Effect of antitumor agents on cytotoxicity induction by sodium fluoride.

We have recently found that sodium fluoride (NaF) induced apoptotic cell death in tumor cell lines. We investigated here whether 6 popular antitumor compounds modify the cytotoxic activity of NaF against human squamous cell carcinoma (HSC-2) and human promyelocytic leukemia (HL-60) cell lines. Cytotoxic concentrations of cisplatin, etoposide, doxorubicin or peplomycin (tentatively termed as Group I compounds), but not methotrexate and 5-FU (tentatively termed as Group II compounds), enhanced the cytotoxic activity of NaF. NaF and Group I compounds induced internucleosomal DNA fragmentation in HL-60 cells, whereas Group II compounds were inactive even in the presence of NaF. Most Group I compounds except doxorubicin (which induced DNA fragmentation less effectively than others) activated caspase 3 more efficiently than Group II compounds. Caspase 8 (involved in non-mitochondrial extrinsic pathway) and caspase 9 (involved in mitochondrial intrinsic pathway) were also activated, but to a much lesser extent. NaF reduced the glucose consumption at early stage, possibly by inhibition of glycolysis, whereas cisplatin and etoposide reduced the glucose consumption at later stage, suggesting that early decline of glucose consumption is rather specific to NaF.

Peplomycin, a bleomycin derivative, induces myofibroblasts in pulmonary fibrosis.

To analyse the mechanism by which a bleomycin derivative, peplomycin (PLM) induces pulmonary fibrosis, we investigated differentiation of rat pulmonary fibroblasts to myofibroblasts (MF). In intraperitoneally PLM (5 mg/kg/day)-injected rats, the peripheries of lungs adjacent to the pleura revealed advanced fibrosis with a small number of alpha-smooth muscle actin (alpha-SMA)-positive MF, which ultrastructurally possessed abundant microfilaments and cellular organelles. In the fibrotic tissue, the expression of alpha-SMA-mRNA was detected by in situ reverse transcription-polymerase (RT-PCR). The message was strong just after a 2-week administration of PLM then decreased thereafter, although fibrosis advanced. When pulmonary fibroblasts were separated from saline-injected rats (N-Fib) and cultivated for 7 days in the presence of 5 mg/mL PLM, alpha-SMA protein was weakly expressed, while the majority of pulmonary fibroblasts separated from PLM-injected rats (P-Fib) became positive for alpha-SMA in 7-day cultivation and the expression of alpha-SMA in P-Fib was strongly increased by cultivation in the presence of PLM and transforming growth factor-beta (TGF-beta), but not basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF), although the cell proliferation was most strongly enhanced by bFGF and only slightly by PLM and TGF-beta. The alpha-SMA-positive cells expressed vimentin, but only weakly expressed desmin. Additionally, P-Fib generated larger amounts of TGF-beta and bFGF than were generated by N-Fib. These results indicate that PLM induces pulmonary fibrosis by differentiating fibroblasts to alpha-SMA-positive MF, and that bFGF and TGF-beta play each critical role in the different phases of PLM-induced pulmonary fibrosis by inducing fibroblast proliferation and transformation, respectively.

Correlation between EGF receptor expression and peplomycin cytotoxicity in squamous cell carcinoma cell lines.

The relationship between sensitivity to anti-cancer agents and EGF receptor expression on squamous cell carcinoma (SCC) cells was investigated. The cytotoxicity of peplomycin (PEP) was correlated with the number of the EGF receptors expressed on the cancer cells, but no correlations were found between the cytotoxicity of adriamycin and cisplatin and EGF receptors. Addition of TNFalpha increased the number of EGF receptors in the SCC cell lines 1.5- to 2-fold. The cytotoxic effect of combined administration of PEP and TNFalpha was correlated with the number of EGF receptors, and produced a 2- to 5-fold increase in IC50 compared with administration of PEP alone. These observations suggest that EGF receptor expression is closely associated with the cytotoxic effect of PEP on SCC cells.

Amplification of pepleomycin-mediated DNA cleavage and apoptosis by unfused aromatic cations.

An important approach to improve chemotherapy of members of the bleomycin (BLM) family of antibiotics is to find compounds (amplifiers) that enhance the activity of BLM-mediated DNA cleavage and apoptosis. Using a DNA-sequencing technique and pulsed field gel electrophoresis, we have investigated whether BLM-mediated cleavage of isolated and cellular DNA is amplifed by three compounds (RW-12, LS-20, 1S-5Me) which have a conformationally flexible, unfused polyaromatic system and cationic side chain in the molecules. RW-12 enhanced most effectively both pepleomycin (PEM)-induced cytotoxicity and apoptosis. The order of the maximum enhancing effect of amplifiers on PEM-mediated DNA damage is RW-12 > LS-20 > 1S-5Me. RW-12 amplified PEM-mediated DNA cleavage most effectively not only in vitro but also in cultured cells. We have reported that the order of the DNA binding constants of these compounds is RW-12 > LS-20 > 1S-5Me. In this study, we found a good correlation between PEM-mediated cleavage of isolated DNA and cellular DNA. These results suggest that BLM amplifiers bind to DNA and by doing so enhance drug-mediated DNA degradation, ultimately leading to apoptosis. The present study on amplifiers of anticancer agents shows a novel approach to the potentially effective anticancer therapy.

Over-expression of TSC-22 (TGF-beta stimulated clone-22) markedly enhances 5-fluorouracil-induced apoptosis in a human salivary gland cancer cell line.

We have recently isolated TSC-22 (transforming growth factor-beta-stimulated clone-22) cDNA as an anticancer, drug-inducible (with vesnarinone) gene in a human salivary gland cancer cell line, TYS. We have also reported that TSC-22 negatively regulates the growth of TYS cells and that down-regulation of TSC-22 in TYS cells plays a major role in salivary gland tumorigenesis (Nakashiro et al, 1998). In this study, we transfected TYS cells with an expression vector encoding the TSC-22-GFP (green fluorescent protein) fusion protein, and we established TSC-22-GFP-expressing TYS cell clones. Next, we examined (a) the subcellular localization of the fusion protein, (b) the sensitivity of the transfectants to several anticancer drugs (5-fluorouracil, cis-diaminedichloroplatinum, peplomycin), and (c) induction of apoptotic cell death in the transfectants by 5-fluorouracil treatment. The TSC-22-GFP fusion protein was clearly localized to the cytoplasm, but not to the nucleus. Over-expression of the TSC-22-GFP fusion protein did not affect cell growth, but significantly increased the sensitivity of the cells to the anticancer drugs (p < 0.01; one-way ANOVA). Furthermore, over-expression of the TSC-22-GFP fusion protein markedly enhanced 5-fluorouracil-induced apoptosis. These findings suggest that over-expression of TSC-22-GFP protein in TYS cells enhances the chemosensitivity of the cells via induction of apoptosis.

Reduced toxicity of a new formulation--peplomycin adsorbed on activated carbon particles--in mice.

We studied the acute toxicity and pathological effects of peplomycin adsorbed on fine activated carbon particles (PEP-CH) injected subcutaneously in mice. The 50% lethal dose value was 41.2 mg/kg in terms of peplomycin, which was 1.52 times that of the peplomycin aqueous solution (PEP-AQ) of 27.1 mg/kg. Deaths occurred from 5 to 31 days after administration of PEP-CH and from 5 to 22 days after administration of PEP-AQ solution. These figures are remarkably different from another report in which the mice given PEP-AQ died within 10 days.