Microbial dysbiosis is an important trigger in the development of oral diseases. Oral keratinocytes or gingival epithelial cells (GECs) offer protection against various microbial insults. Recent studies suggest that GECs expressed higher level of bitter taste receptor 14 (T2R14) compared to other taste receptors and toll-like receptors and act as innate immune sentinels. Macroautophagy or autophagy is a cellular conserved process involved in the regulation of host innate immune responses against microbial infection. Here, we describe a robust method for evaluation of T2R14-dependent autophagy flux in GECs. Autophagy flux was detected using Western blot analysis in GECs and further was confirmed using Acridine Orange-dependent flow cytometry analysis.

Macroautophagy (hereafter autophagy) is a lysosomal degradation pathway that functions in nutrient recycling and as a mechanism of innate immunity. Previously, we reported a novel host-bacteria interaction between cariogenic S. mutans and bitter taste receptor (T2R14) in gingival epithelial cells (GECs), leading to an innate immune response. Further, S. mutans might be using the host immune system to inhibit other Gram-positive bacteria, such as S. aureus. To determine whether these bacteria exploit the autophagic machinery of GEC, it is first necessary to evaluate the role of T2R14 in modulating autophagic flux. So far, the role of T2R14 in the regulation of autophagy is not well characterized. Therefore, in this study, for the first time, we report that T2R14 downregulates autophagy flux in GECs, and T2R14 knockout increases acidic vacuoles. However, the treatments of GEC WT with a T2R14 agonist and antagonist did not lead to a significant change in acidic vacuole formation. Transmission electron microscopy morphometric results also suggested an increased number of autophagic vesicles in T2R14-knockout GEC. Further, our results suggest that S. mutans competence stimulating peptide CSP-1 showed robust intracellular calcium release and this effect is both T2R14- and autophagy protein 7-dependent. In this study, we provide the first evidence that T2R14 modulates autophagy flux in GEC. The results of the current study could help in identifying the impact of T2R in regulation of the immuno-microenvironment of GEC and subsequently oral health.

Human myeloid leukemia cells (such as K562) could be used for the study of erythropoiesis, and mature erythroid markers and globins could be induced during leukemia cell differentiation; however, the pathways involved are different compared with those of hematopoietic stem cells (HSCs).We identified the differentially expressed genes (DEGs) of K562 cells and HSCs associated with stem cells and erythroid differentiation. Furthermore, we showed that hemin-induced differentiation of K562 cells could be induced by serum starvation or treatment with the tyrosine kinase inhibitor saracatinib. However, erythroid differentiation of HSCs was inhibited by the deprivation of the important serum component erythropoietin (EPO) or treatment with saracatinib. Finally, we found that the mRNA expression of K562 cells and HSCs was different during saracatinib-treated erythroid differentiation, and the DEGs of K562 cells and HSCs associated with tyrosine-protein kinase were identified.These findings elucidated the cellular phenomenon of saracatinib induction during erythroid differentiation of K562 cells and HSCs, and the potential mechanism is the different mRNA expression profile of tyrosine-protein kinase in K562 cells and HSCs.

Awakening and growth reinitiation by dormant cells may contribute to epithelial ovarian cancer (EOC) relapse. The links between these phenomena are loose because of the limited stock of compelling models of EOC dormancy. Here, we show a simple and convenient dormancy research protocol based on serum starvation. This study was conducted on established EOC cell lines A2780, OVCAR-3, and SKOV-3, as well as on primary EOC cells. Cell growth arrest and proliferation were monitored by assessing the Ki67 antigen, PKH26 fluorescence, and cell cycle distribution. In addition, cells were tested for ERK1/2/p38 MAPK activity ratio, apoptosis, and senescence. The study showed that 72-h serum starvation induces G0/G1 growth arrest of a significant fraction of cells, accompanied by reduced Ki67 and ERK1/2/p38 MAPK activity ratio, without signs of apoptosis or cellular senescence. Moreover, providing cells with 72 h of a medium enriched in 5% serum allows the culture to regain its proliferative potential. At the same time, we attempted to induce and terminate dormancy with Mitomycin C addition and withdrawal, which were unsuccessful. In conclusion, serum starvation is a convenient way to reliably induce dormancy in EOC cells, allowing them to be efficiently awakened for further mechanistic research in vitro.

Platinum-derived chemotherapy medications are often combined with other conventional therapies for treating different tumors, including colorectal cancer. However, the development of drug resistance and multiple adverse effects remain common in clinical settings. Thus, there is a necessity to find novel treatments and drug combinations that could effectively target colorectal cancer cells and lower the probability of disease relapse. To find potential synergistic interaction, we designed multiple different combinations between cisplatin, cannabidiol, and intermittent serum starvation on colorectal cancer cell lines. Based on the cell viability assay, we found that combinations between cannabidiol and intermittent serum starvation, cisplatin and intermittent serum starvation, as well as cisplatin, cannabidiol, and intermittent serum starvation can work in a synergistic fashion on different colorectal cancer cell lines. Furthermore, we analyzed differentially expressed genes and affected pathways in colorectal cancer cell lines to understand further the potential molecular mechanisms behind the treatments and their interactions. We found that synergistic interaction between cannabidiol and intermittent serum starvation can be related to changes in the transcription of genes responsible for cell metabolism and cancer\'s stress pathways. Moreover, when we added cisplatin to the treatments, there was a strong enrichment of genes taking part in G2/M cell cycle arrest and apoptosis.

Clinical development of cellular therapies, including mesenchymal stem/stromal cell (MSC) treatments, has been hindered by ineffective cryopreservation methods that result in substantial loss of post-thaw cell viability and function. Proposed solutions to generate high potency MSC for clinical testing include priming cells with potent cytokines such as interferon gamma (IFNγ) prior to cryopreservation, which has been shown to enhance post-thaw function, or briefly culturing to allow recovery from cryopreservation injury prior to administering to patients. However, both solutions have disadvantages: cryorecovery increases the complexity of manufacturing and distribution logistics, while the pleiotropic effects of IFNγ may have uncharacterized and unintended consequences on MSC function. To determine specific cellular functions impacted by cryoinjury, we first evaluated cell cycle status. It was discovered that S phase MSC are exquisitely sensitive to cryoinjury, demonstrating heightened levels of delayed apoptosis post-thaw and reduced immunomodulatory function. Blocking cell cycle progression at G0/G1 by growth factor deprivation (commonly known as serum starvation) greatly reduced post-thaw dysfunction of MSC by preventing apoptosis induced by double-stranded breaks in labile replicating DNA that form during the cryopreservation and thawing processes. Viability, clonal growth and T cell suppression function were preserved at pre-cryopreservation levels and were no different than cells prior to freezing or frozen after priming with IFNγ. Thus, we have developed a robust and effective strategy to enhance post-thaw recovery of therapeutic MSC.

Macroautophagy/autophagy is a regulated cellular degradation process essential as a pro-survival mechanism and integral to the regulation of diverse cellular processes in eukaryotes. During cellular stress and nutrient sensing, SQSTM1/p62 (sequestosome 1) functions as a key receptor for selective autophagy by shuttling ubiquitinated cargoes toward autophagic degradation making it a useful marker for monitoring autophagic flux. We present a straightforward and rapid flow cytometric assay for the quantitative measurement of intracellular SQSTM1 with improved sensitivity to conventional immunoblotting and with the benefit of higher throughput and reduced requirements for starting cellular materials for adequate analysis. We demonstrate that flow cytometry is able to detect similar trends in the measurement of intracellular SQSTM1 levels following serum starvation, genetic manipulations, and bafilomycin A1/chloroquine treatments. The assays utilizes readily available reagents and equipment without the need for transfection and utilizes standard flow cytometry equipment. In the present studies, expression of reporter proteins was applied to a range of SQSTM1 expression levels generated by genetic and chemical manipulation in both mouse as well as human cells. In combination with appropriate controls and attention to cautionary issues, this assay offers the ability to assess an important measure of autophagic capacity and flux.Abbreviations: ATG5: autophagy related 5 ATG7: autophagy related 7 BafA: bafilomycin A1 BMDM: bone marrow-derived macrophages CQ: chloroquine EBV: Epstein-Barr Virus EDTA: ethylenediaminetetraacetic acid FBS: fetal bovine serum gMFI: geometric mean fluorescent intensity HD: healthy donor MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3 MedianFI: median fluorescent intensity NTC: non-target control PBMC: peripheral blood mononuclear cells RPMI: Roswell Park Memorial Institution SQSTM1/p62: sequestosome 1 WT: wild type.

OBJECTIVE: Although clinical medicine has significantly progressed in treating nasopharyngeal carcinoma (NPC) in recent years, many patients still have poor prognoses due to distant metastasis. It is still relatively unclear why NPC has a highly metastatic ability. Especially whether the tumor microenvironment affects the invasion and metastasis of NPC still needs to be cleared. In this study, serum starvation was used to simulate nutrient deficiency in the tumor microenvironment to explore whether nutrient deficiency affects the malignancy of NPC cells.
METHODS: Semiquantitative reverse transcription-polymerase chain reaction, ELISA, immunoblotting assay, reporter gene assay, and Matrigel invasion assay were carried out.
RESULTS: Under serum starvation, NPC cells could induce the mRNA expression and protein secretion of matrix metalloproteinase 9 (MMP9). The ERK-AP1 pathway was activated under serum starvation in NPC cells, resulting in the expression of MMP9. In contrast, treatment with an MMP9 inhibitor or an MMP9 siRNA inhibited serum starvation-induced invasion.
CONCLUSIONS: Serum starvation could up-regulate MMP9 expression in NPC cells, contributing to NPC invasion. Therefore, serum starvation may promote malignancy of NPC cells but also support MMP9 as a potential therapeutic target to prevent NPC cell invasion and metastasis.

The microenvironment of solid tumors such as breast cancer is heterogeneous and complex, containing different types of cell, namely, cancer stem cells and immune cells. We previously reported the immunoregulatory behavior of the human immune cell in a solid tumor microenvironment-like culture under serum starvation stress for 96 h. Here, we examined the effect of this culture-derived solution on breast cancer development in rats.
Ninety-six-hour starved PBMCs supernatant (96 h-SPS) was collected after culturing human PBMCs for 96 h under serum starvation condition. Breast cancer stem cells, LA7 cell line, was used for in vitro study by analyzing gene expression status and performing cytotoxicity, proliferation, scratch wound healing assays, followed by in vivo tumor induction in three groups of mature female Sprague Dawley rats. Animals were treated with 96 h-SPS or RPMI and normal saline as control, n = 6 for each group. After biochemical analysis of iron, lactate, and pH levels in the dissected tumors, Ki67 antigen expression, angiogenesis, and necrosis evaluation were carried out. Metabolic-related gene expression was assessed using RT-qPCR. Moreover, 96 h-SPS composition was discovered by Nano-LC-ESI-MS/MS.
96 h-SPS solution reduced the LA7 cell viability, proliferation, and migration and Gch1 and Spr genes expression in vitro (p< 0.05), whereas stemness gene Oct4 was upregulated (p< 0.01). The intracellular lactate was significantly decreased in the 96 h-SPS treated group (p = 0.007). In this group, Gch1 and Spr were significantly downregulated (p< 0.05), whereas the Sox2 and Oct4 expression was not changed significantly. The number of vessels and mitosis (Ki67+ cells) in the 96 h-SPS-treated group was significantly reduced (p = 0.024). The increased rate of necrosis in this group was statistically significant (p = 0.04). Last, proteomics analysis revealed candidate effectors\' components of 96 h-SPS solution.
96 h-SPS solution may help to prevent cancer stem cell mediated tumor development. This phenomenon could be mediated through direct cytotoxic effects, inhibition of cell proliferation and migration in association with reduction in Gch1 and Spr genes expression, angiogenesis and mitosis rate, and necrosis augmentation. The preliminary data obtained from the present study need to be investigated on a larger scale and can be used as a pilot for further studies on the biology of cancer development.

Periods of low energy supply are challenging conditions for organisms and cells during fasting or famine. Although changes in nutrient levels in the blood are first sensed by endothelial cells, studies on their metabolic adaptations to diminished energy supply are lacking. We analyzed the dynamic metabolic activity of human umbilical vein endothelial cells (HUVECs) in basal conditions and after serum starvation. Metabolites of glycolysis, the tricarboxylic acid (TCA) cycle, and the glycerol pathway showed lower levels after serum starvation, whereas amino acids had increased levels. A metabolic flux analysis with 13C-glucose or 13C-glutamine labeling for different time points reached a plateau phase of incorporation after 30 h for 13C-glucose and after 8 h for 13C-glutamine under both experimental conditions. Notably, we observed a faster label incorporation for both 13C-glucose and 13C-glutamine after serum starvation. In the linear range of label incorporation after 3 h, we found a significantly faster incorporation of central carbon metabolites after serum starvation compared to the basal state. These findings may indicate that endothelial cells develop increased metabolic activity to cope with energy deficiency. Physiologically, it can be a prerequisite for endothelial cells to form new blood vessels under unfavorable conditions during the process of angiogenesis in vivo.