Marine-derived antimicrobial peptide piscidin-1 triggers extrinsic and intrinsic apoptosis in oral squamous cell carcinoma through reactive oxygen species production and inhibits angiogenesis.

Cancer of the head and neck encompasses a wide range of cancers, including oral and oropharyngeal cancers. Oral cancer is often diagnosed at advanced stages and has a dismal prognosis. Piscidin-1, a marine antimicrobial peptide (AMP) containing approximately 22 amino acids, also exhibits significant anticancer properties. We investigated the possible anti-oral cancer effects of piscidin-1 and clarified the mechanisms underlying these effects. We treated the oral squamous cell carcinoma cell lines OC2 and SCC4 with piscidin-1. Cell viability and the expression of different hallmark apoptotic molecules, including reactive oxygen species (ROS), were tested using the appropriate MTT assay, flow cytometry and western blotting assays, and human umbilical vein endothelial cell (HUVEC) wound healing, migration, and tube formation (angiogenesis) assays. Piscidin-1 increases cleaved caspase 3 levels to induce apoptosis. Piscidin-1 also increases ROS levels and intensifies oxidative stress in the endoplasmic reticulum and mitochondria, causing mitochondrial dysfunction. Additionally, it decreases the oxygen consumption rates and activity of mitochondrial complexes I-V. As expected, the antioxidants MitoTEMPOL and N-acetylcysteine reduce piscidin-1-induced ROS generation and intracellular calcium accumulation. Piscidin-1 also inhibits matrix metalloproteinase (MMP)-2/-9 expression in HUVECs, affecting migration and tube formation angiogenesis. We demonstrated that piscidin-1 can promote apoptosis via both intrinsic and extrinsic apoptotic pathways and findings indicate that piscidin-1 has anti-proliferative and anti-angiogenic properties in oral cancer treatment. Our study on piscidin-1 thus provides a basis for future translational anti-oral cancer drug research and a new theoretical approach for anti-oral cancer clinical research.

STAT3 phosphorylation inhibitor Bt354 exhibits anti-neoplastic activity in glioblastoma multiforme cells.

The high mortality rate of glioblastoma multiforme (GBM), a lethal primary brain tumor, is attributable to postsurgical recurrence. STAT3, an oncogenic protein, is a signal transducer and transcription activator encourages cancer cell migration and proliferation, which results in resistance to therapy. STAT3 inhibition reduces cancer metastasis and improves patient prognosis. Bt354, a small molecule STAT inhibitor, exhibits significant cytotoxic and anti-proliferative activities against certain cancer types. Here, we demonstrated that exposure of GBM cells (U87 MG) to Bt354 had a significant, concentration-dependent growth suppression. Bt354 also induced apoptosis and downregulated the expression of the epithelial-mesenchymal transition genes. Therefore, this study suggests the potential of Bt354 for treating GBM owing to its ability to induce cytotoxicity.

Immunomodulatory and anti-angiogenesis effects of excavatolide B and its derivatives in alleviating atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin condition primarily driven by T helper 2 (Th2) cytokines, resulting in skin barrier defects, angiogenesis, and inflammatory responses. The marine natural product excavatolide B (EXCB), isolated from the Formosan Gorgonian coral Briareum stechei, exhibits anti-inflammatory and analgesic properties. To enhance solubility, EXCB is chemically modified into the derivatives EXCB-61 salt and EXCB-79. The study aims to investigate the therapeutic effects of these compounds on dinitrochlorbenzene (DNCB)-induced skin damage and to elucidate the underlying anti-inflammatory and anti-angiogenesis mechanism. In vitro, using lipopolysaccharide (LPS)-induced RAW 264.7 cells, all compounds at 10 µM significantly inhibited expression of inflammatory proteins (inducible nitric oxide synthase and cyclooxygenase-2), vascular endothelial growth factor (VEGF), and cytokines (interleukin (IL)-1ß, IL-6, and IL-17A). In vivo, topical application of these compounds on DNCB-induced AD mice alleviated skin symptoms, reduced serum levels of IgE, IL-4, IL-13, IL-17, and interferon-γ, and moderated histological phenomena such as hyperplasia, inflammatory cell infiltration, and angiogenesis. The three compounds restored the expression of skin barrier-related proteins (loricrin, filaggrin, and claudin-1) and reduced the expression of angiogenesis-related proteins (VEGF and platelet endothelial cell adhesion molecule-CD31) in the tissues. This is the first study to indicate that EXCB, EXCB-61 salt, and EXCB-79 can treat AD disease by reducing inflammation and angiogenesis. Hence, they may be considered potential candidates for the development of new drugs for AD.

Manoalide Induces Intrinsic Apoptosis by Oxidative Stress and Mitochondrial Dysfunction in Human Osteosarcoma Cells.

Osteosarcoma (OS) is the most common primary malignant bone tumor that produces immature osteoid. Metastatic OS has a poor prognosis with a death rate of >70%. Manoalide is a natural sesterterpenoid isolated from marine sponges. It is a phospholipase A2 inhibitor with anti-inflammatory, analgesic, and anti-cancer properties. This study aimed to investigate the mechanism and effect of manoalide on OS cells. Our experiments showed that manoalide induced cytotoxicity in 143B and MG63 cells (human osteosarcoma). Treatment with manoalide at concentrations of 10, 20, and 40 µM for 24 and 48 h reduced MG63 cell viability to 45.13-4.40% (p < 0.01). Meanwhile, manoalide caused reactive oxygen species (ROS) overproduction and disrupted antioxidant proteins, activating the apoptotic proteins caspase-9/-3 and PARP (Poly (ADP-ribose) polymerase). Excessive levels of ROS in the mitochondria affected oxidative phosphorylation, ATP generation, and membrane potential (ΔΨm). Additionally, manoalide down-regulated mitochondrial fusion protein and up-regulated mitochondrial fission protein, resulting in mitochondrial fragmentation and impaired function. On the contrary, a pre-treatment with n-acetyl-l-cysteine ameliorated manoalide-induced apoptosis, ROS, and antioxidant proteins in OS cells. Overall, our findings show that manoalide induces oxidative stress, mitochondrial dysfunction, and apoptosis, causing the cell death of OS cells, showing potential as an innovative alternative treatment in human OS.

Antinociceptive Effects of Aaptamine, a Sponge Component, on Peripheral Neuropathy in Rats.

Aaptamine, a natural marine compound isolated from the sea sponge, has various biological activities, including delta-opioid agonist properties. However, the effects of aaptamine in neuropathic pain remain unclear. In the present study, we used a chronic constriction injury (CCI)-induced peripheral neuropathic rat model to explore the analgesic effects of intrathecal aaptamine administration. We also investigated cellular angiogenesis and lactate dehydrogenase A (LDHA) expression in the ipsilateral lumbar spinal cord after aaptamine administration in CCI rats by immunohistofluorescence. The results showed that aaptamine alleviates CCI-induced nociceptive sensitization, allodynia, and hyperalgesia. Moreover, aaptamine significantly downregulated CCI-induced vascular endothelial growth factor (VEGF), cluster of differentiation 31 (CD31), and LDHA expression in the spinal cord. Double immunofluorescent staining showed that the spinal VEGF and LDHA majorly expressed on astrocytes and neurons, respectively, in CCI rats and inhibited by aaptamine. Collectively, our results indicate aaptamine's potential as an analgesic agent for neuropathic pain. Furthermore, inhibition of astrocyte-derived angiogenesis and neuronal LDHA expression might be beneficial in neuropathy.

Isoaaptamine increases ROS levels causing autophagy and mitochondria-mediated apoptosis in glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) is a common central nervous system disease with a poor prognosis; its five-year survival rate is <5 %, and its median survival of 15 months. Current treatment includes chemotherapy with temozolomide, which is ineffective against GBM, suggesting an urgent need to develop novel therapies. This study evaluated isoaaptamine and aaptamine in the GBM cell lines for cell viability; GBM 8401, U87 MG, U138 MG, and T98G. Our findings showed that isoaaptamine was more potent than its iso-form aaptamine in these four cell lines, and GBM 8401 was most sensitive to isoaaptamine. The study in GBM 8401 cells showed that apoptosis was induced by isoaaptamine with increased cleaved caspase 3 and poly ADP-ribose polymerase (PARP). Moreover, isoaaptamine enhanced oxidative stress by increasing the levels of reactive oxygen species (ROS), inhibiting mitochondrial and cellular superoxidase dismutases (SOD1&2), peroxidase and an anti-apoptotic protein (Bcl-2), and disrupting mitochondrial membrane potential. In addition, the oxygen consumption rates and activities of mitochondrial complexes I-V were significantly reduced. Mitochondrial dynamics were prone to fission instead of fusion after isoaaptamine treatment, and ATP synthesis was ablated. Also, autophagy-related acidic organelle vesicles were formed, indicating autophagy was triggered. Overall, isoaaptamine-induced ROS overproduction in mitochondria could cause mitochondrial dysfunction, apoptosis, and autophagy in the GBM cells.

Stellettin B-Induced Oral Cancer Cell Death via Endoplasmic Reticulum Stress-Mitochondrial Apoptotic and Autophagic Signaling Pathway.

Oral squamous cell carcinoma (OSCC) affects tens of thousands of people worldwide. Despite advances in cancer treatment, the 5-year survival rate of patients with late-stage OSCC is low at 50-60%. Therefore, the development of anti-OSCC therapy is necessary. We evaluated the effects of marine-derived triterpene stellettin B in human OC2 and SCC4 cells. Stellettin B dose-dependently decreased the viability of both cell lines, with a significant reduction in OC2 cells at ≥0.1 µM at 24 and 48 h, and in SCC4 cells at ≥1 µM at 24 and 48 h. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells were significantly observed at 20 µM of stellettin B at 48 h, with the overexpression of cleaved caspase3 and cleaved poly(ADP-ribose) polymerase (PARP). Moreover, mitochondrial respiratory functions were ablated by stellettin B. Autophagy-related LC3-II/LC3-I ratio and Beclin-1 proteins were increased, whereas p62 was decreased. At 20 µM at 48 h, the expression levels of the endoplasmic reticulum (ER) stress biomarkers calnexin and BiP/GRP78 were significantly increased and mitogen-activated protein kinase (MAPK) signaling pathways were activated. Further investigation using the autophagy inhibitor 3-methyladenine (3-MA) demonstrated that it alleviated stellettin B-induced cell death and autophagy. Overall, our findings show that stellettin B induces the ER stress, mitochondrial stress, apoptosis, and autophagy, causing cell death of OSCC cells.

Sinularin Induces Oxidative Stress-Mediated Apoptosis and Mitochondrial Dysfunction, and Inhibits Angiogenesis in Glioblastoma Cells.

Glioblastoma multiforme (GBM) is a cancer of largely unknown cause that leads to a 5-year survival rate of approximately 7% in the United States. Current treatment strategies are not effective, indicating a strong need for the development of novel therapies. In this study, the outcomes of sinularin, a marine-derived product, were evaluated against GBM. Our cellular studies using GBM cells revealed that sinularin induces cell death. The measured half maximal inhibitory concentrations (IC50) values ranged from 30 to 6 µM at 24-72 h. Cell death was induced via the generation of ROS leading to mitochondria-mediated apoptosis. This was evidenced by annexin V/propidium iodine staining and an upregulation of cleaved forms of the pro-apoptotic proteins caspase 9, 3, and PARP, and supported by CellROXTM Green, MitoSOXTM Red, and CM-H2DCFDA staining methods. In addition, we observed a downregulation of the antioxidant enzymes SOD1/2 and thioredoxin. Upon treatment with sinularin at the ~IC50 concentration, mitochondrial respiration capacities were significantly reduced, as shown by measuring the oxygen consumption rates and enzymatic complexes of oxidative phosphorylation. Intriguingly, sinularin significantly inhibited indicators of angiogenesis such as vessel tube formation, cell migration, and cell mobility in human umbilical vein endothelial cells or the fusion cell line EA.Hy926. Lastly, in a transgenic zebrafish model, intersegmental vessel formation was also significantly inhibited by sinularin treatment. These findings indicate that sinularin exerts anti-brain cancer properties that include apoptosis induction but also antiangiogenesis.

Pardaxin Activates Excessive Mitophagy and Mitochondria-Mediated Apoptosis in Human Ovarian Cancer by Inducing Reactive Oxygen Species.

Most ovarian cancer (OC) patients are diagnosed with stage III or higher disease, resulting in a poor prognosis. Currently, paclitaxel combined with carboplatin shows the best treatment outcome for OC. However, no effective drug is available for patients that do not respond to treatment; thus, new drugs for OC are needed. We evaluated the antimicrobial peptide, pardaxin, in PA-1 and SKOV3 cells. Pardaxin induced apoptosis as determined by MTT and TUNEL assays, as well as activation of caspases-9/3, Bid, t-Bid, and Bax, whereas Bcl-2 was downregulated. The IC50 values for pardaxin were 4.6-3.0 µM at 24 and 48 h. Mitochondrial and intracellular reactive oxygen species (ROS) were overproduced and associated with disrupted mitochondrial membrane potential and respiratory capacity. Additionally, the mitochondrial network was fragmented with downregulated fusogenic proteins, MFN1/2 and L-/S-OPA1, and upregulated fission-related proteins, DRP1 and FIS1. Autophagy was also activated as evidenced by increased expression of autophagosome formation-related proteins, Beclin, p62, and LC3. Enhanced mitochondrial fragmentation and autophagy indicate that mitophagy was activated. ROS-induced cytotoxicity was reversed by the addition of N-acetylcysteine, confirming ROS overproduction as a contributor. Taken together, pardaxin demonstrated promising anticancer activity in OC cells, which warrants further preclinical development of this compound.

Fumagillin Attenuates Spinal Angiogenesis, Neuroinflammation, and Pain in Neuropathic Rats after Chronic Constriction Injury.

INTRODUCTION: Angiogenesis in the central nervous system is visible in animal models of neuroinflammation and bone cancer pain. However, whether spinal angiogenesis exists and contributes to central sensitization in neuropathic pain remains unclear. This study analyzes the impact of angiogenesis on spinal neuroinflammation in neuropathic pain. METHODS: Rats with chronic constriction injury (CCI) to the sciatic nerve underwent the implantation of an intrathecal catheter. Fumagillin or vascular endothelial growth factor-A antibody (anti-VEGF-A) was administered intrathecally. Nociceptive behaviors, cytokine immunoassay, Western blot, and immunohistochemical analysis assessed the effect of angiogenesis inhibition on CCI-induced neuropathic pain. RESULTS: VEGF, cluster of differentiation 31 (CD31), and von Willebrand factor (vWF) expressions increased after CCI in the ipsilateral lumbar spinal cord compared to that in the contralateral side of CCI and control rats from post-operative day (POD) 7 to 28, with a peak at POD 14. Tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 concentrations, but not IL-10 levels, also increased in the ipsilateral spinal cord after CCI. Fumagillin and anti-VEGF-A reduced CCI-induced thermal hyperalgesia from POD 5 to 14 and mechanical allodynia from POD 3 to 14. Fumagillin reduced CCI-upregulated expressions of angiogenic factors and astrocytes. Furthermore, fumagillin decreased TNF-α and IL-6 amounts and increased IL-10 levels at POD 7 and 14, but not IL-1ß concentrations. CONCLUSIONS: Fumagillin significantly ameliorates CCI-induced nociceptive sensitization, spinal angiogenesis, and astrocyte activation. Our results suggest that angiogenesis inhibitor treatment suppresses peripheral neuropathy-induced central angiogenesis, neuroinflammation, astrocyte activation, and neuropathic pain.

Glutathione S-Transferase M3 Is Associated with Glycolysis in Intrinsic Temozolomide-Resistant Glioblastoma Multiforme Cells.

Glioblastoma multiforme (GBM) is a malignant primary brain tumor. The 5-year relative survival rate of patients with GBM remains <30% on average despite aggressive treatments, and secondary therapy fails in 90% of patients. In chemotherapeutic failure, detoxification proteins are crucial to the activity of chemotherapy drugs. Usually, glutathione S-transferase (GST) superfamily members act as detoxification enzymes by activating xenobiotic metabolites through conjugation with glutathione in healthy cells. However, some overexpressed GSTs not only increase GST activity but also trigger chemotherapy resistance and tumorigenesis-related signaling transductions. Whether GSTM3 is involved in GBM chemoresistance remains unclear. In the current study, we found that T98G, a GBM cell line with pre-existing temozolomide (TMZ) resistance, has high glycolysis and GSTM3 expression. GSTM3 knockdown in T98G decreased glycolysis ability through lactate dehydrogenase A activity reduction. Moreover, it increased TMZ toxicity and decreased invasion ability. Furthermore, we provide next-generation sequencing-based identification of significantly changed messenger RNAs of T98G cells with GSTM3 knockdown for further research. GSTM3 was downregulated in intrinsic TMZ-resistant T98G with a change in the expression levels of some essential glycolysis-related genes. Thus, GSTM3 was associated with glycolysis in chemotherapeutic resistance in T98G cells. Our findings provide new insight into the GSTM3 mechanism in recurring GBM.

Sinularin, an Anti-Cancer Agent Causing Mitochondria-Modulated Apoptosis and Cytoskeleton Disruption in Human Hepatocellular Carcinoma.

Liver cancer remains a leading cause of death, despite advances in anti-cancer therapies. To develop novel drugs, natural products are being considered as a good source for exploration. In this study, a natural product isolated from a soft coral was applied to evaluate its anti-cancer activities in hepatocellular carcinoma SK-HEP-1 cells. Sinularin was determined to have half-maximal inhibitory concentration (IC50) values of ~10 µM after 24, 48, and 72 h. The TUNEL assay and annexin V/PI staining results showed that sinularin induced DNA fragmentation and apoptosis, respectively. An investigation at the molecular level demonstrated that the expression levels of cleaved caspases 3/9 were significantly elevated at 10 µM sinularin. Mitochondrial and intracellular reactive oxygen species (ROS) levels were significantly increased following sinularin treatment, which also affected the mitochondrial membrane potential. In addition, it significantly lowered the mitochondrial respiration parameters and extracellular acidification rates at 10 µM. Further investigation showed that sinularin significantly attenuated wound healing, cell migration, and potential colony formation at 10 µM. Fluorescence microscopic observations showed that the distribution of F-actin filaments was significantly altered at 10 µM sinularin. Supported by Western blot analyses, the expression levels of AKT, p-ERK (extracellular-signal-related kinase), vimentin and VEGF were significantly down-regulated, whereas p-p38, pJNK and E-cadherin were significantly increased. Overall, at the IC50 concentration, sinularin was able to significantly affect SK-HEP-1 cells.

The antimicrobial peptide tilapia piscidin 3 induces mitochondria-modulated intrinsic apoptosis of osteosarcoma cells.

Osteosarcoma (OS) is the most common solid tumor of the bone that most often affects adolescents. The introduction of chemotherapy for the treatment of OS has largely improved the survival rates of patients with localized tumors. However, the 5-year survival rate of OS patients with relapsed or metastatic disease is only 10 to 20%. In this study, the antimicrobial peptide tilapia piscidin 3 (TP3), isolated from Nile tilapia (Oreochromis niloticus), was treated to OS MG63 cells. Our findings showed that TP3 concentration as low as 1 µM induced significant inhibition of cell viability and increased DNA fragmentation, as determined by the MTT and TUNEL assays, respectively. The protein expression levels of cleaved caspases 3/9 were increased. An in situ live-cell time-lapse video and cell tomographic microscopy images showed cellular blebbing, shrinkage, nuclear fragmentation, and chromatin condensation, with the formation of beaded apoptopodia. Moreover, there were significant increase in the production of TP3-induced mitochondrial and cellular reactive oxygen species (ROS), as well as down-regulated mitochondrial oxygen consumption and extracellular acidification rates. Additionally, TP3 enhanced mitochondrial fission, whereas fusion was attenuated. Furthermore, after administration of the mitochondria targeted antioxidant mitoTempo, TP3-induced ROS oxidant levels and alterations in cleaved caspases 3/9 expression were rescued. TP3 promoted mitochondria-modulated intrinsic apoptosis through the induction of ROS production, activation of caspases 3/9, and the down-regulation of mitochondrial oxygen consumption and extracellular acidification rates, suggesting that TP3 has potential as an innovative alternative for OS treatment.

TP3, an antimicrobial peptide, inhibits infiltration and motility of glioblastoma cells via modulating the tumor microenvironment.

Glioblastoma multiforme (GBM) is a cancer of the central nervous system with limited therapeutic outcomes. Infiltrating cancer cells are the contributing factor to high GBM malignancy. The intracranial brain cancer cell infiltration is a complex cascade involving adhesion, migration, and invasion. An arsenal of natural products has been under exploration to overcome GBM malignancy. This study applied the antimicrobial peptide tilapia piscidin 3 (TP3) to GBM8401, U87MG, and T98G cells. The cellular assays and microscopic observations showed that TP3 significantly attenuated cell adhesion, migration, and invasion. A live-cell video clip showed the inhibition of filopodia protrusions and cell attachment. Probing at the molecular levels showed that the proteolytic activities (from secretion), the mRNA and protein expression levels of matrix metalloproteinases-2 and -9 were attenuated. This result strongly evidenced that both invasion and metastasis were inhibited, although metastatic GBM is rare. Furthermore, the protein expression levels of cell-mobilization regulators focal adhesion kinase and paxillin were decreased. Similar effects were observed in small GTPase (RAS), phosphorylated protein kinase B (AKT) and MAP kinases such as extracellular signal-regulated kinases (ERK), JNK, and p38. Overall, TP3 showed promising activities to prevent cell infiltration and metastasis through modulating the tumor microenvironment balance, suggesting that TP3 merits further development for use in GBM treatments.

Piscidin-1 Induces Apoptosis via Mitochondrial Reactive Oxygen Species-Regulated Mitochondrial Dysfunction in Human Osteosarcoma Cells.

Osteosarcoma (OSA) is the most common type of cancer that originates in the bone and usually occurs in young children. OSA patients were treated with neoadjuvant chemotherapy and surgery, and the results were disappointing. Marine antimicrobial peptides (AMPs) have been the focus of antibiotic research because they are resistant to pathogen infection. Piscidin-1 is an AMP from the hybrid striped bass (Morone saxatilis × M. chrysops) and has approximately 22 amino acids. Research has shown that piscidin-1 can inhibit bacterial infections and has antinociception and anti-cancer properties; however, the regulatory effects of piscidin-1 on mitochondrial dysfunction in cancer cells are still unknown. We aimed to identify the effects of piscidin-1 on mitochondrial reactive oxygen species (mtROS) and apoptosis in OSA cells. Our analyses indicated that piscidin-1 has more cytotoxic effects against OSA cells than against lung and ovarian cancer cells; however, it has no effect on non-cancer cells. Piscidin-1 induces apoptosis in OSA cells, regulates mtROS, reduces mitochondrial antioxidant manganese superoxide dismutase and mitochondrial transmembrane potential, and decreases adenosine 5'-triphosphate production, thus leading to mitochondrial dysfunction and apoptosis. The mitochondrial antioxidant, mitoTempo, reduces the apoptosis induced by piscidin-1. Results suggest that piscidin-1 has potential for use in OSA treatment.

Therapeutic Effect of Modulating TREM-1 via Anti-inflammation and Autophagy in Parkinson's Disease.

Parkinson's disease (PD) is one of the most common age-related neurodegenerative diseases, and neuroinflammation has been identified as one of its key pathological characteristics. Triggering receptors expressed on myeloid cells-1 (TREM-1) amplify the inflammatory response and play a role in sepsis and cancer. Recent studies have demonstrated that the attenuation of TREM-1 activity produces cytoprotective and anti-inflammatory effects in macrophages. However, no study has examined the role of TREM-1 in neurodegeneration. We showed that LP17, a synthetic peptide blocker of TREM-1, significantly inhibited the lipopolysaccharide (LPS)-induced upregulation of proinflammatory cascades of inducible nitric oxide synthase (iNOS), cyclooxygenase-2, and nuclear factor-kappa B. Moreover, LP17 enhanced the LPS-induced upregulation of autophagy-related proteins such as light chain-3 and histone deacetylase-6. We also knocked down TREM-1 expression in a BV2 cell model to further confirm the role of TREM-1. LP17 inhibited 6-hydroxydopamine-induced locomotor deficit and iNOS messenger RNA expression in zebrafish. We also observed therapeutic effects of LP17 administration in 6-hydroxydopamine-induced PD syndrome using a rat model. These data suggest that the attenuation of TREM-1 could ameliorate neuroinflammatory responses in PD and that this neuroprotective effect might occur via the activation of autophagy and anti-inflammatory pathways.

BA6 Induces Apoptosis via Stimulation of Reactive Oxygen Species and Inhibition of Oxidative Phosphorylation in Human Lung Cancer Cells.

Lung cancer is the leading cause of cancer deaths in the world, with a five-year survival rate of less than 30%. Clinically effective chemotherapeutic treatments at the initial stage may eventually face the dilemma of no drug being effective due to drug resistance; therefore, finding new effective drugs for lung cancer treatment is a necessary and important issue. Compounds capable of further increasing the oxidative stress of cancer cells are considered to have anticancer potential because they possessed the ability to induce apoptosis. This study mainly investigated the effects of BA6 (heteronemin), the marine sponge sesterterpene, on lung cancer cell apoptosis, via modulation of mitochondrial reactive oxygen species (mtROS) and oxidative phosphorylation (OXPHOS). BA6 has cellular cytotoxic activities against a variety of cancer cell lines, but it has no effect on nontumor cells. The BA6-treated lung cancer cells show a significant increase in both cellular ROS and mtROS, which in turn caused the loss of mitochondrial membrane potential (MMP). The increase of oxidative stress in lung cancer cells treated with BA6 was accompanied by a decrease in the expression of antioxidant enzymes Cu/Zn SOD, MnSOD, and catalase. In addition, OXPHOS performed in the mitochondria and glycolysis in the cytoplasm were inhibited, which subsequently reduced downstream ATP production. Pretreatment with mitochondria-targeted antioxidant MitoTEMPO reduced BA6-induced apoptosis through the mitochondria-dependent apoptotic pathway, which was accompanied by increased cell viability, decreased mtROS, enhanced MMP, and suppressed expression of cleaved caspase-3 and caspase-9 proteins. In conclusion, the results of this study clarify the mechanism of BA6-induced apoptosis in lung cancer cells via the mitochondrial apoptotic pathway, suggesting that it is a potentially innovative alternative to the treatment of human lung cancer.

Anti-Invasion and Antiangiogenic Effects of Stellettin B through Inhibition of the Akt/Girdin Signaling Pathway and VEGF in Glioblastoma Cells.

Angiogenesis and invasion are highly related with tumor metastatic potential and recurrence prediction in the most aggressive brain cancer, glioblastoma multiforme (GBM). For the first time, this study reveals that marine-sponge-derived stellettin B reduces angiogenesis and invasion. We discovered that stellettin B reduces migration of glioblastoma cells by scratch wound healing assay and invasion via chamber transwell assay. Further, stellettin B downregulates Akt/Mammalian Target of Rapamycin (Akt/mTOR) and Signal transducer and activator of transcription 3 (Stat3) signaling pathways, which are essential for invasion and angiogenesis in glioblastoma. This study further demonstrates that stellettin B affects filamentous actin (F-actin) rearrangement by decreasing the cross-linkage of phosphor-Girdin (p-Girdin), which attenuates glioblastoma cell invasion. Moreover, stellettin B blocks the expression and secretion of a major proangiogenic factor, vascular endothelial growth factor (VEGF), in glioblastoma cells. Stellettin B also reduces angiogenic tubule formation in human umbilical vein endothelial cells (HUVECs). In vivo, we observed that stellettin B decreased blood vesicle formation in developmental zebrafish and suppressed angiogenesis in Matrigel plug transplant assay in mice. Decreased VEGF transcriptional expression was also found in stellettin B⁻treated zebrafish embryos. Overall, we conclude that stellettin B might be a potential antiangiogenic and anti-invasion agent for future development of therapeutic agents for cancer therapy.

Prodigiosin stimulates endoplasmic reticulum stress and induces autophagic cell death in glioblastoma cells.

Prodigiosin, a secondary metabolite isolated from marine Vibrio sp., has antimicrobial and anticancer properties. This study investigated the cell death mechanism of prodigiosin in glioblastoma. Glioblastoma multiforme (GBM) is an aggressive primary cancer of the central nervous system. Despite treatment, or standard therapy, the median survival of glioblastoma patients is about 14.6 month. The results of the present study clearly showed that prodigiosin significantly reduced the cell viability and neurosphere formation ability of U87MG and GBM8401 human glioblastoma cell lines. Moreover, prodigiosin with fluorescence signals was detected in the endoplasmic reticulum and found to induce excessive levels of autophagy. These findings were confirmed by observation of LC3 puncta formation and acridine orange staining. Furthermore, prodigiosin caused cell death by activating the JNK pathway and decreasing the AKT/mTOR pathway in glioblastoma cells. Moreover, we found that the autophagy inhibitor 3-methyladenine reversed prodigiosin induced autophagic cell death. These findings of this study suggest that prodigiosin induces autophagic cell death and apoptosis in glioblastoma cells.

Soluble delta-like 1 homolog (DLK1) stimulates angiogenesis through Notch1/Akt/eNOS signaling in endothelial cells.

AIM: Delta-like 1 homolog (DLK1) is a non-canonical ligand of Notch signaling, which plays a pivotal role in vascular development and tumor angiogenesis. This study aimed to elucidate the function and mechanism of DLK1 in angiogenesis. METHODS AND RESULTS: By using in situ hybridization and immunohistochemical studies, expression analysis revealed a unique vascular tropism of DLK1 in vasculature of neuroblastoma and vascular tumors. Thus, it was hypothesized that DLK1 may be cleaved and then bound to endothelial cells, thereby regulating the endothelial function. To test such hypothesis, soluble DLK1 encompassing DLK1 extracellular domain (DLK1-EC) was generated and validated by its inhibitory function in adipogenesis assay. Recombinant DLK1-EC exhibited the preferential binding capability toward endothelial cells and stimulated the microvessels sprouting in aorta rings. Above all, implantation of DLK1-EC dose-dependently elicited the cornea neovascularization in rats. By using various angiogenesis assays, it was delineated that DLK1-EC stimulated the angiogenesis by promoting the proliferation, motility and tube formation of endothelial cells. By immunoblot and luciferase analysis, it was elucidated that DLK1-EC enhanced the expression and activities of Notch1/Akt/eNOS/Hes-1 signaling in dose- and time-dependent manners. Pharmaceutical blockage of Notch signaling using γ-secretase inhibitor DAPT abrogated the DLK1-EC-induced endothelial migration and Hes-1-driven luciferase activities. Furthermore, Notch1 inactivation by neutralizing antibodies or RNA interference reversed the DLK1-EC-induced angiogenesis. CONCLUSIONS: The present study unveils the pro-angiogenic function and mechanism of soluble DLK1 through activation of Notch1 signaling in endothelial cells.