Phosphodiesterase 10A (PDE10A) as a novel target to suppress ß-catenin and RAS signaling in epithelial ovarian cancer.

A leading theory for ovarian carcinogenesis proposes that inflammation associated with incessant ovulation is a driver of oncogenesis. Consistent with this theory, nonsteroidal anti-inflammatory drugs (NSAIDs) exert promising chemopreventive activity for ovarian cancer. Unfortunately, toxicity is associated with long-term use of NSAIDs due to their cyclooxygenase (COX) inhibitory activity. Previous studies suggest the antineoplastic activity of NSAIDs is COX independent, and rather may be exerted through phosphodiesterase (PDE) inhibition. PDEs represent a unique chemopreventive target for ovarian cancer given that ovulation is regulated by cyclic nucleotide signaling. Here we evaluate PDE10A as a novel therapeutic target for ovarian cancer. Analysis of The Cancer Genome Atlas (TCGA) ovarian tumors revealed PDE10A overexpression was associated with significantly worse overall survival for patients. PDE10A expression also positively correlated with the upregulation of oncogenic and inflammatory signaling pathways. Using small molecule inhibitors, Pf-2545920 and a novel NSAID-derived PDE10A inhibitor, MCI-030, we show that PDE10A inhibition leads to decreased ovarian cancer cell growth and induces cell cycle arrest and apoptosis. We demonstrate these pro-apoptotic properties occur through PKA and PKG signaling by using specific inhibitors to block their activity. PDE10A genetic knockout in ovarian cancer cells through CRISP/Cas9 editing lead to decreased cell proliferation, colony formation, migration and invasion, and in vivo tumor growth. We also demonstrate that PDE10A inhibition leads to decreased Wnt-induced ß-catenin nuclear translocation, as well as decreased EGF-mediated activation of RAS/MAPK and AKT pathways in ovarian cancer cells. These findings implicate PDE10A as novel target for ovarian cancer chemoprevention and treatment.

Overcoming Temozolomide Resistance in Glioblastoma via Enhanced NAD+ Bioavailability and Inhibition of Poly-ADP-Ribose Glycohydrolase.

Glioblastoma multiforme (GBM) is an incurable brain cancer with an average survival of approximately 15 months. Temozolomide (TMZ) is a DNA alkylating agent for the treatment of GBM. However, at least 50% of the patients treated with TMZ show poor response, primarily due to elevated expression of the repair protein O6-methylguanine-DNA methyltransferase (MGMT) or due to defects in the mismatch repair (MMR) pathway. These resistance mechanisms are either somatic or arise in response to treatment, highlighting the need to uncover treatments to overcome resistance. We found that administration of the NAD+ precursor dihydronicotinamide riboside (NRH) to raise cellular NAD+ levels combined with PARG inhibition (PARGi) triggers hyperaccumulation of poly(ADP-ribose) (PAR), resulting from both DNA damage-induced and replication-stress-induced PARP1 activation. Here, we show that the NRH/PARGi combination enhances the cytotoxicity of TMZ. Specifically, NRH rapidly increases NAD+ levels in both TMZ-sensitive and TMZ-resistant GBM-derived cells and enhances the accumulation of PAR following TMZ treatment. Furthermore, NRH promotes hyperaccumulation of PAR in the presence of TMZ and PARGi. This combination strongly suppresses the cell growth of GBM cells depleted of MSH6 or cells expressing MGMT, suggesting that this regimen may improve the efficacy of TMZ to overcome treatment resistance in GBM.

PMS2 variant results in loss of ATPase activity without compromising mismatch repair.

Hereditary cancer syndromes account for approximately 5%-10% of all diagnosed cancer cases. Lynch syndrome (LS) is an autosomal dominant hereditary cancer condition that predisposes individuals to an elevated lifetime risk for developing colorectal, endometrial, and other cancers. LS results from a pathogenic mutation in one of four mismatch repair (MMR) genes (MSH2, MSH6, MLH1, and PMS2). The diagnosis of LS is often challenged by the identification of missense mutations, termed variants of uncertain significance, whose functional effect on the protein is not known. Of the eight PMS2 variants initially selected for this study, we identified a variant within the N-terminal domain where asparagine 335 is mutated to serine, p.Asn335Ser, which lacked ATPase activity, yet appears to be proficient in MMR. To expand our understanding of this functional dichotomy, we performed biophysical and structural studies, and noted that p.Asn335Ser binds to ATP but is unable to hydrolyze it to ADP. To examine the impact of p.Asn335Ser on MMR, we developed a novel in-cell fluorescent-based microsatellite instability reporter that revealed p.Asn335Ser maintained genomic stability. We conclude that in the absence of gross structural changes, PMS2 ATP hydrolysis is not necessary for proficient MMR and that the ATPase deficient p.Asn335Ser variant is likely benign.

Temporal dynamics of base excision/single-strand break repair protein complex assembly/disassembly are modulated by the PARP/NAD+/SIRT6 axis.

Assembly and disassembly of DNA repair protein complexes at DNA damage sites are essential for maintaining genomic integrity. Investigating factors coordinating assembly of the base excision repair (BER) proteins DNA polymerase ß (Polß) and XRCC1 to DNA lesion sites identifies a role for Polß in regulating XRCC1 disassembly from DNA repair complexes and, conversely, demonstrates Polß's dependence on XRCC1 for complex assembly. LivePAR, a genetically encoded probe for live-cell imaging of poly(ADP-ribose) (PAR), reveals that Polß and XRCC1 require PAR for repair-complex assembly, with PARP1 and PARP2 playing unique roles in complex dynamics. Further, BER complex assembly is modulated by attenuation/augmentation of NAD+ biosynthesis. Finally, SIRT6 does not modulate PARP1 or PARP2 activation but does regulate XRCC1 recruitment, leading to diminished Polß abundance at sites of DNA damage. These findings highlight coordinated yet independent roles for PARP1, PARP2, and SIRT6 and their regulation by NAD+ bioavailability to facilitate BER.

Biphasic Postnatal Umbilical Cord Shortening.

INTRODUCTION: Variations in postnatal length of refrigerated, unfixed umbilical cords were studied over time to elucidate natural changes and times of stability. METHODS: Length was measured in 132 cords following severance, repeated at varying timed intervals and studied by analysis of variance and regression analysis. RESULTS: Data show immediate rapid initial phase shortening (mean 4.2+/-3.9 cm SD); an interval of lengthening; stable length at hours 3-4 following severance, a slower second phase shortening (mean 1.5+/-0.7 cm SD) beginning at 5 hours and peaking at 12 hours; and gradual lengthening to stable length after 23 hours. Overall, there was a significant net mean decrease of 3.49+/-2.29 cm SD. Shortening was greatest for intact long cord segments (p=0.0001), as much as 11 cm. Two highly significant models for predicting umbilical cord length at delivery (OL) were determined using the post-delivery lengths (Length) measured at different times following delivery (Hours), as follows:At ≤ 3 hours following delivery: OL=1.02xLength cm+1.11xHoursAt >3 hours following delivery: OL=1.07xLength+0.44xHours-0.01x(Hours)2. CONCLUSION: Cord lengths stabilized between hours 3-4 and after 23 hours following severance. Phase one shortening resembles vasoconstriction; phase two resembles rigor mortis. The models allow prediction of the original umbilical cord length at delivery, regardless of the time of measurement.

Exosomal markers (CD63 and CD9) expression and their prognostic significance using immunohistochemistry in patients with pancreatic ductal adenocarcinoma.

BACKGROUND: Exosomes are important mediators of intercellular communications and play pivotal roles in cancer progression, metastasis and chemoresistance. CD63 and CD9 are widely accepted exosomal markers. In patients with pancreatic ductal adenocarcinoma (PDAC), positive correlation between CD9 expression and overall survival (OS) was reported. CD63 expression was conserved in all patients with no reported prognostic significance. This study explored the prognostic significance of CD63 and CD9 expression using immunohistochemistry (IHC) in patients with PDAC of mixed racial background. METHODS: Between 2012 and 2016, 49 patients with PDAC had available tissues for CD63 and CD9 staining using IHC. Two pathologists independently scored the CD63 and CD9 expression. Staining intensity was graded from 1-3 and staining percentage was estimated in 10% increments. Mean Quick-score (Q-score) (Intensity X Percentage of staining) was calculated. RESULTS: The mean Q-score for CD63 and CD9 are higher in primary tumor from the pancreas compared to pancreatic tumor from metastatic sites (185 vs. 102, P=0.0002) and (48 vs. 20, P=0.0418) respectively. We fitted Cox proportion hazard regression models to investigate the impact of the covariates CD63 and CD9 on progression free survival (PFS) and OS. CD63 has significant impact on PFS (P=0.0135) and OS (P=0.003). The higher the CD63 Q-score, the longer the PFS and OS. CD9 doesn't have significant impact on PFS (P=0.5734) or OS (P=0.2682). The mean CD63 and CD9 Q-scores are slightly higher in African American (AA) compared to Caucasians (157 vs. 149, P=0.76) and (45 vs. 29, P=0.43) respectively. CONCLUSIONS: CD63 and CD9 expression is higher in primary tumor from the pancreas compared to pancreatic tumor from metastatic sites. There is correlation between CD63 expression (but not CD9 in this cohort) and PFS and OS. To our knowledge, this is the first study to show prognostic significance of CD63 expression in patients with PDAC using IHC. A trend of higher expression of CD63 and CD9 among AA compared to Caucasians was also noticed.

Cancer Immune Checkpoint Inhibitor Therapy and the Gut Microbiota.

The past decade has seen tremendous advances in both our understanding of cancer immunosuppressive microenvironments and colonic bacteria facilitated by immune checkpoint inhibitor antibodies and next generation sequencing, respectively. Because an important role of the host immune system is to communicate with and regulate the gut microbial community, it should not come as a surprise that the behavior of one is coupled to the other. In this review, we will attempt to dissect some of the studies demonstrating cancer immunotherapy modulation by specific gut microbes and discuss possible molecular mechanisms for this effect.

Immunoregulatory protein B7-H3 regulates cancer stem cell enrichment and drug resistance through MVP-mediated MEK activation.

B7-H3 is a tumor-promoting glycoprotein that is expressed at low levels in most normal tissues, but is overexpressed in various human cancers which is associated with disease progression and poor patient outcome. Although numerous publications have reported the correlation between B7-H3 and cancer progression in many types of cancers, mechanistic studies on how B7-H3 regulates cancer malignancy are rare, and the mechanisms underlying the role of B7-H3 in drug resistance are almost unknown. Here we report a novel finding that upregulation of B7-H3 increases the breast cancer stem cell population and promotes cancer development. Depletion of B7-H3 in breast cancer significantly inhibits the cancer stem cells. By immunoprecipitation and mass spectrometry, we found that B7-H3 is associated with the major vault protein (MVP) and activates MEK through MVP-enhancing B-RAF and MEK interaction. B7-H3 expression increases stem cell population by binding to MVP which regulates the activation of the MAPK kinase pathway. Depletion of MVP blocks the activation of MEK induced by B7-H3 and dramatically inhibits B7-H3 induced stem cells. This study reports novel functions of B7-H3 in regulating breast cancer stem cell enrichment. The novel mechanism for B7-H3-induced stem cell propagation by regulating MVP/MEK signaling axis independent of the classic Ras pathway may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.

DNA Repair Molecular Beacon assay: a platform for real-time functional analysis of cellular DNA repair capacity.

Numerous studies have shown that select DNA repair enzyme activities impact response and/or toxicity of genotoxins, suggesting a requirement for enzyme functional analyses to bolster precision medicine or prevention. To address this need, we developed a DNA Repair Molecular Beacon (DRMB) platform that rapidly measures DNA repair enzyme activity in real-time. The DRMB assay is applicable for discovery of DNA repair enzyme inhibitors, for the quantification of enzyme rates and is sufficiently sensitive to differentiate cellular enzymatic activity that stems from variation in expression or effects of amino acid substitutions. We show activity measures of several different base excision repair (BER) enzymes, including proteins with tumor-identified point mutations, revealing lesion-, lesion-context- and cell-type-specific repair dependence; suggesting application for DNA repair capacity analysis of tumors. DRMB measurements using lysates from isogenic control and APE1-deficient human cells suggests the major mechanism of base lesion removal by most DNA glycosylases may be mono-functional base hydrolysis. In addition, development of a microbead-conjugated DRMB assay amenable to flow cytometric analysis further advances its application. Our studies establish an analytical platform capable of evaluating the enzyme activity of select DNA repair proteins in an effort to design and guide inhibitor development and precision cancer therapy options.

Induced pluripotent stem cells derived from human amnion in chemically defined conditions.

Fetal stem cells are a unique type of adult stem cells that have been suggested to be broadly multipotent with some features of pluripotency. Their clinical potential has been documented but their upgrade to full pluripotency could open up a wide range of cell-based therapies particularly suited for pediatric tissue engineering, longitudinal studies or disease modeling. Here we describe episomal reprogramming of mesenchymal stem cells from the human amnion to pluripotency (AM-iPSC) in chemically defined conditions. The AM-iPSC expressed markers of embryonic stem cells, readily formed teratomas with tissues of all three germ layers present and had a normal karyotype after around 40 passages in culture. We employed novel computational methods to determine the degree of pluripotency from microarray and RNA sequencing data in these novel lines alongside an iPSC and ESC control and found that all lines were deemed pluripotent, however, with variable scores. Differential expression analysis then identified several groups of genes that potentially regulate this variability in lines within the boundaries of pluripotency, including metallothionein proteins. By further studying this variability, characteristics relevant to cell-based therapies, like differentiation propensity, could be uncovered and predicted in the pluripotent stage.

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods - flow cytometry, confocal imaging, teratoma formation and transcriptional profiling - is also described. The newly generated lines express markers of embryonic stem cells - Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 - while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

Immunoregulatory Protein B7-H3 Reprograms Glucose Metabolism in Cancer Cells by ROS-Mediated Stabilization of HIF1α.

B7-H3 is a member of B7 family of immunoregulatory transmembrane glycoproteins expressed by T cells. While B7-H3 overexpression is associated with poor outcomes in multiple cancers, it also has immune-independent roles outside T cells and its precise mechanistic contributions to cancer are unclear. In this study, we investigated the role of B7-H3 in metabolic reprogramming of cancer cells in vitro and in vivo We found that B7-H3 promoted the Warburg effect, evidenced by increased glucose uptake and lactate production in B7-H3-expressing cells. B7-H3 also increased the protein levels of HIF1α and its downstream targets, LDHA and PDK1, key enzymes in the glycolytic pathway. Furthermore, B7-H3 promoted reactive oxygen species-dependent stabilization of HIF1α by suppressing the activity of the stress-activated transcription factor Nrf2 and its target genes, including the antioxidants SOD1, SOD2, and PRX3. Metabolic imaging of human breast cancer xenografts in mice confirmed that B7-H3 enhanced tumor glucose uptake and tumor growth. Together, our results illuminate the critical immune-independent contributions of B7-H3 to cancer metabolism, presenting a radically new perspective on B7 family immunoregulatory proteins in malignant progression. Cancer Res; 76(8); 2231-42. ©2016 AACR.

p-21 activated kinase 4 (PAK4) maintains stem cell-like phenotypes in pancreatic cancer cells through activation of STAT3 signaling.

Pancreatic cancer (PC) remains a highly lethal malignancy due to its unusual chemoresistance and high aggressiveness. A subpopulation of pancreatic tumor cells, known as cancer stem cells (CSCs), is considered responsible not only for tumor-maintenance, but also for its widespread metastasis and therapeutic failure. Here we investigated the role of p-21 activated kinase 4 (PAK4) in driving PC stemness properties. Our data demonstrate that triple-positive (CD24(+)/CD44(+)/EpCAM(+)) subpopulation of pancreatic CSCs exhibits greater level of PAK4 as compared to triple-negative (CD24(-)/CD44(-)/EpCAM(-)) cells. Moreover, PAK4 silencing in PC cells leads to diminished fraction of CD24, CD44, and EpCAM positive cells. Furthermore, we show that PAK4-silenced PC cells exhibit decreased sphere-forming ability and increased chemosensitivity to gemcitabine toxicity. PAK4 expression is also associated with enhanced levels of stemness-associated transcription factors (Oct4/Nanog/Sox2 and KLF4). Furthermore, our data show decreased nuclear accumulation and transcriptional activity of STAT3 in PAK4-silenced PC cells and restitution of its activity leads to restoration of stem cell phenotypes. Together, our findings deliver first experimental evidence for the involvement of PAK4 in PC stemness and support its clinical utility as a novel therapeutic target in PC.

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or ß-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

mRNA detection in living cells: A next generation cancer stem cell identification technique.

Cancer stem cells (CSC) are a distinct subpopulation within a tumor shown to drive tumor progression, metastasis, and recurrence. A review of the literature reveals poor consensus, with the use of a wide variety of surface markers and functional assays to identify and isolate cancer stem cells. Utilizing a novel technology that enables live-cell mRNA quantitation, we have demonstrated the ability to identify and sort viable CSC based on markers associated with stemness in pluripotent cells. Fresh tumor samples from a variety of cancer types were examined by flow cytometry for Nanog expression. Levels of CSC detected ranged from 6% to 19%. This method of CSC detection was cross-validated with other commonly used surface markers with good correlation. Matrigel invasion assays confirmed that CSC isolated using this method are both highly motile and invasive. This approach simplifies the process of identifying viable CSC from fresh tumor tissue, providing a level of accuracy not previously available. This method may also provide a valuable tool for screening and validating new CSC biomarkers.

CXCL12/CXCR4 signaling counteracts docetaxel-induced microtubule stabilization via p21-activated kinase 4-dependent activation of LIM domain kinase 1.

Emerging data highlight the significance of chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 (CXCL12/CXCR4) signaling axis in the chemoresistance of several malignancies, including prostate cancer (PCa); however, underlying mechanisms remain largely elusive. Here, we demonstrate that CXCL12 treatment rescues the PCa cells from docetaxel (DTX)-induced toxicity by overriding its effect on cell cycle (G2/M phase arrest). We further demonstrate that the chemoprotective effect of CXCL12 is abolished upon pharmacological inhibition or RNA interference-mediated silencing of CXCR4. Moreover, microtubule stabilization caused by DTX is suppressed in CXCL12-stimulated PCa cells as revealed by immunofluorescence and immunoblot analyses. The effect of CXCL12 on microtubule stabilization is abrogated when PCa cells are pre-treated with a CXCR4 antagonist. In additional studies, we show that the chemoprotective action of CXCL12/CXCR4 signaling is mediated by p21-activated kinase 4 (PAK4)-dependent activation of Lim domain kinase 1 (LIMK1), and inhibition of either PAK4 or LIMK1 leads to re-sensitization of PCa cells to DTX-induced tubulin polymerization and cellular toxicity even in the presence of CXCL12. Altogether, our findings uncover a novel mechanism underlying CXCL12/CXCR4 signaling-induced PCa chemoresistance and suggest that targeting of this signaling axis or its downstream effector pathway could lead to therapeutic enhancement of DTX.

p-21 activated kinase 4 promotes proliferation and survival of pancreatic cancer cells through AKT- and ERK-dependent activation of NF-κB pathway.

Identification of novel molecular targets and understanding the mechanisms underlying the aggressive nature of pancreatic cancer (PC) remain prime focus areas of research. Here, we investigated the expression and pathobiological significance of p21-activated kinase 4 (PAK4), a gene that was earlier shown to be amplified in a sub-set of PC. Our data demonstrate PAK4 overexpression in PC tissues and cell lines with little or no expression in the normal pancreas. PAK4 silencing in two PC cell lines, MiaPaCa and T3M4, by RNA interference causes suppression of growth and clonogenic ability due to decreased cell cycle progression and apoptosis-resistance. PAK4-silenced PC cells exhibit altered expression of proliferation- and survival-associated proteins. Moreover, we observe decreased nuclear accumulation and transcriptional activity of NF-κB in PAK4-silenced PC cells associated with stabilization of its inhibitory protein, IκBα. Transfection of PAK4-silenced PC cells with constitutively-active mutant of IKKß, an upstream kinase of IκBα, leads to restoration of NF-κB activity and PC cell growth. Furthermore, we show that PAK4-induced NF-κB activity is mediated through activation and concerted action of ERK and Akt kinases. Together, these findings suggest that PAK4 is a regulator of NF-κB pathway in PC cells and can serve as a novel target for therapy.

Panepoxydone targets NF-kB and FOXM1 to inhibit proliferation, induce apoptosis and reverse epithelial to mesenchymal transition in breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a highly diverse group that is associated with an aggressive phenotype. Its treatment has been challenging due to its heterogeneity and absence of well-defined molecular targets. Thus, there is an urgent need to identify novel agents with therapeutic application. NF-κB is over-expressed in many breast cancers; thus, inactivation of the NF-κB pathway could serve as a therapeutic target. Here we report for the first time the anti-tumor activity of panepoxydone (PP), a NF-κB inhibitor isolated from an edible mushroom, in several breast cancer cell lines. METHODS: We investigated the effects of PP on cell growth, migration-invasion, apoptosis and EMT-related proteins expression in MCF-7 and TNBC cell lines MDA-MB-231, MDA-MB-468 and MDA-MB-453. RESULTS: Significant antitumor activity was seen in all cell lines, with differential responses noted in cell-line specific manner. Treatment with PP resulted in significant cytotoxicity, decreased invasion, migration and increased apoptosis in all cell lines tested. Up-regulation of Bax and cleaved PARP and down-regulation of Bcl-2, survivin, cyclin D1 and caspase 3 were noted in PP-treated breast cancer cells. The antitumor effect of PP appeared related to its ability to inhibit the phosphorylation of inhibitor of NF-κB (IκBα) with cytoplasmic accumulation. PP treatment also down-regulated FOXM1 which resulted in a reversal of EMT. Similar results were obtained after silencing of NF-kB and FOXM1. CONCLUSION: Altogether, these studies show, for the first time the antitumor activity of PP against breast cancer cells, in particular TNBC cells. Furthermore, it highlights the concept that optimal treatment of TNBC warrants attention to the differential sensitivity of various TNBC subtypes to therapeutic agents. These results suggest that the PP may be a potentially effective chemopreventive or therapeutic agent against breast cancer. However, additional studies are required to more fully elucidate the mechanism of antitumor effect of PP.

An undesired effect of chemotherapy: gemcitabine promotes pancreatic cancer cell invasiveness through reactive oxygen species-dependent, nuclear factor κB- and hypoxia-inducible factor 1α-mediated up-regulation of CXCR4.

Recently, we have shown that CXCL12/CXCR4 signaling plays an important role in gemcitabine resistance of pancreatic cancer (PC) cells. Here, we explored the effect of gemcitabine on this resistance mechanism. Our data demonstrate that gemcitabine induces CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time-dependent manner. Gemcitabine-induced CXCR4 expression is dependent on reactive oxygen species (ROS) generation because it is abrogated by pretreatment of PC cells with the free radical scavenger N-acetyl-L-cysteine. CXCR4 up-regulation by gemcitabine correlates with time-dependent accumulation of NF-κB and HIF-1α in the nucleus. Enhanced binding of NF-κB and HIF-1α to the CXCR4 promoter is observed in gemcitabine-treated PC cells, whereas their silencing by RNA interference causes suppression of gemcitabine-induced CXCR4 expression. ROS induction upon gemcitabine treatment precedes the nuclear accumulation of NF-κB and HIF-1α, and suppression of ROS diminishes these effects. The effect of ROS on NF-κB and HIF-1α is mediated through activation of ERK1/2 and Akt, and their pharmacological inhibition also suppresses gemcitabine-induced CXCR4 up-regulation. Interestingly, our data demonstrate that nuclear accumulation of NF-κB results from phosphorylation-induced degradation of IκBα, whereas HIF-1α up-regulation is NF-κB-dependent. Lastly, our data demonstrate that gemcitabine-treated PC cells are more motile and exhibit significantly greater invasiveness against a CXCL12 gradient. Together, these findings reinforce the role of CXCL12/CXCR4 signaling in gemcitabine resistance and point toward an unintended and undesired effect of chemotherapy.

High-performance execution of psychophysical tasks with complex visual stimuli in MATLAB.

Behavioral, psychological, and physiological experiments often require the ability to present sensory stimuli, monitor and record subjects' responses, interface with a wide range of devices, and precisely control the timing of events within a behavioral task. Here, we describe our recent progress developing an accessible and full-featured software system for controlling such studies using the MATLAB environment. Compared with earlier reports on this software, key new features have been implemented to allow the presentation of more complex visual stimuli, increase temporal precision, and enhance user interaction. These features greatly improve the performance of the system and broaden its applicability to a wider range of possible experiments. This report describes these new features and improvements, current limitations, and quantifies the performance of the system in a real-world experimental setting.