Anticancer Pathways Research Articles

Cyclooxygenase-Dependent Tumor Growth through Evasion of Immunity

SummaryThe mechanisms by which melanoma and other cancer cells evade anti-tumor immunity remain incompletely understood. Here, we show that the growth of tumors formed by mutant BrafV600E mouse melanoma cells in an immunocompetent host requires their production of prostaglandin E2, which suppresses immunity and fuels tumor-promoting inflammation. Genetic ablation of cyclooxygenases (COX) or prostaglandin E synthases in BrafV600E mouse melanoma cells, as well as in NrasG12D melanoma or in breast or colorectal cancer cells, renders them susceptible to immune control and provokes a shift in the tumor inflammatory profile toward classic anti-cancer immune pathways. This mouse COX-dependent inflammatory signature is remarkably conserved in human cutaneous melanoma biopsies, arguing for COX activity as a driver of immune suppression across species. Pre-clinical data demonstrate that inhibition of COX synergizes with anti-PD-1 blockade in inducing eradication of tumors, implying that COX inhibitors could be useful adjuvants for immune-based therapies in cancer patients.

Calcitriol increases Dicer expression and modifies the microRNAs signature in SiHa cervical cancer cells.

MicroRNAs play important roles in cancer biology. Calcitriol, the hormonal form of vitamin D3, regulates microRNAs expression in tumor cells. In the present study we asked if calcitriol would modify some of the components of the microRNA processing machinery, namely, Drosha and Dicer, in calcitriol-responsive cervical cancer cells. We found that calcitriol treatment did not affect Drosha mRNA; however, it significantly increased Dicer mRNA and protein expression in VDR-positive SiHa and HeLa cells. In VDR-negative C33-A cells, calcitriol had no effect on Dicer mRNA. We also found a vitamin D response element in Dicer promoter that interacts in vitro to vitamin D and retinoid X receptors. To explore the biological plausibility of these results, we asked if calcitriol alters the microRNA expression profile in SiHa cells. Our results revealed that calcitriol regulates the expression of a subset of microRNAs with potential regulatory functions in cancer pathways, such as miR-22, miR-296-3p, and miR-498, which exert tumor-suppressive effects. In summary, the data indicate that in SiHa cells, calcitriol stimulates the expression of Dicer possibly through the vitamin D response element located in its promoter. This may explain the calcitriol-dependent modulation of microRNAs whose target mRNAs are related to anticancer pathways, further adding to the various anticancer mechanisms of calcitriol.

Revealing mechanisms of selective, concentration-dependent potentials of 4-hydroxy-2-nonenal to induce apoptosis in cancer cells through inactivation of membrane-associated catalase

Tumor cells generate extracellular superoxide anions and are protected against superoxide anion-mediated intercellular apoptosis-inducing signaling by the expression of membrane-associated catalase. 4-Hydroxy-2-nonenal (4-HNE), a versatile second messenger generated during lipid peroxidation, has been shown to induce apoptosis selectively in malignant cells. The findings described in this paper reveal the strong, concentration-dependent potential of 4-HNE to specifically inactivate extracellular catalase of tumor cells both indirectly and directly and to consequently trigger apoptosis in malignant cells through superoxide anion-mediated intercellular apoptosis-inducing signaling. Namely, 4-HNE caused apoptosis selectively in NOX1-expressing tumor cells through inactivation of their membrane-associated catalase, thus reactivating subsequent intercellular signaling through the NO/peroxynitrite and HOCl pathways, followed by the mitochondrial pathway of apoptosis. Concentrations of 4-HNE of 1.2µM and higher directly inactivated membrane-associated catalase of tumor cells, whereas at lower concentrations, 4-HNE triggered a complex amplificatory pathway based on initial singlet oxygen formation through H2O2 and peroxynitrite interaction. Singlet-oxygen-dependent activation of the FAS receptor and caspase-8 increased superoxide anion generation by NOX1 and amplification of singlet oxygen generation, which allowed singlet-oxygen-dependent inactivation of catalase. 4-HNE and singlet oxygen cooperate in complex autoamplificatory loops during this process. The finding of these novel anticancer pathways may be useful for understanding the role of 4-HNE in the control of malignant cells and for the optimization of ROS-dependent therapeutic approaches including antioxidant treatments.

Self-assembled nanoscale coordination polymers carrying oxaliplatin and gemcitabine for synergistic combination therapy of pancreatic cancer

Gemcitabine has long been the standard of care for treating pancreatic ductal adenocarcinoma (PDAC), despite its poor pharmacokinetics/dynamics and rapid development of drug resistance. In this study, we have developed a novel nanoparticle platform based on nanoscale coordination polymer-1 (NCP-1) for simultaneous delivery of two chemotherapeutics, oxaliplatin and gemcitabine monophosphate (GMP), at 30wt.% and 12wt.% drug loadings, respectively. A strong synergistic therapeutic effect of oxaliplatin and GMP was observed in vitro against AsPc-1 and BxPc-3 pancreatic cancer cells. NCP-1 particles effectively avoid uptake by the mononuclear phagocyte system (MPS) in vivo with a long blood circulation half-life of 10.1±3.3h, and potently inhibit tumor growth when compared to NCP particles carrying oxaliplatin or GMP alone. Our findings demonstrate NCP-1 as a novel nanocarrier for the co-delivery of two chemotherapeutics that have distinctive mechanisms of action to simultaneously disrupt multiple anticancer pathways with maximal therapeutic efficacy and minimal side effects.

Dietary Sulforaphane in Cancer Chemoprevention: The Role of Epigenetic Regulation and HDAC Inhibition.

Sulforaphane, produced by the hydrolytic conversion of glucoraphanin after ingestion of cruciferous vegetables, particularly broccoli and broccoli sprouts, has been extensively studied due to its apparent health-promoting properties in disease and limited toxicity in normal tissue. Recent Studies: Recent identification of a sub-population of tumor cells with stem cell-like self-renewal capacity that may be responsible for relapse, metastasis, and resistance, as a potential target of the dietary compound, may be an important aspect of sulforaphane chemoprevention. Evidence also suggests that sulforaphane may target the epigenetic alterations observed in specific cancers, reversing aberrant changes in gene transcription through mechanisms of histone deacetylase inhibition, global demethylation, and microRNA modulation. In this review, we discuss the biochemical and biological properties of sulforaphane with a particular emphasis on the anticancer properties of the dietary compound. Sulforaphane possesses the capacity to intervene in multistage carcinogenesis through the modulation and/or regulation of important cellular mechanisms. The inhibition of phase I enzymes that are responsible for the activation of pro-carcinogens, and the induction of phase II enzymes that are critical in mutagen elimination are well-characterized chemopreventive properties. Furthermore, sulforaphane mediates a number of anticancer pathways, including the activation of apoptosis, induction of cell cycle arrest, and inhibition of NFκB. Further characterization of the chemopreventive properties of sulforaphane and its capacity to be selectively toxic to malignant cells are warranted to potentially establish the clinical utility of the dietary compound as an anti-cancer compound alone, and in combination with clinically relevant therapeutic and management strategies.

NIAM-deficient mice are predisposed to the development of proliferative lesions including B-cell lymphomas.

Nuclear Interactor of ARF and Mdm2 (NIAM, gene designation Tbrg1) is a largely unstudied inhibitor of cell proliferation that helps maintain chromosomal stability. It is a novel activator of the ARF-Mdm2-Tip60-p53 tumor suppressor pathway as well as other undefined pathways important for genome maintenance. To examine its predicted role as a tumor suppressor, we generated NIAM mutant (NIAMm/m) mice homozygous for a β-galactosidase expressing gene-trap cassette in the endogenous gene. The mutant mice expressed significantly lower levels of NIAM protein in tissues compared to wild-type animals. Fifty percent of aged NIAM deficient mice (14 to 21 months) developed proliferative lesions, including a uterine hemangioma, pulmonary papillary adenoma, and a Harderian gland adenoma. No age-matched wild-type or NIAM+/m heterozygous animals developed lesions. In the spleen, NIAMm/m mice had prominent white pulp expansion which correlated with enhanced increased reactive lymphoid hyperplasia and evidence of systemic inflammation. Notably, 17% of NIAM mutant mice had splenic white pulp features indicating early B-cell lymphoma. This correlated with selective expansion of marginal zone B cells in the spleens of younger, tumor-free NIAM-deficient mice. Unexpectedly, basal p53 expression and activity was largely unaffected by NIAM loss in isolated splenic B cells. In sum, NIAM down-regulation in vivo results in a significant predisposition to developing benign tumors or early stage cancers. These mice represent an outstanding platform for dissecting NIAM's role in tumorigenesis and various anti-cancer pathways, including p53 signaling.

Imaging manifestations of unusual side effects of new anticancer medications

Aim To illustrate imaging manifestations of unusual side effects of some new anti-cancer medications. Many of the new anti-cancer medications target specific biologic pathways and are expected to cause less adverse effects on healthy tissues. Some of these agents are associated with unusual side effects, recognition of which is clinically important. Ipilimumab is a monoclonal antibody against cytotoxic T lymphocyte-associated antigen (CTLA)-4 used for the treatment of metastatic melanoma. Though it has been shown to increase overall survival rates, it also results in various immune-related adverse effects, one of which is hypophysitis. Crizotinib is an anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 inhibitor, approved for treatment of non-small cell lung carcinoma. Its side effects include the unusual formation of complex renal cysts which mimic renal abscesses or malignancy. In this presentation, we illustrate the imaging manifestations of Ipilimumab associated hypophysitis, early recognition of which is important as cessation of the drug in affected patients leads to resolution of hypophysitis, and also demonstrate complex renal cysts associated with the use of Crizotinib, knowledge of which is important to avoid misdiagnosis of renal abscess/ malignancy and consequent inappropriate treatment or stoppage of Crizotinib.

Biochemical analysis and antitumour effect of Abrus precatorius agglutinin derived peptides in Ehrlich's ascites and B16 melanoma mice tumour model

Anticancer and immunostimulatory properties of tryptic digest peptides of Abrus precatorius agglutinin protein (10kDAGP) have already been reported. Here attempt has been made to further validate anticancer properties of 10kDAGP peptides in Ehrlich's ascites carcinoma (EAC) and B16 melanoma (B16M) bearing mice models and to analyze 10kDAGP by anion exchange chromatography and RP-HPLC for obtaining the bioactive fraction from the total peptide pool. 10kDAGP treatment decreased the tumour pack volume by ∼82% for EAC and 58.8% for B16M. It also showed increase in ex vivo proliferation of splenocyte and thymocyte isolated from tumour bearing mice and increase in TNF-α and Interferon-γ in splenocyte culture supernatant. From chromatographic analysis it was found that anionic peptide fraction may be responsible for anti-proliferative activities of 10kDAGP. As most anticancer peptides are cationic in nature, further studies regarding bioactivity of anionic peptide fraction may lead to novel anticancer peptides and pathways of action.

Novel N-substituted sophoridinol derivatives as anticancer agents

Using sophoridine (1) as the lead compound, a series of new N-substituted sophoridinic acid derivatives were designed, synthesized and evaluated for their cytotoxicity. SAR analysis indicated that introduction of a chlorobenzyl on the 12-nitrogen atom of sophoridinol might significantly enhance the antiproliferative activity. Of the newly synthesized compounds, sophoridinol analogue 9k exhibited a potent effect against six human tumor cell lines (liver, colon, breast, lung, glioma and nasopharyngeal). The mode of action of 9k was to inhibit the DNA topoisomerase I activity, followed by the G0/G1 phase arrest. It also showed a moderate oral bioavailability and good safety in vivo. Therefore, compound 9k has been selected as a novel-scaffold lead for further structural optimizations or as a chemical probe for exploring anticancer pathways of this kinds of compounds.

Combinational Delivery of Hydrophobic and Hydrophilic Anticancer Drugs in Single Nanoemulsions To Treat MDR in Cancer

In this study, we developed the core-matched nanoemulsions (NEs) functionalized by vitamin E (VE) and tocopherol poly(ethylene glycol)succinate (TPGS) to codeliver hydrophobic and hydrophilic drugs, paclitaxel (PTX) and 5-fluoroucacil (5-FU), in order to achieve synergistic effects and overcome PTX resistance in a multi-drug-resistant (MDR) human epidermal carcinoma cell line KB-8-5. Antitumor effect of the combination therapy based on core-matched technology (CMT) was evaluated in vitro and in vivo in mice. The core-matched NEs showed entrapment efficiency of >90% and were of nanoscale particle size and negative zeta-potential. The combined core-matched NEs exhibited concentration and time-dependent cytotoxicity against PTX-sensitive KB-3-1 cells and PTX-resistant KB-8-5 cells as well as an obviously increased G2/M phase block. The improvements in therapeutic response over either PTX–VE or 5-FU–TPGS therapy alone were demonstrated by the ability to effectively induce the apoptosis of tumor cells via up-regulation of tumor suppressor p53 and β-tubulin and by the significant inhibition of cell cycle progression. The combination therapy led to dramatic inhibition of tumor growth with little toxicity in vivo, especially in the PTX-resistant KB-8-5 tumors, whereas Taxol had little therapeutic effect. This was mainly ascribed to the synergism of PTX and 5-FU and the reverse of MDR by the inhibition of ATPase activity by VE and TPGS. Coencapsulation of two chemotherapeutic agents with different mechanisms allows simultaneous interruption of diverse anticancer pathways, resulting in increased therapeutic response and low toxicity. The CMT markedly facilitated the long circulation of PTX and 5-FU, which was closely associated with the high accumulation of chemotherapeutic agents within the tumors and the improvement of antitumor efficacy. The current study demonstrated the feasibility of incorporating PTX and 5-FU targeting to different pathways into a single core-matched NE for the reversal of MDR and synergism in cancer therapy.

O-desmethylangolensin inhibits the proliferation of human breast cancer MCF-7 cells by inducing apoptosis and promoting cell cycle arrest

The aim of the present study was to investigate the anticancer effect of O-desmethylangolensin (O-DMA) by assessing cell proliferation, apoptosis and cell cycle distribution, as well as exploring the mechanisms underlying these effects in breast carcinoma MCF-7 cells. The cells were exposed to O-DMA (5–200 μM) for 24, 48 and 72 h. The results revealed that cell proliferation was significantly inhibited in a dose-dependent manner following treatment for 48 and 72 h, but not after 24 h, and resulted in the significant induction of apoptosis and the promotion of cell cycle arrest at the G1/S and G2/M phases. To elucidate these effects of O-DMA, the expression levels of cell cycle regulators were measured in the cells exposed to O-DMA at 150 μM for 72 h. Of the G1/S phase-related proteins, O-DMA modulated the cyclin-dependent kinases (CDKs), with a decrease in CDK2 and CDK4 and an increase in CDK6, and downregulated cyclin D and E. With respect to the G2/M-related proteins, O-DMA caused a reduction in CDK1, together with a slight increase in cyclin A and B. In addition, O-DMA downregulated p21Cip1 and p27Kip1, but not p16INK4a and p15INK4b, and interacted with the CDK6-cyclin D and CDK1-cyclin B complexes. In conclusion, these results indicate for the first time that the regulation of the CDK4/6-cyclin D and CDK1-cyclin B complexes may participate in the anticancer activity pathway of O-DMA in MCF-7 cells.

Combinational delivery of c-myc siRNA and nucleoside analogs in a single, synthetic nanocarrier for targeted cancer therapy

The treatment of aggressive non-small-cell lung cancer (NSCLC) depends on the creation of new therapeutic regimens in clinical settings. In this study, we developed a Lipid/Calcium/Phosphate (LCP) nanoparticle that combines chemotherapy with gene therapy. By encapsulating a chemodrug, gemcitabine monophosphate (GMP), and siRNA specific to the undruggable cMyc oncogene (cMyc siRNA) into a single nano-sized vesicle and systemically administering them to nude mice, we achieved potent anti-tumor activity in both subcutaneous and orthotopic models of NSCLC. The improvements in therapeutic response over either cMyc siRNA or GMP therapy alone, were demonstrated by the ability to effectively induce the apoptosis of tumor cells and the significant reduction of proliferation of tumor cells. The combination therapy led to dramatic inhibition of tumor growth, with little in vivo toxicity. Additionally, the current studies demonstrated the possibility of incorporating both nucleic acid molecules and phosphorylated small molecule drugs into the inner core of a single nanoparticle formulation. Co-encapsulation of an oncogene-modulating siRNA and a chemotherapeutic agent will allow simultaneous interruption of diverse anti-cancer pathways, leading to increased therapeutic efficacy and reduced toxicities.

Genome-wide Identification of IRF1 Binding Sites Reveals Extensive Occupancy at Cell Death Associated Genes.

IRF1 is a transcription factor involved in interferon signaling and has been shown to harbor tumor suppressor activity. In order to comprehensively identify pathways regulated by IRF1, we used chromatin immunoprecipitation followed by massive-parallel sequencing (ChIP-seq) to evaluate the gene targets of IRF1 genome-wide. We identified 17,416 total binding events in breast cancer cells. Functional categorization of the binding sites after IFN-gamma (interferon-gamma) treatment determined that ‘apoptosis’ or ‘cell death’ is the most enriched target process. Motif discovery analysis of the chromosomal regions bound by IRF1 identified a number of unique motifs correlated with apoptosis, DNA damage and immune processes. Analysis of GEO transcriptome data from IRF1-transduced cells or IFN-gamma treated fibroblasts indicates that IRF1-bound targets in IFN-treated cells are associated with a positive transcriptional response. Many of the enriched target genes from the expression analysis are associated with apoptosis. Importantly, this data indicates that a significant function of IRF1 is the regulation of anti-cancer apoptotic pathways and this reinforces IRF1’s role as a tumor suppressor.

Discovery of BRM Targeted Therapies: Novel Reactivation of an Anti-cancer Gene.

Drug discovery in the field of oncology has been advanced mainly through the targeting of receptor tyrosine kinases. Both antibodies and small molecule inhibitors have been found to have successful applications in blocking the proliferative functions of these cell surface receptors. Based on these early successes, additional kinases within the cytoplasm have been found to promote cancer and, as such, have been recognized as feasible targets for additional modes of therapies. Unlike these oncogene targets, most tumor suppressors are irreversibly altered during cancer progression and therefore are not feasible targets for therapy. However, a subset of these genes is reversibly epigenetically suppressed. One such gene is BRM, and when it is re-expressed in cancer cells, this gene halts their growth. Moreover, as the key catalytic subunit of the SWI/SNF complex, BRM is centrally important to a host of anticancer pathways and cellular mechanisms, and its status may serve as a biomarker. Restoring its expression will both reconnect a number of growth-controlling pathways and affect cellular adhesion, DNA repair, and immune functions. For these reasons, restoring BRM expression is not only feasible, but potentially a potent form of anticancer therapy. To identify BRM-restoring compounds, we developed a cell-based luciferase assay. In this review, we discuss some of the challenges we encountered, issues related to this type of drug discovery, and our future ambitions. We hope this review will provide insight to this type of endeavor and lead to more investigations pursuing this type of drug research.

In vivo modulation of 4E binding protein 1 (4E-BP1) phosphorylation by watercress: a pilot study

Dietary intake of isothiocyanates (ITC) has been associated with reduced cancer risk. The dietary phenethyl ITC (PEITC) has previously been shown to decrease the phosphorylation of the translation regulator 4E binding protein 1 (4E-BP1). Decreased 4E-BP1 phosphorylation has been linked to the inhibition of cancer cell survival and decreased activity of the transcription factor hypoxia-inducible factor (HIF), a key positive regulator of angiogenesis, and may therefore contribute to potential anti-cancer effects of PEITC. In the present study, we have investigated the in vitro and in vivo effects of watercress, which is a rich source of PEITC. We first demonstrated that, similar to PEITC, crude watercress extracts inhibited cancer cell growth and HIF activity in vitro. To examine the effects of dietary intake of watercress, we obtained plasma and peripheral blood mononuclear cells following the ingestion of an 80g portion of watercress from healthy participants who had previously been treated for breast cancer. Analysis of PEITC in plasma samples from nine participants demonstrated a mean maximum plasma concentration of 297nm following the ingestion of watercress. Flow cytometric analysis of 4E-BP1 phosphorylation in peripheral blood cells from four participants demonstrated significantly reduced 4E-BP1 phosphorylation at 6 and 8h following the ingestion of watercress. Although further investigations with larger numbers of participants are required to confirm these findings, this pilot study suggests that flow cytometry may be a suitable approach to measure changes in 4E-BP1 phosphorylation following the ingestion of watercress, and that dietary intake of watercress may be sufficient to modulate this potential anti-cancer pathway.

Targeting silibinin in the antiproliferative pathway

Importance of the field: Due to the failure and severe toxicity of cancer chemotherapy, silibinin, a natural flavonoid from the seeds of milk thistle, has recently received more attention for its potential anticancer and nontoxic roles in animals and humans. Silibinin has clearly demonstrated inhibition of multiple cancer cell signaling pathways, including growth inhibition, inhibition of angiogenesis, chemosensitization, and inhibition of invasion and metastasis. Cumulative evidence implicates that silibinin is a potential agent for cancer chemoprevention and chemotherapy.Areas covered in this review: Our aim is to discuss the recent progress of silibinin in regulating multiple anticancer proliferative signaling pathways; the review covers literature mainly from the past 3 – 5 years.What the reader will gain: One of the strategies for tumor therapy is eradication of cancer cells through targeting specific cell-proliferative pathways. This review highlights the current knowledge of silibinin in regulating multiple cellular proliferative pathways in cancer cells, including receptor tyrosine kinases, androgen receptor, STATs, NF-κB, cell cycle regulatory and apoptotic signaling pathways.Take home message: The molecular mechanisms of silibinin-mediated antiproliferative effects are mainly via receptor tyrosine kinases, androgen receptor, STATs, NF-κB, cell cycle regulatory and apoptotic signaling pathways in various cancer cells. Targeting inhibition of proliferative pathways through silibinin treatment may provide a new approach for improving chemopreventive and chemotherapeutic effects.

Emodin inhibits the growth of hepatoma cells: Finding the common anti-cancer pathway using Huh7, Hep3B, and HepG2 cells

Emodin—a major component of Rheum palmatum L.—exerts antiproliferative effects in cancer cells that are regulated by different signaling pathways. Hepatocellular carcinoma has high-incidence rates and is associated with poor prognosis and high mortality rates. This study was designed to evaluate the effects of emodin on human hepatocarcinoma cell viability and investigate its mechanisms of action in Huh7, Hep3B, and HepG2 cells. To define the molecular changes associated with this process, expression profiles were compared in emodin-treated hepatoma cells by cDNA microarray hybridization, quantitative RT-PCRs, and Western blot analysis. G2/M phase arrest was observed in all 3 cell lines. Cell cycle regulatory gene analysis showed increased protein levels of cyclin A, cyclin B, Chk2, Cdk2, and P27 in hepatoma cells after time courses of emodin treatment, and Western blot analysis showed decreased protein levels of Cdc25c and P21. Microarray expression profile data and quantitative PCR revealed that 15 representative genes were associated with emodin treatment response in hepatoma cell lines. The RNA expression levels of CYP1A1, CYP1B1, GDF15, SERPINE1, SOS1, RASD1, and MRAS were upregulated and those of NR1H4, PALMD, and TXNIP were downregulated in all three hepatoma cells. Moreover, at 6 h after emodin treatment, the levels of GDF15, CYP1A1, CYP1B1, and CYR61 were upregulated. Here, we show that emodin treatment caused G2/M arrest in liver cancer cells and increased the expression levels of various genes both in mRNA and protein level. It is likely that these genes act as biomarkers for hepatocellular carcinoma therapy.

Emodin azide methyl anthraquinone derivative triggers mitochondrial-dependent cell apoptosis involving in caspase-8-mediated Bid cleavage

AMAD, an emodin azide methyl anthraquinone derivative, was extracted from the nature giant knotweed rhizome of traditional Chinese herbs. Here, we investigated the anticancer activities and signaling pathways implicated in AMAD-induced apoptosis in human breast cancer cell lines MDA-MB-453 and human lung adenocarcinoma Calu-3 cells. AMAD was found to have a potent cytotoxic effect on both cell lines. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. Moreover, this apoptotic induction was associated with a collapse of the mitochondrial membrane potential and activated caspases (cysteine aspartase) cascade involving in caspase-8, caspase-9, caspase-3, and poly(ADP-ribose) polymerase cleavage in a concentration-dependent manner. It was noteworthy that AMAD also effectively cleaved Bid, a BH3 domain-containing proapoptotic Bcl-2 family member, and induced the subsequent release of cytochrome c from mitochondria into the cytosol. Furthermore, suppression of caspase-8 activity with Z-IETD-FMK partially inhibited release of cytochrome c and Bid cleavage induced by AMAD, whereas exposure to Z-LETD-FMK, a caspase-9 inhibitor, had no effect. Additionally, there was significant change in other mitochondrial membrane proteins triggered by AMAD, such as Bcl-xl and Bad. It was intriguing that AMAD decreased the generation of reactive oxygen species in both cell lines. DNA-binding assay exhibited apoptosis induced by AMAD was not involved in intercalating to DNA. Taken together, these data suggested that AMAD induced apoptosis via a mitochondrial pathway involving caspase-8/Bid activation in both cell lines.

Cell Signaling/Signal Transduction

Cell Signaling/Signal Transduction

Hesperetin Induced G1-Phase Cell Cycle Arrest in Human Breast Cancer MCF-7 Cells: Involvement of CDK4 and p21

This study was to investigate the effects of hesperetin on cell proliferation and cell cycle arrest and explored the mechanism for these effects in breast carcinoma MCF-7 cells. Hesperetin significantly inhibited cell proliferation in a dose-dependent manner after treatment for 48 and 72 h and resulted in significant cell cycle arrest in the G1 phase. In the G1-phase related proteins, hesperetin downregulates the cyclin-dependent kinases (CDKs) and cyclins and upregulates p21Cip1 and p27 Kip1 in cells treated with hesperetin for 48 h and 72 h. After 72 h treatment, these phenomenons were more pronounced. Hesperetin treatment at high concentration for 72 h resulted in a decrease in CDK2 and CDK4 together with cyclin D. In addition, hesperetin increases the binding of CDK4 with p21 Cip1 but not p27 Kip1 or p57K ip2 . Taken together, our data suggest for the first time that the regulation of CDK4 and p21 Cip1 may participate in the anticancer activity pathway of hesperetin in MCF-7 cells.