Abstract
OBJECTIVE To study the differential expression of COP9, JAK2, HSPS and NADH genes and their relationship with taxol resistance in ovarian carcinoma.
METHODS The up-regulated genes for JAK2, HSPs and NADH, and the down-regulated COP9 gene, which have been demonstrated by a micro-array from our previous study, were examined by the reverse transcription-polymerase chain reaction (RT-PCR) and real-time-PCR in 33 tissue samples from ovarian cancer patients who previously had received taxol-based chemotherapy (Group1), and from 21 ovarian cancer cases who had never received chemotherapy before operation (Group2).
RESULTS The expression rate of COP9 in Group 1 was shown to be markedly lower compared to Group 2 (P = 0.000). However the expression rates of JAK2, HSPs and NADH in Group 1 were significantly higher than those in Group 2 (all P = 0.000). The expression of COP9, HSPs and NADH showed no significant differences related to the histological grade. However, significant higher expression of JAK2 was seen in Grade 3 compared to Grade 1~2 (P = 0.000). We performed real-time PCR to confirm the above findings showing that the level of COP9 gene copies of Group 1 was much lower than that of Group 2 (P = 0.007). Similarly, HSPs and NADH had significantly higher copy numbers in Group 1 compared to those patients who had not undergone chemotherapy (Group 2) (P = 0.018, P = 0.024, respectively). We also found that the JAK2 gene copy concentration in a TAX group was higher than in a non-TAX group (P = 0.001). In addition, we confirmed a higher copy concentration of the JAK2 gene in Grade 3 compared to Grade 1~2 tumors (P = 0.000), though no significant difference in the expression rates and gene copy concentrations of the 4 genes were seen among variable tumor types.
CONCLUSION The down-regulation of COP9 and upregulation of JAK2, HSPs, and NADH genes are related to the mechanism of drug-resistance in ovarian cancers.
keywords
Introduction
Taxol (TAX) is a natural anti-tumor agent with a unique anti-tumor mechanism. It was first extracted from the Taxus brevifolia found in western America in 1971. Tubulin and microtubular systems are generally believed to be the major targets of TAX. TAX prevents tubulin from disassembling and halts the cell proliferation cycle in the G2 and M phases, eventually leading to cell death. From the clinical applications of taxol in the 1990s. It’s use has not only improved efficacy in second-line treatment, but has also led to the combination of taxol with cisplatin instead of with cyclophos phamide (CTX), this has become the standardized first-line chemotherapy in post-surgical ovarian epithelial cancer (OEC) patients. Such development has shown to prolong the medium survival time of advanced-stage OEC patients. However, the longer taxol is clinically administered, the more apparent the resistance taxol. This has been a major obstacle in the chemotherapy for OEC patients. It is thus urgent to elucidate the mechanism responsible for TAX resistance, and to improve the effectiveness of chemotherapy.
In our previous study, a taxol-resistant ovarian cancer cell-line, OC3/Tax300, was set up and differential gene expression was studied using cDNA microarrays[1]. The results showed that there was markedly decreased expression of the cellular signal transduction protein, COP9, and up-regulation of JAK2 (Janus kinase 2), HSPs and NADH genes in taxol-resistant ovarian cancer cells, i.e., in the artificially cultured taxol-resistant cell line OC3/Tax300. Here we aim to examine the gene expression in ovarian cancer tissues and aberrant gene co-expression in those cells, as such research may facilitate clinical treatment. Driven by these questions, we selected COP9, JAK2, HSPs and NADH genes for further study in cancer tissue specimens. In our research, we examined the expression of these genes in samples from different groups of ovarian cancer patients, analyzed their relationship with the clinical pathology and finally demonstrated the relationship between these genes and taxol resistance.
Materials and Methods
Clinical samples
Fifty-four ovarian cancer samples (all cryopreserved in liquid nitrogen) were taken from March 2000 to May 2005 in the Beijing Shijitan Hospital. The samples were divided into two sets: 33 cases with taxol chemotherapy before their first operation, or second operation because of relapse (Group 1), and 21 cases without taxol chemotherapy before operation (Group 2). The histological types were 26 serous ovarian cancers, 21 mucous ovarian cancers, and 7 endometrioid cancers. The histological grades were 30 low-differentiated cancers (Grade 3), and 24 moderate-to well-differentiated cancers (Grade 1~2). See Table 1.
The pathological parameters of 54 ovarian cancers.
Primers used for the PCR
Primers were designed using primer premier 5.0 software. The primer sequences of COP9, NADH, JAK2, HSPs and human 18s (as an internal control) genes are detailed in Table 2.
Primer sequences for COP9, NADH, JAK2, HSPs and human 18s genes.
Reverse transcription of RNA samples and preexperimental quantitative fluorescence PCR
RNA was extracted following the protocol of the TRIzol Reagent kit (Life Technologies) and RNA reverse transcription was performed using a TAKATA kit (Takara Biotechnology [Dalian] Co., Ltd.). The reverse transcribed cDNA was diluted 10 times compared to the previous volume, and then used as a template. A quantitative fluorescence PCR pre-experiment was conducted with primers of the target genes and HS Ex Taq DNA polymerase. The PCR reaction included denaturation at 94°C for 2 min followed by 30 cycles with a 94°C denaturation for 30 s, a 61°C annealing for 30 s and a 72 °C extension for 20 s. The last 5 min of the reaction was kept at 20°C. Products of the PCR reaction were analyzed by agarose-gel electrophoresis and pure samples without interference of primer dimers were selected for further quantitative PCR reaction.
Quantitative fluorescence PCR reaction
The SYBR Premix Ex Taq kit (TAKARA) was employed to perform the quantitative fluorescence PCR reaction which was conducted on the Rotor-Gene RG-3000 Real-Time Thermal Cycler (Corbett Research, Sydney, Australia). The data were processed using the Rotor-Gene 5.0 software with Excel 7.0. The reaction buffer contained 12.5 μl SYBR Premix Ex Taq, 0.5 μl upstream and 0.5 μl downstream primer, 0.5 μl ROX Reference Dye, 2.0 μl DNA template and 9 μl deionized water for a total volume of 25 μl. The PCR reaction involved denaturation at 94°C for 2 min followed by 40 cycles with a 94°C denaturation for 15 s, a 60°C annealing for 20 s and a 72°C extension for 20 s.
The standard curve
A series of dilutions of the pre-experiment PCR products was performed. The reference cDNA was diluted 1:1,000; 1:10,000; 1:100,000; 1:1,000,000 and was amplified in the quantitative fluorescence PCR. The standard curve was generated from the fluorescence intensity, and the concentration of the cDNA templates determined with the Rotor-Gene 5.0 software. The PCR samples tested were quantified against this standard curve.
Statistical analysis
The Pearson Chi-Square and t-test were performed using the SPSS, Version 11.5 software.
Results
The results of RT-PCR indicated that 4 of the genes had the same exchange as that detected by the gene chip. The overexpression rate of COP9 in the group treated with TAX (39.4%) was markedly lower (P = 0.000) compared to the group not treated with TAX (95.2%). The overexpression rates of JAK2, HSPs and NADH in patients treated with TAX were 90.9%, 96.9% and 93.9% respectively, which were significantly higher than those in patients without TAX treatment, namely, 38.1%, 47.6% and 42.8% (all P = 0.000). When it came to the histological grade, the over expression of COP9, HSPs and NADH showed no significant differences among Grade 3 and Grade 1~2 OEC patients; however, significantly higher expression of JAK2 was seen in Grade 3 (90.0%) compared to Grade 1~2 cases (37.5%, P = 0.000, Table 3).
Over-expression of 4 genes determined by RT-PCR in OEC.
We performed real-time PCR to confirm, that the concentration of COP9 gene copies of patients who had received TAX treatment was much lower compared to patients not previously treated with TAX. Similarly, HSPs and NADH had significantly higher copy concentrations in patients treated with TAX in contrast to those not treated with chemotherapy. We also found a higher JAK2 gene copy concentration in the TAX treated group compared to the non-TAX treated group. In addition, we found a higher copy concentration of the JAK2 gene in grade 3 tumors compared to Grade 1~2 tumors (Table 4), though no significant differences in the over-expression rates and gene-copy concentrations of these 4 genes were seen among the variable tumor types. For quantitation data for Cycling A.FAM/Sybr of the 4 genes see Figs. 1A~D, and for the standard curve of the 4 genes see Figs. 2A~D.
The copy concentration of the 4 genes by realtime PCR in OEC.
A, Quantitation data for Cycling A.FAM/Sybr of the JAK2 gene. B, Quantitation data for Cycling A.FAM/Sybr of NADH the gene. C, Quantitation data for Cycling A.FAM/Sybr of the COP9 gene. D, Quantitation data for Cycling A.FAM/Sybr of the HSPs gene.
A, Standard curve of the JAK2 gene; B, Sandard curve of the NADH gene; C, Standard curve of the COP9 gene; D, Standard curve of the HSPs gene.
Discussion
At present, the mechanism of resistance to TAX is not fully understood, though we believe it to be a complicated process involving multiple factors, multiple steps and multiple gene components. Some investigators have observed over-expression of the P-gp protein in TAX-resistant ovarian cancer cell lines[2], while other researchers considered the resistance to be related to the alterations in the expression of the β-tubulin gene in ovarian cancer patients[3]. It has been confirmed by a series of investigations that multidrug resistance 1 (MDR1), taxol resistance associated gene-3 (TRAG-3) and antiapoptotic genes, etc. contribute to the resistance to TAX resistance[4,5].
Our previous study using cDNA microarrays indicated that the taxol-resistant ovarian cancer cell line, OC3/Tax300, had markedly decreased expression of the COP9 cellular signal-transduction protein. Among those up-regulated genes, JAK2, HSPs and NADH were the most representative[1]. The RT-PCR and quantitative analysis by realtime PCR was in agreement with the expression of COP9, JAK2, HSPs and NADH in cDNA microarray assays. In the taxol-chemotherapy group, COP9 was significantly reduced, while JAK2, HSPs and NADH were markedly upregulated.
The COP9 signalosome (CSN) protein complex and the RB system probably coregulate the stability of the cell cycle. It has recently been suggested that the proteins interacting with CSN are substrates of this kinase, including c-Jun, p53, ICSBP and p27[6,7]. JAK2 is a protein phosphatase consisting of 1,132 amino acids with a molecular weight of 131 kDa, which functions as an important cellular signal-transduction protein and triggers the phosphorylation of a protein tyrosine. Besides, JAK2 and transcription binding factor STATS, comprise a new cellular signal-transduction pathway, which leads to cell transformation when over activated. Xie et al.[8] reported that JAK2 mediated the up-regulated expression of the proliferation gene, c-Myc, induced by BCR/ABL. Activated JAK2, elevated the expression of the c-myc protein, a sequence-specific transcription factor which modulated gene expression during the process of cell proliferation and differentiation, promoted the transformation from G0 phase to G1 phase and participated in the anti-apoptosis effects. Our data indicated that up-regulated JAK2 assisted the drug resistance in ovarian cancer cells through its anti-apoptotic effects, though the responsible mechanism remains to be elucidated.
Heat shock proteins (HSPs) are part of a multi-gene family, which play a crucial role in localizing proteins, and maintaining normal pleated sheets so as to form stable structures as well as modulating the balance between protein synthesis and degradation. Following cellular stimulation, the expression of HSPs rapidly increases in order to protect the cells from endogenous stress, enhance their tolerance to stress and reinforce cellular repair. Nevertheless, they participate in many oncogenic pathways. Ciocca et al.[9] reported that breast cancer cells overexpressing HSPs were resistant to chemotherapeutic agents like taxol and cisplatin, resulting in poor curative efficacy. This finding was confirmed by the upregulated HSPC expression in the present study.
Reduced nicotinamide adenine dinucleotide (NADH), a coenzyme essential for cell energy metabolism, is generated in mitochondria and is used to produce ATP via the electron transfer chain. NADH plays a crucial role in the process of cell growth, differentiation and energy metabolism. Zhang et al.[10-12] showed that NADH inhibited apoptosis induced by cisplatin in liver cells. NADH regulated the transmembrane potential of the mitochondria, cytoplasmic-free calcium ions, the pH value as well as the reactive oxygen species (ROS) level in liver cells. Therefore these responses suppressed the activation of the caspase-3 and caspase-8 pathways, prevented mitochondria from releasing cytochrome C into the cytoplasm, and maintained the integrity of polymerases. In addition, NADH helped to down-regulate p53 and up-regulate the apoptosis-inhibiting gene bcl-2. There is a close correlation between cell apoptosis and drug resistance, namely, the absence of any apoptosispromoting genes and the overexpression of any antiapoptotic genes may contribute to the generation of drug resistance.
Footnotes
This work was supported by a grant from Beijing Captial Medicine Development Fundation (No.ZD199915).
- Received February 15, 2008.
- Accepted July 30, 2008.
- Copyright © 2008 by Tianjin Medical University Cancer Institute & Hospital and Springer
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