Identification of a Potent Cytotoxic Pyrazole with Anti-Breast Cancer Activity That Alters Multiple Pathways

In this study, we identified a novel pyrazole-based derivative (P3C) that displayed potent cytotoxicity against 27 human cancer cell lines derived from different tissue origins with 50% cytotoxic concentrations (CC50) in the low micromolar and nanomolar range, particularly in two triple-negative breast cancer (TNBC) cell lines (from 0.25 to 0.49 µM). In vitro assays revealed that P3C induces reactive oxygen species (ROS) accumulation leading to mitochondrial depolarization and caspase-3/7 and -8 activation, suggesting the participation of both the intrinsic and extrinsic apoptotic pathways. P3C caused microtubule disruption, phosphatidylserine externalization, PARP cleavage, DNA fragmentation, and cell cycle arrest on TNBC cells. In addition, P3C triggered dephosphorylation of CREB, p38, ERK, STAT3, and Fyn, and hyperphosphorylation of JNK and NF-kB in TNBC cells, indicating the inactivation of both p38MAPK/STAT3 and ERK1/2/CREB signaling pathways. In support of our in vitro assays, transcriptome analyses of two distinct TNBC cell lines (MDA-MB-231 and MDA-MB-468 cells) treated with P3C revealed 28 genes similarly affected by the treatment implicated in apoptosis, oxidative stress, protein kinase modulation, and microtubule stability.

Genome-Wide Identification and Characterization of the RCI2 Gene Family in Allotetraploid Brassica napus Compared with Its Diploid Progenitors

Brassica napus and its diploid progenitors (B. rapa and B. oleracea) are suitable for studying the problems associated with polyploidization. As an important anti-stress protein, RCI2 proteins widely exist in various tissues of plants, and are crucial to plant growth, development, and stress response. In this study, the RCI2 gene family was comprehensively identified and analyzed, and 9, 9, and 24 RCI2 genes were identified in B. rapa, B. oleracea, and B. napus, respectively. Phylogenetic analysis showed that all of the identified RCI2 genes were divided into two groups, and further divided into three subgroups. Ka/Ks analysis showed that most of the identified RCI2 genes underwent a purifying selection after the duplication events. Moreover, gene structure analysis showed that the structure of RCI2 genes is largely conserved during polyploidization. The promoters of the RCI2 genes in B. napus contained more cis-acting elements, which were mainly involved in plant development and growth, plant hormone response, and stress responses. Thus, B. napus might have potential advantages in some biological aspects. In addition, the changes of RCI2 genes during polyploidization were also discussed from the aspects of gene number, gene structure, gene relative location, and gene expression, which can provide reference for future polyploidization analysis.

Protection of catalpol against triptolide-induced hepatotoxicity by inhibiting excessive autophagy via the PERK-ATF4-CHOP pathway

Catalpol significantly reduces triptolide-induced hepatotoxicity, which is closely related to autophagy. The aim of this study was to explore the unclear protective mechanism of catalpol against triptolide. The detoxification effect of catalpol on triptolide was investigated in HepaRG cell line. The detoxification effects were assessed by measuring cell viability, autophagy, and apoptosis, as well as the endoplasmic reticulum stress protein and mRNA expression levels. We found that 5–20 µg/L triptolide treatments increased the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), as well as the expression of autophagy proteins including LC3 and Beclin1. The expression of P62 was downregulated and the production of autophagosomes was increased, as determined by transmission electron microscope and monodansylcadaverine staining. In contrast, 40 µg/L catalpol reversed these triptolide-induced changes in the liver function index, autophagy level, and apoptotic protein expression, including Cleaved-caspase3 and Cleaved-caspase9 by inhibiting excessive autophagy. Simultaneously, catalpol reversed endoplasmic reticulum stress, including the expression of PERK, which regulates autophagy. Moreover, we used the PERK inhibitor GSK2656157 to prove that the PERK-ATF4-CHOP pathway of the unfolded protein response is an important pathway that could induce autophagy. Catalpol inhibited excessive autophagy by suppressing the PERK pathway. Altogether, catalpol protects against triptolide-induced hepatotoxicity by inhibiting excessive autophagy via the PERK-ATF4-CHOP pathway. The results of this study are beneficial to clarify the detoxification mechanism of catalpol against triptolide-induced hepatotoxicity and to promote the application of triptolide.

Introgression of SbERD4 Gene Encodes an Early-Responsive Dehydration-Stress Protein That Confers Tolerance against Different Types of Abiotic Stresses in Transgenic Tobacco

Salicornia brachiata is an extreme halophyte that commonly grows on marsh conditions and is also considered a promising resource for drought and salt-responsive genes. To unveil a glimpse of stress endurance by plants, it is of the utmost importance to develop an understanding of stress tolerance mechanisms. ‘Early Responsive to Dehydration’ (ERD) genes are defined as a group of genes involved in stress tolerance and the development of plants. To increase this understanding, parallel to this expedited thought, a novel SbERD4 gene was cloned from S. brachiata, characterized, and functionally validated in the model plant tobacco. The study showed that SbERD4 is a plasma-membrane bound protein, and its overexpression in tobacco plants improved salinity and osmotic stress tolerance. Transgenic plants showed high relative water, chlorophylls, sugars, starch, polyphenols, proline, free amino acids, and low electrolyte leakage and H2O2 content compared to control plants (wild type and vector control) under different abiotic stress conditions. Furthermore, the transcript expression of antioxidant enzyme encoding genes NtCAT, NtSOD, NtGR, and NtAPX showed higher expression in transgenic compared to wild-type and vector controls under varying stress conditions. Overall, the overexpression of a novel early responsive to dehydration stress protein 4-encoding gene (SbERD4) enhanced the tolerance of the plant against multiple abiotic stresses. In conclusion, the overexpression of the SbERD4 gene mitigates plant physiology by enduring stress tolerance and might be considered as a promising key gene for engineering salinity and drought stress tolerance in crops.

Production of Human Pancreatitis-Associated Protein (hPAP) in Komagataella phaffii (Pichia pastoris)

Pancreatitis-associated protein (PAP) is a pancreatic stress protein that is not produced in a healthy pancreas but is highly synthesized in pancreatic acinar cells in response to acute and chronic pancreatitis, hypoxia, toxins, diabetes, lipopolysaccharides hypotransferrinemia and organ transplantation. Changes in the PAP levels in serum are an important biological marker in the early stage of pancreatic diseases. In this study, the recombinant human PAP protein, which has the potential to be used as a diagnostic marker and as research material in proliferation, apoptosis, cell migration, cell invasion, and immunoassay studies, was expressed efficiently under the control of the AOX1 gene promoter in the Komagataella phaffii (Pichia pastoris) (K. phaffii) X33 strain. We describe the conditions required for the efficient production of PAP protein by methanol induction and its use without purification. The produced unpurified protein was tested in sandwich ELISA and showed consistent results with the commercial product. These results are encouraging that the protein produced can be used as a biomarker standard in ELISA tests without the cost and labor of purification.

Molecular Mechanisms and Regulation of Mammalian Mitophagy

Mitochondria in the cell are the center for energy production, essential biomolecule synthesis, and cell fate determination. Moreover, the mitochondrial functional versatility enables cells to adapt to the changes in cellular environment and various stresses. In the process of discharging its cellular duties, mitochondria face multiple types of challenges, such as oxidative stress, protein-related challenges (import, folding, and degradation) and mitochondrial DNA damage. They mitigate all these challenges with robust quality control mechanisms which include antioxidant defenses, proteostasis systems (chaperones and proteases) and mitochondrial biogenesis. Failure of these quality control mechanisms leaves mitochondria as terminally damaged, which then have to be promptly cleared from the cells before they become a threat to cell survival. Such damaged mitochondria are degraded by a selective form of autophagy called mitophagy. Rigorous research in the field has identified multiple types of mitophagy processes based on targeting signals on damaged or superfluous mitochondria. In this review, we provide an in-depth overview of mammalian mitophagy and its importance in human health and diseases. We also attempted to highlight the future area of investigation in the field of mitophagy.

CHOP is Dispensable for Exercise-Induced Increases in GDF15

Growth differentiating factor-15 (GDF15) is expressed, and secreted, from a wide range of tissues and serves as a marker of cellular stress. A key transcriptional regulator of this hormone is the endoplasmic reticulum stress protein, CHOP (C/EBP Homologous Protein). Exercise increases GDF15 levels but the underlying mechanisms of this are not known. To test whether CHOP regulates GDF15 during exercise we used various models of altered ER stress. We examined the effects of acute exercise on circulating GDF15 and GDF15 mRNA expression in liver, triceps skeletal muscle, and epididymal white adipose tissue and examined the GDF15 response to acute exercise in lean and high-fat diet-induced obese mice, sedentary and exercise trained mice, and CHOP deficient mice. We found that obesity augments exercise-induced circulating GDF15 although ER stress markers were similar in lean and obese mice. Exercise-induced GDF15 was increased in trained and sedentary mice that ran at the same relative exercise intensity, despite trained mice being protected against increased markers of ER stress. Finally, exercise-induced increases in GDF15 at the tissue and whole-body level were intact in CHOP deficient mice. Together, these results provide evidence that exercise-induced GDF15 expression and secretion occurs independent of ER stress/CHOP.