scholarly journals The AHI-1-BCR-ABL-DNM2 Complex Mediates Mitochondrial Dynamics in Drug-Resistant BCR-ABL+ Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 189-189
Author(s):  
Ryan Yen ◽  
Lambert Yue ◽  
Steven Pelech ◽  
Xiaoyan Jiang
Keyword(s):  
Confocal Microscopy ◽  
Tyrosine Kinase ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mitochondrial Dynamics ◽  
Sh3 Domain ◽  
Negative Control ◽  
Tki Resistance ◽  
Tki Treatment

Chronic myeloid leukemia (CML) is driven by the BCR-ABL1 oncoprotein with constitutively active protein-tyrosine kinase activity, perturbing multiple signaling pathways. Although therapies with tyrosine kinase inhibitors (TKIs) can effectively treat early phase CML, relapses and emergence of TKI resistance are problematic, due to BCR-ABL kinase domain mutations and TKI unresponsive quiescent leukemic stem cells (LSCs). These observations point towards a need for alternate treatment strategies to prevent the development of resistant LSCs. We previously demonstrated that Abelson helper integration site-1 (AHI-1) is a highly deregulated protein in CML LSCs and that its WD40-repeat domain physically interacts with BCR-ABL, enhancing leukemia-initiating activity. AHI-1 also contains an SH3 domain, which mediates TKI resistance in LSCs. This domain interacts with dynamin-2 (DNM2) and forms a complex with BCR-ABL, to enhance the phosphorylation and activity of DNM2. The AHI-1-BCR-ABL-DNM2 complex is shown to regulate leukemic properties in patient LSCs, including increased ROS production, endocytosis and autophagy. Interestingly, deletion of the Ahi-1 SH3 domain (Ahi-1 SH3Δ) results in a defect in Ahi-1 localization, with most being present in the nucleus. To test whether Ahi-1 SH3 domain activity directly affects cytoplasmic anchoring and localization, we have generated two Ahi-1 mutants, using site-directed mutagenesis: a mutation in the key tryptophan residue (W939A) involved in SH3 domain binding and in a non-conserved surface residue (M906A), as a negative control, based on the crystal structure of the AHI-1 SH3 domain. Interestingly, the cytoplasm-to-nucleus signal ratio of Ahi-1 W939A was significantly reduced compared to the negative control or wildtype Ahi-1, as assessed by immunofluorescence and confocal microscopy (70% reduction, p<0.0001), indicating that changes in localization of Ahi-1 SH3Δ may result in disruption of the complex and allow for new interactions with nuclear proteins. Investigating changes in the proteome may help uncover downstream effects of the AHI-1-BCR-ABL-DNM2 complex and its biological role in mediating TKI resistance. Advanced antibody microarray analysis was then used to investigate differences in the proteome and phosphorylation landscape of BCR-ABL+ cells co-transduced with wildtype Ahi-1 or Ahi-1 SH3Δ. This system quantifies the differences in expression and phosphorylation states of key signaling proteins simultaneously, using 878 antibodies in duplicate. Twenty leads were identified by the following criteria: a large signal difference of at least 1.5-fold change, high signal strength for high expression, and low error between duplicates. These leads were validated by Western blot analysis and several of them were confirmed. Particularly, phosphorylation of cyclin-dependent kinase 1 (CDK1), a key player in cell cycle control and mitochondrial dynamics, was greatly reduced in cells expressing wildtype Ahi-1 compared to Ahi-1 SH3Δ, indicating that AHI-1-mediated phosphorylation changes in CDK1 may contribute to regulation of mitochondrial functions. Indeed, BCR-ABL-transduced cells co-expressing wildtype Ahi-1 showed increased mitochondria potential in response to TKI treatment or serum starvation, in MitoTracker analysis (p<0.05). However, this was not observed in BCR-ABL-transduced cells co-expressing the Ahi-1 SH3Δ mutant. A similar trend was also observed in immunofluorescence confocal microscopy analysis of the mitochondrial importer receptor, TOM20. To further study the role of DNM2 in mediating mitochondrial dynamics associated with AHI-1 and BCR-ABL, CRISPR-Cas9 mediated DNM2 knockdown was performed in TKI-resistant cells, using two different DNM2-targeting guide RNAs; these resulted in significant reduction in DNM2 (78% & 75%) in Western blot analysis. The knockdown cells showed a reduction in viability (60% reduction) and increased sensitivity to TKI treatment compared to the control (90% vs. 30% reduction) after 48 hours and changes in mitochondrial activity were also observed in these cells. These results support a role for mitochondrial dynamics in the AHI-1-BCR-ABL-DNM2 complex-mediated TKI response and that targeting key biological processes regulated by the AHI-1-BCR-ABL-DNM2 complex and its pathways may lead to new therapeutic strategies to overcome TKI resistance in CML. Disclosures Pelech: Kinexus Bioinformatics Corporation: Equity Ownership.

scholarly journals Characterization of Human Immunoglobulin (Ig) Isotype and IgG Subclass Response to Bartonella henselae Infection

1998 ◽  
Vol 66 (12) ◽  
pp. 5915-5920 ◽  
Author(s):  
Svena L. McGill ◽  
Russell L. Regnery ◽  
Kevin L. Karem
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Bartonella Henselae ◽  
Negative Control ◽  
Cross Reactivity ◽  
Antibody Activity ◽  
Igg Subclass ◽  
Banding Patterns ◽  
Bartonella Quintana

ABSTRACT Serologic parameters of cat scratch disease (CSD) were evaluated by Western blot analysis. Sera from patients with serologically confirmed CSD antigen were screened for immunoglobulin (Ig) isotype-specific as well as IgG subclass-specific reactivity against Bartonella henselae whole-cell antigen. Bartonella-negative control sera were used to determine baseline antibody activity. Heterogeneous B. henselae-specific IgG reactivity with numerous protein bands, ranging from >150 to <17 kDa, was observed. Though individual banding patterns were variable, one approximately 83-kDa B. henselae protein (Bh83) was immunoreactive with all CSD sera tested, suggesting it is a conserved antigen during infection. Bh83 was not recognized by reference human antisera againstRickettsia rickettsii, Chlamydia group positive, Treponema pallidum, Orientia tsutsugamushi, Fransciscella tularensis,Ehrlichia chaffeensis, Mycoplasma pneumoniae, and Escherichia coli, although other cross-reactive proteins were evident. Significantly, CSD sera failed to recognize the 83-kDa protein when tested against Bartonella quintanaantigen, though sera from B. quintana-infected patients did react to Bh83. This cross-reactivity suggests epitope conservation during infection with B. henselae or B. quintana. Western blot analysis further revealed similar banding patterns when B. henselae was reacted against the Ig isotypes IgG and IgG1 and both secretory and alpha chains of IgA. Neither IgM nor IgE reacted significantly toBartonella antigen by our Western blot analysis. Dissection of the antibody response at the IgG subclass level indicated that prominent antigen recognition was limited to IgG1. These observations provide insight into induced immunity during CSD and provide evidence for conserved epitope expression during infection withB. henselae or B. quintana.

scholarly journals A39 NOVEL GAIN OF FUNCTION NON-RECEPTOR TYROSINE KINASE MUTATIONS ARE LINKED TO THE PATHOGENESIS OF VEOIBD

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 46-48
Author(s):  
M Mehta ◽  
L Wang ◽  
C Guo ◽  
N Warner ◽  
Q Li ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Western Blot ◽  
Receptor Tyrosine Kinase ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Intestinal Inflammation ◽  
De Novo ◽  
Single Gene ◽  
Gain Of Function

Abstract Background Very early-onset inflammatory bowel disease (VEOIBD) is an emerging global disease, that results in inflammation of the digestive tract. Severe forms of VEOIBD can be caused by mutations in a single gene (monogenic variants) and, can result in death. A candidate gene which codes for a non-receptor tyrosine kinase (nRTK) has recently been implicated as a monogenic cause of IBD (unpublished). Whole exome sequencing was performed in two unrelated children who presented with symptoms of IBD identifying two distinct de novo gain of function mutations (S550Y and P342T). Both mutations are located in the highly conserved region of the nRTK, and were predicted to have similar downstream effects. Furthermore, four other patients with a variety of adult-onset immune disorders have recently been identified with rare variants in the same gene (M450I, R42P, A353T, V433M, S550F) but, their potential gain of function status remains to be determined. Studies show that this nRTK is an essential mediator in inflammation. It is expressed in both intestinal epithelial and immune cells however, its role in infantile IBD is unclear. This protein is first activated by phosphorylation and is linked to activating downstream transcription factors such as ERK and JNK. All these target proteins play a meaningful role in intestinal inflammation in patients with IBD. Aims Since we identified P342T and S550Y to be gain of function, we wanted to determine if the new variants exhibit a similar downstream impact on target protein expression levels when compared with S550Y and P342T. We also wanted to identify if all variants can be rescued with a known nRTK inhibitor. It is hypothesized that the new variants are gain of function and that all variants can be rescued with the inhibitor. Methods Using western blot analysis, the activation of ERK, JNK and nRTK was compared between wildtype (WT) and mutants. This in vitro method helped identify the degree of activation. For the second part of the study, HEK293T cells were treated with inhibitor to test for a rescue of phenotypes via western blot analysis. Results Results show an increased activation of nRTK, ERK and JNK in all variants with S550Y and S550F having the highest activation. Furthermore, pharmacological inhibition using small molecular kinase inhibitors resulted in decreased activation of nRTK, ERK and JNK suggesting a rescue of phenotypes. Conclusions Characterizing the downstream functional impact of these nRTK variants is an important first step to determine if gain of function nRTK mutations drive IBD. With a rising prevalence of IBD worldwide, these findings may lead to the development of pharmacological nRTK inhibitors as a novel personalized therapeutic approach for these patients and possibly for the broader IBD population. Funding Agencies CIHR

FLT3 Inhibitor Resistant Cells Show Different Downstream Signaling Pathway Changes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4381-4381
Author(s):  
Kyu-Tae Kim ◽  
Obdulio Piloto ◽  
Donald Small
Keyword(s):  
Tyrosine Kinase ◽  
Western Blot ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mechanisms Of Resistance ◽  
Downstream Signaling ◽  
Flt3 Inhibitor ◽  
Downstream Signaling Pathway

Abstract Receptor tyrosine kinase FLT3 plays an important role in leukemogenesis, especially in acute myeloid leukemia (AML). Tyrosine kinase inhibitors (TKI) targeting wild-type and mutant FLT3 have been developed and shown to have activity in clinical trials. However, as seen with Gleevac in CML, prolonged incubation with TKIs can select for resistant clones that may contribute to disease progression. To study resistance to TKIs against FLT3 we developed FLT3 inhibitor resistant cell lines by co-culturing MOLM14 and BaF3/ITD cells, expressing FLT3/ITD mutants with increasing concentrations of the FLT3 inhibitor CEP-701. The resulting cell lines, MOLM14(R) and BaF3/ITD(R) are resistant to CEP-701 induced cytotoxicity. MOLM14(R) is also resistant to other selective FLT3 TKIs including CEP-5214 and PKC412. In contrast, BaF3/ITD(R) cells were still sensitive to CEP5214 and PKC412. Western blot analysis reveals that CEP-701, CEP-5214 and PKC412 all still inhibit FLT3 in MOLM14(R) cells implying selection of a clone no longer dependent on FLT3 signaling. FLT3 phosphorylation is not inhibited by CEP-701 in BaF3/ITD(R) cells but is still inhibited by CEP-5214 and PKC412. Thus the BaF3/ITD(R) cells appear to remain FLT3-dependent. Sequencing of FLT3 from the resistant clones showed that the resistance was not the result of drug resistance mutations in FLT3/ITD. To investigate possible mechanisms of resistance in FLT3-dependent and FLT3-independent FLT3 inhibitor resistant cells, we examined pathways downstream of FLT3. Previously, we and others reported that constitutive FLT3 activation results in specific changes in gene expression in myeloid leukemic cells. As expected for cells with continued FLT3/ITD activation, Western blot analysis of BaF3/ITD(R) cells treated with CEP-701 show that they maintain activation of Erk/MAPK, Akt, and STAT5 pathways and induction of FLT3 dependent genes including Pim-1 and cMyc. In the apparently FLT3-independent MOLM-14(R) clones, inhibition of FLT3 activity resulted in decreased phosphorylation of downstream Akt and Stat5. However, we found Erk/MAPK phosphorylation and cMyc expression were not decreased in response to FLT3 TKI. This implies that whatever pathway has been selected for the ability to grow in this inhibitor is still feeding into this part of the downstream signaling pathway normally activated by FLT3/ITD. Thus, BaF3/ITD(R) FLT3-dependent and MOLM-14(R) FLT3 independent cells differ in response to several FLT3 inhibitors that results from the differences in their mechanisms of resistance.

scholarly journals Aquaporin-1 Protein Levels Elevated in Fresh Urine of Renal Cell Carcinoma Patients: Potential Use for Screening and Classification of Incidental Renal Lesions

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Shilpa Sreedharan ◽  
John A. Petros ◽  
Viraj A. Master ◽  
Kenneth Ogan ◽  
John G. Pattaras ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Western Blot ◽  
Cell Carcinoma ◽  
Blot Analysis ◽  
Renal Cell ◽  
Western Blot Analysis ◽  
Negative Control ◽  
Aquaporin 1 ◽  
Protein Levels ◽  
Fresh Urine

Introduction and Objectives. There are over 65,000 new cases of renal cell carcinoma (RCC) each year, yet there is no effective clinical screening test for RCC. A single report claimed no overlap between urine levels of aquaporin-1 (AQP1) in patients with and without RCC (Mayo Clin Proc. 85:413, 2010). Here, we used archived and fresh RCC patient urine to validate this report.Methods. Archived RCC, fresh prenephrectomy RCC, and non-RCC negative control urines were processed for Western blot analysis. Urinary creatinine concentrations were quantified by the Jaffe reaction (Nephron 16:31, 1976). Precipitated protein was dissolved in 1x SDS for a final concentration of 2 μg/µL creatinine.Results. Negative control and archived RCC patient urine failed to show any AQP1 protein by Western blot analysis. Fresh RCC patient urine is robustly positive for AQP1. There was no signal overlap between fresh RCC and negative control, making differentiation straightforward.Conclusions. Our data confirms that fresh urine of patients with RCC contains easily detectable AQP1 protein. However, archival specimens showed an absence of detectable AQP1 indistinguishable from negative control. These findings suggest that a clinically applicable diagnostic test for AQP1 in fresh urine may be useful for detecting RCC.

Exposure to UV light causes increased biotinylation of histones in Jurkat cells

2002 ◽  
Vol 283 (3) ◽  
pp. C878-C884 ◽  
Author(s):  
Dorothea M. Peters ◽  
Jacob B. Griffin ◽  
J. Steven Stanley ◽  
Mary M. Beck ◽  
Janos Zempleni
Keyword(s):  
Dna Damage ◽  
Confocal Microscopy ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Uv Light ◽  
Jurkat Cells ◽  
Uv Exposure ◽  
Thymine Dimers

Biotin in breakdown products of biotinylated carboxylases serves as substrate for biotinylation of histones by biotinidase. Here we determined whether biotinylation of histones might play a role in repair of damaged DNA and in apoptosis. Jurkat cells were exposed to UV light to induce DNA damage. Abundance of thymine dimers increased about three times in response to UV exposure, consistent with DNA damage. Biotin-containing carboxylases were degraded in response to UV exposure, as judged by Western blot analysis and carboxylase activities. Mitochondrial integrity decreased in response to UV exposure (as judged by confocal microscopy), facilitating the release of breakdown products of carboxylases from mitochondria. Biotinylation of histones increased in response to UV exposure; biotinylation of histones did not occur specifically at sites of newly repaired DNA. UV exposure triggered apoptosis, as judged by caspase-3 activity and analysis by confocal microscopy. In summary, this study provided evidence that increased biotinylation of histones in DNA-damaged cells might either be a side product of carboxylase degradation or a step during apoptosis.

Study on the extract of Huangqi and Ezhu for the crosstalk of exosomes mediating tamoxifen resistance on LCC9 cells.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e12517-e12517
Author(s):  
Shasha Cui ◽  
Dongni Zhang ◽  
Wenping Lu
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Cancer Stem Cells ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Tamoxifen Resistance ◽  
Negative Control ◽  
Flow Cytometry Analysis

e12517 Background: Estrogen receptor (ER)-positive breast cancer is the most frequent subtype accounting for about 70% of all cases. Tamoxifen (TAM) is still the most effective drug for treating ER+/PR+ breast cancer. However, recurrence and metastasis still occur in 30-50% of patients due to TAM resistance. Furthermore, studies have shown that breast cancer stem cells (BCSCs) play an important role in TAM resistance, and exosomes regulate dynamic changes of BCSCs in tumor microenvironment. Additionally, the extract of Huangqi and Ezhu (EHE) has been shown to inhibit multiple TAM-associated resistance signaling pathways. In the current study, we investigated the effect of EHE to reverse TAM-resistance in LCC9 cells via crosstalk of distinct types of exosomes. Methods: Exosomes were extracted from LCC9 cells by ultracentrifugation and characterized by particle size detection, transmission electron microscopy and western blot analysis. Subsequently, isolated LCC9 exosomes were used to treat bone marrow stem cells (BMSCs) with EHE only, TAM only, EHE and TAM (EHETAM), and no treatment (negative control, NC). After treatment, exosomes of BMSCs were isolated and incubated further with LCC9 cells. CCK-8 assay and flow cytometry analysis were performed, and protein expression in treated LCC9 cells was tested by western blot analysis. Results: Data from CCK-8 assay showed that compared with the NC group, proliferation of LCC9 cells was significantly reduced in the presence of exosomes of BMSCs treated with TAM (82.9%, 77.1% , 75.4%), EHE (73.2 %, 69.5% , 68.2 %) and EHETAM (68.9 %, 52.3%，54.1% ) after 24h, 48h, and 72 h, respectively (p < 0.001). EHE and EHETAM groups have more effective inhibition on cell proliferation than TAM only group on the different time separately(p < 0.001).Flow cytometry analysis demonstrated that compared with the NC group(6.1%), increased apoptosis was observed for groups of TAM (8.1%), EHE (12.5%) and EHETAM (12.2%) (p < 0.001). Compared with TAM only group, EHE and EHETAM groups also have significantly higher cell death rate (p < 0.001). Lower CD44 and HER2 expression and higher ERα expression were observed in EHE, TAM, and EHETAM than those of NC group by western blot analysis of treated LCC9 cells. Conclusions: EHE treatment can reverse TAM-resistance by interfering with the crosstalk between BMSCs and breast cancer cells exosomes to induce more active apoptosis and reduce TAM-resistant CD44+ cancer stem cells.

scholarly journals Proteomic Analysis and Cell Viability of Nine Amnion, Chorion, Umbilical Cord, and Amniotic Fluid–Derived Products

Cartilage ◽  
2020 ◽  
pp. 194760352097676
Author(s):  
Liliya Becktell ◽  
Andrea M. Matuska ◽  
Stephanie Hon ◽  
Michelle L. Delco ◽  
Brian J. Cole ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Western Blot ◽  
Cell Viability ◽  
Blot Analysis ◽  
Proteomic Analysis ◽  
Western Blot Analysis ◽  
Antibody Binding ◽  
Matrix Metalloproteinase 2 ◽  
Live Cells ◽  
Enzyme Linked Immunosorbent Assay

Objective Amnion products are used in various musculoskeletal surgeries and as injections for joint pain with conflicting reports of cell viability and protein contents. The objective of this study was to determine the full proteome and examine cell viability in 9 commercial amnion products using an unbiased bottom-up shotgun proteomics approach and confocal microscopy. Design Products were subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis and searched against a UniProt Homo sapiens database. Relative protein abundance was determined for each sample. Based on proteomics results, lumican was measured by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis was performed for interleukin-1 receptor antagonist (IL-1Ra) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2). Cell viability was determined by calcein AM (live) and ethidium homodimer (dead) staining and confocal microscopy. Results Proteomic analysis revealed 919 proteins in the nine products. Proteins were primarily collagens, keratin, and albumin. Lumican, a small leucine-rich proteoglycan (SLRP) was found in all samples. Western blot analysis for IL-1Ra and TIMP-2 indicated presence of both proteins, with nonspecific antibody binding also present in all samples. No live cells were identified in any product. Conclusions Several novel proteins were identified through proteomics that might impart the beneficial effects of amnion products, including SLRPs, collagens, and regulators of fibroblast activity. IL-1Ra and TIMP-2 were identified, but concentrations measured by ELISA may be falsely increased due to nonspecific antibody binding. The concept that the amnion tissues provide live cells to aid in tissue regeneration cannot be supported by the findings of this study.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

2020 ◽  
Vol 20 (9) ◽  
pp. 1147-1156
Author(s):  
Hanrui Li ◽  
GeTao Du ◽  
Lu Yang ◽  
Liaojun Pang ◽  
Yonghua Zhan
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Tumor Size ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Bioluminescence Imaging ◽  
Antitumor Effects ◽  
In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

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