Human Tumor-derived Cell Research Articles

Multifunctional host-defense peptides isolated from skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae)

Five host-defense peptides (figainin 2PL, hylin PL, raniseptin PL, plasticin PL, and peptide YL) were isolated from norepinephrine-stimulated skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae) collected in Trinidad.Raniseptin PL (GVFDTVKKIGKAVGKFALGVAKNYLNS.NH2) and figainin 2PL (FLGTVLKLGKAIAKTVVPMLTNAMQPKQ. NH2) showed potent and rapid bactericidal activity against a range of clinically relevant Gram-positive and Gram-negative ESKAPE + pathogens and Clostridioides difficile. The peptides also showed potent cytotoxic activity (LC50 values < 30 μM) against A549, MDA-MB-231 and HT29 human tumor-derived cell lines but appreciably lower hemolytic activity against mouse erythrocytes (LC50 = 262 ± 14 μM for raniseptin PL and 157 ± 16 μM for figainin 2PL). Hylin PL (FLGLIPALAGAIGNLIK.NH2) showed relatively weak activity against microorganisms but was more hemolytic. The glycine-leucine-rich peptide with structural similarity to the plasticins (GLLSTVGGLVGGLLNNLGL.NH2) and the non-cytotoxic peptide YL (YVPGVIESLL.NH2) lacked antimicrobial and cytotoxic activities. Hylin PL, raniseptinPL and peptide YL stimulated the rate of release of insulin from BRIN-BD11 clonal β-cells at concentrations ≥100 nM. Peptide YL was the most effective (2.3-fold increase compared with basal rate at 1 μM concentration) and may represent a template for the design of a new class of incretin-based anti-diabetic drugs.

Abstract A010: Assay development to assess the efficiency and stability of candidate molecules for transcriptional gene silencing of FOXP3, using a human tumor-derived cell line

Abstract Regulatory T cells play an important role to modulate the balance between immunotolerance and immunosurveillance. These cells have the property of inhibiting effector lymphocytes and may antagonize antitumor immunity. Regulatory T cells are originated in the thymus, or even converted from peripheral lymphocytes by factors produced in the tumor microenvironment. Clinical data suggests that regulatory T cell infiltration correlates with poor prognosis in the treatment of solid tumors. The FOXP3 transcription factor is considered a master key to control the phenotype of regulatory T cells. It was previously shown, that the ectopic expression of FOXP3 can induce an immunosuppressive phenotype in lymphocytes. In contrast, it has been observed that mutations in the FOXP3 gene can cause impaired immunosuppressive activity mediated by regulatory T cells, such as the autoimmunity syndrome known as IPEX. In this sense, the FOXP3 transcription factor may be an interesting target, looking for inactivation of the immunosuppressive phenotype of regulatory T cells, to potentiate antitumor response. In this work, we present an assay development to investigate the potential of transcriptional interference RNA candidates to silence FOXP3 expression. In contrast to post-transcriptional gene silencing method, which targets messenger RNA and depends on a constant supply or efficient turn-over of the inhibitory molecule, the transcriptional gene silencing targets the cellular DNA genome, inducing epigenetic changes that may control the target gene transcription. We employed the human MCF-7 tumor cell line, which has endogenous and constitutive expression of FOXP3 as a target model to test transcriptional gene silencing candidates. This cell line is transduced with lentiviral vectors harboring interfering RNA sequences driven to the FOXP3 promoter region. The RNA interference candidates are then evaluated for their ability to induce DNA methylation, by bisulfite method, and transcriptional gene silencing of FOXP3, by qPCR. We performed the screening of candidates for FOXP3 transcriptional silencing, in comparison to a post-transcriptional interference RNA control. The lentiviral transduced cells are easily expanded and allow a temporal analysis of target gene expression. In this model, we observed the possibility of finding transcriptional interference RNA candidates that exhibited high efficiency and stability, that may be used for research purposes, or even for the investigation of new therapeutic possibilities in immuno-oncology. Citation Format: Carolinne T Fogagnolo, Daniela S Mizobuti, Marcio C Bajgelman. Assay development to assess the efficiency and stability of candidate molecules for transcriptional gene silencing of FOXP3, using a human tumor-derived cell line [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A010.

Purification, conformational analysis and cytotoxic activities of host-defense peptides from the Tungara frog Engystomops pustulosus (Leptodactylidae; Leiuperinae)

The amphibian family Leptodactylidae is divided into three sub-families: Leiuperinae, Leptodactylinae, and Paratelmatobiinae. Host-defense peptides (HDPs) present in the skins of frogs belonging to the Leptodactylinae have been studied extensively, but information is limited regarding peptides from Leiuperinae species. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Tungara frog Engystomops pustulosus (Leiuperinae) collected in Trinidad led to the isolation and structural characterization of previously undescribed pustulosin-1 (FWKADVKEIG KKLAAKLAEELAKKLGEQ), [Q28E] pustulosin-1 (pustulosin-2), and pustulosin-3 (DWKETAKELLKKIGAKVAQVISDKLNPAPQ). The primary structures of these peptides do not resemble those of previously described frog skin HDPs. In addition, the secretions contained tigerinin-1EP (GCKTYLIEPPVCT) with structural similarity to the tigerinins previously identified in skin secretions from frogs from the family Dicroglossidae. Pustulosin-1 and -3 adopted extended α-helical conformations in 25% trifluoroethanol–water and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). Pustulosin-1 and -3 displayed cytotoxic activity against a range of human tumor-derived cell lines (A549, MDA-MB-231, and HT29), but their therapeutic potential for development into anti-cancer agents is limited by their comparable cytotoxic activity against non-neoplastic human umbilical vein endothelial cells. The peptides also displayed weak antimicrobial activity against Escherichia coli (MIC = 125 µM) but were inactive against Staphylococcus aureus. Tigerinin-1EP was inactive against both the tumor-derived cells and bacteria.

Purification, Conformational Analysis and Cytotoxic Activities of Host-Defense Peptides from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae).

Frogs from the extensive amphibian family Hylidae are a rich source of peptides with therapeutic potential. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae) collected in Trinidad led to the isolation and structural characterization of five host-defense peptides with limited structural similarity to figainin 2 and picturin peptides from other frog species belonging to the genus Boana. In addition, the skin secretions contained high concentrations of tryptophyllin-BN (WRPFPFL) in both C-terminally α-amidated and non-amidated forms. Figainin 2BN (FLGVALKLGKVLG KALLPLASSLLHSQ) and picturin 1BN (GIFKDTLKKVVAAVLTTVADNIHPK) adopt α-helical conformations in trifluroethanol-water mixtures and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). The CD data also indicate contributions from turn structures. Both peptides and picturin 2BN (GLMDMLKKVGKVALT VAKSALLP) inhibited the growth of clinically relevant Gram-negative and Gram-positive bacteria with MIC values in the range 7.8-62.5 µM. Figainin 2BN was potently cytotoxic to A549, MDA-MB-231 and HT-29 human tumor-derived cells (LC50 = 7-14 µM) but displayed comparable potency against non-neoplastic HUVEC cells (LC50 = 15 µM) indicative of lack of selectivity for cancer cells.

Crosstalk between protein kinases AKT and ERK1/2 in human lung tumor-derived cell models.

There is no doubt that cell signaling manipulation is a key strategy for anticancer therapy. Furthermore, cell state determines drug response. Thus, establishing the relationship between cell state and therapeutic sensitivity is essential for the development of cancer therapies. In the era of personalized medicine, the use of patient-derived ex vivo cell models is a promising approach in the translation of key research findings into clinics. Here, we were focused on the non-oncogene dependencies of cell resistance to anticancer treatments. Signaling-related mechanisms of response to inhibitors of MEK/ERK and PI3K/AKT pathways (regulators of key cellular functions) were investigated using a panel of patients' lung tumor-derived cell lines with various stemness- and EMT-related markers, varying degrees of ERK1/2 and AKT phosphorylation, and response to anticancer treatment. The study of interactions between kinases was the goal of our research. Although MEK/ERK and PI3K/AKT interactions are thought to be cell line-specific, where oncogenic mutations have a decisive role, we demonstrated negative feedback loops between MEK/ERK and PI3K/AKT signaling pathways in all cell lines studied, regardless of genotype and phenotype differences. Our work showed that various and distinct inhibitors of ERK signaling - selumetinib, trametinib, and SCH772984 - increased AKT phosphorylation, and conversely, inhibitors of AKT - capivasertib, idelalisib, and AKT inhibitor VIII - increased ERK phosphorylation in both control and cisplatin-treated cells. Interaction between kinases, however, was dependent on cellular state. The feedback between ERK and AKT was attenuated by the focal adhesion kinase inhibitor PF573228, and in cells grown in suspension, showing the possible role of extracellular contacts in the regulation of crosstalk between kinases. Moreover, studies have shown that the interplay between MEK/ERK and PI3K/AKT signaling pathways may be dependent on the strength of the chemotherapeutic stimulus. The study highlights the importance of spatial location of the cells and the strength of the treatment during anticancer therapy.

Abstract 4222: Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway

Abstract Oral cancer is the sixth most prevalent malignancy in the world and oral squamous cell carcinoma accounts for majority of all oral malignancies. Upregulated NLRP3 inflammasome innate immune pathway is of importance to tumor development. Current efforts are being focused on identifying small molecules that exhibit anti-cancer activity as inflammasome pathway inhibitors. Our previously published work on phenstatin based indole linked chalcone scaffold 9a with 1-methyl, 2- and 3-methoxy substituents in the aromatic ring revealed 9a as an anti-oral cancer compound. 9a was found to act through inhibiting tubulin polymerization at protein level, using in vitro models oral cancer cell line/spheroid cells and in vivo animal oral cancer xenograft model. 9a had also shown significant reduction in radiolabeled-glucose uptake in xenograft mice model. Current study was undertaken to evaluate if small molecule inhibitor 9a acts through regulating the NLRP3 pathway. Using computation approach, we predicted the binding of 9a with NLRP3NACHT domain, which revealed stable interaction as similar to that exhibited by NLRP3 inhibitors MCC950 and ADP. Further, we checked immune mechanistic activity of 9a on NLRP3 pathway intermediates in oral cancer cells. AW13516 cell line which was human tongue squamous tumor-derived cell line; indigenously developed at our department previously, was activated for NLRP3 inflammasome pathway using LPS and activator Nigericin in presence of 9a. MCC950 treated cells and only LPS or LPS/Nigericin treated cells served as controls. NLRP3, caspase-1 and mitochondrial protein expression was analyzed in these cells by immunofluorescence (IF) and found to be increased upon LPS/NIG activation and reduced significantly upon MCC950 and 9a treatment. Activation led to puncta formation which was found diffused after MCC950/9a treatment. Similarly treated AW13516 cells were also validated using western blotting experiments. Expression of 118kDa NLRP3 protein was found increased upon inflammasome activation that was significantly reduced in 9a treated cells and reduction was dose dependent. 9a had shown significant reduction in oral cancer xenograft of AW13516 in NOD-SCID mice model. We tested formalin-fixed paraffin sections of these tumors by immunohistochemistry. Tumor areas were assessed for expression of NLRP3 pathway markers and there was significant reduction in NLRP3, Caspase-1, GSDMD and IL-1β in 9a treated tumors compared to control tumors. This reduction was at par with that shown by Adriamycin. Summarizingly, 9a has been found to be regulating inflammatory immune mechanisms and can be developed further as immunomodulatory anti-cancer agent. Since macrophages are major resource immune cells of NLRP3, further studies are underway to test 9a on macrophages from nlrp3-/- and caspase-/- mice in comparison with normal mice. Citation Format: Jyoti Kode, Jitendra Maharana, K. Nirmal Kumar, Trupti Pradhan, Arvind Ingle, Madan Barkume, Meena Patkar, Namitha Thampi, Ankita Patil, Anand Vaibhaw, Jeshma Kovvuri, Ahmed Kamal. Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4222.

Combinations of Toll-like receptor 8 agonist TL8-506 activate human tumor-derived dendritic cells

BackgroundDendritic cells (DCs) are professional antigen presenting cells that initiate immune defense to pathogens and tumor cells. Human tumors contain only few DCs that mostly display a non-activated phenotype. Hence,...

Pan-cancer analysis identifies RNA helicase DDX1 as a prognostic marker.

The online version contains supplementary material available at 10.1007/s43657-021-00034-x.

Senescent Thyrocytes, Similarly to Thyroid Tumor Cells, Elicit M2-like Macrophage Polarization In Vivo.

Simple SummarySeveral studies including ours suggest a pro-tumoral role of senescent thyrocytes in thyroid tumors. On the other hand, M2-like tumor associated macrophages infiltration increases with thyroid cancer aggressiveness. In this work, we used senescent thyrocytes and thyroid tumor cells as models of early and late tumor stages, respectively, and demonstrated their in vivo capability to recruit and polarize macrophages toward a pro-tumoral M2-like phenotype. These findings pave the way for the design of new therapeutic approaches for thyroid tumors based on the elimination or activity modulation of senescent cells and/or infiltrating macrophages.Inflammation plays a critical role in thyroid cancer onset and progression. We previously characterized the in vitro interplay between macrophages and senescent human thyrocytes and thyroid tumor-derived cell lines, modeling the early and the late thyroid tumor phases, respectively. We reported that both models are able to induce pro-tumoral M2-like macrophage polarization, through the activation of the COX2-PGE2 axis. Here, we investigated the presence of macrophage infiltrating cells in mouse xenografts derived from the above described cells models. We showed that subcutaneous injection in immunodeficient mice of both senescent human thyrocytes and thyroid tumor-derived cell lines elicits macrophage recruitment. Furthermore, considering the type of macrophage infiltrate, we observed a stronger infiltration of Arginase I positive cells (M2-like). Overall, these results demonstrate the in vivo capability of senescent and tumor thyroid cells to recruit and polarize macrophages, suggesting that the promotion of a pro-tumoral microenvironment through tumor associated macrophages may occurs in late as well as in early thyroid tumor stages, favoring tumor onset and progression.

Microalgal Co-Cultivation Prospecting to Modulate Vitamin and Bioactive Compounds Production.

Microalgal biotechnology is gaining importance. However, key issues in the pipeline from species selection towards large biomass production still require improvements to maximize the yield and lower the microalgal production costs. This study explores a co-cultivation strategy to improve the bioactive compounds richness of the harvested microalgal biomass. Based on their biotechnological potential, two diatoms (Skeletonema marinoi, Cyclotella cryptica) and one eustigmatophyte (Nannochloropsis oceanica) were grown alone or in combination. Concentrations of ten vitamins (A, B1, B2, B6, B12, C, D2, D3, E and H), carotenoids and polyphenols, together with total flavonoids, sterols, lipids, proteins and carbohydrates, were compared. Moreover, antioxidant capacity and chemopreventive potential in terms inhibiting four human tumor-derived and normal cell lines proliferation were evaluated. Co-cultivation can engender biomass with emergent properties regarding bioactivity or bioactive chemical profile, depending on the combined species. The high vitamin content of C. cryptica or N. oceanica further enhanced (until 10% more) when co-cultivated, explaining the two-fold increase of the antioxidant capacity of the combined C. cryptica and N. oceanica biomass. Differently, the chemopreventive activity was valuably enhanced when coupling the two diatoms C. cryptica and S. marinoi. The results obtained in this pilot study promote microalgal co-cultivation as a valuable strategy aiming to boost their application in eco-sustainable biotechnology.

Changes in Wnt and TGF-β Signaling Mediate the Development of Regorafenib Resistance in Hepatocellular Carcinoma Cell Line HuH7.

Hepatocellular carcinoma (HCC) is an aggressive, chemo resistant neoplasm with poor prognosis and limited treatment options. Exploring activated pathways upon drug treatment can be used to discover more effective anticancer agents to overcome therapy resistance and enhance therapeutic outcomes for patients with advanced HCC. Human tumor-derived cell lines recapitulate HCC diversity and are widely used for studying mechanisms that drive drug resistance in HCC. In this study, we show that regorafenib treatment activates Wnt/β-catenin signaling only in hepatoblast-like HCC cell lines and induces enrichment of markers associated with hepatic stem/progenitor cells. Moreover, activation of Wnt/β-catenin signaling via Wnt3a/R-Spo1 treatment protects these cells from regorafenib induced apoptosis. On the other hand, regorafenib resistant cells established by long-term regorafenib treatment demonstrate diminished Wnt/β-catenin signaling activity while TGF-β signaling activity of these cells is significantly enhanced. Regorafenib resistant cells (RRCs) also show increased expression of several mesenchymal genes along with an induction of CD24 and CD133 cancer stem cell markers. Moreover, regorafenib resistant cells also exhibit significantly augmented in vitro and in vivo migration capacity which could be reversed by TGF-β type 1 receptor (TGFb -R1) inhibition. When combined with regorafenib treatment, TGFβ-R1 inhibition also significantly decreased colony formation ability and augmented cell death in resistant spheroids. Importantly, when we knocked down TGFβ-R1 using a lentiviral plasmid, regorafenib resistant cells entered senescence indicating that this pathway is important for their survival. Treatment of RRCs with TGFβ-R1 inhibitor and regorafenib significantly abolished pSTAT3, pSMAD2 and pERK (44/42) expression suggesting the involvement of both canonical and non-canonical pathways. In conclusion, our data suggest that HCC tumors with aberrant activation in the Wnt/β-catenin pathway, might have higher intrinsic regorafenib resistance and the inhibition of this pathway along with regorafenib administration might increase regorafenib-induced cell death in combinational therapies. However, to resolve acquired regorafenib resistance developed in HCC patients, the combined use of TGF-β pathway inhibitors and Regorafenib constitute a promising approach that can increase regorafenib sensitization and prevent tumor recurrence.

Propylparaben concentrations in the urine of women and adverse effects on ovarian function in mice in vivo and ovarian cells in vitro.

Female reproduction is precisely regulated by hormones, and the ovary is easily affected by environmental endocrine disruptors (EDCs), which are ubiquitous in industrialized societies. Parabens are EDCs that are used as antibacterial preservatives in cosmetics, personal care products (PCPs), medicines, and food. We used ultrahigh-performance liquid chromatography-mass spectrometry to quantitatively detect methyl-, ethyl-, butyl-, and propylparaben (PP) concentrations in urine samples from 74 women of childbearing age. Balb/c mice were subcutaneously injected with 100 mg/kg/day of PP for 21 consecutive days or 100 or 1,000 mg/kg/day of PP during superovulation. Various concentrations of PP (ranging from 1 to 1,000 nM) were added to a human ovarian granulosa tumor-derived cell line (KGN) culture for 24 h. The urinary paraben concentrations of women who used cosmetics and other PCPs within 48 h prior to sample collection were significantly elevated, and the PP concentration was significantly positively correlated with the basal estradiol concentration. After PP injection, the mouse serum estradiol concentrations were significantly increased, estrus cycles were disordered, corpus luteum number was reduced, and number of oocytes retrieved was significantly reduced. In in vitro experiments, PP treatment increased estradiol synthesis and the expression levels of aromatase enzyme (CYP19A1) and steroidogenic acute regulatory protein. This study demonstrates the adverse effects of PP on ovarian estradiol secretion and ovulation, further evaluates the safety of PP as a preservative, and provides guidance for the use of PCPs and cosmetics by women of childbearing age.

Abstract 1917: Immunomodulatory effects of a novel, enhanced potency gibbon ape leukaemia virus (GALV) fusogenic membrane glycoprotein-expressing herpes simplex virus platform with increased efficacy combined with anti PD-1 therapy

Abstract Oncolytic viral immunotherapies are attractive because they are self-amplifying, can kill tumors through multiple, immunologically visible mechanisms and have the ability to promote anti-tumor immune responses. RP1 is a novel, enhanced potency oncolytic HSV that has been genetically engineered to express human GM-CSF and the gibbon ape leukemia virus fusogenic membrane glycoprotein with the R sequence deleted (GALV-GP R-), providing constitutive fusion activity. GALV-GP R- binds pit-1 receptors on human and rat, but not mouse, cells to mediate cell-cell fusion. RP1 potently kills human tumor-derived cells, including a panel of melanoma and head and neck cell lines, as demonstrated here. In vivo, using a human melanoma model in nude mice, higher titres (2-3 log) of virus were retrieved from tumors treated with the virus expressing GALV-GP R- (i.e. RP1) versus an equivalent non-GALV-GP R- expressing virus. No virus was retrieved from uninjected tumors. In addition to enhanced direct effects on injected tumors, contralateral uninjected tumors were significantly smaller in groups treated with RP1 versus the GALV-GP R- non-expressing virus despite restriction of virus replication to the injected tumor. This effect was seen in nude mice, i.e. without an adaptive immune system, suggesting contribution of innate (e.g. NK cell-mediated) immunity to the contralateral effects observed. In the immunocompetent mouse 4434 melanoma model, RP1 also reduced tumor burden in both injected and uninjected tumors, again with absolute restriction of virus replication to injected tumors. Animals treated with RP1 had splenomegaly and a population of tumor-infiltrating PD-L1+ cells within injected tumors at one week post-treatment, albeit without a contribution of fusion mediated by GALV-GP R- in this model. FACS-based analysis identified these PD-L1+ cells as predominantly CD45+/CD11b(med/low)/LY6G+ neutrophils. Co-treatment with RP1 and an anti-mouse PD-1 antibody increased anti-tumor effects and the levels of CD3+ cells in uninjected tumors. Data from detailed RNAseq transcriptional analyses from injected and uninjected tumors will also be presented. Citation Format: Victoria Roulstone, Joan Kyula, Suzanne Thomas, Linta Kuncheria, Praveen K. Bommareddy, Henry Smith, Harriet Whittock, Robert S. Coffin, Kevin Harrington. Immunomodulatory effects of a novel, enhanced potency gibbon ape leukaemia virus (GALV) fusogenic membrane glycoprotein-expressing herpes simplex virus platform with increased efficacy combined with anti PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1917.

Pharmacological Assessment of the Carvacrol Chemotype Essential Oil FromPlectranthus amboinicusGrowing in Cuba

Plant-derived products are employed in various public health practices and have been considered as a major source of medicines. The genus Plectranthus (Lamiaceae) has been widely respected for its nutritional properties, its essential oil, and its therapeutic values. In the present work, the chemical characterization, antimicrobial, antiparasitic, and cytotoxic properties of the essential oil from Plectranthus amboinicus (Lour.) Spreng and its main compound carvacrol were studied. Twenty-one components were identified in the oil by gas chromatography coupled with a mass spectrometric detector. In this oil, carvacrol constitutes the major compound (71%), which represented the more abundant chemotype. The essential oil did not inhibit growth of Escherichia coli, Staphylococcus aureus, Candida albicans, Trypanosoma cruzi, or Leishmania infantum, but displayed activity against Plasmodium falciparum (half-maximal inhibitory concentration [IC50] = 5.9 µg/mL), Trypanosoma brucei (IC50= 34.9 µg/mL), and Leishmania amazonensis (IC50= 58.2 µg/mL), and the human tumor-derived cell lines MCF-7 (IC50= 29.1 µg/mL), MDA-MB-231 (IC50= 41.5 µg/mL), and 22Rv1 (IC50= 29.6 µg/mL), but no cytotoxicity was observed against nonmalignant macrophages. The antiproliferative activity of the oil could be attributed to carvacrol. However, this compound showed certain level of cytotoxicity, which suggests unspecific activity. This study provides evidence about antimicrobial and anticancer potential of the essential oil from P. amboinicus against protozoa and neoplastic diseases, particularly as an antimalarial natural product.

A disintegrin and metalloproteinase domain 17-epidermal growth factor receptor signaling contributes to oral cancer pain.

Cancer cells secrete pronociceptive mediators that sensitize adjacent sensory neurons and cause pain. Identification and characterization of these mediators could pinpoint novel targets for cancer pain treatment. In this study, we identified candidate genes in cancer cell lines that encode for secreted or cell surface proteins that may drive nociception. To undertake this work, we used an acute cancer pain mouse model, transcriptomic analysis of publicly available human tumor-derived cell line data, and a literature review. Cancer cell line supernatants were assigned a phenotype based on evoked nociceptive behavior in an acute cancer pain mouse model. We compared gene expression data from nociceptive and nonnociceptive cell lines. Our analyses revealed differentially expressed genes and pathways; many of the identified genes were not previously associated with cancer pain signaling. Epidermal growth factor receptor (EGFR) and disintegrin metalloprotease domain 17 (ADAM17) were identified as potential targets among the differentially expressed genes. We found that the nociceptive cell lines contained significantly more ADAM17 protein in the cell culture supernatant compared to nonnociceptive cell lines. Cytoplasmic EGFR was present in almost all (>90%) tongue primary afferent neurons in mice. Monoclonal antibody against EGFR, cetuximab, inhibited cell line supernatant-induced nociceptive behavior in an acute oral cancer pain mouse model. We infer from these data that ADAM17-EGFR signaling is involved in cancer mediator-induced nociception. The differentially expressed genes and their secreted protein products may serve as candidate therapeutic targets for oral cancer pain and warrant further evaluation.

Isolation and characterization of cytotoxic and insulin-releasing components from the venom of the black-necked spitting cobra Naja nigricollis (Elapidae).

Four peptides with cytotoxic activity against BRIN-BD11 rat clonal β-cells were purified from the venom of the black-necked spitting cobra Naja nigricollis using reversed-phase HPLC. The peptides were identified as members of the three-finger superfamily of snake toxins by ESI-MS/MS sequencing of tryptic peptides. The most potent peptide (cytotoxin-1N) showed strong cytotoxic activity against three human tumor-derived cell lines (LC50 = 0.8 ± 0.2 μM for A549 non-small cell lung adenocarcinoma cells; LC50 = 7 ± 1 μM for MDA-MB-231 breast adenocarcinoma cells; and LC50 = 9 ± 1 μM for HT-29 colorectal adenocarcinoma cells). However, all the peptides were to varying degrees cytotoxic against HUVEC human umbilical vein endothelial cells (LC50 in the range 2–22 μM) and cytotoxin-2N was moderately hemolytic (LC50 = 45 ± 3 μM against mouse erythrocytes). The lack of differential activity against cells derived from non-neoplastic tissue limits their potential for development into anti-cancer agents. In addition, two proteins in the venom, identified as isoforms of phospholipase A2, effectively stimulated insulin release from BRIN-BD11 cells (an approximately 6-fold increase in rate compared with 5.6 mM glucose alone) at a concentration (1 μM) that was not cytotoxic to the cells suggesting possible application in therapy for Type 2 diabetes.

Cinnamon-flavored electronic cigarette liquids and aerosols induce oxidative stress in human osteoblast-like MG-63 cells

As noncombustible nicotine delivery devices, electronic cigarettes (e-cigarettes) are the most popular tobacco product among youth. The widespread popularity of e-cigarettes combined with possible health consequences suggest a need to further research health hazards associated with e-cigarette use. Since conventional tobacco use is a risk factor for osteoporosis, this study investigates the impact of nicotine-free, cinnamon-flavored e-cigarette liquid (e-liquid) on bone-forming osteoblasts compared to flavorless e-liquid. Human tumor-derived osteoblast-like MG-63 cells were exposed for 24 h or 48 h to 0.0.4 %, 0.04 %, 0.4 % or 1 % of unvaped e-liquid or 0.0025 %, 0.025 %, 0.25 %, 1 % or 2.5 % of aerosol condensate in addition to a culture medium only control. Changes in cell viability were assessed by MTT assay, and the expression of a key bone protein, collagen type I, was analyzed by immunofluorescence. Production of reactive oxygen species (ROS) was detected by fluorometry to assess oxidative stress. Cell viability decreased in a dose-dependent manner, and ROS production increased, which was most pronounced with cinnamon-flavored e-liquids. There were no detectable changes in collagen type I protein following exposure to any of the aerosol condensates. This study demonstrates osteoblast-like cells are sensitive to both e-liquids and aerosol condensates and suggests the cytotoxicity of cinnamon-flavored e-liquids might be associated with oxidative stress rather than changes in collagen type I protein expression. This in vitro study provides insight into the potential impacts of e-cigarette use on bone cells.

Transcriptional analysis and differentially expressed gene screening of spontaneous liver tumors in CBA/CaJ mice

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide due to its frequent metastasis, tumor recurrence, and lack of curative treatment. However, the underlying molecular mechanisms involved in HCC progression remain unclear. Here, we analyzed the global gene expression of spontaneous liver tumor tissue from CBA/CaJ mice by RNA-Seq and identified 10,706 and 10,374 genes in the normal and liver tumor groups, respectively. Only 9793 genes were expressed in both, 913 genes were identified in only the liver tumor group, and 581 genes were found in normal liver tissues. There were 2054 differentially expressed genes (DEGs), with 975 down-regulated genes and 1079 up-regulated genes. Gene ontology (GO) term enrichment analysis showed that 43 up-regulated genes were significantly associated with cell cycle regulation and hundreds of up-regulated genes were related to cell migration, adhesion, or metabolic processes. KEGG pathway enrichment also demonstrated that some DEGs were tightly associated with the cell cycle, extracellular matrix (ECM)-receptor interactions, as well as protein digestion and absorption pathways, indicating that the activation of these oncogenic cascades was closely related to tumor liver progression in CBA/CaJ mice. Ninety-three genes with elevated expression levels preferentially localized in microtubules, kinetochores, and spindles play an important role during mitosis and meiosis and are associated with the reorganization of the cytoskeleton in cancer cells during migration and invasion. Some ECM-related genes were significantly different in the tumor group, including collagen types I, III, IV, V, and VI, non-collagenous glycoproteins, laminin, and fibronectin. We further validated the functions of upregulated genes, such as cyclin-dependent kinase 1 (CDK1) and polo-like kinase 1 (PLK1), with regards to cell cycle regulation, apoptosis, and proliferation in normal human liver or liver tumor-derived cell lines. Our results indicated that the cell cycle dysregulation, ECM-receptor interaction, and cytoskeleton-associated genes in mouse livers may promote HCC progression and deciphering the function of the genes will help investigators understand the underlying molecular mechanism of HCC.

The MYC Oncogene Cooperates with Sterol-Regulated Element-Binding Protein to Regulate Lipogenesis Essential for Neoplastic Growth

Lipid metabolism is frequently perturbed in cancers, but the underlying mechanism is unclear. We present comprehensive evidence that oncogene MYC, in collaboration with transcription factor sterol-regulated element-binding protein (SREBP1), regulates lipogenesis to promote tumorigenesis. We used human and mouse tumor-derived cell lines, tumor xenografts, and four conditional transgenic mouse models of MYC-induced tumors to show that MYC regulates lipogenesis genes, enzymes, and metabolites. We found that MYC induces SREBP1, and they collaborate to activate fatty acid (FA) synthesis and drive FA chain elongation from glucose and glutamine. Further, by employing desorption electrospray ionization mass spectrometry imaging (DESI-MSI), we observed invivo lipidomic changes upon MYC induction across different cancers, for example, aglobal increase in glycerophosphoglycerols. After inhibition of FA synthesis, tumorigenesis was blocked, and tumors regressed in both xenograft and primary transgenic mouse models, revealing the vulnerability of MYC-induced tumors to the inhibition of lipogenesis.

RUNX3 Promotes the Tumorigenic Phenotype in KGN, a Human Granulosa Cell Tumor-Derived Cell Line.

Granulosa cell tumors of the ovary (GCT) are the predominant type of ovarian sex cord/stromal tumor. Although prognosis is generally favorable, the outcome for advanced and recurrent GCT is poor. A better understanding of the molecular pathogenesis of GCT is critical to developing effective therapeutic strategies. Here we have examined the potential role of the runt-related transcription factor RUNX3. There are only two GCT cell lines available. While RUNX3 is silenced in the GCT cell line KGN cells, it is highly expressed in another GCT cell line, COV434 cells. Re-expression of RUNX3 promotes proliferation, anchorage-independent growth, and motility in KGN cells in vitro and tumor formation in mice in vivo. Furthermore, expression of a dominant negative form of RUNX3 decreases proliferation of COV434 cells. To address a potential mechanism of action, we examined expression of cyclin D2 and the CDK inhibitor p27Kip1, two cell cycle regulators known to be critical determinants of GCT cell proliferation. We found that RUNX3 upregulates the expression of cyclin D2 at the mRNA and protein level, and decreases the level of the p27Kip1 protein, but not p27Kip1 mRNA. In conclusion, we demonstrate that RUNX proteins are expressed in GCT cell lines and human GCT specimens, albeit at variable levels, and RUNX3 may play an oncogenic role in a subset of GCTs.