Distamycin A enhances the cytotoxicity of duocarmycin A and suppresses duocarmycin A-induced apoptosis in human lung carcinoma cells

Duocarmycin A (Duo), which is one of well-known antitumor antibiotics, efficiently alkylates adenine N3 at the 3' end of AT-rich sequences in the DNA. The addition of a minor groove binder, distamycin A (Dist), not only accerelates the reactivity of Duo with oligonucleotide duplex but also switches the DNA-alkylation site to guanine in GC-rich sequences. Here we examined cytotoxic effect of Duo in the coexistence of Dist using human lung carcinoma (HLC-2) cells. The cytotoxicity of Duo to HLC-2 cells was enhanced 10 times by the addition of 0.5microg/ml Dist, which was much lower than the IC(50) value of 16microg/ml. Addition of Duo alone to HLC-2 cells resulted in typically apoptotic changes, including chromatin condensation, sub-G1 accumulation in DNA histogram pattern, and decrease in procaspase-3 and 9 levels. Interestingly, these apoptotic characteristics in Duo-treated cells were suppressed by the addition of 0.5microg/ml Dist, and the G2/M population in the cell cycle progression of HLC-2 cells was largely unchanged in the coexistence of Dist along with the extremely low accumulation of p53 and higher induction of p21. In contrast, the treatment of HLC-2 cells with Dist (16microg/ml) alone was observed to induce the accumulation of p53 and cell cycle arrest at the G1 phase. These results indicate that Dist suppresses apoptosis induced by Duo as well as enhances Duo-induced cytotoxicity in living cells, and may contribute to chemotherapy for tumors resistant to inducing apoptotic cell death.     

The CD95/CD95 ligand system is not the major effector in anticancer drug-mediated apoptosis

Many anticancer drugs are able to induce apoptosis in tumor cells but the mechanisms underlying this phenomenon are poorly understood. Some authors reported that the p53 tumor suppressor gene may be responsible for drug-induced apoptosis; however, chemotherapy-induced apoptosis can also be observed in p53 negative cells. Recently, doxorubicin (DXR) was reported to induce CD95L expression to mediate apoptosis through the CD95/CD95L system. Thus, an impairment of such a system may be involved in drug resistance. We evaluated the in vitro antitumor activity of several cytotoxic drugs on two human p53-negative T-cell lymphoma cell lines, the HUT78-B1 CD95L-resistant cell line and the HUT78 parental CD95L-sensitive cell line. We demostrated by Western blotting assay that DXR and etoposide (VP-16) were able to induce CD95L expression after 4 h of treatment. In contrast, they were unable to induce the expression of p53. DXR, at concentrations ranging from 0.001 - 1 microg/ml, and VP16, at concentrations ranging from 0.05 - 1 microg/ml, were equally cytotoxic and induced apoptosis in both cell lines as assessed by fluorescence microscopy and flow cytometry analyses. Although we observed a slightly reduced percentage of apoptotic cells in HUT78B1 when compared with the parental HUT78 cells after few hours of drug exposure, this difference was no longer evident at 48 or 72 h. Similarly, the exposure of HUT78 cells to a CD95-blocking antibody partially reduced early apoptosis (24 h) without affecting the long-term effects of the drugs including cytotoxicity. Furthermore, as observed with DXR and VP-16, both the CD95L-sensitive and the CD95L-resistant cell lines resulted equally sensitive to the cytotoxic effects of a number of different cytotoxic drugs (vincristine, camptothecin, 5-fluorouracil and methotrexate). The treatment with the Caspase-3 tetrapeptide aldehyde inhibitor, Ac-DEVD-CHO, did not affect the DXR-induced apoptosis whereas it only modestly inhibited apoptosis and cytotoxicity of VP-16, while Z-VAD.FMK, a Caspase inhibitor that prevents the processing of Caspase-3 to its active form, was able to block DXR-induced apoptosis at 24 h but not at 48 h. Thus, our results do not confirm a crucial role for the CD95/CD95L system in drug-induced apoptosis and suggest the involvement of alternative p53-independent pathways at least in this experimental model system.     

Induction of apoptosis in K562/ADM cells by gamma-linolenic acid involves lipid peroxidation and activation of caspase-3

Numerous studies have revealed that gamma-linolenic acid (GLA) possesses effective tumoricidal properties while not inducing damage to normal cells or creating harmful systemic side effects. It can exert anti-tumor efficacy against a variety of cancers including leukemia. However, little is known about the effects of GLA on leukemia resistant to chemotherapy, emerging as a serious clinical problem. The present study tested GLA-induced apoptosis in K562/ADM multidrug-resistant (MDR) leukemic cells and investigated its possible mechanisms. Using cell viability, fluorescent staining of nuclei, flow cytometric Annexin V/PI double staining and lactate dehydrogenase (LDH) release, we found that GLA could inhibit cell growth and induce apoptosis and secondary necrosis. The results showed that incubation with GLA concentrations of 10-60 microg/ml caused a dose- and time-dependent decrease of K562/ADM cell viability, and the IC50 value was 50.5 microg/ml at 24 h and 31.5 microg/ml at 48 h. Flow cytometry using Annexin V/PI double staining assessed apoptosis, necrosis and viability. Typical apoptotic nuclei were shown by staining of K562/ADM cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. On the other hand, after treated K562/ADM cells with 20 microg/ml GLA for 48 h and with 40 microg/ml GLA for 12 h, the LDH release significantly increased, indicated losses of plasma membrane integrity and presence of necrosis. Further, the inhibition of GLA-induced apoptosis by a pan-caspase inhibitor (z-VAD-fmk) suggested the involvement of caspases. The increase of caspase-3 activity with GLA concentration confirmed its role in the process. The results also showed that the malondialdehyde (MDA) content was also significantly elevated, and antioxidant BHT could block GLA cytotoxity, indicating the cytotoxity induced by GLA may be due to lipid peroxidation.     

Insulin and IGF-1 mediated inhibition of apoptosis in CHO cells grown in suspension in a protein-free medium

When Chinese hamster ovary (CHO) cells were grown in suspension and deprived of serum, 40% of them became apoptotic after 72 hours, as determined by flow cytometry analysis of TUNEL-labelled cells. Cell viability, assessed by erythrocin B staining, decreased correspondingly. An increase in the total fraction of cells expressing interleukin converting enzyme (ICE; caspase 1), B-cell lymphoma 2 protein (Bcl-2,) and Bcl-2 associated x protein (Bax) was shown by antibody probing and subsequent flow cytometry. The p53 tumour suppressor gene product level remained low within the cell population. Insulin-like growth factor-1 (IGF-1) inhibited cell death in a concentration-dependent manner, and at 20 ng/ml, cell viability was maintained close to 100% and no apoptotic cells were detected. Also, insulin was shown to inhibit cell death - at 1.0 microg/ml, cell viability was 95%, whereas 10% of the cells stained for apoptosis. At the highest concentrations of IGF-1 and insulin, the expression of ICE, Bcl-2 and Bax was fully suppressed, whereas the p53 product level increased, despite still being detectable in a minority of cells. Under these conditions, IGF-1 may increase p53 expression to restrain abnormal cell proliferation. It is concluded that special attention should be paid to exposure and culture conditions that induce acquired susceptibility to a toxic insult, during the development and validation of cell-based assays.     

The response of malignant B lymphocytes to ionizing radiation: cell cycle arrest, apoptosis and protection against the cytotoxic effects of the mitotic inhibitor nocodazole

Ionizing radiation and mitotic inhibitors are used for the treatment of lymphoma. We have studied cell cycle arrest and apoptosis of three human B-lymphocyte cell lines after X irradiation and/or nocodazole treatment. Radiation (4 and 6 Gy) caused arrest in the G(2) phase of the cell cycle as well as in G(1) in Reh cells with an intact TP53 response. Reh cells, but not U698 and Daudi cells with defects in the TP53 pathway, died by apoptosis after exposure to 4 or 6 Gy radiation (>15% apoptotic Reh cells and <5% apoptotic U698/Daudi cells 24 h postirradiation). Lower doses of radiation (0.5 and 1 Gy) caused a transient delay in the G(2) phase of the cell cycle for the three cell lines but did not induce apoptosis (<5% apoptotic cells at 24 h postirradiation). Cells of all three cell lines died by apoptosis after exposure to 1 microg/ml nocodazole, a mitotic blocker that acts by inhibiting the polymerization of tubulin (>25% apoptotic cells after 24 h). When X irradiation with 4 or 6 Gy was performed at the time of addition of nocodazole to U698 and Daudi cells, X rays protected against the apoptosis-inducing effects of the microtubule inhibitor (<5% and 15% apoptotic cells, respectively, 24 h incubation). U698 and Daudi cells apparently have some error(s) in the signaling pathway inducing apoptosis after irradiation, and our results suggest that the arrest in G(2) prevents the cells from entering mitosis and from apoptosis in the presence of microtubule inhibitors. This arrest was overcome by caffeine, which caused U698 cells to enter mitosis (after irradiation) and become apoptotic in the presence of nocodazole (26% apoptotic cells, 24 h incubation). These results may have implications for the design of clinical multimodality protocols involving ionizing radiation for the treatment of cancer.     

Comparison of the apoptosis-inducing abilities of various protein synthesis inhibitors in U937 cells

We compared the abilities of ricin, diphtheria toxin, cycloheximide, and anisomycin to induce apoptosis, using human myeloid leukemia U937 cells at the concentration of each toxin at which almost complete protein synthesis inhibition was attained within 3 h. Among these toxins, anisomycin was found to be the most potent apoptosis inducer. After a 6-h exposure to anisomycin (1 microg/ml), nearly 95% of the cells had apoptotic nuclear morphological changes, while 53%, 30%, and 10% of the cells showed apoptotic changes after exposure to ricin (0.1 microg/ml), diphtheria toxin (10 microg/ml), and cycloheximide (10 microg/ml), respectively. Furthermore, a rapid increase in caspase-3-like activity was observed in anisomycin-treated cells. A similar increase in caspase-3-like activity was also observed in ricin-treated cells on a slower time schedule. However, only a slight increase in the protease activity was induced by diphtheria toxin or cycloheximide even after 6 h of incubation. Since both ricin and anisomycin are known to act on 28S ribosomal RNA, our results suggest that this action mechanism may be responsible for their potent apoptosis induction, and protein synthesis inhibition alone is not sufficient to induce apoptosis.     

Heterocyclic amine induced apoptotic response in the human lymphoblastoid cell line TK6 is linked to mismatch repair status.

The human lymphoblastoid cell, TK6, exhibited a dose-dependent cytotoxic and apoptotic response following treatment with the food borne heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Augmentation of the p53 protein and increases in p21-WAF1 levels were also observed. Comparison of the survival by clonogenic assays and the percentage of apoptotic cells (cells containing subG1 DNA or condensed nuclei) revealed that only 10-20% of the PhIP-induced cell death could be attributed to apoptosis that occurred in the first 24h after treatment. MT1, a derivative of TK6 that contains mutations in both alleles of its hMSH6 gene and is mismatch repair deficient, showed a decreased apoptotic response. A significant increase (P<0.05) in apoptosis was observed in TK6 and not in MT1 following treatment with 2.5microg/ml PhIP. A five- to six-fold increase and less than a two-fold increase in the fraction of apoptotic cells were observed in TK6 and MT1, respectively. Treatment with 5microg/ml PhIP resulted in significant increases in apoptosis (P<0.05) in TK6 and MT1. The percentages of apoptotic cells were, however, two- to three-fold higher in TK6 than in MT1. HCT116, a hMLH1 defective mismatch repair deficient colorectal carcinoma cell line, also exhibited lower PhIP-induced apoptosis than its mismatch repair proficient chromosome transfer cell line (HCT116+chr3) following PhIP treatment. These results show that PhIP-induced apoptosis is mediated through a mismatch repair dependent pathway. Accumulation of p53 in TK6 and MT1 were evident in samples taken 24h after PhIP treatment. Increases in p21-WAF1 were also observed in both cell lines confirming that the p53 was functional. The lower apoptotic response of MT1 but similar p53 accumulation in TK6 and MT1 suggest that the mismatch repair protein(s) are involved downstream of p53 or that PhIP-induced apoptosis is p53-independent.     

Differential effects of non-genotoxic carcinogens and proliferating agents on cell growth, survival and apoptosis in hepatic cells in vitro.

Many nongenotoxic carcinogen's (ngc) produce hyperplastic lesions from which neoplastic foci may arise. Modulation of the rate of apoptosis by some ngc's within these lesions may be critical to their mechanism of tumour promotion but some may be cytotoxic. To establish if these compounds are apoptotic or necrotic in vitro, three ngc's (12-0-tetradecanoyl phorbol-13-acetate (TPA); nickel, and di(2-ethylhexyl-phthalate (DEHP), two noncarcinogenic hepatoproliferating agents (1,4-dichlorobenzene (DCB; HGF) and an in vitro genotoxic reference compound (7-hydroxy-2-acetylaminofluorene (70H2AAF) were used to induce mitogenic or growth responses in two liver cell-lines HepG2 and JTC-15. MTT and 3H-thymidine incorporation assays were used to measure cell growth and DNA replicative activity respectively. Rates of apoptosis were assayed using FITC-annexin V with propidium iodide staining and flow cytometry. Responses in HepG2 cells were HGF (proliferation at > or = 3 ng/ml), TPA (cell growth at > or = 8 ng/ml), DEHP (proliferation at > or = 0.05 microg/ml). NiCl2 and 70H-2AAF were cytotoxic above 0.001 microg/ml and 100 ng/ml respectively. An equivocal result was obtained for DCB. Responses in JTC-15 cells were HGF (proliferation, 3 ng/ml), TPA (DNA replication, 10 ng/ml), and DEHP (cell mass, 2.5 microl/ml). NiCl2 and 70H-2AAF were cytotoxic above 0.01 microg/ml and 110 mg/ml respectively. Equivocal results were obtained for DCB. In flow cytometry assays apoptotic and necrotic populations were not clearly separable. Approximate rates of apoptosis in HepG2 were: control 8.7%; DEHP, 10.19%. NiCl2, 12.67%; 70H2AAF, 16.56%; TPA, 19.72%; HGF, 23.73%; DCB, 24.59%; positive apoptotic control (taxol) 26.94%. These data show apoptosis was increased in chemically activated populations of HepG2. The ngc, DEHP, unexpectedtly produced proliferation in HepG2 and almost totally suppressed apoptosis in vitro in HepG2 relative to the non-carcinogenic hepatoproliferators. The rate of apoptosis induced by the ngc TPA was not considered to be sufficiently different to the rates of apoptosis induced by the noncarcinogenic hepatoproliferators. The results emphasize the importance of considering necrotic reactions from effects on apoptosis in detecting non-genotoxic carcinogens.     

beta-lapachone induces cell cycle arrest and apoptosis in human colon cancer cells

BACKGROUND: Human colon cancers have a high frequency of p53 mutations, and cancer cells expressing mutant p53 tend to be resistant to current chemo- and radiation therapy. It is thus important to find therapeutic agents that can inhibit colon cancer cells with altered p53 status. beta-Lapachone, a novel topoisomerase inhibitor, has been shown to induce cell death in human promyelocytic leukemia and prostate cancer cells through a p53-independent pathway. Here we examined the effects of beta-lapachone on human colon cancer cells. MATERIALS AND METHODS: Several human colon cancer cell lines, SW480, SW620, and DLD1, with mutant or defective p53, were used. The antiproliferative effects of beta-lapachone were assessed by colony formation assays, cell cycle analysis, and apoptosis analysis, including annexin V staining and DNA laddering analysis. The effects on cell cycle and apoptosis regulatory proteins were examined by immunoblotting. RESULTS: All three cell lines, SW480, SW620, and DLD1, were sensitive to beta-lapachone, with an IC(50) of 2 to 3 microM in colony formation assays, a finding similar to that previously reported for prostate cancer cells. However, these cells were arrested in different stages of S phase. At 24 hr post-treatment, beta-lapachone induced S-, late S/G2-, and early S-phase arrest in SW480, SW620, and DLD1 cells, respectively. The cell cycle alterations induced by beta-lapachone were congruous with changes in cell cycle regulatory proteins such as cyclin A, cyclin B1, cdc2, and cyclin D1. Moreover, beta-lapachone induced apoptosis, as demonstrated by annexin V staining, flow cytometric analysis of DNA content, and DNA laddering analysis. Furthermore, down-regulation of mutant p53 and induction of p27 in SW480 cells, and induction of pro-apoptotic protein Bax in DLD1 cells may be pertinent to the anti-proliferative and apoptotic effects of beta-lapachone on these cells. CONCLUSIONS: beta-Lapachone induced cell cycle arrest and apoptosis in human colon cancer cells through a p53-independent pathway. For human colon cancers, which often contain p53 mutations, beta-lapachone may prove to be a promising anticancer agent that can target cancer cells, especially those with mutant p53.     

Cytotoxicity and apoptosis induction of some selected marine bacteria metabolites

AIMS: To study the potential apoptosis effects of cytotoxic marine bacterial metabolites on human HeLa cell line. METHODS AND RESULTS: After HeLa cells were routinely cultured, tetrazolium-based colorimetric assay for cytotoxicity was performed to screen the marine bacteria extracts showing 12 strains active. To find the potential active strain with apoptosis mechanism, a battery of apoptosis assays, including AO/EB staining, TUNEL assay (terminal-deoxynucleotidyl transferase mediated nick end labelling), gel electrophoresis and flow cytometry, were used to determine whether apoptosis was involved in HeLa cell cytotoxicity of marine bacterial extracts. The results indicated that four strains could induce cell shrinkage, cell membrane blebbing, formation of apoptotic body and DNA fragmentation. CONCLUSIONS: Crude extracts of 12 of 153 strains of marine bacteria showed cytotoxic effects with ID50 ranged from 77.20 to 199.84 microg ml(-1), in which eight strains of bacteria were associated bacteria. The metabolites in the strains of QD1-2, NJ6-3-1, NJ1-1-1 and SS6-4 were able to induce HeLa cells apoptosis. Furthermore, the assessment by flow cytometry indicated that the hypodiploid apoptotic cells increased in a time-dependent manner, suggesting that induced apoptosis occurred from 24 h to 48 h after the extracts treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggested that the compounds from fermentation in these four marine bacterial strains could be candidates for developing apoptosis specific anti-tumour agents with lower toxicity. This study indicated that associated marine bacteria could be good source to find cytotoxic metabolites, and some cytotoxic marine bacterial metabolites could have apoptosis mechanisms.     

Different cell cycle mechanisms between UV-induced and X-ray-induced apoptosis in WiDr colorectal carcinoma cells

Although DNA-damaging agents such as ultraviolet (UV) and X-ray can induce apoptosis, the difference in the apoptotic mechanism is not clearly understood. In the present study, we investigated the effects of these two genotoxic agents on the induction of DNA damage and subsequent apoptotic cell death from the viewpoint of cell cycle regulation by using WiDr cells. Transient G1 arrest was observed after UV exposure, whereas G2 but not G1 arrest was induced after X-ray irradiation. UV-exposure could induce G1 arrest in both mutant-type (mt-p53) and wild-type p53 (wt-p53) cells, but obvious G1 arrest was not observed in the cells lacking in p53 expression. An increase in the DNA fragmentation was observed at S phase in UV-irradiated cells and at G2 phase in X-irradiated cells, respectively. UV-irradiated cells showed an increase production of p53 protein and accumulation of p21 protein. On the contrary, both p53 and p21 proteins remained at a low level in X-irradiated cells. Treatment with aphidicolin, an S phase blocking agent, prolonged cell cycle arrest and reduced the rate of apoptotic cell death in both UV-irradiated and X-irradiated cells. From these results, it is suggested that UV-induced apoptosis occurs mainly at S phase and is regulated by increased production of p53 and p21 proteins, while X-ray-induced apoptosis occurs after G2 blockade and may be independent of p53.     

Immunologic and biochemical effects of the fermented wheat germ extract Avemar

Avemar (MSC) is a nontoxic fermented wheat germ extract demonstrated to have antitumor effects. Avemar has the potential to significantly improve the survival rate in patients suffering from malignant colon tumors. We studied its effects in the HT-29 human colon carcinoma cell line. Avemar had an inhibiting effect on colonies of HT-29 cells with an IC50 value of 118 microg/ml (7 days of incubation); this value could be decreased to 100 and 75 microg/ml in the presence of vitamin C. In the cell line examined, Avemar induced both necrosis and apoptosis, as demonstrated by Hoechst/propidium iodide staining. The incubation of cells with 3200 microg/ml Avemar for 24 hrs caused necrosis in 28% and the induction of apoptosis in 22% of the cells. Avemar inhibited the cell-cycle progression of HT-29 cells in the G1 phase of the cell cycle. In addition, Avemar inhibited the activity of the key enzyme of de novo DNA synthesis, ribonucleotide reductase. In addition, we determined the effects of Avemar on the activity of cyclooxygenase-1 and -2. Both enzymes were significantly inhibited by Avemar with IC50 values of 100 and 300 microg/ml, respectively. We outline new explanations for its antitumor activity, which might serve as the basis for further studies using Avemar.     

Role of apoptosis in photodynamic sensitivity of human tumour cell lines

Photodynamic therapy (PDT) using a photosensitizer, such as haematoporphyrin derivative (HpD), in conjunction with visible light is a promising new modality to treat localized cancer. Cell death caused by PDT (through the generation of reactive oxygen species) can occur either by apoptosis (interphase death or as a secondary event following mitosis) and/or necrosis depending on the cell type, concentration and intracellular localization of the sensitizer, and the light dose. Since, apoptosis induced by PDT treatment plays an important role in determining the photodynamic efficacy, in the present work we have investigated the role of apoptotic cell death in relation to the observed differences in sensitivity to HpD-PDT between a human glioma cell line (BMG-1) carrying wild-type tumour suppressor gene p53 and a human squamous carcinoma cell line (4451) with mutated p53. HpD (photosan-3; PS-3) -PDT induced apoptosis was studied by: [A] flow-cytometric analysis of DNA content (sub G0/G1 population); [B] phosphatidylserine externalization (Annexin-V +ve cells); [C] cell size and cytoskeleton reorganization (light-scatter analysis); and [D] fluorescence microscopy (morphological features). PS-3-PDT induced a significantly higher level of apoptosis in BMG-1 cells as compared to 4451 cells. This was dependent on the concentration of PS-3 as well as post-irradiation time in both the cell lines. At 2.5 microg/ml of PS-3 the fraction of BMG-1 cells undergoing apoptosis (60%) was nearly 6 folds higher than 4451 cells (10%). In BMG-1 cells the induction of apoptosis increased with PS-3 concentration up to 5 microg/ml (>80%). However, a decrease was observed at a concentration of 10 microg/ml, possibly due to a shift in the mode of cell death from apoptosis to necrosis. In 4451 cells, on the other hand, the increase in apoptosis could be observed even up to 10 microg/ml of PS-3 (60%). Present results show that the higher sensitivity to PS-3-PDT in glioma cells arise on account of a higher level of apoptosis and suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in certain cell types.     

Anti-Fas antibody-induced apoptosis in human colorectal carcinoma cell lines: role of the p53 gene

The cell surface Fas antigen transducts an apoptotic signal by its crosslinking with Fas ligand or anti-Fas antibody in a variety of human cultured cells. In this study, we examined the expression of Fas antigen and its mediation of apoptosis in six human colorectal carcinoma cell lines. A flow cytometric analysis revealed that LoVo, DLD-1, WiDr and SW837 cell lines showed higher expression levels of Fas antigen, in contrast to lower expression in COLO201 and COLO320DM. Interferon- enhanced the expression of Fas antigen in all of the cell lines examined. Both Fas ligand and Fas-associated phosphatase-1 (FAP-1) were expressed only in COLO320DM. Anti-Fas antibody induced apoptosis in LoVo carrying wild-type p53 gene, but not in the other five cell lines carrying mutated p53 gene. The transfection of wild-type p53 gene using an adenovirous vector upregulated P53 protein in WiDr and SW837 cells, both of which showed, however, no increase in apoptotic cells by anti-Fas antibody treatment. These results indicated that (1) Fas antigen was variably expressed, regardless of the p53 gene status and (2) the susceptibility to anti-Fas antibody-mediated apoptosis did not correlate to Fas, Fas ligand or FAP-1 expression levels. Therefore, we conclude that wild-type P53 expression might not necessarily be essential for Fas-mediated apoptosis in human colorectal carcinoma cell lines.     

Phenylethyl isothiocyanate induces apoptotic signaling via suppressing phosphatase activity against c-Jun N-terminal kinase

Dietary isothiocyanates induce apoptosis in various cancer cell lines through a c-Jun N-terminal kinase (JNK)-dependent mechanism. We found that phenylethyl isothiocyanate (PEITC) was capable of inducing JNK activation and apoptosis in prostate cancer cell lines with distinct p53 statuses. PEITC induced JNK-mediated apoptotic signaling via a different pathway than that used by DNA-damaging agents, because genotoxicresistant LNCaP prostate cancer cells were equally sensitive to PEITC as parental LNCaP cells. PEITC did not induce significant MKK4 or MKK7 activation and did not activate JNK directly, suggesting that JNK and JNK upstream kinases are not primary targets of PEITC. The JNK dephosphorylation and inactivation rates were decreased in cells exposed to PEITC. Expression levels of M3/6, a JNK-specific phosphatase, were down-regulated by PEITC via a proteasome-dependent mechanism. Taken together, our data suggest that PEITC activates JNK through suppression of JNK dephosphorylation and that PEITC may be an alternative therapeutic agent for cancers that are resistant to genotoxic agents. This study also reveals that JNK phosphatases are potential targets for the development of novel cancer therapeutic agents.     

Stable overexpression of specific segments of the P2P-R protein in human MCF-7 cells promotes camptothecin-induced apoptosis

The stable overexpression of near full-length P2P-R protein in human Saos 2 cells restricts cell cycle progression by inducing mitotic arrest at prometaphase and mitotic apoptosis (Gao and Scott, 2002). Those effects of P2P-R were observed in Saos-2 cells that lack p53 and employ a caspase-3-dependent apoptotic signaling pathway. The current studies were performed to evaluate if overexpression of specific segments of the P2P-R protein promote apoptosis in human MCF-7 cells that contain p53 and employ a different apoptotic signaling pathway. Since segments of P2P-R were found not to induce apoptosis independently, the ability of three different P2P-R segments to promote camptothecin-induced apoptosis was evaluated following their stable transfection and expression in MCF-7 cells. Relative to full-length P2P-R (1-1560 aa), the three P2P-R segments used in these studies included: P2P-R-2 (761-1560 aa), P2P-R-3 (1156-1560 aa), and P2P-R-4 (1314-1560 aa). The results document that overexpression of P2P-R-2 and P2P-R-3 promotes camptothecin-induced apoptosis by three to fivefold when assayed by flow cytometric analysis of apoptotic sub 2n cell populations or by TUNEL assays. In contrast, P2P-R-4 had no effect on apoptosis. These results suggest that the ability of P2P-R to promote camptothecin-induced apoptosis in MCF-7 cells involves a specific region (1156-1314 aa) that exists within P2P-R. The data presented also show that the p53 binding domain of P2P-R overlaps with the apoptosis-associated region and previous studies documented that this region of P2P-R also binds single-strand nucleotides (Witte and Scott, 1997). Therefore, P2P-R-promoted apoptosis induced by camptothecin may be influenced by such interactions.     

Taxol-induced mitotic block triggers rapid onset of a p53-independent apoptotic pathway.

BACKGROUND: At therapeutic concentrations, the antineoplastic agent taxol selectively perturbs mitotic spindle microtubules. Taxol has recently been shown to induce apoptosis, similar to the mechanism of cell death induced by other antineoplastic agents. However, taxol has shown efficacy against drug-refractory cancers, raising the possibility that this pharmacological agent may trigger an alternative apoptotic pathway. MATERIALS AND METHODS: The kinetics and IC50 of mitotic (M) block, aberrant mitosis, and cytotoxicity following taxol treatment were analyzed in human cell lines as well as normal mouse embryo fibroblasts (MEFs) and MEFs derived from p53-null mice. Apoptosis was followed by DNA gel electrophoresis and by in situ DNA end-labeling (TUNEL). RESULTS: Taxol induced two forms of cell cycle arrest: either directly in early M at prophase or, for those cells progressing through aberrant mitosis, arrest in G1 as multimininucleated cells. TUNEL labeling revealed that DNA nicking occurred within 30 min of the arrest in prophase. In contrast, G1-arrested, multimininucleated cells became TUNEL positive only after several days. In the subset of cells that became blocked directly in prophase, both wt p53-expressing and p53-null MEFs responded similarly to taxol, showing rapid onset of DNA nicking and apoptosis. However, p53-null MEFs progressing through aberrant mitosis failed to arrest in the subsequent G1 phase or to become TUNEL positive, and remained viable. CONCLUSIONS: Taxol induces two forms of cell cycle arrest, which in turn induce two independent apoptotic pathways. Arrest in prophase induces rapid onset of a p53-independent pathway, whereas G1-block and the resulting slow (3-5 days) apoptotic pathway are p53 dependent.     

A novel adamantane thiadiazole derivative induces mitochondria-mediated apoptosis in lung carcinoma cell line

The interaction of organic compounds with apoptosis regulatory proteins is an attractive field of research because of its relevance in the development of new chemotherapeutic agents for cancer treatment. Our group designed four new adamantane thiadiazole derivatives (ATDs). The four ATDs were theoretically tested for their binding affinities to a model of an apoptosis inhibitor protein using molecular modeling. ATD-4 which interacted with the highest binding affinity was synthesized and characterized. The in vitro cytotoxicity of ATD-4 against different cancer cell lines as well as normal cell line was determined and compared with 5-fluorouracil as a standard positive control. The lung carcinoma cell line that showed the highest cytotoxic activity due to ATD-4 treatment was chosen to further study if ATD-4 can perform its cytotoxic activity through the induction of apoptosis as expected from molecular modeling. Inducing apoptosis by ATD-4 in lung carcinoma cell line was assessed by various biochemical and morphological characteristics. Biochemically: The effect of ATD-4 on cell cycle and its ability to induce apoptosis were checked through flow cytometry. Caspase-3 activity was detected by a colorimetric method. Real time-polymerase chain reaction (q-PCR) was used to detect p53, caspase-3, bcl-2 and bax gene expression. Morphologically: Changes in cell surface morphology, granulation and average surface roughness were detected using atomic force microscopy (AFM). Cell shrinkage, increase in cytoplasmic organelles, changes in mitochondrial number and morphology, chromatin condensation, membrane blebbing and formation of apoptotic bodies were detected using transmission electron microscopy (TEM). The obtained results suggest that ATD-4 exerted its antitumor activity against A549 cells through the induction of the intrinsic (mitochondrial) apoptotic pathway.