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-specific cytotoxicity and type of cell death induced by beta-cyclodextrin benzaldehyde inclusion compound.

The cytotoxicity of beta-cyclodextrin benzaldehyde inclusion compound (CDBA) against human normal and cancer cell lines was investigated. CDBA showed slightly higher cytotoxicity against human tumor cell lines, as compared to normal cells, with a tumor-specificity index of 2.2. Human myelogenous leukemia cell lines (HL-60, ML-1, KG-1) were the most sensitive to CDBA, followed by human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4) and human glioblastoma (T98G, U87MG). Human normal cells (gingival fibroblasts, pulp cells, periodontal ligament fibroblasts) were the most resistant. CDBA induced internucleosomal DNA fragmentation in HL-60 cells and caspase-3, -8, -9 activation, but to a much lesser extent than that attained by UV irradiation or actinomycin D. On the other hand, CDBA did not induce DNA fragmentation, nor caspase activation in HSC-2, HSC-4 or T98G cells. Electron microscopy demonstrated that CDBA induced the destruction of mitochondrial structure and digestion of broken organelles by secondary lysosomes in all of these cells. CDBA also increased the number of acidic organelles as judged by acridine orange staining. The present study suggests that CDBA induces autophagic cell death in cancer cell lines.

Mitochondrial control of cell death induction by sodium 5,6-benzylidene-L-ascorbate.

Previous studies have demonstrated the dramatic antitumor activity of sodium 5,6-benzylidene-L-ascorbate (SBA). However, the molecular mechanism of this antitumor action is unclear. We investigated the changes in the fine structures of a human submandibular gland carcinoma cell line, HSG, during the cell death induced by SBA. When HSG cells were incubated for 6 hours with non-cytotoxic concentrations of SBA, changes in the mitochondria, such as the disassembly of cristae and decrease in the electron density, were discrenible. At cytotoxic concentrations, the swelling and vacuolization of the mitochondria became apparent, while the nuclear architecture including the profile and ratio of heterochromatin and euchromatin and thickness of the nuclear membrane were intact. These data suggest that the mitochondria, not the nucleus, may be the target organelle of SBA, and support the ability of SBA to induce non-apoptotic cell death in HSG cells.

Sodium 5, 6-benzylidene-L-ascorbate induces in vitro neuronal cell differentiation accompanying apoptosis and necrosis.

Antiproliferative activity through induction of differentiation by chemotherapeutic agents is required for certain types of cancers. Here, we report that a potent antitumor agent, sodium 5, 6-benzylidene-L-ascorbate (SBA), could induce morphological change of human neuroblastoma IMR-32 cells into a ganglion-like cell aggregate (pseudoganglion) having many neurites and the property of cholinergic neurons. Simultaneously with neuronal differentiation, substantial apoptosis and necrosis/type 2 physiological cell death, which is independent of apoptosis and resistant to a broad-spectrum caspase inhibitor, Z-Asp-CH2-DCB, were also observed. These data indicated that SBA could suppress tumor cell growth through the induction of three different physiological pathways such as differentiation, apoptosis and necrosis by which tissues and organs regulate their own development and maintenance.

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.