Ranjan Bhadra

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.

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.

HYDROALCOHOLIC HUMAN PLACENTAL EXTRACT: SKIN PIGMENTING ACTIVITY AND GROSS CHEMICAL COMPOSITION

Background. Vitiligo is a pigmentary disorder of the skin of unknown etiology. It is thought to be of autoimmune origin after demonstration of antibody‐mediated destruction of melanocytes. Photochemotherapeutic PUVA therapy is widely used in vitiligo with about 33% success. Aqueous or hydroalcoholic extracts of human placenta of ill‐defined composition have also been used therapeutically for vitiligo. A hydroalcoholic human placental extract has been developed by us with pigmenting activity based on experimental therapies. Its chemical analysis was the primary objective of this study.

Human placental lipid induces mitogenesis and melanogenesis in B16F10 melanoma cells.

A hydroalcoholic extract of fresh term human placenta was found to be mitogenic as well as melanogenic on B16F10 mouse melanoma in anin vitro culture. The extract, a reservoir of a large number of bioactive molecules, was resolved to get the lipid fraction. Its activity was evaluated on B16F10 mouse melanoma by assessing the change in cellular morphology, growth and melanin induction. The lipid fraction, placental total lipid fraction (PTLF) tested in the study employed doses of 0.01 to 200 μg/ml; optimum growth and melanization accompanied by morphological changes were recorded at 10 and 100 μg/ml respectively. At intermediate doses growth and melanization were found to show a pattern of change over between growth and melanization and finally reached at an inverse relation at the respective optimal dose of response. Compared with defined sphingolipids, C2 ceramide and sphingosine-1-phosphate, the results were mostly corroborative. The duality of biological response of sphingolipids as reported in numerous studies was comparable for the PTLF suggesting that its active component is a sphingolipid and showing its use for pigment recovery in vitiligo.

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.

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.

Immunomodulatory stimulation of an invertebrate circulatory lectin by its haptenic molecules of pathogenic origin.

2-keto-3-deoxy octonate and beta-glycerophosphate two important bacterial cell wall constituent molecules, haptenic in nature to an invertebrate (crab) circulatory lectin, carcinoscorpin, gave lectin induction after immunization of the crab, Carcinoscorpius rotunda-cauda. With the exception of erythrocyte, no other sialoglycoconjugate-containing substances (mucin, fetuin) sialodisaccharide O-[N-acetylneuraminyl-(2----6)2-acetamido-2-deoxy galactitol] and free sialic acid were effective in lectin induction. This induction of circulatory lectin failed to appear when, concanavalin A, epinephrine, cytochalasin B, methyl-alpha-D-mannoside and bovine serum albumin were used for immunization. But mechanical injury to crabs were extremely effective in such lectin induction by live crabs. LPS is lethal if used for immunization. The immunoregulatory induction of the circulatory lectin by pathogen-originated cell wall constituent molecules and mechanical injury may be considered as a humoral immune response.

Attenuated Leishmanial sphingolipid induces apoptosis in A375 human melanoma cell via both caspase-dependent and -independent pathways

A fraction of attenuated Leishmanial lipid (ALL) rich in sphingolipids, previously shown to have apoptosis inducing activity in mouse melanoma (B16F10) and human melanoma (A375) cells, was resolved to isolate the bioactive sphingolipid. The mechanism of apoptosis induction by this bioactive attenuated Leishmanial sphingolipid (ALSL) was studied in A375 cells. Apoptosis induced by ALSL in A375 cells was found to be dose and time-dependent. Exposure of cells to ALSL resulted in a rapid increase in reactive oxygen species generation. Pretreatment of cells with the antioxidant N-acetyl-cystein reduced ROS generation and attenuated apoptosis induced by ALSL. Again, ALSL sensitization resulted in the activation of caspase-3 and -9 but not caspase-8. However, inhibitors of these caspases could not protect the cells completely from ALSL-induced apoptosis. N-acetyl-cystein pretreatment was again found to attenuate the activation of caspase-3 and -9. ALSL treatment also resulted in the alteration of mitochondrial membrane potential, and release of pro-apoptotic factors such as cytochrome c and apoptosis inducing factor (AIF) from mitochondria. Furthermore, c-Jun N-terminal kinase was activated that resulted in apoptosis of A375 cells, whereas p38 MAPK was activated to counteract the stress generated in cells in response to ALSL treatment. Taken together, our results indicate that ALSL-induced apoptosis of A375 cells is mediated by both mitochondrial caspase-dependent and -independent pathways and it involves ROS and JNK activation in the mitogen-activated protein kinase cascade.

The effect of epinephrine and serotonin on hepatic poly (A)+ RNA synthesis

In vivo administration of epinephrine or serotonin has been shown to stimulate the incorporation of 14C-orotic acid into Poly(A)+ RNA. However, only epinephrine and not serotonin could stimulate DNA dependent RNA polymerase activity of isolated hepatic nuclei in in vitro experiments.

Effect of amines on fibrinogen synthesis

L-Epinephrine, serotonin, and isoproterenol stimulate the incorporation of [14C]leucine into thrombin-induced clottable protein; this stimulation was abolished by actinomycin D. The incorporation of 32P into total RNA of rat liver, the site of fibrinogen synthesis, was stimulated by epinephrine and was highest at 2 h after 32P administration. [14C]Orotic acid incorporation into polysomal RNA of liver was also increased significantly by epinephrine and serotonin. The immunoprecipitation of newly synthesized protein by monospecific antibody raised against pure rat fibrinogen clearly demonstrates that L-epinephrine increased fibrinogen formation in vivo under the experimental condition. Translation of poly (A)-containing RNA from total polysomal RNA clearly indicates that L-epinephrine increased mRNA specific for fibrinogen.