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1.  Antagonistic Effect of Small Molecule Inhibitors of Wnt/β-catenin in Multiple Myeloma 
Anticancer research  2012;32(11):4697-4707.
Background
Development and progression of multiple myeloma is dependent on the bone marrow (BM) microenvironment, and within the BM, a number of factors are secreted, including the Wnt ligands. Bone marrow stromal cells (BMSC) secrete Wnt ligands that activate Wnt signaling in multiple myeloma. The canonical Wnt pathway which is, mediated through the transcriptional effector β-catenin (β-cat) is commonly deregulated in many cancers. Cells with active β-cat-regulated transcription (CRT) are protected against apoptosis; conversely inhibition of CRT may prevent cell proliferation.
Materials and Methods
In this study, we tested the efficacy of recently described inhibitors of CRT (iCRTs; oxazole and thiazole) for their selective antagonistic effect on Wnt-β-cat response in MM cells MM1, U266, BMSC and primary BMMC obtained from patient samples (n=16).
Results
We demonstrate that iCRTs we used block Wnt/β-cat reporter activity, down regulate β-cat expression and inhibit cell proliferation in a dose dependent manner with an optimal dose closer to 15 µM. Our data further indicate that iCRTs do not influence the expression of the upstream components of the Wnt pathway DKK1 at the optimal dose, suggesting that iCRTs may specifically target β-cat in MM cells. Additionally, iCRT-treatment of MM cells co-cultured with BMSC showed an inhibitory effect on VEGF and cell migration.
Conclusion
This study provides the first in vitro data evaluation of newly described iCRTs as potential Wnt-β-cat/VEGF pathway antagonists in multiple myeloma.
PMCID: PMC3983472  PMID: 23155232
Multiple myeloma; Wnt; β-catenin; transcription; VEGF and DKK1
2.  Effect of an Extract of Withania somnifera Root on Estrogen Receptor-positive Mammary Carcinomas 
Anticancer research  2013;33(4):1519-1523.
The chemopreventive activity of an extract of Withania somnifera (WS) roots was examined in female Sprague-Dawley rats that received the mammary carcinogen methylnitrosourea (MNU). The dose of the extract, administered by gavage, was 150 mg/kg body weight daily for 155 days after injection of MNU. Rats in the treated group (N=15) had an average of 3.47 tumors, and rats in the control group (N=15) had 4.53, a reduction of 23%. The average weights of tumors were 4.98 g for rats in the treated group and 6.30 g for the controls, a difference of 21%. Labeling indices for Ki67 and proliferating cell nuclear antigen (PCNA) markers in cancers of the treated group were 42% and 38% lower, respectively, than those of the corresponding indices for the control group. These results indicate that the root extract significantly reduced the rate of cell division in the mammary tumors.
PMCID: PMC3675906  PMID: 23564793
Withania; chemoprevention; mammary cancer; rats; Ki67; PCNA
3.  Understanding and Improving Platinum Anticancer Drugs – Phenanthriplatin 
Anticancer research  2014;34(1):471-476.
Approximately half of all patients who receive anticancer chemotherapy are treated with a platinum drug. Despite the widespread use of these drugs, the only cure that can be claimed is that of testicular cancer following cisplatin treatment. This article reviews some of our recent work on phenanthriplatin, a cisplatin derivative in which a chloride ion is replaced by phenanthridine, and one of its analogues, the previously reported pyriplatin. These cationic complexes form monofunctional adducts on DNA that do not significantly distort the duplex yet efficiently block transcription. Cell-based assays reveal altered cellular uptake properties and a cancer cell-killing profile different from those of established platinum drugs. Mechanistic work, including a crystal structure analysis of platinum-modified DNA in the active site of RNA polymerase II, is discussed.
PMCID: PMC3937549  PMID: 24403503
cytotoxicity; transcription; RNA polymerase II; X-ray structure; monofunctional platinum; pyriplatin; review
4.  siRNA-Mediated Down-regulation of Ceramide Synthase 1 Leads to Apoptotic Resistance in Human Head and Neck Squamous Carcinoma Cells After Photodynamic Therapy 
Anticancer research  2012;32(7):2479-2485.
Background
The effectiveness of photodynamic therapy (PDT) for cancer treatment correlates with apoptosis. We previously observed that the knockdown of ceramide synthase 6, an enzyme from the de novo sphingolipid biosynthesis pathway, is associated with marked reduction in C18-dihydroceramide and makes cells resistant to apoptosis post-PDT. Down-regulation of ceramide synthase 1 (CERS1) can also render cells resistant to anticancer drugs.
Aim
To explore the impact of CERS1 knockdown on apoptosis and the sphingolipid profile, post-PDT, with the silicone phthalocyanine Pc 4, in a human head and neck squamous carcinoma cell line.
Materials and Methods
Besides siRNA transfection and PDT treatment, the following methods were used: immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectroflurometry and flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation.
Results
CERS1 knockdown led to inhibition of PDT-induced caspase 3-like (DEVDase) activation, of apoptosis and cell death. CERS1 knockdown was associated with global and selective decreases in ceramides and dihydroceramides, in particular C18-, C18:1- and C20-ceramide post-PDT.
Conclusion
Our novel findings are consistent with the notion that CERS1 regulates apoptotic resistance to PDT, partly via C18- and C20-ceramide, and that CERS1 is a molecular target for controlling resistance to PDT.
PMCID: PMC3934872  PMID: 22753704
Apoptosis; ceramide; ceramide synthase 1; dihydroceramide; PDT; sphingolipids; head and neck squamous carcinomas cells UM-SCC-22A
5.  Uncertainty in the Utility of Immunohistochemistry in Mismatch Repair Protein Expression in Epithelial Ovarian Cancer 
Anticancer research  2012;32(11):4963-4969.
Background
Utility of immunohistochemistry (IHC) for mismatch repair (MMR) protein expression has been demonstrated in colorectal cancer but remains incompletely defined in ovarian cancer. We evaluated MMR protein expression in three population-based samples of epithelial ovarian cancers.
Methods
IHC staining was performed on full section (FS) or tissue microarray (TMA) slides for MLH1, MSH2, and MSH6 expression.
Results
Of 487 cases, 147 and 340 were performed through FS and TMA, respectively. Overall, Loss of Expression (LoE) of at least one MMR protein was observed in 12.7% based on an expression score of ≤3 (on a scale of 9). Notably, LoE was significantly higher in TMAs (17.9%) compared to FS cases (0.7%) (p <0.001).
Conclusions
A substantial proportion of epithelial ovarian cancers have a loss of MMR protein expression. Protein expression results vary significantly by the tissue sampling methodology utilized, raising concerns about the clinical utility of this test for ovarian tumors.
PMCID: PMC3919039  PMID: 23155266
immunohistochemistry; IHC; ovarian cancer; mismatch repair; MLH1; MSH2; MSH6
6.  Fenbendazole as a Potential Anticancer Drug 
Anticancer research  2013;33(2):355-362.
Background/Aims
To evaluate the anticancer activity of fenbendazole, a widely used antihelminth with mechanisms of action that overlap with those of the hypoxia-selective nitroheterocyclic cytotoxins/radiosensitizers and the taxanes.
Materials and Methods
We used EMT6 mouse mammary tumor cells in cell culture and as solid tumors in mice to examine the cytotoxic and antitumor effects of fenbendazole as a single agent and in combination regimens.
Results
Intensive treatments with fenbendazole were toxic to EMT6 cells in vitro; toxicity increased with incubation time and under conditions of severe hypoxia. Fenbendazole did not alter the dose-response curves for radiation or docetaxel; instead, the agents produced additive cytotoxicities. Febendazole in maximally-intensive regimens did not alter the growth of EMT6 tumors, or increase the antineoplastic effects of radiation.
Conclusion
These studies provided no evidence that fenbendazole would have value in cancer therapy, but suggested that this general class of compounds merits further investigation.
PMCID: PMC3580766  PMID: 23393324
Fenbendazole; benzamidazoles; tumor microenvironments; radiation therapy; hypoxia; EMT6 cells
7.  Dihydroceramide Desaturase Knockdown Impacts Sphingolipids and Apoptosis after Photodamage in Human Head and Neck Squamous Carcinoma Cells 
Anticancer research  2013;33(1):77-84.
Background
Dihydroceramide desaturase 1 (DES) is the enzyme responsible for converting dihydroceramide into ceramide in the de novo sphingolipid biosynthesis pathway. Dihydroceramide can inhibit ceramide channel formation to interfere with apoptosis. We have shown that following ceramide synthase knockdown, photodynamic therapy (PDT), a cancer treatment modality, is associated with decreased levels of ceramides and dihydroceramides in cells that are resistant to apoptosis.
Aim
Here we investigated the effect of DES knockdown on the sphingolipid profile and apoptosis in human head and neck squamous carcinoma cells after PDT with the silicon phthalocyanine Pc 4.
Materials and Methods
Following siRNA transfection and PDT treatment, quantitative real-time polymerase chain reaction for quantification of DES mRNA, immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectrofluorometry for caspase 3-like (DEVDase) activity, flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation, were performed.
Results
Down-regulation of DES led to a substantial increase in levels of dihydroceramides without affecting ceramide levels. PDT-induced accumulation of individual dihydroceramides and global ceramides was increased by DES knockdown. Concomitantly, mitochondrial depolarization, DEVDase activation, late-apoptosis and cell death were attenuated by DES knockdown. Early apoptosis, however, was enhanced.
Conclusion
Our findings support the following: (i) dihydroceramide reduces pro-apoptotic effects of ceramide; (ii) cells adapt to DES knockdown to become more sensitive to ceramide and early-apoptosis; (iii) DES is a potential molecular target for regulating apoptotic resistance to PDT.
PMCID: PMC3905466  PMID: 23267130
Apoptosis; ceramide; dihydroceramide desaturase; dihydroceramide; PDT; sphingolipids; UM-SCC-22A cells; HNSCC
8.  The Role of Notch and Gamma-secretase Inhibition in an Ovarian Cancer Model 
Anticancer research  2013;33(3):801-808.
Background
The Notch pathway is dysregulated in ovarian cancer. We sought to examine the role of Notch and gamma-secretase (GS) inhibition in ovarian cancer.
Materials and Methods
Established ovarian cancer cell lines were used. Quantitative polymerase chain reaction (qPCR) was used to determine the relative expression of Notch receptor and ligands. Effects of GS inhibition on proliferation, colony formation, and downstream effectors were examined via methylthiazole tetrazolium (MTT) and Matrigel assays, and qPCR, respectively. In vivo experiments with a GS inhibitor and cisplatin were conducted on nude mice. Tumors were examined for differences in microvessel density, proliferation, and apoptosis.
Results
Notch3 was the most up-regulated receptor. The ligands JAGGED1 and DELTA-LIKE4 were both up-regulated. GS inhibition did not affect cellular proliferation or anchorage-independent cell growth over placebo. The GS inhibitor Compound-E reduced microvessel density in vivo.
Conclusion
GS inhibition does not directly affect cellular proliferation in ovarian carcinoma, but Notch pathway blockade may result in angiogenic alterations that may be therapeutically important.
PMCID: PMC3893696  PMID: 23482747
Ovarian cancer; Notch; gamma-secretase inhibitor; model
9.  Neurofibromatosis Type 2 Protein, NF2: An uncoventional cell cycle regulator 
Anticancer research  2013;33(1):1-11.
Neurofibromatosis type 2 protein (NF2) is an underappreciated tumor suppressor involved in a broad range of nervous system tumors. Inactivation of the NF2 gene leads to neurofibromatosis type 2, which is characterized by multiple benign nervous system tumors and mutations in the gene have been demonstrated in many other tumor types as well. All tumors, regardless of location or grade, lack a fundamental control over cell cycle progression. Historically, NF2 is an unconventional tumor suppressor protein in that it does not directly influence the cell cycle. NF2 links receptors at the plasma membrane to their cytoplasmic kinases to facilitate contact inhibition. However, NF2 can also interact with an array of cytoplasmic and nuclear proteins that affect cell cycle progression. Further, through some of these pathways, NF2 may reverse the functional inhibition of conventional tumor suppressor pathways. Here we review mechanisms utilized by NF2 to regain control of the cell cycle.
PMCID: PMC3725758  PMID: 23267122
NF2; merlin; tumor suppressor; cell cycle
10.  Cyclin D1b in Human Breast Carcinoma and Coexpression with Cyclin D1a Is Associated with Poor Outcome 
Anticancer research  2010;30(4):1279-1285.
Background/Aim
Cyclin D1 is a mediator of cell-cycle control that is frequently overexpressed in primary ductal breast carcinomas, but its role is controversial. A polymorphism in the CCND1 gene, G870A, results in an aberrantly spliced protein (cyclin D1b) lacking the Thr-286 phosphorylation site necessary for nuclear export. Studies of murine fibroblasts have shown that although overexpression of canonical cyclin D1 (cyclin D1a) alone is not sufficient to drive malignant transformation, expression of nuclear cyclin D1b is oncogenic. Our objectives were to determine whether cyclin D1b is expressed in human breast carcinomas and to characterize the relationship of this protein to both cyclin D1a and clinical outcome in breast cancer patients.
Patients and Methods
We performed a prospective cohort study of women with early-stage breast cancer and analyzed cyclin D1a and D1b expression in primary breast tumor sections. Expression was tested for correlation with other breast cancer prognostic factors and clinical outcome, including recurrence or death.
Results
A total of 118 patients were included in this analysis, with a median follow-up of 44 months. Cyclin D1b was expressed in 26% of tumors and cyclin D1a was overexpressed in 27%; co-expression occurred in 4%. Cyclin D1a and/or D1b expression were not significantly associated with estrogen or progesterone receptor negativity, Her2 overexpression, young age, lymph node positivity, high tumor grade, nor large tumor size. The risk of recurrence was higher in those co-expressing D1a and D1b compared to the expression of either alone (relative risk=5.3, 95% confidence interval 1.27 to 22.1, p=0.02). The hazard ratio for those with co-expression compared with those without was 6.05 (p=0.04).
Conclusion
Expression of cyclin D1b occurs in primary human breast carcinomas and its coexpression with cyclin D1a may be a marker for increased recurrence risk, independently of other factors.
PMCID: PMC3874215  PMID: 20530440
Breast cancer; cyclin D1; cyclin D1b; cyclin-dependent kinase inhibitor; recurrence
11.  Sunlight, Polymorphisms of Vitamin D-related Genes and Risk of Breast Cancer 
Anticancer research  2013;33(2):543-551.
Background/Aim
Geographic gradients in breast cancer incidence and mortality suggest that vitamin D may reduce risk. The enzyme 25-hydroxyvitamin D 24-hydroxylase (CYP24A1), which degrades the active form of vitamin D, and the vitamin D receptor (VDR) are both found in breast tissue. We investigated six polymorphisms in CYP24A1 and two in the VDR gene in association with breast cancer risk.
Materials and Methods
We conducted a case--control study within the nationwide U.S. Radiologic Technologists cohort, including 845 controls and 484 incident breast cancer cases. Associations of polymorphic variants and ecologic and personal measures of sun exposure with breast cancer risk were assessed using unconditional logistic regression.
Results
Two polymorphisms in CYP24A1 were associated with increased breast cancer risk (rs34043203, Ptrend = 0.03; rs2762934, Ptrend = 0.005) and one with reduced breast cancer risk (rs1570669, Ptrend=0.048). Risk was inversely associated with minor alleles for the VDR Bsm1 polymorphism (rs1544410, Ptrend = 0.05) but not Fok1 (rs2228570). Sunlight measures were not associated with breast cancer risk, however significant interactions between time outdoors in the teen years and three unlinked genotypes were found for VDR (rs1544410, rs2228570) and CYP24A1 (rs1570669).
Conclusion
In this nation-wide breast cancer case--control study, we found the vitamin D pathway was involved in disease etiology and further suggest that reduced cancer risk in association with sunlight may depend on timing of exposure and genetic background. These findings merit further investigation.
PMCID: PMC3866631  PMID: 23393347
Vitamin D; sunlight; polymorphisms; breast cancer; gene; case—control
12.  Synergistic Activity of Histone Deacetylase and Proteasome Inhibition Against Pancreatic and Hepatocellular Cancer Cell Lines 
Anticancer research  2011;31(4):1093-1103.
Aim
To determine the phenotypic effects of belinostat (bel) and bortezomib (bor) against pancreatic cancer (PC) and hepatocellular cancer (HCC) cell lines.
Materials and Methods
Antiproliferative effects were assessed using a sulforhodamine B assay. Synergy was evaluated using the Chou and Talalay method. Apoptosis was measured by caspase −31 −7 activity and PARP cleavage. Downstream effector proteins were detected via immunoblotting. Quantitative nuclear magnetic resonance (NMR)-based metabolomics analysis was performed.
Results
There were single-agent antiproliferative effects against PC and HCC cell lines; the combination of bel and bor (bel+bor) had a synergistic effect. There was up to a 45-fold induction of apoptosis over the control. Post-treatment cell death was associated with p21 up-regulation, more pronounced with treatment with bel+bor. Treatment with bel+bor enhanced hyperacetylation of histone H3 over single-agent bel. A metabolic signature was established for treatments with bor and bel+bor.
Conclusion
The combination of bel+bor displayed significant antiproliferative activity against PC and HCC cell lines, with exhibiting synergistic antiproliferative and proapoptotic patterns even at suboptimal single-agent doses.
PMCID: PMC3866806  PMID: 21508352
Belinostat; bortezomib; pancreas cancer; histone deacetylase inhibitor; proteasome inhibitor; hepatocellular cancer; apoptosis; metabolomics
13.  Growth Inhibitory and Apoptosis-inducing Effects of Xanthohumol, a Prenylated Chalcone Present in Hops, in Human Prostate Cancer Cells 
Anticancer research  2010;30(9):3333-3339.
Promotion of apoptosis in cancer cells could potentially lead to the regression and improved prognosis of hormone-refractory prostate cancer. Xanthohumol (XN), a prenylated chalcone-derived from hops, has shown strong antitumorigenic activity towards diverse types of cancer cells. In the present study, the growth-inhibitory and apoptosis-inducing activity of XN was tested in hormone-sensitive and hormone-refractory human prostate cancer cells lines. Cell growth/viability assay (MTS) demonstrated that prostate cancer cells are highly sensitive to XN at a concentration range of 20-40 μM. The primary mode of tumor cell destruction was apoptosis as demonstrated by the binding of annexin V-FITC, cleavage of PARP-1, activation of procaspases -3, -8, and -9, mitochondrial depolarization and release of cytochrome c from mitochondria. Induction of apoptosis by XN was associated with the inhibition of prosurvival Akt, NF-κB and mTOR signaling proteins and NF-κB-regulated anti-apoptotic Bcl-2 and survivin. These studies provide a rationale for clinical evaluation of XN for the treatment of hormone-refractory metastatic prostate cancer.
PMCID: PMC3846352  PMID: 20944105
Xanthohumol; prostate cancer; apoptosis; prosurvival signaling proteins
14.  Highly Efficient Cationic Ethylphosphatidylcholine siRNA Carrier for GFP Suppression in Modified Breast Cancer Cells 
Anticancer research  2012;32(7):2563-2566.
Aim
Cationic ethylphosphatidylcholines (ePCs) were evaluated for the delivery of siRNA in modified breast cancer cells.
Materials and Methods
Dimyristoleoyl-ePC (C14), dioleoyl-ePC (C18), and dilauroyl-ePC (C12) nanoparticles were complexed with siRNA for green fluorescent protein (GFP) suppression in modified MCF-7 breast cancer cells. The kinetics of GFP suppression were followed over the course of 72 hours.
Results
C14, which has been previously found to be particularly effective in gene transfection into primary human umbilical artery endothelial cells, was also remarkably effective as siRNA carrier, with an efficacy exceeding that of Lipofectamine RNAiMAX. The C14 toxicity remained comparable to that of RNAiMAX. The efficacy of the other tested cationic ePC formulations was less than that of C14 and RNAiMAX.
Conclusion
The cationic lipid C14 is a highly efficient siRNA carrier that could be used for the development of new formulations for siRNA delivery into cancer cells. A valuable advantage of the C14 formulations is the fact that they are simple, and do not require adjuvants or complex preparation procedures.
PMCID: PMC3838928  PMID: 22753714
Cationic lipid; ethylphosphatidylcholine; non-viral vector; siRNA; MCF-7 breast cancer cells
15.  Curcumin Targets FOLFOX-surviving Colon Cancer Cells via Inhibition of EGFRs and IGF-1R 
Anticancer research  2010;30(2):319-325.
Curcumin (diferuloylmethane), which has no discernible toxicity, inhibits initiation, promotion and progression of carcinogenesis. 5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal cancer chemotherapeutics, but produces an incomplete response resulting in survival of cells (chemo-surviving cells) that may lead to cancer recurrence. The present investigation was, therefore, undertaken to examine whether addition of curcumin to FOLFOX is a superior therapeutic strategy for chemo-surviving cells. Forty-eight-hour treatment of colon cancer HCT-116 and HT-29 cells with FOLFOX resulted in 60–70% survival, accompanied by a marked activation of insulin like growth factor-1 receptor (IGF-1R) and minor to moderate increase in epidermal growth factor receptor (EGFR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (HER-2) as well as v-akt murine thymoma viral oncogene homolog 1 (AKT), cyclooxygenase-2 (COX-2) and cyclin-D1. However, inclusion of curcumin to continued FOLFOX treatment for another 48 h greatly reduced the survival of these cells, accompanied by a concomitant reduction in activation of EGFR, HER-2, IGF-1R and AKT, as well as expression of COX-2 and cyclin-D1. More importantly, EGFR tyrosine kinase inhibitor gefitinib or attenuation of IGF-1R expression by the corresponding si-RNA caused a 30–60% growth inhibition of chemo-surviving HCT-116 cells. However, curcumin alone was found to be more effective than both gefitinib and IGF-1R si-RNA mediated growth inhibition of chemo-surviving HCT-116 cells and addition of FOLFOX to curcumin did not increase the growth inhibitory effect of curcumin. Our data suggest that inclusion of curcumin in conventional chemotherapeutic regimens could be an effective strategy to prevent the emergence of chemoresistant colon cancer cells.
PMCID: PMC3836443  PMID: 20332435
Chemo-surviving cells; curcumin; colon cancer; EGFRs; IGF-1R
16.  Synthetic Triterpenoids Inhibit Growth, Induce Apoptosis and Suppress Pro-survival Akt, mTOR and NF-κB Signaling Proteins in Colorectal Cancer Cells 
Anticancer research  2010;30(3):785-792.
Lack of apoptotic cell death has been implicated in malignant transformation and resistance to anticancer therapies. The promotion of apoptosis in cancer cells could potentially lead to the regression and improved prognosis of refractory colorectal cancer. Synthetic triterpenoids have shown strong antitumorigenic activity towards diverse cancer cell types, but have not been investigated for colorectal cancer. In the present study, we tested the apoptosis-inducing activity of oleanane triterpenoid 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oic acid (CDDO) and its C-28 methyl ester (CDDO-Me) and C-28 imidazole (CDDO-Im) derivatives in colorectal cancer cells lines. Cell growth/viability assay (MTS) demonstrated that colorectal cancer cells are highly sensitive to CDDO-Me at concentrations of 1.25 to 10 μM. The primary mode of tumor cell destruction was apoptosis as demonstrated by the cleavage of PARP-1, activation of procaspases-3, -8, and -9 and mitochondrial depolarization. Induction of apoptosis by CDDO-Me was associated with the inhibition of pro-survival Akt, NF-κB and mTOR signaling proteins and NF-κB-regulated anti-apoptotic Bcl-2, Bcl-xL, Bad and survivin. These studies provide rationale for clinical evaluation of CDDO-Me for the treatment of advanced chemotherapy refractory colorectal cancer.
PMCID: PMC3816511  PMID: 20392997
Synthetic triterpenoids; colorectal cancer; apoptosis; signaling proteins
17.  Protein Kinase C Regulates Bombesin-induced Rapid VEGF Secretion in Neuroblastoma Cells 
Anticancer research  2012;32(11):4691-4696.
Background
Intracellular signaling responsible for gastrin-releasing peptide (GRP) receptor-mediated neovascularization is not clearly understood. We sought to determine the cellular mechanisms involved in the GRP receptor regulation of vascular endothelial growth factor (VEGF) release in neuroblastoma cells.
Materials and Methods
BE(2)-C cells were treated with bombesin (BBS), the amphibian equivalent of GRP, Phorbolmyristate acetate (PMA) a PKC agonist, or GF109293X (GFX), and analyses were performed for VEGF secretion, phosphorylated protein kinase B (AKT), extracellular signal-regulated kinases (ERK) and protein kinase D (PKD) expression.
Results
BBS rapidly increased VEGF secretion at 30 min. Pre-treatment with PMA alone produced similar results; this effect was synergistic with the addition of GRP. Conversely, GFX blocked PMA-stimulated increase in VEGF secretion. Immunofluorescent staining for VEGF was correlated to BBS, PMA and GFX.
Conclusions
PKC is critically responsible for rapid VEGF secretion by GRP receptor signaling in neuroblastoma cells. Inhibition of VEGF significantly reduced GRP-mediated cell proliferation, suggesting its crucial role in neuroblastoma tumorigenesis.
PMCID: PMC3668683  PMID: 23155231
PKC; BBS; GRP; VEGF; neuroblastoma
18.  Association between COX-2 Expression and Effectiveness of COX-2 Inhibitors in Phase II Trial in Patients with Metastatic Colorectal Adenocarcinoma 
Anticancer research  2012;32(8):3559-3563.
Aim
The role for the cyclooxygenase-2 (COX-2) pathway in colorectal carcinogenesis has been suggested in pre-clinical models. In a previously reported phase II trial, the addition of COX-2 inhibitor celecoxib to irinotecan and capecitabine did not appear to significantly increase the activity of chemotherapy in patients with metastatic colorectal carcinoma (mCRC). We evaluated COX-2 expression in the available tumors from patients enrolled by immunohistochemistry and its correlation with clinical outcome.
Patients and Methods
Fifty-one patients with mCRC were enrolled in the phase II study between June 2002 and November 2005. Patients received a combination of irinotecan 70 mg/m2 over 30 min i.v. on days 1 and 8, capecitabine 1,000 mg/m2 twice per day orally on days 1-14 and the COX-2 inhibitor celecoxib at a daily dose of 800 mg continuously. Cycles were repeated every 21 days. Formalin-fixed paraffin-embedded tumor tissue samples were available for 17 patients enrolled on that study. COX-2 expression was evaluated by immunohistochemistry and correlated with clinical outcome.
Results
In the phase II study, the objective response rate was 41%. The median time to progression was 7.7 months and median survival time was 21.2 months. Tumor COX-2 expression by immunohistochemistry was assessed for 17 patients enrolled in that same phase II study. While not statistically significant, the response rate was better for patients in the low COX-2 expression group, while time to progression and overall survival was longer in patients in the high COX-2 expression group. This discrepancy can be partially attributed to the small sample size.
Conclusion
In the previously published phase II study, the addition of celecoxib to irinotecan and capecitabine did not appear to significantly increase the activity of chemotherapy. COX-2 expression by immunohistochemistry was neither prognostic nor predictive for response.
PMCID: PMC3807874  PMID: 22843946
COX-2; metastatic colorectal cancer; celecoxib
19.  Regulation of the Proliferation of Colon Cancer Cells by Compounds that Affect Glycolysis, Including 3-Bromopyruvate, 2-Deoxyglucose and Biguanides 
Anticancer research  2013;33(2):401-407.
In previous studies we observed that 2-deoxyglucose blocked the acidification of the medium used for culture of colon cancer cells caused by incubation with biguanides and had an additive inhibitory effect on growth. In the present work, we found that 3-bromopyruvate can also prevent the lowering of pH caused by biguanide treatment. 3-Bromopyruvate inhibited colonic cancer cell proliferation but the effect was not always additive to that of biguanides and an additive effect was more notable in combined treatment with 3-bromopyruvate and 2-deoxyglucose. The induction of alkaline phosphatase activity by butyrate was not consistently affected by combination with other agents that modified glucose metabolism. The drug combinations that were examined inhibited proliferation of wild-type and P53 null cells and affected colonic cancer lines with different growth rates.
PMCID: PMC3805264  PMID: 23393330
Colon cancer; 3-bromopyruvate; metformin; phenformin; 2-deoxyglucose
20.  Microfluidic Assembly of Lipid-based Oligonucleotide Nanoparticles 
Anticancer research  2011;31(3):771-776.
Background
Oligonucleotides (ONs) have shown great promise as therapeutic agents for various diseases. It is necessary to provide a protocol for preparation of ON-loaded lipid nanoparticles (LNPs) in a reproducible manner on a laboratory scale.
Materials and Methods
A 3-inlet microfluidic (MF) chip-based device was used to synthesize LNPs at the lipid/ON ratio of 10/1 (w/w) and at flow rates ranging from 50 to 1100 µl/min. A series of LNPs containing either antisense oligodeoxyribonucleotide (AS-ODN) or small-interfering RNA (siRNA) were synthesized. Bulk mixing was used as control.
Results
The MF method was shown to be particularly useful for synthesis of LNPs loaded with AS-ODN. The optimal range of flow rates for AS-ODN LNPs was found to be 100 to 200 µl/min. MF synthesis produced LNPs with lower polydispersity values. However, the MF was less effective in preparing LNPs loaded with siRNA, which may have been due to greater rigidity of double-stranded siRNA comparing to single-stranded AS-ODN.
Conclusion
MF technology is a simple, affordable and reproducible method for production of ON-LNPs.
PMCID: PMC3791325  PMID: 21498694
Microfluidics; oligonucleotide; lipid nanoparticles
21.  Disulfide-linked Liposomes: Effective Delivery Vehicle for Bcl-2 Antisense Oligodeoxyribonucleotide G3139 
Anticancer research  2010;30(1):31-37.
Background
Disulfide-linked oligodeoxyribonucleotide (ODN) liposomes were formulated and evaluated for the delivery of antisense ODN G3139 in KB human oral carcinoma cells.
Materials and Methods
Liposomes composed of 1,2-di-(9Z-octadecenoyl)-3-trimethylammo-nium-propane (DOTAP)/egg phosphatidylcholine/α-tocopheryl polyethylene glycol 1000 succinate were incorporated with hydrophobized disulfide-linked ODN. Disulfide-linked ODN liposomes were characterized for their size, ODN intracellular delivery, Bcl-2 mRNA and protein expression, growth inhibition, and chemosensitization.
Results
Intracellular delivery of ODN with disulfide-linked ODN liposomes was more efficient than that with non-liposomal hydrophobized disulfide-linked ODN. Treatment of the cells with disulfide-linked ODN liposomes resulted in efficient Bcl-2 down-regulation greater than that with hydrophobized disulfide-linked ODN and consistent with that of cellular growth inhibition and the sensitization to daunorubicin in KB cells. Disulfide-linked ODN liposomes exhibited superior colloidal stability during 5-week storage.
Conclusion
Disulfide-linked liposomes are effective delivery vehicles for antisense ODN.
PMCID: PMC3790264  PMID: 20150614
Liposomes; disulfide; antisense oligonucleotide; G3139; Bcl-2; drug delivery
22.  Ultrasound-enhanced Microfluidic Synthesis of Liposomes 
Anticancer research  2010;30(2):463-466.
Background
Liposomes have been successfully used as delivery vehicles for anticancer drugs. Both sonication and microfluidic technologies have been used to produce liposomes. The combination of the two methods was evaluated in this study.
Materials and Methods
The microfluidic devices, mainly comprising micro-dispensers and a sonicator, were used to produce liposomal nanoparticles. Sonication was used to enhance the reduction of liposome size.
Results
Sonication significantly reduced the size of the liposomes. The particle size also decreased as the buffer to solvent flow rate ratio increased. The smallest particle sizes were achieved with a volumetric flow rate of lipids at 0.374 ml/min.
Conclusion
The microfluidic devices in combination with ultrasound are simple and may be used to produce liposomal nanoparticles with narrow size distribution.
PMCID: PMC3789511  PMID: 20332455
Liposomes; microfluidics; ethanol injection; sonication
23.  Cytotoxic Effects of N-(3-Chloro-1,4-dioxo 1,4-dihydronaphthalen-2-yl)-benzamide on Androgen-dependent and -independent Prostate Cancer Cell Lines 
Anticancer research  2010;30(2):519-527.
Background
Worldwide among men, prostate cancer ranks third in cancer occurrence and sixth in cancer mortality. A number of 1, 4-naphthoquinone derivatives have been identified that possess significant pharmacological effects associated with antitumor activities. In this study, the in vitro effects of N-(3-chloro-1,4-dioxo 1,4-dihydronaphthalen-2-yl)-benzamide (NCDDNB) were evaluated on androgen-dependent (CWR-22) and androgen-independent (PC-3, DU-145) human prostate cancer cell lines, and on a normal bone marrow cell line (HS-5). Specifically, the in vitro activity of this compound on cell cycle regulation and apoptosis was evaluated.
Materials and Methods
Established methods of cell viability, cell cycle, Western blot and apoptosis were used.
Results
The effect of NCDDNB on CWR-22, PC-3, DU-145 and HS-5 cells revealed significant anti-tumor activities with IC50s, of 2.5, 2.5, 6.5, and 25 μM respectively. The results of cell cycle analysis showed that NCDDNB arrested PC-3, DU-145, and CWR-22 cells in the G1-phase of the cell cycle. The compound showed no effect on the cell cycle progression in the HS-5 bone marrow cell line. These findings were further validated using Western blot analysis. NCDDNB showed the greatest amount of apoptosis in the androgen-independent PC-3 cells in a time-dependent manner with the apoptotic apex at day 5 of treatment. Furthermore, NCDDNB induced-apoptosis in DU-145 and CWR-22 cells peaked at day 3 of treatment.
Conclusion
Although the mechanism of action of this compound has not been completely elucidated, the effect on the cell cycle and the induction of apoptosis in different prostate cancer cell lines prompted us to carry out a more in-depth preclinical evaluation. This study suggests that NCDDNB may have an impact on treatment of prostate cancer while protecting the bone marrow.
PMCID: PMC3776453  PMID: 20332464
1,4-Naphthoquinone; human prostate cancer cell lines; cytotoxicity; cell cycle; apoptosis
24.  Role of Caspases in 5-FU and Selenium Induced Growth Inhibition of Colorectal Cancer Cells 
Anticancer research  2008;28(0):3579-3592.
Background
The mechanisms that could explain the poor sensitivity to 5-FU in certain colorectal cancer (CRC) cells were investigated and whether or not co-treatment with low doses of selenium would offer a therapeutic benefit, was explored.
Materials and Methods
Four CRC cell lines (Caco2, RKO, DLD1 and HT-29), with defined tumor signatures and seven different chemical forms of selenium were tested.
Results
5-FU partially inhibited the HT-29 and RKO cells, but had a weak effect on the DLD1 and almost none on the Caco2 cells. Selenous acid and sodium selenite induced growth inhibition of the DLD1, RKO and HT-29 cells, with a marginal effect on the Caco2 cells. The Caco2 cells with mutant p53, failure to activate caspase-8, −9, −7 and −3 and with hypermethylated caspase-8 were resistant to 5-FU. Conversely, RKO cells expressing wild type p53, proteolytically activated caspase-8, −9, −7 and −3 and unmethylated caspase-8 were more responsive to 5-FU and selenous acid induced apoptosis.
Conclusion
Combination treatment with selenous acid may offer an efficacious strategy to overcome 5-FU resistance in certain CRC cells.
PMCID: PMC3771536  PMID: 19189638
Colorectal cancer; caspases; selenium; selenous acid; 5FU; apoptosis
25.  Aryl Hydrocarbon Receptor and Lung Cancer 
Anticancer research  2013;33(4):1247-1256.
The leading cause of lung cancer is exposure to cigarette smoke and other environmental pollutants, which include formaldehyde, acrolein, benzene, dioxin, and polycyclic aromatic hydrocarbons (PAHs). PAHs and dioxins are exogenous ligands that directly bind to the aryl hydrocarbon receptor (AhR), a transcription factor that activates xenobiotic metabolism, histone modification (an important step in DNA methylation), and, ultimately, tumorigenesis. Here we summarize the current understanding of AhR and its role in the development of lung cancer, including its influence on cell proliferation, angiogenesis, inflammation, and apoptosis.
PMCID: PMC3771678  PMID: 23564762
Aryl hydrocarbon receptor; lung cancer; tumorigenesis; dioxin; polycyclic aromatic hydrocarbons; cigarette smoke

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