Defects in apoptotic signal transduction in cisplatin-resistant non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) often shows intrinsic multidrug resistance, which is one of the most serious problems in cisplatin-based adjuvant chemotherapy. Anticancer drugs exert at least part of their cytotoxic effect by triggering apoptosis. In order to understand the molecular alterations leading to heterogeneous cisplatin sensitivity and apoptosis inducibility in NSCLC cells, we analyzed various apoptotic pathways, including the activation of caspase-8, -9 and -3, the release of cytochrome c from mitochondria and the expression levels of pro- and anti-apoptotic proteins such as Bax, Bad, Bcl-2, Bcl-xL, Fas and p53 using heterogeneously apoptosis-sensitive cells (Ma-10, Ma-31 and Ma-46). Cisplatin treatment induced the activation of caspase-8, -9 and -3 and the release of cytochrome c in apoptosis-sensitive Ma-46. The expression of Bcl-xL was the highest and p53 was not expressed in apoptosis-resistant Ma-31, and Fas was not expressed in Ma-46. These expression levels were not correlated with the apoptosis inducibility of the three cell lines. These results suggest that blockage of the apoptotic signal from mitochondria is responsible for apoptosis resistance in NSCLC cell lines. Our findings also indicate that anti-apoptotic Bcl-xL and pro-apoptotic p53 are necessary but not sufficient for resistance to cisplatin-induced apoptosis in NSCLC cells.

Overexpression of constitutive signal transducer and activator of transcription 3 mRNA in cisplatin-resistant human non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) often shows intrinsic multidrug resistance, which is one of the most serious problems in cisplatin-based adjuvant chemotherapy. Recently, the constitutive activation of signal transducer and activator of transcription (STAT) factors has been found in a variety of human cancers. In the present study, the mRNA expression of STATs in various human NSCLC cell lines was investigated by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to determine whether STATs can be implicated in cisplatin resistance and apoptosis inducibility. Cisplatin triggered apoptosis in Ma-46 based on biochemical and morphological findings, but not in Ma-31. The mRNA expression of STAT3 was highest in cisplatin-resistant Ma-31 and lowest in cisplatin-sensitive Ma-46. A 6-hour exposure of cancer cells to cisplatin failed to stimulate STAT3 mRNA expression. Therefore, an increased transcriptional level of constitutive STAT3 may be related to the suppressive regulation of the apoptotic pathway in intrinsically chemo-resistant NSCLC cells.

A selective plasmin inhibitor, trans-aminomethylcyclohexanecarbonyl-L-(O-picolyl)tyrosine-octylamide (YO-2), induces thymocyte apoptosis.

The treatment of rat thymocytes with YO-2, a novel inhibitor of plasmin, resulted in an increase in DNA fragmentation. DNA fragmentation was also induced by another YO compounds such as YO-0, -3, -4 and -5. These YO compounds are the inhibitor of plasmin activity. On the other hand, YO-1, -6 and -8 that hardly inhibit plasmin activity had no effect on DNA fragmentation. Analysis of fragmented DNA from thymocytes treated with YO-2 by agarose gel electrophoresis revealed that the compound caused internucleosomal DNA fragmentation. In addition, judging from a laser scanning microscopy, annexin V-positive and propidium iodide-negative cells were increased by the treatment of the cells with the compound. Moreover, chromatin condensation was observed in thymocytes treated with the compound. These results demonstrated that YO-2 induces thymocyte apoptosis. There seemed to be some correlation between the apoptosis induced by YO compounds and their plasmin inhibitory effect. However, because the other protease inhibitors including pepstatin A, leupeptin, AEBSF, DFP and E-64-d did not affect DNA fragmentation, YO compounds are likely to have unique mechanism on plasmin or to show the effect on the other plasmin-like proteases. The plasmin inhibitory activity may have an important role in YO-2-induced apoptosis. Furthermore, the stimulations of caspase-8, -9 and -3-like activities were observed in thymocytes treated with YO-2. These results suggest that YO-2 induces thymocyte apoptosis via activation of caspase cascade.

Involvement of histone phosphorylation in apoptosis of human astrocytes after exposure to saline solution.

We have previously found using inhibitors of protein phosphatase that phosphorylation of histones may be involved in thymocyte apoptosis. In this study, we examined whether histone modification occurs in astrocyte apoptosis induced by a pathological condition in the absence of drug. Incubation of cultured human astrocytes with growth medium for 24 h after exposure to saline solution for 30 min induced an increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and nuclear condensation, biochemical and morphological hallmarks of apoptotic cell death. Acetic acid-urea-Triton X-100 (AUT) gel electrophoresis of the nuclear histone fraction and N-terminal peptide analysis showed that the treatment with saline solution caused rapid changes in phosphorylation of H2A subfamilies, but not in histone acetylation. The phosphorylation of the two subtypes increased markedly, whereas the phosphorylation of one subtype decreased. In contrast, exposure to ACF-95, an artificial cerebrospinal fluid (CSF), was associated with little induction of apoptotic cell death and induced less changes in histone phosphorylation. These results support the previous idea that chemical modification of histones is involved in the DNA fragmentation in astrocytes undergoing apoptosis.

Expression of multidrug resistance proteins and accumulation of cisplatin in human non-small cell lung cancer cells.

In order to understand and overcome multidrug resistance (MDR) of human non-small cell lung cancer (NSCLC), mRNA and protein expression levels of P-glycoprotein (MDR1), multidrug resistance-associated protein 1 (MRP1), and lung resistance-related protein (LRP) were investigated and compared with the chemosensitivity and the intracellular/intranuclear cisplatin accumulation of three NSCLC cell lines (Ma-10, Ma-31, and Ma-46). Ma-31 was more resistant than Ma-10 and Ma-46 to cisplatin, carboplatin, etoposide, and paclitaxel. The mRNA level of MDR1 was extremely low, and MDR1 protein was not detected in all cell lines. MRP1 mRNA expression was highest in Ma-31 and lowest in Ma-10, but there was no notable difference between the MRP1 protein expression in three cell lines. LRP mRNA/protein was equally expressed in Ma-10 and Ma-31, but was nominal in Ma-46. The intracellular/intranuclear cisplatin accumulation of the cells was determined to be Ma-31>Ma-46>Ma-10. Thus, MDR1, MRP1, and LRP mRNA and protein expression levels were not correlated with the chemosensitivity or the intracellular/intranuclear cisplatin accumulation of each cell line. The present results indicate that MDR proteins (MDR1, MRP1, and LRP) may not play an important role in the chemoresistance and drug efflux of NSCLC cells.