Actin cytoskeleton remodeling drives epithelial-mesenchymal transition for hepatoma invasion and metastasis in mice.

High invasiveness is a hallmark of human hepatocellular carcinoma (HCC). Large tumors predict invasion and metastasis. Epithelial-mesenchymal transition (EMT) is crucial for cancer invasion and metastasis. However, the mechanisms whereby large tumors tend to undergo EMT remain unclear. We conducted a subgenome-wide screen and identified KLHL23 as an HCC invasion suppressor by inhibiting EMT. KLHL23 binds to actin and suppresses actin polymerization. KLHL23 silencing induced filopodium and lamellipodium formation. Moreover, EMT was suppressed by KLHL23 through its action on actin dynamics. Traditionally, actin cytoskeleton remodeling is downstream of EMT reprogramming. It is therefore intriguing to ask why and how KLHL23 inversely regulates EMT. Activation of actin cytoskeleton remodeling by either KLHL23 silencing or treatment with actin cytoskeleton modulators augmented cellular hypoxic responses in a cell-density-dependent manner, resulting in hypoxia-inducible factor (HIF) and Notch signals and subsequent EMT. Environmental hypoxia did not induce EMT unless actin cytoskeleton remodeling was simultaneously activated and only when cells were at high density. The resulting EMT was reversed by either adenosine 5'-triphosphate supplementation or actin polymerization inhibitors. Down-regulation of KLHL23 was associated with invasion, metastasis, and poor prognosis of HCC and pancreatic cancer. Correlations of tumor size with EMT and inverse association of expression of KLHL23 with HIF/Notch signals were further validated in patient-derived xenograft HCCs in mice. CONCLUSION: Simultaneously activation of actin cytoskeleton remodeling by intrinsic (such as KLHL23 down-regulation) or microenvironment cues is crucial for cell-density-dependent and hypoxia-mediated EMT, providing a mechanistic link between large tumor size and invasion/metastasis. Our findings provide a means of developing the prevention and treatment strategies for tumor invasion and metastasis. (Hepatology 2018;67:2226-2243).

Functional genomics identified a novel protein tyrosine phosphatase receptor type F-mediated growth inhibition in hepatocarcinogenesis.

UNLABELLED: It is unclear how proliferating cells elicit suppression on cell proliferation and how cancer cells evade this growth suppression. Using a loss-of-function screening of the human kinome and phosphatome to identify genes suppressing tumor initiation in human hepatocellular carcinoma (HCC), we identified 19 genes and characterized one of the top-scoring tumor suppressor candidates, protein tyrosine phosphatase receptor type F (PTPRF). We found that PTPRF was induced during cell proliferation by cell-cell contact. Ectopic expression of wild-type PTPRF, but not the phosphatase-inactive mutant, suppressed cell proliferation and colony formation in soft-agar assays. In contrast, PTPRF silencing led to cell hyperproliferation, enhanced tumor colony formation in soft agar, and increased xenograft tumor growth in nude mice. Mechanistically, PTPRF silencing showed aberrant ERK-dependent signaling including the phosphorylation/stabilization of v-myc avian myelocytomatosis viral oncogene homolog (MYC) through the direct activation of v-src avian sarcoma viral oncogene homolog (SRC) and suppression of PP2A. This PTPRF-mediated growth suppression during cell proliferation functioned independently of the Hippo-Yap pathway. Clinically, PTPRF was down-regulated in 42% HCC (37/89), 67% gastric cancer (27/40), and 100% colorectal cancer (40/40). PTPRF up-regulation was found in 24% HCC (21/89) and associated with better clinical outcomes. CONCLUSION: A novel PTPRF-mediated growth suppression pathway was identified by way of a functional genomics screening in human hepatoma cells. Induction of PTPRF by cell-cell contact during cell proliferation quenched the activated ERK-dependent proliferation signaling to prevent cell hyperproliferation and tumor initiation. PTPRF down-regulation in HCC facilitated tumor development. Our findings shed light on how cancer cells can evade growth suppression and open a new avenue for future development of anticancer therapies.

Neuregulin/erythroblastic leukemia viral oncogene homolog 3 autocrine loop contributes to invasion and early recurrence of human hepatoma.

UNLABELLED: Intrahepatic metastasis is the primary cause of the high recurrence and poor prognosis of human hepatocellular carcinoma (HCC). However, neither its molecular mechanisms nor markers for its prediction before hepatectomy have been identified. We recently revealed up-regulation of erythroblastic leukemia viral oncogene homolog 3 (ERBB3) in human HCC. Here we examined the clinical and biological significance of ERBB3 in HCC. Up-regulation of ERBB3 in HCC was strongly associated with male gender (P < 0.001), chronic hepatitis B (P = 0.002), microscopic vascular invasion (P = 0.034), early recurrence (P = 0.003), and worse prognosis (P = 0.004). Phosphorylated ERBB3 and its ligands [neuregulins (NRGs)] were detected in both HCC tissues and cells. Phosphorylation of ERBB3 could be induced by conditioned media of HCC cells and abolished by the pretreatment of conditioned media with anti-NRG antibodies or by the silencing of the endogenous NRG expression of the donor HCC cells. Human epidermal growth factor receptor 2 was required for ERBB3 phosphorylation. The downstream phosphoinositide 3-kinase/v-akt murine thymoma viral oncogene homolog pathways were primarily elicited by NRG1/ERBB3 signaling, whereas the mitogen-activated protein kinase/extracellular signal-regulated kinase pathways were elicited by both epidermal growth factor/epidermal growth factor receptor and NRG1/ERBB3 signaling. The activation and silencing of ERBB3-dependent signaling had potent effects on both the migration and invasion of HCC cells, but neither had significant effects on the proliferation of HCC cells, tumor formation, or tumor growth in vitro and in vivo. CONCLUSION: The constitutive activation of ERBB3-dependent signaling via the NRG1/ERBB3 autocrine loop plays a crucial role in the regulation of cell motility and invasion, which contribute to intrahepatic metastasis and early recurrence of HCC. ERBB3 is a marker for the prediction of intrahepatic metastasis and early recurrence. ERBB3-dependent signaling is a candidate target for the treatment of microscopic vascular invasion and for the prevention of HCC recurrence.

Secreted ERBB3 isoforms are serum markers for early hepatoma in patients with chronic hepatitis and cirrhosis.

Most hepatocellular carcinoma (HCC) is generated from chronic hepatitis and cirrhosis. To discover new markers for early HCC in patients with chronic hepatitis and cirrhosis, we initiated our search in the interstitial fluid of tumor (TIF) via differential gel electrophoresis and antibody arrays and identified secreted ERBB3 isoforms (sERBB3). The performance of serum sERBB3 in diagnosis of HCC was analyzed using receiver operating characteristic curves (ROC). The serum sERBB3 level was significantly higher in HCC than in cirrhosis (p < 0.001) and chronic hepatitis (p < 0.001). The accuracy of serum sERBB3 in detection of HCC was further validated in two independent sets of patients. In discrimination of early HCC from chronic hepatitis or cirrhosis, serum sERBB3 had a better performance than alpha-fetoprotein (AFP) (areas under ROC [AUC]: sERBB3 vs AFP = 93.1 vs 81.0% from chronic hepatitis and 70.9 vs 62.7% from cirrhosis). Combination of sERBB3 and AFP further improved the accuracy in detection of early HCC from chronic hepatitis (AUC = 97.1%) or cirrhosis (AUC = 77.5%). Higher serum sERBB3 levels were associated with portal-vein invasion and extrahepatic metastasis of HCC (p = 0.017). Therefore, sERBB3 are serum markers for early HCC in patients with chronic hepatitis and cirrhosis.

Clonal evolution and clinical implications of genetic abnormalities in blastic transformation of chronic myeloid leukaemia.

Blast crisis (BC) predicts dismal outcomes in patients with chronic myeloid leukaemia (CML). Although additional genetic alterations play a central role in BC, the landscape and prognostic impact of these alterations remain elusive. Here, we comprehensively investigate genetic abnormalities in 136 BC and 148 chronic phase (CP) samples obtained from 216 CML patients using exome and targeted sequencing. One or more genetic abnormalities are found in 126 (92.6%) out of the 136 BC patients, including the RUNX1-ETS2 fusion and NBEAL2 mutations. The number of genetic alterations increase during the transition from CP to BC, which is markedly suppressed by tyrosine kinase inhibitors (TKIs). The lineage of the BC and prior use of TKIs correlate with distinct molecular profiles. Notably, genetic alterations, rather than clinical variables, contribute to a better prediction of BC prognosis. In conclusion, genetic abnormalities can help predict clinical outcomes and can guide clinical decisions in CML.

Eicosapentaenoic and docosahexaenoic acids enriched polyunsaturated fatty acids from the coastal marine fish of Bay of Bengal and their therapeutic value.

Eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) enriched polyunsaturated fatty acids (PUFA) significantly present in marine fish oil emerge as preventive agents for combating many health problems specially in chronic or metabolic disorders. The fish in the coastal area of Bay of Bengal has remained unexplored with respect to EPA/DHA enriched PUFA content in its oils, although it may be a potential source in harnessing the health benefit. In this study, seven varieties of the coastal fish were analysed for the content of EPA/DHA. The one locally known as lotte, (Harpadon nehereus) though has low content of total lipids, was found to have high EPA/DHA in its oil. The phospholipids rich fraction was extracted from the total fish oil. The EPA/DHA enriched PUFA was isolated to investigate the potential use for health benefits. EPA/DHA is found to act as protective agent against mercury poisoning studied in cell culture as well as in animal mode. It is found to be highly preventive in diabetes. The lotte is available in the coastal area of Bay of Bengal adjoining West Bengal, India in large scale and it is the first report showing EPA/DHA enriched PUFA in these fish oil that can be availed to harness in important health benefits.

DNMT3A mutants provide proliferating advantage with augmentation of self-renewal activity in the pathogenesis of AML in KMT2A-PTD-positive leukemic cells.

Acute myeloid leukemia (AML) with partial tandem duplication of histone-lysine N-methyltransferase 2A (KMT2A-PTD) is a subtype of AML and is associated with adverse survival, yet the molecular pathogenesis of KMT2A-PTD is not fully understood. DNA methyltransferase 3A (DNMT3A) is mutated in various myeloid neoplasms including AML, especially at the Arg882. Recently, it has been found that DNMT3A mutations frequently coexisted with KMT2A-PTD and are associated with inferior outcomes. We aimed to understand the biological role of DNMT3A mutation in KMT2A-PTD-positive cells. Herein, we found that overexpression of DNMT3A mutants (MT) in KMT2A-PTD-positive EOL-1 cells augmented cell proliferation and clonogenicity. Serial colony replating assays indicated that DNMT3A-MT increased the self-renewal ability of Kmt2a-PTD-expressing mouse bone marrow cells with immature morphology. At 10 months post bone marrow transplantation, mice with the combined Kmt2a-PTD and DNMT3A-MT showed hepatosplenomegaly and leukocytosis with a shorter latency compared to control and DNMT3A-wild-type. Gene expression microarray analyses of bone marrow samples from human AML with KMT2A-PTD/DNMT3A-MT showed a stem cell signature and myeloid hematopoietic lineage with dysregulation of HOXB gene expression. In addition, human bone marrow AML cells carrying KMT2A-PTD/DNMT3A-MT showed abnormal growth and augmented self-renewal activity in primary cell culture. The present study provides information underlying the pathogenic role of DNMT3A-MT with KMT2A-PTD in proliferating advantage with augmentation of self-renewal activity in human leukemia, which may help to better understand the disease and to design better therapy for AML patients with these mutations.

RUNX1 mutations promote leukemogenesis of myeloid malignancies in ASXL1-mutated leukemia.

BACKGROUND: Additional sex combs-like 1 (ASXL1) mutations have been described in all forms of myeloid neoplasms including chronic myelomonocytic leukemia (CMML) and associated with inferior outcomes, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) remains poorly understood. Transformation of CMML to secondary AML (sAML) is one of the leading causes of death in CMML patients. Previously, we observed that transcription factor RUNX1 mutations (RUNX1-MT) coexisted with ASXL1-MT in CMML and at myeloid blast phase of chronic myeloid leukemia. The contribution of RUNX1 mutations in the pathogenesis of myeloid transformation in ASXL1-mutated leukemia, however, remains unclear. METHODS: To evaluate the leukemogenic role of RUNX1-MT in ASXL1-mutated cells, we co-expressed RUNX1-MT (R135T) and ASXL1-MT (R693X) in different cell lines and performed immunoblot, co-immunoprecipitation, gene expression microarray, quantitative RT-PCR, cell proliferation, differentiation, and clonogenic assays for in vitro functional analyses. The in vivo effect was investigated using the C57BL/6 mouse bone marrow transplantation (BMT) model. RESULTS: Co-expression of two mutant genes increased myeloid stem cells in animal model, suggesting that cooperation of RUNX1 and ASXL1 mutations played a critical role in leukemia transformation. The expression of RUNX1 mutant in ASXL1-mutated myeloid cells augmented proliferation, blocked differentiation, and increased self-renewal activity. At 9 months post-BMT, mice harboring combined RUNX1 and ASXL1 mutations developed disease characterized by marked splenomegaly, hepatomegaly, and leukocytosis with a shorter latency. Mice transduced with both ASXL1 and RUNX1 mutations enhanced inhibitor of DNA binding 1 (ID1) expression in the spleen, liver, and bone marrow cells. Bone marrow samples from CMML showed that ID1 overexpressed in coexisted mutations of RUNX1 and ASXL1 compared to normal control and either RUNX1-MT or ASXL1-MT samples. Moreover, the RUNX1 mutant protein was more stable than WT and increased HIF1-α and its target ID1 gene expression in ASXL1 mutant cells. CONCLUSION: The present study demonstrated the biological and functional evidence for the critical role of RUNX1-MT in ASXL1-mutated leukemia in the pathogenesis of myeloid malignancies.

Studies on the interaction of isoxazolcurcumin with calf thymus DNA.

The interaction of isoxazolcurcumin (IOC), a synthetic derivative of curcumin, with calf thymus-DNA (ct-DNA) has been investigated by UV-Vis, fluorescence, circular dichroism spectroscopies, viscosity measurements and docking studies. From these analyses, the binding constant, number of binding sites and mode of binding of IOC to ct-DNA has been determined. The binding constant of IOC to DNA calculated from both UV-Vis and CD spectra was found to be in the 10(4)M(-1) range. Analyses of fluorescence spectra, viscosity measurements and molecular modeling of IOC-DNA interactions indicate that IOC is a minor groove binder of ct-DNA and preferentially binds to AT rich regions. Ethidium bromide displacement studies revealed that IOC did not have any effect on ethidium bromide bound DNA which is indicative of groove binding. To elucidate the preferred region of binding of IOC to DNA, docking studies have been performed and changes in accessible surface area (DeltaASA) of nucleobases determined due to IOC-DNA complexation.

Genetic and Epigenetic Perturbations by DNMT3A-R882 Mutants Impaired Apoptosis through Augmentation of PRDX2 in Myeloid Leukemia Cells.

DNA methyltransferase 3A (DNMT3A) is mutated in various myeloid neoplasms including acute myeloid leukemia (AML), especially at the Arg882 and associated with inferior outcomes. Here, we report that the DNMT3A-Arg882His/Cys (R882H/C) mutations led to inactivation of apoptosis through DNA damage signaling following the impairment of differentiation of myeloid leukemia cells. Gene expression profiling analysis revealed aberrant expression of several cell-cycle and apoptosis-related genes, and the DNA methylation assay identified both hypo- and hypermethylation features in different regions throughout the whole genome of DNMT3A mutants-transduced myeloid cells. We found that DNMT3A-R882H/C mutations upregulated the expression of an antioxidant protein, pyroxiredoxin-2 (PRDX2), at the mRNA and protein levels with decreased accumulation of reactive oxygen species (ROS). Augmentation of ROS generation by ROS accumulating agent or by knockdown of PRDX2 from myeloid cells effectively increased drug sensitivity and apoptosis as a consequence of reduced cell proliferation. DNMT3A-R882C/H mutations decreased apoptosis induction in part by increasing the antioxidant capacity of the cell owing to upregulation of PRDX2. Molecularly, both DNMT3A-WT and R882H/C mutants interacted with PRDX2; and R882C/H mutation-induced hypomethylation increased PRDX2 expression which enhanced cell proliferation and growth with impairment of apoptosis, thereby contributing to leukemogenesis.

Immunobiological activities of a new nontoxic lipopolysaccharide from Acidiphilium GS18h/ATCC55963, a soil isolate from an Indian copper mine.

A novel nontoxic lipopolysaccharide (LPS) was purified from Acidiphilium strain GS18h/ATCC55963. The chemical composition of the lipid A part of this LPS is distinctly different from that of known lipid A molecules. The LPS was investigated to determine its capacity to provide protection against toxic LPS or endotoxic shock, as has been reported for other nontoxic LPSs (Rhodobacter sphaeroides and Rhodobacter capsulatus), and also the extent and type of immunomodulatory response in terms of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-beta), and IL-6 release as well as NO secretion by stimulated monocyte-macrophage systems. This study demonstrates clearly that mice immunized or primed with this LPS are fully protected against challenge with toxic Escherichia coli LPS. Unlike most of the extensively studied nontoxic LPSs, this LPS induced reactive nitrogen intermediates and released TNF-alpha, IL-beta and IL-6 in both mouse and human monocyte-macrophage systems. However, the extent of the cytokine and lymphokine releasing response was well below the range of the toxic LPS, for example that of E. coli. Owing to its capacity to provide immunostimulation of the host without causing any lethality to ensure protection against endotoxic shock, this LPS appears to have potential therapeutic value.

Isolation and characterisation of the lipopolysaccharide from Acidiphilium strain GS18h/ATCC55963, a soil isolate of Indian copper mine.

The lipopolysaccharide (LPS) of the Gram-negative Acidiphilium strain GS18h/ATCC55963, a new soil isolate, exhibited very low endotoxic activity as determined by Limulus gelation activity, lethal toxicity in galactosamine (GalN) sensitised mice, and level of tumor necrosis factor alpha (TNFalpha) in the blood serum of BALB/c mice. Analysis of the LPS, specially of lipid A which usually accounts for the toxicity, revealed the latter to contain glucosamine and phosphate besides fatty acids, of which 14:0(3-OH), 18:0(3-OH), 18:1 and 19:0(cyclo) are the major components, while 12:0, 16:0, 19:1, 20:0(3-OH) and 20:1(3-OH) are present in small amounts. The 14:0(3-OH) and 18:0(3-OH) fatty acids are amide-linked, whereas the rest are ester bound. Glucose, galactose, mannose, rhamnose, heptose, galacturonic acid and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) were present in the polysaccharide part of this LPS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the LPS showed a macromolecular heterogeneity distinctly different from those of Escherichia coli or Salmonella. The toxicity of this LPS being extremely low attributed to fatty acid composition of its lipid A, promises potential therapeutic application.

Sphingolipid-mediated restoration of Mitf expression and repigmentation in vivo in a mouse model of hair graying.

Recent advances in the identification and characterisation of stem cell populations has led to substantial interest in understanding the precise triggers that would operate to induce activation of quiescent stem cells. Melanocyte stem cells (MSCs) reside in the bulge region of the hair follicles and are characterised by reduced expression of the microphthalmia-associated transcription factor (Mitf) and its target genes implicated in differentiation. Vitiligo is characterised by progressive destruction of differentiated melanocytes. However, therapies using UV irradiation therapy can induce a degree of repigmentation, suggesting that MSCs may be activated. As Mitf is implicated in control of proliferation, we have explored the possibility that inducing Mitf expression via lipid-mediated activation of the p38 stress-signalling pathway may represent a re-pigmentation strategy. Here we have isolated from placental extract a C18:0 sphingolipid able to induce Mitf and tyrosinase expression via activation of the p38 stress-signalling pathway. Strikingly, in age-onset gray-haired C57BL/6J mice that exhibit decaying Mitf expression, topical application of placental sphingolipid leads to increased Mitf in follicular melanocytes and fresh dense black hair growth. The results raise the possibility that lipid-mediated activation of the p38 pathway may represent a novel approach to an effective vitiligo therapy.

Activation of the Mitf promoter by lipid-stimulated activation of p38-stress signalling to CREB.

The microphthalmia-associated transcription factor Mitf plays a critical role in regulating many aspects of melanocyte biology. It is required for melanoblast and postnatal melanocyte survival, regulates proliferation, and activates genes associated with differentiation such as tyrosinase and related genes involved in melanogenesis. Identifying the signals that regulate Mitf expression is crucial if we are to understand how cells of the melanocyte lineage respond to environmental cues. Here we show that the Mitf promoter is induced by lipid signalling via the p38 stress-activated kinase pathway that is also activated by a wide range of receptors as well as UV irradiation. Signalling via p38 leads to increased phosphorylation and activation of cyclic adenosine monophosphate response element-binding (CREB) that binds and activates the Mitf promoter via the cyclic adenosine monophosphate (cAMP) response element. Moreover, we also show that activation of p38 mediated by lipids is potentiated by inhibition of the PI3kinase pathway but not by inhibition of protein kinase A (PKA). The results identify a mechanism in which stress signalling via p38 leads to activation of CREB, enhanced Mitf expression and consequently increased tyrosinase expression. The results are relevant for the regulation of melanocytes by Mitf, but also raise the possibility that lipid mediated activation of p38 signalling may represent a potential therapy for vitiligo.

Transcriptional activation of tyrosinase gene by human placental sphingolipid.

The sphingolipids, a class of complex bioactive lipids, are involved in diverse cellular functions such as proliferation, differentiation, and apoptosis as well as growth inhibition. Recently sphingosylphosphorylcholine (SPC), sphingosine-1-phosphate (S1P), and C2-ceramide (C2-Cer), sphingolipid containing acetic acid are emerging as melanogenic regulators. A bioactive sphingolipid (PSL) was isolated from hydroalcoholic extract of fresh term human placenta and it induced melanogenesis in an in vitro culture of mouse melanoma B16F10 cells. Tyrosinase, the rate-limiting enzyme for melanogenesis, is required to be upregulated for the increased melanin production. The expression of tyrosinase, both at protein as well as mRNA level, was higher in the PSL treated B16F10 cells as evidenced by Western blot and RT-PCR analysis. Actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, inhibited PSL-induced tyrosinase activity and its protein expression showing decrease in melanogenesis, correspondingly. The activity of GFP coupled tyrosinase promoter was upregulated in transfected B16F10 cells after treating with PSL as determined by fluorescence microscopy, fluorometric analysis, and Western blot. These results, thus, suggested that PSL upregulated tyrosinase gene expression at transcription level through promoter activation to show increased melanogenesis. Therefore, PSL as an inducer of melanogenesis might account for the recovery of pigment in depigmentation disorder.

Human placental protein/peptides stimulate melanin synthesis by enhancing tyrosinase gene expression.

Placental protein/peptides as biological response modifier are well documented, but not much known about melanogenesis. We possibly for the first time, demonstrated melanogenesis in B16F10 mouse melanoma by a placental protein/peptide fraction (PPPF) prepared from a hydroalcoholic extract of fresh term human placenta. This study described the effect of PPPF on the induction of tyrosinase; the key enzyme of melanogenesis to investigate the basis of PPPF induced pigmentation in primary melanocyte and B16F10 melanoma. Tyrosinase induction by PPPF in B16F10 cells was found dose- and time dependent at the level of activity. Tyrosinase, at the level of transcription and protein expression when assessed by RT-PCR and Western blot analyses found to have considerable induction over untreated control. PPPF led to enhanced activation of tyrosinase promoter resulting higher transcription thus substantiating the role of PPPF as a stimulator of melanogenesis. Actinomycin D, the transcriptional inhibitor of protein synthesis, blocked the stimulatory action of PPPF since the induction of tyrosinase and melanin was markedly reduced in presence of this inhibitor. Thus the results suggested that PPPF mediated increase in tyrosinase expression occurred through transcriptional upregulation to stimulate melanogenesis in B16F10 cells and in primary melanocyte also.

A sphingolipid rich lipid fraction isolated from attenuated Leishmania donovani promastigote induces apoptosis in mouse and human melanoma cells in vitro.

Lipids, especially sphingolipids, are emerging as inducer of apoptosis in a wide range of immortal cells, potentiating their therapeutic application in cancer. In the present study, a sphingolipid rich lipid fraction (denoted here as ALL), isolated from an attenuated strain of Leishmania donovani promastigote, was tested for its tumoricidal activity taking melanoma, the dreaded form of skin cancer cells, as model. ALL was found to induce chromatin condensation, internucleosomal DNA fragmentation and phosphatidylserine externalization with enhanced cell population in sub-G1 region in both mouse and human melanoma systems, namely B16F10 and A375 respectively. These are the hallmarks of cells undergoing apoptosis. Further analysis demonstrated that ALL treated melanoma cells showed significant increase in ROS generation, mitochondrial membrane potential depolarization, release of cytochrome c, and caspase-3 activation, which are the events closely involved in apoptosis. These findings indicate that one or more bioactive sphingolipid(s)/ceramide(s) present in ALL could be the causative agent(s) for the induction of apoptosis in melanoma cells. Further studies are thus necessary to identify these specific bioactive sphingolipid(s)/ceramide(s) and to establish their mechanism of action, in order to explore their use as anticancer agents.

Human placental lipid induces melanogenesis through p38 MAPK in B16F10 mouse melanoma.

Melanogenesis is one of the characteristic functional activities of melanocyte/melanoma and is regulated via mitogen-activated protein kinase (MAPK) and Akt/protein kinase B (PKB) pathways. Placental total lipid fraction (PTLF), prepared from a hydroalcoholic extract of fresh term human placenta contains sphingolipids and was recently shown to stimulate melanogenesis via up-regulation of the key enzyme tyrosinase in B16F10 mouse melanoma cells. How such lipids mediate their effects on pigmentation and tyrosinase expression is a particularly important aspect of melanogenesis. To study the signaling that leads to tyrosinase expression, we have investigated the roles of the MAPK and Akt/PKB pathways in B16F10 melanoma cells in melanogenesis in response to PTLF. Treatment of cells with PTLF led to the time dependent phosphorylation of p38 MAPK. SB203580, a p38 MAPK inhibitor, completely blocked the PTLF-induced melanogenesis by inhibiting promoter activity and subsequent expression of tyrosinase. Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 a blocker of the Akt signaling pathway, or an inhibitor of MEK (MAPK/ERK Kinase), PD98059 when included along with PTLF was found to potentiate PTLF-induced phosphorylation of p38 MAPK together with tyrosinase expression and melanogenesis. The results suggest that the activation of p38 MAPK plays a crucial role in PTLF-induced B16F10 melanogenesis by up-regulating tyrosinase expression.

A human placental extract: in vivo and in vitro assessments of its melanocyte growth and pigment-inducing activities.

BACKGROUND: The authenticity of various prototype human placental extracts with biological activity, such as that inducing vitiligo repigmentation, is under serious criticism, mainly due to a lack of demonstration at the cellular level. Considering the present worldwide scenario with regard to the occurrence and treatment of vitiligo, a thorough scientific exploration of such extracts should be undertaken. METHOD: One such prototype placental preparation was prepared, and was evaluated with regard to its melanogenic action in C57BL/6J mice in vivo and its mitogenic and melanogenic activity on B16F10 mouse melanoma cells and normal human melanocytes in vitro. The extract was applied topically to mice with age-induced prolonged telogenic phase of hair growth (grey body coat hair). Standard 3H-thymidine incorporation and spectrophotometric methods were followed to illustrate mitogenic and melanogenic effects at the cellular level. RESULTS: The resurgence of blue skin, followed by shiny black hair, at the regions of application of the extract demonstrated the reversal of the age-induced prolonged telogenic phase of hair growth to the anagenic phase after topical application of the extract on C57BL/6J mice. Further support was obtained from histology where, at the extract-treated sites, the development of new melanogenic centers and hair follicles was observed. During in vitro studies, the vehicle-free extract constituents stimulated both mitogenesis and melanogenesis of B16F10 mouse melanoma cells in a concentration-dependent manner. The cell morphology and extent of melanogenesis also showed significant changes. In addition, two known melanocyte activity-modulating peptides, endothelin-1 (ET-1) and adrenocorticotropic hormone (ACTH), were determined in the extract, chiefly in the total lipid fraction, indicating their effective cutaneous permeation. CONCLUSIONS: The extract was found to be a potent mitogen in the in vitro condition and a potent melanogen in both the in vitro and in vivo situations. This strongly suggests its therapeutic potential for the repigmentation of vitiligo patches.