Treatment with decitabine induces the expression of stemness markers, PD-L1 and NY-ESO-1 in colorectal cancer: potential for combined chemoimmunotherapy.

BACKGROUND: The mechanism of tumor immune escape and progression in colorectal cancer (CRC) is widely investigated in-vitro to help understand and identify agents that might play a crucial role in response to treatment and improve the overall survival of CRC patients. Several mechanisms of immune escape and tumor progression, including expression of stemness markers, inactivation of immunoregulatory genes by methylation, and epigenetic silencing, have been reported in CRC, indicating the potential of demethylating agents as anti-cancer drugs. Of these, a chemotherapeutic demethylating agent, Decitabine (DAC), has been reported to induce a dual effect on both DNA demethylation and histone changes leading to an increased expression of target biomarkers, thus making it an attractive anti-tumorigenic drug. METHODS: We compared the effect of DAC in primary 1076 Col and metastatic 1872 Col cell lines isolated and generated from patients' tumor tissues. Both cell lines were treated with DAC, and the expression of the NY-ESO-1 cancer-testis antigen, the PD-L1 immunoinhibitory marker, and the CD44, Nanog, KLF-4, CD133, MSI-1 stemness markers were analyzed using different molecular and immunological assays. RESULTS: DAC treatment significantly upregulated stemness markers in both primary 1076 Col and meta-static 1872 Col cell lines, although a lower effect occurred on the latter: CD44 (7.85 fold; ***p = 0.0001 vs. (4.19 fold; *p = 0.0120), Nanog (4.1 fold; ***p < 0.0001 vs.1.69 fold; ***p = 0.0008), KLF-4 (4.33 fold; ***p < 0.0001 vs.2.48 fold; ***p = 0.0005), CD133 (16.77 fold; ***p = 0.0003 vs.6.36 fold; *p = 0.0166), and MSI-1 (2.33 fold; ***p = 0.0003 vs.2.3 fold; ***p = 0.0004), respectively. Interestingly, in the metastatic 1872 Col cells treated with DAC, the expression of both PD-L1 and NY-ESO-1 was increased tenfold (*p = 0.0128) and fivefold (***p < 0.0001), respectively. CONCLUSIONS: We conclude that the upregulation of both stemness and immune checkpoint markers by DAC treatment on CRC cells might represent a mechanism of immune evasion. In addition, induction of NY-ESO-1 may represent an immuno-therapeutic option in metastatic CRC patients. Finally, the combination of DAC and anti-PD-1/anti-PD-L1 antibodies treatment should represent a potential therapeutic intervention for this group of patients.

TRPV2: A Cancer Biomarker and Potential Therapeutic Target.

The Transient Receptor Potential Vanilloid type-2 (TRPV2) channel exhibits oncogenicity in different types of cancers. TRPV2 is implicated in signaling pathways that mediate cell survival, proliferation, and metastasis. In leukemia and bladder cancer, the oncogenic activity of TRPV2 was linked to alteration of its expression profile. In multiple myeloma patients, TRPV2 overexpression correlated with bone tissue damage and poor prognosis. In prostate cancer, TRPV2 overexpression was associated with the castration-resistant phenotype and metastasis. Loss or inactivation of TRPV2 promoted glioblastoma cell proliferation and increased resistance to CD95-induced apoptotic cell death. TRPV2 overexpression was associated with high relapse-free survival in triple-negative breast cancer, whereas the opposite was found in patients with esophageal squamous cell carcinoma or gastric cancer. Another link was found between TRPV2 expression and either drug-induced cytotoxicity or stemness of liver cancer. Overall, these findings validate TRPV2 as a prime candidate for cancer biomarker and future therapeutic target.

Phosphatidylinositol-4,5-Biphosphate (PI(4,5)P2) Is Required for Rapid Endocytosis in Chromaffin Cells.

OBJECTIVE: Phosphoinositides play a regulatory role in clathrin-mediated endocytosis. However, their involvement in clathrin-independent endocytosis termed rapid endocytosis (RE), which is the mode of vesicle recycling during neurotransmitter release by transient fusion (known as kiss-and-run), has not been investigated. Here, we used patch-clamp recording of whole-cell membrane capacitance in adrenal chromaffin cells (ACC) to monitor changes of RE kinetics in response to pharmacological alteration of phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) level by phenylarsine oxide (PAO) or antibody against phosphatidylinositol 4-kinase (AbPI4K). RESULTS: We found that PAO and AbPI4K significantly abrogated RE kinetics. Infusion of PI(4,5)P2 through the patch pipette potentiated RE kinetics and reversed PAO- and AbPI4K-induced blockade of RE. Similarly, the application of the bifunctional thiol dithiothreitol (DTT) to PAO-treated cells completely prevented the inhibitory effect of PAO on RE. These findings indicate that PI(4,5)P2 is implicated in the signaling (mechanistic) process of RE in ACC.

Curcumin-Mediated Apoptotic Cell Death in Papillary Thyroid Cancer and Cancer Stem-Like Cells through Targeting of the JAK/STAT3 Signaling Pathway.

The constitutive activation of Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signal transduction is well elucidated in STAT3-mediated oncogenesis related to thyroid cancer and is considered to be a plausible therapeutic target. Hence, we investigated whether curcumin, a natural compound, can target the JAK/STAT3 signaling pathway to induce cytotoxic effects in papillary thyroid cancer (PTC) cell lines (BCPAP and TPC-1) and derived thyroid cancer stem-like cells (thyrospheres). Curcumin suppressed PTC cell survival in a dose-dependent manner via the induction of caspase-mediated apoptosis and caused the attenuation of constitutively active STAT3 (the dephosphorylation of Tyr705-STAT3) without affecting STAT3. Gene silencing with STAT3-specific siRNA showed the modulation of genes associated with cell growth and proliferation. The cotreatment of PTC cell lines with curcumin and cisplatin synergistically potentiated cytotoxic effects via the suppression of JAK/STAT3 activity along with the inhibition of antiapoptotic genes and the induction of proapoptotic genes, and it also suppressed the migration of PTC cells by downregulating matrix metalloproteinases and the inhibition of colony formation. Finally, thyrospheres treated with curcumin and cisplatin showed suppressed STAT3 phosphorylation, a reduced formation of thyrospheres, and the downregulated expression of stemness markers, in addition to apoptosis. The current study's findings suggest that curcumin synergistically enhances the anticancer activity of cisplatin in PTC cells as well as in cancer stem-like cells by targeting STAT3, which suggests that curcumin combined with chemotherapeutic agents may provide better therapeutic outcomes.

The Role of Extracellular Vesicles as Modulators of the Tumor Microenvironment, Metastasis and Drug Resistance in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common cancers worldwide, with high morbidity and mortality rates. A number of factors including modulation of the tumor microenvironment, high metastatic capability, and resistance to treatment have been associated with CRC disease progression. Recent studies have documented that tumor-derived extracellular vesicles (EVs) play a significant role in intercellular communication in CRC via transfer of cargo lipids, proteins, DNA and RNAs to the recipient tumor cells. This transfer influences a number of immune-related pathways leading to activation/differentiation/expression of immune cells and modulation of the tumor microenvironment that plays a significant role in CRC progression, metastasis, and drug resistance. Furthermore, tumor-derived EVs are secreted in large amounts in biological fluids of CRC patients and as such the expression analysis of EV cargoes have been associated with prognosis or response to therapy and may be a source of therapeutic targets. This review aims to provide a comprehensive insight into the role of EVs in the modulation of the tumor microenvironment and its effects on CRC progression, metastasis, and drug resistance. On the other hand, the potential role of CRC derived EVs as a source of biomarkers of response and therapeutic targets will be discussed in detail to understand the dynamic role of EVs in CRC diagnosis, treatment, and management.

Evaluation of cationic channel TRPV2 as a novel biomarker and therapeutic target in Leukemia-Implications concerning the resolution of pulmonary inflammation.

Patients treated during leukemia face the risk of complications including pulmonary dysfunction that may result from infiltration of leukemic blast cells (LBCs) into lung parenchyma and interstitium. In LBCs, we demonstrated that transient receptor potential vanilloid type 2 channel (TRPV2), reputed for its role in inflammatory processes, exhibited oncogenic activity associated with alteration of its molecular expression profile. TRPV2 was overexpressed in LBCs compared to normal human peripheral blood mononuclear cells (PBMCs). Additionally, functional full length isoform and nonfunctional short form pore-less variant of TRPV2 protein were up-regulated and down-regulated respectively in LBCs. However, the opposite was found in PBMCs. TRPV2 silencing or pharmacological targeting by Tranilast (TL) or SKF96365 (SKF) triggered caspace-mediated apoptosis and cell cycle arrest. TL and SKF inhibited chemotactic peptide fMLP-induced response linked to TRPV2 Ca2+ activity, and down-regulated expression of surface marker CD38 involved in leukemia and lung airway inflammation. Challenging lung airway epithelial cells (AECs) with LBCs decreased (by more than 50%) transepithelial resistance (TER) denoting barrier function alteration. Importantly, TL prevented such loss in TER. Therefore, TRPV2 merits further exploration as a pharmacodynamic biomarker for leukemia patients (with pulmonary inflammation) who might be suitable for a novel [adjuvant] therapeutic strategy based on TL.

Anticancer Activity of Camel Milk via Induction of Autophagic Death in Human Colorectal and Breast Cancer Cells

Background/ Objective: Camel milk is traditionally known for its human health benefits and believed to be a remedy for various human ailments including cancer. The study was aimed to evaluate the inhibitory effects of commercially available camel milk on cancer cells and its underlying mechanism(s). Materials and Methods: Two cell lines: colorectal cancer HCT 116 and breast cancer MCF-7 were cultured with different doses of camel milk. The effects of camel milk on cell death were determined by MTT assay, viability by trypan blue exclusion assay and migration by in vitro scratch assay. The mechanism was elucidated by western blotting and confocal microscopy was used to confirm autophagy. Results: Camel milk significantly reduced proliferation, viability as well as migration of both the cells. The accumulation of LC3-II protein along with reduction in expression of p62 and Atg 5-12, the autophagy proteins implied induction of autophagy. The (GFP)-LC3 puncta detected by confocal microscopy confirmed the autophagosome formation in response to camel milk treatment. Conclusion: Camel milk exerted antiproliferative effects on human colorectal HCT 116 and breast MCF-7 cancer cells by inducing autophagy.

Pyrolysis of waste tires: A modeling and parameter estimation study using Aspen Plus®.

This paper presents a simulation flowsheet model of a waste tire pyrolysis process with feed capacity of 150kg/h. A kinetic rate-based reaction model is formulated in a form implementable in the simulation package Aspen Plus, giving the flowsheet model the capability to predict more than 110 tire pyrolysis products as reported in experiments by Laresgoiti et al. (2004) and Williams (2013) for the oil and gas products respectively. The simulation model is successfully validated in two stages: firstly against experimental data from Olazar et al. (2008) by comparing the mass fractions for the oil products (gas, liquids (non-aromatics), aromatics, and tar) at temperatures of 425, 500, 550 and 610°C, and secondly against experimental results of main hydrocarbon products (C7 to C15) obtained by Laresgoiti et al. (2004) at temperatures of 400, 500, 600, and 700°C. The model was then used to analyze the effect of pyrolysis process temperature and showed that increased temperatures led to chain fractions from C10 and higher to decrease while smaller chains increased; this is attributed to the extensive cracking of the larger hydrocarbon chains at higher temperatures. The utility of the flowsheet model was highlighted through an energy analysis that targeted power efficiency of the process determined through production profiles of gasoline and diesel at various temperatures. This shows, through the summation of the net power gain from the plant for gasoline plus diesel that the maximum net power lies at the lower temperatures corresponding to minimum production of gasoline and maximum production of diesel. This simulation model can thus serve as a robust tool to respond to market conditions that dictate fuel demand and prices while at the same time identifying optimum process conditions (e.g. temperature) driven by process economics.

Airway surface liquid volume expansion induces rapid changes in amiloride-sensitive Na+ transport across upper airway epithelium-Implications concerning the resolution of pulmonary edema.

During airway inflammation, airway surface liquid volume (ASLV) expansion may result from the movement of plasma proteins and excess liquid into the airway lumen due to extravasation and elevation of subepithelial hydrostatic pressure. We previously demonstrated that elevation of submucosal hydrostatic pressure increases airway epithelium permeability resulting in ASLV expansion by 500 µL cm(-2) h(-1). Liquid reabsorption by healthy airway epithelium is regulated by active Na(+) transport at a rate of 5 µL cm(-2) h(-1). Thus, during inflammation the airway epithelium may be submerged by a large volume of luminal liquid. Here, we have investigated the mechanism by which ASLV expansion alters active epithelial Na(+) transport, and we have characterized the time course of the change. We used primary cultures of tracheal airway epithelium maintained under air interface (basal ASLV, depth is 7 ± 0.5 µm). To mimic airway flooding, ASLV was expanded to a depth of 5 mm. On switching from basal to expanded ASLV conditions, short-circuit current (Isc, a measure of total transepithelial active ion transport) declined by 90% with a half-time (t1/2) of 1 h. 24 h after the switch, there was no significant change in ATP concentration nor in the number of functional sodium pumps as revealed by [(3)H]-ouabain binding. However, amiloride-sensitive uptake of (22)Na(+) was reduced by 70% upon ASLV expansion. This process is reversible since after returning cells back to air interface, Isc recovered with a t1/2 of 5-10 h. These results may have important clinical implications concerning the development of Na(+) channels activators and resolution of pulmonary edema.

Coagulation of highly turbid suspensions using magnesium hydroxide: effects of slow mixing conditions.

Laboratory experiments were carried out to study the effects of slow mixing conditions on magnesium hydroxide floc size and strength and to determine the turbidity and total suspended solid (TSS) removal efficiencies during coagulation of highly turbid suspensions. A highly turbid kaolin clay suspension (1,213 ± 36 nephelometric turbidity units (NTU)) was alkalized to pH 10.5 using a 5 M NaOH solution; liquid bittern (LB) equivalent to 536 mg/L of Mg(2+) was added as a coagulant, and the suspension was then subjected to previously optimized fast mixing conditions of 100 rpm and 60 s. Slow mixing speed (20, 30, 40, and 50 rpm) and time (10, 20, and 30 min) were then varied, while the temperature was maintained at 20.7 ± 1 °C. The standard practice for coagulation-flocculation jar test ASTM D2035-13 (2013) was followed in all experiments. Relative floc size was monitored using an optical measuring device, photometric dispersion analyzer (PDA 2000). Larger and more shear resistant flocs were obtained at 20 rpm for both 20- and 30-min slow mixing times; however, given the shorter duration for the former, the 20-min slow mixing time was considered to be more energy efficient. For slow mixing camp number (Gt) values in the range of 8,400-90,000, it was found that the mixing speed affected floc size and strength more than the time. Higher-turbidity removal efficiencies were achieved at 20 and 30 rpm, while TSS removal efficiency was higher for the 50-rpm slow mixing speed. Extended slow mixing time of 30 min yielded better turbidity and TSS removal efficiencies at the slower speeds.

Rab2 utilizes glyceraldehyde-3-phosphate dehydrogenase and protein kinase C{iota} to associate with microtubules and to recruit dynein.

Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical protein kinase Ciota (aPKCiota) for retrograde vesicle formation from vesicular tubular clusters that sort secretory cargo from recycling proteins returned to the endoplasmic reticulum. However, the precise role of GAPDH and aPKCiota in the early secretory pathway is unclear. GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly, aPKC associates directly with MTs. To learn whether Rab2 also binds directly to MTs, a MT binding assay was performed. Purified Rab2 was found in a MT-enriched pellet only when both GAPDH and aPKCiota were present, and Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2 amino terminus (residues 2-70), which directly interacts with GAPDH and aPKCiota. Because GAPDH binds to the carboxyl terminus of alpha-tubulin, we characterized the distribution of tyrosinated/detyrosinated alpha-tubulin that is recruited by Rab2 in a quantitative membrane binding assay. Rab2-treated membranes contained predominantly tyrosinated alpha-tubulin; however, aPKCiota was the limiting and essential factor. Tyrosination/detyrosination influences MT motor protein binding; therefore, we determined whether Rab2 stimulated kinesin or dynein membrane binding. Although kinesin was not detected on membranes incubated with Rab2, dynein was recruited in a dose-dependent manner, and binding was aPKCiota-dependent. These combined results suggest a mechanism by which Rab2 controls MT and motor recruitment to vesicular tubular clusters.

Two mechanistically distinct forms of endocytosis in adrenal chromaffin cells: Differential effects of SH3 domains and amphiphysin antagonism.

We previously identified two forms of endocytosis using capacitance measurements in chromaffin cells: rapid endocytosis (RE), dynamin-1 dependent but clathrin-independent and slow endocytosis (SE), dynamin-2 and clathrin-dependent. Various recombinant SH3 domains that interact with the proline-rich domain of dynamin were introduced into single cells via the patch pipette. GST-SH3 domains of amphiphysin-1, intersectin-IC, and endophilin-I inhibited SE but had no effect on RE. Grb2-SH3 (N-terminal) or a mutant of amphiphysin-1-SH3 was inactive on either process. These data confirm that dynamin-1 dependent RE is independent of clathrin and show that amphiphysin is exclusively associated with clathrin and dynamin-2-dependent SE.

Double patch clamp reveals that transient fusion (kiss-and-run) is a major mechanism of secretion in calf adrenal chromaffin cells: high calcium shifts the mechanism from kiss-and-run to complete fusion.

Transient fusion ("kiss-and-run") is accepted as a mode of transmitter release both in central neurons and neuroendocrine cells, but the prevalence of this mechanism compared with full fusion is still in doubt. Using a novel double patch-clamp method (whole cell/cell attached), permitting the recording of unitary capacitance events while stimulating under a variety of conditions including action potentials, we show that transient fusion is the predominant (>90%) mode of secretion in calf adrenal chromaffin cells. Raising intracellular Ca2+ concentration ([Ca]i) from 10 to 200 microM increases the incidence of full fusion events at the expense of transient fusion. Blocking rapid endocytosis that normally terminates transient fusion events also promotes full fusion events. Thus, [Ca]i controls the transition between transient and full fusion, each of which is coupled to different modes of endocytosis.

Rates of nitric oxide dissociation from hemoglobin.

Nitric oxide (NO) plays a major role in human physiology and in many pathological states. Although oxyhemoglobin is known to destroy NO activity, NO activity can, in principle, be conserved through iron nitrosylation at vacant hemes. In order for this NO activity to be delivered, the NO must dissociate from the heme. Despite its study over the past few decades, our understanding of NO dissociation from hemoglobin is incomplete. In principle, there are at least four NO dissociation rates: kR(alpha), kR(beta), kT(alpha), and kT(beta), where the subscript refers to the quaternary state and the superscript to the hemoglobin chain. In the T-state, a proportion of the proximal histidine bonds break forming pentacoordinate alpha-nitrosyl hemoglobin. In vivo, alpha-nitrosyl hemoglobin predominates over beta-nitrosyl hemoglobin. In this study we have used a fast NO trap, Fe(II)-proline-dithiocarbamate, to measure NO dissociation rates from hemoglobin. We have varied solution conditions so the rate of dissociation from pentacoordinate alpha-nitrosyl hemoglobin could be definitively measured for the first time; kT(alpha) = 4.2 +/- 1.5 x 10(-4) s(-1). We have also found that the fastest NO dissociation rate is on the order of 10(-3) s(-1) and that NO dissociation from sickle cell hemoglobin is the same as that from normal adult hemoglobin.