Abstract
The metabolism of reactive oxygen species (ROS) in cancer cells is a research area that has not been intensively pursued. It is generally believed that cancer cells are persistently oxidative stressed. The consequence of this phenomenon will result in changes of the characteristic of a cancer cell. Whether or not the oxidative stress has a role in regulating the metastatic potential of a cancer cell is a research area that has been totally neglected. Using a group of human hepatocellular carcinoma (HCC) cell lines with distinct disparity in their differentiation status, established by their morphological observation and the abilities of synthesizing at least 15 plasma proteins, as the experimental model, we proposed to investigate the possibility that the constitutive oxidative stress status of these HCC cells may be modulated by their differentiation status. As a result, varying degrees of oxidative stress status of these cells will affect the propensity of expression of a few redox-sensitive transcription factors, such as NF-kB and AP-1, which may eventually modulate the metastasis- related gene expression. If these proposed hypotheses turn out to be true, we will then investigate the underlying mechanism(s) associated with the phenomena observed. Firstly, we demonstrated previously (Liu et al., 2000) that the total antioxidative capacity (as expressed by the composite propensities of expressing 4 antioxidant enzymes and the intracellular glutathione contents) as well as GSH/GSSG ratios of the HCC cells we studied were excellently correlated with their differentiation status, with an order of HepG2 > Hep3B > J5 > SK-Hep-I. To further confirm this observed phenomenon, we quantified the steady state mRNA expressions of the four antioxidant enzymes by duplex RT-PCR method. In this study, we further confirmed that well-differentiated HCC cells, such as HepG2 and Hep3B expressed higher levels of extracellular GPx (eGPx) and catalase mRNAs. Conversely, the expression of mRNA for both enzymes in a poorly-differentiated HCC cells, such as SK-Hep-I and Mahlavu, was trace or even negligible. Since GSH biosynthesis is controlled by g-glutamylcysteine synthetase (g-GCS), a rate-limiting enzyme composing of a catalytic heavy subunit ( gGCS h ) and a regulatory light subunit ( gGCSl) ,we wanted to further substantiate that differentiation status-mediated upregulation of GSH is regulated by this enzyme. We demonstrated that the g-GCSh expression was again differentiation status regulated, established by using either a HPLC or a duplex RT-PCR method. The order of ranking for the expression of g-GCS was HepG2 > Hep3B > J5 > SK-Hep-I. In contrast, we found that g-GCSl mRNA seemed not to be influenced by the differentiation status.
It has been documented that the transcription factors NF-kB and AP-1 are redox-sensitive. Thus, we wanted to see if both transcription factors could constitutively be activated. Also, is the expression propensity of these transcription factors modulated by the oxidative stress status of these HCC cells? Using EMSA and supershift techniques, we demonstrated that varying degrees of expressions of both transcription factors can be seen, with an order of expression propensity of SK-Hep-I > Mahlavu > J5 > Hep3B > HepG2. Having known that both NF-kB and AP-1 could modulate a group of metastasis-related gene expression, we then investigate if the constitutive metastatic potential of these HCC cells can be varied depending upon how extensive the expression propensity of both NF-kB and AP-1, we used a panel of markers for evaluating the metastatic potential, namely: Matrix metalloproteinase (MMP), interleukin 8 (IL-8) and an adhesion molecular E-cadherin. Using both activity and duplex RT-PCR methods, we demonstrated that only a poorly differentiated HCC cells were capable of expressing MMPs (SK-Hep-I predominately expressed a large amount of MMP-9 and mRNA; Mahlavu predominately expressed MMP-2 along with a trace amount of MMP-9). HepG2, Hep3B and J5 were completely devoid of MMP expression. Using ELISA assay, we measured the secretion of IL-8 in the culture media by these HCC cells and demonstrated that the propensity of secretion having an order of SK-Hep-I > HepG2 > Hep3B (J5 and Mahlavu expressed only a trace amount). Next, we used western blotting and duplex RT-PCR techniques to demonstrate that the expressions of E-cadherin were predominately existed in only well-differentiated cell lines, HepG2 and Hep3B. J5, Mahlavu and SK-Hep-I were all devoid of expression. Finally, for the purpose of demonstrating that the intracellular oxidative stress status does have a role in regulating the above-mentioned metastasis-related gene expression, we transfected g-GCSh cDNA to SK-Hep-I cell and obtained a cell type, termed GCS 30, in which its g-GCSh activity, mRNA and GSH content has been proved to be higher than its untransfected counterpart, SK-Hep-I. We then measured the oxidative stress status of GCS 30 using DCFDA-flowcytometric method. From our data, we did demonstrate that the oxidative-stress status of GCS 30 was shown to be decreased as compared to its counterpart. However, we were surprised to find out that GSH/GSSG ratio remained unchange in GCS-30 as compared to SK-Hep-I cells. Using EMSA technique, we showed that GCS 30 cells only exhibited relative strong binding activity to NF-kB, but not for AP-1 binding. Surprisingly, we found that the MMPs activities in GCS 30 cells were relatively comparable to SK-Hep-I, indicating that MMP expression might be regulated by a pathway other than AP-1. The mechanism(s) underlying this observed phenomenon await further clarification.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0205102-145228 |
| Date | 05 February 2002 |
| Creators | Chern, Chi-Liang |
| Contributors | Ming-Hong Tai, Lea-Yea Chuang, Chung-Lung Cho, Tsan-Zon Liu, Jiin-Tsuey Cheng |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0205102-145228 |
| Rights | off_campus_withheld, Copyright information available at source archive |
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