CD138 Expression Promotes Accumulation and Activation of T Cells in Autoimmune MRL/lpr Mice

CD138+ T cells that accumulated in Fas-deficiency lupus mice, had been identified as autoreactive T cells in SLE which significantly promoted autoantibody secretion. In present study, we found CD138 expression in T cells played a key role in the progression of SLE in MRL/lpr mice. Our results indicated CD138+ T cells apoptosis was in Fas dependent way. However, CD138 expression of T cells in MRL/lpr mice could significantly prevent T cells apoptosis, contribute to accumulation of T cells and DN T cells and simultaneously promote T cells activation. Importantly, CD138 expression in DN T cells significantly increased FasL expression of DN T cells enhancing the cytotoxity of DN T cells. Phorbol 12-myristate 13-acetate and Ionomycin (PI) stimulation could significantly prevent CD138+ T cells accumulation by strikingly inducing their specific apoptosis. Moreover, PI stimulation significantly activated CD138+ T cells with increased CD69 expression in them. Importantly, our results showed CD69 expression in CD138+ T cells could significantly increase the apoptosis level of them. That indicated PI stimulation could induce specific apoptosis of CD138+ T cells via increasing CD69 expression in CD138+ T cells. In addition, our results showed CD138- T cells in MRL/lpr mice had significant defects in activation. However, to activate T cells could significantly prevent CD138 expression in CD3+ T cells of MRL/lpr mice. Our results suggested CD138 expression in CD3+ T cells of MRL/lpr mice was probably caused by the failure of activation in autoreactive T cells before self-antigens exposure to immune system.

Hypofractionated Radiation Induced the Immunogenic Death of Bladder Cancer Cells Leading to the Immune Sensitization of Dendritic Cells

Purpose Radiotherapy is a commonly used method in the treatment of bladder cancer (BC). Radiation induced immunogenic death (ID) and antitumor immune response are related to the prognosis of radiotherapy. As the most powerful antigen-presenting cell in the body, the role of dendritic cells (DCs) is not very clear. Methods Apoptosis level, cell cycle analysis and expression levels of high mobility group protein 1 (HMGB1), calreticulin (CRT) and heat shock protein 70 (HSP70) were performed for bladder cancer cells after hypofractionated radiotherapy. The effects of the conditioned media on DCs for antitumor immune response activation were studied as well. Results The significantly increased apoptosis level, G2/M phase cell cycle arrest and significantly increased HMGB1, CRT and HSP70 expressions, and increased secretion of CCL5 and CCL21 in the supernatant of bladder cancer cells after hypofractionated radiotherapy. The expression of CD80, CD86, CCR5 and CCR7 on DCs was upregulated in the conditioned media of bladder cancer cells after hypofractionated radiotherapy. Conclusion Hypofractionated radiation blocked the cell cycle of BC cells in the G2/M phase and induced ID occurrence, resulting in DCs immune sensitization, which is of great clinical significance in understanding the radiotherapy of BC and the immunoregulation function of DCs.

MicroRNA-139-5p Suppresses Cell Malignant Behaviors in Breast Cancer through Targeting MEX3A

Objective. The study was designed to evaluate the underlying mechanism of microRNA-139-5p in breast cancer (BC). Methods. Expression statuses of microRNA-139-5p and MEX3A were measured by qRT-PCR and western blotting. The anticancer effect of microRNA-139-5p in vitro was tested by a set of assays. Interaction between microRNA-139-5p and MEX3A was validated by dual-luciferase detection. Results. MicroRNA-139-5p expression in BC cells was obviously low, while MEX3A was significantly overexpressed. MicroRNA-139-5p restrained proliferative, invasive, and migratory abilities of BC cells and increased apoptosis level of BC cells, while MEX3A exerted a promoting effect on BC cell growth. Dual-luciferase reporter detection confirmed that microRNA-139-5p bound to MEX3A 3 ′ -UTR. Conclusions. MicroRNA-139-5p inhibited the development of BC by targeting MEX3A. MicroRNA-139-5p/MEX3A may be a target for BC therapy.

Nanofluorescence Probes to Detect miR-192/Integrin Alpha 1 and Their Correlations with Retinoblastoma

We developed a novel nanostructure DNA probe for the in situ detection of ITGA1 and miR-192 in retinoblastoma (RB) and to study the correlation between ITGA1 and miR-192 expression and RB development. ITGA1 and miR-192 nanostructure DNA probes were carried by silica particles and coated by dioleoyl-trimethy-lammonium-propane, which enhances their organizational compatibility and infiltration capacity. This probe has stable physicochemical properties and high specificity and sensitivity to detect ITGA1 and miR-192 in situ both in RB cell lines and RB tissues. Using ITGA1 and miR-192 nanostructure DNA probes in RB tissue and cell lines, we found that the expression of ITAG1 drastically increased, but to the contrary, miR-192 was not expressed. After transfection, ITGA1 and miR-192 were overexpressed or silenced in RB116 cells, and we found that ITGA1 could effectively increase the activity and invasion of this RB cell line and reduce its apoptosis level, while miR-192 antagonized this tumor-promoting effect. Therefore, miR-192 can be used as an early biomarker of RB, and ITGA1 may be a new prognostic marker and therapeutic target for the treatment of RB.

Effect of 5-luorouracil Carboxymethyl Chitosan Nanoparticles on the Apoptosis Level of Keloid Fibroblasts

Objective to investigate the effect of 5-fluorouracil (5-FU) loaded carboxymethyl chitosan (CMC) nanoparticles on the apoptosis level of keloid fibroblasts. Methods keloid tissue (n = 80) was taken from inpatient and outpatient patients who were treated in our hospital from January 2020 to December 2020. The fresh keloid specimen tissues were washed with saline three times. After removing the epithelium, they were cut into 2mmx2mm tissue blocks for subculture. CMC nanoparticles loaded with 5-FU were prepared by ionically crosslinking CMC solution with calcium chloride. The viability of fibroblasts was determined by MTT assay. Transwell was used to assess fibroblast invasion. The wound healing test was used to evaluate the migration of fibroblasts. The apoptotic cells were analyzed by Annexin V-FITC and flow cytometry. The expression of apoptotic protein in fibroblasts was determined by Western blot analysis. The expression of transforming growth factor-β (TGF-β) and Smad2/3 were analyzed by immunohistochemistry. The mRNA expression of ERK1/2, protein kinase B (AKT) and nuclear transcription factor-κB (NF-κB) was analyzed by RT-qPCR. Results compared with the control group, there was no difference in the viability of fibroblasts in the nanoparticle group at 0h (P>0.05), and the viability of fibroblasts at 24h, 48h and 72h decreased by 37.29%, 29.58% and 28.38, respectively (P<0.05). Compared with the control group, the fiber cells composed of nanoparticles were less likely to pass through the pores of the basement membrane and their invasiveness decreased (P<0.05). Compared with the control group, the migration ability of fibroblasts in the nanoparticle group decreased (P<0.05). Compared with the control group, the apoptotic rate of fibers composed of nanoparticles increased (P<0.05). Compared with the control group, the expression levels of Bax, Caspase-3 and Caspase-9 apoptotic proteins in the nanoparticle group increased (P<0.05). Compared with the control group, the expression levels of TGF-β and Smad2/3 in the nanoparticle group decreased (P<0.05). Compared with the control group, the expression levels of ERK1/2, Akt and NF-κB mRNA in the nanoparticle group decreased (P<0.05). Conclusion 5-FU loaded CMC nanoparticles could reduce fibrosis in keloids, lower cell proliferation, migration and invasion, and increase cell apoptosis by inhibiting TGF-β related pathways.

ANTIAPOPTOTIC POTENTIAL OF SPIDER TOXINS

Arthropod peptide toxins rich in disulfide bonds are one of the potential sources of bioactive substances. Due to their structure, toxins have increased stability and are able to bind to ion channels, blocking them or changing the gating mechanism. Some spider toxins bind to different types of calcium channels. Calcium ions, in turn, play an important role in many cellular processes, namely, apoptosis. The aim of this paper is to investigate the effect of a number of toxins – arachnid ion-channel blockers in – on intracellular processes associated with the induction of apoptosis in mammalian cells. Materials and Methods. Toxins ω-hexatoxin-Hv1a, ω-theraphotoxin-Hhn2a were used in the study, as they are inhibitors of L- and P/Q-type calcium channels, respectively. Apoptosis was induced using the AC-1001H3 peptide. The authors used fluorescence microscopy to study the effect of toxins on the apoptosis level, oxidative stress, and mitochondrial potential in CHO-K1 cells. Results. The authors observed that incubation of cells with toxins (10 nM) and AC-1001H3 peptide led to increased ROI intracellular concentration, which should have induced apoptotic mechanisms. However, the effect was the opposite. In addition, there was an increase in the mitochondrial potential level. Despite this, the used toxins blocked apoptosis caused by AC-1001H3 and reduced the natural apoptosis level in the CHO-K1 cells. Conclusion. The study demonstrated the antiapoptotic effect of some arthropod peptide toxins. The studied toxins can be used in the treatment of pathologies associated with the activation of apoptotic mechanisms. Keywords: apoptosis, spider toxin, peptide. Пептидные токсины членистоногих, богатые дисульфидными связями, являются одним из потенциальных источников биоактивных веществ. За счет своей структуры токсины обладают повышенной стабильностью и способны связываться с ионными каналами, блокируя их или изменяя механизм стробирования. Ряд токсинов пауков способен связываться с кальциевыми каналами разных типов. Ионы кальция в свою очередь играют важную роль во многих процессах в клетке, одним из которых является апоптоз. Цель работы – исследовать влияние ряда токсинов – блокаторов ионных каналов паукообразных – на внутриклеточные процессы, связанные с индукцией апоптоза в клетках млекопитающих. Материалы и методы. В исследовании использовались токсины ω-hexatoxin-Hv1a, ω-theraphotoxin-Hhn2a, которые являются ингибиторами кальциевых каналов L- и P/Q-типов соответственно. Индукция апоптоза проводилась с использованием пептида AC-1001H3. Изучалось влияние токсинов на уровень апоптоза, оксидативного стресса и митохондриального потенциала в клетках линии CHO-K1 с использованием методов флуоресцентной микроскопии. Результаты. Было установлено, что инкубация клеток с токсинами в концентрации 10 нМ и индуктором апоптоза AC-1001H3 приводила к росту внутриклеточной концентрации активных форм кислорода, что должно индуцировать апоптотические механизмы, однако эффект был противоположным. Кроме того, происходило повышение уровня митохондриального потенциала. Несмотря на это использованные токсины блокировали апоптоз, вызванный AC-1001Н3, и снижали уровень естественного апоптоза в культуре клеток CHO-K1. Выводы. Проведенное исследование продемонстрировало антиапоптотический эффект ряда пептидных токсинов членистоногих. Изученные токсины могут найти применение при лечении патологии, связанной с активацией апоптотических механизмов. Ключевые слова: апоптоз, токсин паука, пептид.

Knockdown of Circular RNA hsa_circ_PVT1 Inhibited Laryngeal Cancer Progression via Preventing wnt4/β-Catenin Signaling Pathway Activation

AimTo explore the function and mechanism of circular has_circ_PVT1 on laryngeal cancer (LC).MethodsMicroarray chip was performed to screen the differential expression of circRNA. Western blot and qRT-PCR was employed to detect the protein and mRNA level. CCK-8, clone formation, cell cycle, wound healing, and Transwell assay were performed to detect the cell proliferation, migration, and invasion ability. Luciferase assay and Fish were used to confirm the relationship between circ_PVT1/CBX4 and miR-21-5p. Flow cytometry and TUNEL assay were carried out to assess the apoptosis level.ResultsThe upregulation of circ_PVT1 was found in LC tissues and cells. Silencing of circ_PVT1 inhibited LC progression via targeting miR-21-5p and indirectly controlling CBX4. Wnt4/β-catenin signal pathway was inactivated by inhibiting the expression of circ_PVT1.ConclusionKnockdown of circ_PVT1 prevented LC progression via targeting miR-21-5p/CBX4 by inhibiting wnt4/β-catenin signal pathway, which could provide a novel therapeutic target for LC.

Overcoming of Microenvironment Protection on Primary Chronic Lymphocytic Leukemia Cells after Treatment with BTK and MDM2 Pharmacological Inhibitors

In B-chronic lymphocytic leukemia (B-CLL), the interaction between leukemic cells and the microenvironment promotes tumor cell survival. The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib is one of the first-in-class molecules for the treatment of B-CLL patients; however, the emerging mechanisms of resistance to ibrutinib call for new therapeutic strategies. The purpose of the current study was to investigate the ability of ibrutinib plus the MDM2-inhibitor nutlin-3 to counteract the tumor microenvironment protective effect. We observed that primary B-CLL cells cultivated in microenvironment mimicking conditions were protected from apoptosis by the up-regulation of c-MYC and of p53. In the same setting, combined treatments with ibrutinib plus nutlin-3 led to significantly higher levels of apoptosis compared to the single treatments, counteracting the c-MYC up-regulation. Moreover, the combination induced high p53 levels and a significant dissipation of the mitochondrial membrane potential, together with BAX cleavage in the more active p18 form and phospho-BAD down-regulation, that are key components of the mitochondrial apoptotic pathway, enhancing the apoptosis level. Our findings propose a new therapeutic strategy to overcome the tumor microenvironment protection involved in B-CLL resistance to drugs, with possible clinical implications also for other hematologic and solid tumors for which ibrutinib is considered a therapeutic option.

Flavonoid Kaempferol inhibits the Proliferation and Survival of Human Leukemia HL60 cells

Background: Acute myeloid leukemia (AML) is an aggressive type of leukemia adversely affecting the normal differentiation and proliferation process of human hematopoietic myeloid lineage. During the last decades, Kaempferol (Kae) (3,4′,5,7-tetrahydroxyflavone) is considered a flavonoid with useful medical significance, capable of inhibiting various types of leukemia (e.g., AML). Objective: To evaluate the Kae effect on the proliferation and apoptosis of a human AML cell line, HL60. Methods: The proliferation capability of the HL60 cells was estimated by MTT assay after 12, 24, and 48 hours after the exposure to Kae at a series of concentrations, including 10, 25, 50, and 75 µM. Also, the apoptosis level of HL60 cells was measured 48 hours after the exposure to various concentrations of Kae (10, 25, and 50 µM) using Annexin-V/PI staining and FACS analysis. Besides, the gene expression of CDK1/2, Bcl-2, survivin, c-FLIP, Mcl-1, XIAP, Bax, and caspase 3 and 8 was assessed following the treatment of HL60 cells with Kae (25 and 50 µM) by Real-Time PCR. Results: The anti-proliferation activity of Kae showed an ascending pattern over time and reached the maximum level after 48 hours of HL60 cells exposure with Kae. Also, it was able to trigger apoptosis of HL60 cells, in particular, at 50 µM concentration. On the other hand, Kae could modify the expression levels of the candidate’s genes in treated cells. Conclusion: The promising results of using Kae against the HL60 cells have made it a good drug candidate to treat AML through the up-regulation of caspases expression and down-regulation of anti-apoptotic proteins.

Environmental Enrichment Protects Offspring of Preeclamptic-Like Rat Model From Cognitive Decline

Background: Preeclampsia affects 5-8% of all pregnancies and contributes to adverse pregnancy and birth outcome. In addition to short-term effects of preeclampsia, it can exert long-term adverse effects on offspring. Numerous studies has demonstrated that offspring of preeclamptic women exhibited cognitive deficit from childhood to old age. However, effective ways to improving their cognitive ability remains to be investigated. The aim of this study was to explore whether Environmental Enrichment in early life could restore cognitive ability of offspring of preeclampsia rat model, as well as cellular and molecular mechanism by which EE improve cognitive ability.Methods: L-NAME was used to establish preeclampsia rat model. The spatial learning and memory ability of 56-day-old offspring was evaluated by Morris Water Maze. Immunofluorescence was performed to evaluate cell proliferation and apoptosis in the DG region of hippocampus. qRT-PCR was performed to examine the expression level of neurogenesis-associated genes and inflammatory cytokines. Enzyme-linked immune absorbent assay was performed to evaluate the concentration of Vascular endothelial growth factor (VEGF) and inflammatory cytokines in the hippocampus.Results: The administration of L-NAME led to increased systolic blood pressure and urine protein level of pregnant rat. Offspring in L-NAME group exhibited impaired spatial learning ability and memory. The hippocampus neurogenesis and synaptic plasticity was impaired in offspring of L-NAME group. Furthermore, the cell apoptosis in hippocampus was increased in L-NAME group. The hippocampus was skewed to a pro-inflammatory profile, reflected by increased inflammatory cytokines level. While EE improved cognitive ability of offspring in L-NAME group and resulted in increased hippocampal neurogenesis and synaptic proteins expression level, decreased apoptosis level and inflammatory cytokines.Conclusions: Environment Enrichment resolves cognitive impairment in offspring of preeclampsia rat model by improving hippocampal neurogenesis and synaptic plasticity, normalizing the apoptosis level and the inflammation balance.