B1a showed the strongest inhibitory activity, with IC50 values of 4.10 ± 0.14 µM and 1.79 ± 0.11 µM, which were 1.5-fold and 2.8-fold more potent than those of positive drugs against Cat L and S, respectively. Further wound-healing and transwell chamber assays demonstrated that B1a presented significant antimetastatic ability in vitro.

Novel pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione (PBD) derivatives as selective HDAC6 inhibitors to suppress tumor metastasis and invasion in vitro and in vivo.

Selective inhibition of histone deacetylase 6 (HDAC6) has been emerged as a promising approach to cancer treatment. As a pivotal strategy for drug discovery,molecular hybridization was introduced in this study and a series of pyrrolo[2,1-c][1,4] benzodiazepine-3,11-diones (PBDs) based hydroxamic acids was rationally designed and synthesizedas novel selective HDAC6 inhibitors. Preliminary in vitro enzyme inhibition assay and structure-activity relationship (SAR) discussion confirmed our design strategy and met the expectation. Several of the compounds showed high potent against HDAC6 enzyme in vitro, and compound A7 with a long aliphatic linker was revealed to have the similar activity as the positive control tubastatin A. Further in vitro characterization of A7 demonstrates the metastasis inhibitory potency in MDA-MB-231 cell line and western blotting showed that A7 could induce the upregulation of Ac-α-tubulin, but not induce the excessive acetylation of histone H3, which indicated that the compound had HDAC6 targeting effect in MDA-MB-231 cells. In vivo study revealed that compound A7 has satisfactory inhibitory effects onliver and lung metastasis of breast cancer in mice. Molecular docking released that A7 could fit well with the receptor and interact with some key residues, which lays a foundation for further structural modifications to elucidate the interaction mode between compounds and target protein. This pharmacological investigation workflow provided a reasonable and reference methodto examine the pharmacological effects of inhibiting HDAC6 with a single molecule, either in vitro or in vivo. All of these results suggested that A7 is a promising lead compound that could lead to the further development of novel selective HDAC6 inhibitors for the treatment of tumor metastasis.

Discovery of a novel and selective cathepsin L inhibitor with anti-metastatic ability in vitro and in vivo against breast cancer cells.

Asperphenamate is a natural product that has attracted considerable interest from researchers worldwide. In the last decade, aiming to increase the biological activity and improve druggability, modifications of the A-ring moiety in asperphenamate have been performed. Our laboratory has also recently reported functional derivatizations of the A ring and studied its effect on the inhibition of cysteine cathepsin L. However, the functional significance of the B-ring fragment toward cathepsin L has not been evaluated thus far. In this paper, forty-four derivatives of the B-ring substituted with different N-phenylsulfonyl groups were designed and synthesized. Among them, the paratrifluromethyl analog B-2a and the 2, 4-difluoro-5-chloro derivative B-11b showed more potent inhibitory activity against cathepsin L than the control compound, ABR, which displayed the strongest inhibitory effect on cathepsin L and S among all reported asperphenamate derivatives. In particular, compound B-2a showed more pronounced selectivity against cathepsin L than the other derivatives. Molecular docking revealed that the N-phenylsulfonylamide moiety was vital for the interactions between B-2a and cathepsin L. Moreover, B-2a displayed no toxicity against normal cells. Therefore, compound B-2a was selected for further studies. Wound-healing assays, Transwell chamber assays and breast cancer lung metastasis mouse models demonstrated that B-2a exhibited antimetastatic ability in vitro and in vivo.

Discovery of specific HDAC6 inhibitor with anti-metastatic effects in pancreatic cancer cells through virtual screening and biological evaluation.

Histone deacetylase 6 (HDAC6) has been demonstrated to play a major role in cell motility and aggresome formation, and HDAC6 inhibition is therefore considered as a promising epigenetic strategy for cancer treatment. At present, only a minority of compounds have been reported as HDAC6 inhibitors, so specific HDAC6 inhibitors with safety profile need to be discovered urgently. In this paper, HDAC6 inhibitors with diverse structures were used to generate the pharmacophore model by ligand-based method, which contained two hydrogen bond acceptors and two hydrophobic groups. A combined virtual screening based on pharmacophore model and molecular docking was adopted to screen potential HDAC6 inhibitors. Subsequently, the HDAC6 inhibitory activity of the hit compounds were evaluated using an in vitro enzyme binding inhibition assay. The experimental results illustrated that cefoperazone sodium had the strongest inhibitory effect on HDAC6 among the six screened compounds, and its IC50 value was 8.59 ± 1.06 µM. Cefoperazone sodium significantly catalyzed the hyperacetylation of α-tubulin but not histone H3, proving that cefoperazone sodium was a selective inhibitor of HDAC6. Since the expression of HDAC6 plays an important role in cancer metastasis, the effects of cefoperazone sodium on migration and invasion of human pancreatic cancer PANC-1 cells were further investigated by wound healing and transwell chamber assays. It was found that cefoperazone sodium could evidently inhibit the migration and invasion of PANC-1 cells. Furthermore, the binding pattern of inhibitor at the active site of the crystal structure was revealed by molecular docking, providing a reference value for the structural design and optimization of HDAC6 inhibitors. This study provides a systematic virtual screening approach for discovering HDAC6 active inhibitors, and by which the specific effect of cefoperazone sodium against HDAC6 was found, suggesting its potential application on cancer therapy.

A novel cathepsin L inhibitor prevents the progression of idiopathic pulmonary fibrosis.

In previous work, the target of asperphenamate as a natural product was successfully determined as cathepsin by the natural product consensus pharmacophore strategy. In order to develop accurate SAR (structure-activity relationship) of asperphenamate-type cathepsin inhibitor, we chose several novel analogs with heterocyclic moiety to perform further study. The molecular simulation showed that 4-pyridyl derivative 3 with the greatest cathepsin inhibitory activity presented new interaction modes with protein utilizing its B-ring moiety. And then molecular dynamics (MD) simulation further revealed that 3 and cathepsin kept stable interaction in the binding site, which validated the molecular docking results. In view that cathepsins play an important role in fibrosis and some cathepsin inhibitors display the therapeutic ability for fibrosis, we investigated the anti-fibrotic effect of 3in vitro and in vivo. The results indicated that 3 displayed the strongest inhibitory effect on the formation of α-SMA and collagen I as the protein markers of fibrosis among all tested derivatives. Further in vivo assay confirmed that 3 indeed showed significant inhibitory ability against pulmonary fibrosis by the method of H&E and Masson staining as well as immunohistochemical staining for characteristic α-SMA proteins.

Discovery of a novel cathepsin inhibitor with dual autophagy-inducing and metastasis-inhibiting effects on breast cancer cells.

Drug resistance and cancer cells metastasis have been the leading causes of chemotherapy failure and cancer-associated death in breast cancer patients. In present, various active molecules either exhibiting novel mechanism of action such as inducing autophagy or inhibiting metastasis have been developed to address these problems. However, the compounds exhibiting such dual functions have rarely been described. Previous work in our group showed that TSA, as a synthetic analog of asperphenamate, induced autophagic cell death in breast cancer cells instead of apoptosis. Furthermore, the target enzyme of TSA was predicted to be cathepin L (Cat L) by natural product consensus pharmacophore strategy. Accumulated evidences have shown that cathepsins are closely associated with migration and invasion of breast cancer cells. It seemed likely that TSA-like molecules may possess the dual functions of inducing autophagy and inhibiting metastasis. Therefore, sixty optically active derivatives were firstly designed and synthesized by replacing the A-ring moiety of TSA with other substituted-phenyl sulfonyl groups. Further cathepsin inhibitory activity assay showed that (S, S) and (S, R) isomers displayed no activity against four kinds of cathepsins (L, S, K, B), while all derivatives tested were inactive toward K and B subtypes. Compound 6a with meta-bromo substituent displayed the greatest inhibitory activity, and its inhibitory capability against Cat L and S was 3.9 and 11.5-fold more potent than that of TSA, respectively. Molecular docking also exhibited that 6a formed more hydrogen bonds or π-π contacts with Cat L or S than TSA. In order to determine whether 6a could play dual roles, its anti-cancer mechanism was further investigated. On the one hand, MDC staining experiment and western blotting analysis validated that 6a can induce autophagy in MDA-MB-231 cells. On the other hand, its metastatic inhibitory ability was also confirmed by wound healing and transwell chamber experiment.

Salvianolic acid B inhibits myofibroblast transdifferentiation in experimental pulmonary fibrosis via the up-regulation of Nrf2.

Salvianolic acid B (SalB) is one of the most bioactive components extracted from Salvia miltiorrhiza, and its antioxidant capacity corresponds with its protective effects against cell injury from oxidative stress. The aim of the present study was to evaluate the effect of SalB on experimental pulmonary fibrosis and its ability to ameliorate the oxidative/antioxidative imbalance during fibrosis pathogenesis. The anti-fibrotic activity of SalB was first confirmed in Transforming growth factor ß1(TGF-ß1)-stimulated MRC-5 cells. The protection of SalB against oxidative stress during fibrogenesis in vitro was verified by detecting ROS production, the levels of glutathione (GSH) and malondialdehyde (MDA). The Western blot and PCR results indicated that SalB could up-regulate nuclear factor erythroid-derived 2-like 2 (Nrf2) at both the protein and mRNA levels and induce Nrf2 nuclear translocation in vitro, which may be the mechanism underlying the anti-fibrotic capacity of SalB. Furthermore, the anti-fibrotic and antioxidant capacities of SalB in vivo were confirmed in rats with BLM-induced pulmonary fibrosis. The immunohistochemistry results showed that Nrf2 was absent in fibroblastic foci (FF) areas, while the SalB treatment could increase the expression of Nrf2 in lung tissues, especially in FF areas.

Discovery of novel cathepsin inhibitors with potent anti-metastatic effects in breast cancer cells.

It is still challenging to determine the potential targets of natural products, which is essential for further drug research and development. Due to its novel mechanism of action of inducing autophagy effects in breast cancer cells, asperphenamate has received our considerable attention. However, its unknown target inevitably impedes further study. In our previous work, the target enzyme of asperphenamate was predicted as cathepsin by the natural product consensus pharmacophore strategy. Then, asperphenamate and its three derivatives were chosen to study in detail by molecular docking calculations with AutoDock 4 suite. The docking results showed the three derivatives interacted more tightly with either cathepsin L or cathepsin S than with asperphenamate. The ortho-benzyloxyl phenylacetyl derivative 1 andp-toluenesulfonyl derivative 3 showed similar interactions with cathepsin L and adopted a better geometric shape within the binding pocket than did the N-CBZ-piperidyl analog 2. On the other hand, compound 2 formed more hydrogen bonds than 1 and 3 to make it tightly bind within cathepsin S. The cathepsin inhibitory activity assay verified the molecular simulation results. Compound 2 was remarkably less active than 1 and 3 against cathepsin L. However, compound 2 showed the strongest potency against cathepsin S with IC50 of 13.12 ±â€¯0.29 µM. Considering that cathepsin S plays a vital role in the process of metastasis in breast cancer cells, the inhibitory effect of 2 on migration and invasion was further studied in human breast cancer MDA-MB-231 cells by wound healing and transwell chamber assays. The results illustrated that 2 exhibited an apparent inhibitory ability to the metastasis of MDA-MB-231 cells. Next, 2 will be chosen as a lead compound to develop novel double functional chemotherapeutic agents with both novel mechanisms of action against apoptosis-resistant cancer cells, such as inducing autophagy and inhibiting cancer metastasis.

Anti-pulmonary fibrotic activity of salvianolic acid B was screened by a novel method based on the cyto-biophysical properties.

Various methods have been used to evaluate anti-fibrotic activity of drugs. However, most of them are complicated, labor-intensive and lack of efficiency. This study was intended to develop a rapid method for anti-fibrotic drugs screening based on biophysical properties. A549 cells in vitro were stimulated with transforming growth factor-ß1 (TGF-ß1), and fibrogenesis was confirmed by conventional immunological assays. Meanwhile, the alterations of cyto-biophysical properties including morphology, roughness and stiffness were measured utilizing atomic force microscopy (AFM). It was found that fibrogenesis was accompanied with changes of cellular biophysical properties. TGF-ß1-stimulated A549 cells became remarkably longer, rougher and stiffer than the control. Then, the effect of N-acetyl-L-cysteine (NAC) as a positive drug on ameliorating fibrogenesis in TGF-ß1-stimulated A549 cells was verified respectively by immunological and biophysical markers. The result of Principal Component Analysis showed that stiffness was a leading index among all biophysical markers during fibrogenesis. Salvianolic acid B (SalB), a natural anti-oxidant, was detected by AFM to protect TGF-ß1-stimulated A549 cells against stiffening. Then, SalB treatment was provided in preventive mode on a rat model of bleomycin (BLM) -induced pulmonary fibrosis. The results showed that SalB treatment significantly ameliorated BLM-induced histological alterations, blocked collagen accumulations and reduced α-SMA expression in lung tissues. All these results revealed the anti-pulmonary fibrotic activity of SalB. Detection of cyto-biophysical properties were therefore recommended as a rapid method for anti-pulmonary fibrotic drugs screening.

Caryophyllene sesquiterpenes from the marine-derived fungus Ascotricha sp. ZJ-M-5 by the one strain-many compounds strategy.

The marine-derived fungus Ascotricha sp. ZJ-M-5, which can produce cyclonerodiol analogues, a 3,4-seco lanostane triterpenoid, and diketopiperazines in an eutrophic medium, was subjected to a one strain-many compounds (OSMAC) analysis. It was found to produce three new caryophyllene derivatives (1-3) and the known 1,3,6-trihydroxy-8-methylxanthone (4) in an oligotrophic medium, Czapek Dox broth with or without Mg(2+). (+)-6-O-Demethylpestalotiopsin A (1) and (+)-6-O-demethylpestalotiopsin C (2), which have a five-membered hemiacetal structural moiety, showed growth inhibitory effects against HL-60 and K562 leukemia cell lines with the lowest GI50 value of 6.9 ± 0.4 µM. It can be concluded that modification of the culture media is still effective in the discovery of novel bioactive fungal secondary metabolites.

Screening approach by a combination of computational and in vitro experiments: identification of fluvastatin sodium as a potential SIRT2 inhibitor.

BACKGROUND: Sirtuins (SIRTs) are NAD+-dependent deacetylases that play various roles in numerous pathophysiological processes, holding promise as therapeutic targets worthy of further investigation. Among them, the SIRT2 subtype is closely associated with tumorigenesis and malignancies. Dysregulation of SIRT2 activation can regulate the expression levels of related genes in cancer cells, leading to tumor occurrence and metastasis. METHODS: In this study, we used computer simulations to screen for novel SIRT2 inhibitors from the FDA database, based on which 10 compounds with high docking scores and good interactions were selected for in vitro anti-pancreatic cancer metastasis testing and enzyme binding inhibition experiments. The results showed that fluvastatin sodium may possess inhibitory activity against SIRT2. Subsequently, fluvastatin sodium was subjected to molecular docking experiments with various SIRT isoforms, and the combined results from Western blotting experiments indicated its potential as a SIRT2 inhibitor. Next, molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations were performed, revealing the binding mode of fluvastatin sodium at the SIRT2 active site, further validating the stability and interaction of the ligand-protein complex under physiological conditions. RESULTS: Overall, this study provides a systematic virtual screening workflow for the discovery of SIRT2 activity inhibitors, identifies the potential inhibitory effect of fluvastatin sodium as a lead compound on SIRT2, and opens up a new direction for developing highly active and selectively targeted SIRT2 inhibitors.

Discovery of novel pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione (PBD) derivatives as selective HDAC6 inhibitors for the efficient treatment of idiopathic pulmonary fibrosis (IPF) in vitro and in vivo.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by a progressive fibrotic phenotype. Immunohistochemical studies on HDAC6 overexpression in IPF lung tissues confirmed that IPF is associated with aberrant HDAC6 activity. We herein developed a series of novel HDAC6 inhibitors that can be used as potential pharmacological tools for IPF treatment. The best-performing derivative H10 showed good selectivity for multiple isoforms of the HDAC family. The structural analysis and structure-activity relationship studies of H10 will contribute to optimizing the binding mode of the new molecules. The pharmacological mechanism of H10 to inhibit pulmonary fibrosis was validated, and its ability to inhibit the IPF phenotype was also demonstrated. Moreover, H10 showed satisfactory metabolic stability. The efficacy of H10 was also determined in a mouse model of bleomycin-induced pulmonary fibrosis. The results highlighted in this paper may provide a reference for the identification of new drug molecules for the treatment of IPF.

ß-Eudesmol induces JNK-dependent apoptosis through the mitochondrial pathway in HL60 cells.

ß-eudesmol, a natural sesquiterpenol present in a variety of Chinese herbs, is known to inhibit the proliferation of human tumor cells. However, the molecular mechanisms of the effect of ß-eudesmol on human tumor cells are unknown. In the present study, we report the cytotoxic effect of ß-eudesmol on the human leukemia HL60 cells and its molecular mechanisms. The cytotoxic effect of ß-eudesmol on HL60 cells was associated with apoptosis, which was characterized by the presence of DNA fragmentation. ß-eudesmol-induced apoptosis was accompanied by cleavage of caspase-3, caspase-9, and poly (ADP-ribose) polymerase; downregulation of Bcl-2 expression; release of cytochrome c from mitochondria; and decrease in mitochondrial membrane potential (MMP). Activation of c-Jun N-terminal kinases (JNK) mitogen-activated protein kinases was observed in ß-eudesmol-treated HL60 cells, and the inhibitor of JNK blocked the ß-eudesmol-induced apoptosis, downregulation of Bcl-2, and the loss of MMP. These data suggest that ß-eudesmol induces apoptosis in HL60 cells via the mitochondrial apoptotic pathway, which is controlled through JNK signaling.

JNK-dependent Atg4 upregulation mediates asperphenamate derivative BBP-induced autophagy in MCF-7 cells.

N-Benzoyl-O-(N'-(1-benzyloxycarbonyl-4-piperidiylcarbonyl)-D-phenylalanyl)-D-phenylalaninol (BBP), a novel synthesized asperphenamate derivative with the increased solubility, showed growth inhibitory effect on human breast carcinoma MCF-7 cells in a time- and concentration-dependent manner. The growth inhibitory effect of BBP was associated with induction of autophagy, which was demonstrated by the development of acidic vesicular organelles, cleavage of LC3 and upregulation of Atg4 in BBP-treated MCF-7 cells. Since the application of Atg4 siRNA totally blocked the cleavage of LC3, we demonstrated a central role of Atg4 in BBP-induced autophagy. The further studies showed that BBP increased the levels of reactive oxygen species (ROS), and pretreatment with NAC effectively blocked the accumulation of ROS, autophagy and growth inhibition triggered by BBP. Moreover, BBP induced the activation of JNK, and JNK inhibitor SP600125 reversed autophagy, the increase of Atg4 levels, conversion of LC3 and growth inhibition induced by BBP. Knockdown of JNK by siRNA efficiently inhibited ROS production and autophagy, but antioxidant NAC failed to block JNK activation induced by BBP, indicating that JNK activation may be a upstream signaling of ROS and should be a core component in BBP-induced autophagic signaling pathway. These results suggest that BBP produces its growth inhibitory effect through induction of the autophagic cell death in MCF-7 cells, which is modulated by a JNK-dependent Atg4 upregulation involving ROS production.

Synthesis and in vitro antitumor activity of asperphenamate derivatives as autophagy inducer.

In an effort to improve the aqueous solubility and the antitumor activity of natural product asperphenamate, we have designed and synthesized three series of asperphenamate derivatives, including series I (simplifying molecular skeleton series), series II (introducing a hydroxyl group to A-phenyl ring series) and series III (disrupting molecular planarity series). All derivatives have displayed a significantly increased solubility compared with asperphenamate. Their growth inhibitory activities in vitro were screened by the standard MTT method in MCF-7, HeLa, and BEL-7402 cell lines. With the exception of the derivatives in series I, most of derivatives in series II and series III showed growth inhibitory activity. Among all derivatives, IM23b in series III showed the greatest potency in human breast cancer MCF-7 cells. The cellular potency of IM23b was approximately 1.5-fold more potent than that of cisplatin. The mechanism of cell death induced by IM23b in human breast cancer MCF-7 cells was further investigated. We concluded that the cell death was induced by autophagy instead of apoptosis or cell cycle arrest.

Isolation and cytotoxic activity of selaginellin derivatives and biflavonoids from Selaginella tamariscina.

Five selaginellin derivatives, including two new selaginellins termed selaginellins M (1) and N (2), and three previously identified compounds, selaginellin (3), selaginellin A (4), and selaginellin C (5), were isolated from the Selaginella tamariscina (Beauv.) Spring plant. In addition, four known biflavonoids, namely neocryptomerin ( 6), hinokiflavone (7), pulvinatabiflavone (8), and 7''- O-methylamentoflavone (9), were also isolated. The structures of new compounds 1 and 2 were elucidated by spectroscopic analysis. The cytotoxic activity of compounds 1- 9 was evaluated against a small panel of human cancer cell lines, including U251 (human glioma cells), HeLa (human cervical carcinoma cells), and MCF-7 (human breast cancer cells). The two new selaginellins, selaginellins M (1) and N (2), showed medium activity against the human cancer cell lines.

Activation of ATM-Chk2 by 16-dehydropregnenolone induces G1 phase arrest and apoptosis in HeLa cells.

The natural pregnane steroid 16-dehydropregnenolone (16-DHP) showed the growth inhibitory activity against human tumor cells; however, the mechanisms of actions of 16-DHP were not clarified. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to investigate the growth inhibitory effect of 16-DHP. Cell cycle distribution was analyzed using flow cytometry. Hoechst 33258 staining and DNA agarose gel electrophoresis were used to detect apoptosis. The levels of proteins were probed by Western blotting, and caspase activities were analyzed using Caspase Activity Kit. We found that 16-DHP inhibited the growth of human cervical carcinoma cells (HeLa cells) in a time- and dose-dependent manner. The growth inhibitory effect of 16-DHP was associated with G1 arrest mediated by ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2)-p53 signaling, as demonstrated by induction of the phosphorylations of ATM, Chk2, and p53 proteins. Followed by G1 arrest, 16-DHP-treated HeLa cells underwent caspase-dependent apoptosis. The inhibitors of caspase-3 and caspase-9 but not caspase-8 inhibitor blocked 16-DHP-induced apoptosis. Moreover, 16-DHP increased the level of Bax protein and the release of cytochrome c from mitochondria, but had no effect on the level of Bcl-2. These results suggested that 16-DHP inhibited the growth of HeLa cells via inducing ATM-Chk2-p53 activation-mediated G1 arrest and mitochondrial cell apoptosis.

ASPER-29 suppresses the metastasis of pancreatic cancer cells by dual inhibition of cathepsin-L and cathepsin-S.

Pancreatic cancer will be the second leading cause of cancer-related mortality worldwide due to its high rate of metastasis. Cathepsins (CATs) are effectors of invasive growth in various cancers. Currently, targeting CATs represents an attractive strategy for the treatment of highly metastatic cancers with high CATs activity, such as pancreatic cancer. To develop a stronger antimetastatic agent, ASPER-29, a novel inhibitor of CATs designed by using the asperphenamate derivative BBP as a lead compound, was synthesized, and its therapeutic potential in pancreatic cancer metastasis was investigated in this study. Molecular docking and enzyme inhibition assays proved that ASPER-29 can inhibit the activity of CAT-L and CAT-S by binding with these enzymes in classical action modes. Furthermore, ASPER-29 significantly inhibited the activity of CAT-L and CAT-S but had no effect on their expression in PANC-1 and BxPC-3 cells. The in vitro antimetastatic activities of ASPER-29 were examined by wound healing and Transwell chamber assays. We found that ASPER-29 inhibited the migration and invasion of PANC-1 and BxPC-3 cells in a concentration-dependent manner. Moreover, the in vivo antimetastatic effects of ASPER-29 were confirmed in a mouse xenotransplantation model. H&E staining and immunohistochemistry assays of Ki67 and CEACAM6 proved that ASPER-29 treatment significantly blocked the metastasis of pancreatic cancer cells to lung and liver tissues. Additionally, the activity of both CAT-L and CAT-S was markedly inhibited in the lung and liver tissues of ASPER-29-administered mice compared with the mice in the model group, suggesting that the metastasis-blocking effect of ASPER-29 should be mediated via inhibition of the activity of CAT-L and CAT-S in pancreatic cancer cells. Together, our results demonstrated that ASPER-29, as a novel inhibitor of CAT-L and CAT-S, possessed the evident ability to block the metastasis of pancreatic cancer cells.

Structural and PK-guided identification of indole-based non-acidic autotaxin (ATX) inhibitors exhibiting high in vivo anti-fibrosis efficacy in rodent model.

In recent decades, pharmacological targeting of the autotaxin (ATX)/lysophosphatidic acid (LPA) axis accounted for excellent disease management benefits. Herein, to extend the scope of structure-activity relationships (SARs), fifteen indole-based carbamate derivatives (1-15) were prepared to evaluate the ATX inhibitory potency. Among them, compound 4 bearing morpholine moiety was identified as the optimal ATX inhibitor (0.41 nM), superior to the positive control GLPG1690 (2.90 nM). To resolve the intractable issue of poor pharmacokinetic (PK) property, urea moiety was introduced as a surrogate of carbamate which furnished compounds 16-30. The dedicated modification identified the diethanolamine entity 30 with satisfactory water solubility and PK profiles with a minimum sacrifice of ATX inhibition (2.17 nM). The most promising candidate 30 was evaluated for anti-fibrosis effect in a bleomycin challenged mice lung fibrosis model. Upon treatment with 30, the in vivo ATX activity in both lung homogenate and broncheoalveolar fluid (BALF) sample was significantly down-regulated. Furthermore, the gene expression of pro-fibrotic cytokines transforming growth factor-ß (TGF-ß), interleukin- 6 (IL-6) and tumor necrosis factor-α (TNF-α) in lung tissue was reduced to normal level. Collectively, the promising biological effects may advocate potential application of 30 in fibrosis relevant diseases.