Phospholipid scramblase activity is involved in the collapse of phospholipid (PL) asymmetry at the plasma membrane leading to externalization of phosphatidylserine. This activity is crucial for initiation of the blood coagulation cascade and for recognition/elimination of apoptotic cells by macrophages. Efforts to identify gene products associated with this activity led to the characterization of PL scramblase (PLSCR) and XKR family members which contribute to phosphatidylserine exposure in response to apoptotic stimuli. Meanwhile, TMEM16 family members were identified to externalize phosphatidylserine in response to elevated calcium in Scott syndrome platelets, which is critical for activation of the coagulation cascade. Herein, we report their mechanisms of gene regulation, molecular functions independent of their scrambling activity, and their potential roles in pathogenic conditions.
BackgroundmiRNAs can regulate cellular survival in various cancer cell types. Recent evidence implicates the formation of lipid droplets as a hallmark event during apoptotic cell death response. It is presently unknown whether MIR494, located at 14q32 which is deleted in renal cancers, reduces cell survival in renal cancer cells and if this process is accompanied by changes in the number of lipid droplets.Methods769-P renal carcinoma cells were utilized for this study. Control or MIR494 mimic was expressed in these cells following which cell viability (via crystal violet) and apoptotic cell numbers (via Annexin V/PI staining) were assessed. By western blotting, MIR494 cellular responses were validated using MIR494 antagomir and Argonaute 2 siRNA. Transmission electron microscopy (TEM) was performed in MIR494-transfected 769-P cells to identify ultrastructural changes. LipidTOX green neutral lipid staining and cholesterol measurements were conducted to assess accumulation of lipids droplets and total cholesterol levels, respectively, in MIR494 expressing 769-P cells. Indirect immunofluorescence and western analyses were also performed to examine changes in mitochondria organization. Co-transfection of MIR494 mimic with siRNA targeting LC3B and ATG7 was conducted to assess their contribution to formation of lipid droplets in MIR494-expressing cells.ResultsMIR494 expression reduces viability of 769-P renal cancer cells; this was accompanied by increased cleaved PARP (an apoptotic marker) and LC3B protein. Further, expression of MIR494 increased LC3B mRNA levels and LC3B promoter activity (2.01-fold; 50 % increase). Interestingly, expression of MIR494 markedly increased multilamellar bodies and lipid droplets (by TEM and validated by LipidTOX immunostaining) while reducing total cholesterol levels. Via immunocytochemistry, we observed increased LC3B-associated endogenous punctae upon MIR494 expression. In contrast to ATG7 siRNA, knockdown of LC3B reduced the numbers of lipid droplets in MIR494-expressing cells. Our results also identified that MIR494 expression altered the organization of mitochondria which was accompanied by co-localization with LC3B punctae, decreased PINK1 protein, and altered Drp1 intracellular distribution.ConclusionCollectively, our findings indicate that MIR494 reduces cell survival in 769-P renal cancer cells which is accompanied by increased lipid droplet formation (which occurs in a LC3B-dependent manner) and mitochondrial changes.
Pancreatic cancer is one of the most recalcitrant and lethal of all cancers. We examined the effects of Anemarrhena asphodeloides ( AA ) and timosaponin‐ AIII ( TAIII ), a steroidal saponin present in AA , on pancreatic cancer cell proliferation and aimed to elucidate their potential apoptotic mechanisms of action. Viability assays and cell cycle analysis revealed that both AA and TAIII significantly inhibited pancreatic cancer cell proliferation and cell cycle progression compared to treatment with gemcitabine, the standard chemotherapeutic agent for advanced pancreatic cancer. We identified a dose‐dependent increase in caspase‐dependent apoptosis and activation of pro‐apoptotic PI 3K/Akt pathway proteins, with a subsequent downregulation of pro‐survival PI 3K/Akt pathway proteins, in pancreatic cancer cells treated with AA or TAIII over those treated with gemcitabine.
Type 1 diabetes (T1D) affects 1.25 million Americans with about 40,000 individuals newly diagnosed each year. T1D is caused by an autoimmune-induced loss of pancreatic β cells and subsequent deterioration of insulin production. Although exogenous insulin is an efficient treatment, there is a strong need to develop therapies that can inhibit the loss of β cells and promote their function. For this purpose, we have been analyzing the antidiabetic capability of Cornus officinalis (CF), which has been utilized in traditional Chinese medicine for over 2,000 years primarily for treatment of T2D. We hypothesize that CF may inhibit β cell destruction and promote β cell function. Using the human 1.1B4 pancreatic β cell line, we have examined the proliferative effect of CF treatment on 1.1B4 cells in a time- and dose-dependent manner via cell viability assays. Within two hours, CF rapidly and significantly induced 1.1B4 proliferation by over 300% as compared to controls. Activation of Th1 cytokines plays a detrimental role in the pathogenesis of T1D. Therefore, we determined whether CF can inhibit cytokine induced β cell death and promote β cell proliferation following treatment with Th1 cytokines of IL-1β, TNF-α, and IFN-γ. CF induced a 200% increase in 1.1B4 proliferation following Th1 cytokine treatment compared to controls. In addition, a metabolic analysis of CF induced 1.1B4 cells was examined using the Agilent Seahorse XF analyzer. At a two-hour time point, there was a remarkable increase in maximal respiratory and glycolytic capacity following CF treatment. Therefore, CF is a strong inducer of metabolism. Ongoing experiments will provide greater detail into the mechanism and metabolic role in which CF is promoting proliferation and metabolism and inhibiting cytokine-induced β-cell death. Disclosure A. Sharp: None. B.R. Burkhardt: None.
Type 1 diabetes (T1D) is an autoimmune disease resulting in the destruction of pancreatic β cells (β-cells) and subsequent loss of insulin production. The only treatment for T1D is using exogenous insulin coupled with continual glucose monitoring following significant autoimmune destruction of β-cells. Novel interventional therapies are needed that can preserve and protect existing pancreatic β cells in individuals with early identified T1D autoimmunity. Our initial in-vitro evidence indicates Cornus officinalis (CO) may be able to serve in this function. What sets ethnopharmacology apart from conventional medicine is the simultaneous targeting of multiple mechanisms using a single herb due to the composition of numerous bioactive ingredients. CO has been used in TCM (traditional Chinese medicine) for over 2 millennia for the therapeutic effect of improved glucose tolerance and has also demonstrated efficacy in animal models but rarely examined in the context of T1D. We hypothesize that CO treatment may provide a β cell restorative and protective therapy for T1D and inhibit progressive cytokine-mediated β cell loss while enhancing existing β cells. Our preliminary evidence demonstrated a dose-dependent exposure of 1.1B4 cells to CO increased proliferation and protected against cytokine-induced cell death. We examined the metabolic effect of CO using the Agilent Seahorse XF Analyzer and at a two-hour time point there was a remarkable increase in maximal respiratory and glycolytic capacity following CO treatment. However, the molecular mechanism in which CO is inducing a proliferative and metabolic effect in 1.1B4 cells has not been elucidated. Therefore, we employed transcriptomics by RNA-Seq to analyze the early initiators of this increased metabolic effect. Our strongest and most significantly differentially expressed transcript was calcium-dependent transcription factor, NFATc. Expression of NFATc was validated by qPCR, which displayed a 2-fold increase in gene expression. NFATc is an essential transcription factor for β cell proliferation, endocrine function, and insulin secretion and may be a mediator of CO induced biological effects. Lastly, to date, 300 compounds have been elucidated from CO; therefore we wanted to analyze the components within our CO extracts via HPLC/MS in order to elucidate the bioactive ingredients. We found over 300 compounds, and of those, we found known bioactive ingredients such as, loganin and morroniside, while also finding novel extracts with potential bioactive properties. Altogether, CO increased β cell metabolism while inducing the NFAT pathway to signal for increased proliferation and endocrine function. Further experiments will examine the anti-diabetic effects of individual CO constituents that were identified via HPLC/MS, and the full molecular mechanism related to the NFAT signaling pathway.
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