In this study, the researchers aimed to confirm the importance of calcium dysregulation in cancer development and investigate the role of Orai3 as an upstream activator of AKT in chemoresistance in non-small cell lung cancer (NSCLC). To assess the impact of calcium dysregulation on cancer development, the researchers conducted several experiments. They first performed a formazan assay to measure cell viability. Living cells were transferred to a 96-well plate containing formazan, and absorbance at 550nm was measured using a Nanodrop 2000 spectrophotometer. The results were recorded in an Excel file. Next, they used fura-2 probe and Metafluor software for calcium imaging. H23 cells were incubated with the fura-2 probe and fluorescence intensity was measured at 512nm while exciting the probe at 340nm and 380nm. The ratio of fluorescence intensities (R) during excitations at 340nm and 380nm indicated intracellular calcium concentrations. By infusing calcium into the extracellular medium for 60 seconds followed by infusion of calcium-free medium with thapsigargin (an inhibitor of SERCA pumps), they activated SOC channels like Orai3 at the plasma membrane. This led to an increase in R due to higher cytoplasmic calcium concentration. Various players involved in calcium homeostasis then caused a reduction in R.
Furthermore, quantitative PCR reactions were performed on samples using a LightCycler® from Roche and ABsolut kitTMqPCR SYBR® Green Mixes. This allowed quantification of cDNA sequences related to interest using SYBR green as a DNA intercalator emitting fluorescence upon contact with double-stranded DNA. The results showed that calcium dysregulation is important for cancer development and demonstrated that Orai3 acts as an upstream activator of AKT which is involved in chemoresistance in NSCLC. This expanded understanding may have implications for developing targeted therapies for lung cancer and other cancers alike.
- Calcium dysregulation means that there is a problem with how calcium is controlled in the body, which is important in cancer development. Calcium is a mineral that helps with many functions in our bodies.
- Orai3 is a protein that starts a process called AKT activation, which helps cancer cells become resistant to chemotherapy in a type of lung cancer called NSCLC.
- Cell viability means how alive and healthy cells are. The formazan assay is a test used to measure this.
- Calcium imaging means using special tools like the Fura-2 probe and Metafluor software to see how much calcium is inside cells.
- SOC channels like Orai3 are activated when there is an increase in calcium concentration inside the cell's cytoplasm, which can affect how cells work.
Important words- Calcium: A mineral that helps with many functions in our bodies, like building strong bones and helping muscles work properly.
- Dysregulation: When something isn't working as it should or isn't being controlled properly.
- Activator: Something that starts or triggers a process.
- Chemoresistance: When cancer cells become resistant to chemotherapy, which makes it harder to treat them.
- NSCLC: A type of lung cancer called non-small cell lung cancer.
- Viability: How alive and healthy something is, like cells being able to function properly.
- Assay: A test or experiment used to measure or study something specific.
- Imaging: Using
Calcium Dysregulation and Orai3 in Non-Small Cell Lung Cancer (NSCLC)
Non-small cell lung cancer (NSCLC) is one of the most common types of cancer, accounting for approximately 85% of all lung cancers. In a recent study, researchers sought to investigate the importance of calcium dysregulation in NSCLC development and to explore the role of Orai3 as an upstream activator of AKT in chemoresistance. To do this, they conducted several experiments that allowed them to measure cell viability, intracellular calcium concentrations, and quantify cDNA sequences related to interest.
Measuring Cell Viability
The first experiment involved measuring cell viability using a formazan assay. Living cells were transferred into a 96-well plate containing formazan and absorbance at 550nm was measured with a Nanodrop 2000 spectrophotometer. The results were recorded in an Excel file.
Measuring Intracellular Calcium Concentrations
To measure intracellular calcium concentrations, researchers used fura-2 probe and Metafluor software for calcium imaging. H23 cells were incubated with the fura-2 probe and fluorescence intensity was measured at 512nm while exciting the probe at 340nm and 380nm. The ratio between these two intensities (R) indicated cytoplasmic calcium concentration levels within the cells. To activate SOC channels like Orai3 at the plasma membrane, they infused calcium into the extracellular medium for 60 seconds followed by infusion of calcium-free medium with thapsigargin (an inhibitor of SERCA pumps). This led to an increase in R due to higher cytoplasmic calcium concentration but various players involved in calcium homeostasis caused it to reduce again afterwards.
Quantifying cDNA Sequences Related To Interest
The final experiment involved quantifying cDNA sequences related to interest using quantitative PCR reactions on samples from LightCycler® from Roche and ABsolut kitTMqPCR SYBR® Green Mixes which emit fluorescence upon contact with double stranded DNA when illuminated by UV light.. The results showed that increased levels of Orai 3 expression leaded to higher AKT activity which is associated with chemoresistance in NSCLC patients suggesting that targeting Orai 3 may be beneficial for treating this type of cancer or other cancers alike where similar mechanisms are present .
Conclusion
This research confirms that increased levels of Orai 3 expression leadsed to higher AKT activity which is associated with chemoresistance in NSCLC patients suggesting that targeting Orai 3 may be beneficial for treating this type or other cancers alike where similar mechanisms are present . Furthermore , it has expanded our understanding regarding how important calciums dysregulation can be when it comes down developing targeted therapies for lung cancer or any other type os cancer .
This is the second part of the previous review. In the previous review we suspected that Orai3 channels were involved in lung cancer and more precisely in several cancers. Here we confirm that calcium dysregulation is important for cancer development. in this paper we show that Orai3 is an upstream activator of AKT and we prove that AKT is involved in chemoresistance in NSCLC.
