[Show abstract][Hide abstract] ABSTRACT: The occurrence of intestinal polyps in people at high risk for developing colorectal cancer provides an opportunity to test the efficacy of chemoprevention agents. In this situation of treating otherwise healthy people, the potential for toxicity must be minimal. The small molecule flexible heteroarotinoid (Flex-Het), called SHetA2, has chemoprevention activity in organotypic cultures in vitro and lack of toxicity at doses capable of inhibiting xenograft tumor growth in vivo. The objective of this study was to evaluate SHetA2 chemoprevention activity and toxicity in the APCMin/+ murine model. Oral administration of SHetA2 at 30 and 60 mg/kg five days per week for 12 weeks significantly reduced development of intestinal polyps by 40 to 60% depending on the dose and sex of the treatment group. Immunohistochemical and Western blot analysis of polyps demonstrated reduced levels of cyclin D1 and proliferating cell nuclear antigen (PCNA) in both SHetA2 treatment groups. Western blot analysis also demonstrated SHetA2 induction of E-cadherin, Bax and caspase 3 cleavage along with reduction in Bcl-2, cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF), consistent with SHetA2 regulation of apoptosis, inflammation and angiogenesis. Neither dose caused weight loss nor gross toxicity in APCMin/+ or wild type littermates. Magnetic resonance imaging (MRI) of cardiac function showed no evidence of SHetA2 toxicity. SHetA2 did not alter left ventricular wall thickness. In summary, SHetA2 exerts chemoprevention activity without overt or cardiac toxicity in the APCMin/+ model. SHetA2 modulation of biomarkers in colon polyps identifies potential pharmacodynamic endpoints for SHetA2 clinical trials.
Full-text · Article · Jul 2013 · Cancer Prevention Research
[Show abstract][Hide abstract] ABSTRACT: Progression of colon cancer is associated with the up-regulation of cyclooxygenase-2 (COX-2) and hydroxymethyl glutaryl CoA reductase (HMG-R). Clinical and preclinical evidence shows that a combination of COX-2 and HMG-R inhibitors provide additive/synergistic chemopreventive effects against colorectal cancer. However, the mechanism by which statins and NSAIDs inhibit cancer growth is not yet fully understood. We aimed to identify critical molecules and signal pathways modulated by a combination of lovastatin and celecoxib in the human HCT-116 colon cancer cell line. HCT-116 cells were exposed to 50 microM celecoxib, 25 microM lovastatin or a combination of both to assess their effect in modulating caveolin-1 expression and its down-stream signaling pathways. Our results suggest that a combination of lovastatin and/or celecoxib suppressed caveolin-1 expression and membrane localization profoundly when compared to either agent alone. Lovastatin and/or celecoxib also inhibited caveolin-1-dependent cell survival signals mediated through Akt activation as well as its down-stream effectors such as phosphorylated ERK and STAT3 in HCT-116 cells. Treatment with lovastatin or celecoxib decreased the levels of cyclin D1, CDK2, pRb and E2F1, while the combination treatment showed more pronounced suppression. In addition, lovastatin and celecoxib also decreased the amount of cholesterol rich cytoplasmic lipid bodies (storehouses of esteridied arachidonates) by 80%, while the combination showed a complete inhibition. Overall, our data suggest that a combination of COX-2 and HMG-R inhibitors synergistically inhibits caveolin-1 and its associated signaling pathways.
Preview · Article · Nov 2009 · International Journal of Oncology
[Show abstract][Hide abstract] ABSTRACT: Tumor suppressor p53 plays a major role in colorectal cancer development. The present study explores the effects of p53-modulating agent CP-31398 alone and combined with celecoxib on azoxymethane-induced aberrant crypt foci (ACF) and colon adenocarcinomas in F344 rats. Maximum tolerated doses were 400 and 3,000 ppm for CP-31398 and celecoxib, respectively. ACF and tumor efficacy endpoints were carried out on azoxymethane-treated 7-week-old rats (48 per group) fed the control AIN-76A diet. Two weeks after carcinogen treatment, rats were fed the diets containing 0, 150, or 300 ppm CP-31398, 300 ppm celecoxib, or 150 ppm CP-31398 plus 300 ppm celecoxib. ACF and colon adenocarcinomas were determined at 8 and 48 weeks after azoxymethane treatment, respectively. Dietary CP-31398 was shown to suppress mean colonic total ACF by 43% and multicrypt ACF by 63%; dietary CP-31398 at 150 and 300 ppm suppressed adenocarcinoma incidence by 30.4% (P < 0.02) and 44% (P < 0.005), respectively, and adenocarcinoma multiplicity by 51% (P < 0.005) and 65% (P < 0.0001), respectively. Dietary celecoxib suppressed colon adenocarcinoma incidence (60%; P < 0.0003) and multiplicity (70%; P < 0.0001). Importantly, combination of low-dose CP-31398 and celecoxib suppressed colon adenocarcinoma incidence by 78% and multiplicity by 90%. Rats that were fed the high-dose CP-31398 or a combination of low-dose CP-31398 and celecoxib showed considerable enhancement of p53 and p21(WAF1/CIP) expression, apoptosis, and reduced tumor cell proliferation in colonic tumors. These observations show, for the first time, that CP-31398 possesses significant dose-dependent chemopreventive activity in a well-established colon cancer model and that a combination of low-dose CP-31398 and celecoxib significantly enhanced colon cancer chemopreventive efficacy.
[Show abstract][Hide abstract] ABSTRACT: Colorectal cancer is the leading cause of cancer related deaths in the United States. Although it is preventable, thousands of lives are lost each year in the U.S. to colorectal cancer than to breast cancer and AIDS combined. In colon cancer, the formation and progression of precancerous lesions like aberrant crypt foci and polyps is associated with the up-regulation of cycloxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and hydroxy methyl glutaryl CoA reductase (HMG-CoA reductase). The current review will focus on the signaling pathway involving COX-2 and HMG-CoA reductase enzymes and their downstream effectors in signaling mechanism. Cancer cells need huge pools of both cholesterol and isoprenoids to sustain their unlimited growth potential. Cholesterol by modulating caveolae formation regulates several signaling molecules like AKT, IGFR, EGFR and Rho which are involved in cell growth and survival. Cholesterol is also essential for lipid body formation which serves as storage sites for COX-2, eicosanoids and caveolin-1. Experimental studies have identified important mechanisms showing that COX-2, caveolin-1, lipid bodies and prenylated proteins is involved in carcinogenesis. Therefore multi-target, multi-drug approach is the ideal choice for effective colon cancer chemoprevention. This review will give an overview of the two pathways, their signaling networks, and the interactions between the components of the two networks in the activation and regulation of cell signaling involving growth/survival and explain the rationale for colon cancer chemoprevention using COX-2 inhibitors and statins.
Full-text · Article · May 2008 · Gene regulation and systems biology
[Show abstract][Hide abstract] ABSTRACT: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation initiated by dedifferentiation and proliferation of renal tubular epithelial cells. Renal tubular epithelial cells (RTC, derived from normal kidney tissue) in primary cultures exhibit both homogeneous expression of gamma-glutamyl transferase and low molecular weight cytokeratin, two different markers for proximal and distal renal epithelial cells, respectively. RTC in cultures also abnormally express the dedifferentiation markers vimentin and PAX-2, which are proteins normally expressed in epithelial cells lining cysts in ADPKD kidneys but not tubular cells in normal kidneys. In contrast, different cultures of cystic epithelial cells (CEC, derived from the cysts walls of polycystic kidneys) display variable expression of cytokeratin, gamma-glutamyl transferase, and PAX-2, but a constant level of vimentin. Importantly, RTC and CEC exhibit the capacity to convert to their respective original structures by forming tubules and cysts, respectively, when cultured in a three-dimensional gel matrix, whereas HK-2, LLC-PK1, and MDCK renal epithelial cell lines form cell aggregates or cysts. Our study demonstrates that the marker expression of the various epithelial cell types is not highly stable in primary cultures. Their modulation is different in cells originating from normal and ADPKD kidneys and in cells cultured in monolayer and three-dimensions. These results indicate the plasticity of epithelial cells that display a mixed epithelial/dedifferentiated/mesenchymal phenotype during their expansion in culture. However, RTC and CEC morphogenic epithelial properties in three-dimensional cultures are similar to those in vivo. Thus, this model is useful for studying the mechanisms leading to tubulogenesis and cystogenesis.
No preview · Article · Mar 2008 · Cell and Tissue Research
[Show abstract][Hide abstract] ABSTRACT: S-adenosyl L-methionine (SAM) is a universal methyl group donor to various intermediary metabolites, hormones, proteins, neurotransmitters, phospholipids and nucleic acids. Deficiency of folate, which plays a role in the synthesis of SAM leads to increased risk for colon cancer. This study tested the effectiveness of SAM supplementation in protecting against azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. We also tested the effect of SAM on cyclooxygenase-2 (COX-2) in a macrophage cell line. Further, we developed a 3-D culture model using Caco-2 cells to test the effect of SAM on tumor spheroid size and number. Groups of rats were given the experimental diet containing either 0-, 400- or 800-ppm SAM, 1 week before the first AOM injection and continued until 8 weeks. In the control group, AOM produced a substantial number of aberrant crypt foci (ACF) (96 +/- 8). Dietary administration of SAM significantly reduced the number of total ACF (400 ppm SAM, 68 +/- 7.3, p < 0.01 and 800 ppm SAM, 57 +/- 7.1, p < 0.001). SAM significantly decreased AOM-induced colonic multicrypt foci in a dose-dependent manner. Suppression of Lipopolysaccharide (LPS) induced COX-2 protein expression was observed in a RAW264.7 cell line. We established growth of Caco-2 cells as spheroids, in a 3D matrix of collagen and matrigel. Treatment with SAM decreased both size and number of spheroids in a dose-dependent manner (p < 0.0001). These observations demonstrate for the first time that SAM can reduce the occurrence of ACF in AOM treated male F344 rats and suppress formation of human tumor spheroids and expression of COX-2.
Full-text · Article · Jan 2008 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by formation of cysts from tubular epithelial cells. Previous studies indicate that secretion of prostaglandin E2 (PGE2) into cyst fluid and production of cAMP underlie cyst expansion. However, the mechanism by which PGE2 directly stimulates cAMP formation and modulates cystogenesis is still unclear, because the particular E-prostanoid (EP) receptor mediating the PGE2 effect has not been characterized. Our goal is to define the PGE2 receptor subtype involved in ADPKD. We used a three-dimensional cell-culture system of human epithelial cells from normal and ADPKD kidneys in primary cultures to demonstrate that PGE2 induces cyst formation. Biochemical evidence gathered by using real-time RT-PCR mRNA analysis and immunodetection indicate the presence of EP2 receptor in cystic epithelial cells in ADPKD kidney. Pharmacological evidence obtained by using PGE2-selective analogs further demonstrates that EP2 mediates cAMP formation and cystogenesis. Functional evidence for a role of EP2 receptor in mediating cAMP signaling was also provided by inhibiting EP2 receptor expression with transfection of small interfering RNA in cystic epithelial cells. Our results indicate that PGE2 produced in cyst fluid binds to adjacent EP2 receptors located on the apical side of cysts and stimulates EP2 receptor expression. PGE2 binding to EP2 receptor leads to cAMP signaling and cystogenesis by a mechanism that involves protection of cystic epithelial cells from apoptosis. The role of EP2 receptor in mediating the PGE2 effect on stimulating cyst formation may have direct pharmacological implications for the treatment of polycystic kidney disease.
Preview · Article · Dec 2007 · American journal of physiology. Renal physiology
[Show abstract][Hide abstract] ABSTRACT: Treatment of ovarian cancer with cisplatin-based chemotherapy is highly toxic and is often followed by cancer recurrence. Repeated treatments with cisplatin frequently result in the development of resistance to this drug. Drugs with low toxicity that could enhance the tumor cell killing effects of cisplatin could potentially reduce the toxicity and enhance the efficacy of cisplatin. The mechanism of cell kill by cisplatin is partially due to induction of apoptosis through the p53 pathway. Retinoids can regulate apoptosis through nuclear retinoic acid receptors, but the role of p53 in the mechanism varies with the individual retinoid and cell type. The objective of this study was to evaluate the potential of receptor-active and -independent retinoids as chemosensitization of ovarian cancer cell lines with different p53 status. The growth of two ovarian cancer cell lines, OVCAR-3 that has wild type p53 and Caov-3 that has mutant type, was evaluated in the presence and absence of various combinations of cisplatin, a series of retinoids and a chemical inhibitor of p53 transactivation activity (pifithrinα). Both receptor active and receptor independent retinoids chemosensitized the two cell lines to cisplatin. PFTα partially attenuated growth inhibition by cisplatin, slightly enhanced the growth inhibition of retinoids and did not affect growth inhibition when retinoids were used in combination with cisplatin. RNase protection demonstrated that PFTα did not alter retinoid effects on p53 regulated genes. In conclusion, retinoids chemosensitize both sensitive and resistant ovarian cancer cells to cisplatin through mechanisms independent of nuclear retinoid receptors and p53.
No preview · Article · Apr 2006 · American journal of pharmacology and toxicology
[Show abstract][Hide abstract] ABSTRACT: The anti-cancer activities and toxicities of retinoic acid (RA) and synthetic retinoids are mediated through nuclear RA receptors (RARs) and retinoid X receptors (RXRs) that act as transcription factors. Heteroarotinoids (Hets), which contain a heteroatom in the cyclic ring of an arotinoid structure, exhibit similar anti-cancer activities, but reduced toxicity in vivo, in comparison to parent retinoids and RA. A new class of Flexible Hets (Flex-Hets), which contain 3-atom urea or thiourea linkers, regulate growth and differentiation similar to RA, but do not activate RARs or RXRs. In addition, Flex-Hets induce potent apoptosis in ovarian cancer and in head and neck cancer cell lines through the intrinsic mitochondrial pathway. In this study, 4 cervical cancer cell lines were growth inhibited by micromolar concentrations of Flex-Hets to greater extents than RAR/RXR active retinoids. The most potent Flex-Het (SHetA2) inhibited each cell line of the National Cancer Institute's human tumor cell line panel at micromolar concentrations. Oral administration of Flex-Hets (SHetA2 and SHetA4) inhibited growth of OVCAR-3 ovarian cancer xenografts to similar extents as administration of a RAR/RXR-panagonist (SHet50) and Fenretinide (4-HPR) in vivo. None of these compounds induced evidence of skin, bone or liver toxicity, or increased levels of serum alanine aminotransferase (ALT) in the treated mice. Topical application of Flex-Hets did not induce skin irritation in vivo, whereas a RAR/RXR-panagonist (NHet17) and a RARgamma-selective agonist (SHet65) induced similar irritancy as RA. In conclusion, Flex-Hets exhibit improved therapeutic ratios for multiple cancer types over RAR and/or RXR agonists.
No preview · Article · Nov 2005 · Investigational New Drugs
[Show abstract][Hide abstract] ABSTRACT: Current therapy for cervical cancer includes radiation therapy. Retinoic acid (RA) can increase the sensitivity of cervical cancer cell lines to radiation. The mechanism of this sensitization may not involve the p53 protein because the human papillomavirus (HPV) E6 protein, which is present in the majority of cervical cancers, promotes p53 degradation. The objective of this study was to determine if p53 is involved in the mechanism of RA radiosensitization.
The effects of radiation on cervical (SiHa, CC-1, and C33a) and vulvar (SW962) cancer cell lines under various experimental conditions were evaluated using clonogenic, Coulter Counter, electrophoretic mobility shift (EMSA) and a multi-probe RNase protection assay of p53-inducible genes.
RA (5 microM 9-cis-RA) radiosensitized the SiHa and CC-1 cell lines that contain HPV-degraded p53, but did not radiosensitize the SW962 cell line, which is HPV negative and contains wild-type p53, nor the C33a cell line, which contains mutant p53 (R273C). Expression of mutant p53 (R273H) in SiHa cells increased the growth rate, but did not prevent RA-induced differentiation or radiosensitization at clinically relevant doses. Inhibition of p53 transactivation with pifithirin alpha did not prevent RA radiosensitization of SiHa at 5 Gy. RA repressed c-fos mRNA expression in control and irradiated SiHa cultures, but did not repress bcl-x(L), p53, GADD45, p21, bax, bcl-2, or mcl-1 mRNA expression.
The mechanism of RA radiosensitization does not require functional p53 and may involve c-fos in cervical cancer cell lines.
No preview · Article · May 2005 · Gynecologic Oncology
[Show abstract][Hide abstract] ABSTRACT: Regulation of growth, differentiation, and apoptosis by synthetic retinoids can occur through mechanisms that are dependent and independent of their ability to bind and activate nuclear retinoic acid receptors. The objective of this study was to determine if increasing flexibility of the heteroarotinoid structure would affect the specificity of the synthetic retinoids for the receptors and for their regulation of cancerous and nonmalignant cells. Methods were developed to produce the first examples of heteroarotinoids 15a-15h, which contain urea and/or thiourea linking groups between two aryl rings. Substituents at the para position of the single phenyl ring were either an ester, a nitro group, or a sulfonamide group. Ovarian cancer cell lines Caov-3, OVCAR-3, SK-OV-3, UCI-101, and 222 were utilized, and the inhibitory prowess of the heteroarotinoids was referenced to that of 4-HPR (25). Similar to 4-HPR (25), the heteroarotinoids inhibited growth of all cell lines at micromolar concentrations. Although the heteroarotinoids did not activate retinoic acid receptors, the agents induced potent growth inhibition against the cancer cells with weak activity against normal and benign cells. The growth inhibition was associated with cell loss and induction of reactive oxygen species.
Full-text · Article · Mar 2004 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: To better define determinants of survival and optimal management strategies for patients with ovarian cancer and brain metastases.
A review of literature using Medline identified 15 case series of ovarian cancer patients with brain metastases (OBM). Each article was abstracted for survival data, and in all cases, the intervals between ovarian cancer diagnosis and brain metastasis identification, and between brain metastasis identification and last follow-up were recorded. Cases were categorized by patient characteristics and treatment modality for brain metastases. Estimated survival probabilities were plotted using the Kaplan-Meier method with differences between subgroups analyzed by the log-rank test. Cox proportional hazards model was used to identify independent prognostic factors age, number of metastasis, and treatment modality associated with survival.
The median interval from ovarian cancer diagnosis to brain metastasis in 104 identified patients was 19.5 months. Brain metastasis was single in 43%, multiple in 41%, and not reported in 16% of cases. About 81.7% of patients were treated for their brain metastases using external radiation therapy (XRT), chemotherapy, and surgery. XRT was utilized in 76% of 104 patients and in 93% of treated patients. The most commonly used modalities were XRT alone (40%) and craniotomy and XRT (17%). The median survival (MS) for all patients regardless of treatment type was 6 months. Patients who received any treatment lived longer than those not receiving surgery/chemotherapy/XRT (MS; 7 months vs. 2 months, P = 0.0001). Patients with single brain metastasis had a longer median survival (21 months vs. 6 months, P = 0.049) when treated with craniotomy plus radiation and/or chemotherapy compared to treatment regimens that excluded craniotomy. In a multivariate analysis, only treatment type was significant in predicting survival.
OBM portends a poor prognosis, however, long-term survival is possible. Patients appear to benefit from therapy, especially selected groups of OBM patients with single brain metastasis treated with radiation therapy and surgery.
No preview · Article · Nov 2001 · Journal of Surgical Oncology
[Show abstract][Hide abstract] ABSTRACT: Retinoic acid analogues, called retinoids, have shown promise in clinical trials in preventing breast and ovarian cancers. Classic retinoids bind to retinoic acid receptors, which regulate cell growth. Some novel retinoids, such as fenretinide, i.e., N-(4-hydroxyphenyl)retinamide (4-HPR), induce apoptosis through retinoic acid receptor-independent mechanisms; however, they appear to do so only at concentrations above those achieved in clinical chemoprevention trials. At lower concentrations (< or =1 microM), 4-HPR acts like classic retinoids, by inducing differentiation through a receptor-dependent mechanism. Our goal was to compare the effects of novel receptor-independent (apoptotic) retinoids with those of classic growth-inhibitory retinoids at clinically achievable doses on growth, differentiation, and apoptosis in ovarian tissue.
Four receptor-independent (apoptotic) and seven growth-inhibitory retinoids, including synthetic, low-toxicity compounds called heteroarotinoids, were administered at concentrations of 1 microM to organotypic cultures of ovarian primary and cancer cell lines: OVCAR-3, Caov-3, and SK-OV-3. After fixation, embedding, and sectioning, the growth fraction was quantified by measuring expression of the proliferation marker Ki-67/myb, differentiation was assessed by expression of mucin, and apoptosis was evaluated by the TUNEL assay. Spearman correlation analysis was performed on the data, and all P values were two-sided.
All 11 retinoids reversed characteristics associated with the cancerous phenotype in all neoplastic cultures. Glandular structures were observed consistently in retinoid-treated, but not in untreated, OVCAR-3 and Caov-3 cultures. All retinoids decreased growth fractions, and some increased mucin expression. All receptor-independent retinoids and two receptor-dependent retinoids induced apoptosis, and the induction correlated significantly with increased expression of the mucin MUC1 (r =.83; P =.03). Retinoids with ester-linking groups did not induce apoptosis but decreased the growth fraction in correlation with MUC1 induction (r = -.93; P =.02).
At clinically achievable concentrations, all retinoids tested decrease the growth fraction, induce differentiation and apoptosis. Induction of MUC1 expression is implicated in the mechanisms of action.
Full-text · Article · Apr 2001 · JNCI Journal of the National Cancer Institute
[Show abstract][Hide abstract] ABSTRACT: We have previously demonstrated that important regulatory elements responsible for regulated expression of the human GLUT4 promoter are located between -1154 and -412 relative to transcription initiation (Olson, A. L., and Pessin, J. E. (1995) J. Biol. Chem. 270, 23491-23495). Through further analysis of this promoter regulatory region, we have identified a perfectly conserved myocyte enhancer factor 2 (MEF2)-binding domain (-CTAAAAATAG-) that is necessary, but not sufficient, to support tissue-specific expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice. Biochemical analysis of this DNA element demonstrated the formation of a specific DNA-protein complex using nuclear extracts isolated from heart, hindquarter skeletal muscle, and adipose tissue but not from liver. DNA binding studies indicated that this element functionally interacted with the MEF2A and/or MEF2C MADS family of DNA binding transcription factors. MEF2 DNA binding activity was substantially reduced in nuclear extracts isolated from both heart and skeletal muscle of diabetic mice, which correlated with decreased transcription rate of the GLUT4 gene. MEF2 binding activity completely recovered to control levels following insulin treatment. Together these data demonstrated that MEF2 binding activity is necessary for regulation of the GLUT4 gene promoter in muscle and adipose tissue.
[Show abstract][Hide abstract] ABSTRACT: We have previously demonstrated that important regulatory elements responsible for regulated expression of the humanGLUT4 promoter are located between −1154 and −412 relative to transcription initiation (Olson, A. L., and Pessin, J. E. (1995)
J. Biol. Chem. 270, 23491–23495). Through further analysis of this promoter regulatory region, we have identified a perfectly conserved
myocyte enhancer factor 2 (MEF2)-binding domain (-CTAAAAATAG-) that is necessary, but not sufficient, to support tissue-specific
expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice. Biochemical analysis of this DNA element
demonstrated the formation of a specific DNA-protein complex using nuclear extracts isolated from heart, hindquarter skeletal
muscle, and adipose tissue but not from liver. DNA binding studies indicated that this element functionally interacted with
the MEF2A and/or MEF2C MADS family of DNA binding transcription factors. MEF2 DNA binding activity was substantially reduced
in nuclear extracts isolated from both heart and skeletal muscle of diabetic mice, which correlated with decreased transcription
rate of theGLUT4 gene. MEF2 binding activity completely recovered to control levels following insulin treatment. Together these data demonstrated
that MEF2 binding activity is necessary for regulation of the GLUT4 gene promoter in muscle and adipose tissue.
No preview · Article · Jun 1998 · Journal of Biological Chemistry