Hareesh B. Nair

Thymoquinone poly (lactide-co-glycolide) nanoparticles exhibit enhanced anti-proliferative, anti-inflammatory, and chemosensitization potential

Thymoquinone (TQ), derived from the medicinal spice Nigella sativa (also called black cumin), has been shown to exhibit anti-inflammatory and anti-cancer activities. In this report we employed polymer-based nanoparticle approach to improve upon its effectiveness and bioavailability. TQ was encapsulated with 97.5% efficiency in biodegradable nanoparticulate formulation based on poly (lactide-co-glycolide) (PLGA) and the stabilizer polyethylene glycol (PEG)-5000. Dynamic laser light scattering and transmission electron microscopy confirmed particle diameter between 150 and 200nm. Electrophoretic gel shift mobility assay showed that TQ nanoparticles (NP) were more active than TQ in inhibiting NF-kappaB activation and in suppressing the expression of cyclin D1, matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), those are markers of cell proliferation, metastasis and angiogenesis, respectively. TQ-NP were also more potent than TQ in suppressing proliferation of colon cancer, breast cancer, prostate cancer, and multiple myeloma cells. Esterase staining for plasma membrane integrity revealed that TQ-NP were more potent than TQ in sensitizing leukemic cells to TNF- and paclitaxel-induced apoptosis. Overall our results demonstrate that encapsulation of TQ into nanoparticles enhances its anti-proliferative, anti-inflammatory, and chemosensitizing effects.

Induction of Aromatase Expression in Cervical Carcinomas: Effects of Endogenous Estrogen on Cervical Cancer Cell Proliferation

Epidemiologic studies have implicated estrogenic exposure as well as human papilloma virus (HPV) infection in cervical carcinogenesis, and some studies have suggested that estrogen and HPV may play synergistic roles in cervical tumorigenesis. In this study, we report a novel finding that approximately 35% of cervical carcinomas tested (n = 19) express aromatase, the enzyme responsible for converting androgen to estrogen, the rate-limiting and final step in estrogen biosynthesis. On the other hand, no aromatase expression was detected in precancerous (n = 42) or normal cervical (n = 17) tissue samples. Increased aromatase was associated with increases in estrogen receptors (ER-alpha and ER-beta) and a decrease in progesterone receptor levels, suggesting that in situ estrogen signaling via ER may be involved in tumor growth. Stable overexpression of aromatase in HPV+ cervical cancer cells resulted in increased cellular proliferation, anchorage-independent growth, and ER expression and activity. In contrast, little change in ER was observed in HPV- cells. Steroid hormone receptor expression observed in vitro paralleled that seen in cervical carcinomas expressing aromatase. Aromatase overexpression also induced the expression of cyclin D1, proliferating cell nuclear antigen, and the HPV oncogenes, E6 and E7. Furthermore, the data underscores the importance of steroid receptor (estrogen and progesterone receptors) regulation in cervical carcinogenesis. To our knowledge, this is the first report demonstrating the induction of aromatase expression in cervical carcinomas, and opens the possibility that aromatase inhibitors may be potential therapeutic agents in cervical carcinomas expressing aromatase.

Elevated Expression of the Oncogene c-fms and Its Ligand, the Macrophage Colony-Stimulating Factor-1, in Cervical Cancer and the Role of Transforming Growth Factor-β1 in Inducing c-fms Expression

Cervical cancer is the third most common gynecologic cancer in the United States. The presence and possible involvement of several cytokines have been studied in cervical cancer; however, very little data, if any, are available on whether cervical tumors are responsive to stimulation by the macrophage colony-stimulating factor-1 (CSF-1). Given the involvement of c-fms and its ligand CSF-1 in gynecologic cancers, such as that of the uterus and the ovaries, we have examined the expression of c-fms and CSF-1 in cervical tumor (n = 17) and normal cervix (n = 8) samples. The data show that c-fms and its ligand are significantly higher in cervical carcinomas compared with normal samples. Immunohistochemistry not only showed that tumor cells expressed significantly higher levels of c-fms but also c-fms levels were markedly higher in tumor cells than tumor-associated stromal cells. Blocking c-fms activity in cervical cancer cells, which express CSF-1 and c-fms, resulted in increased apoptosis and decreased motility compared with control, suggesting that CSF-1/c-fms signaling may be involved in enhanced survival and possibly invasion by cervical cancer cells via an autocrine mechanism. Combined, the data show for the first time the induction of CSF-1 and c-fms in cervical carcinomas and suggest that c-fms activation may play a role in cervical carcinogenesis. Additionally, our data suggest that transforming growth factor-beta1 may be a factor in inducing the expression of c-fms in cervical cancer cells. The data suggest that c-fms may be a valuable therapeutic target in cervical cancer.

Overexpression of the Colony-Stimulating Factor (CSF-1) and/or Its Receptor c-fms in Mammary Glands of Transgenic Mice Results in Hyperplasia and Tumor Formation

A number of recent studies have suggested that the colony-stimulating factor (CSF-1) and its receptor c-fms may be involved in the development of mammary glands during lactation and breast cancer. To study the role of CSF-1 or its receptor in initiation of mammary tumorigenesis, we have generated two independent lines of transgenic mice that overexpress either CSF-1 or c-fms under the control of the mouse mammary tumor virus promoter. Mammary glands of the virgin CSF-1 transgenic mice show increased ductal branching, hyperplasia, dysplasia, and other preneoplastic changes, which are indicative of increased cellular proliferation. Similar changes were also evident in the mammary glands of the c-fms transgenic mice. These changes became more prominent with age and resulted in mammary tumor formation. Moreover, secondary events like dimethylbenz(a)anthracene treatment accelerated mammary tumor formation in these mice. Although the expression of estrogen receptor α was not significantly changed in either of the transgenic mouse strains, progesterone receptor levels was higher in both transgenic lines as compared with the nontransgenic littermates. Expression of G1 cyclins was prominently increased in the mammary glands of both the CSF-1 and c-fms transgenic lines, suggesting increased cell cycle progression in these strains. In addition, the proliferation marker proliferating cell nuclear antigen (PCNA) and the mitogen-responsive transcription factor c-jun were also increased as compared with the nontransgenic controls. These findings, along with the histological data, support the hypothesis that CSF-1 and its receptor are involved in the etiology of breast cancer.

Modeling the early endometriotic lesion: mesothelium-endometrial cell co-culture increases endometrial invasion and alters mesothelial and endometrial gene transcription

OBJECTIVE To determine the role of peritoneal mesothelial cells (PMCs) in the process of endometrial invasion into the peritoneum and to evaluate gene expression after endometrial-PMC co-culture. DESIGN In vitro study. SETTING University laboratory. PATIENT(S) Reproductive-age women without endometriosis. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The rate of endometrial invasion through modeled peritoneum in the presence and absence of PMCs was evaluated. The influence of endometrial-PMC attachment on the expression of target genes, implicated in the pathogenesis of endometriosis, was examined by using reverse transcription polymerase chain reaction. RESULT(S) Endometrial stromal cell (ESC) invasion through invasion chambers coated with Matrigel (MTGL) and with growth factor-reduced Matrigel (GFR-MTGL) was increased 10-fold when a PMC monolayer was present. Endometrial epithelioid cell (EM42) invasion increased greater than threefold through the MTGL and GFR-MTGL-coated membranes when a PMC monolayer was present. Endometrial stromal cell, EM42, and PMC transcription of extracellular signal-related kinase, colony stimulating factor-1, c-fms, and c-Met was increased after endometrial-PMC attachment. Similar changes were not seen when endometrial cells were exposed to PMC-conditioned media and when PMCs were exposed to endometrial cell conditioned media. CONCLUSION(S) Peritoneal mesothelial cells increased invasion of ESCs and EM42s through modeled peritoneum. Endometrial-PMC co-culture led to alterations in gene transcription by endometrial cells and PMCs. This study suggests that PMCs contribute to the process of endometrial invasion into the peritoneum.

Modeling ductal carcinoma in situ: A HER2-Notch3 collaboration enables luminal filling

A large fraction of ductal carcinoma in situ (DCIS), a non-invasive precursor lesion of invasive breast cancer, overexpresses the HER2/neu oncogene. The ducts of DCIS are abnormally filled with cells that evade apoptosis, but the underlying mechanisms remain incompletely understood. We overexpressed HER2 in mammary epithelial cells and observed growth factor-independent proliferation. When grown in extracellular matrix as three-dimensional spheroids, control cells developed a hollow lumen, but HER2-overexpressing cells populated the lumen by evading apoptosis. We demonstrate that HER2 overexpression in this cellular model of DCIS drives transcriptional upregulation of multiple components of the Notch survival pathway. Importantly, luminal filling required upregulation of a signaling pathway comprising Notch3, its cleaved intracellular domain and the transcriptional regulator HES1, resulting in elevated levels of c-MYC and cyclin D1. In line with HER2–Notch3 collaboration, drugs intercepting either arm reverted the DCIS-like phenotype. In addition, we report upregulation of Notch3 in hyperplastic lesions of HER2 transgenic animals, as well as an association between HER2 levels and expression levels of components of the Notch pathway in tumor specimens of breast cancer patients. Therefore, it is conceivable that the integration of the Notch and HER2 signaling pathways contributes to the pathophysiology of DCIS.

Estrogen receptor alpha is required for mammary development and the induction of mammary hyperplasia and epigenetic alterations in the aromatase transgenic mice.

Aromatase transgenic mice exhibit hyperplastic and dysplastic changes, attesting to the importance of local estrogen in breast carcinogenesis. These mice also show increased levels of the estrogen receptor alpha and beta (ERalpha, ERbeta) suggesting that this receptor may play an important role in the initiation of estrogen-mediated mammary hyperplasia observed in these mice. To address the specific role of ERalpha in the mammary development and in the induction of estrogen-mediated hyperplasia in aromatase transgenic mice, we have generated MMTV-aromatase x ERalpha knockout cross (referred as aromatase/ERKO). Even though ERbeta is expressed in aromatase/ERKO mice, lack of ERalpha leads to impaired mammary growth in these mice. The data suggest that ERalpha plays an important role in the mammary gland development as well as in the induction of mammary hyperplasia in aromatase transgenic mice. Lack of ERalpha expression in the aromatase/ERKO mice resulted in a decrease in the expression of Cyclin D1, PCNA and TGFbeta relative to the aromatase parental strain. The studies involving aromatase/ERKO mice show that lack of ERalpha results in impaired mammary development even in the presence of continuous tissue estrogen, suggesting estrogen/ERalpha-mediated actions are critical for mammary development and carcinogenesis.

Induction of endometrial epithelial cell invasion and c-fms expression by transforming growth factor beta

Transforming growth factor beta 1 (TGF-beta1) levels are increased in the peritoneal fluid of endometriosis patients, and endometrial cells express TGF-beta signaling components; however, little is known regarding the role of TGF-beta in endometriosis. Our objective was to examine the effects of TGF-beta1 on (i) the expression of macrophage colony-stimulating factor receptor encoded by the c-fms gene, (ii) transmesothelial invasiveness of endometrial cells, (iii) cellular proliferation and (iv) attachment to peritoneal mesothelial cells (PMCs). Effects of TGF-beta1 on c-fms mRNA expression were determined by real-time RT-PCR and c-fms cell-surface expression by flow cytometry. Effects of TGF-beta1 on the invasiveness of the immortalized endometrial epithelial cell (EEC) line EM42 and primary EECs were examined using a three-dimensional in vitro system modeling the peritoneum. Cellular proliferation and attachment to PMCs were also examined using established techniques. TGF-beta1 had little or no effect on cellular proliferation and endometrial cell attachment to PMCs. TGF-beta1 significantly induced the expression of c-fms mRNA and c-fms cell-surface expression. TGF-beta1 enhanced transmesothelial invasion by EM42 cells and EECs. Antagonists of TGF-beta1 signaling significantly inhibited both the induction of c-fms expression and cellular invasiveness, suggesting that additional studies are warranted to assess the therapeutic potential of TGF-beta antagonists in endometriosis.

HER-2/neu × Aromatase Double Transgenic Mice Model: The Effects of Aromatase Overexpression on Mammary Tumorigenesis

A majority of breast cancers are hormone-responsive, and require estrogen for growth, and respond to hormonal therapy that blocks estrogen receptor action. Breast tumors with low levels of or completely lacking estrogen receptor fail to respond to antiestrogen therapy yet require estrogen for tumor initiation. To address the importance of local estrogen in oncogene-mediated breast tumorigenesis, we have crossed MMTV-aromatase with MMTV-HER2/neu and examined the incidence of breast cancer in double transgenic mice in comparison with parental strains. Double transgenic mice show normal mammary development and express both transgenes at similar levels to that of parental strains. Tumor incidence in double transgenic mice (<5%) decreased compared to HER2/neu mice (>65%). In addition to a significant decrease in tumorigenesis, these mice expressed ERalpha as well as high levels of ERbeta along with decreased levels of cyclin D1 and phosphorylated pRb among other changes. Furthermore, experiments using THC (ERalpha-agonist and ERbeta-antagonist) clearly demonstrate the critical role of ERbeta in HER2/neu-mediated tumorigenesis. These studies provide the first genetic evidence that estrogen receptor, mainly ERbeta than ERalpha and its dependent changes play an important role in regulating mammary tumorigenesis. These findings provide further evidence for development and testing of novel therapeutic approaches based on selective regulation of estrogen receptors (ERalpha and beta)-dependent actions for the treatment and prevention of breast cancers.

Estrogen receptor-β activation in combination with letrozole blocks the growth of breast cancer tumors resistant to letrozole therapy

Treatment with anti-estrogens or aromatase inhibitors (AI) is the main therapeutic strategy used against estrogen receptor ERα-positive breast cancer. Resistance to these therapies presents a major challenge in the management of breast cancer. Little is known about ERβ in breast carcinogenesis. Our aim in this study is to examine potential novel strategies utilizing ERβ activity to overcome AI resistance. We provide evidence that ERβ agonist can reduce the growth of AI-resistant breast cancer cells. Our data further confirm that therapeutic activation of ERβ by DPN, an ERβ agonist, blocks letrozole-resistant tumor growth in a xenograft model. Interestingly, DPN exerted tumor growth inhibition only in the presence of the AI letrozole, suggesting that combination therapy including ERβ activators and AI may be used in the clinical setting treating AI resistant breast cancer. An increase in ERβ levels, with diminished ERα/ERβ ratio, was observed in the tumors from mice treated with DPN/letrozole combination compared to single agents and control. Decreased Cyclin D1 and increased CyclinD1/CDK inhibitors p21 and p27 levels in DPN/letrozole treated tumors were observed, suggesting that the combination treatment may inhibit tumor growth by blocking G1/S phase cell cycle progression. Our data show a decrease in MAPK phosphorylation levels without affecting total levels. In addition to providing evidence suggesting the potential use of ERβ agonists in combination with letrozole in treating AI resistant breast cancer and prolonging sensitivity to AI, we also provide mechanistic evidence supporting the role of ERβ in altering the expression profile associated with resistance.