Correction to: Daphnane diterpenes inhibit the metastatic potential of B16F10 murine melanoma cells in vitro and in vivo.

ᅟ.

Daphnane diterpenes inhibit the metastatic potential of B16F10 murine melanoma cells in vitro and in vivo.

BACKGROUND: Melanoma is one of the most invasive and aggressive types of cancer with a very poor prognosis. Surgery remains the most efficient treatment prior melanoma invasion and metastasis formation. However, therapy becomes a challenge once the cancer cells colonized other tissues. At present, there are two main classes of therapies acting with a certain efficiency on metastatic melanoma: immune check point inhibitors (anti-PD1/PDL1) and targeted therapy such as Vemurafenib. Unfortunately, these therapies are not fully responsive, induce resistance and/or generate unwanted side effects. In this respect, it is important to continue to discover new cancer therapeutics. Here, we show that daphnane diterpenes type of compounds can prevent melanoma metastasis by inhibiting metastasis-associated matrix metalloproteinases expression without cytotoxicity. METHODS: Evaluation of the anti-metastasis effect of daphnane diterpenes-rich Thymelaea hirsuta extract (TH) and its bioactive component gnidilatidin was carried out in vitro using B16 murine melanoma cells and in vivo using male C57BL/6 J mice. Global gene expression in B16 cells was done using DNA microarray, validated using real-time PCR, to further understand the effect of daphnane diterpenes, specifically daphnane diterpenoid gnidilatidin. RESULTS: Oral administration of daphnane diterpenes-rich Thymelaea hirsuta extract (TH) suppressed MMP2 and MMP9 expression, decreasing lung tumor in mice injected with B16 murine melanoma cells. Validation of these observations in vitro showed reduced B16 cells migration, adhesion, and invasion. Results of microarray analysis of B16 cells treated with daphnane diterpenoid gnidilatidin from TH revealed an upregulation of tumor suppressor Egr1 while inhibiting metastasis-associated genes Id2 and Sytl2 expression. A downregulation of the melanoma oncogene microphthalmia-associated transcription factor (Mitf) was observed, and most likely caused by the inhibition of Id2, a gene that regulated HLH transcription factors such as MITF and also reported to promote tumor cell migration and invasion. CONCLUSIONS: Daphnane diterpenes have inhibitory effect on the metastatic potential of B16 melanoma cells, and the results of this study provided evidence for their potential for use in the prevention and inhibition of melanoma metastasis.

Lupenone from Erica multiflora leaf extract stimulates melanogenesis in B16 murine melanoma cells through the inhibition of ERK1/2 activation.

Hypopigmentation diseases are usually managed using UVB light which increases the patients' risk for skin cancer. Here, we evaluated the melanogenesis stimulatory effects of leaf extracts of Erica multiflora, a medicinal plant from the Mediterranean region, and its active component, lup-20(29)-en-3-one, as possible therapeutic agents to address hypopigmentation disorders. B16 murine melanoma cells were treated with E. multiflora extracts or its active component lupenone to evaluate their effects on melanin biosynthesis. The mechanism underlying the observed effects was also determined. Bioactivity-guided fractionation of fifteen ethyl acetate fractions identified fraction 2 to have melanogenesis stimulatory effects due to its ability to decrease mitogen-activated protein kinase phosphorylated extracellular signal-regulated kinases 1 and 2 activation. Preparative TLC of ethyl acetate fraction 2 revealed the presence of lup-20(29)-en-3-one as the major bioactive component. B16 cells treated with lup-20(29)-en-3-one increased melanin content without cytotoxicity. To determine the mechanism for the observed effects of lup-20(29)-en-3-one, the tyrosinase enzyme activity, the tyrosinase protein expression, and the activation of phosphorylated extracellular signal-regulated kinases 1 and 2 were determined. In addition, the expression of the tyrosinase mRNA was quantified using real-time PCR. Results showed that lup-20(29)-en-3-one has no effect on the tyrosinase enzyme activity but can increase tyrosinase expression at both the transcriptional and translational levels. The increase in the tyrosinase mRNA expression was most likely due to the inhibited mitogen-activated protein kinase phosphorylated extracellular signal-regulated kinases 1 and 2 activation. We report for the first time that E. multiflora ethyl acetate extract and its active compound lup-20(29)-en-3-one stimulate melanogenesis by increasing the tyrosinase enzyme expression via mitogen-activated protein kinase phosphorylated extracellular signal-regulated kinases 1 and 2 phosphorylation inhibition, making it a possible treatment for hypopigmentation diseases.

PTPδ is a presynaptic organizer for the formation and maintenance of climbing fiber to Purkinje cell synapses in the developing cerebellum.

Functionally mature neural circuits are shaped during postnatal development by eliminating redundant synapses formed during the perinatal period. In the cerebellum of neonatal rodents, each Purkinje cell (PC) receives synaptic inputs from multiple (more than 4) climbing fibers (CFs). During the first 3 postnatal weeks, synaptic inputs from a single CF become markedly larger and those from the other CFs are eliminated in each PC, leading to mono-innervation of each PC by a strong CF in adulthood. While molecules involved in the strengthening and elimination of CF synapses during postnatal development are being elucidated, much less is known about the molecular mechanisms underlying CF synapse formation during the early postnatal period. Here, we show experimental evidence that suggests that a synapse organizer, PTPδ, is required for early postnatal CF synapse formation and the subsequent establishment of CF to PC synaptic wiring. We showed that PTPδ was localized at CF-PC synapses from postnatal day 0 (P0) irrespective of the expression of Aldolase C (Aldoc), a major marker of PC that distinguishes the cerebellar compartments. We found that the extension of a single strong CF along PC dendrites (CF translocation) was impaired in global PTPδ knockout (KO) mice from P12 to P29-31 predominantly in PCs that did not express Aldoc [Aldoc (-) PCs]. We also demonstrated via morphological and electrophysiological analyses that the number of CFs innervating individual PCs in PTPδ KO mice were fewer than in wild-type (WT) mice from P3 to P13 with a significant decrease in the strength of CF synaptic inputs in cerebellar anterior lobules where most PCs are Aldoc (-). Furthermore, CF-specific PTPδ-knockdown (KD) caused a reduction in the number of CFs innervating PCs with decreased CF synaptic inputs at P10-13 in anterior lobules. We found a mild impairment of motor performance in adult PTPδ KO mice. These results indicate that PTPδ acts as a presynaptic organizer for CF-PC formation and is required for normal CF-PC synaptic transmission, CF translocation, and presumably CF synapse maintenance predominantly in Aldoc (-) PCs. Furthermore, this study suggests that the impaired CF-PC synapse formation and development by the lack of PTPδ causes mild impairment of motor performance.

mGluR5 Is Substitutable for mGluR1 in Cerebellar Purkinje Cells for Motor Coordination, Developmental Synapse Elimination, and Motor Learning.

Group I metabotropic glutamate receptors (mGluRs) include mGluR1 and mGluR5, which are coupled to the Gq family of heterotrimeric G-proteins and readily activated by their selective agonist 3,5-dihydroxyphenilglycine (DHPG). mGluR1 and mGluR5 exhibit nearly complementary distributions spatially or temporally in the central nervous system (CNS). In adult cerebellar Purkinje cells (PCs), mGluR1 is a dominant group I mGluR and mGluR5 is undetectable. mGluR1 expression increases substantially during the first three weeks of postnatal development and remains high throughout adulthood. On the other hand, mGluR5 expression is observed during the first two postnatal weeks and then decreases. However, functional differences between mGluR1 and mGluR5 in the CNS remains to be elucidated. To address this issue, we generated "mGluR5-rescue" mice in which mGluR5 is specifically expressed in PCs in global mGluR1-knockout (KO) mice. mGluR5-rescue mice exhibited apparently normal motor coordination, developmental elimination of redundant climbing fiber (CF)-PC synapses, and delay eyeblink conditioning, which were severely impaired in mGluR1-KO mice. We concluded that mGluR5 is functionally comparable with mGluR1 in cerebellar PCs.

Phospholipase C ß3 is Required for Climbing Fiber Synapse Elimination in Aldolase C-positive Compartments of the Developing Mouse Cerebellum.

In the cerebellum of neonatal mice, multiple climbing fibers (CFs) form excitatory synapses on each Purkinje cell (PC). Only one CF is strengthened in each PC from postnatal day 3 (P3) to P7, whereas the other weaker CFs are eliminated progressively from ∼P7 to ∼P11 (early phase of CF elimination) and from ∼P12 to ∼P17 (late phase of CF elimination). Type 1 metabotropic glutamate receptor (mGluR1) triggers a canonical pathway in PCs for the late phase of CF elimination. Among downstream signaling molecules of mGluR1, phospholipase C ß3 (PLCß3) and ß4 (PLCß4) are expressed complementarily in PCs of aldolase C (Aldoc)-positive (+) and Aldoc-negative (-) cerebellar compartments, respectively. PLCß4 is reported to mediate the late phase of CF elimination in the anterior half of the cerebellar vermis which corresponds to the Aldoc (-) region. However, roles of PLCß3 and Aldoc in CF synapse elimination are unknown. Here, we investigated CF innervation of PCs in Aldoc-tdTomato knock-in mice that underwent lentivirus-mediated knockdown (KD) of PLCß3 in PCs during postnatal development. By recording CF-mediated excitatory postsynaptic currents from PCs and immunostaining CF synaptic terminals, we found that significantly higher percentage of PCs with PLCß3-KD remained multiply innervated by CFs in Aldoc (+) compartments after P12, which was accompanied by impaired elimination of somatic CF synapses and reduced dendritic CF translocation. In contrast, deletion of Aldoc had no effect on CF synapse elimination. These results suggest that PLCß3 is required for the late phase of CF elimination in Aldoc (+) PCs.

Effect of Tunisian Capparis spinosa L. extract on melanogenesis in B16 murine melanoma cells.

The effect of Tunisian Capparis spinosa L. aromatic plant extract on melanogenesis regulation in B16 murine melanoma cells was investigated. B16 cells were treated with 0.0005, 0.005, and 0.05% (w/v) C. spinosa extract after which the melanin content and cell viability were measured. To clarify the mechanism behind melanogenesis regulation, the expression of tyrosinase was determined. Results showed that the extract had a significant stimulative effect on melanogenesis in B16 cells in a dose-dependent manner without cytotoxicity. Western blot analysis showed that expression of tyrosinase in cells treated with 0.03% (w/v) C. spinosa extract increased by 12.5- and 20-fold after 24 and 48 h of incubation, respectively, compared with untreated cells. HPLC analysis of the extract revealed the presence of 1% quercetin, a known melanogenesis stimulator, indicating that our findings may be attributed to quercetin; however, other compounds present in the extract may also have an effect on the overall ability of the extract to stimulate melanogenesis. We report here that Tunisian C. spinosa leaf extract can stimulate melanogenesis in a dose-dependent manner without cytotoxicity by increasing tyrosinase protein expression and has the potential to be used as a possible tanning agent or as a treatment for hair depigmentation.

Antimelanogenesis effect of Tunisian herb Thymelaea hirsuta extract on B16 murine melanoma cells.

Skin pigmentation is the result of melanogenesis that occurs in melanocytes and/or melanoma cells. Although melanogenesis is necessary for the prevention of DNA damage and cancer caused by UV irradiation, excessive accumulation of melanin can also cause melanoma. Thus, we focused on the antimelanogenesis effect of an extract from Thymelaea hirsuta, a Tunisian herb. Murine melanoma B16 cells were treated with T. hirsuta extract, and then cell viability and synthesized melanin content were measured. We found that the T. hirsuta extract decreased the synthesized melanin content in B16 cells without cytotoxicity. Tyrosinase is a key enzyme of melanogenesis and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation is known to be related to melanogenesis inhibition. To clarify its mechanism, we also determined ERK1/2 phosphorylation and tyrosinase expression level. ERK1/2 was immediately phosphorylated in cells just after treatment with the extract. The tyrosinase expression was inhibited after 24 h of stimulation with the extract. The T. hirsuta extract was fractionated, and we found that one fraction considerably decreased the melanin synthesis in B16 cells and that this fraction contains daphnanes as the main component. This indicates that our findings might be attributable to daphnanes.