Research Progress of Pyroptosis in Diabetic Kidney Disease.

Pyroptosis, known as one typical mode of programmed cell death, is generally characterized by the cleaved gasdermin family (GSDMs) forming pores in the cell membrane and inducing cell rupture, and the activation of aspartate-specific proteases (caspases) has also been found during this process. Diabetic Kidney Disease (DKD) is caused by the complication of diabetes in the kidney, and the most important kidney's function, Glomerular Filtration Rate (GFR), happens to drop to less than 90% of its usual and even lead to kidney failure in severe cases. The persistent inflammatory state induced by high blood glucose implies the key pathology of DKD, and growing evidence shows that pyroptosis serves as a significant contributor to this chronic immune-mediated inflammatory disorder. Currently, the expanded discovery of GSDMs, pyroptosis, and its association with innate immunity has been more attractive, and overwhelming research is needed to sort out the implication of pyroptosis in DKD pathology. In this review, we comb both classical studies and newly founds on pyroptosis, prick off the novel awakening of pyroptosis in DKD, and center on the significance of pyroptosis in DKD treatment, aiming to provide new research targets and treatment strategies on DKD.

Changing the Landscape of Solid Tumor Therapy from Apoptosis-Promoting to Apoptosis-Inhibiting Strategies.

The many limitations of implementing anticancer strategies under the term "precision oncology" have been extensively discussed. While some authors propose promising future directions, others are less optimistic and use phrases such as illusion, hype, and false hypotheses. The reality is revealed by practicing clinicians and cancer patients in various online publications, one of which has stated that "in the quest for the next cancer cure, few researchers bother to look back at the graveyard of failed medicines to figure out what went wrong". The message is clear: Novel therapeutic strategies with catchy names (e.g., synthetic "lethality") have not fulfilled their promises despite decades of extensive research and clinical trials. The main purpose of this review is to discuss key challenges in solid tumor therapy that surprisingly continue to be overlooked by the Nomenclature Committee on Cell Death (NCCD) and numerous other authors. These challenges include: The impact of chemotherapy-induced genome chaos (e.g., multinucleation) on resistance and relapse, oncogenic function of caspase 3, cancer cell anastasis (recovery from late stages of apoptosis), and pitfalls of ubiquitously used preclinical chemosensitivity assays (e.g., cell "viability" and tumor growth delay studies in live animals) that score such pro-survival responses as "lethal" events. The studies outlined herein underscore the need for new directions in the management of solid tumors.

The Effects of Apricot Kernels and Pure Amygdalin on the Structural, Oxidative, and Inflammatory Characteristics of Rabbit Testicular Tissue.

BACKGROUND: Apricot kernels containing amygdalin (AMG) as the major cyanogenic glycoside are potentially useful as a complementary therapy for the management of several ailments including cancer. Nevertheless, little is known regarding the toxic and therapeutic doses of AMG, particularly in terms of male reproduction. Hence, this study evaluates selected qualitative characteristics of rabbit testicular tissue following in vivo administration of AMG or apricot kernels for 28 days. METHODS: The rabbits were randomly divided into five groups (Control, P1, P2, P3, P4). The Control received no AMG/apricot kernels while the experimental groups P1 and P2 received a daily intramuscular injection of amygdalin at a dose of 0.6 and 3.0 mg/kg of body weight (b.w.) for 28 days, respectively. P3 and P4 received a daily dose of 60 and 300 mg/kg b.w. of crushed apricot kernels mixed with feed for 28 days, respectively. Changes to the testicular structure were quantified morphometrically, while tissue lysates were subjected to the evaluation of reactive oxygen species (ROS) production, total antioxidant capacity, activities of antioxidant enzymes, and glutathione concentration. The extent of damage to the proteins and lipids was quantified as well. Levels of selected cytokines were determined by the enzyme-linked immunosorbent assay while a luminometric approach was used to assess the activity of caspases. RESULTS: Rabbits treated with 3.0 mg/kg b.w. AMG presented a significantly increased protein oxidation (p = 0.0118) accompanied by a depletion of superoxide dismutase (p = 0.0464), catalase (p = 0.0317), and glutathione peroxidase (p = 0.0002). Significantly increased levels of interleukin-1 beta (p = 0.0012), tumor necrosis factors alpha (p = 0.0159), caspase-3/7 (p = 0.0014), and caspase-9 (p = 0.0243) were also recorded in the experimental group P2 when compared to the Control. No effects were observed in the rabbits treated with apricot kernels at the oxidative, inflammatory, and histopathological levels. CONCLUSIONS: Apricot kernels did not induce toxicity in the testicular tissues of male rabbits, unlike pure AMG, which had a negative effect on male reproductive structures carried out through oxidative, inflammatory, and pro-apoptotic mechanisms.

Caspase-8 activity mediates TNFα production and restricts Coxiella burnetii replication during murine macrophage infection.

Coxiella burnetii is an obligate intracellular bacteria that causes the global zoonotic disease Q Fever. Treatment options for chronic infection are limited, and the development of novel therapeutic strategies requires a greater understanding of how C. burnetii interacts with immune signaling. Cell death responses are known to be manipulated by C. burnetii, but the role of caspase-8, a central regulator of multiple cell death pathways, has not been investigated. In this research, we studied bacterial manipulation of caspase-8 signaling and the significance of caspase-8 to C. burnetii infection, examining bacterial replication, cell death induction, and cytokine signaling. We measured caspase, RIPK, and MLKL activation in C. burnetii-infected tumor necrosis factor alpha (TNFα)/cycloheximide-treated THP-1 macrophage-like cells and TNFα/ZVAD-treated L929 cells to assess apoptosis and necroptosis signaling. Additionally, we measured C. burnetii replication, cell death, and TNFα induction over 12 days in RIPK1-kinase-dead, RIPK3-kinase-dead, or RIPK3-kinase-dead-caspase-8-/- bone marrow-derived macrophages (BMDMs) to understand the significance of caspase-8 and RIPK1/3 during infection. We found that caspase-8 is inhibited by C. burnetii, coinciding with inhibition of apoptosis and increased susceptibility to necroptosis. Furthermore, C. burnetii replication was increased in BMDMs lacking caspase-8, but not in those lacking RIPK1/3 kinase activity, corresponding with decreased TNFα production and reduced cell death. As TNFα is associated with the control of C. burnetii, this lack of a TNFα response may allow for the unchecked bacterial growth we saw in caspase-8-/- BMDMs. This research identifies and explores caspase-8 as a key regulator of C. burnetii infection, opening novel therapeutic doors.

Leveraging the redundancy of S-denitrosylases in response to S-nitrosylation of caspases: Experimental strategies and beyond.

Redox-based protein posttranslational modifications, such as S-nitrosylation of critical, active site cysteine thiols have garnered significant clinical attention and research interest, reasoning for one of the crucial biological implications of reactive messenger molecule, nitric oxide in the cellular repertoire. The stringency of the S-(de)nitrosylation-based redox switch governs the activity and contribution of several susceptible enzymes in signal transduction processes and diverse pathophysiological settings, thus establishing it as a transient yet reasonable, and regulated mechanism of NO adduction and release. Notably, endogenous proteases like cytosolic and mitochondrial caspases with a molecular weight ranging from 33 to 55 kDa are susceptible to performing this biochemistry in the presence of major oxidoreductases, which further unveils the enormous redox-mediated regulational control of caspases in the etiology of diseases. In addition to advancing the progress of the current state of understanding of 'redox biochemistry' in the field of medicine and enriching the existing dynamic S-nitrosoproteome, this review stands as a testament to an unprecedented shift in the underpinnings for redundancy and redox relay between the major redoxin/antioxidant systems, fine-tuning of which can command the apoptotic control of caspases at the face of nitro-oxidative stress. These intricate functional overlaps and cellular backups, supported rationally by kinetically favorable reaction mechanisms suggest the physiological relevance of identifying and involving such cognate substrates for cellular S-denitrosylases that can shed light on the bigger picture of extensively proposing targeted therapies and redox-based drug designing to potentially alleviate the side effects of NOx/ROS in disease pathogenesis.

Inflammatory caspase substrate specificities.

Caspases are a family of cysteine proteases that act as molecular scissors to cleave substrates and regulate biological processes such as programmed cell death and inflammation. Extensive efforts have been made to identify caspase substrates and to determine factors that dictate substrate specificity. Thousands of putative substrates have been identified for caspases that regulate an immunologically silent type of cell death known as apoptosis, but less is known about substrates of the inflammatory caspases that regulate an immunostimulatory type of cell death called pyroptosis. Furthermore, much of our understanding of caspase substrate specificities is derived from work done with peptide substrates, which do not often translate to native protein substrates. Our knowledge of inflammatory caspase biology and substrates has recently expanded and here, we discuss the recent advances in our understanding of caspase substrate specificities, with a focus on inflammatory caspases. We highlight new substrates that have been discovered and discuss the factors that engender specificity. Recent evidence suggests that inflammatory caspases likely utilize two binding interfaces to recognize and process substrates, the active site and a conserved exosite.

Inhibition of caspase-8 cascade restrains the osteoclastogenic fate of bone marrow cells.

Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.

Exploring caspase functions in mouse models.

Caspases are enzymes with protease activity. Despite being known for more than three decades, caspase investigation still yields surprising and fascinating information. Initially associated with cell death and inflammation, their functions have gradually been revealed to extend beyond, targeting pathways such as cell proliferation, migration, and differentiation. These processes are also associated with disease mechanisms, positioning caspases as potential targets for numerous pathologies including inflammatory, neurological, metabolic, or oncological conditions. While in vitro studies play a crucial role in elucidating molecular pathways, they lack the context of the body's complexity. Therefore, laboratory animals are an indispensable part of successfully understanding and applying caspase networks. This paper aims to summarize and discuss recent knowledge, understanding, and challenges in caspase knock-out mice.

Evidence That a Peptide-Drug/p53 Gene Complex Promotes Cognate Gene Expression and Inhibits the Viability of Glioblastoma Cells.

Glioblastoma multiform (GBM) is considered the deadliest brain cancer. Conventional therapies are followed by poor patient survival outcomes, so novel and more efficacious therapeutic strategies are imperative to tackle this scourge. Gene therapy has emerged as an exciting and innovative tool in cancer therapy. Its combination with chemotherapy has significantly improved therapeutic outcomes. In line with this, our team has developed temozolomide-transferrin (Tf) peptide (WRAP5)/p53 gene nanometric complexes that were revealed to be biocompatible with non-cancerous cells and in a zebrafish model and were able to efficiently target and internalize into SNB19 and U373 glioma cell lines. The transfection of these cells, mediated by the formulated peptide-drug/gene complexes, resulted in p53 expression. The combined action of the anticancer drug with p53 supplementation in cancer cells enhances cytotoxicity, which was correlated to apoptosis activation through quantification of caspase-3 activity. In addition, increased caspase-9 levels revealed that the intrinsic or mitochondrial pathway of apoptosis was implicated. This assumption was further evidenced by the presence, in glioma cells, of Bax protein overexpression-a core regulator of this apoptotic pathway. Our findings demonstrated the great potential of peptide TMZ/p53 co-delivery complexes for cellular transfection, p53 expression, and apoptosis induction, holding promising therapeutic value toward glioblastoma.

Time and Cost-Effective Genome Editing Protocol for Simultaneous Caspase 8 Associated Protein 2 Gene Knock in/out in Chinese Hamster Ovary Cells Using CRISPR-Cas9 System.

Background: CHO cells are preferred for producing biopharmaceuticals, and genome editing technologies offer opportunities to enhance recombinant protein production. Targeting apoptosis-related genes, such as Caspases 8-Associated Protein 2 (CASP8AP2), improves CHO cell viability and productivity. Integrating robust strategies with the CRISPR-Cas9 system enables its application in CHO cell engineering. Objectives: This study was performed to develop a cost-effective protocol using the CRISPR-Cas9 system combined with the HITI strategy for simultaneous CASP8AP2 gene deletion/insertion in CHO cells and to assess its impact on cell viability and protein expression. Materials and Methods: We developed an efficient protocol for CHO cell engineering by combining CRISPR/Cas9 with the HITI strategy. Two distinct sgRNA sequences were designed to target the 3' UTR region of the CASP8AP2 gene using CHOPCHOP software. The gRNAs were cloned into PX459 and PX460-1 vectors and transfected into CHO cells using the cost-effective PEI reagent. A manual selection system was employed to streamline the process of single-cell cloning. MTT assays assessed gene silencing and cell viability at 24, 48, and 72 hours. Flow cytometry evaluated protein expression in CASP8AP2-silenced CHO cells. Results: The study confirmed the robustness of combining CRISPR-Cas9 with the HITI strategy, achieving a high 60% efficiency in generating knockout clones. PEI transfection successfully delivered the constructs to nearly 65% of the clones, with the majority being homozygous. The protocol proved feasible for resource-limited labs, requiring only an inverted fluorescent microscope. CASP8AP2 knockout (CHO-KO) cells exhibited significantly extended cell viability compared to CHO-K1 cells when treated with NaBu, with IC50 values of 7.28 mM and 14.25 mM at 48 hours, respectively (P-value 24 hours ≤ 0.0001, 48 hours ≤ 0.0001, P-value 72 hours = 0.0007). CHO CASP8AP2-silenced cells showed a 1.3-fold increase in JRed expression compared to native cells. Conclusions: CRISPR-Cas9 and HITI strategy was used to efficiently engineer CHO cells for simultaneous CASP8AP2 gene deletion/insertion, which improved cell viability and protein expression.

Apoptotic and non-apoptotic roles of caspases in placenta physiology and pathology.

Caspases, a family of cysteine proteases, are pivotal regulators of apoptosis, the tightly controlled cell death process crucial for eliminating excessive or unnecessary cells during development, including placental development. Collecting research has unveiled the multifaceted roles of caspases in the placenta, extending beyond apoptosis. Apart from their involvement in placental tissue remodeling via apoptosis, caspases actively participate in essential regulatory processes, such as trophoblast fusion and differentiation, significantly influencing placental growth and functionality. In addition, growing evidence indicates an elevation in caspase activity under pathological conditions like pre-eclampsia (PE) and intrauterine growth restriction (IUGR), leading to excessive cell death as well as inflammation. Drawing from advancements in caspase research and placental development under both normal and abnormal conditions, we examine the significance of caspases in both cell death (apoptosis) and non-cell death-related processes within the placenta. We also discuss potential therapeutics targeting caspase-related pathways for placenta disorders.

Exacerbated Activation of the NLRP3 Inflammasome in the Placentas from Women Who Developed Chronic Venous Disease during Pregnancy.

Chronic venous disease (CVD) comprises a spectrum of morphofunctional disorders affecting the venous system, affecting approximately 1 in 3 women during gestation. Emerging evidence highlights diverse maternofetal implications stemming from CVD, particularly impacting the placenta. While systemic inflammation has been associated with pregnancy-related CVD, preliminary findings suggest a potential link between this condition and exacerbated inflammation in the placental tissue. Inflammasomes are major orchestrators of immune responses and inflammation in different organs and systems. Notwithstanding the relevance of inflammasomes, specifically the NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3)- which has been demonstrated in the placentas of women with different obstetric complications, the precise involvement of this component in the placentas of women with CVD remains to be explored. This study employs immunohistochemistry and real-time PCR (RT-qPCR) to examine the gene and protein expression of key components in both canonical and non-canonical pathways of the NLRP3 inflammasome (NLRP3, ASC-apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain-caspase 1, caspase 5, caspase 8, and interleukin 1ß) within the placental tissue of women affected by CVD. Our findings reveal a substantial upregulation of these components in CVD-affected placentas, indicating a potential pathophysiological role of the NLRP3 inflammasome in the development of this condition. Subsequent investigations should focus on assessing translational interventions addressing this dysregulation in affected patient populations.

Exploring the Efficacy Enhancement Mechanism of Qixue Shuangbu prescription after TCM processing for treating chronic heart failure by regulating ERK/Bcl-2/Bax/Caspases-3 signaling pathway.

Qixue Shuangbu prescription (QSP) has been used for the treatment of chronic heart failure (CHF) with remarkable curative effect. Processed QSP (PQSP) could significantly improve the treatment of CHF after traditional Chinese medicine (TCM) processing. This study elucidated the underlying efficacy enhancement mechanism of QSP after TCM processing for treating CHF in vitro and in vivo. The injury of rat cardiomyoblast H9c2 cells was induced by anoxia/reoxygenation to mimic CHF state in vitro. Sixty Sprague-Dawley rats were used to established CHF model by intraperitoneally injecting doxorubicin (the accumulative dose 15 mg/kg). Biochemical examinations were performed in serum and cellular supernatant, respectively. Cardiac functions and histopathological changes were evaluated in CHF model rats. The protein and mRNA levels of ERK1/2, Bcl-2, Bax and Caspase-3 were evaluated by Western blot and RT-PCR, respectively. All above results of low dose crude QSP-treated group (L-CQSP), high dose CQSP-treated group (H-CQSP), low dose PQSP-treated group (L-PQSP), high dose PQSP-treated group (H-PQSP) were compared to systematically explore correlations between TCM processing and the efficacy enhancement for treating CHF of PQSP. Compared with the model group, the L-CQSP group showed significant improvement in cardiac function at 8th weeks, while no significant improvement in cardiomyocyte apoptosis and fibrosis. Both H-CQSP, L-PQSP and H-PQSP exerted beneficial therapeutic effects in injured H9c2 cardiomyocytes and CHF model rats. L-PQSP and H-PQSP significantly increased cell viability and the activity of SOD, decreased the activities of LDH, MDA and NO, up-regulated the expression of ERK1/2 and Bcl-2, down-regulated the expression of Bax and Caspase-3 compared to the same dosage of CQSP. The efficacy enhancement mechanism of PQSP after TCM processing for treating CHF was directly related to the regulation of ERK/Bcl-2/Bax/Caspases-3 signaling pathway.

Effect of Cannabis sativa L. extracts, phytocannabinoids and their acetylated derivates on the SHSY-5Y neuroblastoma cells' viability and caspases 3/7 activation.

BACKGROUND: There is a need for novel treatments for neuroblastoma, despite the emergence of new biological and immune treatments, since refractory pediatric neuroblastoma is still a medical challenge. Phyto cannabinoids and their hemisynthetic derivatives have shown evidence supporting their anticancer potential. The aim of this research was to examine Phytocannabinoids or hemisynthetic cannabinoids, which reduce the SHSY-5Y, neuroblastoma cell line's viability. METHODS: Hexane and acetyl acetate extracts were produced starting with Cannabis sativa L. as raw material, then, 9-tetrahidrocannabinol, its acid counterpart and CBN were isolated. In addition, acetylated derivatives of THC and CBN were synthesized. The identification and purity of the chemicals was determined by High Performance Liquid Chromatography and 1H y 13C Magnetic Nuclear Resonance. Then, the capacity to affect the viability of SHSY-5Y, a neuroblastoma cell line, was examined using the resazurin method. Finally, to gain insight into the mechanism of action of the extracts, phytocannabinoids and acetylated derivatives on the examined cells, a caspase 3/7 determination was performed on cells exposed to these compounds. RESULTS: The structure and purity of the isolated compounds was demonstrated. The extracts, the phytocannabinoids and their acetylated counterparts inhibited the viability of the SHSY 5Y cells, being CBN the most potent of all the tested molecules with an inhibitory concentration of 50 percent of 9.5 µM. CONCLUSION: Each of the evaluated molecules exhibited the capacity to activate caspases 3/7, indicating that at least in part, the cytotoxicity of the tested phytocannabinoids and their hemi-synthetic derivatives is mediated by apoptosis.

Sensitivity and activation of endoplasmic reticulum stress response and apoptosis in the perinatal sheep heart.

Although the unfolded protein response (UPR) contributes to survival by removing misfolded proteins, endoplasmic reticulum (ER) stress also activates proapoptotic pathways. Changed sensitivity to normal developmental stimuli may underlie observed cardiomyocyte apoptosis in the healthy perinatal heart. We determined in vitro sensitivity to thapsigargin in sheep cardiomyocytes from four perinatal ages. In utero cardiac activation of ER stress and apoptotic pathways was determined at these same ages. Thapsigargin-induced phosphorylation of eukaryotic initiation factor 2 (EIF2A) was decreased by 72% between 135 and 143 dGA (P = 0.0096) and remained low at 1 dPN (P = 0.0080). Conversely, thapsigargin-induced caspase cleavage was highest around the time of birth: cleaved caspase 3 was highest at 1 dPN (3.8-fold vs. 135 dGA, P = 0.0380; 7.8-fold vs. 5 dPN, P = 0.0118), cleaved caspase 7 and cleaved caspase 12 both increased between 135 and 143 dGA (25-fold and 6.9-fold respectively, both P < 0.0001) and remained elevated at 1 dPN. Induced apoptosis, measured by TdT-mediated dUTP nick-end labeling (TUNEL) assay, was highest around the time of birth (P < 0.0001). There were changes in myocardial ER stress pathway components in utero. Glucose (78 kDa)-regulated protein (GRP78) protein levels were high in the fetus and declined after birth (P < 0.0001). EIF2A phosphorylation was profoundly depressed at 1 dPN (vs. 143 dGA, P = 0.0113). In conclusion, there is dynamic regulation of ER proteostasis, ER stress, and apoptosis cascade in the perinatal heart. Apoptotic signaling is more readily activated in fetal cardiomyocytes near birth, leading to widespread caspase cleavage in the newborn heart. These pathways are important for the regulation of normal maturation in the healthy perinatal heart.NEW & NOTEWORTHY Cardiomyocyte apoptosis occurs even in the healthy, normally developing perinatal myocardium. As cardiomyocyte number is a critical contributor to heart health, the sensitivity of cardiomyocytes to endoplasmic reticulum stress leading to apoptosis is an important consideration. This study suggests that the heart has less robust protective mechanisms in response to endoplasmic reticulum stress immediately before and after birth, and that more cardiomyocyte death can be induced by stress in this period.

A Comprehensive Exploration of Caspase Detection Methods: From Classical Approaches to Cutting-Edge Innovations.

The activation of caspases is a crucial event and an indicator of programmed cell death, also known as apoptosis. These enzymes play a central role in cancer biology and are considered one promising target for current and future advancements in therapeutic interventions. Traditional methods of measuring caspase activity such as antibody-based methods provide fundamental insights into their biological functions, and are considered essential tools in the fields of cell and cancer biology, pharmacology and toxicology, and drug discovery. However, traditional methods, though extensively used, are now recognized as having various shortcomings. In addition, these methods fall short of providing solutions to and matching the needs of the rapid and expansive progress achieved in studying caspases. For these reasons, there has been a continuous improvement in detection methods for caspases and the network of pathways involved in their activation and downstream signaling. Over the past decade, newer methods based on cutting-edge state-of-the-art technologies have been introduced to the biomedical community. These methods enable both the temporal and spatial monitoring of the activity of caspases and their downstream substrates, and with enhanced accuracy and precision. These include fluorescent-labeled inhibitors (FLIs) for live imaging, single-cell live imaging, fluorescence resonance energy transfer (FRET) sensors, and activatable multifunctional probes for in vivo imaging. Recently, the recruitment of mass spectrometry (MS) techniques in the investigation of these enzymes expanded the repertoire of tools available for the identification and quantification of caspase substrates, cleavage products, and post-translational modifications in addition to unveiling the complex regulatory networks implicated. Collectively, these methods are enabling researchers to unravel much of the complex cellular processes involved in apoptosis, and are helping generate a clearer and comprehensive understanding of caspase-mediated proteolysis during apoptosis. Herein, we provide a comprehensive review of various assays and detection methods as they have evolved over the years, so to encourage further exploration of these enzymes, which should have direct implications for the advancement of therapeutics for cancer and other diseases.

A Non-Apoptotic Pattern of Caspase-9/Caspase-3 Activation During Differentiation of Human Embryonic Stem Cells into Cardiomyocytes.

In vitro studies have demonstrated that the differentiation of embryonic stem cells (ESCs) into cardiomyocytes requires activation of caspases through the mitochondrial pathway. These studies have relied on synthetic substrates for activity measurements, which can be misleading due to potential none-specific hydrolysis of these substrates by proteases other than caspases. Hence, caspase-9 and caspase-3 activation are investigated during the differentiation of human ESCs (hESCs) by directly assessing caspase-9 and -3 cleavage. Western blot reveals the presence of the cleaved caspase-9 prior to and during the differentiation of human ESCs (hESCs) into cardiomyocytes at early stages, which diminishes as the differentiation progresses, without cleavage and activation of endogenous procaspase-3. Activation of exogenous procaspase-3 by endogenous caspase-9 and subsequent cleavage of chromogenic caspase-3 substrate i.e. DEVD-pNA during the course of differentiation confirmes that endogenous caspase-9 has the potency to recognize and activate procaspase-3, but for reasons that are unknown to us fails to do so. These observations suggest the existence of distinct mechanisms of caspase regulation in differentiation as compared to apoptosis. Bioinformatics analysis suggests the presence of caspase-9 regulators, which may influence proteolytic function under specific conditions.

Marine-derived antimicrobial peptide piscidin-1 triggers extrinsic and intrinsic apoptosis in oral squamous cell carcinoma through reactive oxygen species production and inhibits angiogenesis.

Cancer of the head and neck encompasses a wide range of cancers, including oral and oropharyngeal cancers. Oral cancer is often diagnosed at advanced stages and has a dismal prognosis. Piscidin-1, a marine antimicrobial peptide (AMP) containing approximately 22 amino acids, also exhibits significant anticancer properties. We investigated the possible anti-oral cancer effects of piscidin-1 and clarified the mechanisms underlying these effects. We treated the oral squamous cell carcinoma cell lines OC2 and SCC4 with piscidin-1. Cell viability and the expression of different hallmark apoptotic molecules, including reactive oxygen species (ROS), were tested using the appropriate MTT assay, flow cytometry and western blotting assays, and human umbilical vein endothelial cell (HUVEC) wound healing, migration, and tube formation (angiogenesis) assays. Piscidin-1 increases cleaved caspase 3 levels to induce apoptosis. Piscidin-1 also increases ROS levels and intensifies oxidative stress in the endoplasmic reticulum and mitochondria, causing mitochondrial dysfunction. Additionally, it decreases the oxygen consumption rates and activity of mitochondrial complexes I-V. As expected, the antioxidants MitoTEMPOL and N-acetylcysteine reduce piscidin-1-induced ROS generation and intracellular calcium accumulation. Piscidin-1 also inhibits matrix metalloproteinase (MMP)-2/-9 expression in HUVECs, affecting migration and tube formation angiogenesis. We demonstrated that piscidin-1 can promote apoptosis via both intrinsic and extrinsic apoptotic pathways and findings indicate that piscidin-1 has anti-proliferative and anti-angiogenic properties in oral cancer treatment. Our study on piscidin-1 thus provides a basis for future translational anti-oral cancer drug research and a new theoretical approach for anti-oral cancer clinical research.

Lentivirus-Mediated BCL-XL Overexpression Inhibits Stem Cell Apoptosis during Ex Vivo Expansion and Provides Competitive Advantage Following Xenotransplantation.

Hematopoietic reconstitution after hematopoietic stem cell transplantation (HSCT) is influenced by the number of transplanted cells. However, under certain conditions donor cell counts are limited and impair clinical outcome. Hematopoietic stem and progenitor cell (HSPC) expansion prior to HSCT is a widely used method to achieve higher donor cell counts and minimize transplantation-related risks such as graft failure or delayed engraftment. Still, expansion in a non-physiological environment can trigger cell death mechanisms and hence counteract the desired effect. We have shown earlier that during HSCT a relevant amount of HSPCs were lost due to apoptosis and that cell death inhibition in donor HSPCs improved engraftment in xenotransplantation experiments. Here, we assessed the effect of combined ex vivo expansion and cell death inhibition on HSPC yield and their reconstitution potential in vivo. During expansion with cytokines and the small molecule inhibitor StemRegenin 1, concomitant lentiviral overexpression of antiapoptotic BCL-XL resulted in an increased yield of transduced HSPCs. Importantly, BCL-XL overexpression enhanced the reconstitution potential of HSPCs in xenotransplantation experiments in vivo. In contrast, treatment with caspase and necroptosis inhibitors had no favorable effects on HSPC yields nor on cell viability. We postulate that overexpression of antiapoptotic BCL-XL, both during ex vivo expansion and transplantation, is a promising approach to improve the outcome of HSCT in situations with limited donor cell numbers. However, such apoptosis inhibition needs to be transient to avoid long-term sequelae like leukemia.

Synthesis and biological activity of 11-Oxygenated and heterocyclic estrone analogs in pancreatic cancer monolayers and 3D spheroids.

Pancreatic Ductal Adenocarcinoma (PDAC), representing over 90 % of pancreatic cancer diagnoses, is an aggressive disease with survivability among the worst of all cancers due to its difficulty in detection and its high metastatic properties. Current therapies for PDAC show limited success at extending life expectancies, primarily due to cancer resistance and lack of patient-specific targeted therapies. This work highlights the design and evaluation of estrone-derived analogs with both heterocyclic side-chain functionality and 11-oxygenated functionality for use in pancreatic cancer. First-round heterocyclic analogs show preliminary promise in AsPC-1 and Panc-1 cell lines, with IC50 values as low as 10.16 ± 0.83 µM. Their success, coupled with design choices from other studies, led to the synthesis of novel 11-hydroxyl and 11-keto estrone analogs that show potent in-vitro toxicity against various pancreatic cancer models. The three most cytotoxic analogs, KA1, KA2, and KA9 demonstrated low micromolar activities in both MTT and CellTiter assays in three pancreatic cancer cell lines: AsPC-1, Panc-1, and BxPC-3, as well as in a co-culture of Panc-1 and pancreatic stellate cells. IC50 values for KA9 (4.17 ± 0.90, 5.28 ± 1.87, and 5.70 ± 0.65 µM respectively) shows consistency in all cell lines tested. KA9 is also able to cause an increase in caspases 3 and 7 activity, key markers for apoptosis, at non-cytotoxic concentrations. Additional work was performed by generating 3D pancreatic cancer spheroids to better modulate the pancreatic tumor microenvironment, and KA9 continued to show the best IC50 values (21.0 and 24.3 µM) in both cell types tested. KA9 was also able to prevent the growth of spheroids whereas the standard chemotherapy, Gemcitabine, could not, suggesting that it may be a potent analog for future development of treatments. Molecular dynamic simulations were also performed to confirm biological findings and uncovered that KA9's preferential binding location is in the active site pocket of key proteins involved in cytotoxicity.