Anlotinib combined with temozolomide suppresses glioblastoma growth via mediation of JAK2/STAT3 signaling pathway

Purpose Anlotinib protects against carcinogenesis through the induction of autophagy and apoptosis. The current study evaluated the role and molecular mechanisms of anlotinib in glioblastoma, and the effects of anlotinib in combination with temozolomide (TMZ). Methods Cell Counting Kit-8 and colony-forming assays were used to evaluate cell viability. Cell migration and invasion were assessed by wound-healing, Transwell migration, and Matrigel invasion assays. Cellular apoptosis and cell cycle analysis were determined by flow cytometry. Angiogenesis was assessed using human umbilical vein endothelial cells (HUVECs). Vascular endothelial growth factor A (VEGFA) was measured by enzyme-linked immunosorbent assay. Protein expression was determined by western blotting or immunofluorescence staining. The in vivo anti-glioblastoma effect was assessed with live imaging of tumor xenografts in nude mice. Results Anlotinib restricted the proliferation, migration, and invasion of glioblastoma cells in a dose-dependent manner. Tumor supernatant from glioblastoma cells treated with anlotinib inhibited angiogenesis in HUVECs. Anlotinib induced autophagy in glioblastoma cells by increasing Beclin-1 and microtubule-associated protein 1 light chain 3B (LC3B) levels. Mechanistically, anlotinib inhibited the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3)/VEGFA signaling pathway. STAT3 inhibition by S3I-201 decreased VEGFA and suppressed cellular proliferation and movement. TMZ enhanced the anti-glioblastoma ability of anlotinib. Finally, anlotinib inhibited tumor growth and JAK2/STAT3/VEGFA signaling in xenografts. Conclusion Anlotinib exerts anti-glioblastoma activity possibly through the JAK2/STAT3/VEGFA signaling pathway. TMZ potentiated the anti-glioblastoma effect of anlotinib via the same signaling pathway, indicating the potential application of anlotinib as a treatment option for glioblastoma.

“Endogenous Melatonin Concentration Along with the Expression of Mitochondrial Regulator Genes is Elevated by the Serum Shock Process in the U87-MG Cell Line”

According to the World Health Organization, glioblastoma, also known as the fourth grade in the development of astrocytoma, is a glial tumor limited to the central nervous system with a strong ability to invade the brain parenchyma. Melatonin can be generated outside of the pineal gland tissue, according to new research. Melatonin is produced by mitochondria independently but in concert with cell demands, and it plays an important function in cell cycle and metabolism regulation. As a result, we set out to investigate the association between cell metabolism and the serum shock-induced increase in endogenous melatonin, as well as the percentage of cell proliferation.Background: Melatonin can be produced in the mitochondria organelle of glioblastoma cells without the involvement of the pineal gland, according to new research. Regarding the physiological function of melatonin secreted by the pineal gland in the regulation of rhythmicity, the goal of this study was to see if the glioblastoma cell's melatonin production ability could be influenced using a typical serum shock technique established for cellular rhythm regulator.Material and methods: First, U87-MG glioblastoma cells were cultured in a DMEM medium containing 10% FBS and then cells were treated with a standard serum shock process (no FBS, 8h). The concentration of melatonin was measured using ELISA method in supernatant and cell extracts of Shock and control groups. The cell proliferation was measured by using BrdU staining and flow cytometry assessment. The gene expression levels of some mitochondria or circadian related genes including TFAM, BMAL1, PPARGC1A(PGC1-α), and DNM1L(DRP1) were measured, using qRT-PCR method.Results: In comparison to the control group, serum shock treated U87-MG glioblastoma cells had higher concentrations of cellular and released endogenous melatonin (two times). At the mRNA level, we discovered considerable upregulation of mitochondrial or circadian regulator genes (TFAM, BMAL1, PPARGC1A, and DNM1L); in the shock group compared to the control group (P <0.0002). Furthermore, although the percentage of proliferative cells (Brdu positive) was higher in the shock group, it was not statistically significant.Conclusion: The serum shock procedure has a significant impact on the U87-MG cell line's cellular activity. In terms of the study's findings, it's worth noting that an increase in endogenous melatonin concentration influences several signaling pathways within the U87-MG cell line, as seen by the increased expression of candidate genes.In light of the findings of this study, it's worth noting that further research into the role of endogenous melatonin and its effects on cancer cells is critical, and that comparing the results of normal and cancer cells can reveal the hotspots of the signaling pathways involved, which could facilitate in better understanding the biology of glioblastoma.

RSL3 Drives Ferroptosis through NF-κB Pathway Activation and GPX4 Depletion in Glioblastoma

Glioblastoma, the most aggressive form of malignant glioma, is very difficult to treat because of its aggressively invasive nature and high recurrence rates. RAS-selective lethal 3 (RSL3), a well-known inhibitor of glutathione peroxidase 4 (GPX4), could effectively induce oxidative cell death in glioblastoma cells through ferroptosis, and several signaling pathways are involved in this process. However, the role of the nuclear factor kappa-B (NF-κB) pathway in glioblastoma cell ferroptosis has not yet been investigated. Therefore, we aimed to clarify the underlying mechanism of the NF-κB pathway in RSL3-induced ferroptosis in glioblastoma cells. We found that RSL3 led to an increase in lipid ROS concentration and downregulation of ferroptosis-related proteins such as GPX4, ATF4, and SLC7A11 (xCT) in glioblastoma cells. Additionally, the NF-κB pathway was activated by RSL3, and its inhibition by BAY 11-7082 could alleviate ferroptosis. The murine xenograft tumor model indicated that NF-κB pathway inhibition could mitigate the antitumor effects of RSL3 in vivo. Furthermore, we found that GPX4 knockdown could not effectively induce ferroptosis. However, NF-κB pathway activation coupled with GPX4 silencing induced ferroptosis. Additionally, ATF4 and xCT expression might be regulated by the NF-κB pathway. Collectively, our results revealed that the NF-κB pathway plays a novel role in RSL3-induced ferroptosis in glioblastoma cells and provides a new therapeutic strategy for glioblastoma treatment.

AaTs-1: A Tetrapeptide from Androctonus australis Scorpion Venom, Inhibiting U87 Glioblastoma Cells Proliferation by p53 and FPRL-1 Up-Regulations

Glioblastoma is an aggressive cancer, against which medical professionals are still quite helpless, due to its resistance to current treatments. Scorpion toxins have been proposed as a promising alternative for the development of effective targeted glioblastoma therapy and diagnostic. However, the exploitation of the long peptides could present disadvantages. In this work, we identified and synthetized AaTs-1, the first tetrapeptide from Androctonus australis scorpion venom (Aa), which exhibited an antiproliferative effect specifically against human glioblastoma cells. Both the native and synthetic AaTs-1 were endowed with the same inhibiting effect on the proliferation of U87 cells with an IC50 of 0.56 mM. Interestingly, AaTs-1 was about two times more active than the anti-glioblastoma conventional chemotherapeutic drug, temozolomide (TMZ), and enhanced its efficacy on U87 cells. AaTs-1 showed a significant similarity with the synthetic peptide WKYMVm, an agonist of a G-coupled formyl-peptide receptor, FPRL-1, known to be involved in the proliferation of glioma cells. Interestingly, the tetrapeptide triggered the dephosphorylation of ERK, p38, and JNK kinases. It also enhanced the expression of p53 and FPRL-1, likely leading to the inhibition of the store operated calcium entry. Overall, our work uncovered AaTs-1 as a first natural potential FPRL-1 antagonist, which could be proposed as a promising target to develop new generation of innovative molecules used alone or in combination with TMZ to improve glioblastoma treatment response. Its chemical synthesis in non-limiting quantity represents a valuable advantage to design and develop low-cost active analogues to treat glioblastoma cancer.

Brusatol Inhibits Proliferation and Invasion of Glioblastoma by Down-Regulating the Expression of ECM1

Brusatol (Bru), a Chinese herbal extract, has a variety of anti-tumor effects. However, little is known regarding its role and underlying mechanism in glioblastoma cells. Here, we found that Bru could inhibit the proliferation of glioblastoma cells in vivo and in vitro. Besides, it also had an inhibitory effect on human primary glioblastoma cells. RNA-seq analysis indicated that Bru possibly achieved these effects through inhibiting the expression of extracellular matrix protein 1 (ECM1). Down-regulating the expression of ECM1 via transfecting siRNA could weaken the proliferation and invasion of glioblastoma cells and promote the inhibitory effect of Bru treatment. Lentivirus-mediated overexpression of ECM1 could effectively reverse this weakening effect. Our findings indicated that Bru could inhibit the proliferation and invasion of glioblastoma cells by suppressing the expression of ECM1, and Bru might be a novel effective anticancer drug for glioblastoma cells.