Apoptotic activity of isomalabaricane triterpenes on human promyelocytic leukemia HL60 cells.

Four isomalabaricane triterpenes were isolated from marine sponge Geodia japonica [W.H. Zhang, C.T. Che, Isomalabaricane-type nortriterpenoids and other constituents of the marine sponge Geodia japonica, J. Nat. Prod. 64 (2001) 1489-1492. ] and their cytotoxicity was evaluated using a human promyelocytic leukemia HL60 cell line. Of the four triterpenes tested, geoditin A was the most cytotoxic to HL60 cells [IC50=3 microg/ml (<6.6 microM)], followed by stellettins A and B, whereas geoditin B exhibited relatively weak cytotoxicity. The treated cells manifested nuclear changes characteristic for apoptosis, and associated with dissipation of mitochondrial membrane potential, activation of caspase 3, and decrease of cytoplasmic proliferating cell nuclear antigen (PCNA), as demonstrated by fluorescence and immunofluorescence microscopy. When the HL60 cells were exposed to geoditin A ranging from 1.25 to 25 microg/ml, a dose-dependent increase of reactive oxygen species, a progressive dissipation of mitochondrial membrane potential, and an increase in annexin V-FITC binding were measured by flow cytometry. Taken together our results suggest that geoditin A markedly induced reactive oxygen species, decreased mitochondrial membrane potential and mediated a caspases 3 apoptosis pathway.

Volvariella volvacea lectin activates mouse T lymphocytes by a calcium dependent pathway.

The immunomodulatory lectin, Volvariella volvacea lectin (VVL), isolated from the edible mushroom, Volvariella volvacea, has been shown to stimulate the expression of Th1 cytokines and the proliferative activity of mouse splenocytes (She et al. [1998]: Biochem Biophys Res Comm 247:106-111). In order to elucidate the mechanisms underlying these activities, we conducted a kinetic analysis of the early and late activation markers in mouse T lymphocytes: (1) flow cytometric analysis of calcium influx, (2) induction of activation molecules (CD25 and CD69), (3) expression and DNA-binding activity of the nuclear factor of activated T cells (NFAT), NFkappaB, and activation protein-1 (AP-1), (4) translational production of cytokines (interleukin-2 (IL-2) and interferon-gamma (IFNgamma)), and (5) cell proliferation by expression of proliferating cell nuclear antigen (PCNA) and tritiated thymidine incorporation. All results showed that VVL induced a rapid expression of CD69, CD25, NFAT, IL-2, and PCNA in a dose- and time-dependent manner, leading to lymphocyte proliferation. These effects brought about by VVL were more potent than those stimulated by equimolar concentrations of mitogenic lectin, concanavalin A (Con A). Cell activation and proliferation were mediated through a calcium-dependent pathway as demonstrated by a VVL-induced increase of intracellular calcium influx, and a proliferation inhibition by the Ca-dependent phosphatase calcineurin blocker-cyclosporin A (CsA). Taken all data together, VVL is a lectin which activates lymphocyte through successive calcium influx, nuclear localization of NFAT transcription factor, induction of activation markers, CD25 and CD69, intracellular cytokine production, and cell proliferation.

Fungal polysaccharopeptide inhibits tumor angiogenesis and tumor growth in mice.

Angiogenesis is crucial to tumor growth and metastasis, and interruption of this process is a prime avenue for therapeutic intervention of tumor proliferation. The present study has made use of the S180 tumor-bearing mouse model to investigate the polysaccharopeptide, PSP, isolated from the edible mushroom Coriolus versicolor, a herbal medicine known for its anti-angiogenesis properties. Quantitative analysis of microcorrosion casting of the tumor tissue showed more angiogenic features such as dense sinusoids and hot spots, in control (untreated) than in PSP-treated animals. Immunostaining of tumor tissues with antibody against the endothelial cell marker (Factor VIII) demonstrated a positive correlation in that both the vascular density and tumor weight were lower in mice treated with PSP. Morphometric analysis of corrosion casts revealed that, even though the total amount of new vessel production was reduced, the basic tumor type-specific vascular architecture was retained. However, the expression of vascular endothelial cell growth factor (VEGF) in these tumors was suppressed. In conclusion, anti-angiogenesis should be one of the pathways through which PSP mediated its anti-tumor activity.

Mitogenic activity of edible mushroom lectins.

A special group of lectins were isolated from three popular Asian edible mushrooms: Volvariella volvacea, Pleurotus flabellatus and Hericium erinacium, and their mitogenic activities towards mouse T cells were compared to the extensively investigated Agaricus bisporus lectin (ABL) and the Jack bean lectin, Concanavalin A (Con A). Among the four mushroom lectins tested, V. volvacea lectin (VVL) exhibited strong mitogenic activity as demonstrated by 3H-thymidine incorporation, which was at least 10-fold more effective than that of Con A, and the other mushroom lectins did not exhibit any proliferative activity. Treatment with VVL and ABL resulted in activation of the protein tyrosine kinase, p56lck, and expression of early activation markers, CD69 and CD25, but only VVL induced intracellular calcium influx while ABL triggered cell death. The calcium influx was sensitive to calcium channel antagonists such as nifedipine and verapamil. The P. flabellatus lectin (PFL) and H. erinacium lectin (HEL) did not stimulate p56lck expression and cell proliferation. Neither of these lectins interfered with Con A-mediated lymphocyte proliferation, which further indicated that both PFL and HEL were non-mitogenic. Taken all results together, VVL induced mitogenesis through T cell receptors and the subsequent calcium signaling pathway.

Wheat germ lectin induces G2/M arrest in mouse L929 fibroblasts.

Wheat germ lectin (WGA) is a cytotoxic lectin for many cell lines [Wang et al., 2000], but its underlying mechanism is not clear. In this report, we found that incubation of synchronized mouse L929 fibroblasts with WGA resulted in a dose-dependent reduction of intracellular incorporation of 3H-thymidine and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)-conversion activity (IC50 congruent with 0.4 microM). Fluorescein-conjugated WGA was demonstrated to transport from the cell surface into the paranuclear region of cultured L929 cells within 30 min, and subsequently evoked lipid peroxidation of plasma membrane and vacuolation in the cytoplasm of these cells. Studies with tritiated thymidine incorporation, immunofluorescence microscopy, immunoblotting analysis and flow cytometry revealed that WGA inhibited cell cycle progression after one replication, resulting in G2/M arrest and alteration of cell cycle regulatory proteins, particularly activation of p21Cip1/WAF1 and suppression of cyclin B and cdc 2. Although there was an increase of cytosolic caspase 3 and bax protein expression, no apoptotic bodies were observed by both fluorescence and transmission electron microscopy. These results suggest that WGA arrested L929 proliferation after one cell cycle in the G2/M phase through activation of the p21Cip1/WAF1 and suppression of Cyclin B-Cdc2.

Jolkinolide B induces neuroendocrine differentiation of human prostate LNCaP cancer cell line.

Euphorbia fischeriana is a Chinese herbal medicine which has been reported to possess chemotherapeutic effects, yet the underlying mechanism is unclear. In order to understand its possible anti-tumor property, we have isolated a number of chemical compounds from the roots of this plant [Phytochemistry 52 (1999) 117] and studied their in vitro effects by using human prostate LNCaP cancer cell line. Among the six compounds tested, jolkinolide B exhibited the most potent anti-proliferative activity (IC(50)=12.5 microg/mL=40 microM) and it inhibited DNA synthesis by down-regulating bromodeoxyuridine (BrdU) incorporation in LNCaP cells in a dose-dependent manner. Jolkinolide B, at concentrations up to 25 microg/mL, induced G1 arrest and neuroendocrine differentiation of LNCaP cells. Immunoblotting analysis confirmed the increased expression of neuroendocrine markers, keratin 8/18 (K8/18) and neuron specific enolase (NSE), in these cells. Apoptotic bodies and DNA fragmentation were observed by fluorescence microscopy and flow cytometry when the cells were exposed to a concentration higher than 25 microg/mL jolkinolide B. Taken all data together, jolkinolide B seems to play a role in the regulation of proliferation, differentiation, and apoptosis of LNCaP cells.