scholarly journals Increased Musashi-2 and Decreased NUMB Protein Levels Observed in Human Colorectal Cancer are reverted to Normal Levels by ATRA-Induced Cell Differentiation

2018 ◽  
Vol 3 (2) ◽  
Keyword(s):  
Colorectal Cancer ◽  
Protein Expression ◽  
Cell Lines ◽  
Cellular Differentiation ◽  
Tissue Samples ◽  
Colorectal Tumorigenesis ◽  
Protein Levels ◽  
Colon Tumorigenesis ◽  
Epithelial Homeostasis ◽  
All Trans Retinoic Acid

Background: Musashi stem cell (SC) proteins (MSI-1 & MSI-2) are known to become over expressed during colorectal tumorigenesis in humans and mice. MSI-1 overexpression induces tumorigenesis through Notch activation via inactivation of NUMB. Previous studies also show that MSI-2 overexpression in mice induces intestinal tumorigenesis but the mechanism is independent of NUMB. However, whether the MSI-2/NUMB pathway contributes to colorectal cancer (CRC) development in humans is still undetermined. Methods: We evaluated expression of MSI-2 and NUMB proteins in matched normal and CRC patient samples, as well as in human CRC cell lines. We also determined whether induction of cellular differentiation by all-trans retinoic acid (ATRA) influences MSI-2 and NUMB expression. Results: Analysis of matched patient tissue samples and CRC cell lines showed that MSI-2 protein expression is significantly increased and NUMB expression is decreased in CRCs compared to the normal colonic tissue. Immunostaining of normal and adenomatous colonic epithelium revealed that MSI-1+ and MSI-2+ SCs reside in the SC niche and they become overpopulated during colon tumorigenesis. Moreover, promoting cellular differentiation by ATRA reduces MSI-2 protein levels, while increasing NUMB protein levels in human CRC cell lines. Conclusions: MSI-2/NUMB protein expression is altered during colon tumorigenesis, and indicates that MSI-2/ NUMB signaling in human colonic stem cells is closely linked to normal colonic epithelial homeostasis. Implications: The ability to normalize MSI-2/NUMB signaling by inducing differentiation of cancer SCs suggests a novel therapeutic approach for CRC treatment.

scholarly journals Dihydropyrimidine Dehydrogenase Levels in Colorectal Cancer Cells Treated with a Combination of Heat Shock Protein 90 Inhibitor and Oxaliplatin or Capecitabine

2019 ◽  
Vol 9 (3) ◽  
pp. 439-444 ◽  
Author(s):  
Mahshid Mohammadian ◽  
Shima Zeynali-Moghaddam ◽  
Mohammad Hassan Khadem Ansari ◽  
Yousef Rasmi ◽  
Anahita Fathi Azarbayjani ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Protein Expression ◽  
Cell Lines ◽  
Dihydropyrimidine Dehydrogenase ◽  
Heat Shock Protein 90 ◽  
Water Soluble ◽  
Chemotherapy Response ◽  
Expression Levels ◽  
Protein Levels ◽  
Gene And Protein Expression

Purpose: Dihydropyrimidine dehydrogenase (DPD) is the principal enzyme in the catabolism of fluoropyrimidine drugs including capecitabine. A recent report has suggested that oxaliplatin chemotherapy is associated with elevated DPD levels and chemoresistance pattern. As a newly developed chemotherapeutic agent, 17-allyloamino-17-demethoxy-geldanamycin (17-AAG) can be effective in combination therapy with oxaliplatin and capecitabine in colorectal cancer (CRC). DPD expression level can be a predictive factor in oxaliplatin and capecitabine-based chemotherapy. We evaluated DPD in mRNA and protein levels with new treatments: 17-AAG in combination with oxaliplatin and capecitabine in HT-29 and HCT-116 cell lines. Methods: Drug sensitivity was determined by the water-soluble tetrazolium-1 assay in a previous survey. Then, we evaluated the expression levels of DPD and its relationship with the chemotherapy response in capecitabine, oxaliplatin, and 17-AAG treated cases in single and combination cases in two panels of CRC cell lines. DPD gene and protein expression levels were determined by real-time polymerase chain reaction and western blotting assay, respectively. Results: DPD gene expression levels insignificantly increased in single-treated cases versus untreated controls in both cell lines versus controls. Then, the capecitabine and oxaliplatin were added in double combinations, where DPD gene and protein expression increased in combination cases compared to pre-chemotherapy and single drug treatments. Conclusion: The elevated levels of cytotoxicity in more effective combinations could be related to a different mechanism apart from DPD mediating effects or high DPD level in the remaining resistance cells (drug-insensitive cells), which should be investigated in subsequent studies.

scholarly journals HERV-K Gag RNA and Protein Levels Are Elevated in Malignant Regions of the Prostate in Males with Prostate Cancer

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 449
Author(s):  
Simin D. Rezaei ◽  
Joshua A. Hayward ◽  
Sam Norden ◽  
John Pedersen ◽  
John Mills ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Lines ◽  
Endogenous Retrovirus ◽  
Human Endogenous Retrovirus ◽  
Normal Prostate ◽  
Tissue Samples ◽  
Gag Protein ◽  
Protein Levels ◽  
Prostate Epithelial Cells ◽  
Prostate Cancers

Heightened expression of human endogenous retrovirus (HERV) sequences has been associated with a range of malignancies, including prostate cancer, suggesting that they may serve as useful diagnostic or prognostic cancer biomarkers. We analysed the expression of HERV-K (Gag and Env/Np9 regions), HERV-E 4.1 (Pol and Env regions), HERV-H (Pol) and HERV-W (Gag) sequences in prostate cancer cells lines and normal prostate epithelial cells using qRT-PCR. HERV expression was also analysed in matched malignant and benign prostate tissue samples from men with prostate cancer (n = 27, median age 65.2 years (range 47–70)) and compared to prostate cancer-free male controls (n = 11). Prostate cancer epithelial cell lines exhibited a signature of HERV RNA overexpression, with all HERVs analysed, except HERV-E Pol, showing heightened expression in at least two, but more commonly all, cell lines analysed. Analysis of primary prostate material indicated increased expression of HERV-E Pol but decreased expression of HERV-E Env in both malignant and benign regions of the prostate in men with prostate cancer as compared to those without. Expression of HERV-K Gag was significantly higher in malignant regions of the prostate in men with prostate cancer as compared to matched benign regions and prostate cancer-free men (p < 0.001 for both), with 85.2% of prostate cancers donors showing malignancy-associated upregulation of HERV-K Gag RNA. HERV-K Gag protein was detected in 12/18 (66.7%) malignant tissues using immunohistochemistry, but only 1/18 (5.6%) benign tissue sections. Heightened expression of HERV-K Gag RNA and protein appears to be a sensitive and specific biomarker of prostate malignancy in this cohort of men with prostate carcinoma, supporting its potential utility as a non-invasive, adjunct clinical biomarker.

scholarly journals TOP2A is overexpressed and is a therapeutic target for adrenocortical carcinoma

2013 ◽  
Vol 20 (3) ◽  
pp. 361-370 ◽  
Author(s):  
Meenu Jain ◽  
Lisa Zhang ◽  
Mei He ◽  
Ya-Qin Zhang ◽  
Min Shen ◽  
...  
Keyword(s):  
Protein Expression ◽  
Anticancer Activity ◽  
Cell Lines ◽  
Antiproliferative Activity ◽  
Adrenocortical Carcinoma ◽  
Cellular Proliferation ◽  
Locally Advanced ◽  
Tissue Samples ◽  
Anchorage Independent Growth ◽  
Mrna And Protein Expression

Adrenocortical carcinoma (ACC) is a rare but aggressive malignancy with no effective therapy for patients with unresectable disease. The aim of the current study was i) to evaluate TOP2A expression and function in human adrenocortical neoplasm and ACC cells and ii) to determine the anticancer activity of agents that target TOP2A. TOP2A mRNA and protein expression levels were evaluated in 112 adrenocortical tissue samples (21 normal adrenal cortex, 80 benign adrenocortical tumors, and 11 ACCs). In vitro siRNA knockdown of TOP2A in ACC cell lines (NCI-H295R and SW13) was used to determine its effect on cellular proliferation, cell cycle, anchorage-independent growth, and cellular invasion. We screened 14 TOP2A inhibitors for their anticancer activity in ACC cells. TOP2A mRNA and protein expression was significantly higher in ACC than in benign and normal adrenocortical tissue samples (P<0.05). Knockdown of TOP2A gene expression in ACC cell lines significantly decreased cell proliferation, anchorage-independent growth, and invasion (P<0.05). A screening assay in NCI-H295R cells showed that 11 of 14 TOP2A inhibitors had antiproliferative activity, 5 of the 14 TOP2A inhibitors had a higher antiproliferative activity than mitotane, and aclarubicin was the agent with the highest activity. Aclarubicin was validated to significantly decrease proliferation and tumor spheroid size in both NCI-H295R and SW13 ACC cell lines (P<0.05). Our results suggest that TOP2A is overexpressed in ACC, regulates cellular proliferation and invasion in ACC cells, and is an attractive target for ACC therapy. Of the TOP2A inhibitors screened, aclarubicin is a good candidate agent to test in future clinical trials for patients with locally advanced and metastatic ACC.

scholarly journals Determination of CYP450 Expression Levels in the Human Small Intestine by Mass Spectrometry-Based Targeted Proteomics

2021 ◽  
Vol 22 (23) ◽  
pp. 12791
Author(s):  
Alexia Grangeon ◽  
Valérie Clermont ◽  
Azemi Barama ◽  
Fleur Gaudette ◽  
Jacques Turgeon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Small Intestine ◽  
Protein Expression ◽  
Mrna Levels ◽  
Targeted Proteomics ◽  
Tissue Samples ◽  
Human Organ ◽  
Expression Levels ◽  
Protein Levels ◽  
Human Small Intestine

The human small intestine can be involved in the first-pass metabolism of drugs. Under this condition, members of the CYP450 superfamily are expected to contribute to drug presystemic biotransformation. The aim of this study was to quantify protein expression levels of 16 major CYP450 isoforms in tissue obtained from nine human organ donors in seven subsections of the small intestine, i.e., duodenum (one section, N = 7 tissue samples), jejunum (three subsections (proximal, mid and distal), N = 9 tissue samples) and ileum (three subsections, (proximal, mid and distal), N = 9 tissue samples), using liquid chromatography tandem mass spectrometry (LC-MS/MS) based targeted proteomics. CYP450 absolute protein expression levels were compared to mRNA levels and enzyme activities by using established probe drugs. Proteins corresponding to seven of sixteen potential CYP450 isoforms were detected and quantified in various sections of the small intestine: CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, CYP3A5 and CYP4F2. Wide inter-subject variability was observed, especially for CYP2D6. CYP2C9 (p = 0.004) and CYP2C19 (p = 0.005) expression levels decreased along the small intestine. From the duodenum to the ileum, CYP2J2 (p = 0.001) increased, and a trend was observed for CYP3A5 (p = 0.13). CYP3A4 expression was higher in the jejunum than in the ileum (p = 0.03), while CYP4F2 expression was lower in the duodenum compared to the jejunum and the ileum (p = 0.005). CYP450 protein levels were better correlated with specific isoform activities than with mRNA levels. This study provides new data on absolute CYP450 quantification in human small intestine that could improve physiologically based pharmacokinetic models. These data could better inform drug absorption profiles while considering the regional expression of CYP450 isoforms.

scholarly journals Down-Regulation of MicroRNA-214 Contributed to the Enhanced Mitochondrial Transcription Factor A and Inhibited Proliferation of Colorectal Cancer Cells

2018 ◽  
Vol 49 (2) ◽  
pp. 545-554 ◽  
Author(s):  
Kaiming Wu ◽  
Jun Ma ◽  
Yanfeng Zhan ◽  
Kuanzhi Liu ◽  
Ziyin Ye ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Transcription Factor ◽  
Cell Lines ◽  
Nuclear Translocation ◽  
Malignant Tumors ◽  
Down Regulation ◽  
Mitochondrial Transcription ◽  
Tissue Samples ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Transcription Factor A

Background/Aims: Colon cancer, also known as colorectal cancer (CRC), is one of the most common malignant tumors globally. Although significant advances have been made for developing novel therapeutics, the mechanisms of progression of colorectal cancer are still poorly understood. Methods: In this study, we identified down-regulation of microRNA-214 (miR-214) as the contributing factor for CRC. Mitochondrial transcription factor A (TFAM) and miR-214 expression in tumor samples from colorectal cancer patients and cancer cell lines were examined by reverse transcription and real-Time PCR (qPCR) or Western Blotting. Results: Our data demonstrated that miR-214 was significantly down-regulated in the tissue samples from CRC patients as well as CRC derived cell lines. TFAM overexpression was also observed in CRC patients and identified as a target for miR-214. Knockdown of TFAM by miR-214 mimics significantly inhibited the proliferation of CRC cell lines. Also, down-regulation of TFAM inhibited nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nuclear translocation and the expression of NF-κB depended genes. Conclusion: In conclusion, our data suggested that down-regulation of MiR-214 contributed to the enhanced TFAM expression and decreased proliferation of CRC cells.

scholarly journals The Effect of Compound Sophora on Fluorouracil and Oxaliplatin Resistance in Colorectal Cancer Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
WeiHua Yin ◽  
GuPing Zhong ◽  
HuiZhen Fan ◽  
HongMei Xia
Keyword(s):  
Colorectal Cancer ◽  
Drug Resistance ◽  
Cancer Cells ◽  
Cell Lines ◽  
Sw480 Cell ◽  
Gastric Cancer Cells ◽  
Glycoprotein P ◽  
Chemotherapy Drugs ◽  
Protein Levels ◽  
Enhanced Expression

Fluorouracil (5-FU) and oxaliplatin (L-OHP) are the most commonly used chemotherapy drugs for colorectal cancer, though resistance is common. Compound Sophora injection is a traditional Chinese medicine that can protect the liver against oxidation, improve immunity, and enhance sensitivity to chemotherapy; it may have an effect of reversing resistance in 5-FU- and L-OHP-resistant gastric cancer cells (5-FU/SW480 and L-OHP/SW480, respectively). A concentration gradient experiment was performed to identify a nontoxic dose of compound Sophora injection. 5-FU/SW480 and L-OHP/SW480 cells were treated with the nontoxic dose of compound radix Sophorae injection for 48 h, and changes in drug resistance to 5-FU and L-OHP were detected. Alterations in apoptosis and the cell cycle were assessed, as were the mRNA and protein levels of permeability glycoprotein (P-gp), annexin A1 (ANXA1), and ATP-binding cassette superfamily G member 2 (ABCG2). Flow cytometry showed a reduction in the number of cells in the G1 phase and an increase of cells in the S phase (P<0.05). mRNA and protein expression of P-gp and ABCG2 was significantly higher in 5-FU/SW480 and L-OHP/SW480 cell lines, and ANXA1 expression decreased significantly (P<0.05). Compound Sophora injection can reverse the drug resistance of 5-FU/SW480 and L-OHP/SW480 cell lines to 5-FU and L-OHP, respectively, possibly through a mechanism involving reduced expression of P-gp and ABCG2 but enhanced expression of ANXA1, which is the basis for the identification of clinical drug resistance in colorectal cancer.

Protein Expression Patterns of Candidate Genes Involved in Apoptosis and Cell Cycle Control in Genetic Subgroups of Chronic Lymphocytic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2796-2796
Author(s):  
Christof Schneider ◽  
Dirk Winkler ◽  
Meike Loddenkemper ◽  
Alexander Krober ◽  
Peter Lichter ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Expression ◽  
Cyclin D1 ◽  
Candidate Genes ◽  
Cell Lines ◽  
Normal Karyotype ◽  
Lymphocytic Leukemia ◽  
Expression Levels ◽  
Protein Levels ◽  
Atm Protein

Abstract Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a highly variable clinical course. Genomic aberrations (such as 13q−, 11q−, +12q, 17p−) can be found in about 80% of CLL cases and define pathogenic as well as clinical subgroups. Similarly, the mutational status of the variable region of the immunoglobulin heavy-chain gene (VH) identifies subgroups with different maturation stage and clinical outcome. In this study protein expression levels of candidate genes involved in cell cycle and apoptosis control (p53, ATM, Akt1, PI3-K, p21, p27, cdk4, Cyclin-D1, D2, D3, Bax, Bcl-2, Apaf-1, Smac, XIAP, cIAP2, survivin) were examined by Western Blotting. A total of 87 CLL cases derived from the subgroups with 11q- (n=22), 17p-/p53 mutation (n=18), +12q (n=24), 13q- (n=8) or a normal karyotype (n=15) were studied and compared to the cell lines EHEB and JVM-2. VH-mutation status was available for 65 cases (unmutated n=48, mutated n=17). Due to limitations in sample availability not all proteins could be examined in all cases. A highly homogenous expression pattern for all the proteins studied was observed in the CLL subgroup with a normal karyotype. This pattern was independent of the VH-status. CLL samples with normal karyotype, +12q and 13q deletion showed equal levels of ATM as compared to EHEB and JVM-2. As compared to cases with a normal karyotype the ATM level within the 11q- subgroup was reduced in 5 cases and absent in 1 case among 11 evaluable 11q- cases. The 17p- subgroup was comprised of 3 cases with concomitant 17p- and 11q- and 15 cases with 17p- but no 11q-. The latter group showed ATM protein levels comparable to the levels of the normal karyotype group. In the group with 17p- and 11q- there was an ATM expression level similar to the groups with 17p- and normal karyotype in two cases while one case had a reduced ATM protein level comparable to the 11q- subgroup. All cases with 17p- exhibited a stronger expression of p53 as compared to the cell lines and all other cases, except for one case with normal karyotype and one with an 11q-. No p53 mutations could be detected in exons 5–9 by sequencing in these two cases. High levels of survivin protein were found in all cases with 17p- and/or 11q-, 13q-, +12q while the subgroup with a normal karyotype showed lower levels. High levels of cdk4 protein were expressed in cases with 17p-, 11q- and 13q- while cdk4 protein levels were low in the subgroup with +12q and normal karyotype. Regarding p21, p27, Bcl2, Bax, Smac, Apaf-1, Cyclin D1–D3, cIAP2, XIAP, Akt1 and PI3K no variation in the expression levels were observed across the genetic CLL subgroups. Comparing the CLL cases to the cell lines the differences in expression levels were found for the cell cycle regulators Cyclin D1, D2, D3, p21 and p27. While the cell lines showed strong protein levels for Cyclin D1, D2, D3 and p21, they were nearly absent in the CLL cases. Expression of p27 was higher in all CLL cases as compared to JVM-2 and EHEB. In conclusion, the 17q- subgroup was the only group with a high level of p53 protein expression indicating that p53 is the affected gene in this subgroup. In contrast, the ATM protein levels are reduced only in a part of the 11q- cases indicating a possible role of additional candidate genes. Cases with +12q and normal karyotype showed weak expression of cdk4 pointing out a possible function in these subgroups.

Correlation of BMAL1 expression in colorectal cancer with resistance to anti-VEGFA therapy with bevacizumab.

2018 ◽  
Vol 36 (4_suppl) ◽  
pp. 705-705
Author(s):  
Elke Burgermeister ◽  
Fagr Eladly ◽  
Wen Wu ◽  
Nadine Schulte ◽  
Johannes Betge ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Protein Expression ◽  
Tumor Cells ◽  
Cell Lines ◽  
T Test ◽  
Rank Test ◽  
Trend Test ◽  
Fisher Exact Test ◽  
Exact Test ◽  
Armitage Trend Test

705 Background: Mechanisms underlying failure of colorectal cancer (CRC) patients to respond to anti-vascular endothelial growth factor A (VEGFA) therapy with bevacizumab in combination with chemotherapy are largely unknown, and novel predictive markers required. Methods: Tumours from patients/mice and CRC cell lines were analysed by IHC, PCR, EMSA, ChIP, ELISA and Western blot. Statistics of (pre)clinical data was calculated with SAS and Graphpad Prism. Response outcomes including progression-free survival (PFS) and restaging according to RECIST (PR/SD/PD) were analyzed. Results: Circadian rhythm transcription factor and heme receptor REVERBA and its target gene BMAL1 promoted binding to and activation of the -700kB RORE DNA-element in the VEGFA promoter resulting in increased VEGFA mRNA expression and VEGFA protein secretion from human CRC cell lines. Conversely, REVERBA siRNA and its antagonist (Fe3+) hemin inhibited VEGFA synthesis. In C57BL/6J Apcmin/+ mice treated with murinized VEGFA Ab (n=11 mu_chimeric_B20-4.1 vs. n=42 mock; 10 mg/kg*week; i.p.; 4 weeks), Vegfa mRNA was reduced, as was incidence (*p=0.0411 Fisher Exact Test) and multiplicity (*p=0.0157 Cochran Armitage Trend Test) of vascularized CRCs. However, Bmal1 mRNA was up-regulated, and high BMAL1 protein expression in tumor cells positively correlated with Ki67+ proliferation (n=3 B20 vs. n=3 mock; *p<0.05 t-test) in treated Apcmin/+ CRCs. BMAL1 protein was also induced in xenografts from BALB/c nude mice s.c. implanted with the human CRC cell line HCT116 and treated with humanized VEGFA Ab (n=4 bevacizumab vs. n=4 vehicle; 10 mg/kg*week; i.p.; 3 weeks; *p<0.05 t-test). In CRC patients, high BMAL1 protein expression in tumor cells was associated with clinical non-response to bevacizumab (n=15 SD vs. n=29 PD: BMAL1- vs. BMAL1+, *p=0.0061 Cochran Armitage Trend Test,*p=0.0130 Fisher Exact Test) and reduced PFS (BMAL1- [671 days] vs. BMAL1+ [368 days], *p=0.0030 log rank test, HR=0.4792 [95%CI 0.3103-0.7871], n=74). Conclusions: BMAL1 may represent a predictive marker for bevacizumab non-response. Due to its drugability, the REVERBA-BMAL1-VEGFA axis may be a potential target to prevent resistance to anti-angiogenic therapy in CRC.

scholarly journals Protein profiling in cancer cell lines and tumor tissue using reverse phase protein arrays

2017 ◽  
Author(s):  
Xiaohong Jing ◽  
Weiqing Wang ◽  
Nicholas P. Gauthier ◽  
Poorvi Kaushik ◽  
Alex Root ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Protein Profiling ◽  
Reverse Phase ◽  
Tissue Samples ◽  
Protein Levels ◽  
Phase Protein ◽  
Pros And Cons ◽  
High Throughput Assay

AbstractReverse phase protein array (RPPA) technology is an antibody-based high-throughput assay for protein profiling of biological specimens that allows for many measurements with very small amounts of cell lysate. Here, we report the sensitivity, reproducibility, and accuracy of a particular RPPA platform called Zeptosens. We customized the RPPA protocol for our in-house setup, and measured more than 80 total protein and phospho-protein levels in various cancer samples, including cell lines, organoids, tumor chunks, core needle biopsies, and laser-capture microdissected tissue samples. We discuss pros and cons of the RPPA platform, and describe results from profiling 15 cancer cell line cells using RPPA.

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