Mitochondrial dysfunction-induced amphiregulin upregulation mediates chemo-resistance and cell migration in HepG2 cells.

The aim of this study was to investigate the contribution of mitochondrial dysfunction to chemoresistance and migration of hepatoma cells. We found that inhibition of mitochondrial respiration and mitochondrial DNA (mtDNA) depletion resulted in induction of amphiregulin (AR) expression in HepG2 cells. Upon oligomycin treatment of HepG2 cells, the cytosolic Ca(2+) was significantly raised after 30 min, and the intracellular level of reactive oxygen species (ROS) was elevated 2.2-fold after 4 h. Moreover, the condition medium of oligomycin-treated HepG2 cells was found to stimulate the migration of SK-Hep-1 cells. On the other hand, oligomycin-induced cisplatin-resistance and cell migration of HepG2 cells were attenuated by AR-specific RNA interference (#L-017435, Dharmacon) and a neutralizing antibody (MAB262, R&D Systems), respectively. Together, these findings suggest that mitochondrial dysfunction induced Ca(2+) mobilization, and ROS overproduction, which modulated the chemo-resistance and migration of hepatoma cells through the induction and activation of AR.

Alteration of the copy number and deletion of mitochondrial DNA in human hepatocellular carcinoma.

Somatic mutations in mitochondrial DNA (mtDNA) have been detected in hepatocellular carcinoma (HCC). However, it remains unclear whether mtDNA copy number and mitochondrial biogenesis are altered in HCC. In this study, we found that mtDNA copy number and the content of mitochondrial respiratory proteins were reduced in HCCs as compared with the corresponding non-tumorous livers. MtDNA copy number was significantly reduced in female HCC but not in male HCC. Expression of the peroxisome proliferator-activated receptor gamma coactivator-1 was significantly repressed in HCCs (P<0.005), while the expression of the mitochondrial single-strand DNA-binding protein was upregulated, indicating that the regulation of mitochondria biogenesis is disturbed in HCC. Moreover, 22% of HCCs carried a somatic mutation in the mtDNA D-loop region. The non-tumorous liver of the HCC patients with a long-term alcohol-drinking history contained reduced mtDNA copy number (P<0.05) and higher level of the 4977 bp-deleted mtDNA (P<0.05) as compared with non-alcohol patients. Our results suggest that reduced mtDNA copy number, impaired mitochondrial biogenesis and somatic mutations in mtDNA are important events during carcinogenesis of HCC, and the differential alterations in mtDNA of male and female HCC may contribute to the differences in the clinical manifestation between female and male HCC patients.

Accumulation of mitochondrial DNA deletions in human oral tissues -- effects of betel quid chewing and oral cancer.

Accumulation of mitochondrial DNA (mtDNA) mutations in human tissues has been associated with intrinsic aging and environmental insult. Recently, mtDNA mutations have been detected in various tumors, including head and neck tumors. However, the factors affecting the occurrence and accumulation of mtDNA deletions in tumor tissues are poorly understood. In Taiwan, betel quid chewing is a major risk factor for oral cancer. Using polymerase chain reaction (PCR) techniques, we examined large-scale deletions of mtDNA in 53 pairs of tumor and non-tumor oral tissues from the patients with or without betel quid chewing history. The results revealed that irrespective of the history of betel quid chewing, the incidences of the 4977bp deletion and other deletions of mtDNA were lower in the tumor portion as compared with the non-tumor portion. The average proportions of the 4977bp deleted mtDNA in the tumor tissues of the betel quid chewers and non-betel quid chewers were 13- and 5-fold, respectively, lower than those in the corresponding non-tumor tissues. Moreover, the average proportion of 4977bp deleted mtDNA was significantly higher (P<0.05) in the non-tumor oral tissues of the patients with betel quid chewing history than that of the patients without the history of betel quid chewing. These results suggest that betel quid chewing may increase mtDNA mutation in human oral tissues and that accumulation of mtDNA deletions and subsequent cytoplasmic segregation of these mutations during cell division could be an important contributor to the early phase of oral carcinogenesis.

Adamantyl maleimide induced changes in adhesion molecules and ROS are involved in apoptosis of human gastric cancer cells.

BACKGROUND: We have previously found that N-1-adamantyl maleimide (AMI) inhibited the growth of SC-M1 tumors in vitro and in vivo. The cytotoxicity of AMI on SC-M1 was accompanied by a decrease of adherent cells and the suppressive effect was associated with conformational changes in cell membrane protein. In order to determine the cellular targets of AMI in human gastric cancer SC-M1 cells, we examined AMI-induced changes in the levels of adhesion molecules CD29 (beta 1 integrin) and CD54 (ICAM-1) and GSH. In addition, we also analyzed changes of apoptosis markers such as annexin V binding to membrane and caspase 3 activity in SC-M1 cells after treatment with AMI. MATERIALS AND METHODS: Changes in CD29, CD54, annexin V binding and GSH levels were examined using FITC-conjugated antibodies or fluorescence probes and flowcytometry. Caspase 3 activity was assayed with spectrofluorometry. RESULTS: We found that the expression of CD29 and CD54 on SC-M1 was decreased and the caspase 3 activity was increased during the early apoptosis induced by AMI. Moreover, it was found that the GSH content of the cell was depleted within 30 minutes and then recovered. CONCLUSION: These results suggest that the cell membrane proteins, such as adhesion molecules (CD29, CD54) and intracellular GSH, were the targets of AMI on SC-M1 cells. Since these membrane alterations were prior to apoptosis they may have transduced a death signal to SC-M1 cells.

Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells.

Mitochondrial respiratory function is impaired in the target tissues of patients with mitochondrial diseases and declines with age in various human tissues. It is generally accepted that respiratory-chain defects result in enhanced production of reactive oxygen species and free radicals in mitochondria. Recently, we have demonstrated that the copy number of mitochondrial DNA (mtDNA) is increased in the lung tissues of elderly human subjects. The mtDNA copy number was suggested to be increased by a feedback mechanism that compensates for defects in mitochondria harbouring mutated mtDNA and a defective respiratory system. However, the detailed mechanism remains unclear. In this study, we treated a human lung fibroblast cell line, MRC-5, with H(2)O(2) at concentrations of 90-360 microM. After the treatment for 24-72 h, we found that cells were arrested at G(0) and G(1) phases but that mitochondrial mass and mtDNA content were significantly increased in a concentration- and time-dependent manner. Moreover, the oxidative stress induced by buthionine sulphoximine was also found to cause an increase in mitochondrial mass of the treated cells. Increased uptake of a vital mitochondrial dye Rhodamine 123 and enhanced tetrazolium [MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] reduction revealed that the mitochondria increased by H(2)O(2) treatment were functional. In addition, the increase in the mitochondrial mass was also observed in cell-cycle-arrested cells induced by mimosine, lovastatin and genistein. Taken together, these findings suggest that the increase in mitochondrial mass and mtDNA content are the early molecular events of human cells in response to endogenous or exogenous oxidative stress through cell-cycle arrest.

Oxidative stress is insignificant in N1S1-transplanted hepatoma despite markedly declined activities of the antioxidant enzymes.

It has been proposed that persistent oxidative stress accounts for the increased levels of DNA damage in cancer tissues. We have examined the profile of anti-oxidant enzymes in a transplanted hepatic tumor model by injecting N1S1 rat hepatoma cells into the liver of Sprague-Dawley rats. The transplanted N1S1 tumors displayed characteristics resembling human hepatocellular carcinoma. The immunoreactivities of catalase (CAT), manganese-superoxide dismutase (Mn SOD), copper/zinc-SOD (Cu/Zn SOD), and glutathione peroxidase (GPx) were found to decrease significantly. The enzyme activity in tumors decreased 26.2-, 4.2-, 4.5-, and 5.4-fold for CAT, Mn SOD, Cu/Zn SOD, and GPx, respectively, relative to those in normal liver tissue from the same animals. In contrast, the mRNA levels of CAT and GPx in tumors decreased only 5- and 2-fold, respectively, and the mRNA levels of Cu/Zn SOD and Mn SOD showed either no change or an increase as compared to those of normal liver tissue. The contents of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and thiobarbituric acid-reactive substances (TBARS) were comparable to those of normal controls. Furthermore, mitochondrial production of superoxide in tumors was 4 times lower than that in normal tissues. In conclusion, the data indicate that the reduced activities of anti-oxidant enzymes in the N1S1 tumor did not cause significant oxidative stress.

Up-regulation of beta-actin, cyclophilin and GAPDH in N1S1 rat hepatoma.

Beta-actin, cyclophilin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are all constantly expressed proteins that regulate cellular structures and endogenous cytoarchitectural functions. In this study, we used an in vivo N1S1 rat hepatoma model to examine changes in the expression levels of these housekeeping genes in normal and tumor liver samples. The beta-actin, cyclophilin and GAPDH genes were all up-regulated in tumor groups as compared to the controls. Our results suggest that up-regulation of beta-actin, cyclophilin and GAPDH genes may be essential for oncogenesis in hepatoma.

[Studies on the centrosome antiserum from a scleroderma patient].

Using ascites cells as screening system and by means of indirect immunofluorescence microscopy, several antisera against centrosome from scleroderma patients were discovered. Since centrosome is chemically complex cellular structure and the autoimmune antiserum is polyclonal, further investigation was made using one of the antisera against centrosome. L929 cultured cells were also employed for the antigen localisation. It was found that the antiserum decorated microtubules, mitotic spindle, centrosome as well as some nuclear structure. Immunoblots of the cell lysate with the antiserum revealed that in addition to the main bands of tubulin there were several less distinct bands. This result confirmed the indirect immunofluorescence observation.