The combination of ADI-PEG20 and TRAIL effectively increases cell death in melanoma cell lines.

Current treatment for advanced, metastatic melanoma is not very effective, and new modalities are needed. ADI-PEG20 is a drug that specifically targets ASS-negative malignant melanomas while sparing the ASS-expressing normal cells. Although laboratory research and clinical trials showed promising results, there are some ASS-negative cell lines and patients that can develop resistance to this drug. In this report, we combined ADI-PEG20 with another antitumor drug TRAIL to increase the killing of malignant melanoma cells. This combination can greatly inhibit cell growth (to over 80%) and also enhanced cell death (to over 60%) in four melanoma cell lines tested compared with control. We found that ADI-PEG20 could increase the cell surface receptors DR4/5 for TRAIL and that caspase activity correlated with the increased cell death. These two drugs could also increase the level of Noxa while decrease that of survivin. We propose that these two drugs can complement each other by activating the intrinsic and extrinsic apoptosis pathways, thus enhance the killing of melanoma cells.

Roles of multidrug resistance genes in breast cancer chemoresistance.

ATP binding cassette (ABC)-containing drug efflux transporters play important roles in regulating intracellular drug concentrations that determine cell sensitivity to chemotherapeutic agents. Of particular relevance to cancer chemotherapy are the transporters P-glycoprotein (Pgp) encoded by multidrug resistance 1 gene, multidrug resistance protein (MRP), and breast cancer resistance protein (BCRP). More than 80% of currently used antitumor agents can be transported by these three transporters, and overexpression of these transporters renders multidrug resistance to a broad spectrum of antitumor agents. Elevated expression of these transporters is frequently found in breast cancers and correlations with elevated expression of Pgp or MRP1 to chemotherapeutic outcomes have been observed in some cases, suggesting that these transporters may contribute to chemoresistance in breast cancers. However, attempts to modulate the activities of these transporters using reversal agents have met with limited success. Future studies should focus on better understanding of the upregulation mechanisms ofABC transporter genes in breast cancers, and of the pharmacologic mechanisms of transporter-reversal agent interactions. These studies may lead to novel strategies for improving chemotherapeutic efficacies through targeted interventions of these ABC transporters.

Molecular cloning of Chinese hamster 1q31 chromosomal fragile site DNA that is important to mdr1 gene amplification reveals a novel gene whose expression is associated with spermatocyte and adipocyte differentiation.

DNA amplification plays important roles in the development of drug resistance and tumor progression. One mechanism of DNA amplification involves the breakage-fusion-bridge (BFB) cycle. We previously reported that in Chinese hamster ovary (CHO) cell line, breakage at fragile site 1q31 was associated with mdr1 gene amplification through the BFB mechanism. To elucidate the molecular basis of BFB-mediated DNA amplification, we cloned 1q31 fragile site DNA from a Chinese hamster cell line containing an integrated neomycin-resistance marker. Sequence analyses revealed many characteristics similar to those in other common fragile sites. Moreover, this fragile site contains an evolutionarily conserved novel gene, designated fragile site-associated (FSA) gene. FSA encodes a approximately 16-kb mRNA, from which an unusually large open reading frame (orf) of 5005 amino acids can be deduced. The C-terminal portion of FSA shares a striking sequence similarity to that of Caenorhabditi elegans lipid depleted-3 (lpd-3) gene whose function has been demonstrated to involve in lipid storage. We also demonstrated that expression of FSA is associated with the developmental programs of spermatogenesis and adipogenesis. Our results suggest that the Chinese hamster 1q31 fragile site has many important functions including regulation of mdr1 amplification and differentiation of adipocytes and spermatocytes.

Association of fragile site-associated (FSA) gene expression with epithelial differentiation and tumor development.

A novel gene designated as fragile site-associated (FSA) gene was recently identified by positional cloning from the CHO 1q31 fragile site which plays an important role in regulating amplification of multidrug resistance (mdr1) gene in multidrug-resistant cells. FSA produces a message of approximately 16 kb which encodes an open-reading frame of 5005 amino acids. FSA shares sequence similarity with that in Caenorhabditis elegans lpd-3, a lipid storage gene. Using immunohistochemical staining and RNA in situ hybridization we report here that expression of FSA is associated with developmental programs of spermatogenesis and mammary gland in mice. Real-time RT-PCR results also support the upregulation of FSA expression in mammary gland development. Expression of FSA in many tissues including colon, skin, ovary, prostate, and bladder is mainly in the postmitotic, well-differentiated compartments. Moreover, levels of FSA expression are downregulated in tumors of these tissue origins. These results suggest that FSA also plays important roles in regulating mammalian epithelial growth and differentiation and tumor development.

Procollagen alpha 1 type 1: a potential aide in histopathological grading of glioma.

Collagen type I production has been shown to play a role in malignant transformation. We examined procollagen type I expression in brain tumors and with histopathological grading. Expression levels of procollagen alpha 1 type 1 were determined in 5 glioma cell lines by RT-PCR, Northern, and Western blot analysis. In addition, 41 primary brain tumors and 2 metastatic lung cancers to the brain were examined by PCR. Of the 5 glioma cell line analyzed, 3 (glioma 1, SW-1783 and U-118) expressed procollagen alpha 1 type I and were sensitive to vitamin D3 (VD3). In contrast, 2 of the cell lines (U-373 and T-98G) lacked procollagen alpha 1 type 1 expression. In patients' samples, 14 of 15 anaplastic and low grade gliomas expressed procollagen alpha 1 type I, and 12 of the 14 expressed high levels. In contrast, only 12 of 21 high grade gliomas from patients expressed procollagen alpha 1 type1 and among these, only 4 of the 12 expressed high levels. Thus, there is an inversed correlation between procollagen alpha 1 type 1 expression and histopathological grading (R2=- 0.56, p=0.0005). Our data suggest that procollagen alpha 1 type I expression occurs more commonly in intermediate and low grade gliomas and may assist in histopathological grading.

Delayed mechanism for induction of gamma-glutamylcysteine synthetase heavy subunit mRNA stability by oxidative stress involving p38 mitogen-activated protein kinase signaling.

Expression of the gamma-glutamylcysteine synthetase heavy subunit (gamma-GCSh), which encodes the rate-limiting enzymes for glutathione biosynthesis, is regulated by many cytotoxic agents. Moreover, gamma-GCSh mRNA expression is elevated in colorectal cancer, but how gamma-GCSh expression is regulated is not completely understood. By using actinomycin D, which inhibits new RNA synthesis, we showed that treatment of human colorectal cancer cells with the prooxidant sulindac increased the half-life of gamma-GCSh mRNA. By using a tetracycline-regulated gamma-GCSh mRNA assay system, we systematically dissected the cis-acting sequence and trans-acting factors that regulate the stability of gamma-GCSh by cytotoxic prooxidants. We demonstrated that a HuR recognition sequence, AUUUA, in the 3'-untranslated region is responsible for the decay of gamma-GCSh mRNA. Oxidative stress enhanced cytoplasmic content of HuR. Overexpression of HuR by transfection stabilized gamma-GCSh mRNA, whereas overexpression of a dominant-negative HuR mutant suppressed the induced stability. Furthermore, prooxidant-induced gamma-GCSh mRNA stabilization and HuR binding were blocked by p38 mitogen-activated protein kinase inhibitors. We provide the first evidence that reduction-oxidation regulation of gamma-GCSh expression, itself a reduction-oxidation sensor and regulator, is mediated at least in part by the p38 mitogen-activated protein kinase signaling through the HuR RNA-binding protein.

Human ATP-binding cassette transporter ABCC10: expression profile and p53-dependent upregulation.

Human ATP-binding cassette (ABC) transporter genes are classified into seven sub-families, where "C" subfamily comprises a total of 13 gene members. The ABCC10 cDNA was cloned in the human full-length cDNA project at the Kazusa DNA Research Institute. However, current information is limited regarding its physiological function and gene expression. In the present study, we have investigated the expression of the ABCC10 gene to gain insight into its biological nature. By quantitative PCR, ABCC10 gene expression is demonstrated to be highest in pancreas among the adult and fetal tissues and tumors presently tested. Decreased expression was observed when resting T- and B-cells were activated. Furthermore, when we examined its expression under apoptotic conditions, we found that ABCC10 mRNA levels remarkably increased in doxorubicin-treated MCF7 cells, whereas its up-regulation was suppressed in p53-dominant-negative MCF7 cells. These results suggest that expression of the ABCC10 gene is regulated in a p53-dependent manner during DNA-damage-related apoptosis.

Prolonged stability and sustained prodrug cell killing activity using receptor-mediated delivery of malarial circumsporozoite-cytosine deaminase fusion protein into liver cancer cells.

An effective strategy of delivering recombinant DNA or protein by nonviral vectors faces two major challenges: (a) the selective delivery to the specific target tissue; and (b) a long-term expression of the protein once inside the cells. The present study describes a receptor-mediated delivery strategy using recombinant fusion protein consisting of malaria circumsporozoite (CS) protein as a ligand and bacterial cytosine deaminase (CD), which catalyzes the production of 5-fluorouracil from its prodrug 5-fluorocytosine. We demonstrate that the CD-CS fusion protein can be internalized in a receptor-mediated manner, providing a target delivery. The internalized CD-CS is capable of synthesizing 5-fluorouracil from the exogenously added 5-fluorocytosine and elicits cell killing with bystander activities. Most importantly, the internalized recombinant protein is stable and remains functional for at least several days, probably because of the entrapment of the fusion protein in particular cytoplasmic compartments that are free from cytoplasmic degradation machinery. Thus, it is possible to use a simple recombinant fusion strategy to enhance intracellular protein stability for manufacturing biological active product in a cell type-specific manner. The application of this strategy in the treatment of liver cancers and liver metastasis of colorectal cancers is discussed.

Cyclooxygenase-2 overexpression reduces apoptotic susceptibility by inhibiting the cytochrome c-dependent apoptotic pathway in human colon cancer cells.

The cyclooxygenase-2 (COX-2) gene encodes an inducible enzyme that converts arachidonic acid to prostaglandins and is up-regulated in colorectal neoplasms. Evidence indicates that COX-2 may regulate apoptosis and can influence the malignant phenotype. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX enzymes and induce apoptosis in colorectal cancer cell lines, which may contribute to their antitumor effects. To determine whether forced COX-2 expression modulates susceptibility to drug-induced apoptosis, HCT-15 colon carcinoma cells were stably transfected with the COX-2 cDNA, and two clones overexpressing COX-2 were isolated. Selective COX-2 (NS398) and nonselective (sulindac sulfide) COX inhibitors, as well as 5-fluorouracil (5-FU), induced apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling in a dosage-dependent manner. Forced COX-2 expression significantly attenuated induction of apoptosis by all three of the drugs compared with parental HCT-15 cells. NSAIDs and 5-FU induced the mitochondrial release of cytochrome c as well as caspase-3 and -9 activation, and to a much lesser extent, caspase-8. COX-2-overexpressing cells showed reduced cytochrome c and caspase activation, relative to parental cells. A specific inhibitor of caspase-3 restored cell survival after drug treatment. COX-2 transfectants were found to overexpress the antiapoptotic Bcl-2 mRNA and protein relative to parental cells. In conclusion, forced COX-2 expression significantly attenuates apoptosis induction by NSAIDs and 5-FU through predominant inhibition of the cytochrome c-dependent apoptotic pathway. COX-2-mediated up-regulation of Bcl-2 suggests a potential mechanism for reduced apoptotic susceptibility.

Cyclooxygenase-2 overexpression inhibits death receptor 5 expression and confers resistance to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human colon cancer cells.

The inducible cyclooxygenase-2 (COX-2) gene regulates prostaglandin biosynthesis,is up-regulated in colorectal cancers, and can influence apoptotic susceptibility. We determined whether forced COX-2 expression modulates apoptosis induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of tumor necrosis factor ligand family, and examined determinants of the apoptotic pathway, including membrane death receptors (DR-4 and DR-5). HCT-15 colon cancer cells lacking endogenous COX-2 proteins were stably transfected with the COX-2 cDNA and incubated with TRAIL. Forced COX-2 expression significantly attenuated TRAIL-induced apoptosis and was associated with transcriptional repression of DR-5 and up-regulation of Bcl-2. COX-2 transfectants showed reduced DR-5 mRNA and protein expression as well as reduced caspase-8, caspase-3, and caspase-9 activation relative to parental cells. Sulindac sulfide treatment restored DR-5 expression and, when combined with TRAIL, reduced cell viability to a greater extent than did either drug alone. In summary, modulation of DR-5 and Bcl-2 levels by COX-2 attenuates TRAIL-induced apoptosis and represents a novel mechanism of intrinsic drug resistance in human colon cancer cells.

Induction of multidrug resistance proteins MRP1 and MRP3 and gamma-glutamylcysteine synthetase gene expression by nonsteroidal anti-inflammatory drugs in human colon cancer cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been demonstrated to suppress colorectal tumorigenesis. NSAIDs have also been used to treat inflammatory illnesses. However, the underlying mechanisms of action by NSAIDs have not been completely elucidated. In this study, we reported that among the six members of the multidrug resistance protein gene (MRP1 to MRP6) family which encode membrane transporters for a diverse group of antitumor agents, expression of MRP1 and MRP3 but not the others in human colorectal cancer cell lines was induced by sulindac. This induction profile is consistent with the results using prooxidants which produce reactive oxygen species (ROS) and generate oxidative stress as previously reported. Moreover, treatment of colorectal cancer cells with sulindac induced ROS. Suppression of ROS formation by antioxidant N-acetylcysteine (NAC) downregulated the induction of MRP1 and MRP3 expression. Expression of another oxidative stress-sensitive gene, gamma-glutamylcysteine synthetase heavy subunit gene (gamma-GCSh), which encodes the rate-limiting enzyme in glutathione biosynthesis, was also induced by sulindac. However, the suppression of sulindac-induced gamma-GCSh expression by NAC was less sensitive compared with that of MRP1 and MRP3. We also demonstrated that induction of MRP3 and gamma-GCSh was independent of intracellular COX-2 levels. These results, collectively, suggest a ROS-related, COX-2-independent mechanism for the induction of drug resistance gene expression that bears important implications to the roles of NSAIDs in colorectal carcinogenesis and inflammatory response.

Expression of heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCSh) in human colorectal carcinoma.

Gamma-glutamylcysteine synthetase (gamma-GCS) is a heterodimer consisting of heavy (gamma-GCSh) and light (gamma-GCSl) subunits. gamma-GCS catalyzes the rate-limiting de novo biosynthesis of glutathione (GSH), an abundant physiological antioxidant that plays important roles for regulating oxidative stress. Expression of gamma-GCSh and gamma-GCSl are sensitive to oxidative stress. To investigate whether expression of gamma-GCS is correlated with tumor progression, we used immunohistochemical approaches to examine 16 human colorectal adenomas and resected 57 carcinomas from untreated patients. In adjacent normal colorectal epithelium, levels of gamma-GCSh expression were low. Strong cytoplasmic staining for gamma-GCSh was detected in 3 (18.8%) adenoma and 48 (84.2%) carcinomas. The frequency of gamma-GCSh expression in carcinoma was significantly higher than in adenoma (p<0.0001). We used RNase protation assay and Western blot to determine levels of gamma-GCSh mRNA and protein from 10 pairs of matched carcinomas with adjacent normal controls. Elevated expression of both gamma-GCSh mRNA and protein were found in 6 cases, suggesting that transcriptional and/or posttranscriptional regulation play an important role in the upregulation of gamma-GCS during colorectal carcinogenesis. We also examined the expression of another redox-regulated gene, multidrug resistance protein 1 (MRP1). Strong staining for MRP1 was detected in 1 (6.3%) adenoma and 40 (70.2%) carcinomas. The frequency of MRP1 expression in carcinoma was significantly higher than in adenoma ( p<0.0001). Nuclear p53 expression was detected in 30 (52.6%) of carcinomas. There is a significant correlation between gamma-GCSh and MRP1 expression (p=0.013) but not between gamma-GCSh and p53. Since gamma-GCS is a sensor of oxidative stress, these results are consistent with the notion that oxidative stress is associated with colorectal tumor progression.