[Show abstract][Hide abstract] ABSTRACT: The labdane diterpene sclareol has demonstrated significant cytotoxicity against human tumor cell lines and human colon cancer xenografts. Therefore, there is need to elucidate the mode of action of this compound as very little information is known for the anticancer activity of sclareol and other labdane diterpenes, in general. COMPARE analysis of GI(50) values for a number of human cancer cell lines was initially implicated in an effort to assign a putative mechanism of action to the compound. Sclareol-induced cell cycle arrest and apoptosis were assessed by flow cytometry and Western blot analyses. Finally, the anticancer ability of sclareol in vivo was assessed by using human colon cancer xenograft/mouse models. Sclareol arrested in vitro the growth of p53-deficient (HCT116(p53-/-)) human colon cancer cells and subsequently induced apoptosis by activating both caspases-8 and -9. Intraperitoneal administration of liposome-encapsulated sclareol at the maximum tolerated dose induced a marked growth suppression of HCT116(p53-/-) tumors established as xenografts in immunodeficient NOD/SCID mice. In conclusion, we demonstrate herein that sclareol kills human tumor cells by inducing arrest at the G(1)-phase of the cell cycle followed by apoptosis that involves activation of caspases-8, -9 and -3 via a p53-independent mechanism. These findings suggest that liposome-encapsulated sclareol possesses chemotherapeutic potential for the treatment of colorectal and other types of human cancer regardless of the p53-status.
Full-text · Article · May 2011 · European journal of pharmacology
[Show abstract][Hide abstract] ABSTRACT: A major factor that impedes the clinical success of cisplatin-based chemotherapy for cancer is cisplatin resistance by cancer cells.
The sensitivity of parental HCT116 human colon cancer cell line and its isogenic gene-knockout sub-lines to cisplatin was determined by clonogenicity assay; furthermore, p53 activation, p21 expression, cell cycle arrest and senescence in these cells after cisplatin treatment were investigated.
Parental cells were six times more resistant than 14-3-3sigma-knockout (sigma-KO) cells to cisplatin. Moreover, activation of p53, p53-dependent expression of p21 and p21-dependent senescence were observed in sigma-KO, but not parental cells after a treatment with a low cisplatin dose.
A 14-3-3sigma-dependent mechanism inhibits p53 activation in parental cells treated with a low cisplatin dose, thereby blocking p21 expression that is essential for senescence and consequently conferring to the parental cells a significant degree of resistance to cisplatin.
No preview · Article · Jul 2009 · Anticancer research
[Show abstract][Hide abstract] ABSTRACT: The plant-derived compound curcumin has shown promising abilities as a cancer chemoprevention and chemotherapy agent in vitro and in vivo but exhibits poor bioavailability. Therefore, there is a need to investigate modified curcumin congeners for improved anticancer activity and pharmacokinetic properties.
The synthetic curcumin analogue dimethoxycurcumin was compared with curcumin for ability to inhibit proliferation and apoptosis of human HCT116 colon cancer cells in vitro by estimating the GI(50) and LC(50) values and detecting the extent of apoptosis by flow cytometry analysis of the cell cycle. Metabolic stability and/or identification of metabolites were evaluated by recently developed mass spectrometric approaches after incubation with mouse and human liver microsomes and cancer cells in vitro. Additionally, circulating levels of dimethoxycurcumin and curcumin were determined in mice following i.p. administration.
Dimethoxycurcumin is significantly more potent than curcumin in inhibiting proliferation and inducing apoptosis in HCT116 cells treated for 48 h. Nearly 100% of curcumin but <30% of dimethoxycurcumin was degraded in cells treated for 48 h, and incubation with liver microsomes confirmed the limited metabolism of dimethoxycurcumin. Both compounds were rapidly degraded in vivo but dimethoxycurcumin was more stable.
Compared with curcumin, dimethoxycurcumin is (a) more stable in cultured cells, (b) more potent in the ability to kill cancer cells by apoptosis, (c) less extensively metabolized in microsomal systems, and (d) more stable in vivo. It is likely that the differential extent of apoptosis induced by curcumin and dimethoxycurcumin in vitro is associated with the metabolite profiling and/or the extent of stability.
Full-text · Article · Feb 2007 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: The eukaryotic proteasome is a 26S ATP-dependent proteolytic complex, which possesses chymotrypsin-like, trypsin-like and peptidyl glutamyl peptide hydrolase (PGPH) activities, which enable the proteasome to degrade all short-lived and many long-lived proteins, and consequently regulate a myriad of activities in cells. In this study, we observed that inhibition of the proteasome, and more specifically, inhibition of the chymotrypsin-like activity of the proteasome, in MCF-7 human breast cancer cells resulted in selective down-regulation of the nuclear estrogen receptor-alpha (ERalpha). Our data indicated that estrogen had no effect, whereas the ERalpha antagonist, tamoxifen, reduced the amount of ERalpha that could be subjected to down-regulation after proteasome inhibition. Furthermore, our data demonstrated that protein synthesis was required for the down-regulation of ERalpha to occur. Collectively, these data indicate the existence of a proteasome-dependent mechanism that is utilized by MCF-7 cells to maintain a steady-state level of ERalpha.
No preview · Article · Sep 2006 · Biochemical Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Treatment of cells with estrogens and several pure ERα antagonists rapidly induces down-regulation of the α-type estrogen receptor (ERα) in the nucleus by mechanisms that are sensitive to the proteasome inhibitors, MG132 and clasto-lactacystin-β-lactone. Hence, it is believed that these ER ligands induce down-regulation of ERα by proteasome-dependent mechanisms, which serve to control both the amount of transcriptional activity and the level of ligand-bound ERα in cells. In this study, we observed that treatment of cultured MCF-7 and T47D human breast cancer cells with the low affinity ER ligand, 4,4′-dihydroxy-trans-stilbene (4,4′-DHS), inhibited the transcriptional activity of ERα and induced slow and gradual decrease in the amount of ERα protein (henceforth referred to as down-regulation of ERα). The 4,4′-DHS-induced down-regulation of ERα in MCF-7 cells involved a mechanism that was insensitive to the two most specific proteasome inhibitors, clasto-lactacystin-β-lactone and epoxomycin, but sensitive to MG132 at concentrations exceeding that required for maximal inhibition of the proteasome in MCF-7 cells. Therefore, 4,4′-DHS appears to induce down-regulation of ERα by a proteasome-independent mechanism. Here, we present data to show that both 4-OH and 4′-OH are critical for the ability of 4,4′-DHS to induce down-regulation of ERα and suggest that 4,4′-DHS provides a useful scaffold for development of novel ERα antagonists.
No preview · Article · Aug 2006 · Biochemical Pharmacology
[Show abstract][Hide abstract] ABSTRACT: The plant-produced compound, resveratrol (3,5,4'-trihydroxy-trans-stilbene, 3,4,5-THS), induces apoptosis in various human leukemia cell types in vitro, and thus appears to be a promising anti-leukemia agent. In this study, we observed that treatment of resveratrol-resistant Jurkat cells with the resveratrol analogue, 3,4,5-trihydroxy-trans-stilbene (3,4,5-THS), rapidly induced extensive apoptosis, indicating that the apoptotic activity of the analogue differed from that of the parental compound resveratrol. Indeed, we found that treatment of Jurkat cells with 3,4,5-THS, unlike treatment with resveratrol, induced activation of caspase-8 and apoptosis by a Fas-associated death domain (FADD) protein-dependent mechanism without involving the known death ligands CD95 ligand (CD95L), tumor necrosis factor alpha (TNFalpha) and TNF-related apoptosis-inducing ligand (TRAIL). Therefore, 3,4,5-THS induced activation of a FADD-dependent apoptotic mechanism that was unresponsive to the parental compound resveratrol. Therefore, the ability of 3,4,5-THS, but not resveratrol, to induce apoptosis demonstrates a structure-associated apoptotic activity of the resveratrol analogue.
No preview · Article · Feb 2005 · Biochemical Pharmacology
[Show abstract][Hide abstract] ABSTRACT: A hexane extract of the plant product Chios mastic gum (He-CMG) is demonstrated to kill human colon cancer cells in vitro via the process of anoikis. Specifically, the sequence of events includes He-CMG-induced GI-arrest of the cells, detachment of the cells from the substrate and subsequent apoptosis. Anoikis is dependent on the concentration and duration of treatment with He-CMG. Presence of the pan-caspase inhibitor, Z-VAD-fmk, did not prevent cell detachment, but it did prevent apoptosis of the detached cells indicating that the process of cell detachment, but not apoptosis, is independent of caspase activation. He-CMG-induced apoptosis is associated with activation of the initiator caspases-8, and -9 and the effector caspase-3. Caspases are activated in cells at a relatively long time after detachment, and caspase-3 activation may require caspase-8 or caspase-9 activation, as determined by using HCT116 isogenic clones impaired in apoptosis mechanisms that involve these two caspases. Finally, electron microscopy observations indicated a time-dependent appearance of morphological features both typical and non-typical of apoptosis in cells treated with He-CMG for various periods of time. Taken together, the results demonstrated that He-CMG induces apoptosis in HCT116 cells and, therefore, further in vivo and in vitro studies of the anticancer activities of this plant product are warranted.
Full-text · Article · Jan 2005 · In vivo (Athens, Greece)
[Show abstract][Hide abstract] ABSTRACT: We compared the abilities of trans-resveratrol and seven analogs to inhibit an azo compound-induced peroxidation of linoleic acid in vitro and to induce apoptosis in cultured human leukemia cells. The results showed that both the antioxidant and apoptotic activities of the analogs containing 3,4-dihydroxyl groups were significantly higher than those of the trans-resveratrol and the other analogs. Hence, the 3,4-dihydroxyl groups were important for trans-resveratrol analogs to exhibit concurrent high antioxidant and apoptotic activities.
Preview · Article · Mar 2004 · Anticancer research
[Show abstract][Hide abstract] ABSTRACT: The anticancer drug, 9-nitrocamptothecin (9NC), has demonstrated an unprecedented activity against human caner cells grown in cultures and as xenografts in nude mice. 9NC-induced apoptosis of cancer cells is mediated by the nuclear enzyme, topoisomerase I, and executed by pathways that involve cytochrome c release from the mitochondrion and/or activation of death receptors depending on the cell type. Alternatively, 9NC has exhibited ability to induce differentiation or senescence of certain cell types in vitro. In several instances, the 9NC activities can be regulated by Bcl-2 family proteins and cell cycle-associated proteins, p53, p21 and Cdks. Also, 9NC can inhibit HIV replication in infected T- and monocytic cells in vitro. Development of resistance to 9NC, associated with mutations in the topoisomerase I gene, can be overcome by regulating specific proteins, such as RKIP, other than topoisomerase I. Finally, derivatives (i.e., alkyl esters) of 9NC, liposome-encapsulated 9NC and combined treatment of 9NC with ionizing radiation or hyperthermia are other approaches to enhance the apoptotic activity of 9NC against human cancer cells.
No preview · Article · Sep 2003 · Anticancer research
[Show abstract][Hide abstract] ABSTRACT: We have investigated whether variability in the apoptotic pathway may account for the differential susceptibility to apoptosis-induction by 9-nitrocamptothecin (9-NC) in cell subpopulations derived from the human ovarian cancer cell line, SKOV-3. Quantitative differences in the apoptotic fractions of cells were assessed by flow cytometry, whereas major regulatory and executing components of the apoptotic machinery were investigated by Western blot analysis using specific antibodies. The results indicate that indeed the apoptotic pathway was activated by 9-NC in some, but not all, cells of the SKOV-3 cell line, suggesting that 9-NC alone may partially be effective for treatment of patients with ovarian cancer.
No preview · Article · Aug 2003 · Anti-Cancer Drugs
[Show abstract][Hide abstract] ABSTRACT: The ability of p53 to alter, at the transcriptional level, the gene expression of downstream targets is critical for its role as a tumor suppressor. Most models of p53 activation postulate the stepwise recruitment by p53 of coactivators, histone acetyltransferases, and/or chromatin remodeling factors to a promoter region to facilitate the subsequent access of the general transcriptional machinery required for transcriptional induction. We demonstrate here, however, that the promoter regions for the p53 target genes, p21, 14-3-3sigma, and KARP-1, exist in a constitutively open conformation that is readily accessible to DNase I. This conformation was not altered by DNA damage or by whether p53 was present or absent in the cell. In contrast, p53 response elements, which resided outside the immediate promoter regions, existed within DNase I-resistant chromatin domains. Thus, p53 activation of downstream target genes occurs without p53 inducing chromatin alterations detectable by DNase I accessibility at either the promoter or the response element. As such, these data support models of p53 activation that do not require extensive chromatin alterations to support cognate gene expression.
No preview · Article · Apr 2003 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The antibacterial agent taurolidine (TRD) has been used as a lavage antibiotic to prevent development of peritonitis in patients after surgery. We recently showed that TRD induced growth arrest and apoptosis of a variety of cultured cell lines derived from human solid tumors and also significantly inhibited the growth of human ovarian tumors in a mouse model. In this report, we present data to show that TRD, at concentrations below the doses that are used to treat patients in the clinic, induces apoptosis of human leukemia HL-60 cells by a mitochondrial cytochrome c-dependent pathway.
No preview · Article · Jul 2002 · Anticancer research
[Show abstract][Hide abstract] ABSTRACT: Treatment of cells with the anti-cancer drug camptothecin (CPT) induces topoisomerase I (Top1)-mediated DNA damage, which
in turn affects cell proliferation and survival. In this report, we demonstrate that treatment of the wild-type HCT116 (wt
HCT116) human colon cancer cell line and the isogenic p53−/−HCT116 and p21−/− HCT116 cell lines with a high concentration (250 nm) of CPT resulted in apoptosis, indicating that apoptosis occurred by a p53- and p21-independent mechanism. In contrast, treatment
with a low concentration (20 nm) of CPT induced cell cycle arrest and senescence of the wt HCT116 cells, but apoptosis of the p53−/− HCT116 and p21−/− HCT116 cells. Further investigations indicated that p53-dependent expression of p21 blocked apoptosis of wt HCT116 cells
treated with 20 nm, but not 250 nm CPT. Interestingly, blocking of the apoptotic pathway, by Z-VAD-FMK, in p21−/− HCT116 cells following treatment with 20 nm CPT did not permit the cells to develop properties of senescence. These observations demonstrated that p21 was required for
senescence development of HCT116 cells following treatment with low concentrations of CPT.
Preview · Article · Jun 2002 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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 SNC-induced death of human leukemia cells. [(C) 1999 Lippincott Williams & Wilkins.]
No preview · Article · Mar 1999 · Anti-Cancer Drugs