RIPK1 inhibition contributes to lysosomal membrane stabilization in ischemic astrocytes via a lysosomal Hsp70.1B-dependent mechanism.

Receptor-interacting protein kinase 1 (RIPK1) contributes to necroptosis. Our previous study showed that pharmacological or genetic inhibition of RIPK1 protects against ischemic stroke-induced astrocyte injury. In this study, we investigated the molecular mechanisms underlying RIPK1-mediated astrocyte injury in vitro and in vivo. Primary cultured astrocytes were transfected with lentiviruses and then subjected to oxygen and glucose deprivation (OGD). In a rat model of permanent middle cerebral artery occlusion (pMCAO), lentiviruses carrying shRNA targeting RIPK1 or shRNA targeting heat shock protein 70.1B (Hsp70.1B) were injected into the lateral ventricles 5 days before pMCAO was established. We showed that RIPK1 knockdown protected against OGD-induced astrocyte damage, blocked the OGD-mediated increase in lysosomal membrane permeability in astrocytes, and inhibited the pMCAO-induced increase in astrocyte lysosome numbers in the ischemic cerebral cortex; these results suggested that RIPK1 contributed to the lysosomal injury in ischemic astrocytes. We revealed that RIPK1 knockdown upregulated the protein levels of Hsp70.1B and increased the colocalization of Lamp1 and Hsp70.1B in ischemic astrocytes. Hsp70.1B knockdown exacerbated pMCAO-induced brain injury, decreased lysosomal membrane integrity and blocked the protective effects of the RIPK1-specific inhibitor necrostatin-1 on lysosomal membranes. On the other hand, RIPK1 knockdown further exacerbated the pMCAO- or OGD-induced decreases in the levels of Hsp90 and the binding of Hsp90 to heat shock transcription factor-1 (Hsf1) in the cytoplasm, and RIPK1 knockdown promoted the nuclear translocation of Hsf1 in ischemic astrocytes, resulting in increased Hsp70.1B mRNA expression. These results suggest that inhibition of RIPK1 protects ischemic astrocytes by stabilizing lysosomal membranes via the upregulation of lysosomal Hsp70.1B; the mechanism underlying these effects involves decreased Hsp90 protein levels, increased Hsf1 nuclear translocation and increased Hsp70.1B mRNA expression.

Necroptotic kinases are involved in the reduction of depression-induced astrocytes and fluoxetine's inhibitory effects on necroptotic kinases.

The role of astrocytes in major depressive disorder has received great attention. Increasing evidence indicates that decreased astrocyte numbers in the hippocampus may be associated with depression, but the role of necroptosis in depression is unknown. Here, in a chronic unpredictable mild stress (CUMS) mouse model and a corticosterone (Cort)-induced human astrocyte injury model in vitro, we found that mice treated with chronic unpredictable mild stress for 3-5 weeks presented depressive-like behaviors and reduced body weight gain, accompanied by a reduction in astrocytes and a decrease in astrocytic brain-derived neurotropic factors (BDNF), by activation of necroptotic kinases, including RIPK1 (receptor-interacting protein kinase 1)/p-RIPK1, RIPK3 (receptor-interacting protein kinase 3)/p-RIPK3 and MLKL (mixed lineage kinase domain-like protein)/p-MLKL, and by upregulation of inflammatory cytokines in astrocytes of the mouse hippocampus. In contrast, necroptotic kinase inhibitors suppressed Cort-induced necroptotic kinase activation, reduced astrocytes, astrocytic necroptosis and dysfunction, and decreased Cort-mediated inflammatory cytokines in astrocytes. Treatment with fluoxetine (FLX) for 5 weeks improved chronic unpredictable mild stress-induced mouse depressive-like behaviors; simultaneously, fluoxetine inhibited depression-induced necroptotic kinase activation, reversed the reduction in astrocytes and astrocytic necroptosis and dysfunction, decreased inflammatory cytokines and upregulated brain-derived neurotropic factors and 5-HT1A levels. Furthermore, fluoxetine had no direct inhibitory effect on receptor-interacting protein kinase 1 phosphorylation. The combined administration of fluoxetine and necroptotic kinase inhibitors further reduced corticosterone-induced astrocyte injury. In conclusion, the reduction in astrocytes caused by depressive-like models in vivo and in vitro may be associated with the activation of necroptotic kinases and astrocytic necroptosis, and fluoxetine exerts an antidepressive effect by indirectly inhibiting receptor-interacting protein kinase 1-mediated astrocytic necroptosis.

Two cases of special POEMS syndrome without monoclonal protein expression: a case report and literature review.

POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes) syndrome is rare, with polyneuropathy and monoclonal plasma cell disorder generally considered as essential diagnostic symptoms. We report two cases of POEMS syndrome without monoclonal protein expression. The first case was a 72-year-old man who had experienced recurrent edema of the lower limbs for 2 years and abdominal distention for 2 months. The other case was a 62-year-old man with a 5-year history of recurrent numbness of the extremities and muscle weakness, which had become serious over the preceding 3 months. Both patients had various symptoms that matched those of POEMS syndrome, but neither had monoclonal protein expression. However, a diagnosis of POEMS syndrome was made in each case. Both patients were treated with lenalidomide and dexamethasone, after which their symptoms improved and laboratory test results normalized. The findings in these two cases suggest the possibility that POEMS syndrome may occur without monoclonal protein expression. The diagnostic criteria of POEMS syndrome may thus need further investigation.

Primary myelofibrosis with concurrent CALR and MPL mutations: A case report.

BACKGROUND: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by recurrent mutations in the JAK2, CALR, and MPL genes. The CALR and MPL co-mutation is very rare. To our knowledge, no more than five cases have been reported. Here, we report a case of PMF in which a CALR and MPL co-mutation was detected by next-generation sequencing (NGS) technology, and a literature review was performed. CASE SUMMARY: A 73-year-old woman was admitted to our hospital in 2018 due to abdominal distension. The patient had splenomegaly, lymphadenopathy, leukopenia, anemia, and immature granulocytes in peripheral blood. There were dacrocytes and atypical megakaryocytes in bone marrow, and megakaryocytic proliferation was very active, accompanied by reticulin fibrosis grade 2. By NGS analysis of the bone marrow sample, we detected mutations in CALR, MPL, and PIK3RI, while JAK2 V617F and BCR-ABL were negative. Therefore, the patient was diagnosed with PMF and received oral ruxolitinib. However, the spleen and hematologic responses were poor. We review the literature, analyze previous reports of the mutation sites in our patient and differences between our patient and other reported cases of co-mutated CALR and MPL genes, and discuss the reason why the CALR and MPL co-mutations are rare and possible mechanisms and their impact on the prognosis of patients. CONCLUSION: CALR and MPL mutations can be concurrent in MPN, but they are rare. The use of NGS may help to identify more patients with co-mutated CALR and MPL genes. This will help to further explore the mechanism and its impact on these patients to develop appropriate treatment strategies.

Comparison of hemodynamic changes and prognosis between stenting and standardized medical treatment in patients with symptomatic moderate to severe vertebral artery origin stenosis.

This study aimed to compare the clinical efficacy of stenting compared with standardized medical treatment in patients with moderate to severe vertebral artery origin stenosis (VAOS).Patients diagnosed with moderate to severe VAOS and indicated to undergo vertebral artery stenting were enrolled. Patients were divided into stenting group and standardized medical treatment group. All patients underwent transcranial Doppler (TCD) before and after treatment. Incidence of new cerebral infarction, transient ischemic attack (TIA), improvement of clinical symptoms, and National Institutes of Health Stroke Scale (NIHSS) score were observed.A total of 98 patients were enrolled. Vertebral artery stenting implant was accepted by 43 patients. Two weeks after treatment, the NIHSS score in the stenting group decreased significantly compared to that in the standardized medical treatment group. The modified Rankin Scale (mRS) score in the stenting group at three months was significantly lower than that in the medical treatment group (P = .044). The extent of vascular stenosis in the stent group decreased significantly (76.5 ±â€Š10.0% vs. 13.7 ±â€Š5.9%, t = 35.878, P = .000). The adverse events occurred in 9 (16.4%) patients in the medical treatment group and 5 (11.6%) in the stenting group (P = .506). There was one case with new cerebral infarction in the stenting group, whereas the medical treatment group showed 1 case with TIA and three with new cerebral infarction during follow-up after 3 months. The peak systolic velocity (PSV), end diastolic velocity (EDV), pulsatility index (PI) of stenosis vertebral artery, and PSV of basilar artery were significantly higher in the stent group than those in the standardized medical group (P < .05).Stenting for VAOS, rather than standardized medical treatment, can effectively relieve vascular stenosis, alter vertebral-basilar artery hemodynamics, and improve neurological function, with low perioperative complications.

Bortezomib-based chemotherapy to treat refractory angioimmunoblastic T-cell lymphoma: A case report and review of the literature.

The peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of aggressive neoplasms that account for <15% of all non-Hodgkin's lymphoma cases in adults. Angioimmunoblastic T-cell lymphoma (AITL) is a specific subtype of PTCL. The tumor is frequently aggressive and there is currently no general consensus regarding an effective treatment strategy. The present study reports a case in which bortezomib combined with dexamethasone was used to treat refractory AITL. A 63-year-old woman was admitted to Sir Run Run Shaw Hospital, Zhejiang University School of Medicine (Zhejiang, China) on August 17, 2013. The patient had been diagnosed with AITL for 4 months and had experienced a relapse of symptoms for the 4 days prior to admission. The patient demonstrated fever and dyspnea, accompanied by severe edema in the face and lower limbs, which later spread to the right upper limb. The patient was treated with bortezomib plus dexamethasone, which rapidly relieved the symptoms. The patient was subsequently administered an additional 2 cycles of bortezomib-based chemotherapy and survived for an additional 4 months, prior to succumbing to the disease. Only a small number of studies have reported the use of bortezomib in the treatment of T-cell lymphoma. The present study suggested that bortezomib-based treatment may be a reliable, safe and effective alternative for the treatment of relapsed/refractory PTCL. The efficacy of bortezomib as a treatment for PTCL requires additional evaluation in future studies.

DNA methylation in cystathionine-γ-lyase (CSE) gene promoter induced by ox-LDL in macrophages and in apoE knockout mice.

Recent studies suggest that epigenetic alterations such as DNA methylation control many aspects of monocytes/macrophages and participate in the pathogenesis of atherosclerosis, a lipid-driven inflammatory disorder. Our and other groups demonstrated that dysregulation of cystathionine γ-lyase (CSE) -hydrogen sulfide (H2S) pathway was involved in monocyte/macrophages-mediated inflammation and atherosclerosis. However, it remains unknown whether altered cse methylation in macrophages may play a role in linking CSE-H2S dysregulation and atherosclerosis. In the present study, we showed that plasma H2S and H2S production in the peritoneal macrophages of apolipoprotein knockout (apoE(-/-)) mice gradually decreased with ages, and were also lower than that in control mice at 12 weeks older. Moreover, CSE mRNA expressions decreased while DNA methyltransferase (DNMT) expressions increased in the peritoneal macrophages isolated from apoE(-/-) mice, compared to age-matched wildtype mice. Similar observations were obtained in an in vitro study. In oxidized low-density lipoprotein (ox-LDL)-treated raw264.7 macrophages, cse transcription was down-regulated while the expression and activity of DNMT was up-regulated, associated with enhanced DNA methylation in cse promoter. Suppression of DNMT with its inhibitor or siRNA reversed the decrease of CSE mRNA. Therefore, our data suggest that DNA hypermethylation of CpG rich region in cse promoter might contribute to the decrease of cse transcription and H2S production in macrophages, and thus contribute to atherosclerosis development.

Homocysteine Triggers Inflammatory Responses in Macrophages through Inhibiting CSE-H2S Signaling via DNA Hypermethylation of CSE Promoter.

Hyperhomocysteinemia (HHcy) is an independent risk factor of atherosclerosis and other cardiovascular diseases. Unfortunately, Hcy-lowering strategies were found to have limited effects in reducing cardiovascular events. The underlying mechanisms remain unclear. Increasing evidence reveals a role of inflammation in the pathogenesis of HHcy. Homocysteine (Hcy) is a precursor of hydrogen sulfide (H2S), which is formed via the transsulfuration pathway catalyzed by cystathionine ß-synthase and cystathionine γ-lyase (CSE) and serves as a novel modulator of inflammation. In the present study, we showed that methionine supplementation induced mild HHcy in mice, associated with the elevations of TNF-α and IL-1ß in the plasma and reductions of plasma H2S level and CSE expression in the peritoneal macrophages. H2S-releasing compound GYY4137 attenuated the increases of TNF-α and IL-1ß in the plasma of HHcy mice and Hcy-treated raw264.7 cells while CSE inhibitor PAG exacerbated it. Moreover, the in vitro study showed that Hcy inhibited CSE expression and H2S production in macrophages, accompanied by the increases of DNA methyltransferase (DNMT) expression and DNA hypermethylation in cse promoter region. DNMT inhibition or knockdown reversed the decrease of CSE transcription induced by Hcy in macrophages. In sum, our findings demonstrate that Hcy may trigger inflammation through inhibiting CSE-H2S signaling, associated with increased promoter DNA methylation and transcriptional repression of cse in macrophages.

Statins upregulate cystathionine γ-lyase transcription and H2S generation via activating Akt signaling in macrophage.

Hydrogen sulfide (H2S), the third gaseous transmitter, is implicated in various pathophysiologic processes. In the cardiovascular system, H2S exerts effects of cardioprotection, vascular tone regulation, and atherogenesis inhibition. Recent studies demonstrated that atorvastatin, the inhibitor of 3-hydroxyl-3-methyl coenzyme A reductase, affected H2S formation in kidney and other organs. However, the underlying mechanisms are not fully understood. In this study, we examined the effects of three different statins (fluvastatin, atorvastatin and pravastatin) on H2S formation in raw264.7 macrophages. There was a remarkable rise in H2S level in fluvastatin- and atorvastatin-stimulated macrophages, while pravastatin failed to show any significant effect on it. Moreover, fluvastatin and atorvastatin enhanced the mRNA and protein expression of cystathionine γ-lyase (CSE) in dose- and time-dependent manners. Fluvastatin also markedly enhanced the CSE activity. However, fluvastatin did not alter the mRNA or protein expression of another H2S-producing enzyme 3-mercaptopyruvate sulfurtransferase. Blockade of CSE with its inhibitor dl-propargylglycine (PAG) or siRNA markedly reduced the H2S level in fluvastatin-stimulated macrophages. In addition, fluvastatin elevated Akt phosphorylation, which occurred as early as 15 min after treatment, peaked at 1h, and lasted at least 3h. Both PI3K inhibitor LY294002 (10 µM) and Akt inhibitor perifosine (10µM) were able to reverse the increases of CSE mRNA and H2S production in fluvastatin-stimulated macrophages. Last, we showed that fluvastatin reduced the mRNA levels of pro-inflammatory molecules such as IL-1ß and MCP-1 in LPS-treated macrophages, which were completely reversed by CSE inhibitor PAG. Taken together, the findings demonstrate that statins may up-regulate CSE expression/activity and subsequently elevate H2S generation by activating Akt signaling pathway and also imply that CSE-H2S pathway plays a critical role in the anti-inflammation elicited by statins.

4-Chlorobenzoyl berbamine, a novel berbamine derivative, induces apoptosis in multiple myeloma cells through the IL-6 signal transduction pathway and increases FOXO3a-Bim expression.

Multiple myeloma (MM) is an incurable hematopoietic malignancy, although many novel therapeutic agents have been explored. In the present study, we showed that 4-chlorobenzoyl berbamine (BBD9), a novel derivative of berbamine, inhibited the growth of 4 MM cell lines (U266, RPMI 8226, MM1.R and MM1.S). After a 24-h treatment with BBD9, the half maximal inhibitory concentration (IC(50)) values were 1.8, 2.3, 1.5 and 2.4 µg/ml, respectively, using MTT assays. In BBD9-treated U266 and RPMI 8226 cells, Annexin V (AV)-propidium iodide (PI) staining and FACS analysis demonstrated that apoptosis was involved in this inhibition. This was confirmed by western blot analysis indicating activation and cleavage of caspase-3, -8, -9 and PARP. BBD9 also induced G2/M phase cell cycle arrest in these cells. To investigate the mechanisms responsible for BBD9-induced apoptosis, U266 cells were incubated with 0, 1 or 2 µg/ml of BBD9 combined with 0 or 150 ng/ml of interleukin (IL)-6. MTT assays showed that IL-6 partially abrogated the BBD9-induced cell growth inhibition. Furthermore, BBD9 inhibited autocrine IL-6 production, and downregulated membrane IL-6 receptor (IL-6R) expression. Crucial proteins downstream of the IL-6 signaling pathway, including AKT and STAT3, were inactivated in BBD9-treated U266 cells, although exogenous IL-6 did not abrogate this effect. Forkhead transcription factor class 3a (FOXO3a), a nuclear transcription factor downstream from AKT, was upregulated in the nuclei of BBD9-treated U266 cells. Bim, the target gene of FOXO3a, was upregulated at both the protein and mRNA levels, as shown by western blot analysis and quantitative PCR. These results suggest that BBD9 induces apoptosis in MM cells through the inhibition of the IL-6 signaling pathway, leading to FOXO3a activation and upregulation of pro-apoptotic Bim.

4-Chlorobenzoyl berbamine induces apoptosis and G2/M cell cycle arrest through the PI3K/Akt and NF-kappaB signal pathway in lymphoma cells.

Berbamine is an herbal compound derived from Berberis amurensis, which is used in Chinese traditional medicine. However, few studies have investigated this anti-tumor effect or the underlying mechanisms of berbamine on lymphoma cells. We investigate the effect, as well as the mechanism of action, of 4-chlorobenzoyl berbamine (BBD9) on Raji, L428, Namalwa and Jurkat lymphoma cells lines. Our findings show that BBD9 inhibits cell proliferation and induces cell apoptosis in lymphoma cell lines as well as G2/M cell cycle arrest through PI3K/Akt and NF-kappaB signaling pathways in a caspase-dependent manner. These results may provide new insights into the treatment of lymphoma.

Interleukin-6-independent expression of glucocorticoid receptor is upregulated by triptolide in multiple myeloma.

Glucocorticoids are widely used chemotherapeutic agents for multiple myeloma. Drug resistance to steroid therapies is associated with the downregulation or loss of glucocorticoid receptor expression in malignant plasma cells. In this study, we examined the constitutive expression of glucocorticoid receptor in dexamethasone-sensitive and dexamethasone-resistant multiple myeloma cell lines. We found that triptolide increased the amount of the phosphorylated glucocorticoid receptor and enhanced the growth inhibitory effect of dexamethasone. Notably, these effects could not be blocked by interleukin-6, one of the most important growth factors in multiple myeloma.

Casticin induces leukemic cell death through apoptosis and mitotic catastrophe.

Casticin, a component from Vitex rotundifolia, widely used as an anti-inflammatory agent in Chinese traditional medicine, was reported to have anti-tumor activities. This study aims to examine the anti-leukemic activity of casticin on leukemia cells and its molecular mechanism. Cell viability was measured by MTT method; apoptosis and cell cycle arrest were determined by flow cytometry, AV-PI assay, and DNA fragmentation assay. Western blot were performed to measure the protein expression level. The cell morphology alteration was detected with immunofluorescent analysis and DAPI nuclear staining. Our results showed that the proliferation of leukemia cells, including K562, Kasumi-1, and HL-60, were inhibited by casticin in a time- and dose-dependent manner. The IC50, determined after 48 h incubation, was 5.95 microM, 4.82 microM, and 15.56 microM for K562, HL-60, and Kasumi-1, respectively. The cell cycle analysis demonstrated casticin treatment resulted in a significant G2/M accumulation, concomitant with upregulation of P21waf1 and P27kip1. The percentage of cells in G2/M increased with time of exposure and reached to its climax (75.3%) at 12 h after casticin treatment, and subsequently declined to 27% at 48 h. We found that casticin treatment induced remarkable apoptosis, evidenced by increased percentage of AV-positive PI-negative cells as well as the cleavage of PARP and caspase 3. In addition, DNA fragmentation assay showed the typical apoptotic DNA ladder in casticin-treated K562 cells. Mitotic catastrophe and decreased polymeric tubulin can also be observed in casticin-treated K562 cells. In addition, we found that PI3K/AKT pathway was activated; Ly294002, a PI3K/AKT specific inhibitor, can enhance the anti-leukemic effect of casticin. Taken together, these results demonstrated that casticin induced leukemic cell death via apoptosis and mitotic catastrophe, and could synergize with PI3K/AKT inhibitor, suggesting that casticin could be a promising therapeutic agent against leukemia.

[Fluorescence in situ hybridization identifies complex chromosomal aberrations in multiple myeloma].

OBJECTIVE: To explore the value of the technique of multiplex fluorescence in sit hybridization (M-FISH) combined with interphase fluorescence in situ hybridization (FISH) in the identification of the chromosomal aberrations in multiple myeloma (MM) and to investigate the frequency of 13q14 deletion, IgH translocations and 17p13 deletion. METHODS: Seven MM patients with complex chromosomal abnormalities (CCAs) were analyzed by combining the technique of conventional cytogenetics (CC) with M-FISH and FISH. RESULTS: M-FISH identified the aberrations which were undetected by CC, including twelve kinds of numeral aberrations and twenty-nine kinds of structural aberrations, In addition, abnormalities of chromosome 1, chromosomes 13 deletion and IgH translocations were the most frequent aberrations. Using the LSI D13S319 probe specific for 13q14, we observed a deletion of 13q14 in 6 MM patients; using the LSI p53 probe specific for 17p13, we observed p53 deletion in 4 MM patients; using the LSI IGHC/IGHV probe specific for 14q32, we observed a translocation involving 14q32 in 5 MM patients (43.5%), two translocations in two cases (case 6 and 7). CONCLUSION: M-FISH combined with FISH could refine the cytogenetics of MM patients and detect the missed abnormalities or correct the misidentified abnormalities analyzed by CC. It provides an ideal method for the research of chromosomal aberrations in MM.