Aim: To investigate the role of hTERT gene expression and AP-2α in n-butylidenephthalide (n-BP)-induced apoptosis in A549 lung cancer cells. Methods: Viability of A549 cells was measured by MTT assay. Protein expression was determined by Western blot. Telomerase activity was measured using the modified telomere repeat amplification protocol (TRAP) assay. Xenograft mice were used as a model system to study the cytotoxic effect of n-BP in vivo. The morphology of tumor was examined by immunohistochemical staining. Results: The growth of A549 lung cancer cells treated with n-BP was significantly inhibited. Telomerase activity and hTERT mRNA expression were determined by telomeric repeat amplification protocol and reverse transcription-polymerase chain reaction, respectively. n-BP inhibited telomerase activity and hTERT mRNA expression in A549 cells while overexpression of hTERT could abolish BPinduced growth inhibition in the A549 cells. We also showed that hTERT promoter activity in the presence of n-BP was mediated via AP-2α. We saw an inhibition of tumor growth when nude mice carrying A549 subcutaneous xenograft tumors were treated with n-BP. Immunohistochemistry of this tumor tissue also showed a decrease in the expression of hTERT. Conclusion: The antiproliferative effects of n-BP on A549 cells in vitro and in vivo suggest a novel clinical application of this compound in the treatment of lung cancers.

Alcoholic fatty liver disease (AFLD) is the result of an excessive or chronic consumption of alcohol. Nine male Wistar rats per group were randomly assigned to one of the following drinking treatments: a 20% (w/w) alcohol solution (ALC); a 20% (w/w) alcohol solution cotreated with 0.25 g silymarin/kg BW/day; or a 20% (w/w) alcohol solution cotreated with 0.025 g Niuchangchih ( Antrodia camphorata )/kg BW/day for 4 weeks. Rats with cotreatments of silymarin or Niuchangchih had smaller (p < 0.05) relative liver size, less (p < 0.05) liver lipid accumulation, and lower (p < 0.05) liver damage indices [aspartate aminotransferase (AST) and alkaline phosphatase (ALP) values]. In the regulation of alcohol metabolism, the lower serum alcohol level was observed only in alcohol-fed rats supplemented with Niuchangchih. Meanwhile, cotreatment of silymarin or Niuchangchih increased (p < 0.05) CAT and ALDH activities but did not (p > 0.05) affect ADH and CYP2E1 expressions, which accelerate alcohol metabolism in the body. Additionally, neither silymarin nor Niuchangchih (p > 0.05) influenced serum/hepatic MMP-2 activities and NF-κB, AP1, and α-SMA gene expressions, but serum/hepatic MMP-9 activities and TNF-α, KLF-6, and TGF-β1 gene expressions of alcohol-fed rats were down-regulated (p < 0.05) by silymarin or Niuchangchih, which also could explain the lower liver damage observed in rats chronically fed alcohol.

The response of suspension-cultured pear (Pyrus communes cv Bartlett) cells to heat stress was studied using three viability tests: regrowth (culture growth during 10 days after stress); triphenyltetrazolium chloride reduction; and electrolyte leakage. Critical (50% injury) temperatures for a 20-minute exposure were 420, 52°, and 560C, respectively, for these viability tests. Electrolyte Plant responses to superoptimal temperature include inhibition of photosynthesis (2, 4), pollination (5), protein synthesis (1, 4, 11), translocation (6); promotion ofcallose synthesis (6,8), and leakage of cell contents (2)(3)(4). This diversity of responses complicates efforts to study specific mechanisms and to identify meaningful markers of improved heat tolerance. We have begun a study of heat hardening and injury in suspension-cultured plant cells. Beyond its usefulness in clarifying the heat response of whole plant systems, information on cultured cells is important because it may be possible to select for heat tolerance in vitro. Before in vitro techniques can be used to improve heat tolerance, the basic parameters of cellular response to heat must be understood. In the first phase of this project, described here, we examined pear cell culture growth after heat stress treatment and assessed the validity of other viability tests. The availability of a suitable viability test for cultured cells will facilitate: (a) comparison of acclimation by elevated growth temperature to that induced by heat shock (14); (b) correlation of biochemical changes (e.g. fatty acid saturation and heat shock proteins) to changes in heat tolerance; and (c) development of criteria for selection and characterization of cell lines with increased heat tolerance. 30 ml of cell suspension in 125-ml Erlenmeyer flasks; they were aerated with gyrotary shaking at 85 to 90 cycles/min. The normal growing temperature was 220 C. Stock cultures were 110 ml in 500-ml Erlenmeyer flasks; they were batch-propagated by transferring 10 ml of cell suspension to 100 ml fresh medium at 7-d intervals. MATERIALS AND METHODSHeat Stress Treatment. All heat stress treatments were applied to cells which were from 9-d-old cultures. In all cases, cells were separated from culture medium by filtration through Miracloth and washed with 7 volumes of a solution containing 0.22 M sucrose and 4.52 /AM 2,4-D. Washed cells were then suspended in the same solution; there were approximately 12.5 mg (cell dry wt) per ml. To measure electrolyte leakage, it was necessary to suspend the cells in a solution of low conductivity. To standardize the cells' environment for all viability tests, the same solution was used throughout this study. Preliminary experiments (data not shown) indicated that sucrose in the medium had little effect on heat injury.We imposed heat stress to 3-ml samples of the washed cells in 1-x 10-cm test tubes, using a heated water bath. When testing different stress temperatures, the duration of exposure was 20 min. In other experiments, time at a constant tempe...

The pipetting ofpear (Pyrus communis cv Bartlett) suspension cultures was followed by a substantial but transient decrease in heat sensitivity. During a culture cycle, pear cells were most sensitive to heat at day 3, which coincided with the period of most active cell division. To minimize serious artifacts, the influence of culture handling and age on parameters such as heat sensitivity must be standardized.In the study of complex phenomena such as temperature response, the convenience, simplicity, and uniformity of cell cultures make them valuable experimental systems. Although effects ofheat stress on photosynthesis are critical to whole plants, the absence of chloroplasts in simple cell cultures may further facilitate close examination of other metabolic responses. In considering tissue cultures as model systems, it is important to note that certain aspects of cultured cell response to heat are similar to those of intact plant tissues. Heat stress disrupts polysomes in cells of intact pear fruit (10) and in cultured pear fruit cells (Romani, personal communication). In both intact organs and tissue cultures, heat shock induces acclimation (8,12) and the synthesis of heat-shock proteins (1, 7). However, it is clear that the isolation and culture of plant cells alters physiological characteristics which may influence high temperature response. For example, batch-propagated suspension cultures are characterized by stages ofrelatively high mitotic activity (5). This is relevant to the study of heat stress because certain dividing cells are especially susceptible to heat injury (9, 1 1). Therefore, it is necessary to consider the relationship of culture age to heat sensitivity. Another inherent feature of tissue culture systems is the routine handling and manipulation involved in the transfer and treatment ofcells. Tissue handling has been shown to induce subtle but substantial metabolic changes (13,14), some ofwhich could influence heat tolerance.In this paper, we describe effects of suspension culture age and handling of the heat tolerance of pear cells. (13,14). Our procedure (15) involved pipetting aliquots to test tubes for imposition of heat stress. We found that handling (transfer by pipette) influenced heat sensitivity (Fig. 1)

Using cultured pear (Pyrus communis cv Bartlett) cells, heat tolerance induced by heat shock was compared to that developed during growth at high temperature. After growth at 22°C, cells exposed to 38°C for 20 minutes (heat shock) showed maximum increased tolerance within 6 hours. Cells grown at 30°C developed maximum heat tolerance after 5 to 6 days; this maximum was well below that induced by heat shock. Heat shock-induced tolerance was fully retained at 22°C for 2 days and was only partly lost after 4 days. However, pear cells acclimated at 30°C lost all acquired heat tolerance I to 2 days after transfer to 22°C. In addition, cells which had been heat-acclimated by growth at 30°C showed an additional increase in heat tolerance in response to 39°C heat shock. The most striking difference between heat shock and high growth temperature effects on heat tolerance was revealed when tolerance was determined using viability tests based on different cell functions. Growth at 30°C produced a general hardening, i.e. increased heat tolerance was observed with all three viability tests. In contrast, significantly increased tolerance of heat-shocked cells was observed only with the culture regrowth test. The two types of treatment evoke different mechanisms of heat acclimation.are constants). If so, then heat acclimation induced by brief HS and that induced by prolonged but more moderate heat (e.g. 30C) would have similar mechanisms. However, results presented in this paper indicate that HS and prolonged exposure to 30°C increase the heat tolerance of pear cells in clearly different ways. MATERIALS AND METHODSAll experiments were conducted with suspension-cultured cells of pear (Pyrus communis cv Bartlett) which were used in our previous studies (13,14). The culture medium and most methods (culture maintenance, growth conditions and measurement, heat stress treatment, and viability tests) were as described before (13). All stock cultures and experimental controls were grown at 22C.Heat shock was administered to cells from 7-d-old suspension cultures using 5-ml aliquots in 1 x 10 cm test tubes. Heat treatment was accomplished in a water bath at the HS temperature; temperature equilibration in the 5-ml aliquots occurred within 3 min. The HS treatments lasted 20 min after which six of the 5-ml heat-shocked cell suspensions were transferred to empty, sterile 125-ml Erlenmeyer flasks and maintained at 22°C with shaking. Non-heat-shocked controls were subjected to the same sample handling procedures.Heat shock, i.e. brief exposure to supraoptimal temperature, alters gene expression and leads to increased heat tolerance in a wide range of organisms (10) 2Abbreviations: HS, heat shock; TTC, triphenyl-tetrazolium chloride. RESULTS AND DISCUSSIONHS-Induced Tolerance. Pear cells exposed to 38C for 20 min and then incubated for 24 h at 22C showed greatly increased tolerance of a subsequent heat stress treatment (Fig. 1) (8) and soybean seedlings (7). Schroeder (11) showed that heat tolerance was induced in avocado tissue cultu...