Novel 5-oxo-hexahydroquinoline derivatives: design, synthesis, in vitro P-glycoprotein-mediated multidrug resistance reversal profile and molecular dynamics simulation study

Overexpression of the efflux pump P-glycoprotein (P-gp) is one of the important mechanisms of multidrug resistance (MDR) in many tumor cells. In this study, 26 novel 5-oxo-hexahydroquinoline derivatives containing different nitrophenyl moieties at C-4 and various carboxamide substituents at C-3 were designed, synthesized and evaluated for their ability to inhibit P-gp by measuring the amount of rhodamine 123 (Rh123) accumulation in uterine sarcoma cells that overexpress P-gp (MES-SA/Dx5) using flow cytometry. The effect of compounds with highest MDR reversal activities was further evaluated by measuring the alterations of MES-SA/Dx5 cells' sensitivity to doxorubicin (DXR) using MTT assay. The results of both biological assays indicated that compounds bearing 2-nitrophenyl at C-4 position and compounds with 4-chlorophenyl carboxamide at C-3 demonstrated the highest activities in resistant cells, while they were devoid of any effect in parental nonresistant MES-SA cells. One of the active derivatives, 5c, significantly increased intracellular Rh123 at 100 mu M, and it also significantly reduced the IC50 of DXR by 70.1% and 88.7% at 10 and 25 mu M, respectively, in MES-SA/Dx5 cells. The toxicity of synthesized compounds against HEK293 as a noncancer cell line was also investigated. All tested derivatives except for 2c compound showed no cytotoxicity. A molecular dynamics simulation study was also performed to investigate the possible binding site of 5c in complex with human P-gp, which showed that this compound formed 11 average H-bonds with Ser909, Thr911, Arg547, Arg543 and Ser474 residues of P-gp. A good agreement was found between the results of the computational and experimental studies. The findings of this study show that some 5-oxo-hexahydroquinoline derivatives could serve as promising candidates for the discovery of new agents for P-gp-mediated MDR reversal

Antioxidant properties of hydroxycinnamic acids: a review of structure- activity relationships

Hydroxycinnamic acids (HCAs) are important phytochemicals possessing significant biological properties. Several investigators have studied in vitro antioxidant activity of HCAs in detail. In this review, we have gathered the studies focused on the structure-activity relationships (SARs) of these compounds that have used medicinal chemistry to generate more potent antioxidant molecules. Most of the reports indicated that the presence of an unsaturated bond on the side chain of HCAs is vital to their activity. The structural features that were reported to be of importance to the antioxidant activity were categorized as follows: modifications of the aromatic ring, which include alterations in the number and position of hydroxy groups and insertion of electron donating or withdrawing moieties as well as modifications of the carboxylic function that include esterification and amidation process. Furthermore, reports that have addressed the influence of physicochemical properties including redox potential, lipid solubility and dissociation constant on the antioxidant activity were also summarized. Finally, the pro-oxidant effect of HCAs in some test systems was addressed. Most of the investigations concluded that the presence of ortho-dihydroxy phenyl group (catechol moiety) is of significant importance to the antioxidant activity, while, the presence of three hydroxy groups does not necessarily improve the activity. Optimization of the structure of molecular leads is an important task of modern medicinal chemistry and its accomplishment relies on the careful assessment of SARs. SAR studies on HCAs can identify the most successful antioxidants that could be useful for management of oxidative stress-related diseases

Never let it go: Stopping key mechanisms underlying metastasis to fight pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive neoplasm, predicted to become the second leading cause of cancer-related deaths before 2030. This dismal trend is mainly due to lack of effective treatments against its metastatic behavior. Therefore, a better understanding of the key mechanisms underlying metastasis should provide new opportunities for therapeutic purposes. Genomic analyses revealed that aberrations that fuel PDAC tumorigenesis and progression, such as SMAD4 loss, are also implicated in metastasis. Recently, microRNAs have been shown to play a regulatory role in the metastatic behavior of many tumors, including PDAC. In particular, miR-10 and miR-21 have appeared as master regulators of the metastatic program, while members of the miR-200 family are involved in the epithelial-to-mesenchymal switch, favoring cell migration and invasiveness. Several studies have also found a close relationship between cancer stem cells (CSCs) and biological features of metastasis, and the CSC markers ALDH1, ABCG2 and c-Met are expressed at high levels in metastatic PDAC cells. Emerging evidence reveals that exosomes are involved in the modulation of the tumor microenvironment and can initiate PDAC pre-metastatic niche formation in the liver and lungs. In this review, we provide an overview of the role of all these pivotal factors in the metastatic behavior of PDAC, and discuss their potential exploitation in the clinic to improve current therapeutics and identify new drug targets

Cytotoxic furanosesquiterpenoids and steroids from Ircinia mutans sponges

CONTEXT: Ircinia mutans Wilson (Irciniidae) is a sponge with antimicrobial and cytotoxic constituents. OBJECTIVE: Our objective was to characterise the cytotoxic constituents of two seasonal collections of I. mutans. MATERIALS AND METHODS: The sponges were extracted in methanol-dichloromethane and their constituents were purified and characterised using column chromatography, GC-MS, 1 D and 2 D NMR. Anti-proliferative activities of the compounds, were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (0.25–100 μg/mL, 72 h) against leukaemia (MOLT-4), breast (MCF-7) and colon cancer (HT-29) human cells. RESULTS: Three furanosesquiterpoids; furodysin (1), ent-furodysinin (2) and furoircin (3) and ten sterols were characterised in I. mutans, for the first time. Cholesterol (4), cholesta-5, 7-dien-3β-ol (5) and ergosterol (6) were determined in the sponge from the winter collections, while cholesta-5, 22-dien-3β-ol (7), 24-methyldesmosterol (8), campesterol (9), stigmasterol (10), γ-ergostenol (11), chondrillasterol (12) and γ-sitosterol (13) were detected in the summer samples. The steroids from the winter collection exhibited cytotoxic activity with IC(50) values of 13.0 ± 0.9, 11.1 ± 1.7 and 1.1 ± 0.4 µg/mL, against the mentioned cancer cell lines, respectively, while those from the summer sample, showed greater activity, IC(50) = 1.1 ± 0.2 μg/mL against MOLT-4. The purified steroids showed potent MOLT-4 cytotoxic activity, IC(50) values = 2.3–7.8 µg/mL. DISCUSSION AND CONCLUSION: The present study suggests that I. mutans is a rich source of cytotoxic steroids, and introduces 3 as new natural product. Considering the high cytotoxic activity of the steroids, these structures could be candidates for anticancer drug development in future research

GLUT1 and LOX inhibitors as perspective anticancer agents tackling glucose avidity and ECM remodeling in tumors

Introduction Most cancers have large hypoxic regions, which display an increase of the glycolytic metabolism leading to the production of lactate, providing cancer cells with adequate amounts of energy and anabolites. To this end, tumor cells generally overexpress glucose transporters (GLUTs), in particular GLUT1, which results in an increased uptake of glucose to support their less efficient energy production (Warburg effect). Therefore, therapeutic interventions aimed at reducing cancer glycolysis may be implemented by several strategies, including the development of inhibitors of glucose transporters. Furthermore, extracellular matrix (ECM) remodeling is one of the key processes preluding metastatic invasion, and it is promoted by several effectors, such as lysyl oxidase (LOX), an enzyme commonly involved in extracellular matrix maturation. LOX is up-regulated by HIF-1 and plays a critical role in the development of metastasis. Therefore, LOX inhibitors may represent an additional and innovative strategy for the treatment and the prevention of metastatic cancer. Methods We have developed various classes of compounds that are able to interfere with GLUTs (Granchi et al. 2015, Tuccinardi et al. 2013) and LOX (Granchi et al. 2009) by molecular design and chemical synthesis. Their effect on cell proliferation, apoptosis, migration and other key determinants of activity were evaluated by sulforhodamine-B and luciferase assays, FACS, wound-healing assay, and Quantitative PCR. The studies were performed in seven PDAC cells, including five primary-cell-cultures and 3D co-cultures with human stellate cells, in normoxic and hypoxic conditions. Results The IC50s of the tested compounds ranged from 13.9 to 32.0 μM after 72-hour exposure. Notably, these compounds were still active in 3D co-cultures of these tumor cells with pancreatic stellate cells, which showed increased resistance to gemcitabine and are more representative of the dense stromal compartment with core hypoxic areas of this tumor type, as detected by immunohistochemical stainings. Remarkably, one compound (PGL-14) showed a synergistic interaction with gemcitabine, increasing apoptosis induction and accumulation of ROS. Furthermore, the combination of these drugs reduced cell migration and enhanced in vitro sensitivity to anoikis, suggesting the ability of these compounds to inhibit metastasis. Discussion GLUT1 inhibitors were more active in hypoxia, but still active also in normoxia. Conversely, we did not detect cytotoxic effects using the LOX-inhibitors in normoxia (at concentration until 50 μM) since they were designed as bioreductively activated prodrugs, which are therefore activated only under hypoxic conditions. However, at O2 tension of 1%, IC50s were below 10 μM. As reported previously, LOX inhibition was associated with reduction of the mRNA levels of fibronectin, suggesting that it might also have impact on the interaction of tumor cells with the stroma that are mediated by integrins and fibronectin, regulating tissue stiffness (Coppola et al. 2017). Conclusion Interventions aimed at blocking the glycolytic activity or the extracellular matrix remodeling of tumors by means of newly designed molecules proved to exert a synergistic effect with clinically approved drugs, such as gemcitabine. These results seem to support the strategy of the simultaneous GLUT/LOX-inhibition in order to further sensitize hypoxic cancer portions to chemotherapy. Bibliography C. Granchi, et al. ChemMedChem 2009, 4, 1590-1594. C. Granchi, et al. ChemMedChem 2015, 10, 1892-1900. T. Tuccinardi, et al. Bioorg. Med. Chem. Lett. 2013, 23, 6923-6927. Coppola S, et al. Drug Resist Updat. 2017, 31, 43-51

PO-278 Assessment of the influence of stellate cells on primary pancreatic cancer cell growth and drug resistance in a spheroid model: MET inhibitors to the rescue

Introduction Pancreatic ductal adenocarcinoma has one of the lowest 5 year survival rates among all cancers and it is going to be the 2nd leading cause of cancer death in 2030. Drug resistance and early metastasis are among the main causes of this dismal prognosis and tumour microenvironment may give a considerable contribution to this aggressive behaviour. Pancreatic stellate cells (PSCs) are the main source of cancer-associated fibroblasts in stroma, and are suspected to induce drug resistance by paracrine secretion of hepatocyte growth factor (HGF) and activation of the MET receptor in cancer cells. Material and methods We first examined the effect of human PSC conditioned medium on the growth and drug resistance of six different primary cell cultures isolated from PDAC patients by sulforhodamine B (SRB) assay growing as monolayers. Further, we developed a spheroid 3D-co-culture with PDAC5-SSEA4 and immortalised or primary PSC cells and examined the effects of different drugs by luciferase assay, immunofluoresence and confocal microscopy. Results and discussions Conditioned medium of stimulated PSC cells, i.e., primed with PDAC conditioned medium, gave growth advantage to different primary PDAC cells and made them several times more resistant to gemcitabine and oxaliplatin. PDAC5-SSEA4/PSC spheroids were much more resistant to gemcitabine and oxaliplatin compared to PDAC5-SSEA4 spheroids. However, MET inhibitors such as tivantinib and PHA-665752 were equally effective in homo and heterospheroids. Of note, immortalised and primary PSC cells had similar influences on the behaviour of PDAC cells in spheroids. Conclusion We successfully developed a 3D-spheroid model to evaluate the interaction of primary PDAC cells with PSCs. Pharmacological studies provided evidence that spheroids containing PSCs are much more resistant to cytotoxic drugs. Conversely MET inhibitors seem to be valuable tools to overcome the drug resistance of PDAC cells caused by the presence of PSC cells

Lipoxygenases and Poly(ADP-Ribose) Polymerase in Amyloid Beta Cytotoxicity

The 12/15-lipoxygenase(s) (LOX), poly(ADP-ribose) polymerase (PARP-1) activity and mitochondrial apoptosis inducing factor (AIF) protein in the amyloid β (Aβ) toxicity were investigated in PC12 cells that express either wild-type (APPwt) or double Swedish mutation (APPsw) forms of human Aβ precursor protein. Different levels of Aβ secretion and free radicals formation characterize these cells. The results demonstrated a relationship between the Aβ levels and LOX protein expression and activity. High Aβ concentration in APPsw cells correlated with a significant increase in free radicals and LOX activation, which leads to translocation of p65/NF-κB into the nucleus. An increase in AIF expression in mitochondria was observed concurrently with inhibition of PARP-1 activity in the nuclear fraction of APPsw cells. We suggested that AIF accumulation in mitochondria may be involved in adaptive/protective processes. However, inhibition of PARP-1 may be responsible for the disturbances in transcription and DNA repair as well as the degeneration of APP cells. Under conditions of increased nitrosative stress, evoked by the nitric oxide donor, sodium nitroprusside (SNP, 0.5 mM), 70–80% of all cells types died after 24 h, significantly more in APPsw cells. There was no further significant change in mitochondrial AIF level and PARP-1 activity compared to corresponding non-treated cells. Only one exception was observed in PC12 control, where SNP significantly inhibits PARP-1 activity. Moreover, SNP significantly activated gene expression for 12/15-LOX in all types of investigated cells. Inhibitors of all LOX isoforms and specific inhibitor of 12-LOX enhanced the survival of cells that were subjected to SNP. We conclude that the LOX pathways may play a role in Aβ toxicity and in nitrosative-stress-induced cell death and that inhibition of these pathways offers novel protective strategies

Lipoic acid plays a role in scleroderma: insights obtained from scleroderma dermal fibroblasts

Abstract Introduction Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis of the skin and organs. Increase in oxidative stress and platelet-derived growth factor receptor (PDGFR) activation promote type I collagen (Col I) production, leading to fibrosis in SSc. Lipoic acid (LA) and its active metabolite dihydrolipoic acid (DHLA) are naturally occurring thiols that act as cofactors and antioxidants and are produced by lipoic acid synthetase (LIAS). Our goals in this study were to examine whether LA and LIAS were deficient in SSc patients and to determine the effect of DHLA on the phenotype of SSc dermal fibroblasts. N-acetylcysteine (NAC), a commonly used thiol antioxidant, was included as a comparison. Methods Dermal fibroblasts were isolated from healthy subjects and patients with diffuse cutaneous SSc. Matrix metalloproteinase (MMPs), tissue inhibitors of MMPs (TIMP), plasminogen activator inhibitor 1 (PAI-1) and LIAS were measured by enzyme-linked immunosorbent assay. The expression of Col I was measured by immunofluorescence, hydroxyproline assay and quantitative PCR. PDGFR phosphorylation and α-smooth muscle actin (αSMA) were measured by Western blotting. Student’s t-tests were performed for statistical analysis, and P-values less than 0.05 with two-tailed analysis were considered statistically significant. Results The expression of LA and LIAS in SSc dermal fibroblasts was lower than normal fibroblasts; however, LIAS was significantly higher in SSc plasma and appeared to be released from monocytes. DHLA lowered cellular oxidative stress and decreased PDGFR phosphorylation, Col I, PAI-1 and αSMA expression in SSc dermal fibroblasts. It also restored the activities of phosphatases that inactivated the PDGFR. SSc fibroblasts produced lower levels of MMP-1 and MMP-3, and DHLA increased them. In contrast, TIMP-1 levels were higher in SSc, but DHLA had a minimal effect. Both DHLA and NAC increased MMP-1 activity when SSc cells were stimulated with PDGF. In general, DHLA showed better efficacy than NAC in most cases. Conclusions DHLA acts not only as an antioxidant but also as an antifibrotic because it has the ability to reverse the profibrotic phenotype of SSc dermal fibroblasts. Our study suggests that thiol antioxidants, including NAC, LA, or DHLA, could be beneficial for patients with SSc.http://deepblue.lib.umich.edu/bitstream/2027.42/112060/1/13075_2014_Article_411.pd