Dong Ho Hong

Stimulation of humoral and cell mediated immunity by polysaccharide from mushroom phellinus linteus

Polysaccharide was purified from mycelial culture of Phellinus linteus (PL) and its effect on immunocompetence of normal splenocytes was observed. Overall in vitro immune function was enhanced by addition of PL into culture of mouse spleen lymphocyte and i.p. injection into mouse, while beta-glucans and other polysaccharides only raised the level of T lymphocyte-mediated immunity. PL stimulated immune functions of T lymphocytes, such as proliferation of T lymphocyte induced by mixed lymphocytes reaction and cytotoxicity of cytotoxic T cells responding to alloantigen. Nonspecific immune functions mediated by natural killer cells and macrophages were increased by treatment of PL in vivo and in vitro. PL also stimulated humoral immune function positively, such as T-dependent and T-independent primary antibody response, and acted as a polyclonal activator on B cell. PL exhibited a wider range of immunostimulation and antitumor activity than other polysaccharides isolated from Basidiomycetes.

A novel stereo-selective sulfonylurea, 1-[1-(4-aminobenzoyl)-2,3-dihydro-1H-indol-6-sulfonyl]-4-phenyl-imidazolidin-2-one, has antitumor efficacy in in vitro and in vivo tumor models.

The antitumor activities of novel 1-[1-(4-aminobenzoyl)-2,3-dihydro-1H-indol-6-sulfonyl]-4-phenyl-imidazolidin-2-ones were studied to determine the potential of these compounds as antitumor candidates. The agents studied were: DW2143 (1-[1-(4-aminobenzoyl)-2,3-dihydro-1H-indol-6-sulfonyl]-4-phenyl-imidazolidin-2-one), a racemic mixture, and DW2282 [(4S)-1-[1-(4-aminobenzoyl)-2,3-dihydro-1H-indol-6-sulfonyl]-4-phenyl-imidazolidin-2-one], an S-isomer. DW2143 and DW2282 suppressed the in vitro growth of tumor cells at lower concentrations than doxorubicin, but tumor specificity was not observed between the compounds. These compounds when administered orally were not active in syngeneic models of murine Colon 26 adenocarcinoma and L1210 leukemia. However, DW2143 suppressed the growth of SW620 (human colon cancer) and NCI-H23 (human lung cancer) cells in nude mice, inhibiting tumor growth by 87 and 67%, respectively. DW2282 was a more potent inhibitor of SW620 tumor cell growth in nude mice and was also lower in toxicity than DW2143. Moreover, DW2282 did not produce a series of toxic symptoms caused by the aniline metabolites of sulfonylureas, including hypoglycemia. These results suggest that DW2282, an S-isomer, could be a novel antitumor candidate with higher specificity and lower toxicity than other orally active sulfonylureas.

Cytotoxicity of urushiols isolated from sap of Korean lacquer tree (Rhus vernicifera stokes)

Cytotoxicities of four urushiols, congeners isolated from the sap of Korean lacquer tree (Rhus vernicifera Stokes), to 29 human cancer cell lines originated from 9 organs were evaluated. Their values of 50% growth inhibition were below 4 μg/ml, and showed cell line specific cytotoxicity. The present result is the first report on the cytotoxicity of urushiols suggesting that they would have an anticancer activity to human cancer cells.

Efficient fixation procedure of human leukemia cells in sulforhodamine B cytotoxicity assay.

The fixation procedures in sulforhodamine B (SRB) assay for human leukemia cells were modified to produce more reliable results. It was found that the concentration of the fixative agent, trichloroacetic acid (TCA), was critical in the selective fixation of cellular protein. While a TCA solution of 80% fixed both cells and serum proteins, a 50% solution fixed only cells with a very low interference of the serum proteins. Accordingly, we selected 50% TCA as a fixative agent which made the final absorbance of the SRB assay to be exactly matched to the cell density with a small deviation and a low background. Besides the change of TCA concentration, a precentifugation of microplate just before fixation also improved the previous assay procedures in the two points of view. The 2-h standing step was simply substituted for only 1 min of centrifugation. Both the rapid and slow application of TCA solution in fixation produced the same extents of fixation. In an actual application, these two kinds of modifications in the previous SRB assay procedure were also proved to be effective in the determination of cytotoxicities of doxorubicin by using human leukemias.

Facilitation of apoptosis by autologous serum and related immunosuppression in the splenocyte culture

The addition of adult mouse serum (MS) to the culture of mouse splenocytes resulted in an accelerated decrease of viable cell number during initial 24 h of culture as determined by the trypan blue dye exclusion test and the propidium iodide staining method. Furthermore, the extent of DNA fragmentation, the hallmark of apoptosis, determined by agarose gel electrophoresis and the amount of fragmented DNA measured by ELISA method showed that the extent of apoptosis was clearly increased in splenocytes cultured in the presence of MS. Under the scanning and transmission electron microscopic observations, the large portion of splenocytes showed morphological characteristics of apoptotic cells such as apoptotic body, condensed chromatin and shrunken appearance. With the accelerated rate of apoptosis, the immunocompetence of splenocytes such as the antibody production, natural killer cell activity, and proliferation by mitogens was strongly suppressed. When analyzed by surface immunolabelling flow cytometry, the subsets of lymphocytes (B, T, CD4+T and CD8+T cells) were affected in a global non-selective manner. As determined by ultrafiltration, the molecular weights of apoptosis-facilitating factors present in MS appeared to be greater than 10 kDa. Upon fractionation with Sephadex G-200, the apoptotic factors were separated into 2 fractions. In summary, results obtained in the present study indicate that some unidentified endogeneous macromolecules present in MS may produce the stimulatory effect on the apoptosis and cause immunosuppression of splenocytes under culture.

Limitation of Hu-PBL-scid mouse model in direct application to immunotoxicity assessment.

Hu-PBL-scid mice were directly introduced to the methods of immunotoxicity assessments. Human IgG and IgM was detected 1 week after transplantation. Cyclosporin A (CsA) and cyclophosphamide (CP), which were injected i.p. 4 weeks after transplantation, decreased the serum concentration of IgM after 2-4 days of treatment but not that of IgG. Lymphocyte proliferation induced by various mitogens and primary T-dependent antibody responses to sheep red blood cells could not be measured by using splenocytes of hu-PBL-scid mice. These results were correlated with the fact that human cells were not detected in the spleen, thymus, or blood of hu-PBL-scid mouse but were detected in lymph nodes of the intestine, which were observed by flow cytometric and immunohistochemical examinations. The present results suggest using hu-PBL-scid mice in routine immunotoxicity investigations: lymph nodes of intestines could be used as the lymphocyte source. In addition, the determination of serum Ig concentration might be used as a experimental item.

Synthesis, cytotoxicity and antitumor activity of 2,3-Diarylcy-clopent-2-ene-1-ones

Two series of 2,3-diarylcyclopent-2-ene-1-ones including 2-aryl-3-(2,5-dihydroxyphenyl)cyclopent-2-ene-1-ones (2a~2f) and 3-aryl-2-(3′,4′,5′-trimethoxyphenyl)cyclopent-2-ene-1-one (3a~3j) were synthesized and evaluated for the cytotoxicity against three tumor cell lines; B16F10, HCT116 and A431. It was found that the 3,4,5-trimethoxy substituent was optimal for the bioactivity of compounds in series 2. Meanwhile, compounds in series3 exhibited the most potent cytotoxicity with 3-aryl ring being 4-methoxyphenyl (compound3f), (3-hydroxy-4-methoxy)phenyl (compound3e), or (3-amino-4-methoxy)phenyl (compound3j).

Synthesis and cytotoxicity of some rigid derivatives of methyl 2,5-dihydroxycinnamate

Eight rigid compounds designed as esterase-stable analogues of methyl 2,5-dihydroxycin-namate (1) were synthesized. These derivatives include 2-(2′,5′-dihydroxybenzylidene)cyclo-pentenone (3a), 2-(2′,5′;-dihydroxybenzylidene)cyclohexanone (3b), 2,6-bis(2′,5′-dihydroxy-benzy-lidene)cyclohexanone (4b), 2,6-bis(2′,5′-dihydroxybenzylidene)cyclopentenone (4a), (E)-3-(2′,5′-dihydroxybenzylidene)pyrrolidin-2-one (5), (E)-5-(2′,5′-dihydroxybenzylidene)-1,2-isothiazolidine-1, 1 -dioxide (6), 4-(2′,5′-dihydroxyphenyl)-5H-furan-2-one (7), and 3-(2′,5′-dihy-droxyphenyl)cyclopent-2-ene-1-one (8). Among the eight compounds, the furanone7 and cyclopentenone8 showed the most potent cytotoxicity with IC50 values of 0.39-0.98 μg/mL Compound8 was further brominated, phenylated and methylated at the α position to give three corresponding analogues, including 2-bromo-3-(2′,5′-dihydroxyphenyl)cyclopent-2-ene-1-one (24), 3-(2′,5′-dihydroxyphenyl)-2-phenylcyclopent-2-ene-1-one (27), and 3-(2′,5′-dihy-droxyphenyl)-2-methylcyclopent-2-ene-1-one (28). Among the three, the most enhanced activity was observed with the phenylated compound27.

MDR‐1 gene expression is a minor factor in determining the multidrug resistance phenotype of MCF7/ADR and KB‐V1 cells

The relevance of MDR‐1 gene expression to the multidrug resistance phenotype was investigated. Drug‐resistant cells, KB‐V1 and MCF7/ADR, constantly expressed mRNA of the MDR‐1 gene and were more resistant to vinblastine and adriamycin than drug‐sensitive cells, KB‐3–1 and MCF7. The drug efflux rate of KB‐V1 was the same as KB‐3–1 although the MDR‐1 gene was expressed in only the resistant cell. The higher intracellular drug concentration of KB‐3–1 than KB‐V1 was due to the large drug influx. In the case of MCF7 and MCF7/ADR, the influx and efflux of the drug had nearly the same pattern and drug efflux was not affected by verapamil. The amount of ATP, cofactor of drug pumping activity of P‐glycoprotein, was not changed by the resistance. These observations suggested that drug efflux mediated by MDR‐1 gene expression was not a major determining factor of drug resistance in the present cell systems, and that the drug resistance could be derived from the change in drug uptake and other mechanisms.

Effect of extracellular cations on the chemotherapeutic efficacy of anticancer drugs.

Cancer development and the efficiency of chemotherapy relies on the patients calcium-related pathological status such as hyper-or hypocalcemia. In the present study, we investigated the effect of extracellular cations such as calcium and magnesium on the therapeutic efficacy of antitumor drugs. The analytic parameters used were cellular drug uptake/excretion and the chemosensitivity of the human breast cancer cell lines, MCF7 and MCF7/ADR. Both calcium and magnesium ions decreased the membrane permeability of cancer cells, which was determined by cell size analysis. These divalent ions also lowered the drug uptake and the cytoplasmic levels of rhodamine 123 and adriamycin, suggesting that they might interfere with the diffusion of these drugs by modifying the physical properties of the cytoplasmic membrane. The acute cytotoxicity of adriamycin after a short period of incubation correlated with changes in its cytoplasmic level. Our results indicate that these extracellular cations might play an important role in the therapeutic activities of anticancer drugs in cancer patients. These results also provide insight a new aspect of chemotherapy, because they suggest that the therapeutic doses of anti-cancer drugs should be modified in cancer-bearing patients presenting with abnormal blood calcium levels.