γ-Linolenic Acid Induces Apoptosis in B-Chronic Lymphocytic Leukaemia Cells in Vitro

Gamma linolenic acid (GLA) is cytotoxic to many types of human cancer cells. Most chemotherapeutic agents are cytotoxic by inducing apoptosis. We examined the apoptotic activity of GLA on purified B-cells isolated from patients with B-cell chronic lymphocytic leukaemia (B-CLL) and from normal individuals. GLA significantly increased the degree of apoptosis in B-CLL B-cells after 24 hours of culture. The mean percentage of cells undergoing apoptosis when cultured in medium alone (spontaneous apoptosis) was 20% (range: 7 to 31%) (n=25) and in the presence of GLA (5μg-60μg) was: 42%-95%. In the presence of GLA 5μg/ml and dexamethasone the degree of apoptosis was 86% (range: 72 to 100%). GLA induced apoptosis in B-CLL B-cells at a higher level than that observed with normal B-cells at all lower concentrations tested 5, 10 and 15μg/ml: P=0.045; 0.027 and 0.022, respectively. At 30μg/ml of GLA, no significant difference in the percentage of cells displaying apoptosis between B-CLL and normal B-cells was observed (P=0.075). GLA induced apoptosis in B-CLL T-cells at both 10 and 30μg/ml. The degree of apoptosis in normal T-cells with GLA was also significant at the higher concentration of 30μg/ml. Interleukin 4 (IL4), a viability factor in B-CLL, and vitamin E, an anti-oxidant, protected B-CLL B-cells against GLA (20μg/ml)-induced apoptosis. These results demonstrate that GLA induces apoptosis in B-CLL B-and T-cells cells in-vitro and that they are more susceptible to GLA-induced apoptosis than normal peripheral blood B-and T-cells.     

Antileukemic activity of the HSP70 inhibitor pifithrin-μ in acute leukemia

Heat shock protein (HSP) 70 is aberrantly expressed in different malignancies and has emerged as a promising new target for anticancer therapy. Here, we analyzed the in vitro antileukemic effects of pifithrin-μ (PFT-μ), an inhibitor of inducible HSP70, in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cell lines, as well as in primary AML blasts. PFT-μ significantly inhibited cell viability at low micromolar concentrations in all cell lines tested, with IC50 values ranging from 2.5 to 12.7 μ, and was highly active in primary AML blasts with a median IC50 of 8.9 μ (range 5.7–37.2). Importantly, higher IC50 values were seen in normal hematopoietic cells. In AML and ALL, PFT-μ induced apoptosis and cell cycle arrest in a dose-dependent fashion. PFT-μ also led to an increase of the active form of caspase-3 and reduced the intracellular concentrations of AKT and ERK1/2 in NALM-6 cells. Moreover, PFT-μ enhanced cytotoxicity of cytarabine, 17-(allylamino)-17-desmethoxygeldanamycin, suberoylanilide hydroxamic acid, and sorafenib in NALM-6, TOM-1 and KG-1a cells. This is the first study demonstrating significant antileukemic effects of the HSP70 inhibitor PFT-μ, alone and in combination with different antineoplastic drugs in both AML and ALL. Our results suggest a potential therapeutic role for PFT-μ in acute leukemias.     

Plasma MIP-1beta levels and skin toxicity in Japanese non-small cell lung cancer patients treated with the EGFR-targeted tyrosine kinase inhibitor, gefitinib

Gefitinib (Iressa^®) is an orally active, selective EGFR tyrosine kinase inhibitor that blocks signal transduction pathways. Skin toxicity has been reported to be the major toxicity observed in patients treated with the EGFR-targeted tyrosine kinase inhibitors, such as gefitinib and erlotinib. Although the mechanisms underlying the development of the skin toxicity remain to be precisely clarified, immunological mechanisms are considered to be involved. We examined the correlations between the plasma levels of several cytokines and the risk of development of adverse events, especially skin toxicity, induced by the administration of gefitinib as first-line monotherapy in non-small cell lung cancer (NSCLC) patients.

Paired plasma samples were obtained from a total 28 patients of non-small cell lung cancer; the first before the initiation of gefitinib administration (250 mg/day) (24 patients) and the second 2 or 4 weeks after the initiation of treatment (23 patients). The plasma concentrations of 17 major cytokines were measured using a bead-based multiplex assay. The median concentrations of eight of these cytokines before the start of treatment ranged from 0.06 (IL-5) to 58.26 (MIP-1β) (μg/ml). The concentrations of the remaining nine cytokines were under the detectable limit (<0.01 μg/ml) in more than 50% of the samples. Comparisons of the levels before and after treatment showed no significant differences for any of the cytokines measured.

The MIP-1β levels were significantly lower in the patients with skin toxicity (16/24) as compared with those in the patients not showing any skin toxicity (59.1 ± 10.5 versus 119.0 ± 36.8; p = 0.042 by the two-sample t-test). The K-Nearest Neighbor Prediction (K = 3) showed the classification rate to be 75% for the prediction sets containing MIP-1β, IL-4 and IL-8. There were no significant associations between the levels of any of the cytokines measured and any other parameters, including the tumor response to the drug. In conclusion, the plasma MIP-1β level may be a useful predictor of the development of skin toxicity in patients receiving gefitinib treatment.     

Phase I and pharmacokinetic study of preirradiation chemotherapy with BCNU, cisplatin, etoposide, and accelerated radiation therapy in patients with high-grade glioma

Purpose: We conducted a Phase I study of bischloroethylnitrosourea (BCNU), cisplatin, and oral etoposide administered prior to and during accelerated hyperfractionated radiation therapy in newly diagnosed high-grade glioma. Pharmacokinetic studies of oral etoposide were also done.

Methods and Materials: Patients started chemotherapy after surgery but prior to definitive radiation therapy (160 cGy twice daily × 15 days; 4800 cGy total). Initial chemotherapy consisted of BCNU 40 mg/m² days 1–3, cisplatin 30 mg/m² days 1–3 and 29–31, and etoposide 50 mg orally days 1–14 and 29–42, repeated in 8 weeks concurrent with radiation therapy. BCNU 200 mg/m² every 8 weeks × 4 cycles was given after radiation therapy.

Results: Sixteen patients, 5 with grade 3 anaplastic astrocytoma and 11 with glioblastoma were studied. Grade 3–4 leukopenia (38%) and thrombocytopenia (31%) were dose-limiting. Other toxicities were anorexia (81%), nausea (94%), emesis (56%), alopecia (88%), and ototoxicity (38%). The maximum tolerated dose was BCNU 40 mg/m² days 1–3, cisplatin 20 mg/m² days 1–3 and 29–31, and oral etoposide 50 mg days 1–21 and 29–49 prior to radiation therapy and repeated in 8 weeks with the start of radiation therapy followed by BCNU 200 mg/m² every 8 weeks for 4 cycles. Median time to progression and survival were 13 and 14 months respectively. Responses occurred in 2 of 9 (22%) patients with evaluable disease. In pharmacokinetic studies, all patients achieved plasma concentrations of >0.1 μg/ml etoposide (the in vitro radiosensitizing threshold), following a 50 mg oral dose. The mean ± SD 2 hr and 6 hr plasma concentrations were 0.92 ± 0.43 μg/ml and 0.36 ± 0.12 μg/ml, respectively. Estimated duration of exposure to >0.1 μg/ml etoposide was 10–17 hr.

Conclusions: Preirradiation chemotherapy with BCNU, cisplatin, and oral etoposide with accelerated hyperfractionated radiation therapy in high-grade gliomas is feasible and merits further investigation. Sustained radiosensitizing concentrations can be achieved with low oral doses of etoposide.     

TCRδ Gene Rearrangements in Acute Myeloid Leukemia with T-lymphoid Antigen Expression

In this review we present our data concerning T-cell receptor (TCR) δ gene rearrangements in acute myeloid leukemia with cuexpression of T-lymphoid features (CD2/CD4/CD7; Ly+ AML). We found a correlation between TCRδ gene rearrangements and coexpression of these T-lymphoid features. Ten of 66 Ly+ AML and only one of 44 AML cases without this coexpression exhibited TCRδ gene rearrangements (p = .028). In contrast, no correlation was observed between terminal deoxynucleotidyl transferase (TdT) expression and the occurence of TCRδ gene rearrangements in AML. Rearrangements were found in two of 25 AML with and seven of 71 AML cases without TdT expression. Interestingly, nucleotide sequencing of junctional sites revealed up to 36 N-nucleotides in cases without or with only weak TdT expression indicating downregulation of TdT expression after the TCR rearrangement took place. Complete Vδ1Jδ1 and incomplete Dδ2Jδ1 gene rearrangements were observed most frequently in Ly+ AML. These recombination patterns were similar to patterns observed in acute T-lymphoblastic leukemia with coexpression of myeloid features (My+ T-ALL) suggesting transformation of a common myeloid/T-lymphoid progenitor cell in these cases.     

Inhibition of ��-secretase induces G2/M arrest and triggers apoptosis in breast cancer cells

γ-Secretase activity is vital for the transmembrane cleavage of Notch receptors and the subsequent migration of their intracellular domains to the nucleus. Notch overexpression has been associated with breast, colon, cervical and prostate cancers. We tested the effect of three different γ-secretase inhibitors (GSIs) in breast cancer cells. One inhibitor (GSI1) was lethal to breast cancer cell lines at concentrations of 2 μM and above but had a minimal effect on the non-malignant breast lines. GSI1 was also cytotoxic for a wide variety of cancer cell lines in the NCI60 cell screen. GSI1 treatment resulted in a marked decrease in γ-secretase activity and downregulation of the Notch signalling pathway with no effects on expression of the γ-secretase components or ligands. Flow cytometric and western blot analyses indicated that GSI1 induces a G2/M arrest leading to apoptosis, through downregulation of Bcl-2, Bax and Bcl-XL. GSI1 also inhibited proteasome activity. Thus, the γ-secretase inhibitor GSI1 has a complex mode of action to inhibit breast cancer cell survival and may represent a novel therapy in breast cancer.     

PTEN regulates colorectal epithelial apoptosis through Cdc42 signalling

Background:

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) regulation of the Rho-like GTPase Cdc42 has a central role in epithelial polarised growth, but effects of this molecular network on apoptosis remain unclear.

Methods:

To investigate the role of Cdc42 in PTEN-dependent cell death, we used flow cytometry, in vitro pull-down assays, poly(ADP ribose) polymerase (PARP) cleavage and other immunoblots in isogenic PTEN-expressing and -deficient colorectal cells (HCT116PTEN^+/+, HCT116PTEN^−/−, Caco2 and Caco2 ShPTEN cells) after transfection or treatment strategies.

Results:

The PTEN knockout or suppression by short hairpin RNA or small interfering RNA (siRNA) inhibited Cdc42 activity, PARP cleavage and/or apoptosis in flow cytometry assays. Transfection of cells with wild-type or constitutively active Cdc42 enhanced PARP cleavage, whereas siRNA silencing of Cdc42 inhibited PARP cleavage and/or apoptosis. Pharmacological upregulation of PTEN by sodium butyrate (NaBt) treatment enhanced Cdc42 activity, PARP cleavage and apoptosis, whereas Cdc42 siRNA suppressed NaBt-induced PARP cleavage. Cdc42-dependent signals can suppress glycogen synthase kinase-β (GSK3β) activity. Pharmacological inhibition of GSK3β by lithium chloride treatment mimicked effects of Cdc42 in promotion of PARP cleavage and/or apoptosis.

Conclusion:

Phosphatase and tensin homologue deleted on chromosome 10 may influence apoptosis in colorectal epithelium through Cdc42 signalling, thus providing a regulatory framework for both polarised growth and programmed cell death.     

Cytokinetics of a Human Neuroblastoma Cell Line Following Treatment with Dianhydrogalactitol

Studies were carried out on the effect of dianhydrogalactitol on a human neuroblastoma cell line in culture. The drug was cytotoxic at concentrations of 5 μg/ml and 10 μ/ml for 1 hour, but not at 1 μ/ml. Flow cytometry of DNA showed that cell kill was preceded by a transient accumulation of cells in early S at 10 hours, and in late S at 36 hours. Smaller increases in the percent of cells in S phase were seen after treatment with 1 μg/ml, without cell kill. It may be concluded that dianhydrogalactitol causes an S-phase block, followed by death of cells in late S at concentrations above 5 μg/ml.

*Assistant Attending Physician, Solid Tumor Service, Department of Medicine; Research Associate, Laboratory of Biophysics, Recipient, American Cancer Society Junior Faculty Clinical Award.     

Cell transfer regimens in patients with highly advanced surgically unresectable non-small cell lung cancer: significantly improved overall survival in patients with lower levels of serum immunosuppressive acidic protein

Sixty-one non-small cell lung cancer (NSCLC) patients with stage II and III/IV were enrolled and 49 completed immunotherapy. Patients were grouped based on immunosuppressive acidic protein (IAP). All patients received monthly intravenous infusions containing 1 × 10¹⁰ (mean cell number per patient) ex vivo expanded and IFN--treated peripheral blood mononuclear cells. No patients had grade 2 or greater adverse events. The patients with ≤580 μg/ml of serum IAP levels (n = 33) had significantly longer recurrence-free survival than those with >580 μg/ml of serum IAP levels (n = 16). Patients with lower IAP levels are still under immunotherapeutic control after 27 months free of recurrence. The IAP levels may be a prognostic marker for treatment efficacy in NSCLC. This immunotherapeutic regimen was feasible and well tolerated in patients with advanced NSCLC in terms of prolongation of survival.     

Antiproliferative activity of PEP005, a novel ingenol angelate that modulates PKC functions, alone and in combination with cytotoxic agents in human colon cancer cells

PEP005 is a novel ingenol angelate that modulates protein kinases C (PKC) functions by activating PKCδ and inhibiting PKCα. This study assessed the antiproliferative effects of PEP005 alone and in combination with several other anticancer agents in a panel of 10 human cancer cell lines characterised for expression of several PKC isoforms. PEP005 displayed antiproliferative effects at clinically relevant concentrations with a unique cytotoxicity profile that differs from that of most other investigated cytotoxic agents, including staurosporine. In a subset of colon cancer cells, the IC₅₀ of PEP005 ranged from 0.01–140 μM. The antiproliferative effects of PEP005 were shown to be concentration- and time-dependent. In Colo205 cells, apoptosis induction was observed at concentrations ranging from 0.03 to 3 μM. Exposure to PEP005 also induced accumulation of cells in the G1 phase of the cell cycle. In addition, PEP005 increased the phosphorylation of PKCδ and p38. In Colo205 cells, combinations of PEP005 with several cytotoxic agents including oxaliplatin, SN38, 5FU, gemcitabine, doxorubicin, vinorelbine, and docetaxel yielded sequence-dependent antiproliferative effects. Cell cycle blockage induced by PEP005 in late G1 lasted for up to 24 h and therefore a 24 h lag-time between PEP005 and subsequent exposure to cytotoxics was required to optimise PEP005 combinations with several anticancer agents. These data support further evaluation of PEP005 as an anticancer agent and may help to optimise clinical trials with PEP005-based combinations in patients with solid tumours.     

Heterogeneous Effects of G-CSF and GM-CSF on Cell Growth and Ara-C Cytotoxicity in Childhood Leukemias Which Express Myeloid Markers

It is uncertain if acute lymphoblastic leukemia (ALL) cells expressing myeloid makers can respond to granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF). We investigated the effects of G-CSF (0.01 μg/ml) and GM-CSF (0.01 μg/ml) on [³H]thymidine (TdR) uptake, and the cytotoxicity of l-β-D-arabinofuranosylcytosine (ara-C) in leukemia cells from 17 pediatric patients. ALL cells without myeloid markers did not respond to G-CSF or GM-CSF. On the other hand, these cytokines enhanced the [³H]TdR uptake and cell growth, not only of AML cells but also of ALL cells expressing myeloid antigens. However, G-CSF and GM-CSF did not always enhance the growth inhibitory effect of the cell cycle specific drug ara-C when the cells were co-cultured with the drug. There was no relationship between cell growth and the amount of [³H]TdR incorporation or the intracellular ara-CTP level. These results indicate the heterogeneous effects of G-CSF and GM-CSF on cell growth and ara-C sensitivity in childhood leukemia cells.     

Effects of a prospective antitumor agent, 1,4-bis(2''-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate, on cultured mammalian cells

The effects of 1,4-bis(2'-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate (CBH; NSC 57198) on cell viability, growth, progression through the cell cycle, survival, and differentiation were investigated in suspension cultures of murine lymphocytic leukemia (L1210) and erythroleukemic (FL) cells and normal human lymphocytes stimulated with phytohemagglutinin (PHA) and in adherent cultures of Chinese hamster ovary (CHO) cells. CBH was equally cytotoxic toward stationary and exponentially growing CHO cells. Cell viability was diminished by 50% following 24 hr exposure to approximately 50 μg CBH per ml. Treatment of quiescent human lymphocytes for 24 hr with up to 100 μg CBH per ml did not appreciably diminish cell viability though the subsequent stimulation of such lymphocytes with PHA was inhibited in a dose dependent fashion. L1210, FL cells, and PHA stimulated human lymphocytes were equally sensitive to CBH, 50% inhibition of growth was obtained following 24 hr treatment with 25 μg CBH per ml. Incubation for up to 48 hr with CBH did not result in differentiation of FL cells to mature hemoglobin containing cells. Constant exposure of L1210 cells and PHA-stimulated human lymphocytes to 10-50 μg CBH per ml resulted in accumulation of cells in G₂ + M phase; higher drug concentrations resulted in cell arrest in mid to late S phase and G₂ phase. A short 1-hr pulse of the drug resulted in a transient accumulation of L1210 cells in S and G₂ phases. However, cells recovered from a short pulse of drug and by 48 hr, both cell proliferation and the cell cycle distribution appeared normal. A detailed analysis of cell cycle progression of L1210 cells in the presence of the drug indicated that the duration of G₂ phase was extended at low concentrations (10 μg/ml) while the transit of cells through S was retarded with subsequent accumulation in late S and G₂ phase at higher (50 μg/ml) concentrations. Concomitant with cell arrest in S and G₂ phase an increase in cellular RNA content indicating unbalanced growth was observed. This state of unbalanced growth was reversible in cultures exposed to a 1-hr pulse of up to 100 μg CBH per ml; cellular RNA content returned to control values by 48 hr. No effect on nuclear chromatin as assayed by acid denaturation was observed. Though the exact mechanism of drug action is not known, the data are not incompatible with the drug acting as an alkylating agent.     

Tocopherols and saponins derived from Argania spinosa exert, an antiproliferative effect on human prostate cancer

The aim of our study is to evaluate the antiproliferative effect of tocopherols obtained from alimentary virgin argan oil extracted from the endemic argan tree of Morocco and of saponins extracted from argan press cake on three human prostatic cell lines (DU145, LNCaP, and PC3). The results were compared to 2-methoxyestradiol as antiproliferative drug candidates. Cytotoxicity and antiproliferative effects were investigated after cells' treatment with tocopherols and saponins compared to 2-Methoxyoestradiol as the positive control. Tocopherols and saponins extracted from argan tree and 2-methoxyestradiol exhibit a dose-response cytotoxic effect and an antiproliferative action on the tested cell lines. The best antiproliferative effect of tocopherols is obtained with DU145 and LNCaP cell lines (28 μg/ml and 32 μg/ml, respectively, as GI₅₀). The saponins fraction displayed the best antiproliferative effect on the PC3 cell line with 18 μg/ml as GI₅₀. Our results confirm the antiproliferative effect of 2-methoxyestradiol and show for the first time the antiproliferative effect of tocopherols and saponins extracted from the argan tree on hormone-dependent and hormone-independent prostate cancer cell lines. These data suggest that argan oil is of potential interest in developing new strategies for prostate cancer prevention.     

Killing of Human Lung Cancer Cells Using a New [111In]Bleomycin Complex [111In]BLMC

The ability of a [¹¹¹In]bleomycin complex ([¹¹¹In]BLMC) to kill five cell lines of human lung cancer (small cell lung cancer) was investigated. Cells were exposed to either 0.9% NaC1, [¹¹¹In]C1₃, BLM, [¹¹¹In]BLMC, nonradioactive InC1₃, or In-BLMC for 60 minutes, plated in soft agarose, and assessed for colony formation. [¹¹¹In]BLMC (40-200 μ;C) carried by 15-25 μ;g BLM/ml) was more cytotoxic than BLM (15-25 μ;g BLM/ml) by a factor of 1.6-5.3 for five cell lines. The percent survival of N417 cells was 28.4 for [¹¹¹In]BLMC (40 μ;Ci/15 μ;g BLM/ml) and 54.3 for BLM (15 μ;g/ml); 1.9 for [¹¹¹In]BLMC (200 μ;Ci/25 μ;g BLM/ml), and 10.0 for BLM (25 μ;g/ml). ¹¹¹InC1₃ (200 μ;Ci/ml) and nonradioactive InC1₃ failed to inhibit colony formation. The new [¹¹¹In]BLMC may be useful for therapy of some lung cancer patients.     

Successful mobilization of peripheral blood stem cells with the DHAP regimen (dexamethasone, cytarabine, cisplatinum) plus granulocyte colony-stimulating factor in patients with relapsed Hodgkin's disease

High-dose chemotherapy followed by autologous stem cell transplantation can improve the outcome of relapsed and refractory Hodgkin's disease (HD) patients. The objective of the trial was to determine the mobilizing potential of the DHAP salvage regimen (dexamethasone, cytarabine, cisplatin) for the collection of peripheral blood stem cells (PBSC) in patients with relapsed HD. The target yield of harvesting CD34 + cells was ≥ 2 × 10⁶/kg in order to support the subsequent myeloablative chemotherapy. Most of the 105 patients included were intensively pre-treated with different combination chemotherapy regimens prior to mobilization. The use of DHAP followed by granulocyte colony-stimulating factor (G-CSF; 10 μg/kg) resulted in the successful collection of adequate numbers of PBSC in 97.1% of patients (102 of 105) with a median harvest of CD34 + cells of 13 × 10⁶/kg (range 2.6 - 85.1). More than 2.0 × 10⁶ CD34 + cells/kg were achieved in 65 of 103 (63%) patients after 1 apheresis, the maximum number of aphereses for all patients was 3. It was found that the optimal time of PBSC harvest was at days 13 - 16 after initiating the mobilization regimen.

These results demonstrate that the salvage chemotherapy regimen, such as DHAP combined with G-CSF, can be successfully used to mobilize PBSC in HD patients.     

Novel HSP90 inhibitors, NVP-AUY922 and NVP-BEP800, radiosensitise tumour cells through cell-cycle impairment, increased DNA damage and repair protraction

Background:

Heat-shock protein 90 (Hsp90) has a crucial role in both the stabilisation and regulation of various proteins, including those related to radioresistance. Inhibition of Hsp90 may therefore provide a strategy for enhancing the radiosensitivity of tumour cells. This study explores the responses of four tumour cell lines (A549, GaMG, HT 1080 and SNB19) to combined treatment with ionising radiation (IR) and two novel inhibitors of Hsp90, NVP-AUY922 and NVP-BEP800. The techniques used included cell and colony counts, expression of Hsp90, Hsp70, Akt, survivin, cleaved caspase 3, p53, cell-cycle progression and associated proteins. DNA damage was analysed by histone γH2AX and Comet assays.

Results:

We found that NVP-AUY922 and NVP-BEP800 enhanced radiosensitivity in all tested cell lines. In contrast, only two cell lines (HT 1080 and GaMG) exhibited an increased rate of apoptosis after drug pretreatment, as revealed by western blot. In all tested cell lines, the expression of histone γH2AX, a marker of DNA double-strand breaks, after combined drug-IR treatment was higher and its decay rate was slower than those after each single treatment modality. Drug-IR treatment also resulted in impaired cell-cycle progression, as indicated by S-phase depletion and G2/M arrest. In addition, the cell cycle-associated proteins, Cdk1 and Cdk4, were downregulated after Hsp90 inhibition.

Interpretation:

These findings show that the novel inhibitors of Hsp90 can radiosensitise tumour cell lines of different entities through destabilisation and depletion of several Hsp90 client proteins, thus causing the depletion of S phase and G2/M arrest, increased DNA damage and repair protraction and, to some extent, apoptosis. The results might have important implications for the radiotherapy of solid tumours.     

Modulation of the reversibility of actinomycin D cytotoxicity in HeLa cells by verapamil

Actinomycin D treatment (0.001–0.005 μg/ml; 0.5–24 h) induced a dose and time response shifting of nucleolar to nuclear fluorescence. In the presence of verapamil, cells were more responsive to actinomycin D. Translocation of protein B23 occurred with lower doses of actinomycin D and in shorter incubation times in the presence of verapamil. Short exposure (0.5 h) of HeLa cells to actinomycin D (0.05–0.25 μg/ml) induced ‘reversible’ translocation of protein B23 as well as ‘reversible’ inhibition of cell growth and RNA synthesis. Verapamil (5 μM) included in the cell culture after removal of actinomycin D inhibited the recoveries of cell growth, RNA synthesis as well as the corresponding relocalization of protein B23 from the nucleoplasm to nucleoli. These results indicate that verapamil can potentiate the antiproliferating activity of actinomycin D by inhibiting reversibility of its cytotoxicity and suggest clinical application.     

PPAR�� agonists inhibit growth and expansion of CD133+ brain tumour stem cells

Brain tumour stem cells (BTSCs) are a small population of cells that has self-renewal, transplantation, multidrug resistance and recurrence properties, thus remain novel therapeutic target for brain tumour. Recent studies have shown that peroxisome proliferator-activated receptor gamma (PPARγ) agonists induce growth arrest and apoptosis in glioblastoma cells, but their effects on BTSCs are largely unknown. In this study, we generated gliospheres with more than 50% CD133+ BTSC by culturing U87MG and T98G human glioblastoma cells with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). In vitro treatment with PPARγ agonist, 15-Deoxy-Δ^12,14-Prostaglandin J₂ (15d-PGJ2) or all-trans retinoic acid resulted in a reversible inhibition of gliosphere formation in culture. Peroxisome proliferator-activated receptor gamma agonists inhibited the proliferation and expansion of glioma and gliosphere cells in a dose-dependent manner. Peroxisome proliferator-activated receptor gamma agonists also induced cell cycle arrest and apoptosis in association with the inhibition of EGF/bFGF signalling through Tyk2-Stat3 pathway and expression of PPARγ in gliosphere cells. These findings demonstrate that PPARγ agonists regulate growth and expansion of BTSCs and extend their use to target BTSCs in the treatment of brain tumour.     

Avemar, a nontoxic fermented wheat germ extract, induces apoptosis and inhibits ribonucleotide reductase in human HL-60 promyelocytic leukemia cells

Avemar (MSC) is a nontoxic fermented wheat germ extract demonstrated to significantly improve the survival rate in patients suffering from various malignancies. We investigated its effects in human HL-60 promyelocytic leukemia cells. After 24, 48, and 72 h of incubation, Avemar inhibited the growth of HL-60 cells with IC₅₀ values of 400, 190, and 160 μg/ml, respectively. Incubation with MSC caused dose-dependent induction of apoptosis in up to 85% of tumor cells. In addition, Avemar attenuated the progression from G2–M to G0–G1 phase of the cell cycle and was also found to significantly reduce the in situ activity of ribonucleotide reductase, the key enzyme of de novo DNA synthesis. We conclude that Avemar exerts a number of beneficial effects which could support conventional chemotherapy of human malignancies.