scholarly journals Multi-Omics Analysis Defines 5-Fluorouracil Drug Resistance in 3D Hela Carcinoma Cell Model

2021 ◽  
Author(s):  
Lin Wang ◽  
Xueting Wang ◽  
Tong Wang ◽  
Yingping Zhuang ◽  
GUAN WANG
Keyword(s):  
Drug Resistance ◽  
Drug Screening ◽  
Monolayer Culture ◽  
Tricarboxylic Acid Cycle ◽  
Critical Role ◽  
Carcinoma Cells ◽  
Metabolic Reprogramming ◽  
Holistic View ◽  
Ecm Proteins

Abstract Cervical cancer is a serious health problem in women around the globe, with 600 thousand new cases each year. However, the use of clinical drug is seriously dampened by the development of drug resistance, which has been evidenced to be associated with metabolic reprogramming and heterogeneity in tumor cells. Efficient in vitro tumor model is essential to improve the efficiency of drug screening and the accuracy of clinical application. Multicellular tumor spheroids (MTSs) can in a way recapitulates tumor traits in vivo, thereby representing a powerful transitional model between 2D monolayer culture and xenograft. In this study, based on the liquid overlay method, a protocol for rapid generation of the MTSs with uniform size and high reproducibility in a high-throughput manner was established. As expected, the cytotoxicity results showed that there was enhanced 5-FU resistance of HeLa carcinoma cells in 3D MTSs than 2D monolayer culture with a resistance index of 5.72. In the presence of both glucose and glutamine, HeLa carcinoma cells preferentially used glutamine as the bioenergetic substrate to support cell proliferation and maintenance under all conditions, while the ubiquitous by-product ammonium might be only recycled in the 3D MTSs. Furthermore, in order to obtain a holistic view of the molecular mechanisms that drive 5-FU resistance in 3D HeLa carcinoma cells, a multi-omics study was applied to discover hidden biological regularities. It was observed that in the 3D MTSs mitochondrial function-related proteins and the metabolites of the tricarboxylic acid cycle (TCA cycle) were significantly decreased, and the cellular metabolism was shifted towards glycolysis. The differences in the protein synthesis, processing, and transportation between 2D monolayer cultures and 3D MTSs was significant, mainly in the heat shock protein family, with the upregulation of protein folding function in endoplasmic reticulum (ER), which promoted the maintenance of ER homeostasis in the 3D MTSs. In addition, at the transcript and protein level, the expression of extracellular matrix (ECM) proteins (e.g., laminin and collagen) were up-regulated in the 3D MTSs, which enhanced the physical barrier of drug penetration. Summarizing, this study formulates a rapid, scalable and reproducible in vitro model of 3D MTS for drug screening purposes, and the findings establish a critical role of glycolytic metabolism, ER hemostasis and ECM proteins expression profiling in tumor chemoresistance of HeLa carcinoma cells towards 5-FU.

scholarly journals Multi-omics analysis defines 5-fluorouracil drug resistance in 3D HeLa carcinoma cell model

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Lin Wang ◽  
Xueting Wang ◽  
Tong Wang ◽  
Yingping Zhuang ◽  
Guan Wang
Keyword(s):  
Drug Resistance ◽  
Drug Screening ◽  
Molecular Mechanisms ◽  
Monolayer Culture ◽  
Tricarboxylic Acid Cycle ◽  
Critical Role ◽  
Carcinoma Cells ◽  
Holistic View ◽  
Ecm Proteins

AbstractCervical cancer is a serious health problem in women around the globe. However, the use of clinical drug is seriously dampened by the development of drug resistance. Efficient in vitro tumor model is essential to improve the efficiency of drug screening and the accuracy of clinical application. Multicellular tumor spheroids (MTSs) can in a way recapitulates tumor traits in vivo, thereby representing a powerful transitional model between 2D monolayer culture and xenograft. In this study, based on the liquid overlay method, a protocol for rapid generation of the MTSs with uniform size and high reproducibility in a high-throughput manner was established. As expected, the cytotoxicity results showed that there was enhanced 5-fluorouracil (5-FU) resistance of HeLa carcinoma cells in 3D MTSs than 2D monolayer culture with a resistance index of 5.72. In order to obtain a holistic view of the molecular mechanisms that drive 5-FU resistance in 3D HeLa carcinoma cells, a multi-omics study was applied to discover hidden biological regularities. It was observed that in the 3D MTSs mitochondrial function-related proteins and the metabolites of the tricarboxylic acid cycle (TCA cycle) were significantly decreased, and the cellular metabolism was shifted towards glycolysis. The differences in the protein synthesis, processing, and transportation between 2D monolayer cultures and 3D MTSs were significant, mainly in the heat shock protein family, with the up-regulation of protein folding function in endoplasmic reticulum (ER), which promoted the maintenance of ER homeostasis in the 3D MTSs. In addition, at the transcript and protein level, the expression of extracellular matrix (ECM) proteins (e.g., laminin and collagen) were up-regulated in the 3D MTSs, which enhanced the physical barrier of drug penetration. Summarizing, this study formulates a rapid, scalable and reproducible in vitro model of 3D MTS for drug screening purposes, and the findings establish a critical role of glycolytic metabolism, ER hemostasis and ECM proteins expression profiling in tumor chemoresistance of HeLa carcinoma cells towards 5-FU. Graphical Abstract

scholarly journals Lactate secreted by PKM2 upregulation promotes Galectin-9-mediated immunosuppression via inhibiting NF-κB pathway in HNSCC

2021 ◽  
Vol 12 (8) ◽  
Author(s):  
Hanyue Chang ◽  
Qiaoshi Xu ◽  
Jiayi Li ◽  
Mingyu Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Critical Role ◽  
Lactate Production ◽  
Metabolic Reprogramming ◽  
Migration And Invasion ◽  
Histone Deacetylase 3 ◽  
Proliferation And Metastasis ◽  
Immunosuppressive Microenvironment ◽  
Transcriptional Complex

AbstractPyruvate kinase M2 as a key rate-limiting enzyme in glycolysis, it plays a critical role in metabolic reprogramming and carcinogenesis. However, whether PKM2 can promote immunosuppressive microenvironment formation remains unknown in head and neck squamous cell carcinoma (HNSCC). PKM2 expression was detected using immunohistochemical staining. The biological functions of PKM2 were investigated in vitro and in vivo. Lactate production and the expression of Galectin-9, a critical immunosuppression molecule, were detected after PKM2 knockdown and overexpression in HNSCC cells. The mechanism of lactate regulating Galectin-9 expression through NF-κB signaling was explored in vitro. Overexpression of PKM2 correlates with poor prognosis in HNSCC patients. Silencing PKM2 markedly inhibits proliferation and metastasis capacity in vivo and in vitro, and vice versa. The glycolysis and glycolytic capacity are significantly decreased after PKM2 silencing. Lactate secretion induced by PKM2 significantly promotes migration and invasion capacity. Furthermore, a positive correlation between PKM2 and Galectin-9 expression is observed in HNSCC tissues. The induction of Galectin-9 expression by PKM2 can be affected by a lactate transporter inhibitor. Mechanically, lactate impeded the suppressive transcriptional complex formation of NF-κB and histone deacetylase 3 (HDAC3), which released the transcription of Galectin-9 mediated by NF-κB signaling. Our findings demonstrate that lactate produced by PKM2 upregulation promotes tumor progression and Galectin-9-mediated immunosuppression via NF-κB signaling inhibition in HNSCC, which bridges metabolism and immunosuppression. The novel PKM2-lactate-Galectin-9 axis might be a potential therapeutic target in HNSCC.

scholarly journals Obesity Accelerates Age-Associated Defects in Human B Cells Through a Metabolic Reprogramming Induced by the Fatty Acid Palmitate

2022 ◽  
Vol 2 ◽  
Author(s):  
Daniela Frasca ◽  
Maria Romero ◽  
Denisse Garcia ◽  
Alain Diaz ◽  
Bonnie B. Blomberg
Keyword(s):  
Fatty Acid ◽  
B Cells ◽  
Critical Role ◽  
Metabolic Reprogramming ◽  
Autoimmune Antibodies ◽  
Human B Cells ◽  
Initial Hypothesis ◽  
First Time ◽  
Culture Supernatants

We have measured the secretion of autoimmune antibodies in plasma samples and in culture supernatants of blood-derived B cells from four groups of individuals: young lean (YL), elderly lean (EL), young obese (YO) and elderly obese (EO). We found secretion comparable in YO and EL individuals, suggesting that obesity accelerates age-associated defects in circulating B cells. To define at least one possible molecular pathway involved, we used an in vitro model in which B cells from YL and EL individuals have been stimulated with the Fatty Acid (FA) palmitate, the most common saturated FA in the human body. The rationale to use palmitate is that there is a chronic increase in circulating levels of palmitate, due to increased spontaneous lipolysis occurring during aging and obesity, and this may induce autoimmune B cells. Results herein show that in vitro incubation of B cells from YL and EL individuals with the FA palmitate induces mRNA expression of T-bet, the transcription factor for autoimmune antibodies, as well as secretion of autoimmune IgG antibodies, with B cells from YL individuals looking similar to B cells from EL individuals, confirming our initial hypothesis. The generation of autoimmune B cells in the presence of the FA palmitate was found to be associated with a metabolic reprogramming of B cells from both YL and EL individuals. These results altogether show the critical role of the FA palmitate in inducing human B cell immunosenescence and show for the first time the importance of metabolic pathways in this process.

scholarly journals P-selectin glycoprotein ligand regulates the interaction of multiple myeloma cells with the bone marrow microenvironment

Blood ◽  
2012 ◽  
Vol 119 (6) ◽  
pp. 1468-1478 ◽  
Author(s):  
Abdel Kareem Azab ◽  
Phong Quang ◽  
Feda Azab ◽  
Costas Pitsillides ◽  
Brian Thompson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Stromal Cells ◽  
Critical Role ◽  
Loss Of Function ◽  
Downstream Signaling ◽  
Target Organs ◽  
Regulation Of Growth

Abstract Interactions between multiple myeloma (MM) cells and the BM microenvironment play a critical role in the pathogenesis of MM and in the development of drug resistance by MM cells. Selectins are involved in extravasation and homing of leukocytes to target organs. In the present study, we focused on adhesion dynamics that involve P-selectin glycoprotein ligand-1 (PSGL-1) on MM cells and its interaction with selectins in the BM microenvironment. We show that PSGL-1 is highly expressed on MM cells and regulates the adhesion and homing of MM cells to cells in the BM microenvironment in vitro and in vivo. This interaction involves both endothelial cells and BM stromal cells. Using loss-of-function studies and the small-molecule pan-selectin inhibitor GMI-1070, we show that PSGL-1 regulates the activation of integrins and downstream signaling. We also document that this interaction regulates MM-cell proliferation in coculture with BM microenvironmental cells and the development of drug resistance. Furthermore, inhibiting this interaction with GMI-1070 enhances the sensitization of MM cells to bortezomib in vitro and in vivo. These data highlight the critical contribution of PSGL-1 to the regulation of growth, dissemination, and drug resistance in MM in the context of the BM microenvironment.

scholarly journals A Biomaterial Screening Approach Reveals Microenvironmental Mechanisms of Drug Resistance

2017 ◽  
Author(s):  
Alyssa D. Schwartz ◽  
Lauren E. Barney ◽  
Lauren E. Jansen ◽  
Thuy V. Nguyen ◽  
Christopher L. Hall ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Systems Biology ◽  
Drug Screening ◽  
Drug Response ◽  
Drug Sensitivity ◽  
Mek Inhibitor ◽  
Tumor Burden ◽  
Adaptive Resistance

TOC FigureDrug response screening, gene expression, and kinome signaling were combined across biomaterial platforms to combat adaptive resistance to sorafenib.Insight BoxWe combined biomaterial platforms, drug screening, and systems biology to identify mechanisms of extracellular matrix-mediated adaptive resistance to RTK-targeted cancer therapies. Drug response was significantly varied across biomaterials with altered stiffness, dimensionality, and cell-cell contacts, and kinome reprogramming was responsible for these differences in drug sensitivity. Screening across many platforms and applying a systems biology analysis were necessary to identify MEK phosphorylation as the key factor associated with variation in drug response. This method uncovered the combination therapy of sorafenib with a MEK inhibitor, which decreased viability on and within biomaterials in vitro, but was not captured by screening on tissue culture plastic alone. This combination therapy also reduced tumor burden in vivo, and revealed a promising approach for combating adaptive drug resistance.AbstractTraditional drug screening methods lack features of the tumor microenvironment that contribute to resistance. Most studies examine cell response in a single biomaterial platform in depth, leaving a gap in understanding how extracellular signals such as stiffness, dimensionality, and cell-cell contacts act independently or are integrated within a cell to affect either drug sensitivity or resistance. This is critically important, as adaptive resistance is mediated, at least in part, by the extracellular matrix (ECM) of the tumor microenvironment. We developed an approach to screen drug responses in cells cultured on 2D and in 3D biomaterial environments to explore how key features of ECM mediate drug response. This approach uncovered that cells on 2D hydrogels and spheroids encapsulated in 3D hydrogels were less responsive to receptor tyrosine kinase (RTK)-targeting drugs sorafenib and lapatinib, but not cytotoxic drugs, compared to single cells in hydrogels and cells on plastic. We found that transcriptomic differences between these in vitro models and tumor xenografts did not reveal mechanisms of ECM-mediated resistance to sorafenib. However, a systems biology analysis of phospho-kinome data uncovered that variation in MEK phosphorylation was associated with RTK-targeted drug resistance. Using sorafenib as a model drug, we found that co-administration with a MEK inhibitor decreased ECM-mediated resistance in vitro and reduced in vivo tumor burden compared to sorafenib alone. In sum, we provide a novel strategy for identifying and overcoming ECM-mediated resistance mechanisms by performing drug screening, phospho-kinome analysis, and systems biology across multiple biomaterial environments.

scholarly journals Microencapsulated multicellular tumor spheroids:preparation and use as a novel in vitro model for drug screening

2010 ◽  
Vol 56 (6) ◽  
pp. 674-685 ◽  
Author(s):  
A.M. Tsoy ◽  
D.S. Zaytseva-Zotova ◽  
E.F. Edelweiss ◽  
A. Bartkowiak ◽  
J-L. Goergen ◽  
...  
Keyword(s):  
Drug Screening ◽  
In Vitro Model ◽  
Monolayer Culture ◽  
Breast Adenocarcinoma ◽  
Viable Cells ◽  
Adenocarcinoma Cells ◽  
Mean Size ◽  
Vitro Model ◽  
Mcf 7

In the current study a technique for microencapsulation of human breast adenocarcinoma cells MCF-7 in alginate-chitosan microcapsules is used. Microencapsulation is proposed to generate multicellular tumor spheroids (MTS) based on these cells and to test them further as an in vitro model for anti-tumor drug screening. Cytotoxicity of methotrexate (MTX) was studied on the obtained MTS. A set of MTS with mean size of 150, 200 and 300 m was prepared in function of a cultivation time. After incubation of MTS in cultivation medium containing MTX at concentrations of 1, 2, 10, 50 and 100 nM for 48 hs cell viability was evaluated. MTS were shown to be more resistant to MTX than the monolayer culture, and the resistance to MTX was increased with enhancing a spheroid size. At MTX concentration of 100 nM a number of viable cells in MTS with the size of 300 m was 2.5-fold bigger than that one in monolayer culture. It is suggested that the cells in microencapsulated MTS can better mimic cell behavior in a small size solid tumor than the cells in a monolayer culture. In future microencapsulated MTS can be proposed as a novel in vitro model for anticancer drug screening.

scholarly journals Inhibiting adenine synthesis attenuates glioblastoma cell stemness and temozolomide resistance

2021 ◽  
Author(s):  
Simranjit X. Singh ◽  
Rui Yang ◽  
Kristen Roso ◽  
Landon J. Hansen ◽  
Changzheng Du ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mitochondrial Function ◽  
Brain Cancer ◽  
De Novo ◽  
Critical Role ◽  
Glioblastoma Cell ◽  
Respiratory Capacity ◽  
Complementary Approach

Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that homozygous MTAP deletion, a genetic alteration occurring in about half of all GBMs, promotes stemness in GBM cells. Exploiting MTAP loss-conferred deficiency in adenine salvage, we demonstrate that transient adenine blockade via treatment with L-Alanosine (ALA), an inhibitor of de novo adenine synthesis, attenuates stemness of MTAP-deficient GBM cells. This ALA-induced reduction in stemness is accompanied by compromised mitochondrial function, highlighted by diminished spare respiratory capacity. Direct pharmacological inhibition of mitochondrial respiration recapitulates the effect of ALA on GBM cell stemness, suggesting ALA targets stemness partially via affecting mitochondrial function. Finally, in agreement with diminished stemness and compromised mitochondrial function, we show that ALA sensitizes GBM cells to temozolomide (TMZ) in vitro and in an orthotopic GBM model. Collectively, these results identify critical roles of adenine supply in maintaining mitochondrial function and stemness of GBM cells, highlight a critical role of mitochondrial function in sustaining GBM stemness, and implicate adenine synthesis inhibition as a complementary approach for treating MTAP-deleted GBMs.

scholarly journals 3D Quantitative and Ultrastructural Analysis of Mitochondria in a Model of Doxorubicin Sensitive and Resistant Human Colon Carcinoma Cells

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1254 ◽  
Author(s):  
Claudia Moscheni ◽  
Emil Malucelli ◽  
Sara Castiglioni ◽  
Alessandra Procopio ◽  
Clara De Palma ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Colon Carcinoma ◽  
Three Dimensional ◽  
Human Colon ◽  
Carcinoma Cells ◽  
Metabolic Reprogramming ◽  
Cancer Drug ◽  
Mitochondrial Morphology ◽  
Colon Carcinoma Cells ◽  
Resistant Cells

Drug resistance remains a major obstacle in cancer treatment. Because mitochondria mediate metabolic reprogramming in cancer drug resistance, we focused on these organelles in doxorubicin sensitive and resistant colon carcinoma cells. We employed soft X-ray cryo nano-tomography to map three-dimensionally these cells at nanometer-resolution and investigate the correlation between mitochondrial morphology and drug resistance phenotype. We have identified significant structural differences in the morphology of mitochondria in the two strains of cancer cells, as well as lower amounts of Reactive oxygen species (ROS) in resistant than in sensitive cells. We speculate that these features could elicit an impaired mitochondrial communication in resistant cells, thus preventing the formation of the interconnected mitochondrial network as clearly detected in the sensitive cells. In fact, the qualitative and quantitative three-dimensional assessment of the mitochondrial morphology highlights a different structural organization in resistant cells, which reflects a metabolic cellular adaptation functional to survive to the offense exerted by the antineoplastic treatment.

scholarly journals Study on Biological Characteristics and Mechanism of Paclitaxel Induced Drug Resistance in Endometrial Carcinoma Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Jie Ding ◽  
Mengxiong Li ◽  
Liuzhi Deng ◽  
Tian Li
Keyword(s):  
Drug Resistance ◽  
Endometrial Carcinoma ◽  
Inhibitory Effect ◽  
Resistant Cell ◽  
Carcinoma Cells ◽  
Biological Characteristics ◽  
High Dose ◽  
Apoptosis Rate ◽  
P70 S6k

Objective. To study the biological characteristics of paclitaxel resistant endometrial carcinoma cells and its mechanism of drug resistance.Method. The paclitaxel resistant cell lines were established by high-dose paclitaxel (TAX) injection. The IC50 of paclitaxel was determined by CCK-8 assay in Ishikawa and Ishikawa-TAX. The cell cycle and apoptosis rate were detected by flow cytometry. Western blot was used to detect the expression of p-AKT and p-p70S6K. The expression of drug resistance-related genes Pgp and MDR1 was determined by RT-PCR. Cell viability was determined by soft agarose assay and invasive ability in vitro by transwell assay.Results. Paclitaxel and NVP-BEZ235 cotreatment group can further inhibit the clonogenicity and invasion of Ishikawa and Ishikawa-TAX cells compared with paclitaxel alone and NVP-BEZ235 treatment group. Paclitaxel and NVP-BEZ235 cotreated groups increased the apoptosis rate of Ishikawa and increased G0/G1 phase arrest in both cells. Paclitaxel alone significantly inhibited p-AK and p-p70 S6K protein expression in Ishikawa and Ishikawa-TAX cells and the inhibition was enhanced by NVP-BEZ235 when cotreated with paclitaxel.Conclusion. Paclitaxel can inhibit Ishikawa and Ishikawa-TAX cell via PI3K/Akt/mTOR signaling pathway. Paclitaxel and NVP-BEZ235 cotreatment can enhance the inhibitory effect.

Cannabis sativa L. Extracts can reverse drug resistance in colorectal carcinoma cells in vitro

Synergy ◽  
2019 ◽  
Vol 9 ◽  
pp. 100056 ◽  
Author(s):  
Innocensia Mokgohlwe Mangoato ◽  
Chandrashekara Puthanapura Mahadevappa ◽  
Motlalepula Gilbert Matsabisa
Keyword(s):  
Drug Resistance ◽  
Colorectal Carcinoma ◽  
Cannabis Sativa ◽  
Carcinoma Cells
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