Study on mechanism of down-regulating ikca1 molecule affecting the increment of oral squamous cell carcinoma.

This research aimed to explore the mechanism of mediating the down-regulation of the calcium-activated potassium channel (IKCa1) gene expression in human oral squamous cell carcinoma Tca-8113 cells, thereby affecting cell proliferation and apoptosis. The expression level of IKCa1 in Tca-8113 cell line (oral squamous cell carcinoma) and HOEC cell line (human normal oral epithelial cell) was detected by RT-PCR. Then, after IKCa1 was knocked down in Tca-8113 cell line and HOEC cell line by RNA interference, and then cell proliferation levels were detected by cell counting kit 8 (CCK-8) method. Cell cycle distribution was detected by flow cytometry. Apoptosis was detected by membrane linked protein V-FITC/propidium iodide (PI) double-staining apoptosis detection kit. The protein expression level of IKCa1 was detected by Western Blot method. According to RT-PRC results, IKCa1 was significantly more expressed in Tca-8113 cell line than in HOEC cell line (P< 0.01).  In addition, the mRNA expression levels in the normal oral epithelium and oral squamous cell carcinoma showed the same trend. After knocking down IKCa1 in Tca-8113 cell line, the IKCa1siRNA group significantly inhibited cell proliferation compared with the siNC control group. The results of flow cytometry showed that the proportion of apoptotic Tca-8113 cells transfected with IKCa1siRNA was significantly increased. The ratio of early apoptosis and late apoptosis of Tca-8113 cells increased (P< 0.05). To investigate the effect of IKCa1 on apoptosis, we tested the expression levels of apoptosis-related proteins. The results showed that the mRNA level of IKCa1siRNA group was significantly decreased by 44.41% compared with the control group (p< 0.01). Meanwhile, the mRNA level of Bax was significantly increased by 36.0% (p< 0.05). Our results showed that knocking down IKCa1 in Tca-8113 cells could induce cell cycle arrest and apoptosis to produce an anti-proliferation effect, thus inhibiting the expression of IKCa1 has an anti-cancer effect in oral squamous cell carcinoma.

Calcium Influx Kinetics and the Characteristics of Potassium Channels in Peripheral T Lymphocytes in Systemic Sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a chronic, immune-mediated, connective tissue disease causing microvascular abnormalities and fibrosis. The cytoplasmic calcium influx kinetics in T lymphocytes governs lymphocyte activation in this inflammatory process. The inhibition of Kv1.3 and IKCa1 potassium channels reduces calcium influx. METHODS: This study aimed to analyze cytoplasmic calcium influx kinetics following activation in Th1, Th2, and CD8 cells in peripheral blood of 12 healthy individuals and 16 patients with systemic sclerosis using flow cytometry. We also evaluated the effect of the specific inhibition of the Kv1.3 and IKCa1 potassium channels. RESULTS: We observed higher levels of activation in CD8 compared with Th1 cells in SSc. However, the activation of CD8 cells was lower in SSc compared to healthy controls. Moreover, activation of Th1 lymphocytes was slower in SSc than in healthy controls. The inhibition of IKCa1 channels decreased the activation of Th1 cells, while the inhibition of Kv1.3 channels modified the dynamics of activation of Th1 and Th2 lymphocytes in SSc. CONCLUSION: Th1 and CD8 cells demonstrate specific activation dynamics and sensitivity to potassium channel inhibition in SSc, distinguishing this condition both from healthy controls and other autoimmune diseases.

Altered calcium influx of peripheral Th2 cells in pediatric Crohn's disease: infliximab may normalize activation patterns.

OBJECTIVE: Crohn's disease is a chronic inflammation of the gastrointestinal tract with an abnormal immune phenotype. We investigated how intracellular calcium kinetics of Th1 and Th2 lymphocytes alter upon specific inhibition of Kv1.3 and IKCa1 channels in pediatric Crohn's disease. STUDY DESIGN: Blood was taken from 12 healthy and 29 Crohn's disease children. Of those, 6 were switched to infliximab and re-sampled after the 4th infliximab treatment. Intracellular calcium levels were monitored using flow cytometry in the presence or absence of specific inhibitors of Kv1.3 and IKCa1 potassium channels. RESULTS: In Crohn's disease treated with standard therapy, calcium response during activation was higher than normal in Th2 cells. This was normalized in vitro by inhibition of Kv1.3 or IKCa1 potassium channels. After the switch to infliximab, potassium channel function and expression in Th2 lymphocytes were comparable to those in Th1 cells. CONCLUSION: These results may indicate that potassium channels are potential immune modulatory targets in Crohn's disease.

The effects of Kv1.3 and IKCa1 channel inhibition on cytokine production and calcium influx of T lymphocytes in rheumatoid arthritis and ankylosing spondylitis.

Kv1.3 and IKCa1 lymphocyte potassium channels have been implicated as important targets of selective immunomodulation. We compared the alterations in cytokine production upon selective inhibition of Kv1.3 or IKCa1 channels (by MGTX and TRAM, respectively) in healthy donors (HD), RA and AS patients. We also determined calcium influx kinetics and its sensitivity to Kv1.3 and IKCa1 channel inhibition following PHA activation in CD4, Th1, Th2 and CD8 cells as well as monocytes. The application of TRAM resulted in a lower production of TNF-a and IL1-RA in all three study groups. Inhibition by TRAM had contrary effects on the production of IL-1b and IL-5: While their production was increased by PBMCs of RA patients, this effect was not observed in HD and AS PBMCs. While treatment with MGTX resulted in a similar decrease in calcium influx in the CD4 and Th2 subsets across all study groups, TRAM treatment had opposite effects on RA and HD samples: It decreased calcium influx in the Th2 and CD8 subsets in RA, while only Th1 cells were affected in HDs. The effects of IKCa1 channel inhibition are controversial in samples of RA and AS patients, since it shifts the inflammatory balance into the pro-inflammatory direction.

Impact of aging on calcium influx and potassium channel characteristics of T lymphocytes.

Adaptive immunity and T cell function are affected by aging. Calcium influx patterns, regulated by Kv1.3 and IKCa1 potassium channels, influence T cell activation. We aimed to compare calcium influx kinetics in CD8, Th1 and Th2 cells in human peripheral blood samples obtained from five different age groups (cord blood, 10-15 ys, 25-40 ys, 45-55 ys, 60-75 ys).We measured calcium influx using flow cytometry in samples treated with or without specific inhibitors of Kv1.3 and IKCa1 channels (MGTX and TRAM, respectively).Calcium influx was higher in Th1 cells of adults, however, its extent decreased again with aging. Importantly, these changes were not detected in Th2 cells, where the pattern of calcium influx kinetics is similar throughout all investigated age groups. MGTX had a more pronounced inhibitory effect on calcium influx in Th2 cells, while in Th1 cells the same was true for TRAM in the 25-40 ys and 45-55 ys groups. Calcium influx of CD8 cells were inhibited to a similar extent by both applied inhibitors in these groups, and had no effect in the elderly.Altered lymphocyte potassium channel inhibitory patterns, regulators of calcium influx kinetics, might contribute to the development of age-related changes of T cell function.