Cranberry proanthocyanidins mediate growth arrest of lung cancer cells through modulation of gene expression and rapid induction of apoptosis

Cranberries are rich in bioactive constituents purported to enhance immune function, improve urinary tract health, reduce cardiovascular disease and more recently, inhibit cancer in preclinical models. However, identification of the cranberry constituents with the strongest cancer inhibitory potential and the mechanism associated with cancer inhibition by cranberries remains to be elucidated. This study investigated the ability of a proanthocyanidin rich cranberry fraction (PAC) to alter gene expression, induce apoptosis and impact the cell cycle machinery of human NCI-H460 lung cancer cells. Lung cancer is the leading cause of cancer-related deaths in the United States and five year survival rates remain poor at 16%. Thus, assessing potential inhibitors of lung cancer-linked signaling pathways is an active area of investigation.     

Triptolide sensitizes lung cancer cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by inhibition of NF-kappaB activation

TNF-related apoptosis-inducing ligand (TRAIL/Apo- 2L), a newly identified member of the TNF family promotes apoptosis by binding to the transmembrane receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). TRAIL known to activate NF-kappaB in number of tumor cells including A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells exerts relatively selective cytotoxic affects to the human tumor cell lines without much effect on the normal cells. We set out to identify an agent that would sensitize lung cancer cells to TRAIL-induced apoptosis through inhibition of NF-kappaB activation. We found that triptolide, an oxygenated diterpene extracted and purified from the Chinese herb Tripterygium wilfordii sensitized A549 and NCI-H1299 cells to TRAIL-induced apoptosis through inhibition of NF-kappaB activation. Pretreatment with MG132 which is a well-known NF-kappaB inhibitor by blocking degradation of IkappaBalpha also greatly sensitized lung cancer cells to TRAIL-induced apoptosis. Triptolide did not block DNA binding of NF-kappaB activated by TRAIL as in the case of TNF-alpha. It has been already proven that triptolide blocks transactivation of p65 which plays a key role in NF-kappaB activation. These observations suggest that triptolide may be a potentially useful drug to enhance TRAIL-induced tumor killing in lung cancer.     

Sintokamides A to E, chlorinated peptides from the sponge Dysidea sp. that inhibit transactivation of the N-terminus of the androgen receptor in prostate cancer cells

The new chlorinated peptides sintokamides A to E (1-5) have been isolated from specimens of the marine sponge Dysidea sp. collected in Indonesia. Their structures were elucidated by a combination of spectroscopic and single-crystal X-ray diffraction analyses. Sintokamide A (1) is an inhibitor of N-terminus transactivation of the androgen receptor in prostate cancer cells.     

A naphthalimide-based peptide conjugate for concurrent imaging and apoptosis induction in cancer cells by utilizing endogenous hydrogen sulfide

The excessive production of endogenous hydrogen sulfide (H₂S) in cancer cells leads to enhanced tumor growth and metastasis. On the other hand, decreased endogenous H₂S suppresses tumor growth. The reported approaches for inhibiting tumor growth are selective silencing of the tumor-promoting genes and pharmacological inhibition of these proteins. To enhance the antitumor efficacy of frontline chemotherapeutic agents, herein, we synthesized a highly sensitive endogenous H₂S responsive fluorescent probe, i.e., a hydrogen sulfide-sensing naphthalimide-based peptide conjugate (HSNPc), which showed selective inhibition of proliferation of cancer cells due to apoptosis induction. Furthermore, HSNPc suppressed the glycolytic reserve, a critical energy source for the proliferation of cancer cells. HSNPc also decreased the Young''s modulus of HeLa cells compared to the control cells, which demonstrated a direct relation between cell apoptosis and cell stiffness. Taken together, we demonstrated the dual function of detection and killing of cancer cells by HSNPc that can be likened to a theranostic role.

A hydrogen sulfide sensing naphthalimide based peptide conjugate (HSNPc) worked as a novel cancer cell imaging agent and showed selective cell apoptosis.     

Photo-activated 5-hydroxyindole-3-acetic acid induces apoptosis of prostate and bladder cancer cells

5-Hydroxyindole-3-acetic acid (5-HIAA), an indole derivative, is the main metabolite of serotonin in the human body. We determined whether or not ultraviolet B (UVB)-activated 5-HIAA (5-HIAA(UVB)) affects the viability of human prostate (LnCaP and PC-3) and bladder cancer cells (TCCSUP). While 5-HIAA alone had no cytotoxic effect at <1mM, 5-HIAA(UVB) induced LnCaP, PC-3, and TCCSUP cell death in a time- and dose-dependent manner. Cell cycle analysis showed that 5-HIAA(UVB) markedly increased the sub-G(0)/G(1) phase and resulted in cell cycle disruption. To elucidate the death mechanism by 5-HIAA(UVB), we examined the signal transduction pathways related to apoptosis using Western blot analysis. 5-HIAA(UVB) led to phosphorylation of stress-activated signaling proteins, such as c-Jun N-terminal kinase (JNK) and/or p38 mitogen-activated protein kinase (MAPK). Furthermore, 5-HIAA(UVB) activated caspase-8, -9, and -3 and cleaved poly(ADP-ribose) polymerase (PARP), which are indicators of apoptosis. From these findings, the present study demonstrated that 5-HIAA(UVB) induces apoptotic cell death of prostate and bladder cancer cells via stress-mediated signaling and apoptotic pathways. Therefore, we suggest that 5-HIAA might be used as a new photosensitizer for photodynamic cancer therapy.     

Structure-activity relationship of Triterpenes and derived Glycosides against cancer cells and mechanism of apoptosis induction

Triterpenoids possess a wide range of biological effects. Here, the cytotoxic activities of 55 triterpenes and derived glycosides against BEL-7404 and SGC-7901 cells were assessed, and structure-activity relationships were analysed accordingly. Nine of them effectively inhibited the two cell lines. In particular, compounds 49 and 52 inhibited BEL-7404 cells as efficiently as 5’-fluorouracil (IC₅₀ values 0.46 and 1.48, respectively). Moreover, we found that compounds 49 and 52 induced apoptosis in BEL-7404 cells. Indeed, DNA fragmentation assay showed a time-dependent degradation of DNA after treatment of cells with compounds 49 and 52. In addition, Bax gene expression levels were increased after treatment with these compounds, in a concentration-dependent manner. Taken together, our findings suggested that compounds 49 and 52 induce apoptosis in BEL-7404 cells by upregulating the Bax gene without affecting Bcl-2 gene expression.     

Toll-like receptor 9-mediated inhibition of apoptosis occurs through suppression of FoxO3a activity and induction of FLIP expression

Synthetic oligodeoxynucleotides (ODN) with a CpG-motif are recognized by Toll-like receptor 9 (TLR9) and pleiotropic immune responses are elicited. Stimulation of macrophages with TLR9 agonist prevented apoptosis induced by serum deprivation through increased expression of FLICE-like inhibitory protein (FLIP). CpG ODN-mediated anti-apoptosis depended on the TLR9-Akt-FoxO3a signaling pathway. Inhibition of TLR9 by small interfering (si) RNA or an inhibitor suppressed CpG ODN-mediated anti-apoptosis. Analysis of signaling pathways revealed that the anti-apoptotic effect of CpG ODN required phosphorylation of FoxO3a and its translocation from the nucleus to the cytosol. Overexpression of FoxO3a increased apoptosis induced by serum deprivation and CpG ODN blocked these effects through FLIP expression. In contrast, siRNA knock-down of FoxO3a decreased apoptosis by serum deprivation. In addition, Akt activation was involved in CpG ODN-induced phosphorylation of FoxO3a, expression of FLIP, and anti-apoptosis. Taken together, these results demonstrate the involvement of Akt-FoxO3a in TLR9-mediated anti-apoptosis and indicate that FoxO3a is a distinct regulator for FLIP expression.     

Dose-dependent induction of apoptosis or necrosis in human cells by organotin compounds

In the present study, the mode of cell death induced by highly toxic trialkylated tin compounds has been evaluated. Treatment of undifferentiated HL-60 cells with submicromolar to micromolar concentrations of tri-n-butyltin (TBT) led only to a slight decrease in cell viability measured with trypan blue exclusion. Nevertheless, cell membrane blebbing was observed by means of light microscopy and condensation of nuclear chromatin and formation of apoptotic bodies was demonstrated in Hoechst 33342 stained cells. The nuclear chromatin condensation was associated with an extensive DNA fragmentation. Visualized by agarose gel electrophoresis, genomic DNA appeared as a characteristic ladder-like pattern of DNA fragments which is the biochemical hallmark of apoptosis. The typical internucleosomal DNA digestion was concentration-dependent and began within 2 to 3 h of incubation. During the incubation period a persistent and steady elevation of intracellular free calcium concentration ([Ca²⁺]_i) could be detected. Furthermore, the chromatin condensation and DNA fragmentation could be blocked by supplementation of the incubation medium with zinc pointing to an activation of a zinc-sensitive and calcium-dependent endogenous endonuclease. Higher concentrations of tributyltin (≥ 5 μmol/L TBT) led within hours to a cell killing with degenerative changes indicative of necrosis, demonstrated by plasma membrane disruption which was accompanied by random DNA breakdown. Furthermore, these concentrations also provoked a persistent elevation in [Ca²⁺]_i which reached, even after 10 min, higher levels in comparison with apoptosis-inducing concentrations. The loss in membrane integrity observed at these concentrations of TBT could be due to an activation of calcium-dependent phospholipases. Here it is shown that activation of cytosolic phospholipase A₂ (cPLA₂) leads to liberation of arachidonic acid (AA) out of the phospholipid membrane. The results presented here demonstrate that organometals are able to induce different cell death pathways depending on the applied concentration: low concentrations led to apoptosis whereas high concentrations stimulate necrosis. We suggest that there exists a direct correlation between the intracellular free calcium concentration and the mode of cell death. Received: 1 August 1997 / Revised: 8 October 1997 / Accepted: 10 October 1997     

Dose-dependent induction of apoptosis or necrosis in human cells by organotin compounds

In the present study, the mode of cell death induced by highly toxic trialkylated tin compounds has been evaluated. Treatment of undifferentiated HL-60 cells with submicromolar to micromolar concentrations of tri-n-butyltin (TBT) led only to a slight decrease in cell viability measured with trypan blue exclusion. Nevertheless, cell membrane blebbing was observed by means of light microscopy and condensation of nuclear chromatin and formation of apoptotic bodies was demonstrated in Hoechst 33342 stained cells. The nuclear chromatin condensation was associated with an extensive DNA fragmentation. Visualized by agarose gel electrophoresis, genomic DNA appeared as a characteristic ladder-like pattern of DNA fragments which is the biochemical hallmark of apoptosis. The typical internucleosomal DNA digestion was concentration-dependent and began within 2 to 3 h of incubation. During the incubation period a persistent and steady elevation of intracellular free calcium concentration ([Ca²⁺]_i) could be detected. Furthermore, the chromatin condensation and DNA fragmentation could be blocked by supplementation of the incubation medium with zinc pointing to an activation of a zinc-sensitive and calcium-dependent endogenous endonuclease. Higher concentrations of tributyltin (≥ 5 μmol/L TBT) led within hours to a cell killing with degenerative changes indicative of necrosis, demonstrated by plasma membrane disruption which was accompanied by random DNA breakdown. Furthermore, these concentrations also provoked a persistent elevation in [Ca²⁺]_i which reached, even after 10 min, higher levels in comparison with apoptosis-inducing concentrations. The loss in membrane integrity observed at these concentrations of TBT could be due to an activation of calcium-dependent phospholipases. Here it is shown that activation of cytosolic phospholipase A₂ (cPLA₂) leads to liberation of arachidonic acid (AA) out of the phospholipid membrane. The results presented here demonstrate that organometals are able to induce different cell death pathways depending on the applied concentration: low concentrations led to apoptosis whereas high concentrations stimulate necrosis. We suggest that there exists a direct correlation between the intracellular free calcium concentration and the mode of cell death. Received: 1 August 1997 / Revised: 8 October 1997 / Accepted: 10 October 1997     

A study of DNA tethered to a surface by an all-atom molecular dynamics simulation

 In order to understand the structure of DNAs and their interactions when on microarray surfaces, we performed the first all-atom molecular dynamics simulation of DNA tethered to a surface. On the surface, the binding of the DNA was enhanced, and its average equilibrium conformation was the B form. The DNA duplex spontaneously tilted towards its nearest neighbor and settled in a leaning position with a interaxial distance of 2.2 nm. This close packing of the DNAs, which affects both in situ synthesis and deposition of probes on microarray surfaces, can thus be explained by salted-induced colloidlike DNA–DNA attractions. Received: 30 November 2000 / Accepted: 7 February 2001 / Published online: 22 May 2001     

p53 overexpression represses androgen-mediated induction of NKX3.1 in a prostate cancer cell line

Prostate cancer is a disease involving complicated multiple-gene alterations. Both NKX3.1 and p53 are related to prostate cancer and play crucial roles in prostate cancer progression. However, little is known about the relationships and interactions between p53 and NKX3.1 in prostate cancer. We found that NKX3.1 expression is down-regulated by over-expression of wild type (wt) p53 in prostate cancer LNCaP cells. NKX3.1 is down-regulated at both the mRNA and protein levels by p53 over- expression due to either transient transfection of exogenous p53 or induction of endogenous p53. p53 over-expression represses androgen-induced transactivation of NKX3.1 by inhibiting the promoter of the androgen acceptor (AR) gene and by blocking AR-DNA binding activity. In addition, transfection with the p21 expression vector (pPSA-p21) showed that p21 does not reduce NKX3.1 expression, indicating that NKX3.1 expression is not the result of nonspecific effects of cell growth arrest. Our results provide biochemical and cellular biologic evidence that NKX3.1 is down-regulated by p53 over-expression in prostate cancer cells.     

Monitoring intracellular melatonin levels in human prostate normal and cancer cells by HPLC

Melatonin (N-acetyl-5-methoxytryptamine) is a potent endogenous antioxidant and free radical scavenger that has attracted much attention as a consequence of its multiple biological functions. In addition to other physiological properties, it has clear antiproliferative activity in several types of cancer cell. The concentration of melatonin necessary to inhibit cell growth is much higher than its blood physiological concentrations in some tumor types. For years its indolic nature has impeded proper monitoring, by molecular or immunological techniques, of its uptake by cancer cells. In this work we developed a simple, rapid, and validated analytical method for detection and quantification of MEL inside normal and cancer cells. For this purpose we performed high-performance liquid chromatographic analysis after liquid–liquid extraction of the indole from biological samples. The method was validated, and the correlation coefficient for amounts from 0.125 to 1.25 μg was higher than 0.999, with a range of recovery near 100%. Precision was evaluated as repeatibility, and for intermediate precision, the relative standard deviation was less than 5%. The method was used to study the stability of the indole in solution and to determine intracellular melatonin concentrations in normal (PNT1A) and several cancer (LNCaP, DU-145, PC-3) prostate cell lines. Intracellular LOQ/LOD were 7.23/2.83, 23.17/9.07, 4.03/1.83, and 6.51/2.53 nmol L⁻¹, or 1.82/4.66, 0.56/1.45, 3.26/8.34, and 2.02/5.17 attogram in each cell in PNT1A, LNCaP, DU145, and PC-3 cells, respectively. Because there was no information about intracellular levels of melatonin inside normal or tumor prostate cells after treatment with the indole, nor a relationship between its antiproliferative activity and its intracellular concentration, this is the first time that, by using an analytical method combined with measurement of cellular volume by flow cytometry, the intracellular concentration of MEL has been estimated. Also, data obtained here explain why the antiproliferative properties of MEL vary in different cell types. This is, moreover, the first time that by increasing the intracellular concentration of melatonin, its antitumor properties have been promoted in prostate cancer cells. This process can be monitored by the method developed here.     

Exploring a model of a chemokine receptor/ligand complex in an explicit membrane environment by molecular dynamics simulation: the human CCR1 receptor

The seven transmembrane helices G-protein-coupled receptors (GPCRs) form one of the largest superfamilies of signaling proteins found in humans. Homology modeling, molecular docking, and molecular dynamics (MD) simulation were carried out to construct a reliable model for CCR1 as one of the GPCRs and to explore the structural features and the binding mechanism of BX471 as one of the most potent CCR1 inhibitors. In this study, BX471 has been docked into the active site of the CCR1 protein. After docking, one 20 ns MD simulation was performed on the CCR1-ligand complex to explore effects of the presence of lipid membrane in the vicinity of the CCR1-ligand complex. At the end of the MD simulation, a change in the position and orientation of the ligand in the binding site was observed. This important observation indicated that the application of MD simulation after docking of ligands is useful. Explorative runs of molecular dynamics simulation on the receptor-ligand complex revealed that except for Phe85, Phe112, Tyr113, and Ile259, the rest of the residues in the active site determined by docking are changed. The results obtained are in good agreement with most of the experimental data reported by others. Our results show that molecular modeling and rational drug design for chemokine targets is a possible approach.     

Antiproliferative and molecular mechanism of eugenol-induced apoptosis in cancer cells

Phenolic phytochemicals are a broad class of nutraceuticals found in plants which have been extensively researched by scientists for their health-promoting potential. One such a compound which has been comprehensively used is eugenol (4-allyl-2-methoxyphenol), which is the active component of Syzigium aromaticum (cloves). Aromatic plants like nutmeg, basil, cinnamon and bay leaves also contain eugenol. Eugenol has a wide range of applications like perfumeries, flavorings, essential oils and in medicine as a local antiseptic and anesthetic. Increasing volumes of literature showed eugenol possesses antioxidant, antimutagenic, antigenotoxic, anti-inflammatory and anticancer properties. Molecular mechanism of eugenol-induced apoptosis in melanoma, skin tumors, osteosarcoma, leukemia, gastric and mast cells has been well documented. This review article will highlight the antiproliferative activity and molecular mechanism of the eugenol induced apoptosis against the cancer cells and animal models.     

Computer‐aided design of a novel ligand for retinoic acid receptor in cancer chemotherapy

The isotypes of RAR and RXR are retinoic acid and retinoid X acid receptors, respectively, whose ligand‐binding domain contains the ligand‐dependent activation function, with distinct pharmacological targets for retinoids, involved in the treatment of various cancers and skin diseases. Due to the major challenge which cancer treatment and cure still imposes after many decades to the international scientific community, there is actually considerable interest in new ligands with increased bioactivity. We have focused on the retinoid acid receptor, which is considered an interesting target for drug design. In this work, we carried out density functional geometry optimizations and different docking procedures. We performed screening in a large database (hundreds of thousands of molecules which we optimized at the AM1 level) yielding a set of potential bioactive ligands. A new ligand was selected and optimized at the B3LYP/6‐31G* level. A flexible docking program was used to investigate the interactions between the receptor and the new ligand. The result of this work is compared with several crystallographic ligands of RAR. Our theoretically more bioactive new ligand indicates stronger and more hydrogen bonds as well as hydrophobic interactions with the receptor. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005     

Medium benzene-fused oxacycles with the 5-fluorouracil moiety: synthesis, antiproliferative activities and apoptosis induction in breast cancer cells

On the basis of the increase in lipophilicity, novel benzannelated six- and seven-membered derivatives have been synthesized, starting from 2-hydroxybenzyl alcohols, 2-hydroxybenzaldehydes, and catechol. The X-ray structure of (RS)-1-(2,3-dihydro-5H-1,4-benzodioxepin-3-yl)-5-fluorouracil (5) is presented and compared with its conformational analysis at the semiempirical (AM1) and ab initio (6-31G^∗) levels and NOE effects. A good agreement between both experimental and theoretical data was found showing a chair conformation for the 2,3-dihydro-5H-1,4-dioxepin ring and an axial orientation of the 5-FU moiety on the C3 position. Compounds 5 and (RS)-1-(7-methoxy-2,3-dihydro-5H-1,4-benzodioxepin-3-yl)-5-fluorouracil (6) were found to be the most potent inhibitor of MCF-7 cells growth. (RS)-1-(2,3-Dihydrobenzoxepin-2-yl)-5-fluorouracil (8) induced apoptosis up to 57.33% of cell population after 24 h.     

Time course of expression of the retinoid X receptor gene and induction of imposex in the rock shell, Thais clavigera, exposed to triphenyltin chloride

To examine the role of the retinoid X receptor (RXR) in the development of imposex in gastropods, we investigated the time course of expression of the RXR gene in various tissues (ctenidium, ovary or testis, digestive gland, penis-forming area or penis, and head ganglia) of female and male rock shells (Thais clavigera) exposed to triphenyltin (TPT) in a flow-through exposure system for 3 months. Accumulations of TPT in tissues were clearly observed in exposed individuals, whereas no accumulation of TPT was observed in the control groups. In females, 3-month exposure to TPT resulted in the development of imposex, and penis lengths in imposex-exhibiting females were significantly longer in small females (shell height <20 mm) than in large females (shell height ≥20 mm). RXR gene expression in the ovary, penis-forming area or penis, and head ganglia of females exposed for 3 months was significantly higher than expression in control females, and the highest RXR gene expression was found in the penis-forming area or penis. Moreover, RXR gene expression in the penis-forming area or penis of each female exposed to TPT seemed to be associated with an increase in penis length. In males, the ratio of penis length to shell height was significantly larger in the exposed groups than in the controls. Although RXR gene expression in males exposed for 3 months was not significantly higher than expression in control males in any tissues, the highest gene expression was observed in the penis of exposed males. These results suggest that RXR plays an important role in the development of male genitalia (i.e., penis and vas deferens) in gastropods, although RXR might also have other physiological functions.      

Differential effects of FR900482 and FK317 on apoptosis, IL-2 gene expression, and induction of vascular leak syndrome

Vascular leak syndrome (VLS) is a harmful side effect that resulted in withdrawal of the antitumor drug FR900482, but not FK317, from clinical trials. Here we present chromatin immunoprecipitation data showing that FK317, like FR900482, crosslinks minor-groove binding proteins to DNA in vivo. However, these drugs differ in how they induce cell death. We demonstrate that, whereas FR900482 induces necrosis, FK317 induces a necrosis-to-apoptosis switch that is drug concentration dependent. Northern blot analyses of drug-treated cells suggest that this "switch" is mediated, at least in part, by modulation of the expression levels of Bcl-2. Additionally, FR900482, in contrast to FK317, induces the expression of known elicitors of both Bcl-2 gene expression and VLS. These findings provide plausible explanations for why these structurally similar drugs have different biological effects, especially with respect to VLS.