Immunomolecular evaluation of dihydroartemisinin effects on apoptosis in chronic lymphocytic leukemia cell lines.

INTRODUCTION: Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has recently shown to induce apoptosis in many types of cancer cells. In this study, we aimed to determine the effects of DHA on apoptosis in human chronic lymphocytic leukemia (CLL) cell lines. METHODS: The cells were treated separately and combined by DHA and Fludurabine (FLU) during 24, 48 and 72 hours. The cell viabilities determined by XTT method. Following separate and combined treatment of IC50 concentrations of DHA and FLU to the cells during 24 hours, the cells were analyzed by flow cytometry to determine the effects on apopotis staining with AnnexinV FITC and PI. mRNA and protein expression levels of TCTP, Mcl-1, Bcl-2, Bax and Caspase-3 were analyzed to find out the molecular mechanisms of apoptosis by using quantitative real-time PCR and flow cytometric methods. RESULTS: Treatment with DHA alone or in combination with FLU induced apoptosis in a dose dependent manner in CLL cells. DHA alone was more effective than FLU alone or combined treatment with DHA and FLU. Our results suggest that Bcl-2 protein family member Bax was active in the apoptotic response of CLL cells after DHA treatment. Moreover, the apoptotic response induced by DHA was independent from the p53 mutation status of the CLL cells. CONCLUSION: DHA might be a potential anti-cancer therapeutic for CLL.

Prognostic importance of markers for inflammation, angiogenesis and apoptosis in high grade glial tumors during temozolomide and radiotherapy.

INTRODUCTION: Angiogenesis, inflammation and apoptosis have an important place in the carcinogenesis of high-grade gliomas (HGG). We evaluated the postoperative levels and the prognostic importance of tumor necrosis factor-alpha (TNFα), interleukin 6 (IL6), endoglin (CD105), vascular endothelial growth factor (VEGF), M65 and M30 as markers of inflammation, angiogenesis and apoptosis in patients with HGG. METHODS AND RESULTS: Postoperative pretreatment sera were collected from 44 newly diagnosed patients with HGG. The control group was also consisted of 44 healthy people. The median age of all patients with HGG was 59 (range: 30-80). Temozolomide concurrent with radiotherapy was given to 37 patients. Thereafter 24 patients received consolidation temozolomide monotherapy. Mean chemotherapy cycle was 4.2. Progression free survival and overall survival were 6 (95% CI; 5.16-6.83) and 16 months (95% CI; 13.07-18.93) respectively in patients treated with concurrent chemoradiotherapy and consolidation chemotherapy. Relative to the control cohort endoglin (p = 0.000) and TNFα (p = 0.000) levels were significantly lower; however VEGF (p = 0.030) levels were higher in the patient group. In contrast, there were no significant change in IL-6 levels and the plasma apoptotic markers M65 (p = 0.085) and M30 (p = 0.292). In separate log rank tests, these biological markers did not correlate with survival. DISCUSSION AND CONCLUSION: In HGG, a significant decrease in endoglin and TNFα levels was observed, while VEGF levels were significantly increased postoperatively. However, with the power from this patient population, no correlation with survival was observed.

The effect of maternal serum sFAS/sFASL system on etiopathogenesis of preeclampsia and severe preeclampsia.

Abstract Objective: The objective of the study is to evaluate whether there is a difference in serum soluble free acid synthesis (sFas)/sFasL levels between patients with preeclampsia, severe preeclampsia and normal healthy pregnancy. Methods: This is a case control study comparing serum sFas and sFasL concentrations in patients with mild preeclampsia (n=26) and patients with severe preeclampsia (n=22) in the second and the third trimester pregnancy to normal healthy pregnant controls (n=21). Maternal serum sFas and sFasL was collected at the time of diagnosis of preeclampsia or severe preeclampsia and obtained using ELISA assay. The control group's characteristics of gestational age, maternal age, BMI, parity, smoking status, and history of hypertension were matched with the study groups. While evaluating the results, mean±SD for quantitaive data, the statistical methods of Kruskal Wallis and Mann-Whitney U tests for comparements between groups were used. Results: Maternal mean serum levels of sFas and sFasL were evaluated and statistical difference was not found in both preeclamptic pregnancies (846,65±96,74pg/ml and 17,48±15.60 pg/ml) and severe preeclamptic pregnancies ( 864,29±106,30 pg/ml and 99,92±336,69 pg/ml) versus control group (830,58±127,21 pg/ml and 30,13±22,69 pg/ml). For all demographic characteristics, there was also no statistical difference between study groups and control group. Conclusions: The finding of this study showed no difference in maternal serum levels of sFas and sFasL in preeclampsia and severe preeclampcia versus control group.

Novel hexahydropyridoindole derivative as prospective agent against oxidative damage in pancreatic ß cells.

The potential protective effect of (±)-8-methoxy-1,3,4,4a,5,9b-hexahydro-pyrido[4,3-b]indole-2-carboxylic acid ethyl ester (II) was assessed against hydrogen peroxide (H2O2)-cytotoxicity in rat pancreatic INS-1E ß cells and compared with the effect of the related pyridoindole, stobadine (I), a promising indole-type reference antioxidant. Only pre-treatment with the compound (II) led to a significant preservation of the metabolic and secretory functions of the cells exposed to H2O2. The caspase-9 and -3 activities, as well as the early apoptotic changes of plasma membrane, were suppressed in the cells pre-incubated with both of compounds tested. However, only pyridoindole (II) inhibited profoundly the time-delayed apoptotic changes, These results suggest that pyridoindole (II) characterized by enhanced intrinsic antioxidant efficiency, may protect ß cells against cytotoxic effects of H2O2, involved in the development of both type 1 and type 2 diabetes.

Effects of local and sustained release of FGF, IGF, and GH on germ cells in unilateral undescended testis in rats.

OBJECTIVES: To evaluate the effects of the local release of fibroblast growth factor (FGF), insulin-like growth factor (IGF), and growth hormone (GH) on a germ cell population of ipsilateral undescended and contralateral descended testes of rats with a surgically created unilateral abdominal testis for 12 weeks and after the application of growth factors after orchidopexy. METHODS: Forty 4-week old male Wistar albino rats were divided into 5 groups as follows: group 1, sham; group 2, gelatin; group 3, FGF; group 4, IGF; and group 5, GH. In the sham group, the right testis was exposed and sutured with 5-0 silk sutures. In groups 2-5, a right intra-abdominal testis was surgically created. After 12 weeks, orchidopexy was performed, and 1 cm(2) gelatin films were sutured to the right testes, either unloaded (group 2), or containing 2.5 microg FGF (group 3), 5 microg IGF (group 4), or 5 microg GH (group 5). After 30 days, both testes were removed for histopathologic investigation and DNA flow cytometry. The mean seminiferous tubular diameters (MSTDs), mean testicular biopsy scores (MTBSs), and percentages of haploid (1n) cells were calculated. RESULTS: Ipsilateral MSTD and MTBS significantly decreased in the gelatin and FGF groups compared with the sham, IGF, and GH groups. Contralateral MSTDs and MTBSs did not differ among groups. The haploid cell percentage significantly decreased in the ipsilateral and contralateral testes of the gelatin group compared with the sham, FGF, IGF, and GH groups. CONCLUSIONS: Local release of IGF and GH resulted in the preservation of germ cell histology in the ipsilateral testes of rats with a surgically created unilateral undescended testis for 12 weeks and after orchidopexy. The administration of IGF, GH, and FGF increased the haploid germ cell population in both ipsilateral undescended and contralateral descended testes.

Stobadine inhibits doxorubicin-induced apoptosis through a caspase-9 dependent pathway in P815 mastocytoma cells.

Doxorubicin (DOXO), a widely used chemotherapeutic agent, induces apoptosis in transformed and non-transformed cells. The apoptotic effect of DOXO has been linked to the generation of reactive oxygen species (ROS). Antioxidants may be effective in the prevention of DOX-induced apoptosis. In the present study we investigated the effects of stobadine, a pyridoindole antioxidant in a DOXO-induced apoptosis model of P815 cells by flow cytometric analyses and by measuring caspase-3 and caspase-9 activities. Pretreating cells with stobadine significantly increased cell viability and decreased apoptosis rate. Inhibition in apoptosis was observed at maximum levels following treatment of cells with 10(-7)M stobadine as evident from flow cytometric analyses. The antiapoptotic effect of stobadine was further confirmed by inhibition of caspase-3 and caspase-9 activities. We found that the antioxidative effects of stobadine were comparable to the effects of a well known antioxidant, N-acetyl l-cysteine (NAC).

Amplification of virus-induced antimelanoma T-cell reactivity by high-dose interferon-alpha2b: implications for cancer vaccines.

PURPOSE: The therapeutic effectiveness of cancer vaccines, composed of tumor antigens that are also self-antigens, may be limited by the normal mechanisms that preserve immunological tolerance. Consistent with this notion, we found that vaccination of melanoma patients with recombinant viral vaccines expressing gp100 (a melanoma antigen also expressed by normal melanocytes) produced only transient increases in noncytotoxic T cells specific for immunodominant gp100 epitopes. To improve the therapeutic effects of these vaccines, IFN-alpha2b (IFN-alpha) was administered to some high-risk patients. EXPERIMENTAL DESIGN: 7 HLA-A*0201(+) patients were injected with high doses of IFN-alpha (20 MU/m(2) x 20 doses) at various times after completing the vaccination protocol. Clinical toxicity and responses were documented, and the effects on gp100-reactive T cells were measured by IFN-gamma enzyme-linked immunospot assays, tetramers of HLA-A*0201 and gp100 epitopes, and cellular cytotoxicity assays. RESULTS: In patients who had previously responded to vaccination, high doses of IFN-alpha recalled gp100-reactive T cells with the ability to kill gp100-expressing tumor targets in vitro. Concomitant with the reappearance of these cytotoxic T cells, tumor regression was observed in the two patients with clinically evident metastatic disease. CONCLUSIONS: The finding that high-dose IFN recalls previously activated tumor-reactive T cells with potent killing ability suggests a strategy to maintain antitumor responses initiated by cancer vaccines.