The H6PD Knockout KYSE-30 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout cell population in which the H6PD gene has been functionally disrupted in the KYSE-30 human esophageal squamous cell carcinoma line. This polyclonal pool allows researchers to interrogate the loss-of-function effects of hexose-6-phosphate dehydrogenase (H6PD) without clonal selection, capturing the heterogeneity of CRISPR-mediated gene disruption across a bulk cell population. The product is designed as an in vitro model for studying endoplasmic reticulum NADPH production, glucocorticoid metabolism, and redox regulation in esophageal cancer biology.
The parental KYSE-30 cell line was established from a poorly differentiated esophageal squamous cell carcinoma of a 64-year-old Japanese male and is a widely used model in esophageal cancer research. KYSE-30 cells exhibit characteristic features of squamous cell carcinoma, including adherent growth and expression of epithelial markers, making them suitable for investigating tumor biology, drug responses, and metabolic reprogramming. The introduction of a targeted H6PD knockout in this line provides a unique platform to dissect the function of the endoplasmic reticulum-localized pentose phosphate pathway in malignant esophageal cells.
H6PD encodes hexose-6-phosphate dehydrogenase, a microsomal enzyme that catalyzes the first and rate-limiting step of the oxidative pentose phosphate pathway within the endoplasmic reticulum lumen, generating NADPH from NADP+ using glucose-6-phosphate as a substrate. This luminal NADPH pool is essential for the reductase activity of 11??-hydroxysteroid dehydrogenase type 1 (11??-HSD1), which converts inert cortisone to active cortisol. H6PD is regulated by factors including PPAR??, glucocorticoids, and ER stress pathways, and its activity impacts downstream effectors such as 11??-HSD1, the ER luminal redox state, and cellular glucocorticoid activation. The interaction between H6PD and 11??-HSD1, coupled with NADP+/NADPH and ER membrane components, forms a key metabolic node linking glucose metabolism to hormonal signaling.
Disruption of H6PD in KYSE-30 cells abrogates the supply of NADPH in the ER lumen, thereby impairing cortisol production and perturbing redox homeostasis. This knockout model is particularly significant given the growing evidence that cancer cells, including esophageal squamous cell carcinomas, exploit metabolic enzymes and redox control mechanisms to sustain proliferation, evade apoptosis, and resist therapy. By eliminating H6PD function, researchers can investigate how loss of ER NADPH generation affects tumor cell growth, oxidative stress responses, and metabolic adaptation, providing insights into potential vulnerabilities of esophageal cancers.
This knockout cell pool is well-suited for a broad array of functional assays, including Western blotting and RT-qPCR to confirm gene disruption and downstream expression changes, cortisol/cortisone quantification via ELISA or LC-MS, NADPH/NADP+ ratio measurements, cell proliferation and colony formation assays, apoptosis detection (caspase-3/7 activity), and reactive oxygen species (ROS) monitoring. Additionally, the cells can be employed in RNA-seq and metabolomics studies to comprehensively profile transcriptional and metabolic alterations upon H6PD loss. For further details, quotation requests, or assistance with experimental design, please contact Ascent Research.
