Cell death analysis of recombinant mature epsilon toxin on the kidney cell line

Background and Objectives: Epsilon toxin is the third hazardous bacterial toxin causing ABS enterotoxaemia in domestic animal. In addition, epsilon toxin is known as a biological warfare agent. The aim of this study was to produce the recombi- nant mature epsilon toxin to evaluate cell death impact on the kidney cell line. Materials and Methods: For this purpose, the sequence of mature epsilon toxin (46-328 aa) in pET28a was cloned and expressed in Escherichia coli BL21 (DE3) and purified by nickel-nitrilotriacetic acid (Ni-NTA) column and confirmed by western blot analysis using HRP conjugated anti-His antibody. Then, to assess the anti-proliferative effects of different con- centrations of recombinant epsilon toxin, the MTT assay was done on the HEK293 cell line. The annexin V/PI staining was done to investigate the apoptotic and necrotic cell populations after exposure to epsilon toxin. Results: Induction by 1 mM IPTG for 4 h at 37°C was an optimized condition for expressing mature epsilon toxin in E. coli strain BL21 (DE3). Electrophoresis on SDS-PAGE 12% gel showed the desired band approximately at 38 KDa. Our results showed that recombinant epsilon toxin is mainly expressed as an inclusion body. Furthermore, 100, 150, and 200 µg/mL of mature epsilon toxin are significantly reduced the cell viability (P≤0.05). The considerable increase of necrotic cell percent- age was shown after exposing to 100, 150, and 200 µg/mL of mature epsilon toxin (P≤0.05). Conclusion: The recombinant mature epsilon toxin had cytotoxic effects and could induce necrosis.

Inhibition of the Anti-Apoptotic Bcl-2 Family by BH3 Mimetics Sensitize the Mitochondrial Permeability Transition Pore Through Bax and Bak

Mitochondrial permeability transition pore (MPTP)-dependent necrosis contributes to numerous pathologies in the heart, brain, and skeletal muscle. The MPTP is a non-selective pore in the inner mitochondrial membrane that is triggered by high levels of matrix Ca2+, and sustained opening leads to mitochondrial dysfunction. Although the MPTP is defined by an increase in inner mitochondrial membrane permeability, the expression of pro-apoptotic Bcl-2 family members, Bax and Bak localization to the outer mitochondrial membrane is required for MPTP-dependent mitochondrial dysfunction and subsequent necrotic cell death. Contrary to the role of Bax and Bak in apoptosis, which is dependent on their oligomerization, MPTP-dependent necrosis does not require oligomerization as monomeric/inactive forms of Bax and Bak can facilitate mitochondrial dysfunction. However, the relationship between Bax and Bak activation/oligomerization and MPTP sensitization remains to be explored. Here, we use a combination of in vitro and ex vivo approaches to determine the role of the anti-apoptotic Bcl-2 family members, which regulate Bax/Bak activity, in necrotic cell death and MPTP sensitivity. To study the role of each predominantly expressed anti-apoptotic Bcl-2 family member (i.e., Mcl-1, Bcl-2, and Bcl-xL) in MPTP regulation, we utilize various BH3 mimetics that specifically bind to and inhibit each. We determined that the inhibition of each anti-apoptotic Bcl-2 family member lowers mitochondrial calcium retention capacity and sensitizes MPTP opening. Furthermore, the inhibition of each Bcl-2 family member exacerbates both apoptotic and necrotic cell death in vitro in a Bax/Bak-dependent manner. Our findings suggests that mitochondrial Ca2+ retention capacity and MPTP sensitivity is influenced by Bax/Bak activation/oligomerization on the outer mitochondrial membrane, providing further evidence of the crosstalk between the apoptotic and necrotic cell death pathways.

Pseudomonas syringae pv. tomato DC3000 Effector HopG1 is a multi-faceted protein that Triggers Necrotic Cell Death that is attenuated by the Nonhost Resistance 2B (AtNHR2B) Protein

The plant pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) has become a paradigm in plant-bacteria interactions due to its ability to cause disease in the model plant Arabidopsis thaliana. Pst DC3000 uses the type III secretion system to deliver type III secreted effectors (T3SEs) directly into the plant cytoplasm. Pst DC3000 T3SEs contribute to pathogenicity by suppressing plant defense responses and targeting plant’s physiological processes. Although the complete repertoire of effectors encoded in the Pst DC3000 genome have been identified, the specific function for most of them remains to be elucidated. The mitochondrial-localized T3E HopG1, suppresses plant defense responses and promotes the development of disease symptoms. Here, we show that HopG1 triggers necrotic cell death that enables the growth of non-adapted pathogens. We further showed that HopG1 interacts with the plant immunity-related protein AtNHR2B and that AtNHR2B attenuates HopG1- virulence functions.

Resistance to chemical carcinogenesis induction via a dampened inflammatory response in naked mole-rats

Naked mole-rats (NMRs) have a very low spontaneous carcinogenesis rate, which has prompted scientists to study their cancer resistance mechanisms in order to provide clues for human cancer prevention. Although cancer resistance in NMRs has been intensively investigated at the cellular level, it is still unknown how strongly resistant NMR individuals are to carcinogenesis and how NMR tissues respond to experimental carcinogenesis induction. Here, we show that NMRs exhibit extraordinary resistance against potent chemical carcinogenesis induction through a dampened inflammatory response. Although carcinogenic insults damaged skin cells of both NMRs and mice, NMR skin showed markedly lower immune cell infiltration and reduced induction of inflammatory genes. NMRs harbor loss-of-function mutations in receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) genes, which are essential for necroptosis, a type of necrotic cell death that activates strong inflammation. A necroptosis-inducing stimulus did not increase death of NMR cells. After carcinogenic insults, leakage of the HMGB1, a marker of necrotic cell death, was not increased in NMR skin. In mice, inhibition or knockout of RIPK3 reduced immune cell infiltration and delayed the onset of chemical carcinogenesis. Therefore, necroptosis deficiency may serve as a cancer resistance mechanism via attenuating the inflammatory response in NMRs. Our study sheds light on the importance of a dampened inflammatory response as a non-cell-autonomous cancer resistance mechanism in NMRs. Further in vivo study of the unusual tissue immune system and carcinogenesis resistance of NMRs may lead to the development of new strategies to prevent carcinogenesis in humans.

Mycobacterium tuberculosis Exploits Focal Adhesion Kinase to Induce Necrotic Cell Death and Inhibit Reactive Oxygen Species Production

Tuberculosis is a deadly, contagious respiratory disease that is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb). Mtb is adept at manipulating and evading host immunity by hijacking alveolar macrophages, the first line of defense against inhaled pathogens, by regulating the mode and timing of host cell death. It is established that Mtb infection actively blocks apoptosis and instead induces necrotic-like modes of cell death to promote disease progression. This survival strategy shields the bacteria from destruction by the immune system and antibiotics while allowing for the spread of bacteria at opportunistic times. As such, it is critical to understand how Mtb interacts with host macrophages to manipulate the mode of cell death. Herein, we demonstrate that Mtb infection triggers a time-dependent reduction in the expression of focal adhesion kinase (FAK) in human macrophages. Using pharmacological perturbations, we show that inhibition of FAK (FAKi) triggers an increase in a necrotic form of cell death during Mtb infection. In contrast, genetic overexpression of FAK (FAK+) completely blocked macrophage cell death during Mtb infection. Using specific inhibitors of necrotic cell death, we show that FAK-mediated cell death during Mtb infection occurs in a RIPK1-depedent, and to a lesser extent, RIPK3-MLKL-dependent mechanism. Consistent with these findings, FAKi results in uncontrolled replication of Mtb, whereas FAK+ reduces the intracellular survival of Mtb in macrophages. In addition, we demonstrate that enhanced control of intracellular Mtb replication by FAK+ macrophages is a result of increased production of antibacterial reactive oxygen species (ROS) as inhibitors of ROS production restored Mtb burden in FAK+ macrophages to same levels as in wild-type cells. Collectively, our data establishes FAK as an important host protective response during Mtb infection to block necrotic cell death and induce ROS production, which are required to restrict the survival of Mtb.

Bortezomib Inhibits Multiple Myeloma Cells by Transactivating ATF3 to Trigger miR-135a-5p- Dependent Apoptosis

Multiple myeloma (MM) is a malignant cancer with an increasing in incidence that can be alleviated through bortezomib (BTZ) treatment. Activating transcription factor 3 (ATF3) plays a major role in cancer development. Moreover, microRNAs (miRNAs) regulate carcinogenic pathways, apoptosis, and programmed necrotic cell death. However, the detailed mechanism by which ATF3 modulates BTZ drug sensitivity/resistance remains elusive. In the current study, expression of ATF3 was significantly increased under BTZ treatment in a dose-dependent manner in MM cell lines. In addition, ATF3 could regulate cell apoptosis under BTZ treatment. The effect of ATF3 was negatively regulated by its binding miRNA, miR-135a-5p. When either ATF3 was silenced or miR-135a-5p mimics were added to MM cells, they partially lost sensitivity to BTZ treatment. This was accompanied by low levels of Noxa, CHOP, and DR5, and a decrease in mitochondrial membrane potential. These results revealed the combinatorial regulatory patterns of ATF3 and miR-135a-5p in the regulatory protein interactome, which indicated a clinical significance of the miR-135a-5p-ATF3 protein interaction network in BTZ therapy. This study provides potential evidence for further investigation into BTZ resistance.

Examining cellular responses to reconstituted antibody protein liquids

Protein ionic liquids (PIL) are a new class of biologic stabilizers designed to protect the functionality and extend the shelf-life of biotechnological and therapeutic agents making them more readily available, and resistant to austere environments. Protein biorecognition elements such as monoclonal antibodies are commonly utilized therapeutics that require the robust stabilization offered by PILs, but biocompatibility remains an important issue. This study has focused on characterizing the biocompatibility of an antibody based PIL by exposing multiple cells types to a cationized immunoglobulin suspended in an anionic liquid (IgG-IL). The IgG-IL caused no significant alterations in cellular health for all three cell types with treatments < 12.5 µg/mL. Concentrations ≥ 12.5 µg/mL resulted in significant necrotic cell death in A549 and HaCaT cells, and caspase associated cell death in HepG2 cells. In addition, all cells displayed evidence of oxidative stress and IL-8 induction in response to IgG-IL exposures. Therapeutic Ig can be utilized with a wide dose range that extends into concentrations we have found to exhibit cytotoxicity raising a toxicity concern and a need for more extensive understanding of the biocompatibility of IgG-ILs.

Dual Effect Exhibited by Insulin in Myeloma and Lymphoblastoid Cells

Many reports have documented the role of INS (insulin) as growth factors in a variety of cancers. Epidemiological studies revealed that INS therapy causes increased mortality in multiple myeloma (MM) patients with pre-existing or steroid-induced type 2 diabetes. However, there is limited experimental evidence of this association. In the present study, the dual effect of INS on the viability of myeloma RPMI8226 and lymphoblastoid IM9 cells was revealed. In serum-containing medium exogenous INS serves as a growth factor, whereas INS decreases the number of cells under serum-free medium. In the last case, the main mechanism of decreasing the cell population is apoptosis through up-regulation of Cas-3 and downregulation of Bcl-2 expression. INS has also been shown to be involved in the regulation of necrotic cell death.

RIPK3-Dependent Necroptosis Limits PRV Replication in PK-15 Cells

Pigs infected by pseudorabies virus (PRV) display necrotic pathology in multiple organs. The mechanism by which PRV induces cell death is still unclear. Recently, necroptosis was identified as a programmed process dependent on the receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase-like protein (MLKL). In this study, we demonstrated that PRV induced RIPK3-dependent necroptosis in PK-15 cells. The data showed that PRV infection caused cell death with Propidium Iodide (PI)-positive staining. Transmission electron microscopy analysis indicated plasma membrane disruption in PRV-infected cells. A pan-caspase inhibitor did not prevent PRV-induced necrotic cell death. Western blot analysis indicated that caspase-3 and caspase-8 were not cleaved during PRV infection. Although the transcription of tumor necrosis factor-alpha (TNF-α) was increased by PRV infection, RIPK1 was shown to be not involved in PRV-induced necrotic cell death by use of its specific inhibitor. Further experiments indicated that the phosphorylation of RIPK3 and MLKL was upregulated in PRV-infected cells. Stable shRNA knockdown of RIPK3 or MLKL had a recovery effect on PRV-induced necrotic cell death. Meanwhile, viral titers were enhanced in RIPK3 and MLKL knockdown cells. Hence, we concluded that initiation of necroptosis in host cells plays a limiting role in PRV infection. Considering that necroptosis is an inflammatory form of programmed cell death, our data may be beneficial for understanding the necrotic pathology of pigs infected by PRV.