Hector Alila

Acute effects of prostaglandin F2α on inositol phospholipid hydrolysis in the large and small cells of the bovine corpus luteum

The present studies were conducted to determine whether the large or small bovine luteal cell was the site for the stimulatory effect of prostaglandin F2 alpha (PGF) on phospholipase C-catalyzed inositol phospholipid hydrolysis. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and large luteal cells were isolated by flow cytometry using a Becton Dickson FACS 440 cell sorter. PGF provoked rapid (5-30 s) and sustained (up to 30 min) increases in the levels of inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively) in small luteal cells. IP3 was formed more rapidly than IP2 or IP following PGF treatment. The PGF-stimulated increase in IP3 was accompanied by a transient reduction in the levels of 3H-labeled phosphatidylinositol 4,5-bisphosphate. LiCl (10 mM) enhanced inositol phosphate accumulation in response to PGF. Maximal increases in inositol phosphate accumulation were observed with 1-10 microM PGF and half-maximal increases were observed with 60 nM PGF. PGF (1-10 microM) had no effect on cAMP levels but stimulated small increases in progesterone accumulation in 30 min incubations of small luteal cells. PGF also increased the accumulation of inositol phosphates in large luteal cells. The increases were apparent within 5 min of incubation (the earliest time examined) and further increases were observed in incubations lasting 30 min. PGF had no significant effect on cAMP or progesterone in 30 min incubations of large cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of the effects of cyclooxygenase prostanoids on progresterone production by small and large bovine luteal cells

Highly purified preparations of small and large bovine luteal cells were utilized to examine the effects of prostaglandins F2 alpha (PGF2 alpha), E2 (PGE2) and I2 (PGI2) analog on progesterone production. Corpora lutea were obtained from Holstein heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. Progesterone accumulation was determined in 1 x 10(5) small and 5 x 10(3) large cells after 2 and 4 h incubations respectively. Progesterone synthesis was increased (p less than 0.05) in the small cells by the increasing levels of PGF2 alpha, PGE2, carba-PGI2 and LH. PGF2 alpha, but not PGE2 or carba-PGI2 increased (p less than 0.05) LH-stimulated progesterone production. There was no interaction of various combinations of prostaglandins on progesterone production in the small cells. In the large cells, PGF2 alpha had no effect on basal progesterone production. However, it inhibited LH-stimulated progesterone synthesis. In contrast, PGE2 and carba-PGI2 stimulated (p less than 0.05) basal progesterone production in the large cells. In the presence of LH, high levels of carba-PGI2 inhibited (p less than 0.05) progesterone synthesis. The PGE2 and PGI2-stimulated progesterone production in the large luteal cells was also inhibited in the presence of PGF2 alpha. These data suggest all of the prostaglandins used exert a luteotropic action in the small cells. In the large cells only PGE2 and carba-PGI2 are luteotropic, while PGF2 alpha exerts a luteolytic action. The effects of the prostaglandins in the small and large luteal cells suggest that their receptors are present in both cell types.

Arachidonic acid and its metabolites increase cytosolic free calcium in bovine luteal cells

We studied the effects of arachidonic acid and its metabolites on intracellular free calcium concentrations ([Ca2+]i) in highly purified bovine luteal cell preparations. Corpora lutea were collected from Holstein heifers between days 10 and 12 of the estrous cycle. The cells were dispersed and small and large cells were separated by unit gravity sedimentation and flow cytometry. The [Ca2+]i was determined by spectrofluorometry in luteal cells loaded with the fluorescent Ca2+ probe, Fura-2. Arachidonic acid elicited a dose-dependent increase in [Ca2+]i in both small and large luteal cells, having an effect at concentrations as low as 5 microM; and was maximally effective at 50 microM. Several other fatty acids failed to exert a similar response. Addition of nordihydroguaiaretic acid (NDGA) or indomethacin failed to suppress the effects of arachidonic acid. In fact, the presence of both inhibitors resulted in increases of [Ca2+]i, with NDGA exerting a greater stimulation of [Ca2+]i than indomethacin. Prostaglandin F2 alpha (PGF2 alpha) as well as prostaglandin E2 (PGE2) increased [Ca2+]i in the small luteal cells. These results support the idea that arachidonic acid exerts a direct action in mobilizing [Ca2+]i, in the luteal cells. Furthermore, they demonstrate that the cyclooxygenase (PGF2 alpha and PGE2) and lipoxygenase products of arachidonic acid metabolism also play a role in increasing [Ca2+]i in bovine luteal cells. Since the bovine corpus luteum contains large quantities of arachidonic acid, these findings suggest that this compound may regulate calcium-dependent functions of the corpus luteum, including steroid and peptide hormone production and secretion.

Phorbol ester receptors in bovine luteal cells: relationship to protein kinase C

We investigated the binding kinetics of the tumor-promoting phorbol ester, phorbol-12,13-dibutyrate (PBt2) to dispersed total bovine luteal cells, purified small luteal cells, and purified luteal protein kinase C (PKC). Saturation analysis and competitive displacement techniques were used. Binding of [3H]PBt2 to total luteal cell preparations resulted in two distinct affinities. The high affinity component was characterized by a Kd of 4.5 +/- 1.5 nM. Analysis of [3H]PBt2 binding to total cells using competitive displacement demonstrated that the low affinity binding was specific and displaceable but dependent on concentrations of [3H]PBt2 far above the Kd for the high affinity binding. In contrast to the total cell preparations, only high affinity binding was observed in intact purified small luteal cells (Kd = 0.96 +/- 0.04 nM). Partial purification of luteal cytosolic PKC by DEAE-Sephadex chromatography resulted in co-elution of PKC enzyme activity and the [3H]PBt2 binding activity. Under conditions of saturating calcium (0.1 mM) and phosphatidylserine (PS) (100 micrograms/tube) concentrations, binding to the partially purified PKC preparation was found to be of a single high affinity and exhibited a Kd (1.3 +/- 0.2 nM) similar to the high affinity binding observed in intact cells. These results suggest that the primary phorbol ester receptor in luteal cells is PKC. However, a low affinity, high capacity [3H]PBt2 binding site also exists within the corpus luteum, either in the large cells or in the accessory cell fraction which consists mainly of endothelial cells.

Abstract 3715: EP-100 syndergizes with paclitaxel in ovarian, breast and prostate cancer cell lines

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL EP-100 is a targeted anti-cancer drug comprised of Luteinizing Hormone Releasing Hormone (LHRH) fused to a lytic peptide. EP-100 is currently being investigated in human clinical trials. EP-100 seeks and destroys LHRH receptor positive cancer cells via a novel mechanism of action involving direct membrane disruption. Studies were conducted to determine the cytotoxic effects of combining EP-100 with paclitaxel using multi-drug resistant LHRH receptor positive ovarian (OVCAR-3), breast (MDA-MB-231), uterine sarcoma (MES-SA-Dx5), prostate (PC-3) and LHRH receptor negative ovarian (SKOV-3) cancer cells. Cells were cultured in the presence of EP-100 (0.005 nM-50µM, N=8) or paclitaxel (0.0025-500 nM, N=8) alone or in combination for 48 to 72 hours in non-constant and constant ratio formats (N=6). Cell viability was measured by luminometric assays. Data for IC50 values were obtained using the GraphPad Prism and GraphPad Software for the Hill Equation. The synergistic effects of EP-100 and paclitaxel were expressed as Combination Index (CI) using the CompuSyn Software analysis program. EP-100 alone killed LHRH receptor positive OVCAR-3, MDA-MB-231, MES-SA-Dx5 and PC-3 cells at low micromolar concentrations after 48 to 72 h incubation(IC50 values 2.189 ± 0.027 µM, 2.092 ± 0.114 µM, 1.419 ± 0.133 µM and 1.9 ± 0.071 µM respectively). As expected, SKOV-3 cells did not respond well to EP-100 (IC50= 10.3 ±0.36 µM) because they lack LHRH receptors on their surfaces. OVCAR-3, MDA-MB-231, MES-SA-Dx5 and SKOV-3 cells were resistant to paclitaxel (IC50 values = 13.6 ± 0.7 nM 86.2 ± 5.9 nM, 94.1 ± 0.3 nM and 48.52 ± 4.5 nM, for OVCAR-3, MDA-MB-231, MES-SA-Dx5 and SKOV-3, respectively) and PC-3 cells were very sensitive to paclitaxel with IC50 of 5.2 ± 1.1 nM. The combination of EP-100 at 50 nM and paclitaxel at 500 nM resulted in 3620 and 6720- fold increased sensitivity when compared to paclitaxel alone. The median CI values were 0.18, 0.05, 0.25, and 0.6 for for MES-SA-Dx5, OVCAR-3, MDA-MB-231 and PC-3, respectively indicating synergistic responses in the cell lines. No synergy was observed in the LHRH receptor negative SKOV-3 cell line (CI=8.4). These results indicate that a combination of EP-100 and paclitaxel results synergistic responses in LHRH-receptor positive and multi-drug resistant cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3715. doi:1538-7445.AM2012-3715

Abstract 5600: Destruction of prostate cancer cell xenografts by FSH-Lytic peptide conjugates.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: In previous studies (Hansel, et al., Mol. and Cell. Endocrinol. 269:26-33, 2007),we showed that conjugates of membrane destroying lytic peptides with either LHRH or with a 15-amino acid segment of the β chain of human chorionic gonadotropin (hCG) target and destroy human prostate, breast and ovarian cancer cells in tumor bearing nude mice. Recently (Radu et al., N. England J. Med. 363:1621-1630, 2012) reported that endothelial cells in blood vessels supplying cancers express follicle-stimulating hormone (FSH) receptors. Objectives:The objectives of this study were to synthesize a bioconjugate of a lyticpeptide (Phor18) to each of three segments of the β chain of FSH that are known to bind to the FSH receptor, and test these conjugates (FSH90-95-Phor18, FSH81-95-Phor18 and FSH33-53-Phor18) for their ability to target and lyse prostate cancer cells in vitro and in vivo. Results: In in vivo experiments, administration of FSH90-95-Phor18 and FSH81-95-Phor18 significantly (p 0.05). The average tumor volume was maintained at significantly lower levels in the mice treated with FSH90-95-Phor18 (p=0.027), FSH81-95-Phor18 (p=0.029) when compared with vehicle and free peptide treatment groups during the study (p < 0.05). Immunohistochemical analyses for FSHR were performed on the tumor samples. FSHR-positive endothelial cells were found in many vessels supplying the tumors of control mice, but few FSHR-positive endothelial cells were present in tumors of mice treated with FSH90-95-Phor18, FSH81-95-Phor 18 or FSH33-53-Phor 18. FSH81-95-Phor 18 was the most effective of the three conjugates tested in destroying the FSHR-bearing endothelial cells and in inhibiting tumor growth of the PC-3 xenografts in nude mice. Conclusion: These data show that Phor18 conjugates of FSH β chain segments that bind to FSHR expressed by the endothelial cells of the blood vessels supplying the tumors are capable of inhibiting prostate cancer cell tumor growth by inhibiting angiogenesis. These FSH β chain-lytic peptide conjugates may be useful in treating prostate and other FSHR expressing cancers. Citation Format: Sita Aggarwal, Ted Gauthier, Hector Alila, Carola Leuschner, Namrata Karki, Rajasree Solipuram, William Hansel. Destruction of prostate cancer cell xenografts by FSH-Lytic peptide conjugates. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5600. doi:10.1158/1538-7445.AM2013-5600

Abstract 978: Synergistic activity of EP-100 and chemotherapies in cancer cell lines.

EP-100, a targeted anti-cancer drug comprised of a lytic peptide fused to Luteinizing Hormone Releasing Hormone (LHRH), is in Phase 2 clinical trial in tumors that over-express LHRH receptors in combination with paclitaxel. It kills cancer cells by membrane disruption. EP-100 was tested in combination with paclitaxel, vinorelbine, doxorubicin, cisplatinum, vincristine and 5 fluorouracil (5FU) in vitro. The sequence of adding EP-100 to the chemotherapeutics was tested in consideration of their half-lives (hours) than EP-100 (minutes). EP-100 was added to the cells before or after treatment with the chemotherapeutics to determine the order of treatment for maximum synergy. Cells were cultured in the presence of each single agent and in combination with EP-100 for 72h. Data were analyzed as IC50 values for each single drug and the combination with EP-100. Combination indices (CI) on interaction of EP-100 and each chemotherapeutic were determined. EP-100 alone was cytotoxic at low uM concentrations in LHRH receptor (+), multi-drug resistant human uterine sarcoma cell line MES-SA-Dx5 and a human breast cancer cell line MDA-MB-231 after 72 h incubation. Both cell lines were resistant to paclitaxel, vinorelbine, vincristine, 5-FU and cisplatinum. MES-SA-Dx5 cells were resistant to doxorubicin. Addition of EP-100 to each of the chemotherapeutics increased sensitivity to all of the drugs except 5-FU (Table 1). Combinations of EP-100 with paclitaxel, vincristine, vinorelbine, doxorubicin and cisplatinum resulted in potentiation of activity in a synergistic manner for both uterine sarcoma and breast cancer cell lines. The combination effects were synergistic with CIs of 0.2. The sequence of exposure was most potent when EP-100 was given first followed by incubation with paclitaxel, doxorubicin or vinorelbine. These results indicate that EP-100 synergizes with paclitaxel, doxorubicin, vinorelbine, vincristine and cisplatinum but not 5-FU in drug resistant human uterine sarcoma and breast cancer cells. Citation Format: Carola Leuschner, Shashi Gavini, Hector W. Alila. Synergistic activity of EP-100 and chemotherapies in cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 978. doi:10.1158/1538-7445.AM2013-978

Abstract 2829: Activity of EP-100 in Non-Hodgkin's Lymphoma - synergy in combination

EP-100 is a targeted anti-cancer peptide comprised of Luteinizing Hormone Releasing Hormone (LHRH) fused to a membrane-disrupting peptide (MDP). It is currently in Phase 1 clinical trial in tumors that over-express LHRH receptors. EP-100 kills cancer cells directly via membrane disruption. We tested a combination of EP-100 and doxorubicin in multi-drug resistant Non-Hodgkin9s Lymphoma (NHL) cell lines and primary cells from refractory/relapsed NHL patients. Cells were cultured in the presence of EP-100 (0.00001-100 μM) or unconjugated MDP alone or in combination with doxorubicin (0.0000056 - 56.5 µM) and EP 100 at 0.5, 5, 50 and 500 nM. Cytotoxicity was determined by membrane integrity and cell viability assays. LHRH receptor expression was determined by flow cytometry. The effect of EP-100 on purified human recombinant p-glycoprotein (h-pgp) pump was measured by ATPase activity. The IC50 values [µM] for EP-100 alone were 0.52±0.13, 0.95±0.2, 2.8±0.5, 0.9±0.13 and values for unconjugated MDP were 59±1.5, 25.6±1.7, 6.1±0.8 after 5 h of incubation for Daudi, Raji, Toledo, Hut78 cells, respectively. EP-100 specifically killed NHL patient cells and unconjugated MDP was ineffective. The IC50 values for EP-100 were 1.2 ± 0.1µM for cells obtained from three Mantle Cell Lymphoma patients (N=3), 2.3± 0.1 µM for Diffuse Large B Cell Lymphoma patient (N=1), 1.7± 0.3 µM for Follicular Lymphoma patients (N=4), and 1.6 ± 0.1 µM for one Waldenstr[[Unable to Display Character: ő]]m Macroglobunemia patient. EP-100 or unconjugated MDP did not kill B-cells from normal subjects (N=2) after 5 hour incubation. LHRH receptors were over-expressed on cell lines and patient cells. Combination of EP-100 with doxorubicin resulted in synergistic responses after 72 hours of incubation and Combination Index was Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2829. doi:1538-7445.AM2012-2829

Abstract 3522: Pre-treatment with FSH enhances the ability of EP-100 to target and destroy human pancreatic cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The expression of luteinizing hormone releasing hormone (LHRH) receptors by pancreatic cancer cells has been clearly established. We observed that pancreatic cancer cell lines contain different numbers of LHRH receptors (BxPC3>MIAPaCa-2>PANC-1) using relative quantitation of RNA by real time PCR. In an in vitro study, we found that treatment of PANC-1 cells in culture with small amounts (10 or 30 ng/ml) of follicle stimulating hormone (FSH) caused a 3-fold increase in LHRH receptor gene expression. Therefore, we hypothesized that pre-treatment of nude mice bearing PANC-1 tumor xenografts with FSH will increase the number of LHRH receptors which will enhance the ability of the EP-100 (LHRH conjugated lytic peptide) to target and destroy PANC-1 pancreatic cancer cells. Our main objective was to determine in vitro and in vivo if FSH enhances the efficacy of EP-100 in targeting and destroying PANC-1 cells. PANC-1 cells (5000/well) were pretreated with FSH (10 and 30 ng/mL) for 24 h followed by EP-100 (4 and 6 µM) for 24 h in a medium containing 0.2% Dextran charcoal treated serum. MTT cell viability assay results showed that pre-treatment of PANC-1 cells with FSH significantly (P<0.001) enhanced the cytotoxicity of EP-100. PANC-1 tumor xenograft bearing Athymic Balb/C nude mice (n=11) were treated with EP-100 (0.2 and 0.02 mg/kg), iv, once a week for three weeks with or without FSH pre-treatment, sc, (3 mg/kg for three days prior to administering the conjugate). The treatment groups included were baseline (sacrificed at the beginning of treatment), vehicle, FSH alone, EP-100 (0.02 mg/kg) alone, EP-100 (0.02mg/kg) with FSH, EP-100 (0.2 mg/kg) alone, EP-100 (0.2 mg/kg) with FSH. FSH pre-treatment significantly enhanced the ability of EP-100 (0.02 mg/kg – P<0.05 and 0.2 mg/kg – P<0.001 compared to baseline) to regress the growth of PANC-1 tumor xenografts in vivo. Histopathological evaluation revealed an increase in tumor necrosis in FSH pre-treated mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3522. doi:10.1158/1538-7445.AM2011-3522