Hesperidin-CAMKIV interaction and its impact on cell proliferation and apoptosis in the human hepatic carcinoma and neuroblastoma cells

Calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a key regulatory molecule of cell signaling, and thereby controls its growth and proliferation, including expression of certain genes. The overexpression of CAMKIV is directly associated with the development of different types of cancers. Hesperidin is abundantly found in citrus fruits and exhibits wide range of pharmacological activities including anti-inflammatory, antibacterial and anticancerous effects. We have investigated binding mechanism of hesperidin with the CAMKIV using molecular docking methods followed by fluorescence quenching and isothermal titration calorimetric assays. An appreciable binding affinity of hesperidin was observed with CAMKIV during fluorescence quenching and isothermal titration calorimetric studies. Efficacy of hesperidin to inhibit the growth of human hepatic carcinoma (HepG2) and neuroblastoma (SH-SY5Y) cancer cell lines were investigated. Hesperidin has significantly reduced the proliferation of HepG2 and SH-SY5Y cells and induces apoptosis by activating the caspase-3-dependent intrinsic pathway through the upregulation of proapoptotic Bax protein. Hesperidin treatment reduces the mitochondrial membrane potential of HepG2 and SH-SY5Y cells. All these observations clearly anticipated hesperidin a potent inhibitor of CAMKIV which may be further exploited a newer therapeutic approach for the management of different cancer types.     

NSAIDs activate PTEN and other phosphatases in human colon cancer cells: novel mechanism for chemopreventive action of NSAIDs

Studies on chemoprevention of colorectal cancer have generated increasing interest. The mechanisms involved in NSAIDs chemopreventive action are not fully elucidated. In this study, we examined in human colon cancer cells the effect of indomethacin and NS-398 (a pre-clinical selective COX-2 inhibitor) on expression of 96 genes of the EGF/PDGF signaling pathways essential for cell proliferation, migration, and survival. We found that indomethacin and NS-398 treatment significantly upregulated expression of the tumor suppressor gene, PTEN, the MAP kinase phosphatase-3, MKP-3, and the protein tyrosine phosphatase, SHP2. Additionally, NS-398 treatment increased expression of apoptotic genes such as BAD, STAT1, and CASP3. These results suggest that as a consequence of increased expression of phosphatases such as PTEN and the resulting dephosphorylation of kinases, NSAIDs can negatively regulate the EGF/PDGF pathways in colon cancer cells-a novel mechanism for NSAIDs' chemopreventive actions.     

Developmental programming: impact of prenatal testosterone excess on ovarian cell proliferation and apoptotic factors in sheep

Prenatal testosterone (T) excess leads to reproductive dysfunctions in sheep, which include increased ovarian follicular recruitment and persistence. To test the hypothesis that follicular disruptions in T sheep stem from changes in the developmental ontogeny of ovarian proliferation and apoptotic factors, pregnant Suffolk sheep were injected twice weekly with T propionate or dihydrotestosterone propionate (DHT; a nonaromatizable androgen) from Days 30 to 90 of gestation. Changes in developmental expression of proliferating cell nuclear antigen (PCNA), BCL2, BAX, activated CASP3, and FAS/FASLG were determined at Fetal Days 90 and 140, 22 wk, 10 mo, and 21 mo of age by immunocytochemisty. Prenatal T treatment induced changes in expression of proliferative and apoptotic markers in a follicle-, age-, and steroid-specific manner. Changes in BAX were evident only during fetal life and PCNA, BCL2, and CASP3 only postnatally. Prenatal T and not DHT increased PCNA and decreased BCL2 in granulosa/theca cells of antral follicles at 10 and 21 mo but decreased CASP3 in granulosa/theca cells of antral follicles at 22 wk (prepubertal) and 10 and 21 mo. Both treatments decreased BAX immunostaining in granulosa cells of Fetal Day 90 primordial/primary follicles. Neither treatment affected FAS expression at any developmental time point in any follicular compartment. Effects on BAX appear to be programmed by androgenic actions and PCNA, BCL2, and CASP3 by estrogenic actions of T. Overall, the findings demonstrate that fetal exposure to excess T disrupts the ovarian proliferation/apoptosis balance, thus providing a basis for the follicular disruptions evidenced in these females.     

Cloning of the bovine antiapoptotic regulator, BCL2-related protein A1, and its expression in trophoblastic binucleate cells of bovine placenta

This report studied the identification and sequence of a full-length cDNA for the bovine BCL2 antiapoptotic family member, BCL2-related protein A1 (BCL2A1), and its localized and quantitative expression in the placenta to clarify the regulatory mechanism of trophoblast cell proliferation and differentiation during implantation and placental development. We cloned a full-length bovine BCL2A1 cDNA with 725 nucleotides and an open-reading frame corresponding to a protein of 175 amino acids. The predicted amino acid sequence shared 78% homology with human BCL2A1. All BCL2 homology domains (BH1, BH2, BH3, and BH4) in bovine BCL2A1 were conserved as well as in other mammalian BCL2A1. In the placentomes, in situ hybridization demonstrated that the BCL2A1 was limited in binucleate cells expressing various pregnancy-specific molecules like placental lactogen. BCL2-associated X protein (BAX) was also expressed in binucleate cells. Quantitative real-time RT-PCR detection exhibited a high-level expression of BCL2A1 in the conceptus at Day 21 of gestation, and it was expressed and increased in the extraembryonic membrane, cotyledon, and intercotyledon from implantation to term. BAX expression intensity increased with progression of gestation and remained elevated in postpartum. Caspase-3 protein (CASP3) and mRNA (CASP3) were detected from late gestation to postpartum in placenta as well as in the results of TUNEL detection. We believe that the apoptosis of binucleate cells may be regulated by the balance of the BCL2A1 and BAX. BCL2A1 genes produced a BCL2A1 protein in the mammalian cell-expression system. This molecule is a new candidate for antiapoptotic maintenance of the binucleate cells that support placental functions throughout gestation in bovine.     

Phloroglucinol derivative MCPP induces cell apoptosis in human colon cancer

This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (3,6-bis(3-chlorophenylacetyl)phloroglucinol; MCPP) in human colon cancer cells. MCPP induced cell death and antiproliferation in three human colon cancer, HCT-116, SW480, and Caco-2 cells, but not in primary human dermal fibroblast cells. MCPP-induced concentration-dependent apoptotic cell death in colon cancer cells was measured by fluorescence-activated cell sorter (FACS) analysis. Treatment of HCT-116 human colon cancer cells with MCPP was found to induce a number of signature endoplasmic reticulum (ER) stress markers; and up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein (GRP)-78, phosphorylation of eukaryotic initiation factor-2α (eIF-2α), suggesting the induction of ER stress. MCPP also increased GSK3α/β(Tyr270/216) phosphorylation and reduced GSK3α/β(Ser21/9) phosphorylation time-dependently. Transfection of cells with GRP78 or CHOP siRNA, or treatment of GSK3 inhibitor SB216163 reduced MCPP-mediated cell apoptosis. Treatment of MCPP also increased caspase-7, caspase-9, and caspase-3 activity. The inhibition of caspase activity by z-DEVE-FMK or z-VAD-FMK significantly reduced MCPP-induced apoptosis. Furthermore, treatment of GSK3 inhibitor SB216763 also dramatically reversed MCPP-induced GRP and CHOP up-regulation, and pro-caspase-3 and pro-caspase-9 degradation. Taken together, the present study provides evidences to support that GRP78 and CHOP expression, and GSK3α/β activation in mediating the MCPP-induced human colon cancer cell apoptosis.     

Hesperidin Produces Cardioprotective Activity via PPAR-γ Pathway in Ischemic Heart Disease Model in Diabetic Rats

The present study investigated the effect of hesperidin, a natural flavonoid, in cardiac ischemia and reperfusion (I/R) injury in diabetic rats. Male Wistar rats with diabetes were divided into five groups and were orally administered saline once daily (IR-sham and IR-control), Hesperidin (100 mg/kg/day; IR-Hesperidin), GW9962 (PPAR-γ receptor antagonist), or combination of both for 14 days. On the 15^th day, in the IR-control and IR-treatment groups, rats were subjected to left anterior descending (LAD) coronary artery occlusion for 45 minutes followed by a one-hour reperfusion. Haemodynamic parameters were recorded and rats were sacrificed; hearts were isolated for biochemical, histopathological, ultrastructural and immunohistochemistry. In the IR-control group, significant ventricular dysfunctions were observed along with enhanced expression of pro-apoptotic protein Bax. A decline in cardiac injury markers lactate dehydrogenase activity, CK-MB and increased content of thiobarbituric acid reactive substances, a marker of lipid peroxidation, and TNF-α were observed. Hesperidin pretreatment significantly improved mean arterial pressure, reduced left ventricular end-diastolic pressure, and improved both inotropic and lusitropic function of the heart (+LVdP/dt and –LVdP/dt) as compared to IR-control. Furthermore, hesperidin treatment significantly decreased the level of thiobarbituric acid reactive substances and reversed the activity of lactate dehydrogenase towards normal value. Hesperidin showed anti-apoptotic effects by upregulating Bcl-2 protein and decreasing Bax protein expression. Additionally, histopathological and ultrastructural studies reconfirmed the protective action of hesperidin. On the other hand, GW9662, selective PPAR-γ receptor antagonist, produced opposite effects and attenuated the hesperidin induced improvements. The study for the first time evidence the involvement of PPAR-γ pathway in the cardioprotective activity of hesperidin in I/R model in rats.     

Topotecan and methotrexate alter expression of the apoptosis-related genes BCL2, FAS and BCL2L12 in leukemic HL-60 cells

The BCL2 family of genes (B-cell CLL/lymphoma 2; Bcl-2) plays a pivotal role in the highly regulated process of apoptosis. We have recently cloned a newly identified member of this family, BCL2L12, which was found to be differentially expressed in many tumors. It is known that topotecan and methotrexate act through induction of apoptosis in cancer cells. In the present study we investigated the expression profile of the novel apoptotic gene BCL2L12 in relation to other apoptotic genes in the human leukemic cell line HL-60, after treatment with topotecan or methotrexate. The kinetics of apoptosis induction and cell toxicity were investigated by DNA laddering and the MTT method, respectively. Gene expression levels were analyzed by RT-PCR using gene-specific primers. Downregulation of BCL2L12, BCL2 and FAS was observed after treatment of HL-60 cells with topotecan, while treatment with methotrexate led to downregulation of BCL2 and FAS, with no change in BCL2L12 expression. Our results support the significance of mRNA modulations in the expression of apoptosis-related genes during treatment of human leukemic cells with anticancer drugs.     

The lethal effect of bis-type azridinylnaphthoquinone derivative on oral cancer cells (OEC-M1) associated with anti-apoptotic protein bcl-2

Several drugs of aziridinylbenzoquinone analogs have undergone clinical trials as potential antitumor drugs. These bioreductive compounds are designed to kill tumor cells preferentially within the hypoxic microenvironment. From our previous reported data, it was found that the synthesized 2-aziridin-1-yl-3-[(2-[2-[(3-aziridin-1-yl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio]ethoxy]ethyl)thio]naphthoquinone (AZ-1) is a bioreductive compound with potent lethal effect on oral cancer cell, OEC-M1. It was found in this study that the lethal effect of the oral cancer cell lines OEC-M1 induced by AZ-1 was mediated through the cell cycle arrest and apoptosis pathway. The LC50 values of OEC-M1 and KB cells induced by AZ-1 compound were 0.72 and 1.02 microM, respectively, which were much lower than that of normal fibroblast cells (SF with LC50 = 5.6 microM) with more than 90% of normal fibroblasts surviving as compared to control at a concentration of AZ-1 as high as 2 microM. It was interesting to note that the LC50 of monotype diaziridinylbenzoquinone compound, diaziquone (AZQ), was 50 microM on OEC-M1 cells. Comparing the cytotoxicity of AZ-1 and AZQ on OEC-M1 cells, AZ-1 is approximately 70 times more potent than AZQ. By using Western blot, both G2/M phase cell cycle arresting protein, cyclin B, and anti-apoptotic protein, bcl-2, were expressed in OEC-M1 cell when the concentrations of AZ-1 were increased from 0.125 to 0.5 microM and then decreased from 1 to 2 microM of AZ-1 treatment as compared with control for 24 h. Both proteins were expressed most abundantly at 0.5 microM AZ-1. However, the expression of bcl-2 protein in OEC-M1 was significantly decreasing in a dose-dependent manner and was only about 50% protein level at 2 microM AZ-1 for 48h as compared with control. The cell survival check protein p53 increased from 1.72- to 2.8-fold and 1.36- to 2.16-fold at concentrations of AZ-1 from 0.125 to 2.0 microM in a dose-dependently increasing manner on OEC-M1 as compared with control for 24 and48 h treatments, respectively. The apoptotic-related phenomena were observed, which included apoptotic body formation and the enzyme activity change of caspase-3. The apoptotic bodies and caspase-3 activity of OEC-M1 were induced only at 2 microM AZ-1 for a 24h treatment, yet apoptotic body formation was observed at as low as 0.5 microM AZ-1 and in a dose-dependently increasing manner for a 48 h treatment. The caspase-3 activity was increased 20.6%, 26.8%, and 84.2%, respectively, at 0.5, 1, and 2muM concentrations of AZ-1 for a 48 h treatment as compared with control. These results indicate that AZ-1 induced the cell death of OEC-M1 through the G2/M phase arrest of cell cycle and anti-apoptosis first and then apoptosis following a 48 h treatment. All of the pathway might be associated with bcl-2 and p53 protein expression. We propose that the AZ-1 could be used as anti-oral cancer drug for future studies with animal models.     

Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

Butyrate has antitumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration, which is regulated by its transport. We have analysed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT-29), in butyrate-resistant cells (BCS-TC2.BR2) and in normal colonic cells (FHC). The properties of transport were analysed with structural analogues, specific inhibitors and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K(m)=109+/-16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K(m)=17.9+/-4.0 microM in BCS-TC2 cells) proton-monocarboxylate co-transporter that was energy-dependent and activated via PKCdelta (protein kinase Cdelta). All adenocarcinoma cells analysed express MCT (monocarboxylate transporter) 1, MCT4, ancillary protein CD147 and AE2 (anion exchanger 2). Silencing experiments show that MCT1, whose expression increases with butyrate treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate/bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time- and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumour regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes.     

Dual apoptotic effect of Xrel3 c-Rel/NF-kappaB homolog in human cervical cancer cells

Cervical cancer is one of the most common cancers affecting a woman's reproductive organs. Despite its frequency and recurrence, the death rate has been declining over the past 40 years, due to early detection and treatment. In a previous report [Shehata Marlene, Shehata Marian, Shehata Fady, Pater Alan. Apoptosis effects of Xrel3 c-Rel/Nuclear factor-kappa B homolog in human cervical cancer cells. Cell Biology International, in press], we studied the role of the NF-kappaB gene family in HeLa human cervical cancer cells, using the Xrel3 c-Rel homologue of Xenopus laevis. These results showed that the expression of Xrel3/c-Rel slowed cell growth, consistent with an upregulated expression of the cell cycle inhibitor p21 and the activated poly(ADP-ribose) polymerase (PARP) apoptosis effector. However, in this report, we examined more apoptotic and anti-apoptotic factors acting upstream and downstream in apoptosis pathways after cisplatin treatment of HeLa cervical cancer cells. After 1 microM cisplatin treatment, Xrel3 had an anti-apoptotic effect, based on significantly lower levels of apoptotic proteins, including caspase-8, caspase-3 and p21. Anti-apoptotic BAG-1 isoforms were upregulated. After 5 microM cisplatin treatment, expression of HeLa Xrel3 had an apoptotic effect, based on significantly increased expression of the cell cycle inhibitor p21 and apoptotic proteins, including cleaved PARP, caspase-8, and caspase-3. However, anti-apoptotic Bcl-2 and Bcl-X(L) were elevated and the cell cycle regulator cyclin D1 was slightly upregulated with both 1 and 5 microM cisplatin treatment. The HPV E6 oncoprotein showed no significant changes. These results support previous conclusions on the potential anti-apoptotic effects of c-Rel/NF-kappaB in mild stress environments, as opposed to the apoptotic effects associated with high stress conditions [Lake BB, Ford R, Kao KR. Xrel3 is required for head development in Xenopus laevis. Development 2001; 128(2), 263-73.]. Thus, c-Rel/NF-kappaB may potentially be of clinical significance in chemotherapy.     

Hesperidin modulates dextran sulfate sodium‐induced ulcerative colitis in rats: Targeting sphingosine kinase‐1‐ sphingosine 1 phosphate signaling pathway,mitochondrial biogenesis,inflammation, and apoptosis

Ulcerative colitis (UC) is a chronic gastrointestinal disorder interfering with life quality. A total of 60 male Wistar rats were divided into four equal groups: Control (group I), hesperidin only (group II), UC untreated (group III), and UC treated with hesperidin (group IV). Hesperidin had modulatory effects on UC pathogenesis, which might be through alleviating colonic sphingosine phosphate phosphatase 2 messenger RNA expression and sphingosine kinase‐1 levels, thus suppressing the subsequent downstream inflammatory and apoptotic cascades represented by decreased macrophage inflammatory protein‐1α and enhancement of B‐cell lymphoma 2 immunohistochemistry expression. Also, it improved mitochondrial biogenesis by increasing the peroxisome proliferator‐activated receptor‐gamma‐coactivator 1‐α level. It successfully restored redox potential as evidenced by marked alleviations of the nitric oxide and peroxynitrite levels, increasing total antioxidant capacity, and activating the superoxide dismutase enzyme. Also, hesperidin alleviated the UC disease activity index and improved the histopathological picture. These findings may offer a new therapeutic strategy for UC treatment.     

Comparison of growth inhibition profiles and mechanisms of apoptosis induction in human colon cancer cell lines by isothiocyanates and indoles from Brassicaceae

The isothiocyanates sulforaphane and PEITC (beta-phenethyl isothiocyanate) as well as the indoles indole-3-carbinol and its condensation product 3,3'-diindolylmethane are known to inhibit cancer cell proliferation and induce apoptosis. In this study, we compared the cell growth inhibitory potential of the four compounds on the p53 wild type human colon cancer cell line 40-16 (p53(+/+)) and its p53 knockout derivative 379.2 (p53(-/-)) (both derived from HCT116). Using sulforhodamin B staining to assess cell proliferation, we found that the isothiocyanates were strongly cytotoxic, whereas the indoles inhibited cell growth in a cytostatic manner. Half-maximal inhibitory concentrations of all four compounds in both cell lines ranged from 5-15 microM after 24, 48 and 72 h of treatment. Apoptosis induction was analyzed by immunoblotting of poly(ADP-ribose)polymerase (PARP). Treatment with sulforaphane (15 microM), PEITC (10 microM), indole-3-carbinol (10 microM) and 3,3'-diindolylmethane (10 microM) induced PARP cleavage after 24 and 48 h in both 40-16 and the 379.2 cell lines, suggestive of a p53-independent mechanism of apoptosis induction. In cultured 40-16 cells, activation of caspase-9 and -7 detected by Western blotting indicated involvement of the mitochondrial pathway. We detected time- and concentration-dependent changes in protein expression of anti-apoptotic Bcl-x(L) as well as pro-apoptotic Bax and Bak proteins. Of note is that for sulforaphane only, ratios of pro- to anti-apoptotic Bcl-2 family protein levels directly correlated with apoptosis induction measured by PARP cleavage. Taken together, we demonstrated that the glucosinolate breakdown products investigated in this study have distinct profiles of cell growth inhibition, potential to induce p53-independent apoptosis and to modulate Bcl-2 family protein expression in human colon cancer cell lines.     

BRCC2, a novel BH3-like domain-containing protein, induces apoptosis in a caspase-dependent manner

We report here the structure-functional characterization of a novel intronless gene, BRCC2, located on human chromosome 11q24.1. BRCC2 open reading frame (327 bp) codes for an approximately 12-kDa protein (108 amino acids (aa)) localized predominantly in the cytosol and to a lesser extent in the mitochondria. Ectopic expression of BRCC2 cDNA also was found in both the cytosol and mitochondria. Exogenous expression of BRCC2 caused apoptotic cell death in three different cell lines as evidenced by enhanced chromatin condensation, DNA fragmentation, or an enhanced number of cells in the sub-G(1) phase. In human prostate cancer cells (PC-3), BRCC2-induced DNA fragmentation was blocked efficiently by coexpression of the anti-apoptotic molecule, Bcl-X(L). Transient transfection of BRCC2 cDNA into PC-3 cells in the presence of a broad-range caspase inhibitor, Z-VAD-fmk (100 microM, 24 h), abrogated DNA fragmentation. Consistently, BRCC2 expression correlated with the activation of caspase-3 and caspase-9. An N-terminal deletion mutant of BRCC2 (10.2 kDa, Delta1-16 aa) lacking a BH3-like domain (5-12 aa, LPIEGQEI) or BRCC2 containing a mutant BH3-like domain (leucine 5-->glutamate) failed to induce apoptosis, whereas a C-terminal deletion mutant (6.8 kDa, Delta62-108 aa) retained the apoptotic activity comparable to the full-length BRCC2. Finally, the treatment of HeLa cells with doxorubicin or hydrogen peroxide (H(2)O(2)) led to an increase in the mitochondrial (heavy membrane) level of endogenous BRCC2 (doxorubicin (100 ng/ml), 5 h, approximately 2-fold; H(2)O(2) (200 microM), 2 h, approximately 2-fold). These findings demonstrate that BRCC2 functions as a proapoptotic molecule and suggest that BRCC2 induces a caspase-dependent mitochondrial pathway of cell death.     

Prior exposure to oxidized low-density lipoprotein limits apoptosis in subsequent generations of endothelial cells by altering promoter methylation

Oxidized LDL (ox-LDL) plays a critical role in atherogenesis, including apoptosis. As hypercholesterolemia causes epigenetic changes resulting in long-term phenotypic consequences, we hypothesized that repeated and continuous exposure to ox-LDL may alter the pattern of apoptosis in human umbilical vein endothelial cells (HUVECs). We also analyzed global and promoter-specific methylation of apoptosis-related genes. As expected, ox-LDL evoked a dose-dependent increase in apoptosis in the first passage HUVECs that was completely abrogated by lectin-like ox-LDL receptor (LOX-1)-neutralizing antibody. Ox-LDL-induced apoptosis was associated with upregulation of proapoptotic LOX-1, ANXA5, BAX, and CASP3 and inhibition of antiapoptotic BCL2 and cIAP-1 genes accompanied with reciprocal changes in the methylation of promoter regions of these genes. Subsequent passages of cells displayed attenuated apoptotic response to repeat ox-LDL challenge with blunted gene expression and exaggerated methylation of LOX-1, BAX, ANXA5, and CASP3 genes (all P < 0.05 vs. first exposure to ox-LDL). Treatment of cells with LOX-1 antibody before initial ox-LDL treatment prevented both gene-specific promoter methylation and expression changes and reduction of apoptotic response to repeat ox-LDL challenge. Based on these data, we conclude that exposure of HUVECs to ox-LDL induces epigenetic changes leading to resistance to apoptosis in subsequent generations and that this effect may be related to the LOX-1-mediated increase in DNA methylation.     

Heterogeneity of primary glioblastoma cells in the expression of caspase-8 and the response to TRAIL-induced apoptosis

Recent studies suggest that cancer stem cells (CSCs) are responsible for cancer resistance to therapies. We therefore investigated how glioblastoma-derived CSCs respond to the treatment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Neurospheres were generated from glioblastomas, characterized for CSC properties including self-renewal, cell differentiation and xenograft formation capacity, and analyzed for TRAIL-induced apoptosis, CASP8 genomic status, and caspase-8 protein expression. The neurosphere NSC326 was sensitive to TRAIL-induced apoptosis as evidenced by cell death and caspase-8, -3, and -7 enzymatic activities. In contrast, however, the neurosphere NSC189 was TRAIL-resistant. G-banding analysis identified five chromosomally distinguishable cell populations in the neurospheres. Fluorescence in situ hybridization revealed the variation of chromosome 2 copy number in these populations and the loss of CASP8 locus in 2q33-34 region in a small set of cell populations in the neurosphere. Immunohistochemistry of NSC189 cell blocks revealed the lack of caspase-8 protein in a subset of neurosphere cells. Western blotting and immunohistochemistry of human glioblastoma tumors demonstrated the expression of caspase-8 protein in the vast majority of the tumors as compared to normal human brain tissues that lack the caspase-8 expression. This study shows heterogeneity of glioblastomas and derived CSCs in the genomic status of CASP8, expression of caspase-8, and thus responsiveness to TRAIL-induced apoptosis. Clinic trials may consider genomic analysis of the cancer tissue to identify the genomic loss of CASP8 and use it as a genomic marker to predict the resistance of glioblastomas to TRAIL apoptosis pathway-targeted therapies.     

Effects of parathyroid hormone like hormone (PTHLH) antagonist, PTHLH(7-34), on fetoplacental development and growth during midgestation in rats

Parathyroid hormone-like hormone (PTHLH) secretion has been reported in human amnion, chorion, decidual cytotrophoblast, syncytiotrophoblast, endometrium, and myometrium; however, the functions of PTHLH during pregnancy, particularly during placenta formation and fetal development, are not well understood. We examined whether neutralization of PTHLH action using PTHLH antagonist, PTHLH(7-34), in rats during early gestation affects fetal and placental growth. Rats received s.c. a daily dose of either 0, 4, 12, or 36 microg of PTHLH(7-34) infused continuously through mini-osmotic pumps from Day 8 through Day 15 of pregnancy. Fetal weights measured on Day 15 were significantly decreased in rats treated with all the doses of PTHLH(7-34) compared to controls, and decreases in placental weights were significant at the 12-microg dose. TUNEL assay demonstrated an increased number of apoptotic cells in placenta of treated rats, including rats treated with the 4-microg dose. Cleaved caspase 3 (CASP3), caspase 9 (CASP9) (P < 0.05) and poly-ADP-ribose polymerase (PARP1) (P < 0.01) expression was increased and BCL2 (P < 0.01) expression was decreased in rats treated with 4 microg PTHLH(7-34) compared to that in control. Placental cytochrome c expression was increased (P < 0.01) in cytosolic and decreased (P < 0.01) in mitochondrial fraction in PTHLH(7-34)-treated rats. Caspase 8 expression was not affected by the treatment. Immunohistochemical analysis of platelet endothelial cell adhesion molecule (PECAM1) showed higher staining intensity in control than in treated rats. In conclusion, these results suggests that PTHLH plays a role in early pregnancy, and that antagonization of PTHLH action causes fetoplacental growth restriction through activation of mitochondrial pathway of apoptosis in the placenta and through decreased expression of PECAM1.