Hydrolyzable tannins of tamaricaceous plants. III. Hellinoyl- and macrocyclic-type ellagitannins from Tamarix nilotica.

Three new hellinoyl-type ellagitannins, nilotinins M4 (7), D7 (8), and D8 (9), and a new macrocyclic-type, nilotinin D9 (10), together with eight known tannins, hirtellins B (2), C (11), and F (12), isohirtellin C (13), tamarixinin A (3), tellimagrandins I and II, and 1,2,6-tri-O-galloyl-beta-d-glucose (14), were isolated from an aqueous acetone extract of Tamarix nilotica dried leaves. Nilotinin M4 (7) is a monomeric tannin possessing a hellinoyl moiety. The structure of 8 demonstrated replacement of one of the HHDP groups at the glucose core O-4/O-6 in ordinary dimeric tannins with a galloyl moiety at O-6. This is a new structural feature among the tamaricaceous ellagitannins. On the basis of the results, reported spectroscopic assignments for 2, 3, and the macrocyclic tannins 11-13 were revised. Unusual shifts in the NMR spectra of these macrocyclic tannins are also discussed in relation to their conformations. Several tannins isolated from T. nilotica were assessed for possible cytotoxic activity against four human tumor cell lines, and nilotinin D8 (9) and hirtellin A (1) showed high cytotoxic effects.

Cell death induced by nutritional starvation in mouse macrophage-like RAW264.7 cells.

Amino acid utilization of mouse macrophage-like RAW264.7 cells was investigated. During the logarithmic growth stage, RAW264.7 cells grew very fast, with an approximate doubling time of 11 hours, in DMEM supplemented with 10% heat-inactivated fetal bovine serum. RAW264.7 cells consumed glutamine at the fastest rate, followed by serine, leucine, isoleucine, arginine, lysine, valine and other amino acids. When the cell density reached a critical threshold level, cells began to suffer non-apoptotic cell death characterized by mitochondrial damage (revealed by transmission electron microscopy) and a smear pattern of DNA fragmentation (revealed by agarose gel electrophoresis). At this point, glutamine, serine and glucose in the medium were almost completely exhausted, whereas other amino acids remained at more than 40% of their initial concentrations. Based on these data, it is recommended that glutamine, serine and glucose should be supplemented for the long culture of RAW264.7 cells.

Selective antibacterial and apoptosis-modulating activities of mastic.

Mastic is a resinous exudate obtained from the stem and the main leaves of Pistacia lentiscus. We have reported the antiplaque effect of mastic-containing chewing gum on the oral cavity. We hypothesize that mastic may be a multifunctional food which has some beneficial pharmaceutical properties. The aim of this study was to assess the biological activity of solid and liquid types of mastic by cytotoxicity against fibroblasts, radical-scavenging activities and inhibitory effect on cell death of oral polymorphonuclear leukocytes (OPMNs). Mastic showed selective antibacterial action against Porphyromonas gingivalis and Prevotella melaninogenica, but no anti-HIV activity. Among a total of thirteen human cell types, promyelocytic leukemia HL-60 was the most sensitive to the cytotoxicity of mastic, followed by myeloblastic leukemia (ML-1, KG-1), erythroleukemia (K-562), oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4), hepatocellular carcinoma (HepG2), glioblastoma (T98G, U87MG) and normal oral cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast, most resistant). Mastic did not induce the differentiation of myelogenous leukemic cells into maturing cells with higher nitroblue tetrazolium-reducing activity, but induced apoptotic cell death, characterized by internucleosomal DNA fragmentation, caspase-3 activation and a decline in the intracellular concentration of putrescine. The cytotoxicity of mastic against leukemic cells did not diminish during its storage. On the other hand, mastic inhibited the spontaneous apoptosis of OPMNs. Mastic showed hydroxyl radical-scavenging activity. The selective antibacterial and apoptosis-modulating activity of mastic suggests its possible beneficial effects on oral health.

Tumor-specific cytotoxicity and type of cell death induced by sodium 5,6-benzylidene-L-ascorbate.

The cytotoxic activity of sodium 5,6-benzylidene-L-ascorbate (SBA) against eight human cancer cell lines and three human normal cells was investigated, SBA showed slightly higher cytotoxicity against human tumor cell lines, as compared with normal cells, with a tumor-specificity index of 2.0. The human myelogenous leukemia cell lines (HL-60, ML-1, KG-1) were the most sensitive to SBA, followed by human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4) and human glioblastoma (T98G, U87MG). Human oral normal cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast) were the most resistant. In contrast to actinomycin D, SBA induced little or no activation of caspase-3, caspase-8 and caspase-9 in the HSC-2, HSC-4, T98G and HL-60 cells, regardless of incubation time (either 6 or 24 h). SBA induced little or no internucleosomal DNA fragmentation after 6 h in all of these cells. However, prolonged treatment with SBA (24 h) induced a smear pattern of DNA fragmentation in the HSC-2, HSC-4 and T98G cells and a low level of internucleosomal DNA fragmentation in the HL-60 cells. Electron microscopy demonstrated the destruction of mitochondrial structure and autophagocytosis of broken organelles by SBA in the HSC-2, HSC-4 and HL-60 cells. At higher concentrations of SBA, necrotic cell death was observed in the HSC-2 cells, but not in the T98G cells, where the production of acidic organelles (detected by acridine orange staining) was much lower than that attained by nutritional starvation, a well-defined method of inducing autophagy. The present study suggests that SBA induces various degrees of autophagic cell death, followed by either necrosis or apoptosis at laters stage, depending on the cell type.

Tumor-specificity and apoptosis-inducing activity of stilbenes and flavonoids.

A total of eleven stilbenes [1-6] and flavonoids [7-11] were investigated for their tumor- specific cytotoxicity and apoptosis-inducing activity, using four human tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, submandibular gland carcinoma HSG and promyelocytic leukemia HL-60) and three normal human oral cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF). All of the compounds, especially sophorastilbene A [1], (+)-alpha-viniferin [2], piceatannol [5], quercetin [9] and isoliquiritigenin [10], showed higher cytotoxicity against the tumor cell lines than normal cells, yielding tumor-specific indices of 3.6, 4.7, >3.5, >3.3 and 4.0, respectively. Among the seven cell lines, HSC-2 and HL-60 cells were the most sensitive to the cytotoxic action of these compounds. Sophorastilbene A [1], piceatannol [5], quercetin [9] and isoliquiritigenin [10] induced internucleosomal DNA fragmentation and activation of caspases -3, -8 and -9 dose-dependently in HL-60 cells. (+)-alpha-Viniferin [2] showed similar activity, but only at higher concentrations. All the compounds failed to induce DNA fragmentation and activated caspases to much lesser extents in HSC-2 cells. Western blot analysis showed that sophorastilbene A [1], piceatannol [5] and quercetin [9] did not induce any consistent changes in the expression of pro-apoptotic proteins (Bax, Bad) and antiapoptotic protein (Bcl-2) in HL-60 and HSC-2 cells. An undetectable expression of Bcl-2 protein in control and drug-treated HSC-2 cells may explain the relatively higher sensitivity of this cell line to stilbenes and flavonoids.

Changes in fluoride sensitivity during in vitro senescence of normal human oral cells.

We have previously reported that sodium fluoride (NaF) showed slightly higher cytotoxicity against human oral tumor cell lines than normal human oral cells. Possible changes in the NaF sensitivity of three normal human oral cell types (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF) during in vitro ageing were investigated in the present study. When these cells were subcultured at 1:4 split ratio every week, their saturation density declined with increasing population doubling level (PDL), and they ceased to divide when they reached 20 PDL. Mitochondrial function, evaluated by MTT stainability per cell basis, was elevated at the terminal phase. NaF dose-dependently reduced the viable cell number, but did not show any beneficial (growth promoting) effect (so-called "hormesis") at lower concentrations. NaF produced large DNA fragments, without induction of internucleosomal DNA fragmentation, possibly due to weak activation of caspases -3, -8 and -9. Higher concentrations of NaF were required to reduce the number of viable senescent cells than younger cells, indicating that cells become resistant to cytotoxicity of NaF with in vitro ageing.

Analysis of type of cell death induced by topoisomerase inhibitor SN-38 in human oral squamous cell carcinoma cell lines.

Despite frequent use of topoisomerase inhibitors (TIs) as antitumor agents, their application to oral squamous cell carcinoma (OSCC) has not been reported. We investigated three inhibitors of topoisomerase I [camptothecin, irinotecan, SN-38 (active metabolite of irinotecan)] and two inhibitors of topoisomerase II (etoposide, teniposide) for their cytotoxicity towards a total of 15 human tumor cell lines and normal cultured cells. All TIs exhibited higher cytotoxicity towards tumor cell lines (OSCC, glioblastoma, myelogenous leukemia) as compared with normal mesenchymal (gingival fibroblast, pulp cell, periodontal ligament fibroblast) and epithelial cells (skin keratinocytes). Among TIs, SN-38 had the highest cytotoxicity towards OSCC cell lines, with a tumor specificity index of 1321 compared to mesenchymal cells and 22 compared with epithelial cells. SN-38 induced different types of cell death in two OSCC cell lines: apoptosis (caspase-3 activation and internucleosomal DNA fragmentation) in HSC-2 cells and autophagy (formation of autophagosome and secondary lysosome) in HSC-4 cells. The cell death of HSC-2 and HSC-4 cells was significantly inhibited by pre-treatment with caspase inhibitor (Z-VAD-FMK) and autophagy inhibitors (3-methyladenine, bafilomycin A1), respectively. The present study demonstrated that SN-38 is highly cytotoxic to OSCC cell lines, regardless of the type of induced cell death, suggesting its future application for chemotherapy of OSCC.

Tumor-specific cytotoxicity and type of cell death induced by benzaldehyde.

We have previously reported that sodium 5,6-benzylidene-L-ascorbate (SBA) induced dramatic antitumor activity in inoperable cancer patients, but induced only marginal tumor specificity in vitro. Here the tumor specificity and type of cell death induced by benzaldehyde (BA), a degradation product of SBA, was investigated, using human tumor cell lines (oral squamous cell carcinoma [OSCC], glioblastoma, myelogenous leukemia) and human normal oral cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast). BA showed much higher tumor-specific cytotoxicity than SBA. BA induced the formation of autophagosomes, the destruction of mitochondrial structure and digestion of broken organelles, without any apparent induction of internucleosomal DNA fragmentation and caspase activation in an OSCC cell line HSC-2, in a similar manner to SBA. However, pretreatment with 3-methyladenine or bafilomycin A(1), autophagy inhibitors, did not completely rescue the cells from the cytotoxicity induced by BA. The study suggests that BA may play an important role in the induction of antitumor activity of SBA in vivo, although the autophagic phenotypes induced by BA may be involved in both cell death and survival.

Non-apoptotic cell death induced by nutritional starvation in J774.1 mouse macrophage-like cell line.

The growth and amino acid utilization of a mouse macrophage-like cell line J774.1 was investigated in two different culture media supplemented with 10% fetal bovine serum (FBS). The J774.1 cells grew faster, and consumed glutamine and serine at higher rates in DMEM than in RPMI1640 medium. The consumption of other amino acids was much less, while considerable quantities of alanine, glutamic acid and glycine were produced by the J774.1 cells. When the cells became confluent, serine, but not glutamine, was nearly depleted from the culture medium, followed by cell death characterized by smear DNA fragmentation, slight caspase-3 activation and structural damage of the mitochondria. Serine is required for the growth of mouse macrophage-like cell lines, and DMEM is superior to RPMI1640 for long-term cell culture.