Cannabidiol Induces Apoptosis and Perturbs Mitochondrial Function in Human and Canine Glioma Cells

Cannabidiol (CBD), the major non-psychoactive compound found in cannabis, is frequently used both as a nutraceutical and therapeutic. Despite anecdotal evidence as an anticancer agent, little is known about the effect CBD has on cancer cells. Given the intractability and poor prognoses of brain cancers in human and veterinary medicine, we sought to characterize the in vitro cytotoxicity of CBD on human and canine gliomas. Glioma cells treated with CBD showed a range of cytotoxicity from 4.9 to 8.2 μg/ml; canine cells appeared to be more sensitive than human. Treatment with >5 μg/ml CBD invariably produced large cytosolic vesicles. The mode of cell death was then interrogated using pharmacologic inhibitors. Inhibition of apoptosis was sufficient to rescue CBD-mediated cytotoxicity. Inhibition of RIPK3, a classical necroptosis kinase, also rescued cells from death and prevented the formation of the large cytosolic vesicles. Next, cellular mitochondrial activity in the presence of CBD was assessed and within 2 hours of treatment CBD reduced oxygen consumption in a dose dependent manner with almost complete ablation of activity at 10 μg/ml CBD. Fluorescent imaging with a mitochondrial-specific dye revealed that the large cytosolic vesicles were, in fact, swollen mitochondria. Lastly, calcium channels were pharmacologically inhibited and the effect on cell death was determined. Inhibition of mitochondrial channel VDAC1, but not the TRPV1 channel, rescued cells from CBD-mediated cytotoxicity. These results demonstrate the cytotoxic nature of CBD in human and canine glioma cells and suggest a mechanism of action involving dysregulation of calcium homeostasis and mitochondrial activity.

Signaling Pathway and Transcriptional Regulation in Osteoblasts during Bone Healing: Direct Involvement of Hydroxyapatite as a Biomaterial

Bone defects and periodontal disease are pathological conditions that may become neglected diseases if not treated properly. Hydroxyapatite (HA), along with tricalcium phosphate and bioglass ceramic, is a biomaterial widely applied to orthopedic and dental uses. The in vivo performance of HA is determined by the interaction between HA particles with bone cells, particularly the bone mineralizing cells osteoblasts. It has been reported that HA-induced osteoblastic differentiation by increasing the expression of osteogenic transcription factors. However, the pathway involved and the events that occur in the cell membrane have not been well understood and remain controversial. Advances in gene editing and the discovery of pharmacologic inhibitors assist researchers to better understand osteoblastic differentiation. This review summarizes the involvement of extracellular signal-regulated kinase (ERK), p38, Wnt, and bone morphogenetic protein 2 (BMP2) in osteoblastic cellular regulation induced by HA. These advances enhance the current understanding of the molecular mechanism of HA as a biomaterial. Moreover, they provide a better strategy for the design of HA to be utilized in bone engineering.

WNT Ligand Dependencies in Pancreatic Cancer

WNT signaling promotes the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) through wide-ranging effects on cellular proliferation, survival, differentiation, stemness, and tumor microenvironment. Of therapeutic interest is a genetically defined subset of PDAC known to have increased WNT/β-catenin transcriptional activity, growth dependency on WNT ligand signaling, and response to pharmacologic inhibitors of the WNT pathway. Here we review mechanisms underlying WNT ligand addiction in pancreatic tumorigenesis, as well as the potential utility of therapeutic approaches that functionally antagonize WNT ligand secretion or frizzled receptor binding.

Phosphatidic Acid Stimulates Myoblast Proliferation through Interaction with LPA1 and LPA2 Receptors

Phosphatidic acid (PA) is a bioactive phospholipid capable of regulating key biological functions, including neutrophil respiratory burst, chemotaxis, or cell growth and differentiation. However, the mechanisms whereby PA exerts these actions are not completely understood. In this work, we show that PA stimulates myoblast proliferation, as determined by measuring the incorporation of [3H]thymidine into DNA and by staining the cells with crystal violet. PA induced the rapid phosphorylation of Akt and ERK1/2, and pretreatment of the cells with specific small interferin RNA (siRNA) to silence the genes encoding these kinases, or with selective pharmacologic inhibitors, blocked PA-stimulated myoblast proliferation. The mitogenic effects of PA were abolished by the preincubation of the myoblasts with pertussis toxin, a Gi protein inhibitor, suggesting the implication of Gi protein-coupled receptors in this action. Although some of the effects of PA have been associated with its possible conversion to lysoPA (LPA), treatment of the myoblasts with PA for up to 60 min did not produce any significant amount of LPA in these cells. Of interest, pharmacological blockade of the LPA receptors 1 and 2, or specific siRNA to silence the genes encoding these receptors, abolished PA-stimulated myoblast proliferation. Moreover, PA was able to compete with LPA for binding to LPA receptors, suggesting that PA can act as a ligand of LPA receptors. It can be concluded that PA stimulates myoblast proliferation through interaction with LPA1 and LPA2 receptors and the subsequent activation of the PI3K/Akt and MEK/ERK1-2 pathways, independently of LPA formation.

Luks-PV Induce HOXA9 Degradation Through Autophagy in MLL-rearranged Acute Myeloid Leukemia

Background: Our previous studies have demonstrated that Luks-PV have good anti-leukemia ability effects and could possibly be a promising therapy for adult acute myeloid leukemia (AML). Aberrant over-expression of HOXA9 is a prominent feature of AML driven by multiple oncogenes, and therapeutic degrading of HOXA9 may be an effective treatment strategy in AML. This paper focused on the Luks-PV-regulating autophagy pathway, aiming to investigate the role of Luks-PV in mixed-lineage leukemia (MLL)-rearranged AML. Methods: The data of leukemia patients were downloaded from the gene expression profiling of TCGA datasets. Taking primary AML and THP-1 cells as the model system in vitro, Luks-PV-inhibited cell proliferation was determined by CCK‐8 and flow cytometry assays. The role of Luks-PV in autophagy regulation was analyzed using immunoblotting, transfection and immunofluorescence.Results: HOXA9 was over-expressed and associated with a poor prognosis in AML patients bearing MLL rearrangement. After the application of pharmacologic inhibitors of autophagy, Luks-PV induced cytotoxic autophagy in AML cells, as suggested by biochemical and microscopy results. HOXA9 molecules were detectable within autophagosomes after Luks-PV treatment, indicating that autophagy induction accounted for the degradation of HOXA9. Moreover, Luks-PV-induced HOXA9 downregulation inhibited AML cell proliferation, suggesting that HOXA9 could be degraded through Luks-PV-induced autophagy.Conclusion: Luks-PV suppresses AML cell proliferation by inducing HOXA9 degradation.

Targeting the CD73-adenosine axis in COVID-19 immunotherapy

The most serious health issue today is the rapid outbreak of Coronavirus Disease 2019 (COVID-19 or SARS-CoV-2). It is now a global pandemic and is a huge concern for public health. So far, more than 3,500,000 confirmed cases were diagnosed in nearly 212 countries and territories around the world and 2 international conveyances, causing globally over 250,000 deaths. Epidemiology, risk factors, and clinical characteristics of COVID-19 patients have been identified but the factors influencing the immune system against COVID-19 have not been well established. Currently, the adenosine pathway is seen as a major obstacle to the efficacy of immune therapies and is an important therapeutic target for cancer and microbial infections. Pharmacologic inhibitors or antibodies specific to adenosine pathway components or adenosine receptors in microbial and tumor therapy have shown efficacy in pre-clinical studies and are entering the clinical arena. In this review, I propose a novel hypothesis explaining the potential for improving the efficiency of innate and adaptive immune systems by co-inhibition of CD73 and A2AR adenosine Signaling for COVID-19 prevention and control.

Heme induces autophagic cell death via ER stress in neuron

Aims Intracerebral hemorrhage (ICH) is serious medical problem and the effective treatment is limited. Hemorrhaged blood is highly toxic to the brain, and heme mainly released from hemoglobin plays a vital role in neurotoxicity. However, the specific mechanism involved in heme mediated neurotoxicity has not been well studied.Methods In this study, we investigated the neurotoxicity of heme in neurons. Neurons were administrated with heme, and the cell death, autophagy and ER stress were analyzed. In addition, the relationship between autophagy and apoptosis in heme-induced cell death and the downstream effects were also detected.Results We showed that heme induced cell death and autophagy in neurons. The suppression of autophagy using either pharmacologic inhibitors (3-methyladenine) or RNA interference in essential autophagy genes (BECN1 and ATG5) decreased the cell death induced by heme in neurons. Moreover, ER stress activator thapsigargin increased the cell autophagy and cell death ratio following heme treatment. Autophagy promotes cell apoptosis and cell death induced by heme through BECN1/ ATG5 pathway.Conclusions Our findings suggest that heme potentiates neuron autophagy via ER stress, in turn inducing cell death via BECN1/ATG5 pathway. Targeting ER stress mediated autophagy might be a promising therapeutic strategy for ICH.

Effect of UBE2Q on BECN and CEA Protein in Colorectal Cancer Cells

Background: Expression of UBE2Q1, UBE2Q2, and members of the ubiquitinconjugating enzyme family (E2) are affected in colorectal cancer (CRC). The BECN gene plays a key role in CRC cells. In gastrointestinal carcinoma therapy, tumorassociated antigens such as CEA are typically used.To investigate the association between UBE2Q1 and Beclin1 autophagy marker and CEA protein expression in LS180 CRC cell line. Materials and Methods: In this study, changes in the expression of BECN marker in LS180 cell lines with the vector containing UBE2Q1 were investigated using real-time PCR. The expression of CEA protein was also evaluated by western blotting. Statistical analyses were performed with Graph Pad Prism software. Results: The results indicated reduced expression of BECN autophagy marker (P=0). Therefore, in the presence of UBE2Q1, cancer cells have less ability to induce autophagy. However, CEA protein levels in LS180 transfected cells with a UBE2Q1- ORF-containing plasmid decreased when compared to non-transfected cells. Conclusion: The use of pharmacologic inhibitors related to the autophagy mechanism can be a novel approach in cancer therapy.

Nitric Oxide Antagonism to Anti-Glioblastoma Photodynamic Therapy: Mitigation by Inhibitors of Nitric Oxide Generation

Many studies have shown that low flux nitric oxide (NO) produced by inducible NO synthase (iNOS/NOS2) in various tumors, including glioblastomas, can promote angiogenesis, cell proliferation, and migration/invasion. Minimally invasive, site-specific photodynamic therapy (PDT) is a highly promising anti-glioblastoma modality. Recent research in the authors’ laboratory has revealed that iNOS-derived NO in glioblastoma cells elicits resistance to 5-aminolevulinic acid (ALA)-based PDT, and moreover endows PDT-surviving cells with greater proliferation and migration/invasion aggressiveness. In this contribution, we discuss iNOS/NO antagonism to glioblastoma PDT and how this can be overcome by judicious use of pharmacologic inhibitors of iNOS activity or transcription.

MTHFD1 is a genetic interactor of BRD4 and links folate metabolism to transcriptional regulation

The histone acetyl-reader BRD4 is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1. We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression, and pharmacologic inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin-associated suggests a direct role for nuclear metabolism in the control of gene expression.