Samriddhi Shukla

Bioactive Dietary Supplements Reactivate ER Expression in ER-Negative Breast Cancer Cells by Active Chromatin Modifications

Breast cancer is the most common cancer and the leading cause of cancer death in women. Although tamoxifen therapy is successful for some patients, it does not provide adequate benefit for those who have estrogen receptor (ER)-negative cancers. Therefore, we approached novel treatment strategies by combining two potential bioactive dietary supplements for the reactivation of ERα expression for effective treatment of ERα-negative breast cancer with tamoxifen. Bioactive dietary supplements such as green tea polyphenols (GTPs) and sulforaphane (SFN) inhibit DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), respectively, which are of central importance to cancer prevention. In the present study, we have observed that treatment of ERα-negative breast cancer cells with GTPs and SFN alone or in combination leads to the reactivation of ERα expression. The combination of 20 µg/mL GTPs and 5 µM SFN was found to be the optimal dose of ERα-reactivation at 3 days in MDA-MB-231 cells. The reactivation of ERα expression was consistently correlated with ERα promoter hypomethylation and hyperacetylation. Chromatin immunoprecipitation (ChIP) analysis of the ERα promoter revealed that GTPs and SFN altered the binding of ERα-transcriptional co-repressor complex thereby contributing to ERα-reactivation. In addition, treatment with tamoxifen in combination with GTPs and SFN significantly increased both cell death and inhibition of cellular proliferation in MDA-MB-231 cells in comparison to treatment with tamoxifen alone. Collectively, our findings suggest that a novel combination of bioactive-HDAC inhibitors with bioactive-demethylating agents is a promising strategy for the effective treatment of hormonal refractory breast cancer with available anti-estrogens.

Role of adipokines and cytokines in obesity-associated breast cancer: Therapeutic targets

Obesity is the cause of a large proportion of breast cancer incidences and mortality in post-menopausal women. In obese people, elevated levels of various growth factors such as insulin and insulin-like growth factors (IGFs) are found. Elevated insulin level leads to increased secretion of estrogen by binding to the circulating sex hormone binding globulin (SHBG). The increased estrogen-mediated downstream signaling favors breast carcinogenesis. Obesity leads to altered expression profiles of various adipokines and cytokines including leptin, adiponectin, IL-6, TNF-α and IL-1β. The increased levels of leptin and decreased adiponectin secretion are directly associated with breast cancer development. Increased levels of pro-inflammatory cytokines within the tumor microenvironment promote tumor development. Efficacy of available breast cancer drugs against obesity-associated breast cancer is yet to be confirmed. In this review, we will discuss different adipokine- and cytokine-mediated molecular signaling pathways involved in obesity-associated breast cancer, available therapeutic strategies and potential therapeutic targets for obesity-associated breast cancer.

Epigenetics of Cancer Stem Cells: Pathways and Therapeutics

BACKGROUND Epigenetic alterations including DNA methylation and histone modifications are the key factors in the differentiation of stem cells into different tissue subtypes. The generation of cancer stem cells (CSCs) in the process of carcinogenesis may also involve similar kind of epigenetic reprogramming where, in contrast, it leads to the loss of expression of genes specific to the differentiated state and regaining of stem cell-specific characteristics. The most important predicament with treatment of cancers includes the non-responsive quiescent CSC. SCOPE OF REVIEW The distinctive capabilities of the CSCs make cancer treatment even more difficult as this population of cells tends to remain quiescent for longer intervals and then gets reactivated leading to tumor relapse. Therefore, the current review is aimed to focus on recent advances in understanding the relation of epigenetic reprogramming to the generation, self-renewal and proliferation of CSCs. MAJOR CONCLUSION CSC-targeted therapeutic approaches would improve the chances of patient survival by reducing the frequency of tumor relapse. Differentiation therapy is an emerging therapeutic approach in which the CSCs are induced to differentiate from their quiescent state to a mature differentiated form, through activation of differentiation-related signalling pathways, miRNA-mediated alteration and epigenetic differentiation therapy. Thus, understanding the origin of CSC and their epigenetic regulation is crucial to develop treatment strategy against not only for the heterogeneous population of cancer cells but also to CSCs. GENERAL SIGNIFICANCE Characterizing the epigenetic marks of CSCs and the associated signalling cascades might help in developing therapeutic strategies against chemo-resistant cancers.

Epigenetic regulation by selected dietary phytochemicals in cancer chemoprevention

The growing interest in cancer epigenetics is largely due to the reversible nature of epigenetic changes which tend to alter during the course of carcinogenesis. Major epigenetic changes including DNA methylation, chromatin modifications and miRNA regulation play important roles in tumorigenic process. There are several epigenetically active synthetic molecules such as DNA methyltransferase (DNMTs) and histone deacetylases (HDACs) inhibitors, which are either approved or, are under clinical trials for the treatment of various cancers. However, most of the synthetic inhibitors have shown adverse side effects, narrow in their specificity and also expensive. Hence, bioactive phytochemicals, which are widely available with lesser toxic effects, have been tested for their role in epigenetic modulatory activities in gene regulation for cancer prevention and therapy. Encouragingly, many bioactive phytochemicals potentially altered the expression of key tumor suppressor genes, tumor promoter genes and oncogenes through modulation of DNA methylation and chromatin modification in cancer. These bioactive phytochemicals either alone or in combination with other phytochemicals showed promising results against various cancers. Here, we summarize and discuss the role of some commonly investigated phytochemicals and their epigenetic targets that are of particular interest in cancer prevention and cancer therapy.

Synthesis of β-Carboline-Based N-Heterocyclic Carbenes and Their Antiproliferative and Antimetastatic Activities against Human Breast Cancer Cells

A series of novel β-carboline-based N-heterocyclic carbenes was prepared via Mannich reaction between methyl 1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-carboxylate, formaldehyde, and primary amines. All compounds were evaluated for their antiproliferative activity using human breast cancer and lung cancer cell lines. Three compounds, 3c, 3j, and 3h, were discovered to display IC50 less than 10 μM against human breast cancer MDA-MB-231 cells at 24 h of treatment. Pharmacologically these compounds lead to G2/M phase cell cycle arrest and induction of cellular apoptosis by triggering intrinsic apoptotic pathway through depolarization of mitochondrial membrane potential and activation of caspases. At lower concentrations, these compounds also showed antimigratory and antiinvasive effects against highly metastatic human breast cancer MDA-MB-231 cells via aberration of MAP-kinase signaling and by the inhibition of matrix metalloproteinases. However, these analogues lack in vivo effect in mouse model which may be attributed to their strong affinity to HSA that was investigated spectroscopically with compound 3h.

Centchroman suppresses breast cancer metastasis by reversing epithelial–mesenchymal transition via downregulation of HER2/ERK1/2/MMP-9 signaling

Metastatic spread during carcinogenesis worsens disease prognosis and accelerates the cancer progression. Therefore, newer therapeutic options with higher specificity toward metastatic cancer are required. Centchroman (CC), a female oral contraceptive, has previously been reported to possess antiproliferative and proapoptotic activities in human breast cancer cells. Here, we investigated the effect of CC-treatment against breast cancer metastasis and associated molecular mechanism using in vitro and in vivo models. CC significantly inhibited the proliferation of human and mouse mammary cancer cells. CC-treatment also inhibited migration and invasion capacities of highly metastatic MDA-MB-231 and 4T1 cells, at sub-IC50 concentrations. Inhibition of cell migration and invasion was found to be associated with the reversal of epithelial-to-mesenchymal transition (EMT) as observed by the upregulation of epithelial markers and downregulation of mesenchymal markers as well as decreased activities of matrix metalloproteinases. Experimental EMT induced by exposure to TGFβ/TNFα in nontumorigenic human mammary epithelial MCF10A cells was also reversed by CC as evidenced by morphological changes and modulation in the expression levels of EMT-markers. CC-mediated inhibition of cellular migration was, at least partially, mediated through inhibition of ERK1/2 signaling, which was further validated by using MEK1/2 inhibitor (PD0325901). Furthermore, CC-treatment resulted in suppression of tumor growth and lung metastasis in 4T1-syngeneic mouse model. Collectively, our findings suggest that CC-treatment at higher doses specifically induces cellular apoptosis and inhibits cellular proliferation; whereas at lower doses, it inhibits cellular migration and invasion. Therefore, CC could further be developed as an effective drug candidate against metastatic breast cancer.

Epigenetic reactivation of p21CIP1/WAF1 and KLOTHO by a combination of bioactive dietary supplements is partially ERα-dependent in ERα-negative human breast cancer cells.

Available treatment strategies against estrogen receptor (ER)-negative breast cancer patients are limited due to their poor response to hormonal therapy. We have shown previously that the combinations of green tea polyphenols (GTPs), a dietary DNA methyltransferase inhibitor, and sulforaphane (SFN), a dietary histone deacetylase inhibitor, reactivate ERα expression in ERα-negative MDA-MB-231 cells. Here, we investigated the functional significance of ERα reactivation in the reactivation of silenced tumor suppressor genes (TSGs) in ERα-negative human breast cancer cells. We found that the treatment of MDA-MB-231 cells with the combinations of GTPs and SFN leads to the reactivation of silenced TSGs such as p21(CIP1/WAF1) and KLOTHO through active chromatin modifications. Further, GTPs- and SFN-mediated reactivation of TSGs was, at least in part, dependent on ERα reactivation in ERα-negative MDA-MB-231 cells. Collectively, our findings suggest that a novel combination of bioactive dietary supplements could further be explored as an effective therapeutic option against hormonal refractory breast cancer.

Cucurbitacin B inhibits the stemness and metastatic abilities of NSCLC via downregulation of canonical Wnt/β-catenin signaling axis

Lack of effective anti-metastatic drugs creates a major hurdle for metastatic lung cancer therapy. For successful lung cancer treatment, there is a strong need of newer therapeutics with metastasis-inhibitory potential. In the present study, we determined the anti-metastatic and anti-angiogenic potential of a natural plant triterpenoid, Cucurbitacin B (CuB) against non-small cell lung cancer (NSCLC) both in vitro and in vivo. CuB demonstrated a strong anti-migratory and anti-invasive ability against metastatic NSCLC at nanomolar concentrations. CuB also showed significant tumor angiogenesis-inhibitory effects as evidenced by the inhibition of migratory, invasive and tube-forming capacities of human umbilical vein endothelial cells. CuB-mediated inhibition of angiogenesis was validated by the inhibition of pre-existing vasculature in chick embryo chorio-allantoic membrane and matrigel plugs. Similarly, CuB inhibited the migratory behavior of TGF-β1-induced experimental EMT model. The CuB-mediated inhibition of metastasis and angiogenesis was attributable to the downregulation of Wnt/β-catenin signaling axis, validated by siRNA-knockdown of Wnt3 and Wnt3a. The CuB-mediated downregulation of Wnt/β-catenin signaling was also validated using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis model in vivo. Collectively, our findings suggest that CuB inhibited the metastatic abilities of NSCLC through the inhibition of Wnt/β-catenin signaling axis.

Cucurbitacin B inhibits breast cancer metastasis and angiogenesis through VEGF-mediated suppression of FAK/MMP-9 signaling axis

Available breast cancer therapeutic strategies largely target the primary tumor but are ineffective against tumor metastasis and angiogenesis. In our current study, we determined the effect of Cucurbitacin B (CuB), a plant triterpenoid, on the metastatic and angiogenic potential of breast cancer cells. CuB was found to inhibit cellular proliferation and induce apoptosis in breast cancer cells in a time- and dose-dependent manner. Further, CuB-treatment significantly inhibited the migratory and invasive potential of highly metastatic breast cancer MDA-MB-231 and 4T1 cells at sub-IC50 concentrations, where no significant apoptosis was observed. CuB was also found to inhibit migratory, invasive and tube-forming capacities of HUVECs in vitro. In addition, inhibition of pre-existing vasculature in chick embryo chorioallantoic membrane ex vivo further supports the anti-angiogenic effect of CuB. CuB-mediated anti-metastatic and anti-angiogenic effects were associated with the downregulation of VEGF/FAK/MMP-9 signaling, which has been validated by using FAK-inhibitor (FI-14). CuB-treatment resulted in a significant inhibition of VEGF-induced phosphorylation of FAK and MMP-9 expressions similar to the action of FI-14. CuB was also found to decrease the micro-vessel density as evidenced by the decreased expression of CD31, a marker for neovasculature. Further, CuB-treatment inhibited tumor growth, lung metastasis and angiogenesis in a highly metastatic 4T1-syngeneic mouse mammary cancer. Collectively, our findings suggest that CuB inhibited breast cancer metastasis and angiogenesis, at least in part, through the downregulation of VEGF/FAK/MMP-9 signaling.

Cucurbitacin B Alters the Expression of Tumor-Related Genes by Epigenetic Modifications in NSCLC and Inhibits NNK-Induced Lung Tumorigenesis

Non–small cell lung cancer (NSCLC) represents almost 85% of total diagnosed lung cancer. Studies have shown that combination of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors is effective against various cancers, including lung cancer. However, optimizing the synergistic dose regime is very difficult and involves adverse side effects. Therefore, in this study, we have shown that cucurbitacin B (CuB), a single bioactive triterpenoid compound, inhibits both DNMTs and HDACs starting at a very low dose of 60 nmol/L in NSCLC H1299 cells. The CuB-mediated inhibition of DNMTs and HDACs in H1299 cells leads to the reactivation of key tumor suppressor genes (TSG) such as CDKN1A and CDKN2A, as well as downregulation of oncogenes c-MYC and K-RAS and key tumor promoter gene (TPG), human telomerase reverse transcriptase (hTERT). The upregulation of TSGs and downregulation of TPG were consistently correlated with the alterations in their promoter methylation and histone modifications. This altered expression of TPG and TSGs is, at least in part, responsible for the inhibition of cellular proliferation and induction of cellular apoptosis in NSCLC. Furthermore, CuB treatment significantly inhibited the tumor incidence and multiplicity in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice, which was associated with the induction of apoptosis and inhibition of hyperproliferation in the lung tissues. Together, our study provides new insight into the CuB-mediated epigenetic alterations and its chemotherapeutic effects on lung cancer. Cancer Prev Res; 8(6); 552–62. ©2015 AACR.