ABSTRACT
Cadmium (Cd) is a nephrotoxic environmental pollutant that causes a generalized dysfunction of the proximal tubule characterized by polyuria and proteinuria. Even though the effects of Cd on the kidney have been well-characterized, the molecular mechanisms underlying these effects have not been fully elucidated. MicroRNAs (miRNAs) are small non-coding RNAs that regulate cellular and physiologic function by modulating gene expression at the post-transcriptional level. The goal of the present study was to determine if Cd affects renal cortex miRNA expression in a well-established animal model of Cd-induced kidney injury. Male Sprague-Dawley rats were treated with subcutaneous injections of either isotonic saline or CdCl2 (0.6 mg/kg) 5 days a week for 12 weeks. The 12-week Cd-treatment protocol resulted in kidney injury as determined by the development of polyuria and proteinuria, and a significant increase in the urinary biomarkers Kim-1, ß2 microglobulin and cystatin C. Total RNA was isolated from the renal cortex of the saline control and Cd treated animals, and differentially expressed miRNAs were identified using µParafloTM microRNA microarray analysis. The microarray results demonstrated that the expression of 44 miRNAs were significantly increased and 54 miRNAs were significantly decreased in the Cd treatment group versus the saline control (t-test, p ≤ 0.05, N = 6 per group). miR-21-5p, miR-34a-5p, miR-146b-5p, miR-149-3p, miR-224-5p, miR-451-5p, miR-1949, miR-3084a-3p, and miR-3084c-3p demonstrated more abundant expression and a significant two-fold or greater increased expression in the Cd-treatment group versus the saline control group. miR-193b-3p, miR-455-3p, and miR-342-3p demonstrated more abundant expression and a significant two-fold or greater decreased expression in the Cd-treatment group versus the saline control group. Real-time PCR validation demonstrated (1) a significant (t-test, p ≤ 0.05, N = 6 per group) increase in expression in the Cd-treated group for miR-21-5p (2.7-fold), miR-34a-5p (10.8-fold), miR-146b-5p (2-fold), miR-224-5p (10.2-fold), miR-3084a-3p (2.4-fold), and miR-3084c-3p (3.3-fold) and (2) a significant (t-test, p ≤ 0.05, N = 6 per group) 52% decrease in miR-455-3p expression in the Cd-treatment group. These findings demonstrate that Cd significantly alters the miRNA expression profile in the renal cortex and raises the possibility that dysregulated miRNA expression may play a role in the pathophysiology of Cd-induced kidney injury. In addition, these findings raise the possibility that Cd-dysregulated miRNAs might be used as urinary biomarkers of Cd exposure or Cd-induced kidney injury.
ABSTRACT
Liposarcoma is a malignant soft tissue tumor that originates from adipose tissue and is one of the most frequently diagnosed soft tissue sarcomas in humans. There is great interest in identifying novel chemotherapeutic options for treating liposarcoma based upon molecular alterations in the cancer cells. The Aurora kinases have been identified as promising chemotherapeutic targets based on their altered expression in many human cancers and cellular roles in mitosis and cytokinesis. In this study, we investigated the effects of an Aurora kinase A inhibitor (MK-5108), an Aurora kinase B inhibitor (AZD1152-HQPA), and a pan-Aurora kinase inhibitor (AMG 900) on undifferentiated SW-872 and well-differentiated 93T449 human liposarcoma cells. Treatment of the SW-872 and 93T449 cells with MK-5108 (0-1000 nM), AZD1152-HQPA (0-1000 nM), and AMG 900 (0-1000 nM) for 72 h resulted in a dose-dependent decrease in the total viable cell number. Based upon the EC50 values, the potency of the three Aurora kinase inhibitors in the SW-872 cells was as follows: AMG 900 (EC50 = 3.7 nM) > AZD1152-HQPA (EC50 = 43.4 nM) > MK-5108 (EC50 = 309.0 nM), while the potency in the 93T449 cells was as follows: AMG 900 (EC50 = 6.5 nM) > AZD1152-HQPA (EC50 = 74.5 nM) > MK-5108 (EC50 = 283.6 nM). The percentage of polyploidy after 72 h of drug treatment (0-1000 nM) was determined by propidium iodide staining and flow cytometric analysis. AMG 900 caused a significant increase in polyploidy starting at 25 nM in the SW-872 and 93T449 cells, and AZD1152-HQPA caused a significant increase starting at 100 nM in the SW-872 cells and 250 nM in the 93T449 cells. The Aurora kinase A inhibitor MK-5108 did not significantly increase the percentage of polyploid cells at any of the doses tested in either cell line. The expression of Aurora kinase A and B was evaluated in the SW-872 cells versus differentiated adipocytes and human mesenchymal stem cells by real-time RT-PCR and Western blot analysis. Aurora kinase A and B mRNA expression was significantly increased in the SW-872 cells versus the differentiated adipocytes and human mesenchymal stem cells. Western blot analysis revealed a ~ 48 kDa immunoreactive band for Aurora kinase A that was not present in the differentiated adipocytes or the human mesenchymal stem cells. A ~ 39 kDa immunoreactive band for Aurora kinase B was detected in the SW-872 cells, differentiated adipocytes, and human mesenchymal stem cells. A smaller immunoreactive band for Aurora kinase B was detected in the SW-872 cells but not in the differentiated adipocytes and human mesenchymal stem cells, and this may reflect the expression of a truncated splice variant of Aurora kinase B that has been associated with poor patient prognosis. The 93T449 cells demonstrated decreased expression of Aurora kinase A and B mRNA and protein compared to the SW-872 cells, and also expressed the truncated form of Aurora kinase B. The results of these in vitro studies indicate that Aurora kinase inhibitors should be further investigated as possible chemotherapeutic agents for human liposarcoma.
