Detecting Effects of Low Levels of FCCP on Stem Cell Micromotion and Wound-Healing Migration by Time-Series Capacitance Measurement.

Electric cell-substrate impedance sensing (ECIS) has been used as a real-time impedance-based method to quantify cell behavior in tissue culture. The method is capable of measuring both the resistance and capacitance of a cell-covered microelectrode at various AC frequencies. In this study, we demonstrate the application of high-frequency capacitance measurement (f = 40 or 64 kHz) for the sensitive detection of both the micromotion and wound-healing migration of human mesenchymal stem cells (hMSCs). Impedance measurements of cell-covered electrodes upon the challenge of various concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), from 0.1 to 30 µM, were conducted using ECIS. FCCP is an uncoupler of mitochondrial oxidative phosphorylation (OXPHOS), thereby reducing mitochondrial ATP production. By numerically analyzing the time-series capacitance data, a dose-dependent decrease in hMSC micromotion and wound-healing migration was observed, and the effect was significantly detected at levels as low as 0.1 µM. While most reported works with ECIS use the resistance/impedance time series, our results suggest the potential use of high-frequency capacitance time series for assessing migratory cell behavior such as micromotion and wound-healing migration.

Electrochemical Assessment of Anticancer Compounds on the Human Tongue Squamous Carcinoma Cells.

The most common oral cancer is squamous cell carcinoma (SCC) and its highest occurrence is in the tongue. Almost 30% of patients with one primary head and neck tumor will have a second primary malignancy. In recent studies, two novel plant extracts, andrographolide and cannabidiol (CBD), have been exploited for their anticancer effects. Here, we investigated the cytotoxic effects of these two compounds on SCC-25 cells, a human tongue squamous carcinoma cell line, and compared the outcomes with two chemotherapeutic drugs, cisplatin and fluorouracil. Electric cell substrate impedance sensing (ECIS) system was applied to measure frequency- and time-dependent impedance of SCC-25 cell-covered electrodes and to further assess subtle changes in cell morphology and micromotion in response to different concentrations (0, 10, 30, 100, and 300 µM) of these compounds. AlamarBlue and Annexin V/7-AAD binding assays were used to measure the concentration dependent changes in viability and apoptosis of SCC-25 cells. Our results demonstrate that 24 hours after exposure to 30 µM CBD can significantly decrease the micromotion rate, damage the integrity of cell morphology, reduce cell viability, and induce higher apoptosis in treated SCC-25 cells, while the other three drugs attain similar effects at the concentration of 100 µM or higher. The apoptosis-induced changes in cell morphology and micromotion monitored by ECIS correlate well with biochemical assays. Thus, both frequency- and time-dependent impedance measurements using ECIS can be used to real-time follow cancer cell activities in response to anticancer drugs with different temporal cytotoxicity profiles.

Application of Electric Cell-Substrate Impedance Sensing to Investigate the Cytotoxic Effects of Andrographolide on U-87 MG Glioblastoma Cell Migration and Apoptosis.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. In recent studies, the efficacy of suberoylanilide hydroxamic acid (SAHA) has been investigated for GBM. We explored the effects of two exploratory compounds, the histone deacetylase SAHA and the natural product andrographolide, on Uppsala 87 Malignant Glioma (U-87 MG) cell migration and viability in comparison with the clinically used therapeutic agent temozolomide (TMZ). We used the electric cell-substrate impedance sensing (ECIS) system to monitor the migration of U-87 MG cells after treatment with various concentrations of these compounds. Moreover, we used the Alamar blue assay and western blotting to observe the concentration-dependent changes in the viability and apoptosis of U-87 MG cells. Our results demonstrated that both SAHA and andrographolide (10-300 µM) significantly inhibited GBM cell migration in a concentration-dependent manner, and 10 µM SAHA and 56 µM andrographolide demonstrated remarkable inhibitory effects on U-87 MG migration. Western blotting indicated that compared with TMZ, both SAHA and andrographolide induced higher expression levels of apoptosis-related proteins, such as caspase-3, BAX, and PARP in U-87 MG cells. Furthermore, all three drugs downregulated the expression of the antiapoptotic protein Bcl-2. In conclusion, SAHA and andrographolide showed exceptional results in inhibiting cell migration and motility. The ECIS wound healing assay is a powerful technique to identify and screen potential therapeutic agents that can inhibit cancer cell migration.

Application of ECIS to Assess FCCP-Induced Changes of MSC Micromotion and Wound Healing Migration.

Electric cell-substrate impedance sensing (ECIS) is an emerging technique for sensitively monitoring morphological changes of adherent cells in tissue culture. In this study, human mesenchymal stem cells (hMSCs) were exposed to different concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) for 20 h and their subsequent concentration-dependent responses in micromotion and wound healing migration were measured by ECIS. FCCP disrupts ATP synthesis and results in a decrease in cell migration rates. To detect the change of cell micromotion in response to FCCP challenge, time-series resistances of cell-covered electrodes were monitored and the values of variance were calculated to verify the difference. While Seahorse XF-24 extracellular flux analyzer can detect the effect of FCCP at 3 µM concentration, the variance calculation of the time-series resistances measured at 4 kHz can detect the effect of FCCP at concentrations as low as 1 µM. For wound healing migration, the recovery resistance curves were fitted by sigmoid curve and the hill slope showed a concentration-dependent decline from 0.3 µM to 3 µM, indicating a decrease in cell migration rate. Moreover, dose dependent incline of the inflection points from 0.3 µM to 3 µM FCCP implied the increase of the half time for wound recovery migration. Together, our results demonstrate that partial uncoupling of mitochondrial oxidative phosphorylation reduces micromotion and wound healing migration of hMSCs. The ECIS method used in this study offers a simple and sensitive approach to investigate stem cell migration and its regulation by mitochondrial dynamics.

Author Correction: Angiotensin-converting enzyme inhibitors or angiotensin receptor blocker monotherapy retard deterioration of renal function in Taiwanese chronic kidney disease population.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Angiotensin-converting enzyme inhibitors or angiotensin receptor blocker monotherapy retard deterioration of renal function in Taiwanese chronic kidney disease population.

It remains unclear how different uses of angiotensin-converting inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) influence the progression of chronic kidney disease (CKD). This study explored CKD progression in a multicentre, longitudinal cohort study that included 2639 patients with CKD stage 1-5 and hypertension. Patients treated with ACEI or ARB for ≥90 days during a 6-mo period comprised the study group, or no treatment, comprised the control group. The study group was subdivided on the basis of treatment: ACEI monotherapy or ARB monotherapy. Progression of renal deterioration was defined by an average eGFR decline of more than 5 mL/min/1.73 m2/yr or the commencement of dialysis. With at least 1-year follow up, a progression of renal deterioration was demonstrated in 29.70% of the control group and 25.09% of the study group. Patients in the study group had significantly reduced progression of CKD with adjusted odds ratio 0.79 (95% confidence interval: 0.63-0.99). However, when ACEI monotherapy and ARB monotherapy were analyzed separately, none of their associations with CKD progression was statistically significant. In conclusion, ACEI or ARB monotherapy may retard the deterioration of renal function among patients with CKD and hypertension.