HIF-1α drives resistance to ferroptosis in solid tumors by promoting lactate production and activating SLC1A1

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Comparing solid tumors with cell lines: implications for identifying drug resistance genes in cancer

Gene expression arrays allow researchers to profile the differences between cell lines or tissues and they may identify genetic markers of development, organ maturation, or tumor progression. Although a primary tumor that grows in a host and a tumor-cell-line derived from that primary tumor and grown in vitro share similar gene expression profiles, there are, not unexpectedly, some important differences. In fact, Stein and colleagues have found that genes that are differentially expressed in primary tumors as compared to the specific genes expressed in their cell-line derivatives are more reliably predictive of tumor tractability. Thus, sensitivity in vitro might not reflect sensitivity in vivo. Because anti-tumor compounds are largely evaluated in cell culture assays, these compounds' therapeutic utility must be judged in light of genes described by Stein et al. that better predict tractability.     

A mathematical model to study the effects of drug resistance and vasculature on the response of solid tumors to chemotherapy

A mathematical model is developed that describes the reduction in volume of a vascular tumor in response to specific chemotherapeutic administration strategies. The model consists of a system of partial differential equations governing intratumoral drug concentration and cancer cell density. In the model the tumor is treated as a continuum of two types of cells which differ in their proliferation rates and their responses to the chemotherapeutic agent. The balance between cell proliferation and death within the tumor generates a velocity field which drives expansion or regression of the spheroid. Insight into the tumor's response to therapy is gained by applying a combination of analytical and numerical techniques to the model equations.     

Microfluorimetric analysis of dynamics of genomic changes in Chinese hamster fibroblasts CHL V-79 RJK with multiple drug resistance

Results of a karyological analysis of cells CHL V-79 RJK selected for their resistance to ethidium bromide (EB) causing multidrug resistance (MDR) (subline Vebr-5) were compared with data from the microfluorimetric determination of the DNA content in individual chromosomes of the karyotype. The analysis was performed at the 11th and 88th passages. Karyotyping of Vebr-5 has shown the presence of an additional genetic material (AGM) in the form of homogenously or differentially stained regions (HSR or DSR, respectively) in two chromosomes (Z1 and Z6, loci 1p29-31 and 1q26, respectively). HSR in Z6, in the site of localization of the mdr gene of the wild type had unstable length and structure, which is characteristic of the morphological markers of the amplification of genes of the mdr family. During the long cultivation of Vebr-5 in the presence of EB (88 passages), the instability of HSR (DSR) in Z6 increased. Results of a microfluorimetric analysis of Vebr-5 at the 11th passage have shown a statistically significant increase of the DNA content not only in chromosomes Z1 and Z6 marked by HSR (DSR), but also in three chromosomes (Z5, Z12, and Z13) that have no visual morphological changes. The corresponding analysis at the 88th passage has also revealed nonrandom changes in the DNA content in four more chromosomes, including an increase in Z14 and a decrease in chromosomes 8, Z7, and Z9. A decrease in the DNA content in chromosomes is considered to be the result of a partial loss of genetic material, while its increase is considered to be the result of its translocation and/or amplification. While the coefficient of the variation of the DNA content changes about 9% for large chromosomes, it amounted to about 26% for the small ones, indicating that small chromosomes to have a greater potential for instability than large chromosomes. The obtained data not only confirm, but also enlarge, the concept of the directions and character of the destabilization of the cell genetic apparatus in the process of neoplastic transformation due to MDR acquisition by these cells.     

Autophagy promotes resistance to photodynamic therapy-induced apoptosis selectively in colorectal cancer stem-like cells

Recent studies have indicated that cancer stem-like cells (CSCs) exhibit a high resistance to current therapeutic strategies, including photodynamic therapy (PDT), leading to the recurrence and progression of colorectal cancer (CRC). In cancer, autophagy acts as both a tumor suppressor and a tumor promoter. However, the role of autophagy in the resistance of CSCs to PDT has not been reported. In this study, CSCs were isolated from colorectal cancer cells using PROM1/CD133 (prominin 1) expression, which is a surface marker commonly found on stem cells of various tissues. We demonstrated that PpIX-mediated PDT induced the formation of autophagosomes in PROM1/CD133⁺ cells, accompanied by the upregulation of autophagy-related proteins ATG3, ATG5, ATG7, and ATG12. The inhibition of PDT-induced autophagy by pharmacological inhibitors and silencing of the ATG5 gene substantially triggered apoptosis of PROM1/CD133⁺ cells and decreased the ability of colonosphere formation in vitro and tumorigenicity in vivo. In conclusion, our results revealed a protective role played by autophagy against PDT in CSCs and indicated that targeting autophagy could be used to elevate the PDT sensitivity of CSCs. These findings would aid in the development of novel therapeutic approaches for CSC treatment.     

A methodology for elucidating regulatory mechanisms leading to changes in lipid profiles

Introduction

It is difficult to elucidate the metabolic and regulatory factors causing lipidome perturbations.

Objectives

This work simplifies this process.

Methods

A method has been developed to query an online holistic lipid metabolic network (of 7923 metabolites) to extract the pathways that connect the input list of lipids.

Results

The output enables pathway visualisation and the querying of other databases to identify potential regulators. When used to a study a plasma lipidome dataset of polycystic ovary syndrome, 14 enzymes were identified, of which 3 are linked to ELAVL1—an mRNA stabiliser.

Conclusion

This method provides a simplified approach to identifying potential regulators causing lipid-profile perturbations.

     

Up-to-date approaches to clinical estimate of phenotype of multiple drug resistance associated with function of ABC-transporters in human solid tumors]

The data on the discovery, specific features and evolution of the aftereffects of functioning in the cells of the ABC-transporters Pgp, MRP and BCRP, the markers of multiple drug resistance (MDRABC), are discussed. The results of the estimate of the MDRABC phenotype of human solid tumors were critically analyzed using different methodic approaches. It was shown that the frequency of the MDRABC phenotype detection by expression of the genes, encoding the ABC-transporter synthesis, was higher than that in detection of transport proteins in the cell. It was concluded that the only adequate clinical method for diagnosis of the MDRABC phenotype could be differential estimate of the functional activity of the ABC-transporters controlling not only the drug release to the cell, but also the drug intracellular compartmentization or division between the cytoplasm and nucleus.     

Capillary electrophoresis-single strand conformation polymorphism for the detection of multiple mutations leading to tuberculosis drug resistance

For rate determinations of anaerobic metabolism it is essential to maintain strictly anoxic conditions throughout the experiment. However, even if oxygen contamination can be avoided while preparing the incubation containers, it is still possible that the incubation containers themselves contaminate the samples by oxygen diffusing from or through their plastic or rubber components. In this study, we investigated the sources and extent of oxygen contamination during anoxic incubations, and present solutions to minimize oxygen contamination. In particular, we investigated oxygen contamination in Labco® Exetainers, glass vials with a butyl rubber septum in the screw cap, which are frequently used in microbiological experiments. Our results show that significant oxygen contamination occurred at different stages during the incubation. Contamination occurred when Exetainers were either filled or incubated for more than 16 h under oxic atmosphere, but also under an oxygen-free atmosphere due to diffusion of oxygen out of the butyl rubber septum. Therefore, to avoid oxygen contamination during incubations, we suggest (1) filling and incubating the incubation containers under anoxic atmosphere (glove bag) and (2) deoxygenating all elastomers in sample processing and incubation equipment. If initial oxygen contamination cannot be avoided, introduction of an anoxic headspace might help extract oxygen from the incubated sample and present a buffer against oxygen diffusing out of the septum. We modeled the amount of oxygen diffusing out of butyl rubber septa under different conditions, and results fitted well with the observed oxygen contamination. Thus, the model can be used to predict oxygen contamination under varying conditions and for differently sized septa.     

CRISPR technology: A versatile tool to model,screen, and reverse drug resistance in cancer

BackgroundDrug resistance is a serious challenge in cancer treatment that can render chemotherapy a failure. Understanding the mechanisms behind drug resistance and developing novel therapeutic approaches are cardinal steps in overcoming this issue. Clustered regularly interspaced short palindrome repeats (CRISPR) gene-editing technology has proven to be a useful tool to study cancer drug resistance mechanisms and target the responsible genes. In this review, we evaluated original research studies that used the CRISPR tool in three areas related to drug resistance, namely screening resistance-related genes, generating modified models of resistant cells and animals, and removing resistance by genetic manipulation. We reported the targeted genes, study models, and drug groups in these studies. In addition to discussing different applications of CRISPR technology in cancer drug resistance, we analyzed drug resistance mechanisms and provided examples of CRISPR’s role in studying them. Although CRISPR is a powerful tool for examining drug resistance and sensitizing resistant cells to chemotherapy, more studies are required to overcome its disadvantages, such as off-target effects, immunotoxicity, and inefficient delivery of CRISPR/cas9 into the cells.     

Biomarker-guided sequential targeted therapies to overcome therapy resistance in rapidly evolving highly aggressive mammary tumors

Combinatorial targeted therapies are more effective in treating cancer by blocking by-pass mechanisms or inducing synthetic lethality. However, their clinical application is hampered by resistance and toxicity. To meet this important challenge, we developed and tested a novel concept of biomarker-guided sequential applications of various targeted therapies using ErbB2-overexpressing/PTEN-low, highly aggressive breast cancer as our model. Strikingly, sustained activation of ErbB2 and downstream pathways drives trastuzumab resistance in both PTEN-low/trastuzumab-resistant breast cancers from patients and mammary tumors with intratumoral heterogeneity from genetically-engineered mice. Although lapatinib initially inhibited trastuzumab-resistant mouse tumors, tumors by-passed the inhibition by activating the PI3K/mTOR signaling network as shown by the quantitative protein arrays. Interestingly, activation of the mTOR pathway was also observed in neoadjuvant lapatinib-treated patients manifesting lapatinib resistance. Trastuzumab + lapatinib resistance was effectively overcome by sequential application of a PI3K/mTOR dual kinase inhibitor (BEZ235) with no significant toxicity. However, our p-RTK array analysis demonstrated that BEZ235 treatment led to increased ErbB2 expression and phosphorylation in genetically-engineered mouse tumors and in 3-D, but not 2-D, culture, leading to BEZ235 resistance. Mechanistically, we identified ErbB2 protein stabilization and activation as a novel mechanism of BEZ235 resistance, which was reversed by subsequent treatment with lapatinib + BEZ235 combination. Remarkably, this sequential application of targeted therapies guided by biomarker changes in the tumors rapidly evolving resistance doubled the life-span of mice bearing exceedingly aggressive tumors. This fundamentally novel approach of using targeted therapies in a sequential order can effectively target and reprogram the signaling networks in cancers evolving resistance during treatment.     

A robust in vivo positive-readout system for monitoring siRNA delivery to xenograft tumors

Delivering small interfering RNA (siRNA) to tumors is the major technical hurdle that prevents the advancement of siRNA-based cancer therapy. One of the difficulties associated with the development of clinically relevant delivery systems is the lack of reliable tools for monitoring siRNA delivery to tumors in vivo. We describe here a novel, positive-readout system where siRNA-mediated target knockdown elicits a rapid and robust increase of reporter activity. Using the positive-readout system, we created (1) β-galactosidase-based tumor models that allow the detection of target knockdown in 1%-2% of tumor cells and can distinguish between tumor areas where effective target knockdown occurs versus tumor areas that are not accessible to delivery, and (2) luciferase-based tumor models that allow the quantitative assessment of a large number of delivery systems. Using these positive-readout models, we screened a number of literature-described siRNA delivery systems and identified lipid nanoparticles as a promising delivery platform for siRNA-based cancer therapy.     

3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs

Three-dimensional cell culture models, such as spheroids, can be used in the process of the development of new anticancer agents because they are able to closely mimic the main features of human solid tumors, namely their structural organization, cellular layered assembling, hypoxia, and nutrient gradients. These properties imprint to the spheroids an anticancer therapeutics resistance profile, which is similar to that displayed by human solid tumors. In this review, an overview of the drug resistance mechanisms observed in 3D tumor spheroids is provided. Furthermore, comparisons between the therapeutics resistance profile exhibited by spheroids, and 2D cell cultures are presented. Finally, examples of the therapeutic approaches that have been developed to surpass the drug resistance mechanisms exhibited by spheroids are described.     

A mechanism of drug resistance to tamoxifen in breast cancer

Drug resistance to tamoxifen (Tam) is a significant clinical problem but the mechanism through which this occurs remains elusive. We have developed a number of xenograft models of Tam-stimulated growth that model breast cancer progression using estrogen receptor positive MCF-7 or T47D breast cancer cells. When estrogen-stimulated T47D:E2 tumors are treated long term with Tam, Tam-stimulated tumors develop (T47D:Tam) that are stimulated by both estrogen and Tam. When HER-2/neu status is determined, it is clear that the T47D:Tam tumors express significantly higher levels of HER-2/neu protein by immunohistochemistry and mRNA as measured by real-time RT-PCR. The T47D:Tam tumors also express higher levels of estrogen receptor and progesterone receptor protein than their estrogen-stimulated T47D:E2 counterparts. We compared out results to the MCF-7 model of Tam-stimulated growth. The MCF-7:Tam ST (estrogen- and Tam-stimulated) and MCF-7:Tam LT (estrogen-inhibited, Tam-stimulated) were bilaterally transplanted to account for any mouse to mouse variation and characteristic growth patterns were observed. TUNEL staining was performed on MCF-7:Tam LT treated with either estrogen or Tam and it was concluded that estrogen-inhibited tumor growth was a result of increased apoptosis. Three phases of tumor progression are described that involve increases in HER-2/neu expression, de-regulation of estrogen receptor expression and increases in apoptosis which in concert determine the phenotype of drug resistance to Tam.     

Estimation of genetic trends from 1977 to 1998 for farrowing characteristics in the French Large White breed using frozen semen

The objective of the study was to estimate genetic trends from 1977 to 1998 in the French Large White (LW) breed for stillbirth and associated traits measured at farrowing using frozen semen. Two groups of pigs (G77 and G98) were obtained by inseminating LW sows with semen from LW boars born either in 1977 or in 1998. A second generation was produced by inter se mating in each group. Farrowing was thoroughly supervised through both direct observations and video recording all long farrowing on a total of 137 first- and second-parity litters produced by sows from this second generation (68 G77 and 69 G98 litters, respectively). Measurements included birth time, weight and birth characteristics (including orientation, presence of cyanosis or oedema, membrane obstruction, umbilical cord length/content) of each piglet, as well as sow traits (weight and backfat thickness, farrowing duration, litter size and within-litter variation of weights at birth). The data were analysed using linear or generalised linear mixed models, according to the definition of the trait (continuous or binary data). The importance of several effects to piglet probability of stillbirth was then quantified by computing the reduction of variance associated with the addition of each effect in the model. Litter size did not significantly differ in first parity, but was higher in G98 second-parity sows: the differences for global (including pre partum dead piglets) and total numbers of piglets born per litter were +2.3 ± 1.1 and +1.3 ± 0.6, respectively. G98 sows also had a higher number of stillbirths in both parities (+0.7 ± 0.3 stillborn per litter). Piglets from G98 litters were heavier at birth (+130 ± 40 g for birth weight adjusted for litter size), without any increase in within-litter heterogeneity of birth weight. No significant difference was detected between G77 and G88 groups for farrowing length and the distribution of time interval between piglet births. G98 stillborn piglets had longer and more often empty umbilical cords at birth. G98 piglets born alive also had more often umbilical nodes than G77 piglets. These characteristics were considered as indicators of increased farrowing difficulties and risk of hypoxia at birth in G98 pigs. Time of birth of each piglet, sow fatness at farrowing and time of first placenta expulsion were the main factors of variation of the piglet's probability of stillbirth.     

Pathway engineering for healthy phytochemicals leading to the production of novel flavonoids in tomato fruit

Flavonoids are a large family of plant polyphenolic secondary metabolites. Although they are widespread throughout the plant kingdom, some flavonoid classes are specific for only a few plant species. Due to their presumed health benefits there is growing interest in the development of food crops with tailor-made levels and composition of flavonoids, designed to exert an optimal biological effect. In order to explore the possibilities of flavonoid engineering in tomato fruits, we have targeted this pathway towards classes of potentially healthy flavonoids which are novel for tomato. Using structural flavonoid genes (encoding stilbene synthase, chalcone synthase, chalcone reductase, chalcone isomerase and flavone synthase) from different plant sources, we were able to produce transgenic tomatoes accumulating new phytochemicals. Biochemical analysis showed that the fruit peel contained high levels of stilbenes (resveratrol and piceid), deoxychalcones (butein and isoliquiritigenin), flavones (luteolin-7-glucoside and luteolin aglycon) and flavonols (quercetin glycosides and kaempferol glycosides). Using an online high-performance liquid chromatography (HPLC) antioxidant detection system, we demonstrated that, due to the presence of the novel flavonoids, the transgenic tomato fruits displayed altered antioxidant profiles. In addition, total antioxidant capacity of tomato fruit peel with high levels of flavones and flavonols increased more than threefold. These results on genetic engineering of flavonoids in tomato fruit demonstrate the possibilities to change the levels and composition of health-related polyphenols in a crop plant and provide more insight in the genetic and biochemical regulation of the flavonoid pathway within this worldwide important vegetable.     

Stochastic modeling of cellular colonies with quiescence: an application to drug resistance in cancer

Several cancers are thought to be driven by cells with stem cell like properties. An important characteristic of stem cells, which also applies to primitive tumor cells, is the ability to undergo quiescence, where cells can temporarily stop the cell cycle. Cellular quiescence can affect the kinetics of tumor growth, and the susceptibility of the cells to therapy. To study how quiescence affects treatment, we formulate a stochastic birth-death process with quiescence, on a combinatorial cellular mutation network, and consider the pre-treatment (growth) and treatment (decay) regimes. We find that, in the absence of mutations, treatment (if sufficiently strong) will proceed as a biphasic decline with the first (faster) phase driven by the elimination of the cycling cells and the second (slower) phase limited by the process of cell awakening. Other regimes are possible for weaker treatments. We also describe how the process of mutant generation is influenced by quiescence. Interestingly, for single-drug treatments, the probability to have resistance at start of treatment is independent of quiescence. For two or more drugs, the probability to have generated resistant mutants before treatment grows with quiescence. Finally, we study the influence of quiescence on the treatment phase. Starting from a given composition of mutants, the chances of treatment success are not influenced by the presence of quiescence.