G-Protein-Coupled Receptors Mediate Modulations of Cell Viability and Drug Sensitivity by Aberrantly Expressed Recoverin 3 within A549 Cells

Abstract

To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.

Lipid Metabolism Regulators Are the Possible Determinant for Characteristics of Myopic Human Scleral Stroma Fibroblasts (HSSFs)

“…Total RNA extraction (n = 3 each) from 2D cultured HCSFs obtained from various AL eyes as described above using a commercially available RNeasy mini kit (Qiagen, Valencia, CA, USA) was processed according to the manufacturer’s protocol. After confirmation of suitable content and quality of the prepared RNA for the RNA sequencing analysis and quantitative real-time PCR (RIN; RNA integrity number, >8.5), RNA sequencing analysis was performed as described recently [ 64 ]. Then, adapter sequence, ambiguous nucleotides and low-quality sequences were removed, and those clean-up reads were mapped as according to the reference genome sequence (GRCh38) using HISAT2 tools software (HISAT 2.2.1, accessed on 1 November 2023) [ 65 ].…”

Physical Properties and Cellular Metabolic Characteristics of 3D Spheroids Are Possible Definitive Indices for the Biological Nature of Cancer-Associated Fibroblasts

“…During these investigations, since the subjected cells spontaneously formed droplets in the culture medium to form 3D spheroids, we speculated that the formation of such 3D spheroids may be caused by potential unknown biological factors. In fact, we found that almost all of the non-cancerous cells that we tested formed globe-shaped 3D spheroids [30,31], while in contrast, non-globe-shaped 3D spheroids were produced from most of the cancerous cells that we examined [35,36,38]. Furthermore, such cancer-cell-related 3D spheroids varied significantly among various cell lines with different clinical and pathological backgrounds even from the same origin [38,45].…”

“…Therefore, such in vitro 3D cell culture models are now being more frequently used for testing the efficacy as well as suitable dosages of anticancer drugs [23][24][25][26][27]. Among the various in vitro 3D cell culture models [28,29], we focused to the simplest 3D drop culture method, and we successfully established various in vitro 3D spheroid models using non-cancerous cells [30,31] as well as cancerous cells [35,36]. During these investigations, since the subjected cells spontaneously formed droplets in the culture medium to form 3D spheroids, we speculated that the formation of such 3D spheroids may be caused by potential unknown biological factors.…”

“…Among the various in vitro 3D cell culture methods, a simpler model, a 3D spheroid culture model, has been the most widely used [28,29]. In our recent investigations, to characterize the microenvironments of various tissues, we independently developed unique 3D drop cell culture methods using several non-cancerous cells including human conjunctival fibroblasts (HconF) [30], human orbital fibroblasts (HOF) [31], human scleral stromal fibroblasts (HSclF) [32] and human scleral fibroblasts (HSSF) [33], and Graves'-diseaserelated HOF (GOF) [34], as well as cancerous cells including an A549 lung adenocarcinoma cell line [35], various malignant melanoma (MM) cell lines [36] and various OSCC. As expected, our findings indicated that the biological natures of the 3D spheroid models were markedly different and distinct from those of 2D cultured models even though both were prepared under exactly identical experimental conditions except that the culture plates that were used were different [37].…”

Three-Dimensional Spheroid Configurations and Cellular Metabolic Properties of Oral Squamous Carcinomas Are Possible Pharmacological and Pathological Indicators

“…Among the several types of in vitro 3D cell culture models, a simpler form called a 3D spheroid that can be obtained in a nonadherent surface manner has been the most frequently used model [17,18]. We recently and independently succeeded at producing in vitro 3D spheroid models that replicate several disease states using non-cancerous cells including 3T3-L1 preadipocytes, human orbital fibroblasts (HOF) [19], human trabecular meshwork (HTM) cells [20], human conjunctival fibroblasts (HconF) [21] as well as other cancerous cell lines, including an A549 lung adenocarcinoma cell [22], and various malignant melanoma cell lines [23]. Based upon these collective studies, we concluded that the biological aspects of 3D spheroids were significantly different from those of 2D cultured cells even though both are cultured under the exact same conditions, except that different culture plates are used [24].…”