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Physalis plants are commonly used traditional medicinal herbs, and most of their extracts containing withanolides show anticancer effects. Physapruin A (PHA), a withanolide isolated from P. peruviana, shows antiproliferative effects on breast cancer cells involving oxidative stress, apoptosis, and autophagy. However, the other oxidative stress-associated response, such as endoplasmic reticulum (ER) stress, and its participation in regulating apoptosis in PHA-treated breast cancer cells remain unclear. This study aims to explore the function of oxidative stress and ER stress in modulating the proliferation and apoptosis of breast cancer cells treated with PHA. PHA induced a more significant ER expansion and aggresome formation of breast cancer cells (MCF7 and MDA-MB-231). The mRNA and protein levels of ER stress-responsive genes (IRE1α and BIP) were upregulated by PHA in breast cancer cells. The co-treatment of PHA with the ER stress-inducer (thapsigargin, TG), i.e., TG/PHA, demonstrated synergistic antiproliferation, reactive oxygen species generation, subG1 accumulation, and apoptosis (annexin V and caspases 3/8 activation) as examined by ATP assay, flow cytometry, and western blotting. These ER stress responses, their associated antiproliferation, and apoptosis changes were partly alleviated by the N-acetylcysteine, an oxidative stress inhibitor. Taken together, PHA exhibits ER stress-inducing function to promote antiproliferation and apoptosis of breast cancer cells involving oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract Background Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion in Y. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme. Results By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and $$\alpha$$ α -amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion. Conclusions Overall, our combined strategies not only proved effective in improving the protein production in Yarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.

Manoalide provides preferential antiproliferation of oral cancer but is non-cytotoxic to normal cells by modulating reactive oxygen species (ROS) and apoptosis. Although ROS interplays with endoplasmic reticulum (ER) stress and apoptosis, the influence of ER stress on manoalide-triggered apoptosis has not been reported. The role of ER stress in manoalide-induced preferential antiproliferation and apoptosis was assessed in this study. Manoalide induces a higher ER expansion and aggresome accumulation of oral cancer than normal cells. Generally, manoalide differentially influences higher mRNA and protein expressions of ER-stress-associated genes (PERK, IRE1α, ATF6, and BIP) in oral cancer cells than in normal cells. Subsequently, the contribution of ER stress on manoalide-treated oral cancer cells was further examined. ER stress inducer, thapsigargin, enhances the manoalide-induced antiproliferation, caspase 3/7 activation, and autophagy of oral cancer cells rather than normal cells. Moreover, N-acetylcysteine, an ROS inhibitor, reverses the responses of ER stress, aggresome formation, and the antiproliferation of oral cancer cells. Consequently, the preferential ER stress of manoalide-treated oral cancer cells is crucial for its antiproliferative effect. [ABSTRACT FROM AUTHOR]

Background: Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion in Y. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme. Results: By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and α -amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion. Conclusions: Overall, our combined strategies not only proved effective in improving the protein production in Yarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast. [ABSTRACT FROM AUTHOR]

Physalis plants are commonly used traditional medicinal herbs, and most of their extracts containing withanolides show anticancer effects. Physapruin A (PHA), a withanolide isolated from P. peruviana, shows antiproliferative effects on breast cancer cells involving oxidative stress, apoptosis, and autophagy. However, the other oxidative stress-associated response, such as endoplasmic reticulum (ER) stress, and its participation in regulating apoptosis in PHA-treated breast cancer cells remain unclear. This study aims to explore the function of oxidative stress and ER stress in modulating the proliferation and apoptosis of breast cancer cells treated with PHA. PHA induced a more significant ER expansion and aggresome formation of breast cancer cells (MCF7 and MDA-MB-231). The mRNA and protein levels of ER stress-responsive genes (IRE1α and BIP) were upregulated by PHA in breast cancer cells. The co-treatment of PHA with the ER stress-inducer (thapsigargin, TG), i.e., TG/PHA, demonstrated synergistic antiproliferation, reactive oxygen species generation, subG1 accumulation, and apoptosis (annexin V and caspases 3/8 activation) as examined by ATP assay, flow cytometry, and western blotting. These ER stress responses, their associated antiproliferation, and apoptosis changes were partly alleviated by the N-acetylcysteine, an oxidative stress inhibitor. Taken together, PHA exhibits ER stress-inducing function to promote antiproliferation and apoptosis of breast cancer cells involving oxidative stress.

Manoalide provides preferential antiproliferation of oral cancer but is non-cytotoxic to normal cells by modulating reactive oxygen species (ROS) and apoptosis. Although ROS interplays with endoplasmic reticulum (ER) stress and apoptosis, the influence of ER stress on manoalide-triggered apoptosis has not been reported. The role of ER stress in manoalide-induced preferential antiproliferation and apoptosis was assessed in this study. Manoalide induces a higher ER expansion and aggresome accumulation of oral cancer than normal cells. Generally, manoalide differentially influences higher mRNA and protein expressions of ER-stress-associated genes (PERK, IRE1α, ATF6, and BIP) in oral cancer cells than in normal cells. Subsequently, the contribution of ER stress on manoalide-treated oral cancer cells was further examined. ER stress inducer, thapsigargin, enhances the manoalide-induced antiproliferation, caspase 3/7 activation, and autophagy of oral cancer cells rather than normal cells. Moreover, N-acetylcysteine, an ROS inhibitor, reverses the responses of ER stress, aggresome formation, and the antiproliferation of oral cancer cells. Consequently, the preferential ER stress of manoalide-treated oral cancer cells is crucial for its antiproliferative effect.

Chinese hamster ovary (CHO) cell expression systems have been exquisitely developed for the production of recombinant biotherapeutics (e.g. standard monoclonal antibodies, mAbs) and are able to generate efficacious, multi-domain proteins with human-like post translational modifications at high concentration with appropriate product quality attributes. However, there remains a need for development of new CHO cell expression systems able to produce more challenging secretory recombinant biotherapeutics at higher yield with improved product quality attributes. Amazingly, the engineering of lipid metabolism to enhance such properties has not been investigated even though the biosynthesis of recombinant proteins is at least partially controlled by cellular processes that are highly dependent on lipid metabolism. Here we show that the global transcriptional activator of genes involved in lipid biosynthesis, sterol regulatory element binding factor 1 (SREBF1), and stearoyl CoA desaturase 1 (SCD1), an enzyme which catalyzes the conversion of saturated fatty acids into monounsaturated fatty acids, can be overexpressed in CHO cells to different degrees. The amount of overexpression obtained of each of these lipid metabolism modifying (LMM) genes was related to the subsequent phenotypes observed. Expression of a number of model secretory biopharmaceuticals was enhanced between 1.5-9 fold in either SREBF1 or SCD1 engineered CHO host cells as assessed under batch and fed-batch culture. The SCD1 overexpressing polyclonal pool consistently showed increased concentration of a range of products. For the SREBF1 engineered cells, the level of SREBF1 expression that gave the greatest enhancement in yield was dependent upon the model protein tested. Overexpression of both SCD1 and SREBF1 modified the lipid profile of CHO cells and the cellular structure. Mechanistically, overexpression of SCD1 and SREBF1 resulted in an expanded endoplasmic reticulum (ER) that was dependent upon the level of LMM overexpression. We conclude that manipulation of lipid metabolism in CHO cells via genetic engineering is an exciting new approach to enhance the ability of CHO cells to produce a range of different types of secretory recombinant protein products via modulation of the cellular lipid profile and expansion of the ER. • We have engineered lipid metabolism gene expression, SREBF1 and SCD1 , in CHO cells. • The amount of lipid metabolism gene overexpression is phenotype related. • Secretory biopharmaceutical yield is enhanced 1.5–9 fold in lipid engineered cells. • Overexpression of SCD1 and SREBF1 modifies the lipid profile of CHO cells. • Overexpression of SCD1 and SREBF1 results in an expanded ER. [ABSTRACT FROM AUTHOR]

Although recent evidence has shown that IL‐6 is involved in enhanced alternative activation of macrophages toward a profibrotic phenotype, the mechanisms leading to their increased secretory capacity are not fully understood. Here, we investigated the effect of IL‐6 on endoplasmic reticulum (ER) expansion and alternative activation of macrophages in vitro. An essential mediator in this ER expansion process is the IRE1 pathway, which possesses a kinase and endoribonuclease domain to cleave XBP1 into a spliced bioactive molecule. To investigate the IRE1‐XBP1 expansion pathway, IL‐4/IL‐13 and IL‐4/IL‐13/IL‐6‐mediated alternative programming of murine bone marrow‐derived and human THP1 macrophages were assessed by arginase activity in cell lysates, CD206 and arginase‐1 expression by flow cytometry, and secreted CCL18 by ELISA, respectively. Ultrastructural intracellular morphology and ER biogenesis were examined by transmission electron microscopy and immunofluorescence. Transcription profiling of 128 genes were assessed by NanoString and Pharmacological inhibition of the IRE1‐XBP1 arm was achieved using STF‐083010 and was verified by RT‐PCR. The addition of IL‐6 to the conventional alternative programming cocktail IL‐4/IL‐13 resulted in increased ER and mitochondrial expansion, profibrotic profiles and unfolded protein response‐mediated induction of molecular chaperones. IRE1‐XBP1 inhibition substantially reduced the IL‐6‐mediated hyperpolarization and normalized the above effects. In conclusion, the addition of IL‐6 enhances ER expansion and the profibrotic capacity of IL‐4/IL‐13‐mediated activation of macrophages. Therapeutic strategies targeting IL‐6 or the IRE1‐XBP1 axis may be beneficial to prevent the profibrotic capacity of macrophages. IL‐6 is known to increase the polarization of alternatively activated macrophages. Here, we show that this hyperpolarization is associated with a dramatic increase in the endoplasmic reticulum, an organelle required for folding of secreted proteins. We show further that the inhibition of a known activator of the Unfolded Protein Response, IRE1, prevents both ER expansion and alternative activation of macrophages, suggesting that targeting the process of ER expansion in alternatively activated macrophages may be considered to prevent the known profibrotic activity of alternatively activated macrophages. [ABSTRACT FROM AUTHOR]

Background: Endoplasmic reticulum (ER) stress induces ER expansion. The expansion of the intracisternal space of the ER was found in macrophages associated with human atherosclerotic lesions. We also previously reported that palmitate induces cisternal ER expansion and necrosis in RAW 264.7 cells. In this study, we report on an investigation of the likely mechanism responsible for this palmitate-induced cisternal ER expansion in a mouse macrophage cell line, RAW 264.7 cells. Methods: RAW 264.7 cells were pre-treated with the designated inhibitor or siRNA, followed by treatment with palmitate. Changes in the ER structure were examined by transmission electron microscopy. The induction of ER stress was confirmed by an increase in the extent of phosphorylation of PERK, the expression of BiP and CHOP, and the splicing of XBP-1 mRNA. Phospholipid staining was performed with the LipidTOX Red phospholipidosis detection reagent. Related gene expressions were detected by quantitative real time-RT-PCR or RT-PCR. Results: Palmitate was found to induce ER stress and cisternal ER expansion. In addition, palmitate-induced cisternal ER expansion was attenuated by ER stress inhibitors, such as 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA). The findings also show that palmitate induced-mRNA expression of CCTα, which increases phospholipid synthesis, was attenuated by the down-regulation of XBP-1, a part of ER stress. Furthermore, palmitate-induced phospholipid accumulation and cisternal ER expansion were attenuated by the down-regulation of XBP-1 or CCTα. Conclusions: The findings reported herein indicate that palmitate-induced cisternal ER expansion is dependent on the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) of eukaryotic cells is involved in the synthesis and processing of proteins and lipids in the secretory pathway. These processing events that proteins undergo in the ER may present major limiting steps for recombinant protein production. Increased protein synthesis, accumulation of improperly processed or mis-folded protein can induce ER stress. To cope with ER stress, the ER has quality control mechanisms, such as the unfolded protein response (UPR) and ER-associated degradation to restore homeostasis. ER stress and UPR activation trigger multiple physiological cellular changes. Here we review cellular mechanisms that cope with ER stress and illustrate how this knowledge can be applied to increase the efficiency of recombinant protein expression. [ABSTRACT FROM AUTHOR]

Autophagy and endoplasmic reticulum (ER) stress response is important for cancer cells to maintain malignancy and resistance to therapy. trans-Resveratrol (RSV), a non-flavonoid agent, has been shown to induce apoptosis in human nasopharyngeal carcinoma (NPC) cells. In this study, the involvements of tumor-specific ER stress and autophagy in the RSV-mediated apoptosis were investigated. In addition to traditional autophagosomes, the images of transmission electron microscopy (TEM) indicated that RSV markedly induced larger, crescent-shaped vacuoles with single-layered membranes whose the expanded cisternae contains multi-lamellar membrane structures. Prolonged exposure to RSV induced a massive accumulation of ER expansion. Using an EGFP-LC3B transfection and confocal laser microscopy approach, we found RSV-induced EGFP-LC3 puncta co-localized with ER-tracker red dye, implicating the involvement of LC3II in ER expansion. The proapoptotic effect of RSV was enhanced after suppression of autophagy by ATG7 siRNA or blocking the autophagic flux by bafilomycin A1, but that was not changed after targeted silence of IRE1 or CHOP by siRNA. Using caspase inhibitors, we demonstrated the upregulation of caspase-12 (casp12) and the activation of casp4 were associated with the proapoptotic induction of RSV through the caspase-9/caspase-3 pathway. Intriguingly, siRNA knockdown of casp12, but not caspase-4, decreased the susceptibility of the NPC cells to RSV-mediated apoptosis. Further, we showed that RSV dose-dependently increased the ceramide accumulation as assessed by LC-MS/MS system. Using serine palmitoyltransferase (SPT, a key enzyme of de novo ceramide biosynthesis) inhibitors ( l-cycloserine and myriocin), we found the increased ceramide accumulation was strongly correlated with the proapoptotic potential of RSV. This study revealed the ER expansion and upregulation of ER casp12 together may indicate profound biological effects of RSV and contributed to NPC cell death. Targeting the different status of ER stress may provide a possible strategy for cancer treatments. [ABSTRACT FROM AUTHOR]

• High Ca2+ turnover is distinctive for the Ca2+ homeostasis in secretory cells. • The Ca2+ leak from ER is enhanced in secretory cells. • High expression of Sec61 translocons enhances the Ca2+ leak from ER. • Massive Ca2+ entry backs up the large Sec61-mediated Ca2+ leak in secretory cells. The endoplasmic reticulum (ER) is extensively remodelled during the development of professional secretory cells to cope with high protein production. Since ER is the principal Ca2+ store in the cell, we characterised the Ca2+ homeostasis in NALM-6 and RPMI 8226 cells, which are commonly used as human pre-B and antibody secreting plasma cell models, respectively. Expression levels of Sec61 translocons and the corresponding Sec61-mediated Ca2+ leak from ER, Ca2+ storage capacity and store-operated Ca2+ entry were significantly enlarged in the secretory RPMI 8226 cell line. Using an immunoglobulin M heavy chain producing HeLa cell model, we found that the enlarged Ca2+ storage capacity and Ca2+ leak from ER are linked to ER expansion. Our data delineates a developmental remodelling of Ca2+ homeostasis in professional secretory cells in which a high Sec61-mediated Ca2+ leak and, thus, a high Ca2+ turnover in the ER is backed up by enhanced store-operated Ca2+ entry. [Display omitted] [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) is extensively remodelled during the development of professional secretory cells to cope with high protein production. Since ER is the principal Ca 2+ store in the cell, we characterised the Ca 2+ homeostasis in NALM-6 and RPMI 8226 cells, which are commonly used as human pre-B and antibody secreting plasma cell models, respectively. Expression levels of Sec61 translocons and the corresponding Sec61-mediated Ca 2+ leak from ER, Ca 2+ storage capacity and store-operated Ca 2+ entry were significantly enlarged in the secretory RPMI 8226 cell line. Using an immunoglobulin M heavy chain producing HeLa cell model, we found that the enlarged Ca 2+ storage capacity and Ca 2+ leak from ER are linked to ER expansion. Our data delineates a developmental remodelling of Ca 2+ homeostasis in professional secretory cells in which a high Sec61-mediated Ca 2+ leak and, thus, a high Ca 2+ turnover in the ER is backed up by enhanced store-operated Ca 2+ entry., (Copyright © 2021. Published by Elsevier Ltd.)