Iqbal Unnisa Ali

Progress in cancer chemoprevention.

ABSTRACT More than 40 promising agents and agent combinations are being evaluated clinically as chemopreventive drugs for major cancer targets. A few have been in vanguard, large‐scale intervention trials‐for example, the studies of tamoxifen and fenretinide in breast, 13‐cis‐retinoic acid in head and neck, vitamin E and selenium in prostate, and calcium in colon. These and other agents are currently in phase II chemoprevention trials to establish the scope of their chemopreventive efficacy and to develop intermediate biomarkers as surrogate end points for cancer incidence in future studies. In this group are fenretinide, 2‐difluoromethylornithine, and oltipraz. Nonsteroidal anti‐inflammatories (NSAID) are also in this group because of their colon cancer chemopreventive effects in clinical intervention, epidemiological, and animal studies. New agents are continually considered for development as chemopreventive drugs. Preventive strategies with antiandrogens are evolving for prostate cancer. Anti‐inflammatories that selectively inhibit inducible cyclooxygenase (COX)‐2 are being investigated in colon as alternatives to the NSAID, which inhibit both COX‐1 and COX‐2 and derive their toxicity from COX‐1 inhibition. Newer retinoids with reduced toxicity, increased efficacy, or both (e.g., 9‐cis‐retinoic acid) are being investigated. Promising chemopreventive drugs are also being developed from dietary substances (e.g., green and black tea polyphenols, soy isoflavones, curcumin, phenethyl isothiocyanate, sulforaphane, lycopene, indole‐3‐carbinol, perillyl alcohol). Basic and translational research necessary to progress in chemopreventive agent development includes, for example, (1) molecular and genomic biomarkers that can be used for risk assessment and as surrogate end points in clinical studies, (2) animal carcinogenesis models that mimic human disease (including transgenic and gene knockout mice), and (3) novel agent treatment regimens (e.g., local delivery to cancer targets, agent combinations, and pharmacodynamically guided dosing).

Molecular Profiling of Angiogenesis Markers

The goal of this study was to develop a sensitive, simple, and widely applicable assay to measure copy numbers of specific mRNAs using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and identify a profile of gene expression closely associated with angiogenesis. We measured a panel of nine potential angiogenesis markers from a mouse transgenic model of prostate adenocarcinoma (TRAMP) and a mouse skin model of vascular endothelial growth factor (VEGF)-driven angiogenesis. In both models, expression of VEGF correlated with expression of mRNAs encoding other angiogenic cytokines (angiopoietin-1 and angiopoietin-2), endothelial cell receptor tyrosine kinases (Flt-1, KDR, Tie-1), and endothelial cell adhesion molecules (VE-cadherin, PECAM-1). Relative to control, in dermis highly stimulated by VEGF, the Ang-2 mRNA transcript numbers increased 35-fold, PECAM-1 and VE-cadherin increased 10-fold, Tie-1 increased 8-fold, KDR and Flt-1 each increased 4-fold, and Ang-1 increased 2-fold. All transcript numbers were correspondingly reduced in skin with less VEGF expression, indicating a relationship of each of these seven markers with VEGF. Thus, this study identifies a highly efficient method for precise quantification of a panel of seven specific mRNAs that correlate with VEGF expression and VEGF-induced neovascularization, and it provides evidence that real-time quantitative RT-PCR offers a highly sensitive strategy for monitoring angiogenesis.

Inhibition of the mammary carcinoma angiogenic switch in C3(1)/SV40 transgenic mice by a mutated form of human endostatin

Cancer therapies based on the inhibition of angiogenesis by endostatin have recently been developed. We demonstrate that a mutated form of human endostatin (P125A) can inhibit the angiogenic switch in the C3(1)/Tag mammary cancer model. P125A has a stronger growth‐inhibitory effect on endothelial cell proliferation than wild‐type endostatin. We characterize the angiogenic switch, which occurs during the transition from preinvasive lesions to invasive carcinoma in this model, and which is accompanied by a significant increase in total protein levels of vascular endothelial growth factor (VEGF) and an invasion of blood vessels. Expression of the VEGF188 mRNA isoform, however, is suppressed in invasive carcinomas. The VEGF receptors fetal liver kinase‐1 (Flk‐1) and Fms‐like tyrosine kinase‐1 (Flt‐1) become highly expressed in epithelial tumor and endothelial cells in the mammary carcinomas, suggesting a potential autocrine effect for VEGF on tumor cell growth. Angiopoietin‐2 mRNA levels are also increased during tumor progression. CD‐31 (platelet‐endothelial cell adhesion molecule [PECAM]) staining revealed that blood vessels developed in tumors larger than 1 mm The administration of P125A human endostatin in C3(1)/Tag females resulted in a significant delay in tumor onset, decreased tumor multiplicity and tumor burden and prolonged survival of the animals. Endostatin treatment did not reduce the number of preinvasive lesions, proliferation rates or apoptotic index, compared with controls. However, mRNA levels of a variety of proangiogenic factors (VEGF, VEGF receptors Flk‐1 and Flt‐1, angiopoietin‐2, Tie‐1, cadherin‐5 and PECAM) were significantly decreased in the endostatin‐treated group compared with controls. These results demonstrate that P125A endostatin inhibits the angiogenic switch during mammary gland adenocarcinoma tumor progression in the C3(1)/Tag transgenic model.

Effect of a 4-month tea intervention on oxidative DNA damage among heavy smokers: role of glutathione S-transferase genotypes.

Glutathione S-transferase (GST), a member of the phase II group of xenobiotic metabolizing enzymes, has been intensively studied at the levels of phenotype and genotype. The GST μ 1 (GSTM1) and GST θ 1 (GSTT1) genes have a null-allele variant in which the entire gene is absent. The null genotype for both enzymes has been associated with many different types of tumors. The aim of this study was to determine the possible differences in increased oxidative stress susceptibility to smoking within the GSTM1 and GSTT1 genotypes and the impact of high tea drinking on this. We designed a Phase II randomized, controlled, three-arm tea intervention trial to study the effect of high consumption (4 cups/day) of decaffeinated green or black tea, or water on oxidative DNA damage, as measured by urinary 8-hydroxydeoxyguanosine (8-OHdG), among heavy smokers over a 4-month period and to evaluate the roles of GSTM1 and GSTT1 genotypes as effect modifiers. A total of 133 heavy smokers (100 females and 33 males) completed the intervention. GSTM1 and GSTT1 genotype statuses were determined with a PCR-based approach. Multiple linear regression models were used to estimate the main effects and interaction effect of green and black tea consumption on creatinine-adjusted urinary 8-OHdG, with or without adjustment for potential confounders. Finally, we studied whether the effect of treatment varied by GSTM1 and GSTT1 status of the individual. Although there were no differences in urinary 8-OHdG between the groups at baseline, the between-group 8-OHdG levels at month 4 were statistically significant for GSTM1-positive smokers (P = 0.05) and GSTT1-positive smokers (P = 0.02). GSTM1-positive and GSTT1-positive smokers consuming green tea showed a decrease in urinary 8-OHdG levels after 4 months. Assessment of urinary 8-OHdG after adjustment for baseline measurements and other potential confounders revealed significant effect for green tea consumption (P = 0.001). The change from baseline was significant in both GSTM1-positive (t = −2.99; P = 0.006) and GSTT1-positive (P = 0.004) green tea groups, but not in the GSTM1-negative (P = 0.07) or GSTT1-negative (P = 0.909) green tea groups. Decaffeinated black tea consumption had no effect on urinary 8-OHdG levels among heavy smokers. Our data show that consumption of 4 cups of tea/day is a feasible and safe approach and is associated with a significant decrease in urinary 8-OHdG among green tea consumers after 4 months of consumption. This finding also suggests that green tea intervention may be effective in the subgroup of smokers who are GSTM1 and/or GSTT1 positive.

Cyclooxygenase-2 Polymorphisms, Aspirin Treatment, and Risk for Colorectal Adenoma Recurrence—Data from a Randomized Clinical Trial

Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the production of prostaglandins, potent mediators of inflammation. Chronic inflammation plays an important role in the development and progression of colorectal cancer. Aspirin inhibits COX-2 activity and lowers the risk for colorectal adenomas and cancer. We investigated whether common genetic variation in COX-2 influenced risk for colorectal adenoma recurrence among 979 participants in the Aspirin/Folate Polyp Prevention Study who were randomly assigned to placebo or aspirin and followed for 3 years for the occurrence of new adenomas. Of these participants, 44.2% developed at least one new adenoma during follow-up. Adjusted relative risks and 95% confidence intervals (95% CI) were calculated to test the association between genetic variation at six COX-2 single-nucleotide polymorphisms and adenoma occurrence and interaction with aspirin treatment. Two single-nucleotide polymorphisms were significantly associated with increased adenoma recurrence: for rs5277, homozygous carriers of the minor C allele had a 51% increased risk compared with GG homozygotes (relative risk, 1.51; 95% CI, 1.01-2.25), and for rs4648310, heterozygous carriers of the minor G allele had a 37% increased risk compared with AA homozygotes (relative risk, 1.37; 95% CI, 1.05-1.79). (There were no minor allele homozygotes.) In stratified analyses, there was suggestive evidence that rs4648319 modified the effect of aspirin. These results support the hypothesis that COX-2 plays a role in the etiology of colon cancer and may be a target for aspirin chemoprevention and warrant further investigation in other colorectal adenoma and cancer populations.(Cancer Epidemiol Biomarkers Prev 2009;18(10):2726–33)

Proteomic Profiling Identifies Cyclooxygenase-2-Independent Global Proteomic Changes by Celecoxib in Colorectal Cancer Cells

Celecoxib, a selective inhibitor of the enzyme cyclooxygenase-2 (COX-2), has been shown to be a promising chemoprevention agent. The chemopreventive efficacy of celecoxib is believed to be a consequence of its COX-2-dependent and COX-2-independent effects on a variety of cellular processes including proliferation, apoptosis, angiogenesis, and immunosurveillance. In an attempt to identify proteomic markers modulated by celecoxib that are independent of its inhibitory effect on COX-2, the colorectal cancer cell line HCT-116, a nonexpresser of COX-2, was treated with celecoxib. We used the powerful, state-of-the-art two-dimensional difference gel electrophoresis technology coupled with mass spectrometric sequencing to compare global proteomic profiles of HCT-116 cells before and after treatment with celecoxib. Among the differentially expressed proteins identified following celecoxib treatment were proteins involved in diverse cellular functions including glycolysis, protein biosynthesis, DNA synthesis, mRNA processing, protein folding, phosphorylation, redox regulation, and molecular chaperon activities. Our study presents a comprehensive analysis of large-scale celecoxib-modulated proteomic alterations, at least some of which may be mechanistically related to the COX-2-independent chemopreventive effect of celecoxib. (Cancer Epidemiol Biomarkers Prev 2006;15(9):1598–606)

Comparative molecular genetic profiles of anaplastic astrocytomas/glioblastomas multiforme and their subsequent recurrences.

Malignant glial tumors (anaplastic astrocytomas and glioblastomas multiforme) arise mostly either from the progression of low grade precursor lesions or rapidly in a de novo fashion and contain distinct genetic alterations. There is, however, a third subset of malignant gliomas in which genetic lesions remain to be identified. Following surgical resection, all gliomas appear to have an inherent tendency to recur. Comparative molecular analysis of ten primary malignant gliomas (three anaplastic astrocytomas and seven glioblastomas multiforme) with their recurrences identified two distinct subgroups of recurrent tumors. In one group, primary tumors harbored genetic aberrations frequently associated with linear progression or de novo formation pathways of glial tumorigenesis and maintained their genetic profiles upon recurrence. In the other subset with no detectable known genetic mutations at first presentation, the recurrent tumors sustained specific abnormalities associated with pathways of linear progression or de novo formation. These included loss of genes on chromosomes 17 and 10, mutations in the p53 gene, homozygous deletion of the DMBTA1 and p16 and/or p15 genes and amplification and/or overexpression of CDK4 and alpha form of the PDGF receptor. Recurrent tumors from both groups also displayed an abnormal expression profile of the metalloproteinase, gel A, and its inhibitor, TIMP-2, consistent with their highly invasive behavior. Delineation of the molecular differences between malignant glioblastomas and their subsequent recurrences may have important implications for the development of rational clinical approaches for this neoplasm that remains refractory to existing therapeutic modalities.

Vitamins B2, B6, and B12 and Risk of New Colorectal Adenomas in a Randomized Trial of Aspirin Use and Folic Acid Supplementation

Background: Folate, other vitamin B cofactors, and genes involved in folate-mediated one-carbon metabolism all may play important roles in colorectal neoplasia. In this study, we examined the associations between dietary and circulating plasma levels of vitamins B2, B6, and B12 and risk colorectal adenomas. Methods: The Aspirin/Folate Polyp Prevention Study is a randomized clinical trial of folic acid supplementation and incidence of new colorectal adenomas in individuals with a history of adenomas (n = 1,084). Diet and supplement use were ascertained through a food frequency questionnaire administered at baseline. Blood collected at baseline was used to determine plasma B-vitamin levels. We used generalized linear regression to estimate risk ratios (RR) and 95% confidence intervals (95% CI) as measures of association. Results: We found a borderline significant inverse association with plasma B6 [pyridoxal 5′-phosphate (PLP)] and adenoma risk (adjusted RR Q4 versus Q1, 0.78; 95% CI, 0.61-1.00; Ptrend = 0.08). This association was not modified by folic acid supplementation or plasma folate. However, the protective association of PLP with adenoma risk was observed only among subjects who did not drink alcohol (Pinteraction = 0.03). Plasma B2 (riboflavin) was inversely associated with risk of advanced lesions (adjusted RR Q4 versus Q1, 0.51; 95% CI, 0.26-0.99; Ptrend = 0.12). No significant associations were observed between adenoma risk and plasma vitamin B12 or dietary intake of vitamin B2 and B6. When we examined specific gene-B-vitamin interactions, we observed a possible interaction between methylenetetrahydrofolate reductase -C677T and plasma B2 on risk of all adenomas. Conclusion: Our results suggest that high levels of PLP and B2 may protect against colorectal adenomas. (Cancer Epidemiol Biomarkers Prev 2008;17(8):2136–45)

Angiogenesis as a potential biomarker in prostate cancer chemoprevention trials.

Prostate cancer is a multistep process in which progression rather than initiation may be the rate-limiting step. A strong possibility is that prostatic intraepithelial neoplasia lesions that switch to angiogenic phenotype eventually progress to cancer. However, it is a challenging task to quantitate angiogenesis in preneoplastic lesions. A promising approach to measuring angiogenesis involves real-time TaqMan polymerase chain reaction to quantitate mRNAs encoding a panel of angiogenesis markers. This highly sensitive molecular technique has potential for quantitating angiogenesis in clinical settings and can be used as a high-throughput screening procedure in prostate cancer clinical trials.

Heterogeneity of genetic alterations in primary human breast tumors.

The etiology of human breast cancer is thought to involve a complex interplay among genetic, hormonal, and dietary factors that are superimposed on the physiological status of the host [1, 2]. As a consequence, several different types of breast tumors can be distinguished by histopathological criteria, chromosomal abnormalities, hormone receptor status, and other biochemical characteristics. However, attempts to derive a cohesive picture of how the various factors participate in the etiology of breast cancer have been confounded by a lack of information on specific genetic mutations associated with the initiation or progression of the disease. Three general approaches have been used to attempt to identify genetic mutations associated with breast cancer.