Showing papers in "International Journal of Oncology in 2005"

Expression of epithelial cell adhesion molecule in carcinoma cells present in blood and primary and metastatic tumors

Abstract: The epithelial cell adhesion molecule (EpCAM) is involved in homophilic cell-cell adhesion in normal epithelia and is frequently overexpressed in primary and metastatic adenocarcinomas. It has been postulated that during detachment and dissemination of tumor cells, EpCAM may be down-regulated. Circulating tumor cells (CTC) may demonstrate this phenomenon as they have successfully escaped their local microenvironment and entered the circulation. EpCAM expression of CTC was compared to tumor cells in paraffin-embedded tissue arrays containing various benign diseases and carcinomas. EpCAM expression on CTC was determined by flow cytometry (FCM) and by immunohistochemistry (IHC) in paraffin-embedded tissue. To permit comparison of FCM results to those derived by IHC, EpCAM was quantified on cancer cell lines by FCM and then paraffin-embedded cell-blocks of these lines were used as staining guides for IHC analysis of tissue arrays. By IHC, 97% (384/397) of solid tissues analyzed had detectable EpCAM, with 72% of tissues showing antigen expression levels of > or =400,000 EpCAM molecules per cell. FCM analysis of CTC from 100 metastatic carcinoma patients with > or =2 CTC/90 microl blood showed EpCAM expression ranging from 9,900 to 246,000 (mean 49,700) antigens per cell. EpCAM expression was approximately 10-fold lower on CTC as compared to primary and metastatic tissues, suggesting that EpCAM expression is transient and dependent upon the local micro-environment. This supports the hypothesis that this adhesion molecule is down-regulated on carcinoma cells in the circulation.

Radiation-induced autophagy is associated with LC3 and its inhibition sensitizes malignant glioma cells.

Abstract: Autophagy is a novel response of cancer cells to ionizing radiation (IR) or chemotherapy, but its significance or mechanism remains largely elusive. Autophagy is characterized with the prominent formation of autophagic vacuoles in the cytoplasm. It is a protein degradation system that involves autophagic/lysosomal compartment. The process begins with sequestering a portion of the cytoplasm, forming the autophagosome. The autophagosome then fuses with the lysosome and lyses its contents. To study radiation-induced autophagy with specific molecules, we assessed changes in the expression of microtubule-associated protein light chain 3 (LC3) and its intracellular distribution after IR in comparison with starvation-induced autophagy. First, we showed that IR induced cell cycle arrest and autophagy, but not apoptosis, in human malignant glioma U373-MG cells. Type II LC3, that is specifically associated with the membrane of the autophagosome, increased after IR and amino acid starvation. Exogenous LC3 distributed on punctate structures, indicative of the formation of autophagosomes. Autophagy inhibitors, 3-methyladenine and bafilomycin A1, radiosensitized U373-MG cells. Furthermore, gammaH2AX foci, that show the extent of DNA double-strand breaks, were more pronounced and prolonged in the cells treated with IR and autophagy inhibitors than in those cells treated with IR only. Our results suggest that autophagy inhibitors may represent a new application of radiosensitization for malignant glioma cells.

Overexpression of G protein-coupled receptors in cancer cells: involvement in tumor progression.

Abstract: G protein-coupled receptors (GPCRs) play important roles in a variety of biological and pathological processes. They are considered among the most desirable targets for drug development. Recent studies have demonstrated that many GPCRs, such as endothelin receptors, chemokine receptors and lysophosphatidic acid receptors have been implicated in the tumorigenesis and metastasis of multiple human cancers. In this study, we conducted an in silico analysis of GPCR gene expression in primary human tumors by analyzing some publicly available gene expression profiling data. Statistical analysis was performed on eight microarray data sets of non-small cell lung cancer, breast cancer, prostate cancer, melanoma, gastric cancer and diffused large B cell lymphoma to identify GPCRs that are up-regulated in primary or metastatic cancer cells. Our analysis has demonstrated overexpression of several GPCRs in primary tumor cells, including chemokine receptors and protease-activated receptors that were shown to be important for tumorigenesis by previous studies. In addition, we have uncovered several GPCRs, such as neuropeptide receptors, adenosine A2B receptor, P2Y purinoceptor, calcium-sensing receptor and metabotropic glutamate receptors, that are expressed at a significantly higher level in some cancer tissue and may play a role in cancer progression. Analysis of cancer samples in different disease stages also suggests that some GPCRs, such as endothelin receptor A, may be involved in early tumor progression and others, such as CXCR4, may play a critical role in tumor invasion and metastasis. The present study demonstrates the value of publicly available microarray data as a resource to gain more understanding of cancer biology, to validate previous findings from in vitro experiments, and to identify potential novel anticancer targets and biomarkers.

Silymarin and skin cancer prevention: anti-inflammatory, antioxidant and immunomodulatory effects (Review).

Abstract: Several environmental and genetic factors are involved in skin cancer induction, however exposure to chemical carcinogens and solar ultraviolet (UV) radiation are primarily responsible for several skin diseases including skin cancer. Chronic exposure of solar UV radiation to the skin leads to basal cell and squamous cell carcinoma, and melanoma. Chemoprevention of skin cancer by consumption of naturally occurring botanicals appears a practical approach and therefore world-wide interest is considerably increasing to use these botanicals. Sunscreens are useful but their protection is not ideal because of inadequate use, incomplete spectral protection and toxicity. Silymarin, a plant flavonoid isolated from the seeds of milk thistle (Silybum marianum), has been shown to have chemopreventive effects against chemical carcinogenesis as well as photocarcinogenesis in various animal tumor models. Topical treatment of silymarin inhibited 7,12-dimethylbenz(a)anthracene-initiated and several tumor promoters, like 12-O-tetradecanoylphorbol-13-acetate, mezerein, benzoyal peroxide and okadaic acid, induced skin carcinogenesis in mouse models. Similarly, silymarin also prevented UVB-induced skin carcinogenesis. Wide range of in vivo mechanistic studies indicated that silymarin possesses antioxidant, anti-inflammatory and immunomodulatory properties which may lead to the prevention of skin cancer in in vivo animal models. The available experimental information suggests that silymarin is a promising chemopreventive and pharmacologically safe agent which can be exploited or tested against skin cancer in human system. Moreover, silymarin may favorably supplement sunscreen protection and provide additional anti-photocarcinogenic protection.

COX-2 overexpression increases motility and invasion of breast cancer cells.

Abstract: Cyclooxygenase-2 (COX-2), an inducible enzyme involved in prostaglandin (including PGE(2)) biosynthesis, is overexpressed in several epithelial malignancies including breast cancer. We tested the hypothesis that COX-2 overexpression in breast cancer cells results in increased cell motility and invasion. COX-2 overproducing cells were generated by stable transfection of several human breast cancer cells with pSG5-COX2 vector. We confirmed the overexpression of COX-2 protein by western blotting, and by measuring PGE(2) in the medium with an immunoassay. We measured cell motility by counting the number of cells crossing an 8-micron pore size PET membrane, and cell invasion by counting the number of cells invading through a Matrigel-coated membrane that simulates basement membrane. COX-2 transfected MDA-231 cells produced 30-43-fold more PGE2 as compared to parental cells. COX-2 overexpression increased cell migration approximately 2.2-fold and cell invasion through Matrigel approximately 5.1-fold. Addition of 50 microM NS-398, a COX-2 inhibitor, inhibited Matrigel invasion of MDA-231 cells by 54% as compared to solvent confirming the role of COX-2 in cell invasion. It is known that an increase in cell migration and invasion can be brought about by cytoskeletal alterations and basement membrane degradation due to increased expression of pro-urokinase plasminogen activator (pro-uPA). To investigate the mechanism of our observed increase in cell invasion by COX-2, we found by western blotting that the level of pro-uPA was significantly higher (approximately 5-fold) in COX-2 transfected MDA-231 cells than untransfected MDA-231 cells. Similar to our observations in cell culture, we found evidence that increased COX-2 activity correlates with uPA in a mouse model of breast cancer metastasis to bone. In this study, we conclude that COX-2 overexpression in human breast cancer cells enhances cell motility and invasiveness thus suggesting a mechanism of COX-2 mediated metastasis.

The disintegrin-metalloproteinases ADAM9, ADAM12, and ADAM15 are upregulated in gastric cancer.

Abstract: The ADAMs (A disintegrin and metalloproteinase) are a family of cell-surface membrane glycoproteins, whose multidomain structure enables diverse roles in a wide range of cellular processes. Accumulating evidence associates an increased expression of individual ADAM family members with various types of cancer, and we investigated the possible involvement of ADAM9, 12 and 15 in the pathogenesis of gastric cancer (GC). Using immunohistochemistry and quantitative RT-PCR, we examined the transcription and expression pattern of ADAM9, 12, and 15 in GCs and the corresponding non-tumor tissue, and in GC cell lines (AGS, MKN45, MKN28, NCI-N87, KATOIII). All three ADAMs were found to be significantly upregulated in GC compared to non-neoplastic foveolar epithelium, with ADAM12 expression being higher in intestinal- than in diffuse-type tumors. In vitro proliferation assays were used to evaluate the effects of ADAM-specific antibodies on the growth of GC cell lines. The administration of anti-ADAM9 and anti-ADAM15 antibodies inhibited cell growth, whereas anti-ADAM12 enhanced the proliferation of the GC cell lines. ADAM9, 12 and 15 are implicated in the malignant growth of GC cells, perhaps via the interaction with adhesion molecules, or the proteolytic 'shedding' of signaling molecules and the consequent transactivation of their receptors, such as the epithelial growth factor receptor and its ligands. The resultant modulation of the tumor-host interface may contribute to the pathogenesis, development or progression of gastric cancer.

DJ-1 restores p53 transcription activity inhibited by Topors/p53BP3

Abstract: DJ-1 is a multi-functional protein that plays roles in transcriptional regulation and anti-oxidative stress, and loss of its function is thought to result in onset of Parkinson's disease. Here, we report that DJ- 1 bound to Topors/p53BP3, a ring finger protein binding to both topoisomerase I and p53, in vitro and in vivo and that both proteins were colocalized in cells. DJ-1 and p53 were then found to be sumoylated by Topors in cells. It was also found that DJ-1 bound to p53 in vitro and in vivo and that colocalization with and its binding to p53 were stimulated by UV irradiation of cells. Transcription activity of p53 was found to be abrogated by Topors concomitant with sumoylation of p53 in a dose-dependent manner, and DJ-1 restored its repressed activity by releasing the sumoylated form of p53. These findings suggest that DJ-1 positively regulates p53 through Topors-mediated sumoylation.

Expression of transcription factor Yin Yang 1 in prostate cancer.

Abstract: The transcription repressor Yin Yang 1 (YY1) is expressed in several human cancer cell lines and its expression correlates with resistance to immune-mediated apoptosis. This study used tissue microarrays to investigate the expression and localization of YY1 in 1364 representative tissue samples from 246 hormone naive prostate cancer patients who underwent radical prostatectomy. Staining intensity and frequency measures for both YY1 nuclear and cytoplasmic expression were higher in neoplastic tissues and in PIN samples compared to matched benign cells (p < 0.0001 for all comparisons). Expression of YY1 is predominantly elevated in early malignancy (PIN), as well as in tumors of intermediate to high morphologic grade (Gleason's grade 3-5). Using multivariate Cox proportional hazards analysis, we observed that low nuclear YY1 staining is an independent predictor of a shorter time to recurrence (p = 0.012). Based on these results, we hypothesize that YY1 may play a role in prostate cancer development; however, decreased YY1 may give metastatic cells a survival advantage. These results may also implicate YY1 as a useful diagnostic and prognostic marker.

Epithelial-mesenchymal transition in gastric cancer (Review).

Abstract: Endoscopic mucosal resection (EMR), endoscopic submucosal dissection (ESD), surgical gastrectomy, and chemotherapy are therapeutic options of gastric cancer; how-ever, prognosis of advanced gastric cancer patients is still poor. Gastric cancer cells with fibroblastoid morphological changes show increased motility and invasiveness due to decreased cell-cell adhesion, which are reminiscent of epithelial-mesenchymal transition (EMT) during embryonic development. Here, EMT signaling networks in gastric cancer were reviewed. E-cadherin at adherens junction is a key molecular target of EMT. CDH1 gene at human chromosome 16q22.1 encodes E-cadherin. Familial diffuse type gastric cancer occurs due to germ-line mutations of the CDH1 gene. Down-regulation of E-cadherin function due to mutation, deletion, CpG hyper-methylation, and SNAIL (SNAI1)- or SIP1-mediated transcriptional repression of the CDH1 gene leads to EMT in gastric cancer. Amplification of ERBB2, MET, FGFR2, PIK3CA, AKT1 genes, up-regulation of WNT2, WNT2B, WNT8B, and down-regulation of SFRP1 lead to EMT in gastric cancer through GSK3beta inhibition and following SNAIL-mediated CDH1 repression. Claudin (CLDN) and PAR3/PAR6/aPKC complex at tight junction are other key molecular targets of EMT. CLDN23 gene is down-regulated in intestinal type gastric cancer. Down-regulation of PAR3/PAR6/aPKC complex also leads to EMT. Single nucleotide polymorphisms (SNPs) and copy number polymorphisms (CNPs) of genes encoding EMT signaling molecules will be identified as novel risk factors of gastric cancer. In addition, antibodies, RNAi compounds, and small molecular inhibitors for EMT signaling molecules will be developed as novel therapeutic agents for gastric cancer. Personalized medicine based on the combination of genetic screening and novel therapeutic agents could dramatically improve the prognosis of gastric cancer patients in the future.

Bone morphogenetic protein 2 (BMP-2) induces in vitro invasion and in vivo hormone independent growth of breast carcinoma cells.

Abstract: Breast cancer cell lines migrated towards a BMP-2 source depending on BMP-2 concentration. After a short exposure to BMP-2, the cells were able to migrate through matrigel. MCF-7 cells transfected with the BMP-2 gene also showed enhanced migratory properties and high expression of the metastasis-related gene BCSG1. In a xenograft model without estrogen supplementation MCF-7/BMP-2 cells formed tumors. These tumors were characterised by an enhanced vasculature and the formation of chondroid and osseous structures. In conclusion elevated levels of BMP-2 enhance the tumorigenic properties of breast carcinoma cells and drive the cells towards a more aggressive phenotype with estrogen independent growth.

Suppression of tumor growth in human gastric cancer with HER2 overexpression by an anti-HER2 antibody in a murine model

Abstract: New modalities are necessary for the treatment of patients with unresectable gastric cancer. The aim of this study was to investigate whether or not anti-HER2 antibody could suppress the growth of human gastric cancer cells with HER2 overexpression in vitro and in vivo. Four human gastric cancer cell lines, NCI-N87, MKN-45P, Kato-III, and MKN-1, were used in this study. The suppression of cell proliferation in vitro and of subcutaneous tumor growth in a nude mouse model after treatment with trastuzumab was examined. The expression of HER2 protein was investigated by Western blot analysis. The effect of trastuzumab on the survival rate of nude mice with peritoneal dissemination was examined. Trastuzumab significantly reduced proliferative activity in NCI-N87, a HER2-overexpressing human gastric cancer cell line, in vitro. In the nude mouse model with transplanted subcutaneous tumor, trastuzumab significantly suppressed the tumor growth of NCI-N87 cells, and then HER2 expression was reduced. Trastuzumab improved the survival rate of mice with peritoneal dissemination of MKN-45P cells. Trastuzumab therapy is a potential candidate for a novel treatment of HER2-overexpressing gastric cancer.

Systemic delivery of RafsiRNA using cationic cardiolipin liposomes silences Raf-1 expression and inhibits tumor growth in xenograft model of human prostate cancer.

Abstract: Raf-1, a protein serine-threonine kinase, plays a critical role in mitogen-activated protein kinase kinase (MKK/ MEK)- mitogen-activated protein kinase (extracellular signal-regulated kinase) (MAPK/ERK) pathways. We show here that systemically delivered novel cationic cardiolipin liposomes (NeoPhectin-AT) containing a small interfering RNA (siRNA) against Raf-1 silence the expression of Raf-1 in tumor tissues and inhibit tumor growth in xenograft model of human prostate cancer. The knockdown of Raf-1 expression by siRNA is also associated with down-regulation of cyclin D1 expression in vivo.

The high expression of Fractalkine results in a better prognosis for colorectal cancer patients.

Abstract: Local and systemic immune responses are impaired in patients with colorectal cancer (CRC) and it is known that the number of tumor infiltrating lymphocytes (TILs) is considerably few. On the other hand, some CRC cases in which many TIL were observed, survived longer than those cases with a small number of TIL. Considerable attention has been recently paid to the relationship between chemokines and tumor cells. Some chemokines recruit lymphocytes for tumor lesions. We made a hypothesis that Fractalkine, a CX3C chemokine, would recruit lymphocytes in the CRC and play an important role in anti-tumor immunity. We analyzed the expression level of Fractalkine in CRC cell lines as well as in clinical samples (n=80). The expression level of Fractalkine was thus found to correlate with the density of TIL (p<0.05). The CRC cases with a strong Fractalkine expression (n=50) showed a significantly better prognosis than those with a weak expression (n=30) (p<0.05). In addition, the Fractalkine expression was found to be an independent prognostic factor (p<0.05). We furthermore clarified that some of the tumor-infiltrating cells were natural killer cells and cytotoxic T cells expressed Fractalkine receptor. These data suggest that Fractalkine expressed in the tumor appears to recruit cytotoxic T cells and NK cells to the tumor site and these cytotoxic cells result in a better prognosis mediated by tumor cell cytotoxicity using a perforin and granzyme B mechanism. The expression level of Fractalkine was an essential biomarker for predicting the prognosis of patients with CRC. Fractalkine is considered to be one of the biomarkers for detecting patients with a high risk for recurrence, and who might therefore benefit from additional therapeutic strategies such as adjuvant therapy.

Apigenin induces cell cycle arrest and p21/WAF1 expression in a p53-independent pathway.

Abstract: Apigenin, a common dietary flavonoid, has been shown to induce cell growth-inhibition and cell cycle arrest in many cancer cell lines. One important effect of apigenin is to increase the stability of the tumor suppressor p53 in normal cells. Therefore, apigenin is expected to play a large role in cancer prevention by modifying the effects of p53 protein. However, the mechanisms of apigenin's effects on p53-mutant cancer cells have not been revealed yet. We assessed the influence of apigenin on cell growth and the cell cycle in p53-mutant cell lines. Treatment with apigenin resulted in growth-inhibition and G2/M phase arrest in two p53-mutant cancer cell lines, HT-29 and MG63. These effects were associated with a marked increase in the protein expression of p21/WAF1. We have shown that p21/WAF1 mRNA expression was also markedly increased by treatment with apigenin in a dose- and time-dependent manner. However, we could not detect p21/WAF1 promoter activity following treatment with apigenin. Similarly, promoter activity from pG13-Luc, a p53-responsive promoter plasmid, was not activated by treatment with apigenin with or without p53 protein expression. These results suggest that there is a p53-independent pathway for apigenin in p53-mutant cell lines, which induces p21/WAF1 expression and growth-inhibition. Apigenin may be a useful chemopreventive agent not only in wild-type p53 status, but also in cancer with mutant p53.

Vascular endothelial growth factor-D induces lymphangiogenesis and lymphatic metastasis in models of ductal pancreatic cancer.

Abstract: The presence of lymphatic metastases is a strong indicator for poor prognosis in patients with ductal pancreatic cancer. In order to better understand the mechanisms controlling lymphatic growth and lymph node metastasis in human ductal pancreatic cancer, we analyzed the expression pattern of the vascular endothelial growth factor-D (VEGF-D), its receptor VEGF-receptor-3 (VEGFR-3) and the lymphatic endothelium-specific hyaluronan receptor LYVE-1 in a panel of 19 primary human ductal pancreatic tumors and 10 normal pancreas specimens. We further addressed the biological function of VEGF-D for induction of lymphatic metastasis in a nude mouse xenograft model using two human ductal pancreatic cancer cell lines with overexpression of VEGF-D. Compared to normal human pancreas, pancreatic cancer tissue showed overexpression of VEGF-D and VEGFR-3 in conjunction with a high lymphatic vascularization as determined by immunohistochemistry and in situ hybridization. Tumors derived from VEGF-D-overexpressing cells had a higher microvessel density compared to their mock-controls, as determined based on CD31 immunohistochemistry. Importantly, these tumors also revealed a significant induction of intra- and peritumoral lymphatics, as judged from immunohistochemical detection of LYVE-1 expression. This was associated with a significant increase in lymphatic vessel invasion by tumor cells and an increased rate of lymphatic metastases, as indicated by pan-cytokeratin reactive cells in lymph nodes. Our results suggest that VEGF-D plays a pivotal role in stimulating lymphangiogenesis and lymphatic metastasis in human ductal pancreatic cancer, and therefore represents a novel therapeutic target for this devastating disease.

Genistein suppresses the invasive potential of human breast cancer cells through transcriptional regulation of metalloproteinases and their tissue inhibitors.

Abstract: Progression of breast cancer implicates the degradation of extracellular matrix (ECM) by metalloproteinases (MMPs), a process with important consequences on the growth and invasiveness of cancer cells in adjacent and distant sites. The isoflavone, genistein - a natural inhibitor of protein tyrosine kinase pathway - inhibits the growth of a wide range of cancer cells in vitro. The aim of this study was to investigate: i) the expression of mRNAs encoded for MMPs and their endogenous inhibitors (TIMPs) associated with pathogenesis and metastatic potential of breast cancer cells; and ii) the effect of genistein on the transcription of MMPs and TIMPs and the invasive potential of breast cancer cells. Gene expression at transcriptional level was examined in cell cultures of two epithelial breast cancer cell lines, the high invasive (ER-negative) MDA-MB-231 and the low invasive (ER-positive) MCF-7, as well as the normal mammary cells (MCF-12A) following RNA isolation and reversed transcriptase polymerase chain reaction (RT-PCR). The inhibitory effect of genistein on functional invasiveness was examined by a cell invasion assay. Cell cycle distribution showed that genistein arrested breast cancer MDA-MB-231, MCF-7 and BT-20 cells in the G 2 /M phase. Both normal and breast cancer cell lines express the genes of MMP-2, -9, MT1-, MT2-, MT3-MMP and TIMP-1, -2 and -3. MCF-7 express notably less MMPs than MDA-MB-231 cell line. The addition of genistein resulted in down-regulation of the transcription of all MMP genes in MDA-MB-231 and most of MMPs in MCF-7 cells. The inhibitory effect of genistein on MMPs was functionally confirmed, since it significantly reduced the invasion properties of cancer cells in vitro. The obtained results indicate that genistein may be of great value in prevention of breast cancer cell metastasis, since it represents both a transcriptional modulator of genes involved in this pathogenetic process and a suppressor of breast cancer cell invasiveness.

The cytostatic and cytotoxic effects of oridonin (Rubescenin), a diterpenoid from Rabdosia rubescens, on tumor cells of different lineage

Abstract: Rabdosia rubescens is a herbal medicine used to treat esophageal cancer in China. In this study, the sesquiterpene oridonin, an isoprenoid, was isolated from Rabdosia rubescens. Mass spectroscopy and carbon 13 NMR spectroscopy were used to identify the structure of the purified compound. It was then evaluated for biological activity against human cell lines derived from prostate (DU-145, LNCaP), breast (MCF-7), and ovarian (A2780 and PTX10) cancers. Oridonin exhibited anti-proliferative activity toward all cancer cell lines tested, with an IC50 estimated by the MTT cell viability assay ranging from 5.8+/-2.3 to 11.72+/-4.8 microM. Flow cytometric analysis demonstrated that oridonin induced a G1 phase arrest in androgen receptor-positive LNCaP cells containing wt p53, while it blocked the cell cycle at G2 and M phases in androgen receptor-negative DU-145 cells with mutated p53; the arrest in M was verified by examination of cell morphology and by the increased frequency of cells with Ser-10 phosphorylated histone H3. The increased incidence of apoptosis, identified by characteristic changes in cell morphology, was seen in tumor lines treated with oridonin. Notably, at concentrations that induced apoptosis among tumor cells, oridonin failed to induce apoptosis in cultures of normal human fibroblasts. Western blot analysis was used to determine the protein expression of cancer suppressor genes, p53 (wt) and Bax, and the proto-oncogene, Bcl-2 in LNCaP cells following treatment with oridonin. Oridonin up-regulated p53 and Bax and down-regulated Bcl-2 expression in a dose-dependent manner. To further explore the possible interaction between oridonin and DNA, its absorption spectrum was measured in the presence and absence of double stranded (ds) DNA. Spectral shifts and an increase in absorption band intensity were observed indicating interaction of oridonin with DNA bases. The nature of the binding is not clear at present though no evidence of histone H2AX phosphorylation on Ser-139 was apparent in DU-145 cells treated with oridonin that would indicate the induction of ds DNA breaks. In conclusion, oridonin inhibits cancer cell growth in a cell cycle specific manner and shifts the balance between pro- and anti-apoptotic proteins in favor of apoptosis. The present data suggest that further studies are warranted to assess the potential of oridonin in cancer prevention and/or treatment.

Heterogeneous expression of the aquaporin 1 (AQP1) water channel in tumors of the prostate, breast, ovary, colon and lung: A study using high density multiple human tumor tissue microarrays

Abstract: Aquaporin 1 (AQP1) water channels are membrane proteins that control the permeability of endothelial and epithelial barriers by facilitating water movement across cell membranes. Recent studies suggest that AQP1 may be responsible for the high vascular permeability and interstitial fluid pressure in tumors of the brain, colon, breast and pancreas. AQP1 may also play a role in tumor angiogenesis and may be involved in development of effusions or edema fluid. The aim of the present study was to use immunohistochemistry and semi-quantitative histomorphometric analysis to compare the distribution and relative abundance of AQP1 on NCI TARP human multiple tumor tissue microarrays (TMAs) with normal tissues represented on the CHTN TMAs. Immunohistochemistry and semi-quantitative histomorphometric analysis were used to compare the distribution of AQP1 in tumors of the prostate, colon, lung, breast and ovary represented on TARP TMAs with their normal counterparts on CHTN TMAs. AQP1 was expressed in capillary endothelia of all normal tissues. In most tumors AQP1 was confined to endothelial barriers. AQP1 expression was marginally higher in microvascular structures in prostate and ovarian tumors and was higher in advanced mammary and colorectal carcinomas where AQP1 immunoreactivity was also seen in some neoplastic tumor cells. In conclusion, the AQP1 water channel is an excellent marker of microvasculature but it is heterogeneously expressed in different human tumors and not necessarily expressed in all neoplastic cells. Increased AQP1 expression in some human adenocarcinomas may be a consequence of angiogenesis and important for the formation or clearance of tumor edema.

In vivo and in vitro effect of baicalein on human prostate cancer cells

Abstract: We investigated the in vitro effects of baicalein and baicalin on human umbilical vein endothelial cells (HUVECs) and on human prostate tumor cells (DU-145 and PC3) as well as the effect of orally administered baicalein on the growth of DU-145 cells after subcutaneous injection into SCID mice. In vitro effects of baicalein and baicalin treatment on human prostate cancer cell lines DU-145 and PC-3 were assessed by employing cell proliferation (MTS) assay, cytotoxicity (LIVE/DEAD) assay, and TUNEL assay. In vitro anti-proliferative and anti-angiogenic properties of baicalein and baicalin were studied on HUVECs by sprout assay. The effect of orally administered baicalein on tumor growth in SCID mice was studied in four groups (n=10) of animals injected subcutaneously with DU-145 cells and treated daily for 28 days. The control group received only vehicle (carboxymethylcellulose), whereas the other three groups received escalating doses of baicalein (10, 20, and 40 mg/kg per day). Baicalein and baicalin exhibit dose-dependent growth inhibitory effects on human prostate cancer cells and umbilical vein endothelial cells in vitro. Also, treatment by these two flavonoid compounds significantly decreased the average number and length of sprouts formed by the endothelial cell aggregates in a dose-dependent manner. In vivo, treatment of mice with baicalein demonstrated a statistically significant tumor volume reduction (p<0.01) when compared to the control. This is the first study demonstrating an in vivo growth inhibitory effect of orally administered baicalein on human prostate tumors in mice.

The inhibitory effect and possible mechanisms of D-allose on cancer cell proliferation

Abstract: Rare sugars are monosaccharides distributed rarely in nature, because of its very limited amount and cost, the biological effect has hardly been studied. Recently, an effective strategy for mass production of rare sugars has been developed. As a result, a wide range of study of rare sugars from the basic to applied research has become possible. For biological application of rare sugars, it is necessary to fundamentally investigate the relationships between rare sugars and living cells in terms of physiology. Therefore, we firstly examined the effect of rare sugars including D-psicose, D-allose, D-altrose and D-talitol on cell proliferation using certain cell lines in vitro. Cell growth was evaluated by MTT assay after 24-, 48- and 72-h treatment. The result shows that D-allose has a significant inhibitory effect on cancer cell proliferation in a dose-dependent manner. Although the exact mechanism remains unclear, this finding represents a novel aspect of the biological profile of D-allose and suggests that D-allose may be an effective adjuvant therapeutic agent against cancer in the future.

A combination of docosahexaenoic acid and celecoxib prevents prostate cancer cell growth in vitro and is associated with modulation of nuclear factor-kappaB, and steroid hormone receptors.

Abstract: Epidemiological studies have provided evidence that high intake of saturated fat and/or animal fat increases the risk of prostate cancer, but on the other hand, diets rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs), present in fish oils were found to reduce the risk. There are indications of an increased expression of immunoreactive PPARgamma in prostatic intraepithelial neoplasia (PIN) and prostate cancer, suggesting that PPARgamma ligands may exert their own potent anti-proliferative effect against prostate cancer. The experimental evidence for the role of cyclooxygenase-2 (COX-2) in prostate carcinogenesis is well established through several investigations. It clearly suggests the need for development of strategies to inhibit COX-2 mediated prostate carcinogenesis. However, administration of high doses of COX-2 inhibitors, such as celecoxib, over longer periods may not be devoid of side effects. We assessed the efficacy of DHA and celecoxib individually and in combination at low doses in three prostate cancer cell lines (LNCaP, DU145 and PC-3) measuring cell growth inhibition and apoptosis, and on the levels of expression of COX-2, nuclear factor-kappaB (NF-kappaBp65), and nuclear receptors, such as PPARgamma and retinoid X receptors (RXR), all of which presumably participate in prostate carcinogenesis. A 48-h incubation of prostate cancer cells with 5 microM each of DHA or celecoxib induced cell growth inhibition and apoptosis, and altered the expression of the above molecular parameters. Interestingly, the modulation of these cellular and molecular parameters was more pronounced in cells treated with low doses of DHA and celecoxib (2.5 microM each) in combination than in cells treated with the higher doses of individual agents. In conclusion, the present study demonstrates for the first time that a combination of lower doses of the n-3 PUFA, and DHA with the selective COX-2 inhibitor celecoxib effectively modulates the above cellular and molecular parameters that are relevant to prostate cancer. This raises the intriguing prospect that the use of low doses of a COX-2 inhibitor in combination with an n-3 PUFA could be a highly promising strategy for prostate cancer chemoprevention while minimizing undesired side effects.

In vitro inhibition of matriptase prevents invasive growth of cell lines of prostate and colon carcinoma.

Abstract: Matriptase, also known as membrane-type-serine-protease 1 (MT-SP 1), is a type II transmembrane serine protease involved in the activation of the precursor form of hepatocyte growth factor/scatter factor (pro-HGF/SF). Since HGF/SF is a well-known extracellular signal, which plays a key role in the control of invasive growth, we investigated the effects of matriptase inhibition in cell lines derived from colon (DLD-1) or prostate (PC-3) carcinomas. Biochemical analysis showed that matriptase was very efficient in the proteolytic conversion of the inactive HGF/SF precursor into HGF/SF. Inhibition of endogenous matriptase synthesis in DLD-1 or PC-3 cells by specific small interfering RNAs impaired the conversion of pro-HGF/SF into HGF/SF at the cell surface and inhibited cell scattering upon pro-HGF/SF stimulation. The same effect was observed after treatment of these cells with matriptase inhibitors of the 3-amidinophenylalanine-type, CJ-697 or CJ-730. Inhibition of matriptase significantly reduced invasion of the extracellular matrix as well. Interestingly, this reduction was observed even in the presence of pre-activated HGF/SF. It is concluded that matriptase plays a dual-role in the events unleashing the invasive phenotype, one 'upstream' from the HGF/SF signalling cascade and one 'downstream', most likely at the level of the plasminogen activation system. These data provide a proof of concept for the targeting of matriptase in the search for anti-invasive drugs.

Expression of SIP1 in oral squamous cell carcinomas: implications for E-cadherin expression and tumor progression

Abstract: Loss of E-cadherin expression allows carcinoma cells to liberate from the primary site and enhances invasion and metastasis. The genetic aberration of E-cadherin is a rare event in sporadic carcinomas, and transcription repressors are considered to take a central role in E-cadherin loss. However, expression of E-cadherin repressors is largely dependent on tissue and cell type. To identify the repressor expressed in oral squamous carcinomas, we compared the expression levels of E-cadherin and repressors by real-time RT-PCR. Among the repressors including SNAIL, SLUG, SIP1, E12 and E47, SIP1 was inversely correlated to E-cadherin (P < 0.05). Chromatin immunoprecipitation showed that SIP1 specifically bound to the E-cadherin promoter region. SIP1 expression was immuno-histochemically detected in 27.7% of 47 oral carcinomas, and SIP1-positive carcinomas did not express E-cadherin (P < 0.01). Thirteen patients with SIP1 staining showed a lower disease-specific survival rate (P < 0.05). Multivariate risk factor analysis demonstrated that SIP1 expression was an independent prognostic value for disease-specific overall survival (P < 0.05). These results suggest that SIP1 contributes to the loss of E-cadherin expression and that detection of SIP1 expression is a predictive and prognostic tool in clinical management of oral carcinomas.

Oncolysis of vascular malignant human melanoma tumors by Coxsackievirus A21.

Abstract: Cultured melanoma cell lines despite exhibiting similar in vitro morphology, display significant phenotypic and growth rate differences when propagated as in vivo xenografts. Previously we have shown that Coxsackievirus A21 (CVA21) lytically infects in vitro cultures of malignant melanoma cells and is efficient at reducing the tumor burden of mice bearing slow-growing SK-Mel-28 melanoma xenografts. The oncolytic activity of CVA21 against in vivo melanoma xenografts, which possess rapid growth rates and more extensive vascular structure than SK-Mel-28 xenografts warrants further investigation. In the present study we evaluated the oncolytic action of CVA21 against rapidly growing melanoma xenografts (ME4405) which exhibit a highly vascular phenotype. Flow cytometric analysis indicated that in vitro cultures of ME4405 cells expressed comparable levels of the CVA21 cellular receptors, ICAM-1 (intercellular adhesion molecules-1) and DAF (decay accelerating factor) to SK-Mel-28 cells. Despite similar levels of CVA21 receptor expression, SK-Mel-28 cells appear to be more susceptible to viral lysis than ME4405 cells, even though the kinetics of virus replication in both lines was comparable. Intratumoral, intraperitoneal or intravenous administration of CVA21 were equally effective in reducing the tumor volume of ME4405 xenografts in immunodeficient mice, and provides further evidence for the use of CVA21 as a novel oncolytic agent against varying phenotypes of malignant melanoma.

Mucinous carcinomas of the colorectum have distinct molecular genetic characteristics.

Abstract: We compared the frequency of CpG island methylation phenotype (CIMP), inactivation of APC, p53 and DCC genes and K-ras and BRAF mutations in 39 mucinous carcinomas (MC) and 34 non-mucinous carcinomas (NMC) of the colorectum with different microsatellite instability (MSI) status. The higher incidence of MSI (36% vs. 18%) was observed in MC compared with NMC. APC inactivation and K-ras mutations occurred more frequently in NMC (APC, 88%, p<0.001; K-ras, 58%, p=0.01) than in MC (APC, 24%; K-ras, 28%) regardless of MSI status. BRAF mutation occurred at a higher frequency in MC (18%, p=0.01) than in NMC (0%). However, with respect to inactivation of p53 and DCC, MSI status did matter and in both NMC and MC, more frequent inactivation of p53 and DCC was observed in MSS tumors than in MSI tumors. MSS tumors of NMC had a higher frequency of p53 (58% by IHC, p=0.03 and 83% by LOH, p=0.02) and DCC inactivation (83%, p=0.02) compared to MSI tumors of NMC (p53, 33% by IHC and 20% by LOH; DCC, 20%). MSS tumors of MC also showed a higher frequency of p53 and DCC inactivation (p53, 45% by IHC, p=0.02 and 53% by LOH, p=0.005; DCC, 82%, p=0.001) compared to MSI tumors of MC (p53, 0% by IHC and 0% for LOH; DCC, 17%). MC showed a higher frequency of CIMP compared with NMC (41% vs. 11%, p=0.01). These results indicate that mucinous carcinomas of the colorectum exhibit distinct molecular genetic characteristics and may arise from distinct pathogenic pathways.

Potentiation of the antitumor activity of α, α, α-trifluorothymidine by the co-administration of an inhibitor of thymidine phosphorylase at a suitable molar ratio in vivo

Abstract: Trifluorothymidine (FTD) is a thymidine analog that exhibits an antitumor activity through its inhibition of thymidylate synthase and its incorporation into DNA. However, FTD is rapidly hydrolyzed to an inactive form by thymidine phosphorylase (TP). We attempted to augment the antitumor activity of FTD by combining it with a potent and reversible inhibitor of TP, 5-chloro-6-(2-imino-propyrrolidin-1-yl) methyl-2, 4 (1H, 3H)-pyrimidinedione hydrochloride (TPI) in human tumor xenografts with a low sensitivity to 5-fluorouracil. The optimum ratio of TPI to FTD was determined by measuring the maximum plasma level of FTD after oral administration and the antitumor effect of FTD on human tumor xenografts in mice. When > 0.5 M of TPI and 1 M of FTD (10 mg/kg) were co-administered, the plasma FTD levels in mice and monkeys were elevated, almost reaching a maximal and constant value of 20-30 microg/ml and 15 microg/ml, respectively. When human gastrointestinal cancer cell lines (DLD-1, CO-3 and AZ521) were xenografted into nude mice, the antitumor activity of FTD was augmented by the co-administration of TPI, compared to that of FTD alone, and the ED50 value, used to indicate the antitumor effect, reached a maximum value (about 25, 20, and 10 mg/kg in the DLD-1, CO-3, and AZ521 tumors, respectively) when > 0.5 M of TPI was combined with 1 M of FTD. The oral administration of TPI markedly improved the FTD-induced toxicity, as evaluated by the decrease in the body weight of the mice. These results suggested that the optimum ratio of FTD to TPI was 1:0.5 M, enabling a high antitumor activity and a low toxicity. We further evaluated whether TPI inhibits TP-induced angiogenesis in a gelatin-sponge mouse model, based on the finding that TP is identical to platelet-derived endothelial cell growth factor. Ten and 30 mg/kg administration of TPI significantly inhibited TP-induced neovascularization in a dose-dependent manner in a mouse model. The above results suggest that the combination of TPI and an antitumor nucleoside, FTD, not only enhances the antitumor efficacy and decreases the toxicity of FTD, but also suppresses TP-induced angiogenesis.

Antitumor effects of vitamins K1, K2 and K3 on hepatocellular carcinoma in vitro and in vivo

Abstract: A number of studies have shown that various K vitamins, specifically vitamins K2 and K3, possess antitumor activity on various types of rodent- and human-derived neoplastic cell lines. In the present study, we examined the antitumor effects of vitamins K1, K2 and K3 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vitro and in vivo. Furthermore, we examined the mechanisms of antitumor actions of these vitamins in vitro and in vivo. Although vitamin K1 did not inhibit proliferation of PLC/PRF/5 cells at a 90-microM concentration (the highest tested), vitamins K2 and K3 suppressed proliferation of the cells at concentrations of 90 and 9 microM, respectively. By flow cytometric analysis, it was shown that not only vitamin K1, but also vitamin K2 did not induce apoptosis or cell cycle arrest on PLC/PRF/5 cells. In contrast, vitamin K3 induced G1 arrest, but not apoptosis on PLC/PRF/5 cells. Subsequent in vivo study using subcutaneous HCC-bearing athymic nude mice demonstrated that both vitamins K2 and K3 markedly suppressed the growth of HCC tumors to similar extent. Protein expression of cyclin D1 and cyclin-dependent kinase 4 (Cdk4), but not p16INK4a Cdk inhibitor in the tumor was significantly reduced by vitamin K2 or K3 treatment, indicating that vitamins K2 and K3 may induce G1 arrest of cell cycle on PLC/PRF/5 cells in vivo. Taken collectively, vitamins K2 and K3 were able to induce potent antitumor effects on HCC in vitro and in vivo, at least in part, by inducing G1 arrest of the cell cycle. The results indicate that vitamins K2 and K3 may be useful agents for the treatment of patients with HCC.

Up-regulation of hnRNP A1 gene in sporadic human colorectal cancers.

Abstract: We have previously reported that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a major hnRNP, binds to G-rich repetitive sequences and quadruplex (G4') structures in DNA, including the 5'-TTAGGG-3' telomere repeat and 5'-GGCAG-3' short-tandem-repeat. DNA synthesis arrest at the (GGG) sites within these repeats in vitro was retrieved by the addition of the hnRNP A1 protein or its N-terminal proteolytic product, UP1, in a dose-dependent manner. Therefore, functional perturbation of hnRNP A1 may abrogate the genomic stability of telomere repeats and other G-rich sequences, independent of its major role in transcriptional and translational regulation. In the present study, we conducted genetic and expression analysis of the hnRNP A1 gene in sporadic human colorectal cancers to clarify its possible involvement in human carcinogenesis. Of 30 lesions, one harbored a mutation at the -11 position from the translation initiation site, but none in the coding region. A single nucleotide polymorphism, an A or G-allele, was found in the 5' upstream promoter region of the gene. Quantitative gene expression analysis revealed that 60% (18/30) of cases showed over-expression of hnRNP A1 in cancer tissues by 2-fold or greater, compared to their normal colon tissues, with values of 78, 64 and 40% for clinicopathological stages II, III and IV, respectively. Although the biological consequences of hnRNP A1 overexpression in colorectal cancers remain to be clarified, it could contribute to maintenance of telomere repeats in cancer cells with enhanced cell proliferation. Alternatively, since the variations in the stoichiometry of hnRNP family proteins are considered to affect cell-specific gene expression, quantitative alteration of hnRNP A1 could result in facilitation of transformation of colon epithelial cells as a consequence of transcriptional and translational perturbation.

HMG-CoA reductase inhibitors (statins) as anticancer drugs (review).

Abstract: Apart from their lipid lowering activity, HMG-CoA reductase inhibitors (statins) impair numerous cellular functions associated with metastasis, e.g. gene expression, angiogenesis, cell adhesion, cell motility and invasiveness. Furthermore, statins have impact on apoptotic cell death and modulate cellular susceptibility to cell killing by anticancer drugs and ionizing radiation. Part of the effects provoked by statins are due to the inhibition of the prenylation of low molecular weight GTPases, in particular Ras and Rho, which play key roles in signaling evoked by stimulation of cell surface receptors. C-terminal lipid modification of Ras/Rho GTPases is essential for their correct intracellular localization and function. By depletion of the cellular pool of isoprene precursor molecules, statins reduce the level of membrane-bound active Ras/Rho proteins, thereby impairing corresponding functions. Since broad clinical experience already exists for statins, their incorporation into established tumor-therapeutic regimens would be realizable in a rather short period of time. Here, data available at present arguing for the usefulness of statins in anticancer therapy are summarized and discussed.

Coexpression, prognostic significance and predictive value of EGFR, EGFRvIII and phosphorylated EGFR in colorectal cancer

Abstract: In colorectal cancer, epidermal growth factor receptor (EGFR) expression is reported in 8-100% of the cases examined and there has been no clear association between EGFR expression and prognosis, or response to EGFR inhibitors. In this retrospective study, 87 archival specimens from node positive (Dukes' C) colorectal cancer patients were analysed immunohistochemically, for the expression of EGFR, mutant EGFR (EGFRvIII) and phosphorylated EGFR (pEGFR, tyr1068). Each section was scored on the basis of location and intensity of staining, and the immunostaining was considered positive if greater than 10% of the tumour cells were stained by the antibody. The association between these scores and overall survival was estimated using univariate and multivariate (Cox) analysis. Overall, we found that 76% and 100% of cases were EGFR positive using antibodies to the external or internal domain of EGFR respectively, and 34% of the cases were EGFRvIII positive. However, only 8% of the cases expressed pEGFR and pEGFR immunostaining was never present in more than 10% of tumour cells. The expression of EGFR, EGFRvIII, pEGFR, or coexpression of EGFR and EGFRvIII was not associated with overall survival. Cytoplasmic expression of EGFR (p = 0.0141) or EGFRvIII (p = 0.005) was, however, associated with improved survival in patients receiving radiotherapy. Our results suggest that coexpression of cytoplasmic EGFR and EGFRvIII occurs in a significant proportion (34%) of Dukes' C colorectal cancer and the cytoplasmic expression of EGFR or EGFRvIII is a good indicator of response to radiotherapy.