Mitsuzi Yoshida

Antitumor activity of daphnane-type diterpene gnidimacrin isolated from Stellera chamaejasme L.

The daphnane‐type diterpene gnidimacrin, isolated from the root of the Chinese plant, Stellera chamaejasme L., was found to strongly inhibit cell growth of human leukemias, stomach cancers and non‐small cell lung cancers in vitro at concentrations of 10−9 to 10−10 M. On the other hand, even at 10−8 to 10−5 M, the small cell lung cancer cell line H69 and the hepatoma cell line HLE were refractory to gnidimacrin. The agent showed significant antitumor activity against murine leukemias and solid tumors in an in vivo system. In K562, a sensitive human leukemia cell line, gnidimacrin induced blebbing of the cell surface, which was completely inhibited by staurosporine at concentrations above 10−8 M, and arrested the cell cycle transiently to G2 and finally the G1 phase at growth‐inhibitory concentrations. It inhibited phorbol‐12,13‐dibutyrate (PDBu) binding to K562 cells and directly stimulated protein kinase C (PKC) activity in the cells in a dose‐dependent manner (3–100 nM). Although activation of PKC isolated from refractory H69 cells was observed only with 100 nM gnidimacrin, the degree of activation was lower than that produced by 3 nM in K562 cells. Our results suggest that gnidimacrin acts as a PKC activator for tumor cells and that this mechanism may be responsible for its antitumor activity.

Mechanism of antitumor action of PKC activator, gnidimacrin.

Daphnane‐type diterpene gnidimacrin isolated from the Chinese plant Stellera chamaejasme L. is an antitumor agent that activates protein kinase C (PKC). The mechanism of antitumor action of gnidimacrin and the possible involvement of PKC were examined using sensitive K562 and refractory HLE cells. Gnidimacrin did bind to K562 cells 3 times more than to HLE cells. Immunoblot analyses revealed pronounced PKCβII expression in gnidimacrin sensitive cell lines including K562 cells, while refractory HLE cells strongly expressed PKCα, but not PKCβII. In a 24‐hr exposure of K562 cells to gnidimacrin, G1 phase arrest and inhibition of cdk2 kinase activity was found at growth‐inhibitory concentration (0.0005 μg/ml). Complete inhibition of cdk2 activity and maximum G1 phase arrest were observed at 0.005 μg/ml, however, these biological effects were reduced at 0.05 μg/ml (260 times the 50% inhibitory concentration). Cellular PKC after a 24‐hr exposure was examined by immunoblot analysis and specific binding of [3H]phorbol‐12,13‐dibutyrate as a ligand of PKC. Expression and the amount of functional PKC of K562 cells were not changed at 0.002 μg/ml, but down‐regulated to less than 1/10th of the control at 0.05 μg/ml. The reduction of biological effects at 0.05 μg/ml is most likely due to PKC down‐regulation. Our results suggest that PKC (particularly βII) is one of the major determinants of the ability of cells to respond to gnidimacrin and that the antitumor action might be associated with cell‐cycle regulation through suppression of cdk2 activity. Int. J. Cancer 77:243–250, 1998.© 1998 Wiley‐Liss, Inc.

Phenotypical and functional alterations during the expansion phase of invariant Vα14 natural killer T (Vα14i NKT) cells in mice primed with α-galactosylceramide

Invariant Vα14 natural killer T (Vα14i NKT) cells are a unique immunoregulatory T‐cell population that is restricted by CD1d. The glycolipid α‐galactosylceramide (α‐GalCer) is presented by CD1d and causes robust Vα14i NKT‐cell activation. Three days after injection of α‐GalCer, Vα14i NKT cells vigorously increase in number and then gradually decrease to normal levels. In the present study, we found that the re‐administration of α‐GalCer into mice primed 3 days earlier causes a marked increase in serum interleukin‐4 and interferon‐γ. Intracellular staining revealed that the only expanded Vα14i NKT cells are responsible for the enhanced cytokine production. The enhanced cytokine production was correlated with an increased number of Vα14i NKT cells after priming. Additionally, primed Vα14i NKT cells produced larger amounts of cytokine as compared with naive Vα14i NKT cells when cultured with α‐GalCer‐pulsed dendritic cells. Thus, we considered that a subset of expanded Vα14i NKT cells acquired a strong ability to produce cytokines. In contrast to mice primed 3 days earlier, cytokine production is markedly diminished in mice primed 7 days earlier. The expanded Vα14i NKT cells altered the surface phenotype (NK1.1– CD69–) and contained intracellular interferon‐γ. Additionally, we found that primed Vα14i NKT cells did not disappear or down‐regulate surface TCR expression when re‐injected with α‐GalCer as compared with naive Vα14i NKT cells. These results demonstrate that the function and surface phenotype of Vα14i NKT cells is dramatically altered after α‐GalCer priming.

Involvement of PKC βII in anti-proliferating action of a new antitumor compound gnidimacrin

Daphnane‐type diterpene gnidimacrin (NSC 252940) shows significant antitumor activity against murine tumors and human tumor cell lines. This compound binds to and directly activates protein kinase C (PKC), arresting the cell cycle at the G1 phase through inhibition of cdk2 activity in human K562 leukemia cells. In our study, we examined whether cellular PKC is involved in the antiproliferating effect of gnidimacrin. In a 24‐hr exposure of K562 cells to high concentrations of bryostatin 1 (0.11–3.3 μM), both expression of PKC α and PKC βII was downregulated, and thereafter these cells became resistant to gnidimacrin in response to the degree of PKC downregulation. In addition, PKC α and PKC βII genes were transfected to gnidimacrin‐resistant human hepatoma HLE cells that demonstrated positive expression of PKC α and negative expression of PKC βII. PKC βII gene‐transfected cells became sensitive to gnidimacrin in relation to the degree of PKC βII expression. The most sensitive clone to show 0.001 μg/mL (1.2 nM) as IC50 in a continuous 4‐day exposure was obtained. While PKC α gene‐transfected cells exhibited an increase in PKC α expression and became sensitive to gnidimacrin, sensitivity was one‐hundredth of that in PKC βII gene‐transfected cells. These results suggest that PKC, in particular PKC βII, is necessary in the antitumor effect of gnidimacrin.

Antitumor action of the PKC activator gnidimacrin through cdk2 inhibition

Daphnane‐type diterpene gnidimacrin (NSC252940), isolated from a Chinese plant, exhibited antitumor activity against murine leukemias and solid tumors. At concentrations of 10–9 to 10–10 M, this agent strongly inhibited the growth of human tumor cell lines. In sensitive human leukemia K562 cells, gnidimacrin is a PKC activator that arrests the cell cycle in the G1 phase by inhibiting cdk2 activity. A 4 hr exposure of K562 cells to gnidimacrin induced the CDK inhibitor p21WAF1/Cip1, but this effect was transient and did not correlate temporally with the onset of G1 arrest. Expression of cdc25A, a phosphatase that activates cdk2, was reduced during 24‐hr exposure to gnidimacrin. Moreover, the suppression corresponded in a concentration‐ and time‐dependent manner to both the inhibition of cdk2 activity and the mobility shift observed when cdk2 was electrophoresed on SDS‐PAGE, indicating that the phosphorylation state of cdk2 must change. Cyclin E, the other regulator of cdk2 activity, was not influenced by gnidimacrin. These results suggest that gnidimacrin exerts antitumor activity through suppression of cdc25A and inhibition of cdk2 activity.

Synthesis and biological activities of 22-hydroxy and 22-methoxy derivatives of 1α,25-dihydroxyvitamin D3: Importance of side chain conformation for biological activities

Abstract Both 22-epimers of 22-hydroxy and 22-methoxy derivatives of 1α,25-dihydroxyvitamin D 3 (1) were synthesized from 22-aldehyde (6) to clarify the precise structural requirement for exerting various biological activities. While the synthetic vitamin D derivatives did not show any increase of serum calcium concentration in rats by oral administration, all derivatives induced into nitroblue tetrazolium (NBT)-positive cells at a concentration more than 1 μg/ml in the NBT reduction test for induction of differentiation of HL-60 cells. Especially noteworthy was that 22 S -isomers ( 3 and 5 ) were at least 30 times more effective than corresponding 22 R -isomers ( 2 and 4 ), respectively. Binding affinities of the (22 S )-22-methoxy derivatives (5) to the chick intestinal receptor for 1 was also about 3 times as potent as 22 R -isomer (4) . A major structural difference was their side chain conformations, which were elucidated by molecular mechanics calculation (MM2 force field) and NMR studies. A zig-zag conformation turned out to be sterically most favorable for 22 S -isomers, whereas such a zig-zag conformation is energetically unfavorable for 22 R -isomers due to the interaction between the 22-substituent and the 16-methylene group. The side chain conformation seems to be responsible for the difference of their biological activity and the zig-zag conformation plays an important role for the activities.

Interleukin (IL)-4 promotes T helper type 2-biased natural killer T (NKT) cell expansion, which is regulated by NKT cell-derived interferon-γ and IL-4

CD1d‐restricted natural killer T (NKT) cells can rapidly produce T helper type 1 (Th1) and Th2 cytokines and also play regulatory or pathological roles in immune responses. NKT cells are able to expand when cultured with α‐galactosylceramide (α‐GalCer) and interleukin (IL)‐2 in a CD1d‐restricted manner. However, the expansion ratio of human NKT cells is variable from sample to sample. In this study, we sought to determine what factor or factors are responsible for efficient in vitro expansion of NKT cells from various inbred mouse strains. Although the proportion of NKT cells in the spleen was nearly identical in each mouse strain, the growth rates of NKT cells cultured in vitro with α‐GalCer and IL‐2 were highly variable. NKT cells from the B6C3F1 and BDF1 mouse strains expanded more than 20‐fold after 4 days in culture. In contrast, NKT cells from the strain C3H/HeN did not proliferate at all. We found that cell expansion efficiency correlated with the level of IL‐4 detectable in the supernatant after culture. Furthermore, we found that exogenous IL‐4 augmented NKT cell proliferation early in the culture period, whereas interferon (IFN)‐γ tended to inhibit NKT cell proliferation. Thus, the ratio of production of IL‐4 and IFN‐γ was important for NKT cell expansion but the absolute levels of these cytokines did not affect expansion. This finding suggests that effective expansion of NKT cells requires Th2‐biased culture conditions.

U5A2-13, an antigen originally found on mouse NK-like T cells, is an early inducible cell surface antigen during lymphoid activation

We have previously reported a monoclonal antibody (mAb), U5A2-13 mAb, which originally recognizes a phenotypically and functionally similar population of natural killer (NK)-like T cells. In this study, we found that U5A2-13 antigen (U5A2-13) was expressed not only on NK-like T cells but also on T and B cells during activation. In contrast to the low levels of U5A2-13 on freshly harvested T and B cells, the activation of these cells by various stimuli resulted in high levels of expression of U5A2-13 in vitro and in vivo. Similar to CD69, U5A2-13 is also expressed in most mouse lymphoid cell lines but not in nonhematopoietic cells. U5A2-13 on T cells reached maximal expression by 24h after stimulation and returned to baseline levels after 3 days. However, U5A2-13 differed from CD69 since its expression profile was different on CD4(+)- and CD8(+)-activated T cells, phorbol ester-activated EL-4 cells, and activated splenocytes in CD69-deficient mice. In addition, immunoprecipitation study indicated that U5A2-13 is not identical to CD69. Importantly, the U5A2-13-positive population of CD4(+) T cells exhibited significant levels of cytokine producing activity upon stimulation. Overall, U5A2-13 is an early inducible cell surface antigen that could be involved in lymphocyte activation.

ACTION OF 5-FLUOROCYCLOCYTIDINE ON CULTURED L-5178Y CELLS

Effect of substitution of 5-position of cyclocytidine with fluorine on its antitumor activity in cultured cells was examined. 5-Fluorocyclocytidine was active against cultured L-5178Y cells similar to cyclocytidine. IC50 of the compound was 0.054 mug/ml. This compound inhibited thymidine incorporation into acid-soluble fraction of the cells. Cell growth inhibition by 5-fluorocyclocytidine was reversed by deoxycytidine but not by thymidine and deoxyuridine. On the other hand, cell growth inhibition by 5-fluorouracil was reversed by thymidine and deoxyuridine. As a result, site of action of 5-fluorocyclocytidine was considered to be similar to that of cyclocytidine and not to 5-fluorouracil.