Identification of Bcl2 as a Stably Expressed qPCR Reference Gene for Human Colon Cancer Cells Treated with Cottonseed-Derived Gossypol and Bioactive Extracts and Bacteria-Derived Lipopolysaccharides

Abstract

Cottonseed contains many bioactive molecules including plant polyphenols. Cottonseed value might be increased by providing high-value bioactive polyphenols for improving nutrition and health. However, there was a lack of molecular evidence for cottonseed bioactivity in mammalian cells. One widely used method for evaluating the bioactivity of natural products is quantitative real-time-PCR (qPCR). The selection of stably expressed internal reference genes is a crucial task of qPCR assay for data analysis. The rationale for reference gene selection is that a lower standard deviation of the cycle of threshold (Cq) among the treatments indicates a more stable expression of the gene. The objective of this study was to select reference genes in human colon cancer cells (COLO 205) treated with cottonseed-derived gossypol and bioactive extracts along with bacterial endotoxin lipopolysaccharides (LPS). SYBR Green qPCR was used to analyze the mRNA levels of a wide range of biomarkers involved in glucose transport, lipid biosynthesis, inflammatory response, and cancer development. qPCR data (10,560 Cq values) were generated from 55 genes analyzed from 64 treatments with triplicate per treatment for each gene. The data showed that B-cell lymphoma 2 (Bcl2) mRNA was the most stable among the 55 mRNAs analyzed in the human colon cancer cells. Glyceraldehyde 3 phosphate dehydrogenase (Gapdh) and ribosome protein L32 (Rpl32) mRNAs were not good qPCR references for the colon cancer cells. These observations were consistent regardless of the treatment comparison between gossypol and LPS, glanded and glandless seed extracts, seed coat and kernel extracts, or treatment for 8 and 24 h. These results suggest that Bcl2 is a preferable reference gene for qPCR assays in human colon cancer cells treated with cottonseed-derived gossypol and bioactive extracts as well as LPS. The extensive qPCR results firmly support the conclusion that the Bcl2 gene is stably expressed at the mRNA level in the human colon cancer cells regardless of the treatment, suggesting that Bcl2 gene expression is not regulated at the mRNA level but at the post-transcriptional level. These results should facilitate studies designated to evaluate bioactivity on gene expression regulation by cottonseed molecules and other natural and synthetic molecules for nutrition and health uses.

Plant Polyphenol Gossypol Induced Cell Death and Its Association with Gene Expression in Mouse Macrophages

“…The more metabolic activity in the sample, the higher the signal. The selection of gossypol concentration and duration of treatment was based on our previous study using human colon cancer cells [ 42 , 43 ]. Mouse macrophages were treated with up to 100 µg/mL of gossypol and incubated at 37 °C, 5% CO 2 for 2–24 h. MTT assay reagent was added to the media and incubated at 37 °C, 5% CO 2 for 2 h before adding MTT solubilization solution to each well and shaking at room temperature overnight.…”

Characterization of the Nonpolar and Polar Extractable Components of Glanded Cottonseed for Its Valorization

“…While the three acylamide compounds were detected in the Gd samples, only one was detected in the Gl cottonseed with lower abundance. This information will aid the characterization and valorization of Gd and Gl cottonseed as functional food supplements and biomedical additives [ 31 , 38 , 73 ]. Manipulation of the sample processing could alter the abundance of some compounds.…”

“…Whereas cottonseed is a natural resource of agrochemicals (e.g., fiber, proteins, carbohydrates, and lipids), its nutritional value is hindered by the presence of a toxic chemical (i.e., gossypol) in the conventional glanded (Gd) cottonseed [ 5 , 14 ]. The current strategies of valorization of cottonseed include (1) the development of novel Gl cottonseed varieties for food applications [ 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ], (2) synthesis and formulation of biobased materials from traditional Gd cottonseed and its byproducts for nonfood applications [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ], and (3) exploration of the bioactive components in cottonseed for nutritive, health, and medical applications [ 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. While these bioactive components are assumed to be peptide fragments, polyphenolics, and/or flavonoids, the specific functional groups or compounds are yet to be identified [ 33 , 38 , 39 , 40 , 41 ].…”

Plant Polyphenol Gossypol Induced Cell Death and Its Associ-ation with Gene Expression in Mouse Macrophages

“…The more metabolic activity in the sample, the higher the signal. Selection of gossypol concentration and duration of treatment was based on our previous study using human colon cancer cells [42,43]. Mouse macrophages were treated with up to 100 µg/mL of gossypol and incubated at 37°C, 5% CO2 for 2-24 h. MTT assay reagent was added to the media and incubated at 37 C, 5% CO2 for 2 h before adding MTT solubilization solution to each well, shaken at room temperature overnight.…”