Aberrant regulation of argininosuccinate synthetase by TNF-alpha in human epithelial ovarian cancer

The pro-inflammatory cytokine, tumour necrosis factor-alpha, TNF-alpha, is dysregulated in malignant compared with normal ovarian surface epithelium (OSE). Several epidemiological studies have associated inflammation with ovarian tumorigenesis, with TNF-alpha playing a key role in modulating invasion, angiogenesis and metastasis. Here, we show that TNF-alpha also induces expression of arate-limiting enzyme in arginine synthesis, argininosuccinate synthetase (AS), thereby linking inflammation with several arginine-dependent metabolic pathways, implicated in accelerated carcinogenesis and tumour progression. Having identified AS mRNA induction in TNF-alpha-treated IGROV-1 ovarian cancer cells, using RNA-arbitrarily primed-PCR, we then observed differential regulation of AS mRNA and protein in malignant, compared with normal, OSE cells. A cDNA cancer profiling array with matched normal ovarian and ovarian tumour samples revealed increased expression of AS mRNA in the latter. Moreover, AS protein co-localised with TNF-alpha in ovarian cancer cells, with significantly higher levels of AS in malignant compared with normal ovarian tissue. Increased co-expression of AS and TNF-alpha mRNA was also observed in 2 other epithelial tumours, non-small cell lung and stomach cancer, compared with normal corresponding tissues. In summary, high levels of AS expression, which may be required for several arginine-dependent processes in cancer, including the production of nitric oxide, proline, pyrimidines and polyamines, is regulated by TNF-alpha and may provide an important molecular pathway linking inflammation and metabolism to ovarian tumorigenesis.     

Arginine deprivation and argininosuccinate synthetase expression in the treatment of cancer

Arginine, a semi‐essential amino acid in humans, is critical for the growth of human cancers, particularly those marked by de novo chemoresistance and a poor clinical outcome. In addition to protein synthesis, arginine is involved in diverse aspects of tumour metabolism, including the synthesis of nitric oxide, polyamines, nucleotides, proline and glutamate. Tumoural downregulation of the enzyme argininosuccinate synthetase (ASS1), a recognised rate‐limiting step in arginine synthesis, results in an intrinsic dependence on extracellular arginine due to an inability to synthesise arginine for growth. This dependence on extracellular arginine is known as arginine auxotrophy. Several tumours are arginine auxotrophic, due to variable loss of ASS1, including hepatocellular carcinoma, malignant melanoma, malignant pleural mesothelioma, prostate and renal cancer. Importantly, targeting extracellular arginine for degradation in the absence of ASS1 triggers apoptosis in arginine auxotrophs. Several phase I/II clinical trials of the arginine‐lowering drug, pegylated arginine deiminase, have shown encouraging evidence of clinical benefit and low toxicity in patients with ASS1‐negative tumours. In part, ASS1 loss is due to epigenetic silencing of the ASS1 promoter in various human cancer cell lines and tumours, and it is this silencing that confers arginine auxotrophy. In relapsed ovarian cancer, this is associated with platinum refractoriness. In contrast, several platinum sensitive tumours, including primary ovarian, stomach and colorectal cancer, are characterised by ASS1 overexpression, which is regulated by proinflammatory cytokines. This review examines the prospects for novel approaches in the prevention, diagnosis and treatment of malignant disease based on ASS1 pathophysiology and its rate‐limiting product, arginine.     

Epigenetic silencing of cell cycle regulatory genes during 3‐methylcholanthrene and diethylnitrosamine‐induced multistep rat lung cancer

The rat lung cancers induced by 3‐methylcholanthrene (MCA) and diethylnitrosamine (DEN) are considered to be a good model for illustrating genetic alterations in human lung precancerous and cancerous lesions. Recently, we had reported that the model can also be used to investigate the step‐by‐step dynamic changes in DNA methylation during lung carcinogenesis. In this study, we have used the same animal model to further study the evolution of methylation alterations of cell cycle regulatory genes CDKN1B (p27) and CDKN1C (p57). Our results showed epigenetic alterations in p27 and p57. Promoter hypermethylation of p27 was detected in one sample of carcinoma in situ (CIS) and two samples of infiltrating carcinoma, all three of which lacked expression of the p27 protein. Methylation of the p57 promoter correlated with the loss of protein expression in lung pathologic lesions, with a gradual increase in methylation frequency from 0 sample in the normal epithelium and hyperplasia, to 11.1% in squamous metaplasia, 18.9% in dysplasia, 26.7% in CIS, and finally 36.0% in infiltrating carcinoma samples. Immunohistochemical analysis showed that p27 and p57 protein expression decreased as lung carcinogenesis progressed. Moreover, weak expression of p27 and p57 in methylated primary tumor cell lines increased markedly after treatment with 5‐aza‐2′‐deoxycytidine (5‐aza‐dC), confirming that methylation was indeed responsible for the gene downregulation. These results suggest that the progression of rat lung carcinogenesis induced by MCA/DEN is associated with dynamic changes in promoter hypermethylation of cell cycle regulatory genes, including p27 and p57, accounting for their defective expression. © 2010 Wiley‐Liss, Inc.     

精氨酸琥珀酸合成酶作为乳腺癌个体化治疗新靶点的研究

氨基酸代谢在细胞生理学中有着举足轻重的作用,因为它参与了大量的细胞代谢和信号通路.精氨酸琥珀酸合成酶(ASS1)是尿素循环中的关键酶,在ASS1缺乏或表达较低的乳腺癌细胞中精氨酸缺乏,减少或降低精氨酸体内含量可以有效抑制肿瘤进展.实验结果表明,聚乙二醇化精氨酸脱亚胺酶(ADI-PEG20)促进精氨酸转变为瓜氨酸,从而降低精氨酸含量;同时mTOR抑制剂抑制CAD(氨甲酰磷酸合成酶2、天门冬氨酸转移酶和氨甲酰天冬氨酸脱水酶复合酶)的磷酸化,与ASS1竞争底物天冬氨酸,达到抑制乳腺癌细胞增殖生长的效果.提示ASS1可能成为抗癌新靶点.     

Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase

Eukaryotic cells can synthesize the non-essential amino acid arginine from aspartate and citrulline using the enzyme argininosuccinate synthetase (ASS). It has been observed that ASS is underexpressed in various types of cancers ASS, for which arginine become auxotrophic. Arginine deiminase (ADI) is a prokaryotic enzyme that metabolizes arginine to citrulline and has been found to inhibit melanoma and hepatoma cancer cells deficient of ASS. We tested the hypothesis that pancreatic cancers have low ASS expression and therefore arginine deprivation by ADI will inhibit cell growth. ASS expression was examined in 47 malignant and 20 non-neoplastic pancreatic tissues as well as a panel of human pancreatic cancer cell lines. Arginine deprivation was achieved by treatment with a recombinant form of ADI formulated with polyethylene glycol (PEG-ADI). Effects on caspase activation, cell growth and cell death were examined. Furthermore, the effect of PEG-ADI on the in vivo growth of pancreatic xenografts was examined. Eighty-seven percent of the tumors lacked ASS expression; 5 of 7 cell lines similarly lacked ASS expression. PEG-ADI specifically inhibited growth of those cell lines lacking ASS. PEG-ADI treatment induced caspase activation and induction of apoptosis. PEG-ADI was well tolerated in mice despite complete elimination of plasma arginine; tumor growth was inhibited by approximately 50%. Reduced expression of ASS occurs in pancreatic cancer and predicts sensitivity to arginine deprivation achieved by PEG-ADI treatment. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of pancreatic cancer, a malignancy in which new therapy is desperately needed.     

RNAi沉默RABGEF1基因对人卵巢癌细胞增殖的影响

目的:探讨沉默RABGEF1基因对人卵巢癌细胞(SK-OV-3细胞)增殖的影响.方法:通过小干扰RNA技术沉默SK-OV-3细胞的RABGEF1基因表达,用实时荧光定量RT-PCR法鉴定细胞内RABGEF1基因的表达水平,培养RABGEF1基因被沉默的SK-OV-3细胞,计数不同时间点的细胞数,分析RABGEF1基因对人卵巢癌细胞增殖的影响.结果:实时荧光定量RT-PCR法检测显示所构建的4个siRNA表达载体对RABGEF1基因表达的沉默率分别为52.5％、58.1％、76.2％和87.7％.将RABGEF1基因表达沉默率为87.7％的SK-OV-3细胞继续培养,计数显示细胞数目较未沉默基因减少(P＜0.05).结论:应用RNAi技术沉默RABGEF1基因可抑制SK-OV-3细胞的增殖,提示RABGEF1基因可能参与卵巢癌的发生发展.     

Downregulation of miR‐29 contributes to cisplatin resistance of ovarian cancer cells

Drug resistance is an obstacle to the treatment of ovarian cancer. Using a unique cell model, we have proven previously that a subpopulation of ovarian cancer cells is more resistant to cisplatin than are the original cells. MicroRNAs (miRNAs), small noncoding RNAs, are involved in many biological events in cancer cells. In our study, we explored whether miRNAs are involved in cisplatin resistance of ovarian cancer cells. Cisplatin‐resistant cells expressed a lower level of miR‐29a/b/c. Manipulation of microRNA‐29 (miR‐29) expression modulated cisplatin sensitivity of CP70, HeyC2, SKOV3 and A2780 ovarian cancer cells. Knockdown of miR‐29a/b/c increased the ability of cells to escape cisplatin‐induced cell death partly through upregulation of collagen type I alpha 1 (COL1A1) and increased the activation of extracellular signal‐regulated kinase 1/2 and inactivation of glycogen synthase kinase 3 beta. When combined with cisplatin treatment, knockdown of miR‐29 decreased the amount of the active form of caspase‐9 and caspase‐3. Ectopic expression of miR‐29 alone or in combination with cisplatin treatment efficaciously reduced the tumorigenicity of CP70 cells in vivo. Our data show that downregulation of miR‐29 increases cisplatin resistance in ovarian cancer cells. Taken together, these data suggest that overexpression of miR‐29 is a potential sensitizer to cisplatin treatment that may have therapeutic implications.     

HIPEC ROC I: A phase i study of cisplatin administered as hyperthermic intraoperative intraperitoneal chemoperfusion followed by postoperative intravenous platinum‐based chemotherapy in patients with platinum‐sensitive recurrent epithelial ovarian cancer

This phase I study tested the safety, feasibility, pharmacokinetics and pharmacodynamics of cisplatin administered as hyperthermic intraoperative intraperitoneal chemoperfusion (HIPEC) in patients with platinum‐sensitive recurrent epithelial ovarian cancer (EOC) undergoing secondary cytoreductive surgery followed by postoperative platinum‐based intravenous chemotherapy. Twelve patients with operable, recurrent platinum‐sensitive EOC (recurrence ≥6 months after first‐line therapy) were included according to the classical 3+3 dose‐escalation design at three dose levels—60, 80 and 100 mg/m². After surgical cytoreduction, a single dose of cisplatin was administered via HIPEC for 90 min at 41–43°C. Postoperatively, all patients were treated with standard intravenous platinum‐based combination chemotherapy. One of six patients experienced a dose‐limiting toxicity (grade 3 renal toxicity) at a dose of 100 mg/m². The remaining five patients treated with 100 mg/m² tolerated their treatment well. The recommended phase II dose was established at 100 mg/m². The mean peritoneal‐to‐plasma AUC ratio was 19·5 at the highest dose level. Cisplatin‐induced DNA adducts were confirmed in tumor samples. Common postoperative grade 1–3 toxicities included fatigue, postoperative pain, nausea, and surgical site infection. The ability to administer standard intravenous platinum‐based chemotherapy after HIPEC was uncompromised. Cisplatin administered as HIPEC at a dose of 100 mg/m² has an acceptable safety profile in selected patients undergoing secondary cytoreductive surgery for platinum‐sensitive recurrent EOC. Favorable pharmacokinetic and pharmacodynamic properties of HIPEC with cisplatin were confirmed at all dose levels, especially at 100 mg/m². The results are encouraging to determine the efficacy of HIPEC as a complementary treatment in patients with EOC.     

5 Aza CdR通过DNMT1调控卵巢癌细胞ERCC1基因甲基化及其表达

目的:研究5-氮杂-2'-脱氧胞苷(5-Aza-CdR)在卵巢癌细胞系SKOV3中对核苷酸切除交叉修复互补基因1(exeision repair cross complementation group 1,ERCC1)表达的影响及可能的机制.方法:设计特异性针对DNA甲基转移酶1(DNAmethyhransferase 1,DNMT1)基因的shRNA转染人人卵巢癌细胞系SKOV3细胞中,Western blotting检测SKOV3细胞DNMT1以及ERCC1的表达变化;利用不同浓度5-Aza-CdR于不同时间点处理卵巢癌SKOV3细胞,Western blotting检测DNMTI和ERCC1蛋白在处理前后的变化,利用亚硫酸氢钠法检测ERCC1基因启动子区域甲基化水平.结果:0.5、1.0、2.0、4.0μmol/L的5-Aza-CdR作用于SKOV3细胞后,DNMT1表达水平呈浓度依赖性降低,而ERCC1表达水平呈浓度依赖性升高;使用终浓度为1.0 μmol/L的5-Aza-CdR处理SKOV3细胞12、24、36 h后,DNMT1表达水平呈时间依赖性降低,而ERCC1表达水平呈时间依赖性升高,亚硫酸氢钠法检测示药物处理前ERCC1启动子区域处于高甲基化水平,在用1.0μmol/L的5-Aza-CdR处理后,其启动子发生了去甲基化.结论:5-Aza-CdR通过DNMT1调控卵巢癌SKOV3细胞中ERCC1基因的甲基化及其表达水平.     

Epigenetic silencing of the PRSS3 putative tumor suppressor gene in non-small cell lung cancer

The serine protease family member PRSS3 (trypsinogen-IV) has been implicated as a putative tumor suppressor gene due to its loss of expression, which is correlated with promoter hypermethylation, in esophageal squamous cell carcinoma and gastric adenocarcinoma. As epigenetic alteration is common in non-small cell lung cancer (NSCLC), we sought to determine if promoter hypermethylation of PRSS3 occurred in this disease, and if it was associated with clinical features of NSCLC or tobacco-related exposures in these patients. Using methylation-specific PCR, we determined the promoter hypermethylation status of PRSS3 in a case series study of primary NSCLC, and found methylation of this gene to be common, occurring in 53% (86 of 166) of tumors examined. There was no association of this alteration with patient demographics, tumor features, or exposure histories of the patients. The lack of association is of interest, as it may suggest a lack of specific selection for inactivation of this gene. On the other hand, the high prevalence of this alteration makes PRSS3 methylation an attractive biomarker for use in diagnostic or screening applications in NSCLC.     

Dasatinib induces autophagic cell death in human ovarian cancer

BACKGROUND:

Dasatinib, an inhibitor of Src/Abl family kinases, can inhibit tumor growth of several solid tumors. However, the effect and mechanism of action of dasatinib in human ovarian cancer cells remains unknown.

METHODS:

Dasatinib‐induced autophagy was determined by acridine orange staining, punctate localization of GFP‐LC3, LC3 protein blotting, and electron microscopy. Significance of beclin 1, AKT, and Bcl‐2 in dasatinib‐induced autophagy and growth inhibition was assayed by small interfering RNA (siRNA) silencing and/or overexpression of the gene of interest.

RESULTS:

Dasatinib inhibited cell growth by inducing little apoptosis, but substantial autophagy in SKOv3 and HEY ovarian cancer cells. In vivo studies showed dasatinib inhibited tumor growth and induced both autophagy and apoptosis in a HEY xenograft model. Knockdown of beclin 1 and Atg12 expression with their respective siRNAs diminished dasatinib‐induced autophagy, whereas knockdown of p27Kip1 with specific siRNAs did not. Small hairpin RNA knockdown of beclin 1 expression reduced dasatinib‐induced autophagy and growth inhibition. Dasatinib reduced the phosphorylation of AKT, mTOR, p70S6K, and S6 kinase expression. Constitutive expression of AKT1 and AKT2 inhibited dasatinib‐induced autophagy in both HEY and SKOv3 cells. Dasatinib also reduced Bcl‐2 expression and activity. Overexpression of Bcl‐2 partially prevented dasatinib‐induced autophagy.

CONCLUSIONS:

Dasatinib induces autophagic cell death in ovarian cancer that partially depends on beclin 1, AKT, and Bcl‐2. These results may have implications for clinical use of dasatinib. Cancer 2010. © 2010 American Cancer Society.     

The experience of chemotherapy‐induced alopecia for Australian women with ovarian cancer

This article describes the experience of chemotherapy‐induced alopecia. Data resulted from an ongoing study, which sought to explore the experience of Australian women with a primary diagnosis of ovarian cancer. Phenomenological analysis of written accounts or interviews with 15 Australian women resulted in 13 of these 15 women giving priority to describing their experience of alopecia. The women described alopecia as the most distressing corporeal feature of the ovarian cancer experience. Factors which contributed to women's distress included: loss of sense of self and altered body image; reminder of their illness and potential for an early death; public statement about their private life, practical issues and re‐growth. No literature was located, worldwide, which specifically explores the experience of alopecia for women with ovarian cancer. This article presents the first in‐depth exploration of the experience of alopecia for Australian women with ovarian cancer. Insight gained from this study will inform understanding of the issues associated with alopecia for women with ovarian cancer and may facilitate the provision of optimal supportive care provided by health care professionals for female cancer patients with chemotherapy‐induced alopecia.     

Antisense blocking of BRCA1 enhances sensitivity to plumbagin but not tamoxifen in BG-1 ovarian cancer cells

Previous studies have shown that reduction in BRCA1 mRNA and protein can result in increased proliferation of BG-1 ovarian cancer cells in both in vitro and in vivo conditions, suggesting that BRCA1 may normally act as a growth inhibitor in these cells. Also, there are other reports that suggest that wild-type BRCA1 protein may repress estrogen receptor (ER) function either directly or indirectly. However, response to antiestrogen drugs in BRCA1-blocked ER-positive ovarian cancer cells has not been reported, and this served as the rationale for this study. We analyzed the effect of tamoxifen, emodin, and plumbagin in BRCA1-blocked ER-positive BG-1 ovarian cancer cells. For all three drugs, BRCA1-blocked cells were more sensitive than the corresponding control cells as assessed by MTT assay; however, only plumbagin showed a statistically significant difference in mean viability (P < 0.05). All three drugs induced loss of mitochondrial membrane potential (DeltaPsi(m)), nuclear condensation, DNA fragmentation, and morphological changes, as observed after 6 h of drug treatment, suggesting apoptosis induction in both BRCA1-blocked and control cells. However, apoptosis induction was greater in BRCA1-blocked cells, the efficacy being in the order of plumbagin > tamoxifen > emodin. The dose of plumbagin needed to kill 50% was 5 microM in the control cells and 2.68 microM for the BRCA1-blocked cells, indicating that the latter was about twofold more sensitive to plumbagin than the wild-type cells. This throws light on the fact that plumbagin may have chemotherapeutic potential as an anticancer agent in BRCA1-mutated ovarian cancer patients.     

Epigenetic silencing of ASPP1 confers 5‐FU resistance in clear cell renal cell carcinoma by preventing p53 activation

Inactivation of p53 has been shown to correlate with drug resistance in tumors. However, in clear cell renal cell carcinoma (ccRCC), p53 is rarely mutated, yet the tumors remain highly insensitive to the conventional chemotherapeutic drugs. The underlying mechanisms responsible for the non‐genetic p53 inactivation remain obscure. Here, we report, for the first time, that Apoptosis Stimulating of P53 Protein 1 (ASPP1) was remarkably downregulated at both mRNA (about 3.9‐fold) and protein (about 4.9‐fold) levels in ccRCC human specimens in comparison with the paired normal controls. In addition, lower ASPP1 was closely related to the higher grade of tumors and shorter life expectancy of ccRCC patients, both with p < 0.001. We also find that CpG island hypermethylation at promoter region contributed to the suppression of ASPP1 expression in ccRCC that contained relatively low levels of ASPP1. Further functional studies demonstrated that forced expression ASPP1 not only significantly inhibited the growth rate of ccRCC, but also promoted sensitivity of ccRCC to the conventional chemotherapeutic drug 5‐fluorouracil (5‐FU)‐induced apoptosis. Moreover, ASPP1 expression was accompanied with the apoptosis‐prone alterations of p53 targets expression and p53 target PIG3 luciferase reporter activation. In contrast, ASPP1 knockdown promoted cell growth and prevent 5‐FU‐induced p53 activation and apoptosis. In conclusion, our results suggest that ASPP1 silencing is one of dominate mechanisms in inhibiting wild type p53 in ccRCC. ASPP1, therefore, may be potentially used as a promising biomarker for prognosis and therapeutic intervention in ccRCC.     

Treatment outcomes and clinicopathologic characteristics of triple‐negative breast cancer patients who received platinum‐containing chemotherapy

The aim of this study was to evaluate the role of platinum‐containing chemotherapy for metastatic triple‐negative breast cancer (TNBC) patients in terms of the response rate (RR) and progression‐free survival. A second aim was to characterize the clinical behavior at the time of relapse of TNBC. We retrospectively analyzed the clinical outcomes of patients with metastatic breast cancer who received taxane–platinum chemotherapy as the first‐ or second‐line treatment, focusing on the TN phenotype. In total, 257 patients with metastatic breast cancer received platinum‐containing chemotherapy at Samsung Medical Center from 1999 to 2006. Of these patients, 106 patients with available data on estrogen (ER), progesterone (PgR) and human epidermal growth factor receptor‐2 (HER2) receptor status received taxane–platinum regimen as the first‐ or second‐line treatment. The overall RR of patients with TNBC was 39%. This rate did not differ significantly from those of patients with other phenotypes. The time to death after chemotherapy (19 vs. 50 months, p = 0.037) and overall survival (OS) (21 vs. 56 months, p = 0.030) differed significantly between patients with TNBC and non‐TNBC. TNBC showed a unique locoregional infiltration pattern at relapse, which might reflect its aggressive clinical behavior. Despite the similar response to platinum‐containing chemotherapy, patients with TNBC had a shorter OS than patients with non‐TNBC. The implication of TN phenotype as poor prognostic factor is uncertain, because it needs to be defined whether poor outcome is related to the rapid growing characteristics of tumor itself or the resistance to drug therapy. Further prospective studies are warranted. © 2008 Wiley‐Liss, Inc.