There is growing evidence to support a role for the ceramide-metabolizing enzyme, glucosylceramide synthase (GCS), in resistance to a variety of chemotherapeutic agents. Whether GCS contributes to ...oxaliplatin resistance in colorectal cancer (CRC) has not yet been determined. We have addressed this potentially important clinical issue by examining GCS function in two panels of oxaliplatin-resistant, isogenic CRC cell lines. Compared to parental cell lines, oxaliplatin-resistant cells have increased expression of GCS protein associated with increased levels of the pro-survival ceramide metabolite, glucosylceramide (GlcCer). Inhibition of GCS expression by RNAi-mediated gene knockdown resulted in a reduction in cellular GlcCer levels, with restored sensitivity to oxaliplatin. Furthermore, oxaliplatin-resistant CRC cells displayed lower ceramide levels both basally and after treatment with oxaliplatin, compared to parental cells. GlcCer, formed by GCS-mediated ceramide glycosylation, is the precursor to a complex array of glycosphingolipids. Differences in cellular levels and species of gangliosides, a family of glycosphingolipids, were also seen between parental and oxaliplatin-resistant CRC cells. Increased Akt activation was also observed in oxaliplatin-resistant CRC cell lines, together with increased expression of the anti-apoptotic protein survivin. Finally, this study shows that GCS protein levels are greatly increased in human CRC specimens, compared to matched, normal colonic mucosa, and that high levels of UGCG gene expression are significantly associated with decreased disease-free survival in colorectal cancer patients. These findings uncover an important cellular role for GCS in oxaliplatin chemosensitivity and may provide a novel cellular target for augmenting chemotherapeutic drug effectiveness in CRC.
Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three‐dimensional (3D) bioreactor system where ...cancer cells are grown in Matrigel in modified six‐well plates. Oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells, with microfabricated PDMS pillars to control oxygen delivery. The plates receive 3% oxygen from below and 0% oxygen at the top surface of the media, providing a gradient of 3–0% oxygen. We compared growth and transcriptional profiles for cancer cells grown in Matrigel in the bioreactor, 3D cultures grown in 21% oxygen, and cells grown in a standard hypoxia chamber at 3% oxygen. Additionally, we compared gene expression of conventional two‐dimensional monolayer culture and 3D Matrigel culture in 21% oxygen. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.
Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three‐dimensional (3D) bioreactor system where cancer cells are grown in Matrigel and oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.
Inside Cover Image, Volume 234, Number 11, November 2019 Wulftange, William J.; Rose, Michelle A.; Garmendia‐Cedillos, Marcial ...
Journal of cellular physiology,
November 2019, 2019-11-00, 20191101, Letnik:
234, Številka:
11
Journal Article
Recenzirano
Inside Cover: The cover image is based on the Original Research Article Spatial control of oxygen delivery to 3D cultures alters cancer cell growth and gene expression by William J. Wulftange et al., ...DOI: 10.1002/jcp.28665.
An accurate way to characterize the functional potential of a protein is to analyze recognized protein domains encoded by the genes in a given group. The epsin N-terminal homology (ENTH) domain is an ...evolutionarily conserved protein module found primarily in proteins that participate in clathrin-mediated trafficking. In this work, we investigate the function of the single ENTH-containing protein from the protist Giardia lamblia by testing its function in Saccharomyces cerevisiae. This protein, named GlENTHp (for G. lamblia ENTH protein), is involved in Giardia in endocytosis and in protein trafficking from the ER to the vacuoles, fulfilling the function of the ENTH proteins epsin and epsinR, respectively. There are two orthologs of epsin, Ent1p and Ent2p, and two orthologs of epsinR, Ent3p and Ent5p in S. cerevisiae. Although the expression of GlENTHp neither complemented growth in the ent1Δent2Δ mutant nor restored the GFP-Cps1 vacuolar trafficking defect in ent3Δent5Δ, it interfered with the normal function of Ent3/5 in the wild-type strain. The phenotype observed is linked to a defect in Cps1 localization and α-factor mating pheromone maturation. The finding that GlENTHp acts as dominant negative epsinR in yeast cells reinforces the phylogenetic data showing that GlENTHp belongs to the epsinR subfamily present in eukaryotes prior to their evolution into different taxa.
•The protein GlENTHp (for Giardia lamblia ENTH protein) acts an Ent3/5p dominant-negative protein in Saccharomyces cerevisiae.•GlENTHp impairs the correct clathrin-mediated trafficking between the TGN and the vacuole.•GlENTHp might belong to the epsinR subfamily.•A higher conservation of the protein sequence is required for proper function of the clathrin-coated machinery in yeast cells.
The platinum drugs cisplatin, carboplatin, and oxaliplatin are highly utilized in the clinic and as a consequence have been extensively studied in the laboratory setting, sometimes by generating ...fluorophore-tagged analogs. Here, we synthesized two Pt(II) complexes containing ethane-1,2-diamine ligands linked to a BODIPY fluorophore, and compared their biological activity with previously reported Pt(II) complexes conjugated to carboxyfluorescein and carboxyfluorescein diacetate. The cytotoxicity and DNA damage capacity of Pt–fluorophore complexes was compared to cisplatin, and the Pt–BODIPY complexes were found to be more cytotoxic with reduced cytotoxicity in cisplatin-resistant cells. Microscopy revealed a predominately cytosolic localization, with nuclear distribution at higher concentrations. Spheroids grown from parent and resistant cells revealed penetration of Pt–BODIPY into spheroids, and retention of the cisplatin-resistant spheroid phenotype. While most activity profiles were retained for the Pt–BODIPY complexes, accumulation in resistant cells was only slightly affected, suggesting that some aspects of Pt–fluorophore cellular pharmacology deviate from cisplatin.
Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin‐mediated endocytosis (CME), adaptors bind to cytoplasmic regions of ...transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin‐associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N‐terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME‐deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony‐sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)‐dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin‐independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild‐type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1‐dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT‐dependent trafficking on endocytic recycling and the PM.
In a WT cell (left), endocytosis delivers protein cargoes to late endosomes; given the recently changing views on the structure of the yeast endomembrane system, dotted lines are used here to represent the multiple potential trafficking pathways between multivesicular bodies and the plasma membrane (PM). Cargoes can then be trafficked into vesicles within late endosomes and delivered to the vacuole lumen for degradation. In many endocytosis mutants (center), cargoes accumulate at the PM as a result of decreased endocytic efficiency. In cells that lack Vps4, protein cargoes that are normally trafficked to the vacuole lumen instead accumulate in class E organelles, stacks of membranous tubules adjacent to vacuoles. In the absence of Vps4, internalized cargoes are aberrantly returned to the PM via endocytic recycling. We propose that, when these mutations are combined (right), the redirection of cargo to the PM in an endocytosis mutant, which already has an elevated concentration of cell‐surface proteins, leads to the synthetic lethality observed in this study.
Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three-dimensional bioreactor system where cancer ...cells are grown in Matrigel in modified 6-well plates. Oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells, with microfabricated PDMS pillars to control oxygen delivery. The plates receive 3% oxygen from below and 0% oxygen at the top surface of the media, providing a gradient of 3% to 0% oxygen. We compared growth and transcriptional profiles for cancer cells grown in Matrigel in the bioreactor, 3D cultures grown in 21% oxygen, and cells grown in a standard hypoxia chamber at 3% oxygen. Additionally, we compared gene expression of conventional 2D monolayer culture and 3D Matrigel culture in 21% oxygen. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.
Early diagnosis of colorectal cancer (CRC) and access to optimal treatment achieves optimal cancer outcomes. However, CRC survival inequalities persist with a lower survival rate for older patients ...(≥65 years). Although the reasons for poorer cancer survival in older people are complex, evidence suggests that these patients are less likely to receive best practice care as indicated by access to multidisciplinary team (MDT) care. Three electronic databases were systematically searched to examine factors that affect access to, and clinical decision-making, in the context of MDT care of older people with CRC. We included studies reporting empirical data relating to predictors for a patient's case being discussed at a MDT meeting and/or factors that impact treatment decision-making during the meeting. From 303 returned titles and abstracts, eighteen articles were reviewed. Eight studies specifically selected older patients, with eligibility criteria varying from ≥65 to ≥80 years. Five articles explored predictors of MDT access, with all articles identifying age as a negative, and advanced stage as a positive predictor of MDT discussion. Fourteen studies explored factors that influenced the MDT decision-making process, with older age and presence of comorbid disease negatively influencing treatment decisions (cases less often discussed and/or treatment not recommended). A few studies identified access to a MDT discussion as an independent predictor for CRC treatment. Access to the MDT process for older patients with a CRC diagnosis should be based on relevant geriatric domains rather than on chronological age alone, which is expected to allow more appropriate clinical decision-making and reduce treatment inequities for older patients with cancer.
Restricted number of neuroendocrine tumors (NET) shows overexpression of somatostatin receptors. Therefore, long-acting somatostatin analogues are used in diagnosis and treatment of those tumors. ...Here we present our first case of NET, localized in pancreas treated with DOTA-D-Phe 1-Tyr 3-octreotide (DOTATATE), for receptor-mediated radioisotope therapy. DOTATATE is a newly developed somatostatin analogue labeled with beta-emitter yttrium 90 (90Y) and beta, gamma-emitter lutetium 177 (177Lu). A 34-year old woman was suffering from several years gastrointestinal symptoms. NET of the pancreas with multiple metastases into the liver was diagnosed based on histopatological, biochemical and radiological tests. First, she had chemiotherapy (leucovorin, 5-FU, cisplatin), however there was any positive effects of this therapy. Next, she received four single doses of 90Y DOTATATE at 4-6- week intervals, yielding a cumulative dose of 7.4 GBq/m2. During the 4th cycle the Lu-177 DOTATATE was additionally administered. As a renal protection i.v. infusion of amino acid solution were used during the treatment sessions. To date, patient has shown partial remission with reduction of tumor masses. We observed spectacular clinical, biochemical and radiological improvement. Radioisotope therapy could be a powerful and promising method of treatment at least in patients who had no other treatment option.
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