Mitochondrial complex III bypass complex I to induce ROS in GPR17 signaling activation in GBM

Guanine nucleotide binding protein (G protein) coupled receptor 17 (GPR17) plays crucial role in Glioblastoma multiforme (GBM) cell signaling and is primarily associated with reactive oxidative species (ROS) production and cell death. However, the underlying mechanisms by which GPR17 regulates ROS level and mitochondrial electron transport chain (ETC) complexes are still unknown. Here, we investigate the novel link between the GPR17 receptor and ETC complex I and III in regulating level of intracellular ROS (ROSi) in GBM using pharmacological inhibitors and gene expression profiling. Incubation of 1321N1 GBM cells with ETC I inhibitor and GPR17 agonist decreased the ROS level, while treatment with GPR17 antagonist increased the ROS level. Also, inhibition of ETC III and activation of GPR17 increased the ROS level whereas opposite function was observed with antagonist interaction. The similar functional role was also observed in multiple GBM cells, LN229 and SNB19, where ROS level increased in the presence of Complex III inhibitor. The level of ROS varies in Complex I inhibitor and GPR17 antagonist treatment conditions suggesting that ETC I function differs depending on the GBM cell line. RNAseq analysis revealed that ~ 500 genes were commonly expressed in both SNB19 and LN229, in which 25 genes are involved in ROS pathway. Furthermore, 33 dysregulated genes were observed to be involved in mitochondria function and 36 genes of complex I-V involved in ROS pathway. Further analysis revealed that induction of GPR17 leads to loss of function of NADH dehydrogenase genes involved in ETC I, while cytochrome b and Ubiquinol Cytochrome c Reductase family genes in ETC III. Overall, our findings suggest that mitochondrial ETC III bypass ETC I to increase ROSi in GPR17 signaling activation in GBM and could provide new opportunities for developing targeted therapy for GBM

Novel, isoform-selective, cholecystokinin A receptor antagonist inhibits colon and pancreatic cancers in preclinical models through novel mechanism of action

Colon and pancreatic cancers contribute to 90,000 deaths each year in the USA. These cancers lack targeted therapeutics due to heterogeneity of the disease and multiple causative factors. One important factor that contributes to increased colon and pancreatic cancer risk is gastrin. Gastrin mediates its actions through two G-protein coupled receptors (GPCRs): cholecystokinin receptor A (CCK-A) and CCK-B/gastrin receptor. Previous studies have indicated that colon cancer predominantly expresses CCK-A and responds to CCK-A isoform antagonists. However, many CCK-A antagonists have failed in the clinic due to poor pharmacokinetic properties or lack of efficacy. In the present study, we synthesized a library of CCK-A isoform-selective antagonists and tested them in various colon and pancreatic cancer preclinical models. The lead CCK-A isoform, selective antagonist PNB-028, bound to CCK-A at 12 nM with a 60-fold selectivity towards CCK-A over CCK-B. Furthermore, it inhibited the proliferation of CCK-A-expressing colon and pancreatic cancer cells without affecting the proliferation of non-cancerous cells. PNB-028 was also extremely effective in inhibiting the growth of MAC-16 and LoVo colon cancer and MIA PaCa pancreatic cancer xenografts in immune-compromised mice. Genomewide microarray and kinase-array studies indicate that PNB-028 inhibited oncogenic kinases and angiogenic factors to inhibit the growth of colon cancer xenografts. Safety pharmacology and toxicology studies have indicated that PNB-028 is extremely safe and has a wide safety margin. These studies suggest that targeting CCK-A selectively renders promise to treat colon and pancreatic cancers and that PNB-028 could become the next-generation treatment option

A dual fluorescence protein expression system detects cell cycle dependent protein noise

Inherently identical cells exhibit significant phenotypic variation. It can be essential for many biological processes and is known to arise from stochastic, ‘noisy’, gene expression that is determined by intrinsic and extrinsic components. It is now obvious that the noise varies as a function of inducer concentration. However, its fluctuation over the cell cycle is limited. Applying dual colour fluorescence protein reporter system, Cyan Fluorescent Protein (CFP) and Yellow fluorescent protein (YFP) tagged multi-copy plasmids, we determine variation of the noise components over the phases in lac promoter induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG) and in presence of additional Magnesium, Mg2+ ion. We, also, estimate the how such system deviates from observations of single-copy plasmid. Found 25 % difference between multi-copy system and single-copy system clarifies that observed noise is considerable and estimates population behaviour during the cell cycle. We show that total variation in cells induced with IPTG is determined by higher extrinsic than intrinsic noise. It increases from Lag to Exponential phase and decreases from Retardation to Stationary phase. By observing slow and fast dividing cells, we show that 5 mM Mg2+ increases population homogeneity compared to 2.5 mM Mg2+ in the environment. The experimental data obtained using dual colour fluorescence protein reporter system demonstrates that protein expression noise, depending on intra cellular ionic concentration, is tightly controlled by phase of the cell.Peer reviewe

Marine halophyte derived polyphenols inhibit glioma cell growth through mitogen-activated protein kinase signaling pathway

Plants that are pharmacologically significant require intensive phytochemical characterization for bioactive profiling of the compounds, which has enabled their safe use in ayurvedic medicine. The present study is focused on the phytochemical analyses, quantitative estimation and profiling of secondary metabolites of leaf extract, as well as the antioxidant and cytotoxic activity of the potent halophytes such as Avicennia marina, Ceriops tagal, Ipomoea pes-caprae, and Sonneratia apetala. The in vitro antioxidant property was investigated using DPPH, ferric reducing antioxidant capacity (FRAP) assay. Bioactive compounds such as phenols, flavonoids, saponin and alkaloids were quantitatively estimated from the extracts of A.marina, C.tagal, I.pes-capra and S.apetala, which possessed higher phenol content than the other studied halophytes. The extracts at 200 µg/ml revealed higher antioxidant activity than the standard ascorbic acid and it functions as a powerful oxygen free radical scavenger with 77.37%, 75.35% and 72.84% for S.apetala, I.pes-caprae and C.tagal respectively and with least IC50 for I.pes-caprae (11.95 µg/ml) followed by C.tagal (49.94 µg/ml). Cell viability and anti-proliferative activity of different polyphenolic fractions of C.tagal (CT1 and CT2) and I.pes-caprae fraction (IP) against LN229, SNB19 revealed Ipomoea as the promising anti-cytotoxic fraction. IP-derived polyphenols was further subjected to apoptosis, migration assay, ROS and caspase − 3 and − 7 to elucidate its potentiality as a therapeutic drug. IP-polyphenols was found to have higher percentage of inhibition than the CT1 and CT2 polyphenols of C.tagal on comparison with TMZ. All the above-mentioned in-vitro analysis further validated the ability of IP-polyphenols inducing cell death via ROS-mediated caspase dependent pathway. Further, proteomic and phospho-proteomic analysis revealed the potential role of IP-polyphenols in the regulation of cell proliferation through MMK3, p53, p70 S6 kinase and RSK1 proteins involved in mitogen-activated protein kinase signaling pathway. Our analysis confirmed the promising role of I.pes-caprae derived polyphenols as an anti-metastatic compound against GBM cells.publishedVersionPeer reviewe

Advances in the lung cancer immunotherapy approaches

Despite the progress in the comprehension of LC progression, risk, immunologic control, and treatment choices, it is still the primary cause of cancer-related death. LC cells possess a very low and heterogeneous antigenicity, which allows them to passively evade the anticancer defense of the immune system by educating cytotoxic lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), regulatory T cells (Treg), immune checkpoint inhibitors (ICIs), and myeloid-derived suppressor cells (MDSCs). Though ICIs are an important candidate in first-line therapy, consolidation therapy, adjuvant therapy, and other combination therapies involving traditional therapies, the need for new predictive immunotherapy biomarkers remains. Furthermore, ICI-induced resistance after an initial response makes it vital to seek and exploit new targets to benefit greatly from immunotherapy. As ICIs, tumor mutation burden (TMB), and microsatellite instability (MSI) are not ideal LC predictive markers, a multi-parameter analysis of the immune system considering tumor, stroma, and beyond can be the future-oriented predictive marker. The optimal patient selection with a proper adjuvant agent in immunotherapy approaches needs to be still revised. Here, we summarize advances in LC immunotherapy approaches with their clinical and preclinical trials considering cancer models and vaccines and the potential of employing immunology to predict immunotherapy effectiveness in cancer patients and address the viewpoints on future directions. We conclude that the field of lung cancer therapeutics can benefit from the use of combination strategies but with comprehension of their limitations and improvements

Bias-force guided simulations combined with experimental validations towards GPR17 modulators identification

Glioblastoma Multiforme (GBM) is known to be by far the most aggressive brain tumor to affect adults. The median survival rate of GBM patient's is < 15 months, while the GBM cells aggressively develop resistance to chemo- and radiotherapy with their self-renewal capacity which suggests the pressing need to develop novel preventative measures. We have recently proved that GPR17 —an orphan G protein-coupled receptor— is highly expressed on the GBM cell surface and it has a vital role to play in the disease progression. Despite the progress made on GBM downregulation, there still remain difficulties in developing a promising modulator for GPR17, till date. Here, we have performed robust virtual screening combined with biased-force pulling molecular dynamic (MD) simulations to predict high-affinity GPR17 modulators followed by experimental validation. Initially, the database containing 1379 FDA-approved drugs were screened against the orthosteric binding pocket of the GPR17. The external bias-potentials were then applied to the screened hits during the MD simulations which enabled to predict a spectrum of rupture peak force values that were used to select four approved drugs –ZINC000003792417 (Sacubitril), ZINC000014210457 (Victrelis), ZINC000001536109 (Pralatrexate) and ZINC000003925861 (Vorapaxar)– as top hits. The hits selected turns out to demonstrate unique dissociation pathways, interaction pattern, and change in polar network over time. Subsequently the selected hits with GPR17 were measured by inhibiting the forskolin-stimulated cAMP accumulation in GBM cell lines, LN229 and SNB19. The ex vivo validations shows that Sacubitril drug can act as a full agonist, while Vorapaxar functions as a partial agonist for GPR17. The pEC50 of Sacubitril was identified as 4.841 and 4.661 for LN229 and SNB19, respectively. Small interference of the RNA (siRNA)– silenced the GPR17 to further validate the targeted binding of Sacubitril with GPR17. In the current investigation, we have identified new repurposable GPR17 specific drugs which are likely to increase the opportunity to treat orphan deadly diseases.publishedVersionPeer reviewe

P2Y1 agonist HIC in combination with androgen receptor inhibitor abiraterone acetate impairs cell growth of prostate cancer

P2Y receptors belong to the large superfamily of G-protein-coupled receptors and play a crucial role in cell death and survival. P2Y1 receptor has been identified as a marker for prostate cancer (PCa). A previously unveiled selective P2Y1 receptor agonist, the indoline-derived HIC (1-(1-((2-hydroxy-5-nitrophenyl)(4-hydroxyphenyl)methyl)indoline-4-carbonitrile), induces a series of molecular and biological responses in PCa cells PC3 and DU145, but minimal toxicity to normal cells. Here, we evaluated the combinatorial effect of HIC with abiraterone acetate (AA) targeted on androgen receptor (AR) on the inhibition of PCa cells. Here, the presence of HIC and AA significantly inhibited cell proliferation of PC3 and DU145 cells with time-dependent manner as a synerfistic combination. Moreover, it was also shown that the anticancer and antimetastasis effects of the combinratorial drugs were noticed through a decrease in colony-forming ability, cell migration, and cell invasion. In addition, the HIC + AA induced apoptotic population of PCa cells as well as cell cycle arrest in G1 progression phase. In summary, these studies show that the combination of P2Y1 receptor agonist, HIC and AR inhibitor, AA, effectively improved the antitumor activity of each drug. Thus, the combinatorial model of HIC and AA should be a novel and promising therapeutic strategy for treating prostate cancer.publishedVersionPeer reviewe

Benzenesulfonamide Analogs : Synthesis, Anti-GBM Activity and Pharmacoprofiling

The tropomyosin receptor kinase A (TrkA) family of receptor tyrosine kinases (RTKs) emerge as a potential target for glioblastoma (GBM) treatment. Benzenesulfonamide analogs were identified as kinase inhibitors possessing promising anticancer properties. In the present work, four known and two novel benzenesulfonamide derivatives were synthesized, and their inhibitory activities in TrkA overexpressing cells, U87 and MEF cells were investigated. The cytotoxic effect of benzenesulfonamide derivatives and cisplatin was determined using trypan blue exclusion assays. The mode of interaction of benzenesulfonamides with TrkA was predicted by docking and structural analysis. ADMET profiling was also performed for all compounds to calculate the drug likeness property. Appropriate QSAR models were developed for studying structure–activity relationships. Compound 4-[2-(4,4-dimethyl-2,6-dioxocyclohexylidene)hydrazinyl]-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzenesulfon-amide (AL106) and 4-[2-(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)hydrazinyl]-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzenesulfonamide (AL107) showed acceptable binding energies with the active sites for human nerve growth factor receptor, TrkA. Here, AL106 was identified as a potential anti-GBM compound, with an IC50 value of 58.6 µM with a less toxic effect in non-cancerous cells than the known chemotherapeutic agent, cisplatin. In silico analysis indicated that AL106 formed prominent stabilizing hydrophobic interactions with Tyr359, Ser371, Ile374 and charged interactions with Gln369 of TrkA. Furthermore, in silico analysis of all benzenesulfonamide derivatives revealed that AL106 has good pharmacokinetics properties, drug likeness and toxicity profiles, suggesting the compound may be suitable for clinical trial. Thus, benzenesulfonamide analog, AL106 could potentially induce GBM cell death through its interaction with TrkA and might be an attractive strategy for developing a drug targeted therapy to treat glioblastoma.Peer reviewe

Pharmacologic activation of estrogen receptor α increases mitochondrial function, energy expenditure, and brown adipose tissue

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154285/1/fsb2fj201600787rr.pd

Methanodibenzo[b,f][1,5]dioxocins as Novel Glutaminase Inhibitor with Anti-Glioblastoma Potential

Glutamine metabolism is an important hallmark of several cancers with demonstrated antitumor activity in glioblastoma cancer cells (GBM). GBM cells regulate glutamine and use it as a major energy source for their proliferation through the glutaminolysis process. Enzymes, such as glutaminase in glutaminolysis, can be targeted by small-molecule inhibitors, thus exhibiting promising anticancer properties. The resistance to glutaminolysis demands the development of new therapeutic molecules to overcome drug resistance. Herein, we have reported a novel library of constrained methanodibenzo[b,f][1,5]dioxocin derivatives as glutaminase (GLS) inhibitors and their anti-GBM potential. The library consisting of seven molecules was obtained through self-condensation of 2′-hydroxyacetophenones, out of which three molecules, namely compounds 3, 5, and 6, were identified with higher binding energy values ranging between −10.2 and −9.8 kcal/mol with GLS (PDB ID; 4O7D). Pharmacological validation of these compounds also showed a higher growth inhibition effect in GBM cells than the standard drug temozolomide (TMZ). The most promising compound, 6, obeyed Lipinski’s rule of five and was identified to interact with key residues Arg307, Asp326, Lys328, Lys399, and Glu403 of GLS. This compound exhibited the best cytotoxic effect with IC50 values of 63 µM and 83 µM in LN229 and SNB19 cells, respectively. The potential activation of GLS by the best-constrained dibenzo[b,f][1,5]dioxocin in the tested series increased apoptosis via reactive oxygen species production in both GBM cells, and exhibited anti-migratory and anti-proliferative properties over time in both cell lines. Our results highlight the activation mechanism of a dibenzo[b,f][1,5]dioxocin from the structural basis and demonstrate that inhibition of glutaminolysis may facilitate the pharmacological intervention for GBM treatment.publishedVersionPeer reviewe