Doublecotin-like kinase 1 increases chemoresistance of colorectal cancer cells through the anti-apoptosis pathway
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Abstract Colorectal cancer (CRC) is the third most common cancer diagnosed and the second leading cause of cancer-related deaths in the United States. About 50% of CRC patients relapsed after surgical resection and ultimately died of metastatic disease. Cancer stem cells (CSCs) are believed to be the primary reason for the recurrence of CRC. Specific stem cell marker, doublecortin-like kinase 1 (DCLK1) plays critical roles in initiating tumorigenesis, facilitating tumor progression, and promoting metastasis of CRC. It is up-regulated in CRC and up-regulation of DCLK1 indicates poor prognosis. Whether DCLK1 is correlated with enhanced chemoresistance of CRC cells is unclear. Our research aims to reveal association of DCLK1 with chemoresistance of CRC cells and the underlying molecular mechanisms. In order to achieve our goal, we established stable DCLK1 over-expression cells (DCLK1+) using the HCT116 cells (WT). DCLK1+ and WT cells were treated with 5-Fluorouracil (5-Fu) at different doses for 24 or 48 hours. MTT assay was used to evaluate cell viability and IC 50 of 5-Fu was determined. Quantitative real time PCR was applied to determine gene expression of caspase-3 (casp-3), caspase-4 (casp-4), and caspase-10 (casp-10). Cleaved casp-3 expression was investigated using Western blot and immunofluorescence. Our results demonstrated that IC 50 of 5-Fu for the DCLK1+ cells was significantly higher than that of the WT cells for both 24 and 48-hour treatment ( P =0.002 and 0.048 respectively), indicating increased chemoresistance of the DCLK1+ cells. Gene expression of casp-3, casp-4, and casp-10 were significantly inhibited in the DCLK1+ cells after 5-Fu treatment compared to the WT cells ( P =7.616e-08, 1.575e-05 and 5.307e-08, respectively). Cleaved casp-3 amount and casp-3 positive cells were significantly decreased in the DCLK1+ cells after 5-Fu treatment compared to the WT cells ( P =0.015). In conclusion, our results demonstrated that DCLK1 overexpression enhanced the chemoresistance of CRC cells to 5-Fu treatment by suppressing gene expression of key caspases in the apoptosis pathway and activation of apoptosis pathway. DCLK1 can be an intriguing therapeutic target for the effective treatment of CRC patients.Keywords:
Viability assay
Objective To evaluate the Cambridge Biotech HIV-1 Western blot kit and to assay the Sensitivity and Specificity in the different HIV infected status groups in the fields. Methods To collect the serum specimens from 645 cases, using Cambridge Biotech HIV-1 Western blot kit and using Genelabs Diagnostics HIV blot 2.2 Western blot kit to confirm the presence of HIV antibody through ELISA test. Results On HIV antibody positive individuals, Cambridge Biotech HIV-1 Western blot kit and Genelabs Diagnostics HIV blot 2.2 Western blot kit showed positive reaction. The sensitivity of both western blot kits was 100% . Of 398 serum specimens from ELISA testing on HIV antibody negative individuals, 23 showed indeterminate when tested by Cambridge Biotech HIV-1 Western blot kit and 86 showed indeterminate when tested by Genelabs Diagnostics HIV blot 2.2 Western blot kit. In such HIV screen test negative groups, the rates of specificity of Cambridge kit were 94.22% and 78.39% for Genelabs kit respectively. Conclusion The Cambridge Biotech HIV-1 Western blot kit had much higher specificity then Genelabs Diagnostics HIV blot 2.2 Western blot kit.
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TP53 gene has been found to have the highest correlation with human tumors, and its mutations occurr in about 50% malignant tumors. Its encoded p53 protein is a well-known tumor-suppressor factor in vivo, which is closely related to tumorigenesis. It is found that tumorigenesis has a close relationship with various abnormal biological processes, including cell cycle regulation, apoptosis, DNA damage repair, cell senescence, autophagy, metabolic regulation. This paper reviews the complex network relationship between p53 protein and tumorigenesis from biological processes affecting the tumorigenesis.
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Neoplams; Tumor suppressor protein p53; Biological processes
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Aim: To analysis the HSP70 protein polymorphism with 2-DE technique. Methods: The protein components of 5 volunteers' lymphocytes were analyzed with 2-DE,and the HSP70 components were identified with the specific antibody using Western blot.Results: With 2-DE and Western blot, at least 5 subtypes of HSP70 could be detected. Conclusion: The subtypes of HSP70 can be well separated using 2-DE and Western blot, and the key steps of 2-DE can be optimized.
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HIV-1 antibody determination by ELISA screening takes 4 hr to complete and is not reliable. Regular Western blot can take up to 24 hr. This makes organ transplantation both difficult and risky with regard to HIV-1 transmission. We developed a modification of the Western blot technique that takes under 50 min, is transportable, is definitive on site, and does not delay organ retrieval. This test has been called the Quick Western Blot. We examined 459 serum specimens from referrals; from the AIDS Testing Proficiency Panel, Walter Reed Army Institute of Research; and from 36 organ donors. All specimens were tested by ELISA HIV-1 Ab screening, the regular Western blot, and by the Quick Western Blot. The organ donors were initially tested on-site during organ procurement by the Quick Western Blot and later had complete testing by the reference methods. Compared with regular Western blot, the ELISA showed a specificity of 78.4% and a positive predictive value of 65.5%, whereas the Quick Western Blot was as reliable and specific as the regular Western blot, but much quicker. Because of the rapidity and specificity of the test, this test has particular utility in the screening of organ donors, as was shown in a case of multiple organ donation where the ELISA was negative, but the Quick Western Blot was found positive and thereby prevented the donation of HIV-1 infected organs.
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The Genie HIV-1/2 kit (Sanofi Diagnostics Pasteur, Montreal, Quebec), a synthetic-peptide solid-phase enzyme immunoassay, was evaluated as a confirmatory assay for HIV-1 antibodies in comparison with Western blot (BioRad, Hercules, CA, USA) on 50 stored HIV-1 antibody-positive sera and the 137 sera yielding repeated positive results in the conventional EIA screen out of 13405 fresh patient sera from Saskatchewan in 1993. The stored HIV-1-positive sera were uniformly positive in the Genie test. Of the 137 EIA screen-positive sera, 33 were uniformly positive and 64 were uniformly negative in Genie and Western blot; 36 were Genie-negative and indeterminate by Western blot; and four were Genie indeterminate, of which one was negative and three were indeterminate by Western blot. All HIV-1 Western blot-indeterminate and Genie-interdeterminate sera were negative in radio-immunoprecipitation assay (RIPA) and Western blot for HIV-1 and HIV-2 antibodies performed by a reference laboratory. Genie gave an accurate definitive result for 97% of EIA positive sera compared with 71% for Western blot. There was excellent correlation between Genie, Western blot and RIPA results. However, the Genie assay was faster, less costly and yielded fewer indeterminate results than Western blot in confirmatory testing for HIV-1 antibodies.
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Abstract Cell viability is one of the critical quality attributes (CQAs) for analyzing and characterizing cellular therapeutic products. The characterization of cell viability is essential to ensure quality, safety and efficacy during cell and gene therapy development. In this work, we characterized and compared two commonly used viability dual-staining methods, acridine orange/propidium iodide (AO/PI) and acridine orange/4′,6-diamidino-2-phenylindole (AO/DAPI) using the Cellaca MX high-throughput cell counter. In general, the AO/DAPI method provided higher viability results than AO/PI, with the measured viability difference between these two methods ranging from 0% to 24%. To identify the sources of the measured viability difference, we tested a list of factors including cell dying process, staining time and initial viability levels. First, we observed a larger viability difference from naturally-dying Jurkat cells compared to the result from heat-killed cells, suggesting that the differences may depend on the cell dying process. Second, we found that AO/PI provided more consistent cell count and viability than AO/DAPI during the first 30 min of staining time. Finally, we showed that measured viability difference might be larger for low-viability, naturally-dying Jurkat cells, as compared to healthy samples. The results from our characterization studies may benefit the selection of viability methods that are fit-for-purpose for analyzing cellular therapeutic products.
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The impact of plant extracts and phytochemicals on in vitro cell viability is usually assessed by employing cell viability assays dependent upon the activity of dehydrogenase enzymes. The CellTiter 96® AQueous One Solution Cell Proliferation Assay (CellTiter) was used to measure cell viability in response to antioxidant-rich extracts of Terminalia ferdinandiana fruits. Conflicting results were obtained from this assay whereby higher concentrations of extracts significantly increased cell viability compared to lower concentrations. Intrinsic reductive potential was observed in a cell-free system when extracts were added directly to the CellTiter assay reagent. To confirm this effect in a similar cell proliferation assay, we employed the CellTiter-Blue® Cell Viability Assay and again observed increased viability with increased concentrations of the extracts and direct reduction of the assay reagent by the extracts in cell-free systems. In the search for a cell proliferation assay that would not be directly affected by the plant extracts, we identified the CyQUANT® NF Cell Proliferation Assay that is based on the estimation of DNA content in viable cells. Cell viability decreased with increasing concentrations of the extracts. Accordingly, the results of the present study indicated that cell viability assays reliant upon dehydrogenase activity may lead to false positive results when testing antioxidant-rich plant extracts with intrinsic reductive potential, and alternative cell viability assays should be used to measure the cell viability.
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MTT assay
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