Madhuri Kakarala

Implications of the Cancer Stem-Cell Hypothesis for Breast Cancer Prevention and Therapy

Recent research in breast biology has provided support for the cancer stem-cell hypothesis. Two important components of this hypothesis are that tumors originate in mammary stem or progenitor cells as a result of dysregulation of the normally tightly regulated process of self-renewal. As a result, tumors contain and are driven by a cellular subcomponent that retains key stem-cell properties including self-renewal, which drives tumorigenesis and differentiation that contributes to cellular heterogeneity. Advances in stem-cell technology have led to the identification of stem cells in normal and malignant breast tissue. The study of these stem cells has helped to elucidate the origin of the molecular complexity of human breast cancer. The cancer stem-cell hypothesis has important implications for early detection, prevention, and treatment of breast cancer. Both hereditary and sporadic breast cancers may develop through dysregulation of stem-cell self-renewal pathways. These aberrant stem cells may provide targets for the development of cancer prevention strategies. Furthermore, because breast cancer stem cells may be highly resistant to radiation and chemotherapy, the development of more effective therapies for this disease may require the effective targeting of this cell population.

Phase IIA Clinical Trial of Curcumin for the Prevention of Colorectal Neoplasia

Curcumin is derived from the spice tumeric and has antiinflammatory and antineoplastic effects in vitro and in animal models, including preventing aberrant crypt foci (ACF) and adenomas in murine models of colorectal carcinogenesis. Inhibiting the production of the procarcinogenic eicosanoids prostaglandin E2 (PGE2) and 5-hydroxyeicosatetraenoic acid (5-HETE) can suppress carcinogenesis in rodents. Curcumin reduces mucosal concentrations of PGE2 (via inhibition of cyclooxygenases 1 and 2) and 5-HETE (via inhibition of 5-lipoxygenase) in rats. Although preclinical data support curcumin activity in many sites, the poor bioavailability reported for this agent supports its use in the colorectum. We assessed the effects of oral curcumin (2 g or 4 g per day for 30 days) on PGE2 within ACF (primary endpoint), 5-HETE, ACF number, and proliferation in a nonrandomized, open-label clinical trial in 44 eligible smokers with eight or more ACF on screening colonoscopy. We assessed pre- and posttreatment concentrations of PGE2 and 5-HETE by liquid chromatography tandem mass spectroscopy in ACF and normal-tissue biopsies; ACF number via rectal endoscopy; proliferation by Ki-67 immunohistochemistry; and curcumin concentrations by high-performance liquid chromatography in serum and rectal mucosal samples. Forty-one subjects completed the study. Neither dose of curcumin reduced PGE2 or 5-HETE within ACF or normal mucosa or reduced Ki-67 in normal mucosa. A significant 40% reduction in ACF number occurred with the 4-g dose (P < 0.005), whereas ACF were not reduced in the 2-g group. The ACF reduction in the 4-g group was associated with a significant, five-fold increase in posttreatment plasma curcumin/conjugate levels (versus pretreatment; P = 0.009). Curcumin was well tolerated at both 2 g and 4 g. Our data suggest that curcumin can decrease ACF number, and this is potentially mediated by curcumin conjugates delivered systemically. Cancer Prev Res; 4(3); 354–64. ©2011 AACR.

Pharmacokinetics of Curcumin Conjugate Metabolites in Healthy Human Subjects

Background: Curcumin is a polyphenol, found in the spice turmeric, that has promising anticancer properties, but previous studies suggest that absorption of curcumin may be limited. Methods: This study examined the pharmacokinetics of a curcumin preparation in healthy human volunteers 0.25 to 72 h after a single oral dose. Curcumin was administered at doses of 10 g (n = 6) and 12 g (n = 6). Subjects were randomly allocated to dose level for a total of six subjects at each dose level. Serum samples were assayed for free curcumin, for its glucuronide, and for its sulfate conjugate. The data were fit to a one-compartment absorption and elimination model. Results: Using a high-performance liquid chromatography assay with a limit of detection of 50 ng/mL, only one subject had detectable free curcumin at any of the 14 time points assayed, but curcumin glucuronides and sulfates were detected in all subjects. Based on the pharmacokinetic model, the area under the curve for the 10 and 12 g doses was estimated (mean ± SE) to be 35.33 ± 3.78 and 26.57 ± 2.97 μg/mL × h, respectively, whereas Cmax was 2.30 ± 0.26 and 1.73 ± 0.19 μg/mL. The Tmax and t1/2 were estimated to be 3.29 ± 0.43 and 6.77 ± 0.83 h. The ratio of glucuronide to sulfate was 1.92:1. The curcumin conjugates were present as either glucuronide or sulfate, not mixed conjugates. Conclusion: Curcumin is absorbed after oral dosing in humans and can be detected as glucuronide and sulfate conjugates in plasma. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1411–7)

American Society of Clinical Oncology Position Statement on Obesity and Cancer

Rates of obesity have increased significantly over the last three decades in the United States and globally. In addition to contributing to heart disease and diabetes, obesity is a major unrecognized risk factor for cancer. Obesity is associated with worsened prognosis after cancer diagnosis and also negatively affects the delivery of systemic therapy, contributes to morbidity of cancer treatment, and may raise the risk of second malignancies and comorbidities. Research shows that the time after a cancer diagnosis can serve as a teachable moment to motivate individuals to adopt risk-reducing behaviors. For this reason, the oncology care team--the providers with whom a patient has the closest relationships in the critical period after a cancer diagnosis--is in a unique position to help patients lose weight and make other healthy lifestyle changes. The American Society of Clinical Oncology is committed to reducing the impact of obesity on cancer and has established a multipronged initiative to accomplish this goal by 1) increasing education and awareness of the evidence linking obesity and cancer; 2) providing tools and resources to help oncology providers address obesity with their patients; 3) building and fostering a robust research agenda to better understand the pathophysiology of energy balance alterations, evaluate the impact of behavior change on cancer outcomes, and determine the best methods to help cancer survivors make effective and useful changes in lifestyle behaviors; and 4) advocating for policy and systems change to address societal factors contributing to obesity and improve access to weight management services for patients with cancer.

Obesity, energy balance, and cancer: New opportunities for prevention

Obesity is associated with increased risk and poor prognosis for many types of cancer. The mechanisms underlying the obesity-cancer link are becoming increasingly clear and provide multiple opportunities for primary to tertiary prevention. Several obesity-related host factors can influence tumor initiation, progression and/or response to therapy, and these have been implicated as key contributors to the complex effects of obesity on cancer incidence and outcomes. These host factors include insulin, insulin-like growth factor-I, leptin, adiponectin, steroid hormones, cytokines, and inflammation-related molecules. Each of these host factors is considered in the context of energy balance and as potential targets for cancer prevention. The possibility of prevention at the systems level, including energy restriction, dietary composition, and exercise is considered as is the importance of the newly emerging field of stem cell research as a model for studying energy balance and cancer prevention. Cancer Prev Res; 5(11); 1260–72. ©2012 AACR.

Schoolchildren's consumption of competitive foods and beverages, excluding à la carte.

BACKGROUND Competitive foods/beverages are those in school vending machines, school stores, snack bars, special sales, and items sold à la carte in the school cafeteria that compete with United States Department of Agriculture (USDA) meal program offerings. Grouping à la carte items with less nutritious items allowed in less regulated venues may obfuscate analysis of the school competitive food environment. Excluding à la carte items from competitive foods, the objectives were to: (1) assess competitive food use by gender, ethnicity, eligibility for free or reduced-price meals, and participation in school meals programs, (2) determine differences between grade levels in energy intakes obtained from food sources, (3) determine the nutrient intake derived from competitive foods for students who consumed them, and (4) determine energy-adjusted differences in 24-hour nutrient intakes of protein, calcium, iron, and other selected nutrients between competitive food consumer and nonconsumers. METHODS Competitive foods/beverages use, excluding à la carte items, was examined using the third School Nutrition Dietary Assessment Study (SNDA III), a nationally representative sample of 2309 schoolchildren in grades 1 to 12. Mean nutrient intakes were adjusted for energy intake and other covariates, and differences between consumers and nonconsumers of competitive items were determined using analysis of variance and sudaan. RESULTS Excluding à la carte items, 22% of schoolchildren consumed competitive items in a representative school day and use was highest in high school. Consumers of competitive items other than à la carte had significantly higher mean energy, sugar intakes, and lower sodium, dietary fiber, B vitamins, and iron intakes than nonconsumers. CONCLUSIONS Use of competitive foods/beverages, excluding à la carte, was detrimental to childrens diet quality.

Ultra-low Flow Liquid Chromatography Assay with Ultraviolet (UV) Detection for Piperine Quantitation in Human Plasma

A robust and sensitive ultra-low flow liquid chromatography (UFLC) method that can reproducibly, at reasonable cost, detect low concentrations of piperine from human plasma is necessary. Piperine in plasma was separated and quantified by a gradient method using ultraviolet detection at a maximal absorbance wavelength of 340 nm. An aliquot was injected onto a reversed-phase column Waters SymmetryShield, 2.1 x 100 mm, 3.5 microm, C(18) column, attached to a Waters absorbosphere, 4.6 x 30 mm, C(18) guard column and eluted with a mobile phase containing a mixture of acetonitrile/water/acetic acid (25:74.9:0.1, v/v/v) on line A and acetonitrile/acetic acid (99.9:0.1, v/v) on line B. The flow rate was 0.3 mL/min. The gradient method consisted of an opening condition of 20% pump B, with a linear increase to 37% pump B over 8 min, then a linear increase to 100% pump B at 11 min, 2 min at 100% pump B, and then a return to the opening condition (20% pump B) via a linear gradient over 2 min, followed by 5 min re-equilibration at opening conditions. The total run time was 20 min for each sample. All samples were processed protected from ambient light to avoid isomerization of piperine. The plasma assay was linear with R = 0.9995, with a lower limit of detection [signal-to-noise (S/N) > 5:1] of 100 pg of piperine loaded into the analytical system with acceptable accuracy and precision. Extraction recoveries of piperine from human plasma were 88% for quality control high (QCH), 93% for quality control medium (QCM), and 90% for quality control low (QCL), and the matrix effect was <12%. Piperine was quantifiable from a 50 mg oral dose given to human volunteers. A UFLC method for the rapid assay of human plasma with sensitivity to detect as low as 5 ng/mL piperine was developed. The method sensitivity equals that of liquid chromatography/tandem mass spectrometry (LC/MSMS) methods with much less cost.

Clinical Trial Design for Testing the Stem Cell Model for the Prevention and Treatment of Cancer

The cancer stem cell model introduces new strategies for the prevention and treatment of cancers. In cancers that appear to follow the stem cell model, pathways such as Wnt, Notch and Hedgehog may be targeted with natural compounds such as curcumin or drugs to reduce the risk of initiation of new tumors. Disease progression of established tumors could also potentially be inhibited by targeting the tumorigenic stem cells alone, rather than aiming to reduce overall tumor size. These new approaches mandate a change in the design of clinical trials and biomarkers chosen for efficacy assessment for preventative, neoadjuvant, adjuvant, and palliative treatments. Cancer treatments could be evaluated by assessing stem cell markers before and after treatment. Targeted stem cell specific treatment of cancers may not result in “complete” or “partial” responses radiologically, as stem cell targeting may not reduce the tumor bulk, but eliminate further tumorigenic potential. These changes are discussed using breast, pancreatic, and lung cancer as examples.

Abstract 1286: Pharmacodynamics of oral curcumin in plasma and rectal mucosa in a Phase IIa trial

Background: Curcumin, a dietary polyphenol derivative of turmeric, has potent cancer reductive activity in in vivo colon carcinogenesis models. We administered curcumin at doses of 2 g or 4 g to separate groups of 20 healthy human smokers with > 8 aberrant crypt foci (ACF). The 2 g dose showed no ACF reduction while the 4 g dose showed a 46% reduction (p Methods: Curcuminoid products were extracted from homogenized biopsy tissue and ultacentrifuged plasma and evaporated under argon. The separation was performed on a Symmetry ® C 18 column. The mobile phase used under gradient conditions was 25% acetonitrile with 74.9% containing 0.1% acetic acid (25:74.9:0.1; v/v/v; A) and 100% acetonitrile containing 0.1% acetic acid (99.9:0.1; v/v; B) The flow rate was 0.3 mL/min and injection volume for all samples was 10 µL. The lower limit of detection for curcumin from human plasma was 5 ng/mL and from human colonic mucosa was 50 ng/5mg tissue. Results: See Table below (summarized by pre and post treatment dose for plasma and mucosal concentrations) Conclusions: Native curcumin is not detectable in the majority of biopsy and plasma samples. Conjugated curcumin is detectable in all plasma samples and the majority of mucosal biopsies with a significant increase in plasma concentrations at 4g. We postulate the conjugates detected in the rectal mucosa are delivered systemically from absorption and conjugation higher in the gastrointestinal tract and may have biologic activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1286. doi:10.1158/1538-7445.AM2011-1286

Abstract 1842: Vitamin D compounds induce BRCA1 expression and inhibit breast stem cells

Background: Basal breast cancer which occurs in women with BRCA1 mutations has been suggested to arise from arrested differentiation of stem/luminal progenitor cells, whose gene profile includes Vitamin D receptor prominently. In addition to DNA repair, BRCA1 enhances luminal differentiation of breast stem cells. Vitamin D compounds have long been known to have differentiating function. We hypothesized that Vitamin D receptor exists on stem cells and that vitamin D treatment induces differentiation of stem/progenitor cells, in part due to increased BRCA1 expression. Methods: We treated normal human breast epithelial cells and MCF-7 breast cancer cells with 10 or 100 nM 1, 25 dihydroxyvitamin D3 or the less hypercalcemic vitamin D5 analogue (1alpha(OH)D5) in suspension culture in stem cell media to assess: 1) stem cell self renewal using the mammosphere assay; 2) inhibition of stem cell percentage; 3) induction of BRCA1 mRNA and 4) induction of BRCA1 protein. Results: Teatment with 100nM 1alpha(OH)D5 inhibited stem cell self renewal as assessed by mammosphere formation assay from 23spheres/high power field (HPF) to 1 per HPF at 7 days; inhibited of percentage of stem cells from 17% in vehicle control to 7% with vitamin D5. Both vitamin D3 and D5 caused an 8 fold induction of vitamin D target CYP24 gene expression; 4 fold increase in BRCA1 mRNA by real time PCR; and 4 fold increase in BRCA1 protein expression by western blot compared to actin control. Conclusion: Vitamin D3 and D5 analogue induce BRCA1 expression and inhibit stem cell percentage in normal breast cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1842. doi:10.1158/1538-7445.AM2011-1842