Akash Gupta

Antioxidant and Antiproliferative Activities of Anthocyanin/Ellagitannin-Enriched Extracts From Syzygium cumini L. (Jamun, the Indian Blackberry)

Colored fruits, particularly berries, are highly chemoprotective because of their antioxidant, antiproliferative, and antiinflammatory activities. We report the cancer chemoprotective potential of Syzygium cumini L., commonly known as jamun or Indian blackberry. Anthocyanins and other polyphenolics were extracted with acidic ethanol and enriched by amberlite XAD7/HP20 (1:1). The pulp powder was found to contain 0.54% anthocyanins, 0.17% ellagic acid/ellagitannins, and 1.15% total polyphenolics. Jamun seed contained no detectable anthocyanins but had higher amounts of ellagic acid/ellagitannins (0.5%) and total polyphenolics (2.7%) than the pulp powder. Upon acid hydrolysis, the pulp extract yielded 5 anthocyanidins by HPLC: malvidin (44.4%), petunidin (24.2%), delphinidin (20.3%), cyanidin (6.6%), and peonidin (2.2%). Extracts of both jamun pulp (1,445 ± 64 μmol of trolox equivalent (TE)/g) and seeds (3,379 ± 151 μM of TE/g) showed high oxygen radical absorbance capacity. Their high antioxidant potential was also reflected by 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)- and 2,2-diphenyl-1-picrylhydrazyl-scavenging and ferrous ion-chelating activities. We also analyzed antiproliferative activity of jamun extracts against human lung cancer A549 cells. The hydrolyzed pulp and seed extracts showed significant antiproliferative activity. However, unhydrolyzed extracts showed much less activity. These data showed that in addition to 5 anthocyanidins, jamun contains appreciable amounts of ellagic acid/ellagitannins, with high antioxidant and antiproliferative activities.

Mathematical Analysis of the Normal Anatomy of the Aging Fovea

PURPOSE To mathematically analyze anatomical changes that occur in the normal fovea during aging. METHODS A total of 2912 spectral-domain optical coherence tomography (SD-OCT) normal foveal scans were analyzed. Subjects were healthy individuals, aged 13 to 97 years, with visual acuity ≥20/40 and without evidence of foveal pathology. Using automated symbolic regression software Eureqa (version 0.98), foveal thickness maps of 390 eyes were analyzed using several measurements: parafoveal retinal thickness at 50 μm consecutive intervals, parafoveal maximum retinal thickness at two points lateral to central foveal depression, distance between two points of maximum retinal thickness, maximal foveal slope at two intervals lateral to central foveal depression, and central length of foveal depression. A unique mathematical equation representing the mathematical analog of foveal anatomy was derived for every decade, between 10 and 100 years. RESULTS The mathematical regression function for normal fovea followed first order sine curve of level 10 complexity for the second decade of life. The mathematical regression function became more complex with normal aging, up to level 43 complexity (0.085 fit; P < 0.05). Young foveas had higher symmetry (0.92 ± 0.10) along midline, whereas aged foveas had significantly less symmetry (0.76 ± 0.27, P < 0.01) along midline and steeper maximal slopes (29 ± 32°, P < 0.01). CONCLUSIONS Normal foveal anatomical configuration changes with age. Normal aged foveas are less symmetric along midline with steeper slopes. Differentiating between normal aging and pathologic changes using SD-OCT scans may allow early diagnosis, follow-up, and better management of the aging population.

VITREOMACULAR ADHESION EVOLUTION WITH AGE IN HEALTHY HUMAN EYES.

Purpose: To quantify the changes in vitreomacular interactions that occur with normal aging in normal eyes. Methods: Spectral domain optical coherence tomography (SD-OCT, Spectralis; Heidelberg Engineering, Heidelberg, Germany) foveal scans of subjects with best corrected visual acuity better than 20/40 and no ocular pathology were included in the study. Each scan was analyzed to determine the status of vitreoretinal interface: complete vitreous adhesion, partial posterior vitreous detachment (PVD) with persistent vitreomacular adhesion (VMA), or complete PVD. Area of VMA was delineated using the Spectralis drawing tool and calculated in mm2 for each scan. Subjects, aged 10 years to 97 years, were divided into 9 age groups according to decade of life. Results: Five hundred and sixty-six SD-OCT scans were analyzed. Area of VMA (mm2) decreased sigmoidally (R2 = 0.99) with each decade of life. With aging, percentage of PVD increased while percentage of complete adhesion decreased. Males were found to have significantly larger area of VMA (mm2) compared with females in the fifth through eighth decades of life, P < 0.05. Conclusion: Vitreomacular interface interactions throughout life are age and gender dependent. This adds to our current understanding of the normal aging process undergone by the vitreous, thereby providing assistance in the clinical differentiation between normal and pathologic vitreomacular interactions.

DIABETES ALTERS THE MAGNITUDE OF VITREOMACULAR ADHESION.

Purpose: To quantify the changes in vitreomacular interactions that occur with aging in diabetic eyes in comparison with age-matched control eyes. Methods: Spectral-domain optical coherence tomography (Spectralis; Heidelberg Engineering) foveal scans of diabetic patients, without evidence of cystoid macular edema, were included. Twenty-five raster foveal scans were performed on every subject. Area of vitreomacular adhesion was delineated using the Spectralis drawing tool and calculated in square millimeter. Data collected included gender, race, best-corrected visual acuity, and posterior vitreous detachment status. Subjects were divided into age groups according to decade of life. Results: Spectral-domain optical coherence tomography scans from 141 diabetic patients were analyzed. Area of vitreomacular adhesion (mm2) showed a hyperbolic decline in diabetic patients (35.5 ± 0, 35.0 ± 3, 34.0 ± 3, 33.9 ± 5, 33.7 ± 6, 29.0 ± 11, 23 ± 15, 13 ± 15). With aging, incidence of posterior vitreous detachment increased and incidence of complete attachment decreased. Conclusion: Diabetes affects the magnitude of attachment of the vitreous gel to the macula that results in stronger and longer lasting attachment of the gel throughout life. Gender differences were not noticed in diabetic patients, suggesting that vitreomacular adhesion remains robust in both genders in diabetes despite aging.

Abstract 4619: Chemopreventive activities of the dark-color fruit ‘jamun’, the Indian blackberry

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Colored fruits, particularly berries, are emerging as highly chemoprotective because of their antioxidant, anti-proliferative and anti-inflammatory activities. In this study, we investigated the chemoprotective potential of Eugenia jambolana Lam., commonly known as ‘jamun’ or Indian blackberry. Jamun, a widely available fruit in India and other Asian countries, has also been used in the treatment of various ailments, including diabetes mellitus. To determine its antioxidant potential, jamun pulp was collected from freshly harvested fruits, dried, and the powder was extracted with acidic ethanol, and enriched by amberlite XAD7/HP20 (1:1) column chromatography. The pulp powder was found to contain 0.54% anthocyanins, 0.17% ellagic acid/ellagitannins and 1.15% total phenolics using standard assays. HPLC analysis of acid-hydrolyzed extract showed the presence of five anthocyanidins – malvidin, petunidin, delphinidin, cyanidin and peonidin. The unhydrolyzed extract was found to have significant antioxidant activity as reflected by high ORAC value (1,445±64 μmol of TE/g) as well as high ABTS- and DPPH-scavenging activities, and ferrous ions-chelating activity; the acid-hydrolyzed extract showed higher activity than unhydrolyzed extract. Since some of the jamun bioactives (ellagitannins and anthocyanidins/anthocyanins) have significant antiproliferative activity, we also analyzed these extracts for their antiproliferative potential using MTT assay against human lung cancer A549 cells in cell culture. The hydrolyzed jamun extract showed significant antiproliferative activity (IC50=79 µg/mL). However, unhydrolyzed extract showed almost no activity. These data suggest that the antiproliferative activity may result from anthocyanidins (present in the hydrolyzed extract) rather than anthocyanins (present in unhydrolyzed extract). Together, our data show high antioxidant, and antiproliferative activities in jamun pulp and may be a viable candidate for chemoprevention of lung cancer (Supported from Agnes Brown Duggan Endowment). 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 4619. doi:10.1158/1538-7445.AM2011-4619

P2-17-05: Antiprogestin Proellex Suppresses Proliferation of Aromatase Overexpressing and Letrozole Resistant T47D Breast Cancer Cells.

Background: Aromatase is a cytochrome P450 CYP19A1 enzyme responsible for the conversion of C19 androgens to C18 estrogen. Aromatase overexpression leads to increase in local estrogen concentration in post menopausal women diagnosed for breast cancer. Aromatase inhibitors (AI) are used as one of the first line therapies for the treatment of ER+PR+ breast cancers. However, many patients acquire resistance to AI treatment. Therefore alternative approaches are being sought for patients with AI resistance. Previous reports suggest that upregulation of progesterone receptor can lead to enhanced expression of EGFR/ERK/MAPK involved in acquiring resistance. In this study, we evaluated the effect of a selective progesterone receptor modulator, CDB4124 (Proellex) with low glucocorticoid activity on aromatase overexpressing and Letrozole resistant T47D cells. Methods: Aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines were generated by stable transfection with plasmid containing CYP19A1 coding region, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom for 75 weeks in the presence of 10μM Letrozole. Cell proliferation was determined by MTT or Crystal violet assays. Gene expressions were quantitated by qRT-PCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student9s ‘T’ test or ANOVA followed by Bonferroni9s post hoc test. Results: T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA and protein) and as a result exhibited increased conversion of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone. In T47Darom cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex, Letrozole and other known AI (Anastrozole, Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. The inhibition of cell proliferation was significantly greater when cells were treated with Proellex in combination to other AIs. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex may be due to PR-B/EGFR modulation in ER+PR+, aromatase overexpressing cells. Conclusion: Overall these results suggest that antiprogestin, Proellex could be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for AI resistant breast cancer patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-17-05.

Abstract 5688: Chemopreventive potential of ‘jamun’ (Indian blackberry) against estrogen-mediated mammary carcinogenesis

Colored fruits, particularly berries, are emerging as highly chemoprotective because of their antioxidant, anti-proliferative and anti-inflammatory activities. In this study, we investigated chemoprotective potential of Eugenia jambolana Lam., commonly known as ‘jamun’ or Indian blackberry. Jamun is widely used as a fruit in India and several other Asian countries, besides its use in the treatment of various ailments, including diabetes mellitus. Anthocyanin pigments in the jamun were extracted with acidic ethanol, and the crude anthocyanin extract was purified by ethyl acetate partitioning, amberlite XAD7/HP20 column chromatography, followed by Sephadex LH-20 column Chromatography. Anthocyanin content in jamun pulp powder was found to be 0.08% by HPLC-PDA (at 520nm). Upon hydrolysis, the jamun extract yielded five anthocyanidins, namely delphinidin (20.3%), cyanidin (6.6%), petunidin (24.6%), peonidin (2.8%) and malvidin (44.2%) as detected by HPLC and confirmed by mass spectral analysis. The jamun extract had high antioxidant potential as reflected by ABTS scavenging (IC50, 12 µg/ml), DPPH scavenging (IC50, 110 µg/ml), and ferrous ions-chelating activities (IC50, 50 µg/ml). In vivo chemopreventive effect of jamun was investigated using the estrogen-mediated ACI rat mammary tumorigenesis model. Groups of female rats were given control (AIN 93M) diet or diet supplemented with jamun pulp powder (5%, w/w). Two weeks later, one half of the animals from each group were treated with subcutaneous silastic implants of 17β-estradiol (1.2 cm; 9 mg). Diet and water were provided ad libitum. Twelve weeks later immediately before the appearance of mammary tumors, all groups were euthanized and blood and mammary tissue were collected. No significant difference was observed in body weight gain and diet intake when compared with control, suggesting that jamun diet was well tolerated. Estrogen treatment resulted in substantial (30-fold) elevation of the plasma prolactin, a key factor in mammary tumorigenesis. The plasma prolactin levels were significantly offset by jamun-supplemented diet. To determine molecular mechanisms of its chemopreventive effects, mammary tissues were analyzed for the expression of cyclin D1 and estrogen receptor alpha (ERα). Western blot analysis showed that jamun diet significantly down-regulated the levels of both cyclin D1 and ERα. Together, these data suggest that jamun contains compounds with high antioxidant and antiproliferative activities, suggesting that consumption of jamun fruit may be useful in the prevention/treatment of breast cancer (Supported from CA-125152 and Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5688.