Extracts of Strawberry Fruits Induce Intrinsic Pathway of Apoptosis in Breast Cancer Cells and Inhibits Tumor Progression in Mice

Background: The consumption of berry fruits, including strawberries, has been suggested to have beneficial effects against oxidative stress mediated diseases. Berries contain multiple phenolic compounds and secondary metabolites that contribute to their biological properties. Methodology/Principal Findings: Current study investigates the anticancer activity of the methanolic extract of strawberry (MESB) fruits in leukaemia (CEM) and breast cancer (T47D) cell lines ex vivo, and its cancer therapeutic and chemopreventive potential in mice models. Results of MTT, trypan blue and LDH assays suggested that MESB can induce cytotoxicity in cancer cells, irrespective of origin, in a concentration-and time-dependent manner. Treatment of mice bearing breast adenocarcinoma with MESB blocked the proliferation of tumor cells in a time-dependent manner and resulted in extended life span. Histological and immunohistochemical studies suggest that MESB treatment affected tumor cell proliferation by activating apoptosis and did not result in any side effects. Finally, we show that MESB can induce intrinsic pathway of apoptosis by activating p73 in breast cancer cells, when tumor suppressor gene p53 is mutated. Conclusions/Significance: The present study reveals that strawberry fruits possess both cancer preventive and therapeutic values and we discuss the mechanism by which it is achieved.

An Inhibitor of Nonhomologous End-Joining Abrogates Double-Strand Break Repair and Impedes Cancer Progression

DNA Ligase IV is responsible for sealing of double-strand breaks (DSBs) during nonhomologous end-joining (NHEJ). Inhibiting Ligase IV could result in amassing of DSBs, thereby serving as a strategy toward treatment of cancer. Here, we identify a molecule, SCR7 that inhibits joining of DSBs in cell-free repair system. SCR7 blocks Ligase IV-mediated joining by interfering with its DNA binding but not that of T4 DNA Ligase or Ligase I. SCR7 inhibits NHEJ in a Ligase IV-dependent manner within cells, and activates the intrinsic apoptotic pathway. More importantly, SCR7 impedes tumor progression in mouse models and when coadministered with DSB-inducing therapeutic modalities enhances their sensitivity significantly. This inhibitor to target NHEJ offers a strategy toward the treatment of cancer and improvement of existing regimens.

Oncogenic MicroRNA-155 Down-regulates Tumor Suppressor CDC73 and Promotes Oral Squamous Cell Carcinoma Cell Proliferation IMPLICATIONS FOR CANCER THERAPEUTICS

The CDC73 gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. It negatively regulates beta-catenin, cyclin D1, and c-MYC. Down-regulation of CDC73 has been reported in breast, renal, and gastric carcinomas. However, the reports regarding the role of CDC73 in oral squamous cell carcinoma (OSCC) are lacking. In this study we show that CDC73 is down-regulated in a majority of OSCC samples. We further show that oncogenic microRNA-155 (miR-155) negatively regulates CDC73 expression. Our experiments show that the dramatic up-regulation of miR-155 is an exclusive mechanism for down-regulation of CDC73 in a panel of human cell lines and a subset of OSCC patient samples in the absence of loss of heterozygosity, mutations, and promoter methylation. Ectopic expression of miR-155 in HEK293 cells dramatically reduced CDC73 levels, enhanced cell viability, and decreased apoptosis. Conversely, the delivery of a miR-155 antagonist (antagomir-155) to KB cells overexpressing miR-155 resulted in increased CDC73 levels, decreased cell viability, increased apoptosis, and marked regression of xenografts in nude mice. Cotransfection of miR-155 with CDC73 in HEK293 cells abrogated its pro-oncogenic effect. Reduced cell proliferation and increased apoptosis of KB cells were dependent on the presence or absence of the 3'-UTR in CDC73. In summary, knockdown of CDC73 expression due to overexpression of miR-155 not only adds a novelty to the list of mechanisms responsible for its down-regulation in different tumors, but the restoration of CDC73 levels by the use of antagomir-155 may also have an important role in therapeutic intervention of cancers, including OSCC.

Combination of single walled carbon nanotubes/graphene oxide with paclitaxel: a reactive oxygen species mediated synergism for treatment of lung cancer

Heterogeneity in tumors has led to the development of combination therapies that enable enhanced cell death. Previously explored combination therapies mostly involved the use of bioactive molecules. In this work, we explored a non-conventional strategy of using carbon nanostructures (CNs) single walled carbon nanotube (SWNT) and graphene oxide (GO)] for potentiating the efficacy of a bioactive molecule paclitaxel (Tx)] for the treatment of lung cancer. The results demonstrated enhanced cell death following combination treatment of SWNT/GO and Tx indicating a synergistic effect. In addition, synergism was abrogated in the presence of an anti-oxidant, N-acetyl cysteine (NAC), and was therefore shown to be reactive oxygen species (ROS) dependent. It was further demonstrated using bromodeoxyuridine (BrdU) incorporation assay that treatment with CNs was associated with enhanced mitogen associated protein kinase (MAPK) activation that was ROS mediated. Hence, these results for the first time demonstrated the potential of SWNT/GO as co-therapeutic agents with Tx for the treatment of lung cancer.

Rice LHS1/OsMADS1 Controls Floret Meristem Specification by Coordinated Regulation of Transcription Factors and Hormone Signaling Pathways

SEPALLATA (SEP) MADS box transcription factors mediate floral development in association with other regulators. Mutants in five rice (Oryza sativa) SEP genes suggest both redundant and unique functions in panicle branching and floret development. LEAFY HULL STERILE1/OsMADS1, from a grass-specific subgroup of LOFSEP genes, is required for specifying a single floret on the spikelet meristem and for floret organ development, but its downstream mechanisms are unknown. Here, key pathways and directly modulated targets of OsMADS1 were deduced from expression analysis after its knockdown and induction in developing florets and by studying its chromatin occupancy at downstream genes. The negative regulation of OsMADS34, another LOFSEP gene, and activation of OsMADS55, a SHORT VEGETATIVE PHASE-like floret meristem identity gene, show its role in facilitating the spikelet-to-floret meristem transition. Direct regulation of other transcription factor genes like OsHB4 (a class III homeodomain Leu zipper member), OsBLH1 (a BEL1-like homeodomain member), OsKANADI2, OsKANADI4, and OsETTIN2 show its role in meristem maintenance, determinacy, and lateral organ development. We found that the OsMADS1 targets OsETTIN1 and OsETTIN2 redundantly ensure carpel differentiation. The multiple effects of OsMADS1 in promoting auxin transport, signaling, and auxin-dependent expression and its direct repression of three cytokinin A-type response regulators show its role in balancing meristem growth, lateral organ differentiation, and determinacy. Overall, we show that OsMADS1 integrates transcriptional and signaling pathways to promote rice floret specification and development.

Regulation of protumorigenic pathways by Insulin like growth factor binding protein2 and its association along with beta-catenin in breast cancer lymph node metastasis

Background: Insulin like growth factor binding proteins modulate the mitogenic and pro survival effects of IGF. Elevated expression of IGFBP2 is associated with progression of tumors that include prostate, ovarian, glioma among others. Though implicated in the progression of breast cancer, the molecular mechanisms involved in IGFBP2 actions are not well defined. This study investigates the molecular targets and biological pathways targeted by IGFBP2 in breast cancer. Methods: Transcriptome analysis of breast tumor cells (BT474) with stable knockdown of IGFBP2 and breast tumors having differential expression of IGFBP2 by immunohistochemistry was performed using microarray. Differential gene expression was established using R-Bioconductor package. For validation, gene expression was determined by qPCR. Inhibitors of IGF1R and integrin pathway were utilized to study the mechanism of regulation of beta-catenin. Immunohistochemical and immunocytochemical staining was performed on breast tumors and experimental cells, respectively for beta-catenin and IGFBP2 expression. Results: Knockdown of IGFBP2 resulted in differential expression of 2067 up regulated and 2002 down regulated genes in breast cancer cells. Down regulated genes principally belong to cell cycle, DNA replication, repair, p53 signaling, oxidative phosphorylation, Wnt signaling. Whole genome expression analysis of breast tumors with or without IGFBP2 expression indicated changes in genes belonging to Focal adhesion, Map kinase and Wnt signaling pathways. Interestingly, IGFBP2 knockdown clones showed reduced expression of beta-catenin compared to control cells which was restored upon IGFBP2 re-expression. The regulation of beta-catenin by IGFBP2 was found to be IGF1R and integrin pathway dependent. Furthermore, IGFBP2 and beta-catenin are co-ordinately overexpressed in breast tumors and correlate with lymph node metastasis. Conclusion: This study highlights regulation of beta-catenin by IGFBP2 in breast cancer cells and most importantly, combined expression of IGFBP2 and beta-catenin is associated with lymph node metastasis of breast tumors.

A Novel Anticancer Agent, 8-Methoxypyrimido4 `,5 `: 4,5]thieno(2,3-b) Quinoline-4(3H)-One Induces Neuro 2a Neuroblastoma Cell Death through p53-Dependent, Caspase-Dependent and Independent Apoptotic Pathways

Neuroblastoma is the most common cancer in infants and fourth most common cancer in children. Despite recent advances in cancer treatments, the prognosis of stage-IV neuroblastoma patients continues to be dismal which warrant new pharmacotherapy. A novel tetracyclic condensed quinoline compound, 8-methoxypyrimido 4 `,5 `: 4,5] thieno(2,3-b) quinoline-4(3H)-one (MPTQ) is a structural analogue of an anticancer drug ellipticine and has been reported to posses anticancer property. Study on MPTQ on neuroblastoma cells is very limited and mechanisms related to its cytotoxicity on neuroblastoma cells are completely unknown. Here, we evaluated the anticancer property of MPTQ on mouse neuro 2a and human SH-SY5Y neuroblastoma cells and investigated the mechanisms underlying MPTQ-mediated neuro 2a cell death. MPTQ-mediated neuro 2a and SH-SY5Y cell deaths were found to be dose and time dependent. Moreover, MPTQ induced cell death reached approximately 99.8% and 90% in neuro 2a and SH-SY5Y cells respectively. Nuclear oligonucleosomal DNA fragmentation and Terminal dUTP Nick End Labelling assays indicated MPTQ-mediated neuro 2a cell death involved apoptosis. MPTQ-mediated apoptosis is associated with increased phosphorylation of p53 at Ser15 and Ser20 which correlates with the hyperphosphorylation of Ataxia-Telangiectasia mutated protein (ATM). Immunocytochemical analysis demonstrated the increased level of Bax protein in MPTQ treated neuro 2a cells. MPTQ-mediated apoptosis is also associated with increased activation of caspase-9, -3 and -7 but not caspase-2 and -8. Furthermore, increased level of caspase-3 and cleaved Poly ( ADP Ribose) polymerase were observed in the nucleus of MPTQ treated neuro 2a cells, suggesting the involvement of caspase-dependent intrinsic but not extrinsic apoptotic pathway. Increased nuclear translocation of apoptosis inducing factor suggests additional involvement of caspase-independent apoptosis pathway in MPTQ treated neuro 2a cells. Collectively, MPTQ-induced neuro 2a cell death is mediated by ATM and p53 activation, and Bax-mediated activation of caspase-dependent and caspase-independent mitochondrial apoptosis pathways.

Inhibition of cancer cell proliferation and apoptosis-inducing activity of fungal taxol and its precursor baccatin III purified from endophytic Fusarium solani

Background: Taxol (generic name paclitaxel), a plant-derived antineoplastic agent, used widely against breast, ovarian and lung cancer, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. The limited supply of the drug has prompted efforts to find alternative sources, such as chemical synthesis, tissue and cell cultures of the Taxus species both of which are expensive and yield low levels. Fermentation processes with microorganisms would be the methods of choice to lower the costs and increase yields. Previously we have reported that F. solani isolated from T. celebica produced taxol and its precursor baccatin III in liquid grown cultures J Biosci 33: 259-67, 2008. This study was performed to evaluate the inhibition of proliferation and induction of apoptosis of cancer cell lines by the fungal taxol and fungal baccatin III of F. solani isolated from T. celebica. Methods: Cell lines such as HeLa, HepG2, Jurkat, Ovcar3 and T47D were cultured individually and treated with fungal taxol, baccatin III with or without caspase inhibitors according to experimental requirements. Their efficacy on apoptotic induction was examined. Results: Both fungal taxol and baccatin III inhibited cell proliferation of a number of cancer cell lines with IC50 ranging from 0.005 to 0.2 mu M for fungal taxol and 2 to 5 mu M for fungal baccatin III. They also induced apoptosis in JR4-Jurkat cells with a possible involvement of anti-apoptotic Bcl2 and loss in mitochondrial membrane potential, and was unaffected by inhibitors of caspase-9,-2 or -3 but was prevented in presence of caspase-10 inhibitor. DNA fragmentation was also observed in cells treated with fungal taxol and baccatin III. Conclusions: The cytotoxic activity exhibited by fungal taxol and baccatin III involves the same mechanism, dependent on caspase-10 and membrane potential loss of mitochondria, with taxol having far greater cytotoxic potential.

Endocrine Correlates of Musth in Free-Ranging Asian Elephants (Elephas maximus) Determined by Non-Invasive Faecal Steroid Hormone Metabolite Measurements

The occurrence of musth, a period of elevated levels of androgens and heightened sexual activity, has been well documented for the male Asian elephant (Elephas maximus). However, the relationship between androgen-dependent musth and adrenocortical function in this species is unclear. The current study is the first assessment of testicular and adrenocortical function in free-ranging male Asian elephants by measuring levels of testosterone (androgen) and cortisol (glucocorticoid - a physiological indicator of stress) metabolites in faeces. During musth, males expectedly showed significant elevation in faecal testosterone metabolite levels. Interestingly, glucocorticoid metabolite concentrations remained unchanged between musth and non-musth periods. This observation is contrary to that observed with wild and captive African elephant bulls and captive Asian bull elephants. Our results show that musth may not necessarily represent a stressful condition in free-ranging male Asian elephants.

Sapodilla Plum (Achras sapota) Induces Apoptosis in Cancer Cell Lines and Inhibits Tumor Progression in Mice

Intake of fruits rich in antioxidants in daily diet is suggested to be cancer preventive. Sapota is a tropical fruit grown and consumed extensively in several countries including India and Mexico. Here we show that methanolic extracts of Sapota fruit (MESF) induces cytotoxicity in a dose-dependent manner in cancer cell lines. Cell cycle analysis suggested activation of apoptosis, without arresting cell cycle progression. Annexin V-propidium iodide double-staining demonstrated that Sapota fruit extracts potentiate apoptosis rather than necrosis in cancer cells. Loss of mitochondrial membrane potential, upregulation of proapoptotic proteins, activation of MCL-1, PARP-1, and Caspase 9 suggest that MESF treatment leads to activation of mitochondrial pathway of apoptosis. More importantly, we show that MESF treatment leads to significant inhibition of tumor growth and a 3-fold increase in the life span of tumor bearing animals compared to untreated tumor mice.

MicroRNA-125a Reduces Proliferation and Invasion of Oral Squamous Cell Carcinoma Cells by Targeting Estrogen-related Receptor alpha IMPLICATIONS FOR CANCER THERAPEUTICS

Estrogen-related receptor (ESRRA) functions as a transcription factor and regulates the expression of several genes, such as WNT11 and OPN. Up-regulation of ESRRA has been reported in several cancers. However, the mechanism underlying its up-regulation is unclear. Furthermore, the reports regarding the role and regulation of ESRRA in oral squamous cell carcinoma (OSCC) are completely lacking. Here, we show that tumor suppressor miR-125a directly binds to the 3UTR of ESRRA and represses its expression. Overexpression of miR-125a in OSCC cells drastically reduced the level of ESRRA, decreased cell proliferation, and increased apoptosis. Conversely, the delivery of an miR-125a inhibitor to these cells drastically increased the level of ESRRA, increased cell proliferation, and decreased apoptosis. miR-125a-mediated down-regulation of ESRRA impaired anchorage-independent colony formation and invasion of OSCC cells. Reduced cell proliferation and increased apoptosis of OSCC cells were dependent on the presence of the 3UTR in ESRRA. The delivery of an miR-125a mimic to OSCC cells resulted in marked regression of xenografts in nude mice, whereas the delivery of an miR-125a inhibitor to OSCC cells resulted in a significant increase of xenografts and abrogated the tumor suppressor function of miR-125a. We observed an inverse correlation between the expression levels of miR-125a and ESRRA in OSCC samples. In summary, up-regulation of ESRRA due to down-regulation of miR-125a is not only a novel mechanism for its up-regulation in OSCC, but decreasing the level of ESRRA by using a synthetic miR-125a mimic may have an important role in therapeutic intervention of OSCC and other cancers.

Anticancer Effects of Brominated Indole Alkaloid Eudistomin H from Marine Ascidian Eudistoma viride Against Cervical Cancer Cells (HeLa)

Marine invertebrates called ascidians are prolific producers of bioactive substances. The ascidian Eudistoma viride, distributed along the Southeast coast of India, was investigated for its in vitro cytotoxic activity against human cervical carcinoma (HeLa) cells by the MTT assay. The crude methanolic extract of E. viride, with an IC50 of 53 mu g/ml, was dose-dependently cytotoxic. It was more potent at 100 mu g/ml than cyclohexamide (1 mu g/ml), reducing cell viability to 9.2%. Among nine fractions separated by chromatography, ECF-8 exhibited prominent cytoxic activity at 10 mu g/ml. The HPLC fraction EHF-21 of ECF-8 was remarkably dose- and time-dependently cytotoxic, with 39.8% viable cells at 1 mu g/ml compared to 51% in cyclohexamide-treated cells at the same concentration; the IC50 was 0.49,mu g/ml. Hoechst staining of HeLa cells treated with EHF-2I at 0.5 mu g/ml revealed apoptotic events such an cell shrinkage, membrane blebbing, chromatin condensation and formation of apoptotic bodies. Cell size and granularity study showed changes in light scatter, indicating the characteristic feature of cells dying by apoptosis. The cell-cycle analysis of HeLa cells treated with fraction EHF-21 at 1 mu g/ml showed the marked arrest of cells in G(0)/G(1), S and G(2)/M phases and an increase in the sub G(0)/G(1) population indicated an increase in the apoptotic cell population. The statistical analysis of the sub-G(1) region showed a dose-dependent induction of apoptosis. DNA fragmentation was also observed in HeLa cells treated with EHF-21. The active EHF-2I fraction, a brominated indole alkaloid Eudistomin H, led to apoptotic death of HeLa cells.

Effects of Withania somnifera and Tinospora cordifolia Extracts on the Side Population Phenotype of Human Epithelial Cancer Cells: Toward Targeting Multidrug Resistance in Cancer

Recent reports suggest the existence of a subpopulation of stem-like cancer cells, termed as cancer stem cells (CSCs), which bear functional and phenotypic resemblance with the adult, tissue-resident stem cells. Side population (SP) assay based on differential efflux of Hoechst 33342 has been effectively used for the isolation of CSCs. The drug resistance properties of SP cells are typically due to the increased expression of ABC transporters leading to drug efflux. Conventionally used chemotherapeutic drugs may often leads to an enrichment of SP, revealing their inability to target the drug-resistant SP and CSCs. Thus, identification of agents that can reduce the SP phenotype is currently in vogue in cancer therapeutics. Withania somnifera (WS) and Tinospora cordifolia (TC) have been used in Ayurveda for treating various diseases, including cancer. In the current study, we have investigated the effects of ethanolic (ET) extracts of WS and TC on the cancer SP phenotype. Interestingly, we found significant decrease in SP on treatment with TC-ET, but not with WS-ET. The SP-inhibitory TC-ET was further fractionated into petroleum ether (TC-PET), dichloromethane (TC-DCM), and n-butyl alcohol (TC-nBT) fractions using bioactivity-guided fractionation. Our data revealed that TC-PET and TC-DCM, but not TC-nBT, significantly inhibited SP in a dose-dependent manner. Furthermore, flow cytometry-based functional assays revealed that TC-PET and TC-DCM significantly inhibited ABC-B1 and ABC-G2 transporters and sensitized cancer cells toward chemotherapeutic drug-mediated cytotoxicity. Thus, the TC-PET and TC-DCM may harbor phytochemicals with the potential to reverse the drug-resistant phenotype, thus improving the efficacy of cancer chemotherapy.

Homology and enzymatic requirements of microhomology-dependent alternative end joining

Nonhomologous DNA end joining (NHEJ) is one of the major double-strand break (DSB) repair pathways in higher eukaryotes. Recently, it has been shown that alternative NHEJ (A-NHEJ) occurs in the absence of classical NHEJ and is implicated in chromosomal translocations leading to cancer. In the present study, we have developed a novel biochemical assay system utilizing DSBs flanked by varying lengths of microhomology to study microhomology-mediated alternative end joining (MMEJ). We show that MMEJ can operate in normal cells, when microhomology is present, irrespective of occurrence of robust classical NHEJ. Length of the microhomology determines the efficiency of MMEJ, 5 nt being obligatory. Using this biochemical approach, we show that products obtained are due to MMEJ, which is dependent on MRE11, NBS1, LIGASE III, XRCC1, FEN1 and PARP1. Thus, we define the enzymatic machinery and microhomology requirements of alternative NHEJ using a well-defined biochemical system.

HIF-1 alpha (1772C > T) polymorphism as marker for breast cancer development

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) is an important transcription factor that regulates different cellular responses to hypoxia. HIF-1 alpha is rapidly degraded by von Hippel-Lindau (VHL) protein under normoxic conditions and stabilized under hypoxia. A common variant of HIF-1 alpha (1772C > T) (rs 11549465) polymorphism, corresponding to an amino acid change from proline to serine at 582 position within the oxygen-dependent degradation domain, results in increased stability of the protein and altered transactivation of its target genes. The present study was aimed to find the association between HIF-1 alpha (1772C > T) (rs 11549465) polymorphism and breast cancer development. For this purpose, 348 primary breast cancer patients and 320 healthy and age-matched controls were genotyped through PCR-RFLP method. The genotype frequencies were compared between patients and controls, and their influence on clinical characteristics of breast cancer patients was analyzed. Our study revealed a significant increase of TT genotype in breast cancer patients compared to controls (p = 0.038). Further, TT genotype and T allele were found to be associated with progesterone receptor (PR)-negative status (p < 0.09). None of the clinical variables revealed significant association with HIF-1 alpha (1772C > T) (rs 11549465) polymorphism.