Sesquiterpene lactone dehydroleucodine selectively induces transient arrest in G2 in Allium cepa root meristematic cells

Dehydroleucodine is a sesquiterpene lactone recently isolated from aerial parts of a medicinal herb, Artemisia douglasiana Besser. We have previously shown that 25 and 100 microM dehydroleucodine slowed down onion root growth by 30 and 70%, respectively, affecting neither cell viability nor cell elongation. In the present study we analyze the effect of dehydroleucodine on cell cycle phases in onion (Allium cepa L.) root meristematic cells synchronized with caffeine or caffeine and hydroxyurea. Synchronized root cells treated with 100 and 200 microM dehydroleucodine present an interphase lengthening of 5.2 h and 8.2 h, respectively. The S-phase length, estimated by [3H]thymidine incorporation assay, was 6 h for both control roots and roots that had been immersed in dehydroleucodine. The peak of [3H]leucine incorporation was observed 6 h after release from synchronization in controls and in dehydroleucodine-treated roots, indicating that protein synthesis in G2 was not affected. Thus, these results show that dose-dependently dehydroleucodine selectively induces a transient arrest of meristematic cell in G2 and that dehydroleucodine can be used experimentally as a cell cycle suppressor.     

Fluorescence probes in metastatic B16 melanoma membranes

Fluorescence probe molecules, trans-parinaric acid and 1,6-diphenylhexatriene, were utilized to characterize the structure of plasma membranes, microsomes and mitochondria from B16 melanoma cells. High metastatic B16-F10 and low metastatic B16-F1 melanoma cell lines had markedly different membrane structures. The fluorescence polarization, fluorescence lifetime and limiting anisotropy of trans-parinaric acid were significantly lower ($\text{P < 0.05}$) in all three membrane fractions of the B16-F1 cell line than in the corresponding membranes of the B16-F10 cell line. These data indicated less restriction to rotational motion in the solid lipid domains of B16-F1 cell membranes preferentially sensed by trans-parinaric acid. The limiting anisotropy of both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene was significantly lower in the outer monolayer than the inner monolayer of the plasma membrane of B16-F1 cells but not in B16-F10 cells. A breakpoint in Arrhenius plots of fluorescence near 30–34°C indicated the presence of a phase separation that was assigned to the inner monolayer of the plasma membrane. However, no differences in this breakpoint temperature were noted between the B16-F1 and B16-F10 melanoma membranes. Thus, more fluid solid membrane domains and a distinct transbilayer fluidity difference were characteristic of plasma membranes from low metastatic B16-F1 melanoma cells in contrast to high metastatic B16-F10 melanoma cells.     

Immunization of mice with melanoma cells transfected to secrete the superantigen, staphylococcal enterotoxin A

Immunization of mice with a melanoma vaccine coupled with staphylococcal enterotoxin A (SEA) inhibits the growth of primary melanoma tumors in mice. We have now successfully transfected B16 cells with the sea gene and have immunized C57BL/6 mice subcutaneously once per week for 4 weeks prior to tumor challenge with vaccines of irradiated B16 cells or, 4 weeks following tumor challenge of naïve mice with B16 cells, with irradiated B16 cells transfected with the sea gene. Primary tumor growth following both types of treatments was inhibited significantly. To characterize immune responses to these immunogens, we examined the production of antibodies to the B700 melanoma antigen, the stimulation of endogenous IL-2 production, the expression of CD4, CD8, Vβ and CD25 T cell markers, and the induction of NK activity. At 4 weeks following immunization of mice, there was a significant increase (P<0.05) in levels of interleukin-2 production by splenocytes from mice immunized with SEA-secreting B16 cells or with the parental B16 cells, compared to controls. Levels of antibodies to the B700 melanoma antigen were also significantly higher in mice immunized with the SEA-secreting B16 cells, as was expression of CD4, CD8, CD25 and Vβ T cell antigens, particularly CD4. Natural killer cell activity (at various E:T ratios) was tenfold higher in splenocytes of mice immunized with SEA-secreting B16 cells, and fivefold higher in mice immunized with the parental B16 cells, compared to controls.?These data confirm the possibility of using irradiated murine melanoma cells transfected to secrete SEA in vaccines targeted at preventing the development and growth of melanoma.     

4-氨基苯酚维甲酰胺对肝癌和恶性黑色素瘤细胞的抑制作用

In this paper, a significantly effect of N-(4-hydrophenyl) retinamide (4-HPR), a derivative of retinoic acid, was observed on inhibition of migration, invasion, cell growth, and induction of apoptosis in hepatoma cells and B16 melanoma cells. The number of migratory hepatoma cells reduced significantly from the control 201 +/- 27.2 to 58 +/- 5.03 after 6-hour incubation with 4-HPR (p < 0.01, n = 4). The number of migratory B16 melanoma cells reduced from the control 302 +/- 30.1 to 254 +/- 25.04 (p < 0.05, n = 4). The invasive ability of these cells was also suppressed by 4-HPR treatment. Cells that penetrated the artificial membrane matrigel decreased from 27 +/- 13.1 to 11.2 +/- 3.3 in hepatoma cells, from 67.5 +/- 10.1 to 24.3 +/- 3.2 in B16 melanoma cells (p < 0.05, n = 3). Furthermore, cell growth was significantly inhibited especially in B16 melanoma cells and 37.11 +/- 0.94% cells were induced to apoptosis after 48-hour induction by 4-HPR, which was significantly higher than those by retinoic acid treatment (p < 0.05). Although the mechanism of 4-HPR effects was not very clear, over expression of CST, which was inhibited by 4-HPR in our previous study, could diminish the apoptosis--inducing effect by 4-HPR. We believe that 4-HPR has a strong inhibitory effect on melanoma and hepatocarcinoma cells and might become a potent therapeutic agent.     

Potent anti-metastatic activity of dimeric sesquiterpene thioalkaloids from the rhizome of Nuphar pumilum

The methanolic extract and its alkaloid fraction from the rhizomes of Nuphar pumilum inhibited invasion of B16 melanoma cells across collagen-coated filters in vitro. Dimeric sesquiterpene thioalkaloids with the 6-hydroxyl group, 6-hydroxythiobinupharidine, 6,6′-dihydroxythiobinupharidine, and 6-hydroxythionuphlutine B, showed potent activity with IC₅₀ values of 0.029, 0.087, and 0.36 μM, respectively, but dimeric sesquiterpene thioalkaloids lacking the 6-hydroxyl group (thiobinupharidine, neothiobinupharidine, syn-thiobinupharidine sulfoxide, thionuphultine B β-sulfoxide, and neothiobinupharidine β-sulfoxide) and monomeric sesquiterpene alkaloids (nupharidine, deoxynupharidine, 7-epideoxynupharidine, and nupharolutine) showed weak activity. The alkaloid fraction (20 mg/kg/d, po) and the principal dimeric sesquiterpene thioalkaloid 6-hydroxythiobinupharidine (5 mg/kg/d, po) significantly inhibited lung tumor formation by more than 90% 10 days after injection of B16 melanoma cells in mice.     

Anti-Tumor Effects after Adoptive Transfer of IL-12 Transposon-Modified Murine Splenocytes in the OT-I-Melanoma Mouse Model

Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments. We have previously used the non-viral piggyBac transposon system to gene modify human T cells for potential immunotherapy. However, these previous studies utilized adoptive transfer of modified human T cells to target cancer xenografts in highly immunodeficient (NOD-SCID) mice that do not recapitulate an intact immune system. Currently, only viral vectors have shown efficacy in permanently gene-modifying mouse T cells for immunotherapy applications. Therefore, we sought to determine if piggyBac could effectively gene modify mouse T cells to target cancer cells in a mouse cancer model. We first demonstrated that we could gene modify cells to express murine interleukin-12 (p35/p40 mIL-12), a transgene with proven efficacy in melanoma immunotherapy. The OT-I melanoma mouse model provides a well-established T cell mediated immune response to ovalbumin (OVA) positive B16 melanoma cells. B16/OVA melanoma cells were implanted in wild type C57Bl6 mice. Mouse splenocytes were isolated from C57Bl6 OT-I mice and were gene modified using piggyBac to express luciferase. Adoptive transfer of luciferase-modified OT-I splenocytes demonstrated homing to B16/OVA melanoma tumors in vivo. We next gene-modified OT-I cells to express mIL-12. Adoptive transfer of mIL-12-modified mouse OT-I splenocytes delayed B16/OVA melanoma tumor growth in vivo compared to control OT-I splenocytes and improved mouse survival. Our results demonstrate that the piggyBac transposon system can be used to gene modify splenocytes and mouse T cells for evaluating adoptive immunotherapy strategies in immunocompetent mouse tumor models that may more directly mimic immunotherapy applications in humans.     

The effects of patchouli alcohol and combination with cisplatin on proliferation,apoptosis and migration in B16F10 melanoma cells

Melanoma is a highly metastatic cancer with a low incidence rate, but a high mortality rate. Patchouli alcohol (PA), a tricyclic sesquiterpene, is considered the main active component in Pogostemon cablin Benth, which improves wound healing and has anti-tumorigenic activity. However, the pharmacological action of PA on anti-melanoma remains unclear. Thus, the present study aimed to investigate the role of PA in the proliferation, cell cycle, apoptosis and migration of melanoma cells. These results indicated that PA selectively inhibited the proliferation of B16F10 cells in a dose- and time-dependent manner. It induced cell cycle arrest at the G₀/G₁ phase and typical morphological changes in apoptosis, such as chromatin condensation, DNA fragmentation and apoptotic bodies. In addition, PA reduced the migratory ability of B16F10 cells by upregulating E-cadherin and downregulating p-Smad2/3, vimentin, MMP-2 and MMP-9 expression. PA was also found to strongly suppress tumour growth in vivo. Furthermore, PA combined with cisplatin synergistically inhibited colony formation and migration of B16F10 cells and attenuated the development of resistance to treatment. Therefore, the results of this study indicate that PA may play a pivotal role in inducing apoptosis and reducing the migration of melanoma cells, and may thus be a potential candidate for melanoma treatment.     

LPA is a chemorepellent for B16 melanoma cells: action through the cAMP-elevating LPA5 receptor

Lysophosphatidic acid (LPA), a lipid mediator enriched in serum, stimulates cell migration, proliferation and other functions in many cell types. LPA acts on six known G protein-coupled receptors, termed LPA(1-6), showing both overlapping and distinct signaling properties. Here we show that, unexpectedly, LPA and serum almost completely inhibit the transwell migration of B16 melanoma cells, with alkyl-LPA(18:1) being 10-fold more potent than acyl-LPA(18:1). The anti-migratory response to LPA is highly polarized and dependent on protein kinase A (PKA) but not Rho kinase activity; it is associated with a rapid increase in intracellular cAMP levels and PIP3 depletion from the plasma membrane. B16 cells express LPA(2), LPA(5) and LPA(6) receptors. We show that LPA-induced chemorepulsion is mediated specifically by the alkyl-LPA-preferring LPA(5) receptor (GPR92), which raises intracellular cAMP via a noncanonical pathway. Our results define LPA(5) as an anti-migratory receptor and they implicate the cAMP-PKA pathway, along with reduced PIP3 signaling, as an effector of chemorepulsion in B16 melanoma cells.     

Inhibition of murine melanoma cell-matrix adhesion and experimental metastasis by albolabrin, an RGD-containing peptide isolated from the venom of Trimeresurus albolabris

Albolabrin, a 7.5-kDa cysteine-rich protein isolated from the venom of Trimeresurus albolabris, contains the arginine-glycine-aspartic acid (RGD) cell recognition sequence found in many cell adhesion-promoting extracellular matrix proteins, such as fibronectin and laminin. Albolabrin belongs to a family of RGD-containing peptides, termed disintegrins, recently isolated from the venom of various vipers and discovered to be potent inhibitors of both platelet aggregation and cell-substratum adhesion. Here we report that albolabrin inhibited the attachment of B16-F10 mouse melanoma cells to either fibronectin or laminin absorbed on plastic. When immobilized on plastic, albolabrin promoted B16-F10 melanoma cell attachment; this was inhibited by either RGD-serine (RGDS) or antibodies to integrins, suggesting that albolabrin binds via its RGD amino sequence to integrin receptors expressed on the melanoma cell surface. In an in vivo experimental metastasis system, albolabrin at a concentration of 300-600 nM inhibited C57BL/6 mouse lung colonization by tail vein-injected mouse melanoma cells and was at least 2000 times more active than RGDS in this assay. We propose that albolabrin inhibits tumor cell metastasis by inhibiting integrin-mediated attachment of melanoma cells to RGD-containing components of the extracellular matrix in the mouse lung.     

Nuphar alkaloids with immediately apoptosis-inducing activity from Nuphar pumilum and their structural requirements for the activity

The methanolic extract and its alkaloid fraction from the rhizomes of Nuphar pumilum showed cytotoxic effects on human leukemia cell (U937), mouse melanoma cell (B16F10), and human fibroblast (HT1080). Dimeric sesquiterpene thioalkaloids with the 6-hydroxyl group (6-hydroxythiobinupharidine, 6,6'-dihydroxythiobinupharidine, 6-hydroxythionuphlutine B) showed substantial cytotoxic activity at a concentration of 10 microM, but dimeric sesquiterpene thioalkaloids lacking the 6-hydroxyl group (thiobinupharidine, thionuphlutine B, 6'-hydroxythionuphlutine B, neothiobinupharidine, thionuphlutine B beta-sulfoxide, and neothiobinupharidine beta-sulfoxide) and monomeric sesquiterpene alkaloids (nupharidine, 7-epideoxynupharidine, and nupharolutine) showed weak activity. Next, apoptosis-inducing activity of a principal active constituent, 6-hydroxythiobinupharidine, on U937 was examined using morphological observation and DNA fragmentation assay (TUNEL method). Apoptosis of U937 was immediately observed within 1 h after treatment of 6-hydroxythiobinupharidine at 2.5-10 microM.     

Proteomic studies of B16 lines: involvement of annexin A1 in melanoma dissemination

To identify proteins involved in melanoma metastasis mechanisms, comparative proteomic studies were undertaken on B16F10 and B16Bl6 melanoma cell lines and their subsequent syngenic primary tumours as pulmonary metastases were present only in the mice bearing a B16Bl6 tumour. 2DE analyses followed by MALDI-TOF identification showed variations of 6 proteins in vitro and 13 proteins in vivo. Differential expressed proteins in tumours were related to energy production and storage. Two differentially expressed proteins which had not been previously associated to melanoma progression, annexin A1 (ANXA1) and creatine kinase B (CKB), were found both in cells and in tumours. To characterize ANXA1 involvement in melanoma B16 dissemination, we reduced ANXA1 protein level by siRNA and observed a significant decrease of B16Bl6 cell invasion through Matrigel coated chambers. We further demonstrated that the presence of several formyl peptide receptors (FPR1, FPRrs1 and 2) revealed by qRT-PCR, played a role in B16 invasion: incubation of B16Bl6 cells with the FPR agonist (fMLP) or antagonist (tBOC) enhanced or decreased Matrigel coated chamber invasion respectively, with a correlation of ANXA1 levels in both treatments. As ANXA1 could bind to FPRs, this should amplify invasion and enhance melanoma dissemination.     

Characterization and Inducing Melanoma Cell Apoptosis Activity of Mannosylerythritol Lipids-A Produced from Pseudozyma aphidis

Mannosylerythritol lipids (MELs) are natural glycolipid biosurfactants which have potential applications in the fields of food, cosmetic and medicine. In this study, MELs were produced from vegetable oil by Pseudozyma aphidis. Their structural data through LC/MS, GC/MS and NMR analysis revealed that MEL-A with two acetyls was the major compound and the identified homologs of MEL-A contained a length of C8 to C14 fatty acid chains. This glycolipid exhibited a surface tension of 27.69 mN/m at a critical micelle concentration (CMC), self-assembling into particles in the water solution. It was observed to induce cell growth-inhibition and apoptosis of B16 melanoma cells in a dose-dependent manner, as well as cause cell cycle arrest at the S phase. Further quantitative RT-PCR analysis and western blotting revealed an increasing tendency of both mRNA and protein expressions of Caspase-12, CHOP, GRP78 and Caspase-3, and a down-regulation of protein Bcl-2. Combined with the up regulation of signaling IRE1 and ATF6, it can be speculated that MEL-A-induced B16 melanoma cell apoptosis was associated with the endoplasmic reticulum stress (ERS).     

Effect of Vascular Formed Endothelial Cell Network on the Invasive Capacity of Melanoma Using the In Vitro 3D Co-Culture Patterning Model

In vitro three dimensional (3D) cancer models were developed to observe the invasive capacity of melanoma cell spheroids co-cultured with the vascular-formed endothelial cell network. An array-like multicellular pattern of mouse melanoma cell line B16F1 was developed by magnetic cell labeling using a pin-holder device for allocation of magnetic force. When the B16F1 patterned together with a vascular network of human umbilical vein epithelial cells (HUVEC), spreading and progression were observed along the HUVEC network. The B16F1 cells over 80 µm distance from HUVEC remain in a compact spheroid shape, while B16F1 in the proximity of HUVEC aggressively changed their morphology and migrated. The mRNA expression levels of IL-6, MDR-1 and MMP-9 in B16F1 increased along with the distance the HUVEC network, and these expressions were increased by 5, 3 and 2-fold in the B16F1 close to HUVEC (within 80 µm distance) as compared to that far from HUVEC (over 80 µm distance). Our results clearly show that malignancy of tumor cells is enhanced in proximity to vascular endothelial cells and leads to intravasation.     

Protein kinase C and linoleic acid-induced inhibition of melanogenesis

Linoleic acid has been shown to inhibit melanogenesis in cultured B16 mouse melanoma cells. We report here the possible involvement of protein kinase C (PKC) in linoleic acid-induced inhibition of melanogenesis in B16 cells. A single PKC subspecies (alpha-PKC) was detected in B16 cells. The enzyme was activated by linoleic acid in vitro. The effective concentrations at which PKC was activated (25 microM; maximum response) were consistent with those for the inhibition of melanogenesis in cell culture system. In addition, the permeable diacylglycerol 1-oleoyl-2-acetyl glycerol that activates PKC also inhibits melanogenesis at 100 microM. These results suggest that activation of PKC plays a pivotal role in the linoleic acid-induced inhibition of melanogenesis in B16 cells.     

Differential Antitumor Effects of IgG and IgM Monoclonal Antibodies and Their Synthetic Complementarity-Determining Regions Directed to New Targets of B16F10-Nex2 Melanoma Cells

Malignant melanoma has increased incidence worldwide and causes most skin cancer-related deaths. A few cell surface antigens that can be targets of antitumor immunotherapy have been characterized in melanoma. This is an expanding field because of the ineffectiveness of conventional cancer therapy for the metastatic form of melanoma. In the present work, antimelanoma monoclonal antibodies (mAbs) were raised against B16F10 cells (subclone Nex4, grown in murine serum), with novel specificities and antitumor effects in vitro and in vivo. MAb A4 (IgG2ak) recognizes a surface antigen on B16F10-Nex2 cells identified as protocadherin β₁₃. It is cytotoxic in vitro and in vivo to B16F10-Nex2 cells as well as in vitro to human melanoma cell lines. MAb A4M (IgM) strongly reacted with nuclei of permeabilized murine tumor cells, recognizing histone 1. Although it is not cytotoxic in vitro, similarly with mAb A4, mAb A4M significantly reduced the number of lung nodules in mice challenged intravenously with B16F10-Nex2 cells. The V_H CDR3 peptide from mAb A4 and V_L CDR1 and CDR2 from mAb A4M showed significant cytotoxic activities in vitro, leading tumor cells to apoptosis. A cyclic peptide representing A4 CDR H3 competed with mAb A4 for binding to melanoma cells. MAb A4M CDRs L1 and L2 in addition to the antitumor effect also inhibited angiogenesis of human umbilical vein endothelial cells in vitro. As shown in the present work, mAbs A4 and A4M and selected CDR peptides are strong candidates to be developed as drugs for antitumor therapy for invasive melanoma.     

Heparanase mechanisms of melanoma metastasis to the brain: Development and use of a brain slice model

Heparanase (HPSE-1) is an endo-beta-D-glucuronidase that cleaves heparan sulfate (HS) chains of proteoglycans (HSPG), and its expression has been associated with increased cell growth, invasion, and angiogenesis of tumors as well as with embryogenesis and tissue development. Since metastatic cancer cells express HPSE-1, we have developed an orthotopic brain slice model to study HPSE-1 involvement in brain-metastatic melanoma. This model allows for the characterization of tumor cell invasion at both quantitative and qualitative levels. Brain-metastatic melanoma cells (B16B15b) showed augmenting levels of HPSE-1 protein expression in a time-dependent manner. Secondly, B16B15b cells pre-treated with HPSE-1 showed a significant increase in the number of cells that invaded into the brain tissue. Finally, HPSE-1 exposure-augmented invasion depth in brain sections by brain-metastatic melanoma cells. We concluded that applying this brain slice model can be beneficial to investigate HPSE-1- related in vivo modalities in brain-metastatic melanoma and brain invasion in general. These results also further emphasize the potential relevance of using this model to design therapies for controlling this type of cancer by blocking HPSE-1 functionality.     

Guanosine 5′-triphosphate inhibits growth and stimulates differentiated functions in B16 melanoma cells

Exogenous guanosine 5′-triphosphate (GTP) at a concentration of 0.5 mM causes in vitro growth inhibition and induces morphological and biochemical differentiation of B16 melanoma cells. After two days in the presence of GTP, cell proliferation is markedly reduced. Cessation of cell proliferation is followed by the extension of numerous dendrite-like processes and marked increase in melanin production. Other nucleotides such as guanosine 5′-diphosphate (GDP), guanosine 5′-monophosphate (GMP), guanosine 3′:5′-cyclic-monophosphoric acid (cGMP) or adenosine 5′-triphosphate (ATP) have little or no effect on cell morphology or melanin production in B16 melanoma cells, although these compounds retard cell proliferation similar to GTP. These findings are discussed in light of a possible relationship between cell proliferation and differentiation.     

Diterpenoids and a sesquiterpene lactone from viguiera ladibractate

Eight known diterpene acids, ent-12-oxokaur-9(11),16-dien-19-oic acid, ent-12β-hydroxykaur-9(11),16-dien-19-oic acid, ent-isokaur-15(16)-en-17,19-dioic acid, ent-15α,16-epoxy-17-hydroxykaura-19-oic acid, ent-kaura-17,19-dioic acid, ent-kaur-16-en-19-oic acid, grandifloric acid, angeloyloxygrandifloric acid, as well as a new sesquiterpene lactone, ladibranolide, were isolated from Viguiera ladibractate. The stereochemistry of the sesquiterpene lactone was established by NOE experiments.