Data from Neem Leaf Glycoprotein Disrupts Exhausted CD8<sup>+</sup> T-Cell–Mediated Cancer Stem Cell Aggression
Mohona ChakravartiSaurav BeraSukanya DharAnirban SarkarPritha Roy ChoudhuryNilanjan GangulyJuhina DasJasmine SultanaAishwarya GuhaSouradeep BiswasTapasi DasSubhadip HajraSaptak BanerjeeRathindranath BaralAnamika Bose
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<div>Abstract<p>Targeting exhausted CD8<sup>+</sup> T-cell (T<sub>EX</sub>)–induced aggravated cancer stem cells (CSC) holds immense therapeutic potential. In this regard, immunomodulation via Neem Leaf Glycoprotein (NLGP), a plant-derived glycoprotein immunomodulator is explored. Since former reports have proven immune dependent–tumor restriction of NLGP across multiple tumor models, we hypothesized that NLGP might reprogram and rectify T<sub>EX</sub> to target CSCs successfully. In this study, we report that NLGP’s therapeutic administration significantly reduced T<sub>EX</sub>-associated CSC virulence in <i>in vivo</i> B16-F10 melanoma tumor model. A similar trend was observed in <i>in vitro</i> generated T<sub>EX</sub> and B16-F10/MCF7 coculture setups. NLGP rewired CSCs by downregulating clonogenicity, multidrug resistance phenotypes and PDL1, OCT4, and SOX2 expression. Cell cycle analysis revealed that NLGP educated–T<sub>EX</sub> efficiently pushed CSCs out of quiescent phase (G<sub>0</sub>G<sub>1</sub>) into synthesis phase (S), supported by hyper-phosphorylation of G<sub>0</sub>G<sub>1</sub>–S transitory cyclins and Rb proteins. This rendered quiescent CSCs susceptible to S-phase–targeting chemotherapeutic drugs like 5-fluorouracil (5FU). Consequently, combinatorial treatment of NLGP and 5FU brought optimal CSC-targeting efficiency with an increase in apoptotic bodies and proapoptotic BID expression. Notably a strong nephron-protective effect of NLGP was also observed, which prevented 5FU-associated toxicity. Furthermore, Dectin-1–mediated NLGP uptake and subsequent alteration of Notch1 and mTOR axis were deciphered as the involved signaling network. This observation unveiled Dectin-1 as a potent immunotherapeutic drug target to counter T-cell exhaustion. Cumulatively, NLGP immunotherapy alleviated exhausted CD8<sup>+</sup> T-cell-induced CSC aggravation.</p><p><b>Implications:</b> Our study recommends that NLGP immunotherapy can be utilized to counter ramifications of T-cell exhaustion and to target therapy elusive aggressive CSCs without evoking toxicity.</p></div>AbstractA variety of flow cytometric methods have been developed over the past 25 yr to study how treatment with chemotherapeutic agents affects cell-cycle progression. One of the most commonly used measurements relies on a singletime analysis of the DNA distribution of a cell population (1). This analysis may also be multivariate, for instance, when another cell feature is measured in addition to DNA. The additional feature(s) often provides information about a particular metabolic or molecular feature of the cell that often correlates with the rate of cell progression through the cycle or cell quiescence. Therefore, although such measurements per se cannot reveal whether or not the cell actually progresses through the cycle, the kinetic information is inferred from the DNA content (cell-cycle position) and from the metabolic or molecular profile of that cell.KeywordsFlow Cytometric MethodCyclin ExpressionCentrifuge CellResuspend Cell PelletBrdUrd IncorporationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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The aim of the present study was to determine the expression and function of B cell translocation gene 1 (BTG1) in kidney carcinoma. Kidney samples were obtained from cancer lesions (n=85) and the adjacent normal tissue (n=40) in kidney cancer patients immediately following endoscopic biopsy. The effect of BTG1 overexpression was examined in vitro utilizing a human kidney cancer cell line, ACHN, stably transfected with a recombinant lentivirus (LeBTG1 cells) and compared to empty vector‑transfected controls (LeEmpty). BTG1 protein expression was significantly lower in kidney cancer tissue biopsies compared to normal tissue, as measured by immunohistochemistry (34.1 vs. 77.8% of tissues; P<0.05) and western blotting (0.481±0.051 vs. 0.857±0.081; P<0.05). In vitro analyses revealed that LeBTG1 cells had a reduced survival fraction compared to control LeEmpty cells, with higher rates of apoptosis (16.6±2.5 vs. 6.1±0.7%; P<0.05). The proportion of LeBTG1 cells in G(0)/G(1) stage and S phase was also significantly different from LeEmpty cells (66.8±5.3 and 22.2±1.5% vs. 44.4±3.1 and 34.5±2.3%, respectively; P<0.05), and the migration and invasion of LeBTG1 cells was significantly impaired with respect to LeEmpty cells (74.0±9.0 and 53.0±7.0 vs. 118.0±15.0 and 103.0±13.0, respectively; P<0.05). These effects were accompanied by decreased protein expression of cyclin D1, B‑cell lymphoma 2 and matrix metalloproteinase 9 in LeBTG1 cells (0.118±0.018, 0.169±0.015 and 0.207±0.027, respectively) compared to control LeEmpty cells (0.632±0.061, 0.651±0.063 and 0.443±0.042, respectively; P<0.05). Reduced BTG1 expression is associated with increased disease severity, suggesting it is a negative regulator of kidney cancer and can serve as a prognostic indicator. The results of the present study show that BTG1 protein levels were significantly reduced in kidney cancer biopsy specimens and were associated with disease progression and prognosis.
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Cell size is fundamental to cell physiology. For example, cell size determines the spatial scale of organelles and intracellular transport and thereby affects biosynthesis. Although some genes that affect mammalian cell size have been identified, the molecular mechanisms through which cell growth drives cell division have remained elusive. We show that cell growth during the G
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Objective To examine the pathway of effect of BaP on cell cycle.Methods We exposed BaP to human embryonic lung fibroblasts(HE LF,p53~(+/+)),human non-small cell lung cancer(A549,p53~(+/+)) and human non-snall cell lung cancer(H1299,p53~(-/-)),and monitored the changes in cell growth by cell counting,the cell size and cell cycle distribution by FACScan flow cytometer,and the mRNA levels of p53 by RT-PCR.Results BaP exposure promoted HELF and A549 cell growth,enlarged cell size,enhanced the transition of G1 phase to S and G2/M phase,and did not affect the cell proliferation and cell cycle distribution in H1299 cells.BaP exposure increased p53 mRNA to 4.03 and 2.48 times in HELF and A549 cells in comparison to the untreated cells.Conclusion It is concluded that the effect of BaP on cell proliferation and cell cycle maybe be related with p53 status in cells.
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DNA damage in cells occurs from both endogenous and exogenous sources, and failure to repair such damage is associated with the emergence of different cancers, neurological disorders and aging. DNA damage responses (DDR) in cells are closely associated with the cell cycle. While most of our knowledge of DDR comes from bulk biochemistry, such methods require cells to be arrested at specific stages for cell cycle studies, potentially altering measured responses; nor is cell to cell variability in DDR or direct cell-level correlation of two response metrics measured in such methods. To overcome these limitations we developed a microscopy-based assay for determining cell cycle stages over large cell numbers. This method can be used to study cell-cycle-dependent DDR in cultured cells without the need for cell synchronization. Upon DNA damage γH2A.X induction was correlated to nuclear enrichment of p53 on a cell-by-cell basis and in a cell cycle dependent manner. Imaging-based cell cycle staging was combined with single molecule P53 mRNA detection and immunofluorescence for p53 protein in the very same cells to reveal an intriguing repression of P53 transcript numbers due to reduced transcription across different stages of the cell cycle during DNA damage. Our study hints at an unexplored mechanism for p53 regulation and underscores the importance of measuring single cell level responses to DNA damage.
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Objective To investigate the effects of simulated weightlessness on cell area,cell multiplication and cell cycle of MG-63 cells.Methods The rotating clinostat was used to simulated weightlessness.The change of cell area was measured with image processing and analysis soft(Image J).Cell growth curve was measured with the cytometry.The change of cell cycle was examined with a flow cytometer.Results Compared with control group,the cell area of cell line MG-63 in 24 h,36 h and 48 h duratons of rotating group decreased significantly(P0.05).Compared with 12 h duration,the cell area in 24 h,36 h and 48 h durations of rotating group decreased significantly(P0.05).But in control group,cell area of all 4 experimental durations showed no significant difference among each other.Compared with control group,cell growth curve revealed that cell multiplication was inhibited in rotating group.Compared with control group,the cell cycles of cell line MG-63 in 12 h,36 h,48 h sections of rotating group did not change significantly,but the cell in 24 h duration of rotating group was decreased significantly(P0.05) in G0+G1 phase and increased significantly(P0.05) in S phase.Conclusion Simulated weightlessness in rotating clinostat may lead cell area of cell line MG-63 in 24 h,36 h and 48 h durations to be decreased.The cell multiplication is inhibited in 12 h,24 h,36 h and 48 h durations.The conversion of cell from S phase to G2 phase is inhibited in 24 h duration.
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Enzymes which affect histone acetylation status have been shown to play an important role in determining transcriptional activity in chromatin through conformational modification of its structure. Since the timely presence of such enzymes may be of critical importance, our experiments were designed to determine whether the level of expression of HDAC1 is cell cycle dependent and/or affected by a high cell density. Our results show that in mouse fibroblasts the expression of mHDAC1 is neither affected by cell cycle phases nor by cell density. In contrast, the expression of several hHDACs including hHDAC1 were affected in a cell density dependent fashion in the human prostate adenocarcinoma cell line PC3, paralleling our previously published findings in the hepatocellular carcinoma derived cell line Hep3B. Differential recruitment of HDAC mRNAs suggests that these enzymes may play unique roles in different cell types and under different environmental conditions (i.e., exposure to various cell densities and cell-cell contacts). Our study has implications for the proposed use of HDAC inhibitors in the treatment of human malignancy, highlighting issues of drug action selectivity in tissues and potential secondary effects.
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Summary The mechanism that coordinates cell growth and cell cycle progression remains poorly understood; in particular, whether the cell cycle and cell wall biosynthesis are coordinated remains unclear. Recently, cell wall biosynthesis and cell cycle progression were reported to respond to wounding. Nonetheless, no genes are reported to synchronize the biosynthesis of the cell wall and the cell cycle. Here, we report that wounding induces the expression of genes associated with cell wall biosynthesis and the cell cycle, and that two genes, AtMYB46 in Arabidopsis thaliana and RrMYB18 in Rosa rugosa , are induced by wounding. We found that AtMYB46 and RrMYB18 promote the biosynthesis of the cell wall by upregulating the expression of cell wall‐associated genes, and that both of them also upregulate the expression of a battery of genes associated with cell cycle progression. Ultimately, this response leads to the development of curled leaves of reduced size. We also found that the coordination of cell wall biosynthesis and cell cycle progression by AtMYB46 and RrMYB18 is evolutionarily conservative in multiple species. In accordance with wounding promoting cell regeneration by regulating the cell cycle, these findings also provide novel insight into the coordination between cell growth and cell cycle progression and a method for producing miniature plants.
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Journal Article Functional interplay between the cell cycle and cell phenotypes Get access Wei-Chiang Chen, Wei-Chiang Chen Johns Hopkins Physical Sciences – Oncology Center, The Johns Hopkins University, Baltimore, Maryland 21218, USADepartment of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA Search for other works by this author on: Oxford Academic Google Scholar Pei-Hsun Wu, Pei-Hsun Wu Johns Hopkins Physical Sciences – Oncology Center, The Johns Hopkins University, Baltimore, Maryland 21218, USADepartment of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA Search for other works by this author on: Oxford Academic Google Scholar Jude M. Phillip, Jude M. Phillip Johns Hopkins Physical Sciences – Oncology Center, The Johns Hopkins University, Baltimore, Maryland 21218, USADepartment of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA Search for other works by this author on: Oxford Academic Google Scholar Shyam B. Khatau, Shyam B. Khatau Johns Hopkins Physical Sciences – Oncology Center, The Johns Hopkins University, Baltimore, Maryland 21218, USADepartment of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA Search for other works by this author on: Oxford Academic Google Scholar Jae Min Choi, Jae Min Choi Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA Search for other works by this author on: Oxford Academic Google Scholar Matthew R. Dallas, Matthew R. Dallas Johns Hopkins Physical Sciences – Oncology Center, The Johns Hopkins University, Baltimore, Maryland 21218, USADepartment of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA Search for other works by this author on: Oxford Academic Google Scholar Konstantinos Konstantopoulos, Konstantinos Konstantopoulos Johns Hopkins Physical Sciences – Oncology Center, The Johns Hopkins University, Baltimore, Maryland 21218, USADepartment of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA Search for other works by this author on: Oxford Academic Google Scholar Sean X. Sun, Sean X. Sun Johns Hopkins Physical Sciences – Oncology Center, The Johns Hopkins University, Baltimore, Maryland 21218, USADepartment of Mechanical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA Search for other works by this author on: Oxford Academic Google Scholar Jerry S. H. Lee, Jerry S. H. Lee Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USACenter for Strategic Scientific Initiatives, Office of the Director, National Cancer Institute, National Institute of Health, Bethesda, Maryland 20892, USA Search for other works by this author on: Oxford Academic Google Scholar Didier Hodzic, Didier Hodzic Department of Ophthalmology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA Search for other works by this author on: Oxford Academic Google Scholar ... Show more Denis Wirtz Denis Wirtz Johns Hopkins Physical Sciences – Oncology Center, The Johns Hopkins University, Baltimore, Maryland 21218, USADepartment of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA E-mail: wirtz@jhu.edu Search for other works by this author on: Oxford Academic Google Scholar Integrative Biology, Volume 5, Issue 3, March 2013, Pages 523–534, https://doi.org/10.1039/c2ib20246h Published: 14 January 2013 Article history Received: 14 October 2012 Accepted: 05 December 2012 Published: 14 January 2013
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Heterogeneity of cell phenotypes remains a barrier in progressing cell research and a challenge in conquering cancer-related drug resistance. Cell morphology, the most direct property of cell phenotype, evolves along the progression of the cell cycle; meanwhile, cell motility, the dynamic property of cell phenotype, also alters over the cell cycle. However, a quantifiable research understanding the relationship between the cell cycle and cell migration is missing. Herein, we coordinate the migratory behaviours of NIH 3T3 fibroblasts to their corresponding phases of the cell cycle, the G1, the S, and the G2 phases, and explain the relationship through the spatiotemporal arrangements between the Rho GTPases' signals and cyclin-dependent kinase inhibitors, p21Cip1, and p27Kip1. Taken together, we demonstrate that both cell morphology and the dynamic subcellular behaviour are homogenous within each stage of the cell cycle phases but heterogenous between phases through quantitative cell analyses and an interactive molecular mechanism between the cell cycle and cell migration, posing potential implications in countering drug resistance.
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