秋山 秀彦

OBJECTIVES: Agaritine (AGT) is a hydrazine-containing compound derived from the mushroom Agaricus blazei Murill. We previously reported the antitumor effect of AGT on hematological tumor cell lines and suggested that AGT induces apoptosis in U937 cells via caspase activation. However, the antitumor mechanism of AGT has not been fully understood. METHODS: Four hematological tumor cell lines (K562, HL60, THP-1, H929) were used in this study. The cells were incubated in the presence of 50 μM AGT for 24 h and analyzed for cell viability, annexin V positivity, caspase-3/7 activity, mitochondrial membrane depolarization, cell cycle, DNA fragmentation, and the expression of mitochondrial membrane-associated proteins (Bax and cytochrome c). RESULTS: In HL60, K562, and H929 cells, AGT reduced cell viability and increased annexin V- and dead cell-positive rates; however, it did not affect THP-1 cells. In K562 and HL60 cells, caspase-3/7 activity, mitochondrial membrane depolarization, and expression of mitochondrial membrane proteins, Bax and cytochrome c, were all increased by AGT. Cell cycle analysis showed that only K562 exhibited an increase in the proportion of cells in G2/M phase after the addition of AGT. DNA fragmentation was also observed after the addition of AGT. CONCLUSIONS: These results indicate that AGT induces apoptosis in K562 and HL60 cells, like U937 reported previously, but showed no effect on THP-1 cells. It was suggested that AGT-induced apoptosis involves the expression of Bax and cytochrome c via mitochondrial membrane depolarization.

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells.   Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC).    Results:  Andro reduced the viability of THP-1 and H929 in a dose-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR.  Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for (thus forming: The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.) H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

<p>Objectives: Andrographis paniculata (A. paniculata) is a widely used herb that has potential medical properties. Andrographolide (Andro) is the major component of A. paniculata. We evaluated the anti-tumor activity of Andro using leukemic cell line cells.</p><p>Methods: Leukemic cell lines U937, HL60 or H929 cells were cultured in the presence or absence of Andro and compared with the effects of Ara-C or vincristine. The anti-tumor activity was assessed by morphological observations of the cells, DNA fragmentation, MTT assay, Annexin V positive rate, caspase-3/7 activity, and cell cycle analysis.</p><p>Results: After addition of Andro, the morphology of cells changed to characteristic shapes with apoptotic bodies. Furthermore, the Annexin V positive rate and caspase-3/7 activities were increased compared with untreated cells. The G1 phase of cell cycle was also similarly increased compared with cells treated with Ara-C.</p><p>Conclusions: Our results show that Andro has an anti-tumor activity against leukemic cell lines, very possibly by inducing apoptosis.</p>

Bone marrow stroma cells (MSCs) have been shown to differentiate into multiple lineages and have great potential for regenerative therapy. We have obtained hMSCs from 6 human adults. They were all positive for CD13, CD44, and CD90 and weakly positive for CD49, while negative for CD45, suggesting that they were different from hematopoetic stem cells. hMSCs were induced to become neuronal cells and maintained as long as three weeks in the serum free medium supplemented with N2. An increase in the amount of mRNA was observed for the NeuroD1, neurofilament M and H, MAP2, neuron-specific enolase, tryptophan hydroxylase, Nurr1, and neuron specific Na⁺ channel genes, and the existence of voltage-gated Na⁺ channels that were sensitive to tetrodotoxin was confirmed electro physiologically. These results suggested that hMSCs differentiated into serotonergic neural cells. However, the expression of genes specific for stroma cells, the Big-h3 and vimentin-genes, was observed equally during the induction process, indicating that the expression pattern was not the completely same as in genuine neural cells. hMSCs cultured with serial passages showed aging phenomena at the cellular level under the various conditions examined in this study. Trials to isolate cellular clones proliferating indefinitely have not succeeded.