Induced Apoptosis In Glioma Cells Research Articles

NPS-2143 inhibit glioma progression by suppressing autophagy through mediating AKT-mTOR pathway.

Gliomas are the most common tumours in the central nervous system. In the present study, we aimed to find a promising anti-glioma compound and investigate the underlying molecular mechanism. Glioma cells were subjected to the 50 candidate compounds at a final concentration of 10 μM for 72 h, and CCK-8 was used to evaluate their cytotoxicity. NPS-2143, an antagonist of calcium-sensing receptor (CASR), was selected for further study due to its potent cytotoxicity to glioma cells. Our results showed that NPS-2143 could inhibit the proliferation of glioma cells and induce G1 phase cell cycle arrest. Meanwhile, NPS-2143 could induce glioma cell apoptosis by increasing the caspase-3/6/9 activity. NPS-2143 impaired the immigration and invasion ability of glioma cells by regulating the epithelial-mesenchymal transition process. Mechanically, NPS-2143 could inhibit autophagy by mediating the AKT-mTOR pathway. Bioinformatic analysis showed that the prognosis of glioma patients with low expression of CASR mRNA was better than those with high expression of CASR mRNA. Gene set enrichment analysis showed that CASR was associated with cell adhesion molecules and lysosomes in glioma. The nude mice xenograft model showed NPS-2143 could suppress glioma growth invivo. In conclusion, NPS-2143 can suppress the glioma progression by inhibiting autophagy.

Daurisoline suppress glioma progression by inhibiting autophagy through PI3K/AKT/mTOR pathway and increases TMZ sensitivity

Glioma is one of the most common primary malignant tumors of the central nervous system. Temozolomide (TMZ) is the only effective chemotherapeutic agent, but it easily develops resistance and has unsatisfactory efficacy. Consequently, there is an urgent need to develop safe and effective compounds for glioma treatment. The cytotoxicity of 30 candidate compounds to glioma cells was detected by the CCK-8 assay. Daurisoline (DAS) was selected for further investigation due to its potent anti-glioma effects. Our study revealed that DAS induced glioma cell apoptosis through increasing caspase-3/6/9 activity. DAS significantly inhibited the proliferation of glioma cells by inducing G1-phase cell cycle arrest. Meanwhile, DAS remarkably suppressed the migration and invasion of glioma cells by regulating epithelial-mesenchymal transition. Mechanistically, our results revealed that DAS impaired the autophagic flux of glioma cells at a late stage by mediating the PI3K/AKT/mTOR pathway. DAS could inhibit TMZ-induced autophagy and then significantly promote TMZ chemosensitivity. Nude mice xenograft model revealed that DAS could restrain glioma proliferation and promote TMZ chemosensitivity. Thus, DAS is a potential anti-glioma drug that can improve glioma sensitivity to TMZ and provide a new therapeutic strategy for glioma in chemoresistance.

Characterization of Prognostic Apoptosis-Related Gene Signature to Evaluate Glioma Immune Microenvironment and Experimental Verification.

Purpose: Recently, apoptosis-related genes were shown to modulate cancer immunity. However, the role of apoptosis-related genes in the glioma immune microenvironment (GIME) remains unknown. This study aimed to explore the prognostic value of apoptosis-related genes in glioma. Methods: Doxorubicin was used to induce glioma cell apoptosis, and four differentially expressed apoptosis-related genes were identified: CREM, TNFSF12, PEA15, and PRKCD. Kaplan-Meier analyses, receiver operating characteristic curve analyses, and nomograms were established to determine the relationship between risk markers and the prognosis of patients with glioma. Results: Risk biomarkers were significantly associated with overall survival, immune cell infiltration, and immune checkpoints in patients with glioma. Somatic mutations and anti-PD-1/L1 immunotherapy were associated with worse prognosis in the high-risk group receiving anti-PD-1/L1 therapy. The expression of these four apoptosis-related genes was verified using quantitative polymerase chain reaction and immunohistochemistry, and the relationship between these four genes and apoptosis was examined using flow cytometry. Conclusions: This study suggests that apoptosis-related genes play a critical role in shaping the GIME. Assessing the apoptotic patterns of individual tumors will enhance our understanding of GIME infiltration features and lead to improved strategies for immunotherapy.

Effects of STAT3 Inhibitor BP-1-102 on The Proliferation, Invasiveness, Apoptosis and Neurosphere Formation of Glioma Cells in Vitro.

Malignant glioma, especially glioblastoma (GBM), has historically been associated with a low survival rate. The hyperactivation of STAT3 played a key role in GBM initiation and resistance to therapy; thus, there is an urgent requirement for novel STAT3 inhibitors. BP-1-102 was recently reported as a biochemical inhibitor of STAT3, but its roles and mechanism in biological behavior of glioma cells were still unclear. In this study, the effects of BP-1-102 on proliferation, apoptosis, invasion and neurosphere formation of glioma cell were investigated. Our results indicated that BP-1-102 inhibited the proliferation of U251 and A172 cells, and their IC50 values were 10.51 and 8.534 μM, respectively. Furthermore, BP-1-102 inhibited the invasion and migration abilities of U251 and A172 cells by decreasing the expression of matrix metallopeptidase 9, and induced glioma cell apoptosis by decreasing the expression of B-cell lymphoma-2. BP-1-102 also inhibited the formation of neurosphere. Mechanically, BP-1-102 reduced the phosphorylation of STAT3 and the p-STAT3's nuclear translocation in glioma cells. Thus, this study herein provided a potential drug for glioma therapy.

Suppression of RPL34 Inhibits Tumor Cell Proliferation and Promotes Apoptosis in Glioblastoma.

Accumulating evidence indicates Ribosomal protein 34 (RPL34) promotes tumor malignance and its expression is associated with poor prognosis in multiple cancer cells. However, the physiological role and biological mechanism of RPL34 in glioblastoma (GBM) remain unclear. Hence, this study aimed to investigate the expression and the role of RPL34 in GBM. A total of 59 glioma samples and 12 normal brains for epilepsy surgery were used to determine the underlying mechanisms and the biological behaviors of RPL34 in GBM. In this study, we identified that RPL34 expression was significantly (p < 0.05) enriched in GBM tumors compared with low-grade glioma and normal brain, and its expression was associated with poor survival. Additionally, RPL34 was functionally required for tumor proliferation in vitro. Mechanically, inhibition of RPL34 induced glioma cell apoptosis by activation of Bad/Caspase7/PARP signaling pathway. The RPL34 promotes cell survival in GBM and could be a potential therapeutic target for GBM.

WZY-321 triggers glioma cell apoptosis via XAF1 up-regulation caused by MTM-mediated miR-873 down-regulation.

Gliomas account for the majority of primary malignant brain tumors around the world and are highly aggressive. Evodiamine is one of the main effective components of Evodia rutaecarpa, which can inhibit proliferation and promote apoptosis of tumor cells including glioma cells. The derivative of Evodiamine named WZY-321 was successfully developed, and exhibited significant cytotoxicity and could efficiently induce glioma cell apoptosis; however, the mechanism of WZY-321-induced glioma cell apoptosis is not clear. Our current studies showed that WZY-321 increased X-linked inhibitor of apoptosis-associated factor 1 (XAF1) expression in glioma cells, and up-regulated XAF1 resulted in glioma cell apoptosis. Moreover, WZY-321 treatment decreased miR-873 expression and increased lncRNA MTM expression in glioma cells, and down-regulated miR-873 or up-regulated MTM lead to glioma cell apoptosis. Mechanically, WZY-321 up-regulated XAF1 gene expression via MTM-decreased miR-873 expression, that bound to XAF1 3' UTR and decreased XAF1 mRNA levels. Taken together, these data indicate that WZY-321 triggers glioma cell apoptosis via XAF1 up-regulation caused by MTM-mediated miR-873 down-regulation.

Clavipyrrine A, a unique polycyclic nitrogenous meroterpenoid with promising anti-glioma effects isolated from the fungus Clitocybe clavipes

Clavipyrrine A (1), a novel polycyclic nitrogenous meroterpenoid with a pyrrolo[1,2-a]imidazole and a 10-membered carbocycle fused with an α,β-epoxy-γ-lactone, was isolated from Clitocybe clavipes, a basidiomycete. X-ray crystallography and spectroscopic analysis were used to fully elucidate its structure. The biosynthetic origin of the pyrrole unit in this nitrogenous meroterpenoid was identified by incorporating 15N-labeled γ-aminobutyric acid. Compound 1 displayed promising anti-glioma activities and induced glioma cell apoptosis through inhibiting the JAK/STAT3 pathway and reinforcing SOCS1/3.

KDELR2 knockdown synergizes with temozolomide to induce glioma cell apoptosis through the CHOP and JNK/p38 pathways.

BackgroundThe C-terminal tetrapeptide Lys-Asp-Glu-Leu receptors (KDELRs) are transmembrane proteins that regulate ER stress (ERS) response, growth, differentiation, and immune responses. There is an association between KDELR2and promotion of glioblastoma tumorigenesis. The aim of the present study was to explore the functional mechanism of KDELR2 in glioma and during response to chemotherapy to temozolomide (TMZ).MethodsThe expression of KDELR2 in glioma tissues and cells was evaluated by immunohistochemistry, western blot and RT-qPCR assay. Then role of KDELR2 was demonstrated by CCK8, colony formation, flow cytometry and Hochest 33258 assays. The expression of genes (ATF4, ATF6, PERK, eIF2-α, GRP78 and CHOP) in U373 cells was evaluated by RT-qPCR. The protein expression of genes (cleaved caspase 3, caspase 3, cleaved PARP, PARP, Bax, Bcl-2, JNK, p-JNK, p38, p-p38, ATF4, ATF6, XBP-1s, PERK, p-PERK, GRP78 and CHOP) was measured by western blot assay.ResultsThe expression of KDELR2 was upregulated in high-grade gliomas tissues. KDELR2 knockdown suppressed cell proliferation but increased cell apoptosis. Further, Knockdown of KDELR2 also activated the ER stress (ERS)-dependent CHOP pathway, and resulted in increased levels of phosphorylated c-Jun N-terminal kinase (JNK) and p38. Moreover, the combination of KDELR2 knockdown and TMZ application showed a synergistic cytotoxic effect in U373 cells through the ERS-dependent CHOP and JNK/p38 pathways.ConclusionsKDELR2 knockdown induces apoptosis and sensitizes glioma cells to TMZ, which is mediated by the ERS-dependent CHOP and JNK/p38 pathways.

Pterostilbene promotes mitochondrial apoptosis and inhibits proliferation in glioma cells

Glioma is the most general primary and lethal intracranial malignant tumor. Pterostilbene (PTE), an analog of stilbene and resveratrol, has attracted attention in recent years due to its significant antitumor activity in multiple solid tumors; however, its effect on drug-resistant glioma cells and the underlying mechanism have not yet been reported. In this study, we found that pterostilbene inhibited proliferation, induced intrinsic mitochondria-mediated apoptosis and caused S phase arrest, inhibited migration and excessive invasion in glioma cells. Pretreatment with the pan-caspase-inhibitor Z-VAD-FMK attenuated the PTE-induced apoptosis of glioma cells. Moreover, PTE significantly increased the production of reactive oxygen species (ROS) and reduce the mitochondrial membrane potential (MMP). Inhibition of ROS with N-acetyl-l-cysteine not only rescued PTE-induced reduction of cellular viability but also prevented glioma cell apoptosis. We also discovered ERK 1/2 and JNK signaling pathways were activated by PTE and contributed to induce glioma cell apoptosis. In addition, specific inhibitors of ERK 1/2 and JNK attenuated PTE-induced apoptosis. Besides, PTE significantly reduced tumor volume and prolonged median survival of tumor-bearing rats in vivo. In summary, the results of this study indicate that the anti-tumor effect of PTE on glioma cells may provide a new treatment option for glioma patients.

Corilagin induces apoptosis and autophagy in NRF2‑addicted U251 glioma cell line

Corilagin, extracted from the Euphorbiaceae and Phyllanthus plants, inhibits the growth of a number of types of tumors. Compared with temozolomide, the traditional chemotherapy drug, corilagin has demonstrated stronger antitumor activity. However, the pharmaceutical mechanism of corilagin in glioma remains unclear. Nuclear factor erythroid 2 like 2 (NFE2L2 or NRF2) is positively associated with several types of tumor including glioma. In the present study, NRF2 expression was higher in glioma tissues compared with non-glioma specimens. Therefore, it was hypothesized that corilagin targets NRF2 regulation of U251 cell apoptosis. The present study used Hoechst 33258 staining to demonstrate that corilagin induced glioma cell apoptosis and observed that the expression of the apoptosis-related gene Bcl-2 was reduced. In addition, corilagin induced autophagy and promoted the conversion of light chain 3 (LC3) protein from LC3I to LC3II. NRF2 expression was downregulated by corilagin stimulation. Furthermore, the gene expression pattern following knockdown of NRF2 in U251 cells using siRNA was consistent with corilagin stimulation. Therefore, it was preliminarily concluded that corilagin induces apoptosis and autophagy by reducing NRF2 expression.

Aspirin Induced Glioma Apoptosis through Noxa Upregulation.

Clinically, high cyclooxygenase-2 expression in malignant glioma correlates well with poor prognosis and the use of aspirin is associated with a reduced risk of glioma. To extend the current understanding of the apoptotic potential of aspirin in most cell types, this study provides evidence showing that aspirin induced glioma cell apoptosis and inhibited tumor growth, in vitro and in vivo. We found that the human H4 glioma cell-killing effects of aspirin involved mitochondria-mediated apoptosis accompanied by endoplasmic reticulum (ER) stress, Noxa upregulation, Mcl-1 downregulation, Bax mitochondrial distribution and oligomerization, and caspase 3/caspase 8/caspase 9 activation. Genetic silencing of Noxa or Bax attenuated aspirin-induced viability loss and apoptosis, while silencing Mcl-1 augmented the effects of aspirin. Data from genetic and pharmacological studies revealed that the axis of ER stress comprised an apoptotic cascade leading to Noxa upregulation and apoptosis. The apoptotic programs and mediators triggered by aspirin in H4 cells were duplicated in human U87 glioma cell line as well as in tumor-bearing BALB/c nude mice. The involvement of ER stress in indomethacin-induced Mcl-1 downregulation was reported in our previous study on glioma cells. Therefore, the aforementioned phenomena indicate that ER stress may be a valuable target for intervention in glioma apoptosis.

Lcn2-derived Circular RNA (hsa_circ_0088732) Inhibits Cell Apoptosis and Promotes EMT in Glioma via the miR-661/RAB3D Axis.

Background: Glioma is the most common malignant tumor of the central nervous system, and often displays invasive growth. Recently, circular RNA (circRNA), which is a novel non-coding type of RNA, has been shown to play a vital role in glioma tumorigenesis. However, the functions and mechanism of lipocalin-2 (Lcn2)-derived circular RNA (hsa_circ_0088732) in glioma progression remain unclear.Methods: We evaluated hsa_circ_0088732 expression by fluorescence in situ hybridization (FISH), Sanger sequencing, and PCR assays. Cell apoptosis was evaluated by flow cytometry and Hoechst 33258 staining. Transwell migration and invasion assays were performed to measure cell metastasis and viability. In addition, the target miRNA of hsa_circ_0088732 and the target gene of miR-661 were predicted by a bioinformatics analysis, and the interactions were verified by dual-luciferase reporter assays. RAB3D expression was analyzed by an immunochemistry assay, and E-cadherin, N-cadherin, and vimentin protein expression were examined by western blot assays. A mouse xenograft model was developed and used to analyze the effects of hsa_circ_0088732 on glioma growth in vivo.Results: We verified that hsa_circ_0088732 is circular and highly expressed in glioma tissues. Knockdown of hsa_circ_0088732 induced glioma cell apoptosis and inhibited glioma cell migration, invasion, and epithelial-mesenchymal transition (EMT). We found that hsa_circ_0088732 negatively regulated miR-661 by targeting miR-661, and RAB3D was a target gene of miR-661. In addition, inhibition of miR-661 promoted glioma cell metastasis and suppressed cell apoptosis. Knockdown of RAB3D induced cell apoptosis and suppressed cell metastasis. Moreover, hsa_circ_0088732 accelerated glioma progression through its effects on the miR-661/RAB3D axis. Finally, results from a mouse xenograft model confirmed that knockdown of hsa_circ_0088732 induced miR-661 expression, resulting in suppression of RAB3D expression and inhibition of tumor growth in vivo.Conclusion: We demonstrated that hsa_circ_0088732 facilitated glioma progression by sponging miR-661 to increase RAB3D expression. This study provides a theoretical basis for understanding the development and occurrence of glioma, as well as for the development of targeted drugs.

Interferon-β sensitizes human glioblastoma cells to the cyclin-dependent kinase inhibitor, TG02.

Novel treatments for glioblastoma, the most common malignant primary brain tumor, are urgently required. Type I interferons (IFN) are natural cytokines primarily involved in the defense against viral infections, which may also serve a role in the control of cancer, notably in the suppression of the cancer stem cell phenotype. TG02 is a novel orally available cyclin-dependent kinase 9 inhibitor which induces glioma cell apoptosis without profound caspase activation, which is currently explored in early clinical trials in newly diagnosed and recurrent glioblastoma. In the present study, human glioma-initiating cell line models were used to explore whether IFN-β modulates the anti-glioma activity of TG02. The present study employed immunoblotting to assess protein levels, several viability assays and gene silencing strategies to assess gene function. Pre-exposure to IFN-β sensitized human glioma models to a subsequent exposure to TG02. Combination treatment was associated with increased DEVD-amc cleaving caspase activity that was blocked by the anti-apoptotic protein, BCL2. However, BCL2 did not protect from the synergistic effects of IFN and TG02 on glioma cell growth. Furthermore, although IFN strongly induced pro-apoptotic XIAP-associated factor (XAF) expression, disrupting XAF expression did not abrogate the synergy with TG02. Consistent with that, caspase 3 gene silencing did not abrogate the effects of TG02 or IFN-β alone or in combination. Finally, it was observed that IFN-β may indeed modulate the effects of TG02 upstream in the signaling cascade since inhibition of RNA polymerase II phosphorylation, a direct readout of the pharmacodynamic activity of TG02, was facilitated when glioma cells were pre-exposed to IFN-β. In summary, these data suggest that type I IFN may be combined with TG02 to limit glioblastoma growth, but that the well characterized effects of IFN and TG02 on apoptotic signaling are dispensable for synergistic tumor growth inhibition. Instead, exploring how IFN signaling primes glioma cells for TG02-mediated direct target inhibition may help to design novel and effective pharmacological approaches to glioblastoma.

DDIS-04. INTERFERON-β SENSITIZES HUMAN GLIOBLASTOMA CELLS TO THE CYCLIN-DEPENDENT KINASE INHIBITOR, TG02

Abstract Novel treatments for glioblastoma, the most common malignant primary brain tumor, are urgently needed. Type I interferons (IFN) are natural cytokines primarily involved in defense against viral infections, but may also play a role in the control of cancer, notably in the suppression of the cancer stem cell phenotype. TG02 is a novel orally available cyclin-dependent kinase 9 inhibitor which induces glioma cell apoptosis without profound caspase activation and is currently being explored in early clinical trials in newly diagnosed and recurrent glioblastoma. Here we used human glioma-initiating cell line models to explore whether IFN-β modulate the anti-glioma activity of TG02. Pre-exposure to IFN-β sensitized human glioma models to a variable extent to subsequent exposure to TG02. Combination treatment was associated with increased DEVD-amc cleaving activity that was blocked by BCL2. However, BCL2 did not protect from the synergistic effects of IFN and TG02 on glioma cell growth, furthermore, although IFN strongly induced pro-apoptotic XIAP-associated factor (XAF) expression, disrupting XAF expression did not abrogate the synergy with TG02 either. Consistent with that, caspase 3 gene silencing did not abrogate effects of TG02 or IFN-β alone or in combination. Finally, we observed that IFN-β may indeed modulate the effects of TG02 up-stream in the signaling cascade since inhibition of RNA polymerase II phosphorylation, a direct readout of TG02 pharmacodynamic activity, was facilitated in glioma cells pre-exposed to IFN-β. In summary, these data suggest that type I IFN may be combined with TG02 to limit glioblastoma growth but the well characterized effects of IFN and TG02 on apoptotic signaling are dispensable for synergistic control of tumor growth. Instead, exploring how IFN signaling primes glioma cells for TG02-mediated direct target inhibition may help to design novel, more effective pharmacological approaches to glioblastoma.

Xanthatin induces glioma cell apoptosis and inhibits tumor growth via activating endoplasmic reticulum stress-dependent CHOP pathway.

Xanthatin is a natural sesquiterpene lactone purified from Xanthium strumarium L., which has shown prominent antitumor activity against a variety of cancer cells. In the current study, we investigated the effect of xanthatin on the growth of glioma cells in vitro and in vivo, and elucidated the underlying mechanisms. In both rat glioma C6 and human glioma U251 cell lines, xanthatin (1–15 μM) dose-dependently inhibited cell viability without apparent effect on the cell cycle. Furthermore, xanthatin treatment dose-dependently induced glioma cell apoptosis. In nude mice bearing C6 glioma tumor xenografts, administration of xanthatin (10, 20, 40 mg·kg−1·d−1, ip, for 2 weeks) dose-dependently inhibited the tumor growth, but did not affect the body weight. More importantly, xanthatin treatment markedly increased the expression levels of the endoplasmic reticulum (ER) stress-related markers in both the glioma cell lines as well as in C6 xenografts, including glucose-regulated protein 78, C/EBP-homologous protein (CHOP), activating factor 4, activating transcription factor 6, spliced X-box binding protein-1, phosphorylated protein kinase R-like endoplasmic reticulum kinase, and phosphorylated eukaryotic initiation factor 2a. Pretreatment of C6 glioma cells with the ER stress inhibitor 4-phenylbutyric acid (4-PBA, 7 mM) or knockdown of CHOP using small interfering RNA significantly attenuated xanthatin-induced cell apoptosis and increase of proapoptotic caspase-3. These results demonstrate that xanthatin induces glioma cell apoptosis and inhibits tumor growth via activating the ER stress-related unfolded protein response pathway involving CHOP induction. Xanthatin may serve as a promising agent in the treatment of human glioma.

TRIM44 is indispensable for glioma cell proliferation and cell cycle progression through AKT/p21/p27 signaling pathway.

Glioma is one of the lethal cancers which needs effective therapeutic target. TRIM44 has been found playing a carcinogenic role in human tumors such as breast cancer and ovarian cancer. However, the pathophysiological significance of TRIM44 in glioma is still unclear. Quantitative-PCR and western blot were used to assess the expression of TRIM44 in glioma cells. For cell proliferation, Brdu incorporation and colony formation assays were performed. By Caspase 3 staining and FACS analysis, we revealed that TRIM44 knockdown induced glioma cell apoptosis. A BALB/c nude mouse xenograft model and following immunohistochemical (IHC) staining enables us to explore the effect of TRIM44 deletion on glioma growth in vivo. Western blot of p21, p27 and AKT indicated the possible role of TRIM44 in regulation AKT pathway in glioma. TRIM44 was significantly elevated in glioma cells, and high expression of TRIM44 is related to poor prognostic of glioma patients. TRIM44 knockdown by shRNAs inhibit glioma cell proliferation, migration, induced cell cycle disruption and further cellular apoptosis in vitro. As well, TRIM44 inactivation obviously inhibit tumor growth in xenograft model. Furthermore, the negative cell cycle regulators p21/p27 are significantly upregulated, while AKT which is known as the main regulator of p21/p27 is inactivated in TRIM44-dificient cells. These results suggested that TRIM44 inactivation disrupted cell cycle progression and inhibit cell proliferation through AKT/p21/p27 pathway in glioma. TRIM44 was associated with oncogenic potential of glioma. Targeting TRIM44 might be beneficial for glioma therapy.

Mechanisms of the mixed umbilical cord blood-derived mesenchymal stem cells in inhibiting proliferation and inducing apoptosis of glioma cells

Objective To investigate the possible mechanism of mixed umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) in vitro to inhibit proliferation and induce apoptosis of C6 glioma cells. Methods The expression of B cell lymphoma/leukemia-2 (bcl-2) and cysteinyl aspartate-specific protease (Caspase)-3 proteins in C6 cells was detected by immunohistochemistry and Western blotting imprinting method, and C6 cell cycle was detected by flow cytometry. Results Different sources of UCB-MSCs have a tendency to increase the expression of apoptosis protein Caspase-3 in C6 cells and reduce the expression of apoptosis protein bcl-2. Different sources UCB-MSCs induce C6 cell apoptosis, E/T=0∶1, 5∶1, (2.5+ 2.5)∶1, 10∶1, (5+ 5)∶1 C6 cells acting on, The percentages of G0/G1 were (65.49±1.83)%, (64.83±2.19)%, (70.30±3.21)%, (69.03±2.19)%, (77.63±3.10)%, and the percentage of C6 cells in G0/G1 increased with the increase of the E/T ratio, showing a proportional dependence. Conclusion After phytohemagglutinin (PHA) stimulation, large doses of UCB-MSCs and mixed UCB-MSCs have inhibitory effects on proliferation of C6 cells in vitro, and this inhibition may be related to apoptosis induction Key words: Mesenchymal stem cells; Induce; Apoptosis

Nano hydroxyapatite induces glioma cell apoptosis by suppressing NF-κB signaling pathway.

Nano-sized hydroxyapatite (nHA) particles have been demonstrated to exert anti-cancer effects on multiple cancer cell lines and animal models of cancer biology. However, the molecular mechanism underlying the effects of nHA particles on glioma cells remains unclear. The present study aimed to examine the effects of nHA on the behavior of glioma cells and investigate its underlying molecular mechanism. Rat glioma C6 cells and human glioma U87MG ATCC cells were exposed to nHA (20–100 µg/ml), and its effects on cell morphology, viability, apoptosis, cell cycle, invasion and nuclear factor (NF)-κB signaling were analyzed. Exposure of C6 and U87MG ATCC cells to 20 µg/ml nHA for 24 h caused cell detachment. Viability of C6 and U87MG ATCC cells were significantly reduced by nHA in a dose-dependent manner (P<0.05). Nuclear staining with Hoechst 33258 exhibited clear chromatin condensation in C6 cells following 24 h exposure to ≥25 µg/ml nHA. Flow cytometry revealed that nHA (20–100 µg/ml) significantly induced apoptosis and cell cycle G2/M arrest in C6 and U87MG ATCC cells (P<0.05). Transwell invasion assay demonstrated that nHA (20–60 µg/ml) significantly inhibited invasion of U87MG ATCC cells (P<0.05). Furthermore, western blotting and confocal immunofluorescence microscopy revealed that nHA (20–100 µg/ml) decreased NF-κB p65 protein expression and blocked NF-κB p65 nuclear translocation in C6 cells. The protein expression of NF-κB target molecules, such as B cell lymphoma 2, cyclooxygenase-2 and survivin, were also significantly reduced by nHA in a dose-dependent manner in both C6 and U87MG ATCC cells (P<0.05). In conclusion, it was demonstrated that the inhibitory effect of nHA on glioma cells is likely associated with the downregulation of NF-κB signaling.

Rnd3 induces apoptosis of glioma cells through promoting the ubiquitination of p65

Objective Explore the role and mechanism of Rnd3 during the process of apoptosis in glioma cells. Methods Rnd3 plasmid was transfected to regulate the expression of Rnd3 in U251 glioma cells, Myc plasmid was transfected as control. Flow cytometry was used to detect apoptosis. We detected the expression of Rnd3, p65, B cell lymphoma/leukemia-2 (bcl-2) and cleaved-Caspase-3 in glioma cells by Western blotting. Flag-p65 plasmid was used to restore p65 expression, then detected the expression of bcl-2 and cleaved-Caspase-3 by Western blotting, Flag plasmid was transfected as control. MG-132 was applied to inhibit the ubiquitination of protein after Rnd3 plasmid transfected, then detected the expression of p65 by Western blotting. Results After transfection with eukaryotic expression plasmid, the expression of Rnd3 protein in Myc-Rnd3 group was significantly higher than that in Myc group (P<0.05); the apoptotic rate of Myc-Rnd3 group increased from 7.28% (5.36±0.10, 1.92±0.12) in U251-Myc group to 19.93% (13.51±0.32, 6.42±0.21) in U251-Myc-Rnd3 group, and the apoptotic rate was significantly higher than that in Myc group (P<0.05); compared with U251-Myc group, the expression of p65 protein in U251-Myc-Rnd3 group decreased, and the expression of Rnd3 in U251-Myc group decreased. The expression of p65 was down-regulated, which resulted in the down-regulation of bcl-2 and the up-regulation of cleaved-Caspase-3 (P<0.05). After reversing the function of p65 protein (p65+ , Rnd3-), the expression of p65 was increased (P<0.05), the expression of bcl-2 was increased (P<0.05), the expression of cleaved-Caspase-3 was decreased (P<0.05); (p65+ , Rnd3+ ) the expression of p65+ , Rnd3-) was higher than that of (p65+ , Rnd3-). The expression of p65-, Rnd3-) was higher than that of (p65-, Rnd3-) Rnd3 (P<0.05), p65 expression was lower (P<0.05), bcl-2 expression was lower (P<0.05), cleaved-Caspase-3 expression was higher (P<0.05), and MG-132 inhibited ubiquitination pathway of intracellular protein, which showed that the expression of p65 decreased with the increase of Rnd3 protein expression. In MG-132 treatment group, with the increase of Rnd3 protein expression, there was no significant difference in p65 protein expression. Conclusion Rnd3 induces apoptosis of U251 glioma cells may through promoting the ubiquitination of p65. Key words: Rnd3; p65; Glioma; Ubiquitination

Luteolin suppresses tumor proliferation through inducing apoptosis and autophagy via MAPK activation in glioma.

PurposeGlioma is a malignant tumor that originates in the brain and spine and is difficult to be completely removed. Though glioma patients receive active treatment, the survival rate is still poor. Therefore, it is urgent to discover a new medicine to treat glioma patients in order to improve the survival rate. In this study, we explored the anticancer effect and the potential mechanism of luteolin on glioma in vitro.Materials and methodsCell viability was determined by Cell Counting Kit-8 (CCK-8) assay. Fluorescent microscopy and flow cytometry analysis were used to determine the cellular apoptosis. Western blot analysis was performed to explore the changes in protein expression. Quantitative reverse transcription-PCR (qRT-PCR) analysis was utilized to evaluate the expression level of the tumor suppressor miR-124-3p.ResultsCCK-8 assays indicated that luteolin significantly inhibited glioma cell proliferation in a time- and dose-dependent manner. Fluorescent microscopy and flow cytometry analysis confirmed that luteolin induced glioma cell apoptosis. Western blot analysis showed that luteolin induced cellular apoptosis in glioma cells via MAPK activation (JNK, ERK, and p38). Luteolin stimulated the death receptor (FADD) to regulate the apoptosis proteins (Caspase-8, Caspase-3, and PARP). Luteolin increased the expression levels of LC3B II/I and downregulated the level of p62 that promotes cell autophagy. Finally, qRT-PCR confirmed that luteolin upregulated the expression levels of miR-124-3p.ConclusionThese findings illustrate that luteolin may be a potential drug for glioma treatment.