Kou-Wha Kuo

Anticancer activity evaluation of the Solanum glycoalkaloid solamargine - Triggering apoptosis in human hepatoma cells

Solamargine, an herbal and molluscicidal medicine derived from Solanum incanum, is a steroidal alkaloid glycoside. To characterize the anticancer mechanism of solamargine on human hepatoma cells (Hep3B), changes of cell morphology, DNA content, and gene expression of cells after solamargine treatment were studied. The appearance in solamargine-treated cells of chromatin condensation, DNA fragmentation, and a sub-G(1) peak in a DNA histogram suggests that solamargine induces cell death by apoptosis. The maximum number of dead Hep3B cells was detected within 2 hr of incubation with constant concentrations of solamargine, and no further cell death was observed after an extended incubation with solamargine, indicating that the action of solamargine was irreversible. To determine the susceptibility of cell phases to solamargine-mediated apoptosis, Hep3B cells were synchronized at defined cell cycles by cyclosporin A, colchicine, and genistein, followed by solamargine treatment. The IC(50) values of solamargine for control, G(0)/G(1)-, M-, and G(2)/M-synchronized Hep3B cells were 5.0, > 10, 3.7, and 3.1 microg/mL, implying that cells in the G(2)/M phases are relatively susceptible to solamargine-mediated apoptosis. In addition, a parallel up-regulation of tumor necrosis factor receptor (TNFR)-I and -II on Hep3B cells was detected after solamargine treatment, and the solamargine-mediated cytotoxicity could be neutralized with either TNFR-I or -II specific antibody. Therefore, these results reveal that the actions of TNFR-I and -II on Hep3B cells may be independent, and both are involved in the mechanism of solamargine-mediated apoptosis.

Action of solamargine on human lung cancer cells - enhancement of the susceptibility of cancer cells to TNFs

Solamargine (SM), isolated from Solanum incanum herb, displayed a superior cytotoxicity in four human lung cancer cell lines. The half‐inhibitory concentrations (IC50), of the cell viability assay for H441, H520, H661 and H69 cells were 3, 6.7, 7.2 and 5.8 μM, respectively. SM‐induced apoptosis of these cells by PS externalization in a dose‐dependent manner and increased sub‐G1 fraction were observed. Quenching of the expression of tumor necrosis factor receptors (TNFRs) during the progress of human lung carcinogenesis has been previously reported. SM may induce cell apoptosis via modulating the expression of TNFRs and their subsequent TRADD/FADD signal cascades. Subsequently, SM treatment increased the binding activities of TNF‐α and TNF‐β to the lung cancers, and the intrinsic TNFs‐resistant cancer cells became susceptible to TNF‐α and ‐β. In addition, SM caused release of cytochrome c, downregulation of anti‐apoptotic Bcl‐2 and Bcl‐xL, increase of caspase‐3 activity, and DNA fragmentation. Thus, SM could modulate the expressions of TNFRs and Bcl‐2, and might be a potential anticancer agent for TNFs and Bcl‐2 related resistance of human lung cancer cells.

A novel function of emodin - Enhancement of the nucleotide excision repair of UV- and cisplatin-induced DNA damage in human cells

Nucleotide excision repair (NER) is the main pathway by which mammalian cells remove carcinogenic DNA lesions caused by UV light and many other common mutagens. To explore the effect of emodin on NER, its influence on the repair of UV- and cisplatin-induced DNA damage in human fibroblast cells (WI38) was evaluated. Emodin increased unscheduled DNA synthesis (UDS) of UV-treated cells and reduced cisplatin-induced DNA adducts in WI38 in a concentration-dependent manner, indicating that emodin might promote NER capability in cells. The resultant NER complex is a cooperative assembly of XPF, ERCC1, XPA, RPA, and XPG subunits. The gene regulations of the subunits after emodin treatment were determined by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers. Among the subunits, the expression of ERCC1 in WI38 cells was up-regulated significantly after emodin treatment. All other expressions remained essentially unchanged. In addition, calcium influx in WI38 was increased in proportion to the concentration of emodin. Since UV-induced NER is Ca2+ dependent, elevation of calcium influx may be another mechanism by which emodin facilitates DNA repair. In conclusion, emodin can increase the repair of UV- and cisplatin-induced DNA damage in human cells, and elevated ERCC1 gene expression and Ca2+-mediated DNA repair processes may be involved in the repair mechanism of emodin.

Modulation of Epidermal Terminal Differentiation in Patients after Long-term Topical Corticosteroids

The expression of the various markers for terminal epidermal differentiation in atrophic skin of patients after long‐term topical corticosteroids (TCS) was studied by electron microscopy, immunofluorescence using antibody to profilaggrin/filaggrin (PF/FG), immunoperoxidase staining using antibody to involucrin, and oil red O stain for neutral lipids of the stratum corneum. Thirty‐nine patients were subdivided into two groups: (A) 19 patients suffering from rebound phenomenon after stopping TCS and (B) 20 patients without rebound phenomenon. Biopsy specimens were taken before ending the use of TCS in both groups. In group A, both the morphological markers (including the different epidermal strata, keratohyalin granules, lamellar granules, and cornified cell envelopes) and the molecular markers (including involucrin, PF/FG, and neutral lipids) of terminal epidermal differentiation were significantly suppressed. On the other hand, the differentiational markers in the atrophic skin of patients without rebound phenomena were only slightly altered. These results suggest that potent TCS not only has antiproliferative actions but also inhibits the differentiation of epidermis, resulting in structural defects in the epidermis, especially the stratum corneum.

Downregulation of HER2/neu receptor by solamargine enhances anticancer drug-mediated cytotoxicity in breast cancer cells with high-expressing HER2/neu

Overexpression of HER2/neu is associated with drug resistance and poor outcome in breast cancer. Solamargine (SM), a glycoalkaloid purified from the herb Solanum incanum, exhibits HER2/neu gene modulation of HER2/neu high-expressing human breast cancer cell line ZR-75-1. SM downregulation of HER2/neu gene expression was determined by RT-PCR and Southern hybridization. Additionally, the membrane-bound HER2/neu receptor in highly HER2/neu-expressing breast cancer cells was determined by radioimmunoassay, immunocytochemistry, fluorescent immunocytochemistry, and flow cytometry. SM significantly decreased the number of HER2/neu receptors on the cell membrane. Methotrexate (MTX), 5-florouracil (5-Fu), and cisplatin (CDDP) are commonly used for breast carcinoma treatment in clinics; however, patients with HER2/neu overexpression exhibit resistance to these anticancer drugs. Notably, combination of MTX, 5-Fu, and CDDP with SM individually increased the susceptibility of breast cancer cells to these chemotherapeutic agents. Experimental results indicated that downregulation of HER2/neu by SM might be an effective strategy for enhancing drug susceptibility of breast cancer cells expressing high levels of HER2/neu.

Changes in the activities of mitochondrial enzymes in the progress of tumorigenesis of bladder cancer.

To investigate changes in the activities of mitochondrial enzymes in the progress of tumorigenesis of bladder cancer, N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine (BBN) carcinogen was administered orally to male Sprague‐Dawley rats for eight weeks. The urinary bladders were harvested periodically for the evaluation of tumorigenesis. The activities of NADH cytochrome c reductase (NCCR), succinate cytochrone c reductase (SCCR) and cytochrome c oxidase (CCO) were measured. The NCCR and SCCR activities elevated significantly by the exposure of BBN and decreased rapidly when BBN was withdrawn. However, the CCO activity increased and reached plateau at 18 weeks in spite of the discontinuance of BBN. The results indicated that the NCCR, SCCR and CCO activities were significantly elevated in the tumorigenesis. However, the CCO enzyme may be more related to the progress of tumorigenesis of bladder cancer.

In vitro amplification of human basic fibroblast growth factor mRNA by RNA‐specific exon‐junction primers

Highly sensitive and RNA‐specific primers for the determination of human basic fibroblast growth factor (bFGF) gene expression by RT‐PCR were identified. The RNA‐specific primers could amplify bFGF mRNA from 10 pg to 1 ng of total cellular RNA without interfering with the presence of genomic DNA of the cell. The feasible temperatures of the primers annealed to the template were 5t°C, 60°C and 65°C. In addition, different locations of primers on the bFGF mRNA molecule yielded distinct amounts of RT‐PCR products from the same concentration of RNA, suggesting that the mRNA secondary structure of bFGF affected the RT‐PCR. Owing to high sensitivity and specificity of the primers to bFGF RNA, the RNA‐specific primers may be potentially utilized for the determination of human bFGF gene expression by in situ RT‐PCR.