Using human Pompe disease-induced pluripotent stem cell-derived neural cells to identify compounds with therapeutic potential.

Pompe disease (OMIM # 232300) is a glycogen storage disease caused by autosomal recessive mutations of the gene encoding alpha-1,4-glucosidase (GAA; EC 3.2.1.20). Despite the relatively effective employment of enzyme replacement therapy, some critical medical issues still exist in patients with this disease, including the persistence of abnormalities in the central nervous system (CNS), probably because of the inability of the recombinant GAA to pass through the blood-brain barrier. To address this issue, identification of more therapeutic agents that target the CNS of patients with Pompe disease may be required. In this study, we derived neuronal cells from Pompe disease-induced pluripotent stem cells (Pom-iPSCs) and proved that they are able to recapitulate the hallmark cellular and biochemical phenotypes of Pompe disease. Using the Pom-iPSC-derived neurons as an in vitro drug-testing model, we then identified three compounds, ebselen, wortmannin and PX-866, with therapeutic potential to alleviate Pompe disease-associated pathological phenotypes in the neurons derived from Pom-iPSCs. We confirmed that all three compounds were able to enhance the GAA activity in the Pom-iPSC-derived neurons. Moreover, they were able to enhance the GAA activity in several important internal organs of GAA-deficient mice when co-injected with recombinant human GAA, and we found that intraperitoneal injection of ebselen was able to promote the GAA activity of the GAA-heterozygous mouse brain. Our results prove the usefulness of Pom-iPSC-derived neuronal populations for identifying new compounds with therapeutic potential.

Inhibition of phosphatidylinositol 3-kinase by PX-866 suppresses temozolomide-induced autophagy and promotes apoptosis in glioblastoma cells.

BACKGROUND: Temozolomide (TMZ) is the most commonly used chemotherapeutic agent used to treat glioblastoma (GBM), which causes significant DNA damage to highly proliferative cells. Our observations have added to accumulating evidence that TMZ induces stress-responsive cellular programs known to promote cell survival, including autophagy. As such, targeting these survival pathways may represent new vulnerabilities of GBM after treatment with TMZ. METHODS: Using the T98G human glioma cell line, we assessed the molecular signaling associated with TMZ treatment, the cellular consequences of using the pan-PI3K inhibitor PX-866, and performed clonogenic assays to determine the effect sequential treatment of TMZ and PX-866 had on colony formation. Additionally, we also use subcutaneous GBM patient derived xenograft (PDX) tumors to show relative LC3 protein expression and correlations between survival pathways and molecular markers which dictate clinical responsiveness to TMZ. RESULTS: Here, we report that TMZ can induce autophagic flux in T98G glioma cells. GBM patient-derived xenograft (PDX) tumors treated with TMZ also display an increase in the autophagosome marker LC3 II. Additionally, O6-methylguanine-DNA-methyltransferase (MGMT) expression correlates with PI3K/AKT activity, suggesting that patients with inherent resistance to TMZ (MGMT-high) would benefit from PI3K/AKT inhibitors in addition to TMZ. Accordingly, we have identified that the blood-brain barrier (BBB) penetrant pan-PI3K inhibitor, PX-866, is an early-stage inhibitor of autophagic flux, while maintaining its ability to inhibit PI3K/AKT signaling in glioma cells. Lastly, due to the induction of autophagic flux by TMZ, we provide evidence for sequential treatment of TMZ followed by PX-866, rather than combined co-treatment, as a means to shut down autophagy-induced survival in GBM cells and to enhance apoptosis. CONCLUSIONS: The understanding of how TMZ induces survival pathways, such as autophagy, may offer new therapeutic vulnerabilities and opportunities to use sequential inhibition of alternate pro-survival pathways that regulate autophagy. As such, identification of additional ways to inhibit TMZ-induced autophagy could enhance the efficacy of TMZ.

Down-regulation of hTERT and Cyclin D1 transcription via PI3K/Akt and TGF-ß pathways in MCF-7 Cancer cells with PX-866 and Raloxifene.

Human telomerase reverse transcriptase (hTERT) is the catalytic and limiting component of telomerase and also a transcription factor. It is critical to the integrity of the ends of linear chromosomes and to the regulation, extent and rate of cell cycle progression in multicellular eukaryotes. The level of hTERT expression is essential to a wide range of bodily functions and to avoidance of disease conditions, such as cancer, that are mediated in part by aberrant level and regulation of cell cycle proliferation. Value of a gene in regulation depends on its ability to both receive input from multiple sources and transmit signals to multiple effectors. The expression of hTERT and the progression of the cell cycle have been shown to be regulated by an extensive network of gene products and signaling pathways, including the PI3K/Akt and TGF-ß pathways. The PI3K inhibitor PX-866 and the competitive estrogen receptor ligand raloxifene have been shown to modify progression of those pathways and, in combination, to decrease proliferation of estrogen receptor positive (ER+) MCF-7 breast cancer cells. We found that combinations of modulators of those pathways decreased not only hTERT transcription but also transcription of additional essential cell cycle regulators such as Cyclin D1. By evaluating known expression profile signatures for TGF-ß pathway diversions, we confirmed additional genes such as heparin-binding epidermal growth factor-like growth factor (HB EGF) by which those pathways and their perturbations may also modify cell cycle progression.

Combinatorial PX-866 and Raloxifene Decrease Rb Phosphorylation, Cyclin E2 Transcription, and Proliferation of MCF-7 Breast Cancer Cells.

As a potential means to reduce proliferation of breast cancer cells, a multiple-pathway approach with no effect on control cells was explored. The human interactome being constructed by the Center for Cancer Systems Biology will prove indispensable to understanding composite effects of multiple pathways, but its discovered protein-protein interactions require characterization. Accordingly, we explored the effects of regulators of one protein on downstream targets of the other protein. MCF-7 estrogen receptor-positive (ER+) breast cancer cells were treated with raloxifene to upregulate the TGF-ß pathway and PX-866 to down-regulate the PI3K/Akt pathway. This resulted in highly significant downstream reduction of cell cycle proliferation in breast cancer cells with no significant proliferation reduction following similar treatment of noncancerous MCF10A breast epithelial cells. Reduced phosphorylation of p107 and substantial reduction of Rb phosphorylation were observed in response. The effects of reduced Rb and p107 phosphorylation were reflected in significant decline in E2F-1 transcriptional activity, which is dependent on pocket protein phosphorylation status. The reduced proliferation was related to decreased expression of cyclins, including E2F-1-regulated Cyclin E2, which was also in response to raloxifene and PX-866. All combinations of raloxifene and PX-866 produced significant or highly significant results for reduced MCF-7 cell proliferation, reduced Cyclin E2 transcription, and reduced Rb phosphorylation. These studies demonstrated that uncontrolled proliferation of ER+ breast cancer cells can be significantly reduced by combinational targeting of two relevant pathways. J. Cell. Biochem. 117: 1688-1696, 2016. © 2015 Wiley Periodicals, Inc.

Luteoprotective roles of luteinizing hormone are mediated by not only progesterone production but also glucocorticoid conversion in bovine corpus luteum.

Luteinizing hormone (LH) is known as a key regulator of corpus luteum (CL) function, but the luteoprotective mechanisms of LH in the maintenance of bovine CL function are not well understood. The current study investigated if LH increases cell viability and induces cortisol conversion, and if the luteoprotective action of LH is mediated by stimulating the local production and action of progesterone (P4) and/or cortisol. Cultured bovine luteal cells obtained at the mid-luteal stage (Days 8-12 of the estrous cycle) were treated for 24 hr with LH (10 ng/ml) with/without onapristone (OP, a specific P4 receptor antagonist; 100 µM), cortisone (1 µM), and aminoglutethimide (AGT, a specific inhibitor of cytochrome P450 side-chain cleavage; 100 µM). LH with and without OP significantly increased the mRNA and protein expressions of 11ß-hydroxysteroid dehydrogenase (HSD11B) 1, but did not affect the mRNA or protein expression of HSD11B2. These treatments also significantly increased HSD11B1 activity. Cell viability was significantly increased by LH alone or by LH in combination with cortisone and OP. LH in combination with OP or AGT significantly decreased cell viability as compared to LH alone. The overall results suggest that LH stimulates not only P4 production but also HSD11B1 expression, thereby increasing the cortisol concentration in the bovine CL, and that LH prevents cell death through these survival pathways. LH may consequently support CL function during the luteal phase in cattle.

Roles of cytokines and progesterone in the regulation of the nitric oxide generating system in bovine luteal endothelial cells.

Nitric oxide (NO) produced by luteal endothelial cells (LECs) plays important roles in regulating corpus luteum (CL) function, yet the local mechanism regulating NO generation in bovine CL remains unclear. The purpose of the present study was to elucidate if tumor necrosis factor-α (TNF), interferon γ (IFNG), and/or progesterone (P4) play roles in regulating NO generating system in LECs. Cultured bovine LECs obtained from the CL at the mid-luteal stage (Days 8-12 of the cycle) were treated for 24 hr with TNF (2.9 nM), IFNG (2.5 nM), or P4 (0.032-32 µM). NO production was increased by TNF and IFNG, but decreased by P4 (P < 0.05). TNF and IFNG stimulated the relative steady-state amounts of inducible nitric oxide synthase (iNOS) mRNA and iNOS protein expression (P < 0.05), whereas P4 inhibited relative steady-state amounts of iNOS mRNA and iNOS protein expression (P < 0.05). In contrast, endothelial nitric oxide synthase (eNOS) expression was not affected by any treatment. TNF and IFNG stimulated NOS activity (P < 0.05) and 1400W, a specific inhibitor of iNOS, reduced NO production stimulated by TNF and IFNG in LECs (P < 0.05). Onapristone, a specific P4 receptor antagonist, blocked the inhibitory effect of P4 on NO production in LECs (P < 0.05). The overall findings suggest that TNF and IFNG accelerate luteolysis by increasing NO production via stimulation of iNOS expression and NOS activity in bovine LECs. P4, on the other hand, may act in maintaining CL function by suppressing iNOS expression in bovine LECs.

Inhibition of PI3K by PX-866 prevents transforming growth factor-alpha-induced pulmonary fibrosis.

Transforming growth factor-alpha (TGFalpha) is a ligand for the epidermal growth factor receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. EGFR signaling activates several intracellular signaling pathways including phosphatidylinositol 3'-kinase (PI3K). We previously showed that induction of lung-specific TGFalpha expression in transgenic mice caused progressive pulmonary fibrosis over a 4-week period. The increase in levels of phosphorylated Akt, detected after 1 day of doxycycline-induced TGFalpha expression, was blocked by treatment with the PI3K inhibitor, PX-866. Daily administration of PX-866 during TGFalpha induction prevented increases in lung collagen and airway resistance as well as decreases in lung compliance. Treatment of mice with oral PX-866 4 weeks after the induction of TGFalpha prevented additional weight loss and further increases in total collagen, and attenuated changes in pulmonary mechanics. These data show that PI3K is activated in TGFalpha/EGFR-mediated pulmonary fibrosis and support further studies to determine the role of PI3K activation in human lung fibrotic disease, which could be amenable to targeted therapy.

[Subcellular proteomic analysis of Tetrazanbigen on human hepatocellular carcinoma cell line QGY-7701].

OBJECTIVE: To investigate the clinical efficacy of cryotherapy ablation treatment for advanced hepatocellular carcinoma, and to analyse the predictive factors of cryotherapy ablation treatment. METHODS: 190 patients of hepatitis B-related advanced HCC from 2005 to 2008 in our hospital underwent curative cryoablation. We used clinical cohort method to analyze cryoablation group (147 cases) and control group (43 cases). The median OS (over survival time) and TTP (time to disease progression) were compared. We also evaluated the clinical significance of age, gender, location of portal vein tumor thrombus, HBeAg, tumor histological grade, Child-Pugh classification, end-stage liver disease (MELD) score, advanced liver cancer prediction system (ALCPS) score and the Eastern Cooperative Oncology Group performance status (ECOG PS) score for predicting the efficacy of cryoablation. Two Groups were compared with the x² test. Survival rates were estimated by the Kaplan-Meier method and compared by the log rank test. The Cox proportional hazards model was used to determine the independent factors on survival based on the variables selected in univariate analysis. RESULTS: Median survival time of cryoablation group and Control group were 7.5 (4.2 to 14.6) months and 3.2 (1.2 to 8.6) months, median TTP were 3.5 (2.5 to 4.5) months and 1.5 (1.0 to 3.5 months), the differences between were statistically significant (P < 0.05). Median OS and TTP of advanced HCC patients who had Well-differentiated tumor, Child-pugh A-class and low score of MELD score, ALCPS score; ECOG PS score were significantly longer than that of the poorly differentiated tumor, Child-pugh B-class and the high those scores (P < 0.05). ECOG PS (P less than 0.05, 95% CI 1.074 - 2.143) and ALCPS (P < 0.05, 95% CI 1.005-2.121) were independent predictors for OS of advanced HCC. CONCLUSIONS: Cryoablation treatment can prolong median OS and TTP of advanced HCC. ECOG PS and ALCPS are important predictors for survival time of advanced HCC.

Tetrazanbigen Derivatives as Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) Partial Agonists: Design, Synthesis, Structure-Activity Relationship, and Anticancer Activities.

Tetrazanbigen (TNBG) is a novel sterol isoquinoline derivative with poor water solubility and moderate inhibitory effects on human cancer cell lines via lipoapoptosis induction. Herein, we developed a series of novel TNBG analogues with improved water solubility and antiproliferative activities. The CCK-8 assay enabled us to identify a novel compound, 14g, which strongly inhibited HepG2 and A549 cell growth with IC50 values of 0.54 and 0.47 µM, respectively. The anticancer effects might be explained by the partial activation and upregulation of PPARγ expression, as indicated by the transactivation assay and western blotting evaluation. Furthermore, the in vitro antiproliferative activity was verified in an in vivo xenograft model in which 14g strongly reduced tumor growth at a dose of 10 mg/kg. In line with these positive observations, 14g exhibited an excellent water solubility of 31.4 mg/mL, which was more than 1000-fold higher than that of TNBG (4 µg/mL). Together, these results suggest that 14g is a promising anticancer therapeutic that deserves further investigation.

Peroxisome proliferator-activated receptor gamma agonist pioglitazone prevents the hyperglycemia caused by phosphatidylinositol 3-kinase pathway inhibition by PX-866 without affecting antitumor activity.

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascade is an important component of the insulin signaling in normal tissues leading to glucose uptake and homeostasis and for cell survival signaling in cancer cells. Hyperglycemia is an on-target side effect of many inhibitors of PI3K/Akt signaling including the specific PI3K inhibitor PX-866. The peroxisome proliferator-activated receptor gamma agonist pioglitazone, used to treat type 2 diabetes, prevents a decrease in glucose tolerance caused by acute administration of PX-866. Our studies have shown that pioglitazone does not inhibit the antitumor activity of PX-866 in A-549 non-small cell lung cancer and HT-29 colon cancer xenografts. In vitro studies also showed that pioglitazone increases 2-[1-(14)C]deoxy-D-glucose uptake in L-6 muscle cells and prevents inhibition of 2-deoxyglucose uptake by PX-866. Neither pioglitazone nor PX-866 had an effect on 2-deoxyglucose uptake in A-549 lung cancer cells. In vivo imaging studies using [18F]2-deoxyglucose (FDG) positron emission tomography showed that pioglitazone increases FDG accumulation by normal tissue but does not significantly alter FDG uptake by A-549 xenografts. Thus, peroxisome proliferator-activated receptor gamma agonists may be useful in overcoming the increase in blood glucose caused by inhibitors of PI3K signaling by preventing the inhibition of normal tissue insulin-mediated glucose uptake without affecting antitumor activity.

Progesterone receptor promotes degradation of STAT2 to inhibit the interferon response in breast cancer.

Type I (IFNα/ß) interferon signaling represents a critical transduction pathway involved in recognition and destruction of nascent tumor cells. Downregulation of this pathway to promote a more immunosuppressed microenvironment contributes to the ability of tumor cells to evade the immune system, a known Hallmark of Cancer. The present study investigates the progesterone receptor (PR), which is expressed in the vast majority of breast cancers, and its ability to inhibit efficient interferon signaling in tumor cells. We have shown that PR can block the interferon signaling cascade by promoting ubiquitination and degradation of STAT2. Targeting STAT2 is critical, as we show that it is an essential protein in inducing transcription of interferon-stimulated genes (ISG); shRNA-mediated knockdown of STAT2 severely abrogates the interferon response in vitro. Importantly, we were able to reverse this inhibition by treating with onapristone, an anti-progestin currently being investigated in breast cancer clinical trials. Additionally, we have found that an interferon-related gene signature (composed of ISGs) is inversely correlated with PR expression in human tumors. We speculate that PR inhibition of interferon signaling may contribute to creating an immunosuppressed microenvironment and reversal of this through anti-progestins may present a novel therapeutic target to promote immune activity within the tumor.

Differential expression of calreticulin, a reticuloplasmin in primate endometrium.

BACKGROUND: To our knowledge, there are no data on hormonal regulation of reticuloplasmins in primate endometrium. We report the presence and modulation of expression of three reticuloplasmins in endometrium of bonnet monkeys (Macaca radiata). METHODS: Receptive and non-receptive endometria obtained from vehicle-treated control and onapristone (antiprogestin)-treated animals, respectively, were compared for differentially expressed proteins by two-dimensional proteomics. Mass spectrometric analysis annotated two such proteins as calreticulin and protein disulfide-isomerase (PDI), known to be molecular chaperones in endoplasmic reticulum. We then investigated if endoplasmin, another reticuloplasmin is also differentially expressed. Expression of these reticuloplasmins was also investigated in the endometriuma during pregnancy in bonnet monkeys. Samples were analysed by immunohistochemistry and western blot (calreticulin in human endometrium), and calreticulin transcript levels in Ishikawa cell line were assessed by real time PCR. RESULTS: Immunohistochemical analysis of the functionalis region of non-receptive endometria in monkeys revealed higher expression of (i) calreticulin (P < 0.01) in glandular epithelium and (ii) PDI in stroma (P < 0.0001), but no change in endoplasmin in stroma or glands, compared with receptive endometria. Protein level of all three reticuloplasmins in the stromal region of endometrial functionalis was higher in pregnant than non-pregnant animals (P < 0.05). Human endometrial calreticulin protein was higher in the estrogen-dominant (proliferative) phase than progesterone-dominant (mid-secretory) phase of the cycle. Calreticulin mRNA in Ishikawa cells is up-regulated by estrogen (P < 0.05 versus control), with a trend towards down-regulation by progesterone. CONCLUSION: Our data suggest that endometrial reticuloplasmins are regulated by hormones and embryonic stimuli in a cell-type specific manner. These novel data open up new lines of investigation for elucidating the mechanisms by which hormones or embryonic stimuli influence the sub-cellular physiology of endometrium.

The prostate growth stimulation by progesterone is due to androgenic products and progesterone receptor-mediated mechanisms.

The antiprogestin mifepristone has been demonstrated to inhibit the growth of R3327HI rat prostatic carcinoma. A comparable antitumor effect of onapristone (ON) on rat Dunning tumors was found in our laboratories. We found the localization of progesterone (P4) receptors (PR) in prostate and prostatic tumors. These findings suggest the involvement of P4 in the mechanism of hormone-dependent growth of prostate and tumors. To study the influence of P4 on the growth of ventral (VP) and dorsolateral prostate (DLP), orchiectomized rats were treated (s.c.) daily with P4 (0.3, 1.0, 3.0 or 10.0 mg), dihydrotestosterone (DHT, 0.05 mg), estradiol (E2, 3.0 microg), ON (3.0 mg), ICI 182780 (1.0 mg) or flutamide (FL, 3.0 mg) for 12 days. One day after the last treatment, animals were sacrificed, and the organ weight of VP and DLP was determined. P4 increased the organ weight of VP and DLP in a dose-dependent manner. In contrast to DHT, which preferentially stimulated the growth of VP, P4 led rather to an increase in the weight of DLP. The effect of P4 on the DLP was enhanced by a simultaneous application of DHT or E2. The antiprogestin ON and the pure antiestrogen ICI 182780 had no appreciable effect on the P4-induced growth of VP and DLP. ON inhibited, however, the E2/P4-induced growth of DLP without affecting the growth of the VP. In contrast the antiandrogen FL suppressed the stimulatory effect of P4 on both the VP and DLP. These findings suggest that the stimulatory effect of P4 on the rat DLP may be partly due to androgenic products derived from P4 and may be also mediated by PR.

Effects of tetrazanbigen on the protein expression in human hepatocellular carcinoma cell line QGY-7701.

Tetrazanbigen (TNBG) is a novel synthetic antitumor drug with significant antitumor effects on common solid tumors in vitro and in vivo. It may lead to death of cancer cells through a tumor-associated lipoidosis mechanism, and result in lipid droplets (LDs) accumulation at the cytoplasm. In this study, the effects of TNBG on protein expression in human hepatocellular carcinoma cell line QGY-7701 were studied for elucidating its antitumor mechanism. The proteins extracted from TNBG-treated human hepatocellular carcinoma cell line QGY-7701 were analyzed and compared with control cells by two-dimensional gel electrophoresis. The differential proteins were identified by matrix-associated laser desorption ionization time-of-flight mass (MALDI-TOF-MS) spectrometry. Two proteins of interest, the levels of which were significantly increased in TNBG-treated cells, were further characterized by Western blot analysis. The results showed a total of 846+/-23 spots in control cells and 853+/-30 spots in TNBG-treated cells. Twenty-six up-regulated or down-regulated proteins were found by analyzing differential proteomic 2-DE map. Eleven of them were identified by mass spectrometry. They were protein disulfide-isomerase precursor, 94 kD glucose-regulated protein, heat shock protein (HSP) 90-alpha, ATP-citrate lyase, HMG-CoA reductase, glucose-6-phosphate 1-dehydrogenase, very-long-chain specific acyl-CoA dehydrogenase, squalene synthetase, sterol regulatory element-binding protein 1, fructose-bisphosphate aldolase A, and peroxiredoxin-1. These up-regulated or down-regulated proteins are mostly related to lipid metabolism. The TNBG antitumor mechanism is probably to influence tumor lipid metabolism, resulting in accumulation of LDs in tumor cells.

A Phase II Study of PX-866 in Patients With Recurrent or Metastatic Castration-resistant Prostate Cancer: Canadian Cancer Trials Group Study IND205.

BACKGROUND: In PTEN-loss models, the phosphatidylinositol 3-kinase (PI3K)/AKT and androgen receptor signaling pathways cross-regulate by reciprocal feedback whereby inhibition of one activates the other, creating a rationale for co-targeting. We studied the irreversible, pan-isoform inhibitor of Class I PI-3K PX-866 singly (part A) and with abiraterone acetate (AA) in patients on AA with rising prostate-specific antigen (PSA) (part B). PATIENTS AND METHODS: The primary endpoint was lack of progression at 12 weeks. Exploratory endpoints included changes in circulating tumor cells (CTC), pharmacodynamic studies on platelets (part A), and archival tumor exploration of PTEN as predictor of response (part B). RESULTS: A total of 43 and 25 patients accrued to parts A and B, respectively. In part A, 14 (33%) patients were progression-free at 12 weeks, with 2 partial objective responses and 1 confirmed PSA response. Favorable CTC conversion (< 5 CTC/7.5 mL) occurred in 6 (24%) of 25 evaluable patients. In part B, 11 of 25 patients had measurable disease. Six (24%) patients were progression-free at 12 weeks. No objective or PSA responses were observed. For all 68 patients, the most common toxicities were diarrhea (53 patients), nausea (36), anorexia (24), fatigue (22), and vomiting (20). Among 17 patients for whom PTEN testing was possible, 3 had PTEN homozygous deletion and 14 had no change. No correlation between PTEN status and response was seen. CONCLUSIONS: PX-866 had modest single agent activity. Adding AA to PX-866 showed no evidence of resistance reversal. Strategies to combine PI3K inhibition with androgen receptor-targeted therapies could consider initiation earlier, combination with other agents, and/or recruiting a selected population.

TNBG-5602, a novel derivative of quinoxaline, inhibits liver cancer growth via upregulating peroxisome proliferator-activated receptor γ in vitro and in vivo.

OBJECTIVES: TNBG-5602 is a newly synthesized compound with an isoquinoline structure. In the present study, we demonstrated the anticancer effect of TNBG-5602 in in-vitro and in-vivo models and investigated its possible anticancer mechanism. METHODS: The antiproliferation effect of TNBG-5602 in vitro was evaluated in human liver cancer cell line QGY-7701. The acute toxicity of TNBG-5602 was evaluated in mice. The anticancer activity of TNBG-5602 in vivo was assessed in a xenograft model of human liver cancer cell line QGY-7701. KEY FINDINGS: The results of CCK-8 assay showed that TNBG-5602 can effectively inhibit the proliferation of liver cancer cells in vitro. The acute toxicity test in mice showed that the LD50 of TNBG-5602 was 172 mg/kg. In a xenograft liver cancer model, TNBG-5602 could remarkably inhibit the growth of tumours. During in-vitro and in-vivo studies, we noted that TNBG-5602 could induce lipid accumulation in cancer cells and tissues. Further study indicated that the anticancer effect of TNBG-5602 may be exerted through activating peroxisome proliferator-activated receptor γ (PPARγ) and downregulating proliferating cell nuclear antigen (PCNA). CONCLUSIONS: Our results suggested that TNBG-5602 might exert potent anticancer activity through increasing the expression of PPARγ.

Targeted disruption of PI3K/Akt/mTOR signaling pathway, via PI3K inhibitors, promotes growth inhibitory effects in oral cancer cells.

PURPOSE: The phosphoinositide-3-kinase (PI3K) pathway is the frequently altered in human cancer. This has led to the development and study of novel PI3K inhibitors for targeted therapy and also to overcome resistance to radiotherapy. METHOD: The anti-tumour effects of PI3K inhibitors (PI-828, PI-103 and PX-866) in terms of cell proliferation, colony formation, induction of apoptosis, cell cycle arrest, invasion, autophagy, and pNF-κB/p65 translocation in SCC-4, SCC-9 and SCC-25 cells were studied by performing MTT, clonogenic, DAPI staining, propidium iodide staining, annexin-V binding, matrigel invasion, acridine orange staining and immuno-fluorescence assay. Western blot assay was performed to assess the alteration in the expression of various proteins. RESULT: PI-828 and PI-103 treatment exhibited dose-dependent inhibition of growth and proliferation of OSCC cells with a concomitant induction of apoptosis, altered cell cycle regulation and decreased invasiveness (p < 0.01). PX-866 induced apoptosis, cell cycle arrest, autophagy and a significant decrease in the invasiveness of oral cancer cells as compared to untreated cells (p < 0.01). These compounds significantly reduced expression of COX-2, cyclin-D1 and VEGF in the treated cells besides cytoplasmic accumulation of pNF-κB/p65 protein. In addition to PI3Kα, inactivation of downstream components, i.e. Akt and mTOR was seen. CONCLUSION: PI3K inhibitors such as PI-103, PI-828 and PX-866 may be developed as potential therapeutic agents for effective treatment of oral squamous cell carcinoma (OSCC) patients, associated with activated PI3K/Akt pathway.

Ligand-specific dynamics of the progesterone receptor in living cells and during chromatin remodeling in vitro.

Progesterone receptor (PR), a member of the nuclear receptor superfamily, is a key regulator of several processes in reproductive function. We have studied the dynamics of the interaction of PR with a natural target promoter in living cells through the use of fluorescence recovery after photobleaching (FRAP) analysis and also have characterized the dynamics of the interaction of PR with the mouse mammary tumor virus (MMTV) promoter reconstituted into chromatin in vitro. In photobleaching experiments, PR in the presence of the agonist R5020 exhibits rapid exchange with the MMTV promoter in living cells. Two PR antagonists, RU486 and ZK98299, have opposite effects on receptor dynamics in vivo. In the presence of RU486, PR binds to the promoter and is exchanged more slowly than the agonist-activated receptor. In contrast, PR bound to ZK98299 is not localized to the promoter and exhibits higher mobility in the nucleoplasm than the agonist-bound receptor. Significantly, PR bound to R5020 or RU486 can recruit the SWI/SNF chromatin remodeling complex to the promoter, but PR activated with ZK98299 cannot. Furthermore, we found ligand-specific active displacement of PR from the MMTV promoter during chromatin remodeling in vitro and conclude that the interaction of PR with chromatin is highly dynamic both in vivo and in vitro. We propose that factor displacement during chromatin remodeling is an important component of receptor mobility and that ligand-specific interactions with remodeling complexes can strongly influence receptor nuclear dynamics and rates of exchange with chromatin in living cells.

A new generation of progesterone receptor modulators.

Progesterone receptor (PR) modulators have evolved both structurally and mechanistically over the past half-century. Classical steroidal PR agonists continue to play an important role in women's health such as in oral contraception and post-menopausal hormone therapy whereas steroid-based PR antagonists and selective PR modulators are being evaluated clinically in a wide range of gynecologic conditions. This review will focus primarily on the newer generation of PR modulators derived from structurally unique chemical scaffolds. For example, tanaproget (TNPR) is described as a non-steroidal PR agonist with high affinity and selectivity for the PR that is significantly more potent than many of its steroidal counterparts in a variety of pre-clinical efficacy models. Similarly, we present numerous examples of unique non-steroidal PR antagonists in various stages of characterization and development. A basic understanding of the structural determinants for high affinity binding of these new PR modulators to the PR ligand-binding domain (LBD) is also discussed. Finally, as the biology of the PR becomes further defined, we speculate on the future development of novel PR modulators.

Effects of cyclopenta[c]phenanthrene and its derivatives on zona radiata protein, ERalpha, and CYP1A mRNA expression in liver of rainbow trout (Oncorhynchus mykiss Walbaum).

Despite cyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs) have been detected in the environment, the ability of CP-PAH to induce cellular and tissue responses remains poorly characterized. In this study, xenoestrogen-associated responses (mRNA levels of estrogen receptor alpha, ERalpha, and zona radiata protein, Zrp) and xenobiotic effects (CYP1A mRNA) have been investigated in liver of juvenile rainbow trout after short-term treatment (8 and 24 h) with following compounds administered singly: cyclopenta[c]phenanthrene (CP[c]Ph); its derivatives, 5A-CP[c]Ph; 5A6M-CP[c]Ph; 5A9M-CP[c]Ph; B[c]Ph, a structurally similar polycyclic aromatic hydrocarbon; B[a]P, a model CYP1A inducer; and zearalenone (ZEA), naturally occurring ligand for ER. The CYP1A mRNA expression after 24 h of exposure with CP[c]Ph or its derivatives, except 5A9M-CP[c]Ph, was 3-9-fold higher compared to controls (P<0.05), but it was less than that caused by B[a]P (65-fold up regulation; P<0.01). Moreover, neither of the CP-PAH compounds modulated liver ERalpha or Zrp mRNA levels as compared to effects associated with ZEA. Interestingly, a treatment with this ER-ligand, caused moderate but significant increase of CYP1A mRNA expression (about 2.5-fold; P<0.05). The finding that ZEA is capable of acting as either estrogenic and xenobiotic compound, should be further explored in a more detailed and differently designed experiment.