Antiproliferative activity and induction of apoptosis in human colon cancer cells treated in vitro with constituents of a product derived from Pistacia lentiscus L. var. chia.

In this report, we demonstrate that a 50% ethanol extract of the plant-derived product, Chios mastic gum (CMG), contains compounds which inhibit proliferation and induce death of HCT116 human colon cancer cells in vitro. CMG-treatment induces cell arrest at G(1), detachment of the cells from the substrate, activation of pro-caspases-8, -9 and -3, and causes several morphological changes typical of apoptosis in cell organelles. These events, furthermore, are time- and dose-dependent, but p53- and p21-independent. Apoptosis induction by CMG is not inhibited in HCT116 cell clones expressing high levels of the anti-apoptotic protein, Bcl-2, or dominant-negative FADD, thereby indicating that CMG induces cell death via a yet-to-be identified pathway, unrelated to the death receptor- and mitochondrion-dependent pathways. The findings presented here suggest that CMG (a) induces an anoikis form of cell death in HCT116 colon cancer cells that includes events associated with caspase-dependent pathways; and (b) might be developed into a chemotherapeutic agent for the treatment of human colon and other cancers.

Differential susceptibility to etoposide in clones derived from a human ovarian cancer cell line.

OBJECTIVES: To identify parameters/factors that may contribute to the differential sensitivity to etoposide in two clones isolated from the human ovarian carcinoma SKOV-3 cell line, which does not express p53 and is resistant to platinum-based regimens. METHODS: Differential sensitivity of the cells to etoposide was monitored by microscopy to observe morphological changes, by flow cytometry analyses to detect cell cycle perturbations, and by molecular/biochemical assays to identify events involved in induction of apoptosis. RESULTS: Etoposide treatment (1) induced apoptosis in one clone, ES, but not in another clone, ER, (2) had no effect on the expression of the antiapoptotic proteins Bcl-2 and Bcl-X(L) in both cell clones, whereas the proapoptotic proteins Bak and Bax were dramatically upregulated in ES, but not ER cells, and (3) induced more extensive processing of procaspase-8, procaspase-9, and the caspase-3-targeted substrates, topoisomerase I and PARP, in ES cells. Ectopic overexpression of Bcl-2 in ES cells failed to inhibit etoposide-induced apoptosis. CONCLUSIONS: The differential susceptibility of ES and ER cells to etoposide-induced apoptosis is associated with differences in several events rather than with a specific single genetic regulator of the apoptotic machinery. We propose that the differential response of ovarian cancer patients to etoposide treatment is associated with the number of etoposide-sensitive cells in the tumor.

Helix 6 of tBid is necessary but not sufficient for mitochondrial binding activity.

The apoptosis effector Bid regulates cell death at the level of mitochondrial cytochrome c efflux. Bid consists of 8 alpha-helices (designated H1 through H8, respectively) and is a soluble cytosolic protein in its native state. Proteolysis of the N-terminus (encompassing H1 and H2) of Bid yields activated "tBid" (truncated Bid), which translocates to the mitochondria and induces the efflux of cytochrome c. Here, we demonstrate that helix H6 of tBid is necessary, albeit not sufficient, for mitochondrial binding. In particular, a 33 amino acid long domain, which encompassed H6 and H7, behaved as the minimum domain in tBid that was sufficient for mitochondrial binding. Unexpectedly, the hydrophobic surface of these helices could be mutated without altering the binding activity of the domain, implying that the secondary structure of the helices may be the key determinant of binding. These experiments expand our mechanistic understanding of the apoptotic regulator, tBid.

Induction of apoptosis in 9-nitrocamptothecin-treated DU145 human prostate carcinoma cells correlates with de novo synthesis of CD95 and CD95 ligand and down-regulation of c-FLIP(short).

Stimulation of CD95 leads to oligomerization of this receptor and the recruitment of the Fas-associated death domain (FADD) and procaspase-8 to form the death-inducing signaling complex (DISC). Subsequent proteolytic activation of caspase-8 at the DISC leads to the activation of downstream caspases and execution of apoptosis. The anticancer drug 9-nitrocamptothecin (9NC) inhibits the nuclear enzyme topoisomerase I (Top1), an event followed by apoptosis of cancer cells. We investigated whether other mechanisms downstream of the DNA-Top1-9NC complexing step regulate the apoptotic ability of 9NC in DU145 cells. We demonstrate that induction of apoptosis in DU145 cells, upon exposure to 9NC, is associated with de novo expression of CD95 and CD95L, suggesting that 9NC-induced apoptosis is mediated by the CD95 system. In this line, we observed early activation of procaspase-3, -7, and -8, but not -1, -9, and -10. Moreover, 9NC treatment resulted in the dramatic down-regulation of c-FLIP(short) expression, but not that of c-FLIP(long) or FADD. Furthermore, incubation of DU145 cells with a neutralizing antibody (NOK-1) to CD95L or transient transfection of a c-FLIP(short) expression vector into DU145 cells partially abrogated 9NC-triggered apoptosis. We propose that 9NC triggers apoptosis by driving DU145 cells from a nonapoptotic status (c-FLIP(short)(high), CD95(low), CD95L(low)) toward a proapoptotic status (c-FLIP(short)(low), CD95(high), CD95L(high)). These findings indicate that in addition to a Top1-mediated effect, 9NC can additionally activate a CD95/CD95L-dependent apoptotic pathway.

Raf kinase inhibitor protein interacts with NF-kappaB-inducing kinase and TAK1 and inhibits NF-kappaB activation.

The Raf kinase inhibitor protein (RKIP) acts as a negative regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by Raf-1. RKIP inhibits the phosphorylation of MAP/extracellular signal-regulated kinase 1 (MEK1) by Raf-1 by disrupting the interaction between these two kinases. We show here that RKIP also antagonizes the signal transduction pathways that mediate the activation of the transcription factor nuclear factor kappa B (NF-kappaB) in response to stimulation with tumor necrosis factor alpha (TNF-alpha) or interleukin 1 beta. Modulation of RKIP expression levels affected NF-kappaB signaling independent of the MAPK pathway. Genetic epistasis analysis involving the ectopic expression of kinases acting in the NF-kappaB pathway indicated that RKIP acts upstream of the kinase complex that mediates the phosphorylation and inactivation of the inhibitor of NF-kappaB (IkappaB). In vitro kinase assays showed that RKIP antagonizes the activation of the IkappaB kinase (IKK) activity elicited by TNF-alpha. RKIP physically interacted with four kinases of the NF-kappaB activation pathway, NF-kappaB-inducing kinase, transforming growth factor beta-activated kinase 1, IKKalpha, and IKKbeta. This mode of action bears striking similarities to the interactions of RKIP with Raf-1 and MEK1 in the MAPK pathway. Emerging data from diverse organisms suggest that RKIP and RKIP-related proteins represent a new and evolutionarily highly conserved family of protein kinase regulators. Since the MAPK and NF-kappaB pathways have physiologically distinct roles, the function of RKIP may be, in part, to coordinate the regulation of these pathways.

9-Nitrocamptothecin is an effective drug for the treatment of human lung tumors: comparison of in vitro and in vivo studies.

9-Nitrocamptothecin (9NC) results in complete regression of small-cell lung carcinoma (SCLC) and non-SCLC (NSCLC) growing as xenografts in immunodeficient mice. In this study, we have monitored histological changes in the tumors during 9NC-induced regression, and perturbations in the cell cycle of cells derived from these tumors using flow cytometry. In vivo, 9NC treatment induces dramatic changes in the tumor cells, which die by apoptosis and are ultimately eliminated from the normal tissue. In vitro, 9NC treatment resulted in apoptosis and cytostasis of the NSCLC and SCLC cells, respectively. Further, 9NC induced cytostasis in control, normal human lung fibroblasts. Therefore, the studies in vivo have indicated that 9NC acquires a remarkable antitumor activity against both the SCLC and NSCLC types tested, and that results of studies in vitro may not reflect the results observed in vivo.

A Fas-associated death domain protein-dependent mechanism mediates the apoptotic action of non-steroidal anti-inflammatory drugs in the human leukemic Jurkat cell line.

Non-steroidal anti-inflammatory drugs (NSAIDs) are inhibitors of cyclooxygenase-1 and -2 and are useful for prevention and cure of cancers, especially colon and rectal cancers. The NSAIDs indomethacin and sulindac sulfide have been shown to induce apoptosis of colon epithelial cancer cells by a Bax-dependent mechanism that involves mitochondria-mediated activation of a caspase-9-dependent pathway. In this report, we demonstrate that indomethacin and sulindac sulfide induce apoptosis of human leukemic Jurkat cells by a mechanism that requires the Fas-associated Death Domain Protein-mediated activation of a caspase-8-dependent pathway. Therefore, NSAIDs induce apoptosis by different mechanisms depending on the cell type.

c-Myc is necessary for DNA damage-induced apoptosis in the G(2) phase of the cell cycle.

The c-myc proto-oncogene encodes a transcription factor that participates in the regulation of cellular proliferation, differentiation, and apoptosis. Ectopic overexpression of c-Myc has been shown to sensitize cells to apoptosis. We report here that cells lacking c-Myc activity due to disruption of the c-myc gene by targeted homologous recombination are defective in DNA damage-initiated apoptosis in the G(2) phase of the cell cycle. The downstream effector of c-Myc is cyclin A, whose ectopic expression in c-myc(-/-) cells rescues the apoptosis defect. The kinetics of the G(2) response indicate that the induction of cyclin A and the concomitant activation of Cdk2 represent an early step during commitment to apoptosis. In contrast, expression of cyclins E and D1 does not rescue the apoptosis defect, and apoptotic processes in G(1) phase are not affected in c-myc(-/-) cells. These observations link DNA damage-induced apoptosis with cell cycle progression and implicate c-Myc in the functioning of a subset of these pathways.

Cathepsin B and complement C3 are major comigrants in the estrogen-induced peroxidase fraction of rat uterine fluid.

The luminal fluid of estrogen or DES-stimulated uterus of immature rats contains 10-12 isoforms of peroxidase between pI 4.5-6.0. N-terminal amino acid sequencing of diaminobenzidine-peroxidase bands eluted from IEF and SDS-PAGE gels showed the presence of cathepsin B and the complement family of proteins as the major comigrants. Sequential treatment of uterine fluid by cation, anion, and size exclusion chromatography resulted in a five-fold purification of peroxidase having a specific activity of 273 units/mg. Mass spectrometric studies of bands isolated from SDS-PAGE gels from the size-exclusion purified peroxidase fraction showed the presence of complement C3 along with novel previously uncharacterized proteins. Two dimensional electrophoresis followed by N-terminal amino acid sequencing confirmed the presence of cathepsin B isoforms and isoforms of a novel protein at approximately 87 kDa. Identification by mass spectrometry from the database for this novel protein was inconclusive but could most likely be a candidate for estrogen-induced peroxidase. Results conclusively prove that cathepsin B and complement C3 are major proteins in the estrogen-induced peroxidase fraction of uterine fluid.

Telomerase activity, Myc and Bcl-2: possible indicators of effective therapy of prostate cancer with 9-nitrocamptothecin.

In this report, we demonstrate the down-regulation of telomerase activity and c-Myc and Bcl-2 expression during 9-nitrocamptothecin (9NC)-induced regression of human DU145 prostate tumors grown as xenografts in immunodeficient mice. These changes were not observed in tumors generated by DU145-derived cells resistant to 9NC. We suggest that telomerase activity, c-Myc and Bcl-2 can collectively serve as molecular diagnostic indicators of the effectiveness of 9NC during treatment of human prostate tumors.

Fibronectin/fibroblast growth factor/cell matrix signaling pathways and reciprocal membrane interaction may be the regulators of cell growth and apoptosis in Trypanosoma musculi in co-cultures with fibroblasts.

Trypanosoma musculi cultivated in medium containing serum alone or in the absence of fibroblasts in vitro were transformed into rounded, immotile cells, incapable of division and infectivity. Only in close contact with fibroblasts could the parasites survive and grow indefinitely. This report established the identity of the splenic 'sustentacular' cells as fibroblasts and utilized immunocytochemistry to demonstrate the putative cytoskeletal and membrane-associated molecules that may be involved in the control of growth and division, and apoptosis of T. musculi in vitro. The results indicated that cells that reacted intensely for fibroblast growth factor (FGF) also displayed a complex cytoskeletal system of F-actin bands underlying the plasma membrane of the fibroblast cell body and its numerous processes. Among the cytoskeletal and membrane glycoproteins, fibronectin, I-CAM, laminin, occludin, vinculin and desmin were most prominent. Fibronectin was most highly enhanced on the cell membrane and deposited as 'finger prints or tracks' on the extracellular culture surfaces. Transmission and scanning electron microscopy confirmed the intimate contact between trypanosomes and fibroblasts, however, neither membrane fusion or junctions were apparent. Our results suggested that a fibroblast-derived, membrane-associated factor appeared to be the putative growth regulator and apoptosis inhibitor in co-cultures of spleen-derived fibroblasts and T. musculi.

Susceptibility to apoptosis is restored in human leukemia HCW-2 cells following induction and stabilization of the apoptotic effector Bak.

We demonstrate that treatment of HCW-2 cells, an apoptotic resistant variant of the human HL-60 promyelocytic leukemia cell line with phorbol-12-myristate acetate (PMA), induced differentiation along the monocytic lineage. During this process there was a dramatic increase in the mitochondrial levels of the apoptosis effector, Bak, due to the stabilization of bak mRNA, which was correlated with the sensitization of HCW-2 cells to respond to the apoptotic effect of staurosporine (STS). Treatment of PMA-differentiated, but not undifferentiated, HCW-2 cells induced processing of Bid, substantial efflux of cytochrome c from mitochondria to the cytosol, activation of caspase-3 and apoptosis. The biological significance of the increased mitochondrial Bak in differentiated HCW-2 cells was supported by the finding that transient transfection of a bak cDNA into HCW-2 cells conferred sensitivity to STS-triggered apoptosis, as determined by pro-caspase-3 processing, cytochrome c efflux and DNA fragmentation. Our results suggest that the induction of Bak, upon monocytic differentiation, may be a critical event that regulates the apoptotic sensitivity of differentiated HCW-2 cells. Oncogene (2000) 19, 4108 - 4116

The staurosporine analog, Ro-31-8220, induces apoptosis independently of its ability to inhibit protein kinase C.

A series of bisindolylmaleimide (Bis) compounds were designed as analogs of the natural compound staurosporine (STS), which is a potent inducer of apoptosis. Many of the Bis analogs appear to be highly selective inhibitors of the protein kinase C (PKC) family, including PKC-alpha, -beta, -gamma, -delta, -epsilon, and -zeta, unlike STS, which is an inhibitor of a broad spectrum of protein kinases. In this report we describe the effects of the Bis analogs, Bis-I, Bis-II, Bis-III and Ro-31-8220 on the survival and proliferation of HL-60 cells, which have been widely used as a model cell system for studying the biological roles of PKC. Treatment of HL-60 cells with Bis-I, Bis-II, Bis-III, or Ro-31-8220 blocked phosphorylation of the PKC target protein Raf-1 with equal potency but did not appear to affect the general phosphorylation of proteins by other kinases. However, the biological effects of the Bis compounds were different: Bis-I and Bis-II had no observable effects on either cell survival or proliferation; Bis-III inhibited cell proliferation but not survival, whereas Ro-31-8220 induced apoptosis. These results indicated that the members of the PKC family which could be inhibited by the Bis analogs were required neither for survival nor proliferation of HL-60 cells. Analyses of cells treated with Ro-31-8220 showed that the apoptotic effect of Ro-31-8220 on HL-60 cells was mediated by a well-characterized transduction process of apoptotic signals: i.e., mitochondrial cytochrome c efflux and the activation of caspase-3 in the cytosol. Moreover, the ability of Ro-31-8220 to induce apoptotic activation was completely inhibited by the over-expression of the apoptotic suppressor gene, Bcl-2, in the cells. Interestingly, proliferation of the Bcl-2-over-expressing cells was still sensitive to the presence of Ro-31-8220, suggesting that the inhibitory effects of Ro-31-8220 on viability and cell proliferation were mediated by different mechanisms. In particular, the apoptotic effect of Ro-31-8220 on cells was not altered by the presence of an excess amount of the other Bis analogs, suggesting that this effect is mediated by a factor(s) other than PKC or by a mechanism which was not saturable by the other Bis analogs. Finally, structure-function analyses of compounds related to Ro-31-8220 revealed that a thioamidine prosthetic group in Ro-311-8220 was largely responsible for its apoptotic activity.

Up-regulation of cyclin B1 and cdc2 expression during 9-nitrocamptothecin-induced regression of DU145 prostate tumor.

BACKGROUND: We investigated changes in the content and subcellular localization of the cell cycle regulators, cyclin B1 and cyclin-dependent kinase cdc2, in human prostate DU145 tumor and cultured cells treated with the anticancer drug 9-nitrocamptothecin (9NC). MATERIALS AND METHODS: Proteins of interest were identified by Western blot methodology using specific antibodies. RESULTS: The cyclin B1 and cdc2 contents were dramatically elevated in biopsies of DU145 tumor regressing upon 9NC-treatment. In vitro, 9NC-induced apoptosis of DU145 cells was associated with up-regulation of expression and nuclear accumulation of cyclin B1 and cdc2. No changes were observed in cyclins A and E and the cyclin-dependent kinase cdk2 in 9NC-treated DU145 tumor and cultured cells. CONCLUSION: 9NC-induced apoptosis in DU145 cells in vivo and in vitro is associated with up-regulation of expression and nuclear localization of cyclin B1 and cdc2.

THP-1 monocytic leukemia cells express Fas ligand constitutively and kill Fas-positive Jurkat cells.

Normal human monocytes express Fas and are susceptible to Fas ligand (FasL)-induced apoptosis. Because the myeloid leukemia cell lines HL-60 and THP-1 can be differentiated into functional monocytes and macrophages, we studied their expression of Fas and Fas ligand (FasL) to determine whether there were differentiation-associated changes in these proteins. THP-1, HL-60 and HCW-2, both before and after treatment with PMA, expressed high levels of Fas ligand (FasL), but did not express Fas. The FasL expressed by THP-1 cells was functional as measured by their ability to kill Jurkat T-cells by apoptosis. The THP-1 Fas gene appears to be silent, because bacterial lipopolysaccharide (LPS) induced Fas expression in fully differentiated THP-1 cells. Our results suggest that FasL expression by leukemia cells may account in part for the pathophysiology of myeloid leukemia, and that PMA-differentiated THP-1 cells, while possessing many of the functional properties of normal macrophages, are abnormal with respect to a major apoptotic pathway.

Apoptosis and differentiation in the crypt-villus unit of the rat small intestine.

This investigation utilized immunocytochemical and fluorescent protocols to analyze the roles of cellular proliferation and apoptosis in the regulation of differentiation and senescence of the rat small intestinal mucosa. Specifically, the study localized apoptotic zones of the villus through the use of enzymatic tags; established the transition point between cell growth and differentiation, i.e. the point of no return where crypt cells differentiate into absorptive cells with barrier functions; and the role that plasmalemmal, cytoskeletal, junctional and extracellular matrix (ECM) elements may play in the regulation of differentiation and migration of epithelial cells from crypt to villus. Apoptosis was relegated to the villus tip forming a prominent 'apoptotic cuff' of cells. Close scrutiny of these cuffs reveals the presence of apoptotic cells adjacent to non-apoptotic (healthy) cells. Mid-villus epithelial cells were non-apoptotic and all cells in the crypt-villus unit expressed Bcl-2 activity. Intestinal lactase expression was prominent in post-mitotic cells along the villus, while cells in the crypt and base were negative for lactase activity. In contrast, all the cells of the crypt-villus unit were intensely reactive for F-actin. Close scrutiny of isolated cells and frozen sections indicates specific localization of actin in the microvillus region, apical cytoplasm, basolateral and lateral plasmalemma which was in close proximity to fibronectin in the basement lamina. Occludin positive junctional networks were prominent at villus tips, where senescent and apoptotic cells were also most prominent, suggesting that tight junctional integrity was essential to barrier, digestive and absorptive functions in all regions of the mucosa.

Schedule-dependent efficiency of thermochemotherapy in vitro with etoposide and heating at 43 degrees C.

We have investigated the effect of hyperthermia (HT) treatment on the extent of etoposide-induced apoptosis in human leukemia HL-60 cells. The cells were heated at 43 degrees C for 15 to 60 min. Relative quantitative changes in the apoptotic (Ap) fractions were monitored by flow cytometry, HT treatment for 15 min prior to or concurrently with etoposide exposure had no effect on the Ap fraction generated by the drug alone, whereas, 60 min heating resulted in an increase in the Ap fraction. Heating of the cells at 6 to 24 hr after drug exposure enhanced the Ap fraction. Taken collectively, the results indicate that appropriately scheduled HT and etoposide treatments may lead to a thermochemotherapy protocol that will result in increased etoposide-induced death of human leukemia cells.

Propionate and butyrate esters of camptothecin and 9-nitrocamptothecin as antileukemia prodrugs in vitro.

Six camptothecin (CPT) alkyl esters and four 9-nitrocamptothecin (9NC) alkyl esters were assayed for ability to inhibit proliferation and induce programmed cell death (apoptosis) in human leukemia HL-60 and U-937 cells, which exhibit differential sensitivity to CPT and 9NC. In general, CPT-propionate and CPT-butyrate demonstrated activities, while the other esters were practically inactive. Similarly, 9NC-propionate and 9NC-butyrate were active, while the other 9NC esters exhibited little or no activity. The biologically active esters required metabolic conversion (i.e., de-esterification) to their parental compounds as demonstrated by the conversion of CPT-propionate to CPT in mouse liver homogenate, and the topoisomerase I-inhibition assay. In conclusion, the propionate and butyrate esters of CPT and 9NC are CPT and 9NC prodrugs, that can develop to important chemotherapeutic agents for the effective treatment of human leukemias and other malignancies.

Modulation of 9-nitrocamptothecin-induced apoptosis by hyperthermia in human leukemia HL-60 cells.

We have investigated whether hyperthermia (HT) treatment at 43 degrees C for 15-60 min can affect the extent of apoptosis induced in human leukemia HL-60 cells by the anticancer drug 9-nitrocamptothecin (9NC). Quantitative changes in the apoptotic (Ap) fraction in the cell cultures were monitored by flow cytometry. The results showed that (i) heating for 15 min prior to or concurrently with 9NC exposure had no effect on the Ap fraction generated by the drug alone, whereas 60 min heating resulted in an increase in the Ap fraction; and (ii) heating of the cells at 6-24 h after exposure to the drug enhanced the Ap fraction. These results indicate that appropriate scheduling of HT and 9NC treatments may lead to thermochemotherapy protocols that will result in increased 9NC-induced death of human leukemia cells.

Water-insoluble camptothecin analogues as potential antiviral drugs.

In addition to being causative agents of infectious diseases in animals and humans, DNA viruses have served as models for the study of eukaryotic molecular mechanisms including replication and transcription. Studies of DNA virus functions utilizing cell-free systems and virus-infected cells in culture, in the presence of the anticancer drug camptothecin (CPT), have demonstrated that CPT is a potent inhibitor of replication, transcription and packaging of double-stranded DNA-containing adenoviruses, papovaviruses and herpesviruses, and the single- stranded DNA-containing autonomous parvoviruses. CPT inhibits viral functions by inhibiting topoisomer- ase I, a host cell enzyme required for initiation and completion of the viral functions. These findings indicate that CPT analogues could be developed for use as potent drugs against DNA viruses.