Anlotinib Inhibiting Mantle Cell Lymphoma Proliferation and Inducing Apoptosis through PI3K/AKT/mTOR Pathway.

BACKGROUND: This study investigates the inhibitory mechanism of anlotinib on human Mantle Cell Lymphoma (MCL) cells through in vitro and in vivo experiments. METHODS: In vitro cellular experiments validate the effects of anlotinib on MCL cell proliferation and apoptosis. Moreover, a subcutaneous xenograft nude mice model of Mino MCL cells was established to assess the anti-tumour effect and tumour microenvironment regulation of anlotinib in vivo. RESULTS: The results indicate that MCL cell proliferation was significantly inhibited upon anlotinib exposure. The alterations in the expression of apoptosis-related proteins further confirm that anlotinib can induce apoptosis in MCL cells. Additionally, anlotinib significantly reduced the PI3K/Akt/mTOR phosphorylation level in MCL cells. The administration of a PI3K phosphorylation agonist, 740YP, could reverse the inhibitory effect of anlotinib on MCL. In the xenograft mouse model using Mino MCL cells, anlotinib treatment led to a gradual reduction in body weight and a significant increase in survival time compared to the control group. Additionally, anlotinib attenuated PD-1 expression and elevated inflammatory factors, CD4, and CD8 levels in tumour tissues. CONCLUSION: Anlotinib effectively inhibits proliferation and induces apoptosis in MCL both in vitro and in vivo. This inhibition is likely linked to suppressing phosphorylation in the PI3K/Akt/mTOR pathway.

GLYR1 transcriptionally regulates PER3 expression to promote the proliferation and migration of multiple myeloma.

Period circadian regulator 3 (PER3) functions as a tumor suppressor in various cancers. However, the role of PER3 in multiple myeloma (MM) has not been reported yet. Through this study, we aimed to investigate the potential role of PER3 in MM and the underlying mechanisms. RT-qPCR and western blotting were used to determine the mRNA and protein expression levels of PER3. Glyoxylate reductase 1 homolog (GLYR1) was predicted to be a transcription factor of PER3. The binding sites of GLYR1 on the promoter region of PER3 were analyzed using UCSC and confirmed using luciferase and chromatin immunoprecipitation assays. Viability, apoptosis, and metathesis were determined using CCK-8, colony formation, TUNEL, and transwell assays. We found that PER3 expression decreased in MM. Low PER3 levels may predict poor survival rates; PER3 overexpression suppresses the viability and migration of MM cells and promotes apoptosis. Moreover, GLYR1 transcriptionally activates PER3, and the knockdown of PER3 alleviates the effects of GLYR1 and induces its malignant behavior in MM cells. To conclude, GLYR1 upregulates PER3 and suppresses the aggressive behavior of MM cells, suggesting that GLYR1/PER3 signaling may be a potential therapeutic target for MM.

CALCRL knockdown suppresses cancer stemness and chemoresistance in acute myeloid leukemia with FLT3-ITD and DNM3TA-R882 double mutations.

Acute myeloid leukemia (AML) patients with FLT3 internal tandem duplication (FLT3-ITD) and DNA methyltransferase 3A (DNMT3A) R882 double mutations had a worse prognosis compared with AML with FLT3-ITD or DNMT3A R882 single mutation. This study was designed to explore the specific role of Calcitonin Receptor Like (CALCRL) in AML with FLT3-ITD and DNMT3A R882 double mutations. MOLM13 cells were transduced with CRISPR knockout sgRNA constructs to establish the FTL3-ITD and DNMT3A-R882 double-mutated AML cell model. Quantitative real-time PCR and Western blot assay were carried out to examine corresponding gene and protein expression. Methylation of CALCRL promoter was measured by methylation-specific PCR (MSP). Cell viability, colony formation, flow cytometry, and sphere formation assays were conducted to determine cell proliferation, apoptosis, and stemness. MOLM13 cells were exposed to stepwise increasing concentrations of cytarabine (Ara-C) to generate MOLM13/Ara-C cells. An in vivo AML  animal model was established, and the tumor volume and weight were recorded. TUNEL assay was adopted to examine cell apoptosis in tumor tissues. DNMT3A-R882 mutation upregulated the expression of CALCRL while downregulated the DNA methylation level of CALCRL in MOLM13 cells. CALCRL knockdown greatly inhibited cell proliferation, promoted apoptosis and repressed cell stemness, accompanied with the downregulated Oct4, SOX2, and Nanog in DNMT3A-R882-mutated MOLM13 cells and MOLM13/Ara-C cells. Furthermore, CALCRL knockdown restricted tumor growth and the chemoresistance of AML in vivo, as well as inducing cell apoptosis in tumor tissues. Together, these data reveal that CALCRL is a vital regulator of leukemia cell survival and resistance to chemotherapy, suggesting CALCRL as a promising therapeutic target for the treatment of FTL3-ITD and DNMT3A-R882 double-mutated AML.

A Novel Inflammatory-Nutritional Prognostic Scoring System for Patients with Diffuse Large B Cell Lymphoma.

Purpose: This study aimed to examine the predictive ability of inflammatory and nutritional markers and further establish a novel inflammatory nutritional prognostic scoring (INPS) system. Patients and Methods: We collected clinicopathological and baseline laboratory data of 352 patients with DLBCL between April 2010 and January 2023 at the First affiliated hospital of Ningbo University. Eligible patients were randomly divided into training and validation cohorts (n = 281 and 71, respectively) in an 8:2 ratio. We used the least absolute shrinkage and selection operator (LASSO) Cox regression model to determine the most important factors among the eight inflammatory-nutritional variables. The impact of INPS on OS was evaluated using the Kaplan-Meier curve and the Log rank test. A prognostic nomogram was developed based on the multivariate Cox regression method. Then, we used the concordance index (C-index), calibration plot, and time-dependent receiver operating characteristic (ROC) analysis to evaluate the prognostic performance and predictive accuracy of the nomogram. Results: Seven inflammatory-nutritional biomarkers, including neutrophil-lymphocyte ratio (NLR), prognostic nutritional index (PNI), body mass index (BMI), monocyte-lymphocyte ratio (MLR), prealbumin, C reactive protein, and D-dimer were selected using the LASSO Cox analysis to construct INPS, In the multivariate analysis, IPI-High-intermediate group, IPI-High group, high INPS were independently associated with OS, respectively. The prognostic nomogram for overall survival consisting of the above two indicators showed excellent discrimination. The C-index for the nomogram was 0.94 and 0.95 in the training and validation cohorts. The time-dependent ROC curves showed that the predictive accuracy of the nomogram for OS was better than that of the NCCN-IPI system. Conclusion: The INPS based on seven inflammatory-nutritional indexes was a reliable and convenient predictor of outcomes in DLBCL patients.

CALCRL induces resistance to daunorubicin in acute myeloid leukemia cells through upregulation of XRCC5/TYK2/JAK1 pathway.

Chemotherapy is the main treatment option for acute myeloid leukemia (AML), but acquired resistance of leukemic cells to chemotherapeutic agents often leads to difficulties in AML treatment and disease relapse. High calcitonin receptor-like (CALCRL) expression is closely associated with poorer prognosis in AML patients. Therefore, this study was performed by performing CALCRL overexpression constructs in AML cell lines HL-60 and Molm-13 with low CALCRL expression. The results showed that overexpression of CALCRL in HL-60 and Molm-13 could confer resistance properties to AML cells and reduce the DNA damage and cell cycle G0/G1 phase blocking effects caused by daunorubicin (DNR) and others. Overexpression of CALCRL also reduced DNR-induced apoptosis. Mechanistically, the Cancer Clinical Research Database analyzed a significant positive correlation between XRCC5 and CALCRL in AML patients. Therefore, the combination of RT-PCR and Western blot studies further confirmed that the expression levels of XRCC5 and PDK1 genes and proteins were significantly upregulated after overexpression of CALCRL. In contrast, the phosphorylation levels of AKT/PKCε protein, a downstream pathway of XRCC5/PDK1, were significantly upregulated. In the response study, transfection of overexpressed CALCRL cells with XRCC5 siRNA significantly upregulated the drug sensitivity of AML to DNR. The expression levels of PDK1 protein and AKT/PKCε phosphorylated protein in the downstream pathway were inhibited considerably, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were upregulated. Animal experiments showed that the inhibitory effect of DNR on the growth of HL-60 cells and the number of bone marrow invasions were significantly reversed after overexpression of CALCRL in nude mice. However, infection of XCRR5 shRNA lentivirus in HL-60 cells with CALCRL overexpression attenuated the effect of CALCRL overexpression and upregulated the expression of apoptosis-related proteins induced by DNR. This study provides a preliminary explanation for the relationship between high CALCRL expression and poor prognosis of chemotherapy in AML patients. It offers a more experimental basis for DNR combined with molecular targets for precise treatment in subsequent studies.

Silencing of PD-1 combined with EBV-specific killer T cells for the treatment of EBV-associated B lymphoma.

Epstein-Barr Virus (EBV) infection is closely associated with the development of lymphoma, as it plays a significant role in the malignant transformation of lymphocytes. The expression of programmed death-1 (PD-1), which binds to PD-L1 in tumor cells, can lead to immune evasion by lymphoma cells and promote tumor progression. In this study, immortalized B lymphoblastoid cell lines (B-LCLs) positive for EBV-specific proteins were established from human peripheral mononuclear cells (PBMCs) using EBV induction along with CpG-ODN 2006 and cyclosporin A. EBV-specific T cells (EBVST) were generated by multiple immunizations of CD3+ T lymphocytes using irradiated B-LCLs. Flow cytometry analysis confirmed the activation of EBVST through the detection of CD3+, CD4+, and CD8+ markers. Co-incubation of EBVST with EBV-positive B lymphocyte cell lines resulted in the secretion of perforin by EBVST, leading to granzyme B-mediated cell death and an increase in LDH levels. Silencing PD-1 in EBVST cells enhanced perforin production, increased granzyme B release, and upregulated cell death in co-incubated B lymphocytes. In a nude mice tumor transplantation model, silencing PD-1 in combination with EBV-specific killer T cells exhibited the maximum inhibition of B-lymphoblastoma. This treatment upregulated the expression of proteins associated with apoptosis and immune response, while inhibiting anti-apoptotic protein expression in tumor tissues. Silencing PD-1 also increased the infiltration of EBV-specific killer T cells in the tumor tissues. Overall, PD-1 silencing enhanced the tumor targeting effect of EBV-specific killer T cells on EBV-infected B lymphocytes and attenuated the immune escape effect mediated by the PD-1 pathway.

The Differential Expression of CD47 may be Related to the Pathogenesis From Myelodysplastic Syndromes to Acute Myeloid Leukemia.

Myelodysplastic syndrome (MDS) can lead to the development of peripheral blood cytopenia and abnormal cell morphology. MDS has the potential to evolve into AML and can lead to reduced survival. CD47, a member of the immunoglobulin family, is one molecule that is overexpressed in a variety of cancer cells and is associated with clinical features and poor prognosis in a variety of malignancies. In this study, we analyzed the expression and function of CD47 in MDS and AML, and further analyzed its role in other tumors. Our analysis revealed significantly low CD47 expression in MDS and significantly high expression in AML. Further analysis of the function or pathway of CD47 from different perspectives identified a relationship to the immune response, cell growth, and other related functions or pathways. The relationship between CD47 and other tumors was analyzed from four aspects: DNA methyltransferase, TMB, MSI, and tumor cell stemness. Changes in gene expression levels have a known association with aberrant DNA methylation, and this methylation is the main mechanism of tumor suppressor gene silencing and clonal variation during the evolution of MDS to AML. Taken together, our findings support the hypothesis that the differential expression of CD47 might be related to the transformation of MDS to AML.

Effects of physical activity on clinical and inflammatory markers in diagnosing multiple myeloma patients.

Multiple myeloma (MM) is the second most common hematological disorder. Although several drugs have been developed to treat MM, their efficacy is uncertain. In addition, how normal physical activities can decrease inflammatory responses and clinical biomarkers in MM patients needs to be better defined. Therefore, this study evaluated possible clinical and inflammatory markers to determine the early diagnosis of MM during physical activity. This study selected 30 MM patients with normal or no physical activity with ages of >50 years. This study did not require any specific exercise protocols other than noting patients' daily life activities and considering them as physical activity for 17 days. Then, blood samples were collected to assess clinical and inflammatory markers. Regarding clinical markers, daily life activities in MM patients decreased their LDH, calcium, and ß2-microglobulin levels significantly compared to other clinical biomarkers such as creatine and total protein. Further, this study observed no significant differences between daily life activities of MM patients and normal MM patients regarding levels of immunoglobulins except IgM. Furthermore, IL-6 level was significantly increased with the daily life activities of MM patients, suggesting the role of physical activities in increasing anti-inflammatory response along with altering the biochemical profiles including LDH, calcium and ß2-microglobulin in MM patients.

GPX3 methylation is associated with hematologic improvement in low-risk myelodysplastic syndrome patients treated with Pai-Neng-Da.

OBJECTIVE: The aim of this prospective randomized controlled clinical trial was to explore the relationship between GPX3 methylation and Pai-Neng-Da (PND) in the treatment of patients with low-risk myelodysplastic syndrome (MDS). METHODS: There were 82 low-risk MDS patients who were randomly divided into the following two groups: androl, thalidomide, and PND capsule (ATP group, n = 41); or androl and thalidomide (AT group, n = 41). Hemoglobin and neutrophil and platelet counts and changes in GPX3 methylation level were assessed. RESULTS: The plasma hemoglobin level increased in both groups after treatment. However, the platelet count increased only in the ATP group. Patients in the ATP group had a better platelet response than the AT group, and GPX3 methylation markedly decreased after treatment with ATP but not after treatment with AT. Moreover, male patients had a significantly lower GPX3 methylation level than female patients, while platelet counts from male patients increased dramatically after the ATP regimens compared with female patients. GPX3 methylation changes were negatively correlated with platelet changes in ATP group. CONCLUSION: PND can improve hematological parameters and decrease the GPX3 methylation level. Decreasing GPX3 methylation is associated with the hematologic response that includes platelet in GPX3 methylation.China Clinical Trial Bureau (ChiCTR; http://www.chictr.org.cn/) registration number: ChiCTR-IOR-15006635.

Cytotoxicity of Donor Natural Killer Cells to Allo-Reactive T Cells Are Related With Acute Graft-vs.-Host-Disease Following Allogeneic Stem Cell Transplantation.

Objectives: The mechanism and immunoregulatory role of human natural killer (NK) cells in acute graft-vs.-host-disease (aGVHD) remains unclear. This study quantitatively analyzed the cytotoxicity of donor NK cells toward allo-reactive T cells, and investigated their relationship with acute GVHD (aGVHD). Methods: We evaluated NK dose, subgroup, and receptor expression in allografts from 98 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). A CD107a degranulating assay was used as a quantitative detection method for the cytotoxic function of donor NK cells to allo-reactive T cells. In antibody-blocking assay, NK cells were pre-treated with anti-DNAM-1(CD226), anti-NKG2D, anti-NKP46, or anti-NKG-2A monoclonal antibodies (mAbs) before the degranulating assay. Results: NK cells in allografts effectively inhibited auto-T cell proliferation following alloantigen stimulation, selectively killing alloantigen activated T cells. NKG2A- NK cell subgroups showed higher levels of CD107a degranulation toward activated T cells, when compared with NKG2A- subgroups. Blocking NKG2D or CD226 (DNAM-1) led to significant reductions in degranulation, whereas NKG2A block resulted in increased NK degranulation. Donor NK cells in the aGVHD group expressed lower levels of NKG2D and CD226, higher levels of NKG2A, and showed higher CD107a degranulation levels when compared with NK cells in the non-aGVHD group. Using univariate analysis, higher NK degranulation activities in allografts (CD107ahigh) were correlated with a decreased risk in grade I-IV aGVHD (hazard risk [HR] = 0.294; P < 0.0001), grade III-IV aGVHD (HR = 0.102; P < 0.0001), and relapse (HR = 0.157; P = 0.015), and improved overall survival (HR = 0.355; P = 0.028) after allo-HSCT. Multivariate analyses showed that higher NK degranulation activities (CD107ahigh) in allografts were independent risk factors for grades, I-IV aGVHD (HR = 0.357; P = 0.002), and grades III-IV aGVHD (HR = 0.13; P = 0.009). Conclusions: These findings reveal that the degranulation activity of NK in allografts toward allo-activated T cells was associated with the occurrence and the severity of aGVHD, after allogeneic stem cell transplantation. This suggested that cytotoxicity of donor NK cells to allo-reactive T cells have important roles in aGVHD regulation.

Clinically useful flow cytometry approach to identify immunophenotype in acute leukemia.

OBJECTIVES: Acute leukemia (AL) is a highly heterogeneous malignant disease caused by hematopoietic cell abnormalities. Our study investigated the potential for immunophenotyping of leukemic cells via flow cytometry and the clinical usefulness of this approach in treatment of AL. METHODS: Bone marrow (BM) specimens were collected to detect antigen expression on hematopoietic cells in pre-treatment samples from patients with AL. In addition, fraction survival curves were calculated using the Kaplan-Meier method to explore the effect of markers on prognosis in AL. RESULTS: Expression levels of immunophenotypic markers in patients with acute lymphoblastic leukemia (ALL) were significantly different from those in patients with acute myeloid leukemia (AML). In addition, there was a potential association between the surface marker, cluster of differentiation 2 (CD2), and fraction survival in AML. However, no similar result was found in ALL. Moreover, genetic tests showed greater positive variation of the break point cluster-Abelson tyrosine kinase ( BCR-ABL) fusion gene in samples from patients with ALL than in samples from patients with AML. CONCLUSIONS: We have shown a rapid and effective flow cytometry method that enables the identification of immunophenotype in AL. Moreover, CD2 may constitute a predictive marker for prognosis in patients with AML.

Graphene/h-BN/GaAs sandwich diode as solar cell and photodetector.

In graphene/semiconductor heterojunction, the statistic charge transfer between graphene and semiconductor leads to decreased junction barrier height and limits the Fermi level tuning effect in graphene, which greatly affects the final performance of the device. In this work, we have designed a sandwich diode for solar cells and photodetectors through inserting 2D hexagonal boron nitride (h-BN) into graphene/GaAs heterostructure to suppress the static charge transfer. The barrier height of graphene/GaAs heterojunction can be increased from 0.88 eV to 1.02 eV by inserting h-BN. Based on the enhanced Fermi level tuning effect with interface h-BN, through adopting photo-induced doping into the device, power conversion efficiency (PCE) of 10.18% has been achieved for graphene/h-BN/GaAs compared with 8.63% of graphene/GaAs structure. The performance of graphene/h-BN/GaAs based photodetector is also improved with on/off ratio increased by one magnitude compared with graphene/GaAs structure.

Interface designed MoS2/GaAs heterostructure solar cell with sandwich stacked hexagonal boron nitride.

MoS2 is a layered two-dimensional semiconductor with a direct band gap of 1.8 eV. The MoS2/bulk semiconductor system offers a new platform for solar cell device design. Different from the conventional bulk p-n junctions, in the MoS2/bulk semiconductor heterostructure, static charge transfer shifts the Fermi level of MoS2 toward that of bulk semiconductor, lowering the barrier height of the formed junction. Herein, we introduce hexagonal boron nitride (h-BN) into MoS2/GaAs heterostructure to suppress the static charge transfer, and the obtained MoS2/h-BN/GaAs solar cell exhibits an improved power conversion efficiency of 5.42%. More importantly, the sandwiched h-BN makes the Fermi level tuning of MoS2 more effective. By employing chemical doping and electrical gating into the solar cell device, PCE of 9.03% is achieved, which is the highest among all the reported monolayer transition metal dichalcogenide based solar cells.

Supporting Effect of Umbilical Cord Blood-Derived Mesenchymal Stem Cells on CD34+ Cell Proliferation and Its Mechanism.

OBJECTIVE: To investigate the ability of UCB-derived MSCs to support the expansion of HSCs ex vivo and the possible mechanisms involved in this process. METHODS: HSCs from UCB were co-cultured with UCB-derived MSCs for 14 days, and then the total number of HSCs and colony-forming units (CFU) were detected. Cytokines levels of MSCs supernatant were analyzed using ELISA. RESULTS: The proliferation rate of HSCs co-cultured with MSCs was significantly higher than that of cultured HSCs alone (P<0.05). Furthermore, the addition of exogenous cytokines to the culture system significantly increased the proliferation rate of HSCs (P<0.05). MSCs had secretion of many cytokines, including GM-CSF, IL-7, IL-8, IL-11, SCF and SDF-1α. CONCLUSION: UCB-derived MSCs as a feeder layer can be an alternative approach for ex vivo expansion of HSCs, and the cytokines by secreted UCB-MSCs may mediate the supportive role of MSC to HSC proliferation.

[Effect of umbilical cord blood mesenchymal stem cells on peripheral blood lymphocyte subsets].

OBJECTIVE: This study was aimed to investigate the effect of mesenchymal stem cells (MSCs) on subsets and cytokine secretion of T lymphocytes. METHODS: Umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) were isolated by density gradient and were cultared by amplifying culture. The subsets and cytokine secretion of T lymphocytes were detected by flow cytomety after being co-cultured with UCBMSC. RESULTS: The proliferation of lymphocytes was inhibited. CD4(+)T cell subsets were increased, CD8(+)T cell subsets decreased when co-cultured with UCBMSC; Th1 and Tc1 level significantly reduced, while Th2 and Tc2 level slightly increased. CONCLUSION: The UCBMSC can inhibit the proliferation of lymphocytes, especially CD8(+)T cell subsets. In addition, UCBMSCs can reduce Th1 and Tc1 cells, and increase Th2 and Tc2 cells. UCBMSC may have the clinical application potential for preventing and remedying GVHD.

[Umbilical cord blood-derived mesenchymal stem cells inhibit proliferation of peripheral blood lymphocytes].

OBJECTIVE: To study the effect of umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) on the proliferation of adult peripheral blood lymphocytes. METHODS: MSCs were isolated and amplified from human UCB and identified by detecting the surface markers and the ability of differentiation into osteoblasts and adipocytes. Lymphocytes were stimulated with phytohemagglutinin (PHA) and co-cultured with UCBMSCs, or cultured with UCBMSCs in non-contact Transwell(TM) system. Two culture systems were both divided into negative control group (lymphocytes were cultured alone), positive control group (lymphocytes were stimulated with PHA), and experimental group (lymphocytes were cultured with UCBMSCs and stimulated with PHA). The proliferation of lymphocytes was examined by MTS assay, and the inhibition rate was calculated. RESULTS: In both culture systems, UCBMSCs could inhibit the proliferation of peripheral blood lymphocytes in a dose-dependent manner (P<0.05). But when the proportion of UCBMSCs to lymphocytes of the two systems was the same, the inhibitory effect of co-culture system was significantly stronger than that of Transwell(TM) system (P<0.05). CONCLUSION: UCBMSCs can inhibit the proliferation of peripheral blood lymphocytes, and the inhibitory effect of co-culture system is stronger than that of Transwell(TM) system.