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1.
Int J Mol Sci ; 23(15)2022 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-35955449

RESUMO

Aims: Some gene variants in the sodium channels, as well as calcium channels, have been associated with Brugada syndrome (BrS). However, the investigation of the human cellular phenotype and the use of drugs for BrS in presence of variant in the calcium channel subunit is still lacking. Objectives: The objective of this study was to establish a cellular model of BrS in the presence of a CACNB2 variant of uncertain significance (c.425C > T/p.S142F) using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and test drug effects using this model. Methods and results: This study recruited cells from a patient with Brugada syndrome (BrS) and recurrent ventricular fibrillation carrying a missense variant in CACNB2 as well as from three healthy independent persons. These cells (hiPSC-CMs) generated from skin biopsies of healthy persons and the BrS patient (BrS-hiPSC-CMs) as well as CRISPR/Cas9 corrected cells (isogenic control, site-variant corrected) were used for this study. The hiPSC-CMs from the BrS patient showed a significantly reduced L-type calcium channel current (ICa-L) compared with the healthy control hiPSC-CMs. The inactivation curve was shifted to a more positive potential and the recovery from inactivation was accelerated. The protein expression of CACNB2 of the hiPSC-CMs from the BrS-patient was significantly decreased compared with healthy hiPSC-CMs. Moreover, the correction of the CACNB2 site-variant rescued the changes seen in the hiPSC-CMs of the BrS patient to the normal state. These data indicate that the CACNB2 gene variant led to loss-of-function of L-type calcium channels in hiPSC-CMs from the BrS patient. Strikingly, arrhythmia events were more frequently detected in BrS-hiPSC-CMs. Bisoprolol (beta-blockers) at low concentration and quinidine decreased arrhythmic events. Conclusions: The CACNB2 variant (c.425C > T/p.S142F) causes a loss-of-function of L-type calcium channels and is pathogenic for this type of BrS. Bisoprolol and quinidine may be effective for treating BrS with this variant.


Assuntos
Síndrome de Brugada , Células-Tronco Pluripotentes Induzidas , Potenciais de Ação , Arritmias Cardíacas/metabolismo , Bisoprolol/farmacologia , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Quinidina/farmacologia
2.
Asian J Transfus Sci ; 12(2): 136-140, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30692798

RESUMO

BACKGROUND: Platelet additive solutions (PAS) are crystalloid nutrient media used in place of plasma for platelet storage. They replace 60%-70% of plasma in platelet components, so the amount of storage plasma can be decreased. Platelets in PAS have lower risk for allergic transfusion reactions with equivalent clinical efficacy for controlling bleeding. AIM: The aim of this study is to evaluate the clinical and laboratory efficacy of PAS-platelets. MATERIALS AND METHODS: A total of 1674 single donor platelet (SDP) were collected in PAS in the month of June to September 2016 by different apheresis systems. The quality control tests were done on 356 units in 4 months. Total number of SDP were processed with Amicus device (n = 232), Trima Accel (n = 84), and MCS+ (n = 40). The parameters analyzed were antibody titer of anti-A and anti-B, volume, platelet count, pH, bacterial contamination, and reporting of adverse transfusion reaction. Antibody titers were checked by tube technique, and platelet counts were checked by hematology analyzer Sysmex poch 100i. The swirling was checked manually, and pH was checked with pH strips. RESULTS: Out of 356, 164 units were O group, 113 units were B group, 68 units were of A group, and the remaining 11 units were of AB Group. Anti-A and anti-B titer was significantly reduced in PAS-SDP and found 1:32 or less for all the units. All the units found negative for bacterial contamination. No transfusion reaction was reported of the units transfused. All other quality parameters for platelets also found satisfactory after implementing the additive solution. CONCLUSION: The ABO antibody titers were significantly reduced after addition of PAS. This facilitates the ABO incompatible SDP transfusion and helps in inventory management. The risk of allergic transfusion reaction decreases after reducing the amount of plasma from SDP units. Using PAS-SDP certainly improve the inventory management for platelets with no compromise on clinical and laboratory efficacy.

3.
J Indian Soc Periodontol ; 19(1): 37-42, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25810591

RESUMO

BACKGROUND: The purpose of this study was to assess the clinical and microbiological effects of the local and sub-gingival application of a hyaluronan gel on scaling and root planing (SRP) in the treatment of moderate generalized chronic periodontitis. MATERIALS AND METHODS: In this split mouth study, 72 teeth in 18 patients with generalized chronic periodontitis with moderate severity were chosen for the study. Plaque samples were obtained by paper points at required intervals. Contra-lateral pairs of premolars and canine teeth in the maxilla or the mandible were selected to receive test treatment or serve as controls. Experimental jaw quadrants received sub-gingival administration of 0.2-ml 0.8% hyaluronan gel into selected sites following SRP and 1-week later. Clinical parameters were assessed at baseline, 1(st), 4(th), and 12(th) week. Colony-forming units (CFU) per milliliter were assessed at baseline, after SRP and after 2 weeks of drug insertion Student t-test and repeated measure ANOVA (RMANOVA) were used in this study. RMANOVA was used to find the significance in bleeding on probing (BOP) and plaque index (PI) and t-test for probing pocket depth (PPD) and clinical attachment level (CAL). RESULTS: The results revealed that there was a significant reduction in BOP (P < 0.001) PI (P < 0.001), PPD (P < 0.001) and CAL (P < 0.001) were also observed in experimental jaw quadrant following SRP and insertion of 0.8% hyaluronan when compared with the control group. A statistically significant reduction of CFUs was also found (P < 0.001) in the experimental site when compared with the control site. CONCLUSION: Sub-gingival placement of 0.2-ml of 0.8% hyaluronan along with SRP resulted in a significant improvement in both clinical and microbiological parameters when compared with the control site.

4.
Bioorg Chem ; 37(2): 57-63, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19223058

RESUMO

Structure-functional characterization of vitamin D receptor (VDR) requires identification of structurally distinct areas of VDR-ligand-binding domain (VDR-LBD) important for biological properties of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). We hypothesized that covalent attachment of the ligand into VDR-LBD might alter 'surface structure' of that area influencing biological activity of the ligand. We compared anti-proliferative activity of three affinity alkylating derivatives of 1,25(OH)(2)D(3) containing an alkylating probe at 1,3 and 11 positions. These compounds possessed high-affinity binding for VDR; and affinity labeled VDR-LBD. But, only the analog with probe at 3-position significantly altered growth in keratinocytes, compared with 1,25(OH)(2)D(3). Molecular models of these analogs, docked inside VDR-LBD tentatively identified Ser237 (helix-3: 1,25(OH)(2)D(3)-1-BE), Cys288 (beta-hairpin region: 1,25(OH)(2)D(3)-3-BE,) and Tyr295 (helix-6: 1,25(OH)(2)D(3)-11-BE,) as amino acids that are potentially modified by these reagents. Therefore, we conclude that the beta-hairpin region (modified by 1,25(OH)(2)D(3)-3-BE) is most important for growth inhibition by 1,25(OH)(2)D(3), while helices 3 and 6 are less important for such activity.


Assuntos
Marcadores de Afinidade/química , Calcitriol/análogos & derivados , Reagentes de Ligações Cruzadas/química , Receptores de Calcitriol/química , Vitamina D/análogos & derivados , Células 3T3 , Marcadores de Afinidade/síntese química , Alquilantes/síntese química , Alquilantes/química , Animais , Sítios de Ligação , Calcitriol/síntese química , Calcitriol/química , Linhagem Celular , Simulação por Computador , Reagentes de Ligações Cruzadas/síntese química , Ligantes , Camundongos , Modelos Moleculares , Receptores de Calcitriol/metabolismo , Relação Estrutura-Atividade , Vitamina D/química , Vitamina D/farmacologia
5.
Bioorg Chem ; 36(3): 165-8, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18374965

RESUMO

Vitamin D-binding protein (DBP) and albumin (ALB) are abundant serum proteins and both possess high-affinity binding for saturated and unsaturated fatty acids. However, certain differences exist. We surmised that in cases where serum albumin level is low, DBP presumably can act as a transporter of fatty acids. To explore this possibility we synthesized several alkylating derivatives of (14)C-palmitic acid to probe the fatty acid-binding pockets of DBP and ALB. We observed that N-ethyl-5-phenylisooxazolium-3'-sulfonate-ester (WRK-ester) of (14)C-palmitic acid specifically labeled DBP; but p-nitrophenyl- and N-hydroxysuccinimidyl-esters failed to do so. However, p-nitrophenyl ester of (14)C-palmitic acid specifically labeled bovine ALB, indicating that the micro-environment of the fatty acid-binding domains of DBP and ALB may be different; and DBP may not replace ALB as a transporter of fatty acids.


Assuntos
Albuminas/química , Ácidos Graxos/metabolismo , Proteína de Ligação a Vitamina D/química , Sítios de Ligação , Transporte Biológico , Radioisótopos de Carbono , Ácido Palmítico/metabolismo
6.
Biochem Biophys Res Commun ; 365(4): 746-50, 2008 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-18035050

RESUMO

Serum vitamin D-binding protein (DBP) is structurally very similar to serum albumin (ALB); both have three distinct structural domains and high cysteine-content. Yet, functionally they are very different. DBP possesses high affinity for vitamin D metabolites and G-actin, but ALB does not. It has been suggested that there may be cross-talk among the domains so that binding of one ligand may influence the binding of others. In this study we have employed 2-p-toluidinyl-6-sulfonate (TNS), a reporter molecule that fluoresces upon binding to hydrophobic pockets of DBP. We observed that recombinant domain III possesses strong binding for TNS, which is not influenced by 25-hydroxyvitamin D(3) (25-OH-D(3)), yet TNS fluorescence of the whole protein is quenched by 25-OH-D(3). These results provide a direct evidence of cross-talk among the structural domains of DBP.


Assuntos
Proteína de Ligação a Vitamina D/química , Vitamina D/análogos & derivados , Sítios de Ligação , Humanos , Ligação Proteica , Vitamina D/química
7.
Chem Biol Drug Des ; 70(4): 302-10, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17937776

RESUMO

The cytotoxic, anti-proliferative and apoptotic effects of 3-Bromoacetoxy Calcidiol (B3CD), a derivative of vitamin D3 precursor calcidiol, on human neuroblastoma (NB) cells were examined. NB, predominantly a tumor of early childhood, is the most common extracranial solid tumor. Despite aggressive treatments, survival for advanced stages remains low and novel treatment strategies are needed. B3CD-induced apoptosis in various neuroblastic cells via caspases-3 and -9 activation. B3CD upregulated mitochondrial pro-apoptotic Bax and anti-apoptotic Bcl-2 expression, caused cytochrome c release, downregulated N-Myc expression and activated pro-survival marker Akt. Accordingly, B3CD treatment dose dependently reduced the viability of NB cells with IC50 values between 1 and 3 microm. The cytotoxicity of B3CD was significantly higher than for the calcemic parent-compound vitamin D3 (IC50 between 10 and 30 microm). Further studies revealed that B3CD treatment inhibits the proliferation of NB cells at low concentrations (IC50 between 30 and 100 nm). Cell cycle analysis showed a dramatic increase in the apoptotic sub-diploidal population along with a cell cycle block. In summary, the present study shows that B3CD is toxic to NB cells via suppression of cell proliferation and cell viability by caspase activation and regulation of survival signals. These results suggest that B3CD could be developed as a treatment for NB.


Assuntos
Apoptose/efeitos dos fármacos , Compostos de Bromo , Calcifediol/análogos & derivados , Proliferação de Células , Biomarcadores/metabolismo , Conservadores da Densidade Óssea/química , Conservadores da Densidade Óssea/farmacologia , Conservadores da Densidade Óssea/uso terapêutico , Compostos de Bromo/química , Compostos de Bromo/farmacologia , Compostos de Bromo/uso terapêutico , Calcifediol/química , Calcifediol/farmacologia , Calcifediol/uso terapêutico , Calcitriol/metabolismo , Calcitriol/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Humanos , Estrutura Molecular , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia
8.
J Exp Ther Oncol ; 5(4): 287-300, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17024969

RESUMO

Advanced ovarian cancer (OC) is not curable by surgery alone and chemotherapy is essential for its treatment. Isothiocyanates have been shown to inhibit carcinogen-induced tumorigenesis in animal models, yet no efforts have been made to determine their therapeutic potential in OC. In the present study, we investigated the mechanism of the anti-proliferative and apoptotic activity of benzyl isothiocyanate (BITC) in OC. BITC inhibited the proliferation of OC cells and induced apoptosis in OC cells. Apoptosis was induced by a strong activation of caspase-3 and -9, and cleavage of PARP-1. However, caspase-8 was not activated by BITC. Cytotoxic effects of BITC were reversed by the inhibition caspase-3 and -9 specific inhibitors. BITC showed a concentration dependent decrease in the levels of Bcl-2 with a concomitant increase in Bax levels. In addition, BITC activated proapoptotic signaling by phosphorylation JNK1/2 and p38 while simultaneously inhibiting survival signaling mediated by ERK1/2 and Akt phosphorylation in a dose-dependent manner. While JNK inhibitor SP600125 and p38 inhibitor SB203580, abolished the cytotoxic effect of BITC, MEK inhibitor, PD98059 and PI3 kinase inhibitor, LY294002 failed to show such reversal indicating a critical role played by JNK1/2 and p38 signaling in apoptosis induced by BITC. In summary, our studies demonstrate that BITC inhibits proliferation of OC cells and induces apoptosis via caspase-9 and -3 pathways. BITC inhibits ERK1/2 and Akt survival signaling while simultaneously activating pro-apoptotic p38 and JNK1/2. Therefore, BITC can be potentially developed as a therapeutic agent to treat OC.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Isotiocianatos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Animais , Caspase 3/metabolismo , Caspase 9/metabolismo , Replicação do DNA/efeitos dos fármacos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Feminino , Células HL-60 , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos
9.
J Cell Biochem ; 99(3): 665-70, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16795032

RESUMO

We hypothesized that estrogen receptor (ER) in hormone-sensitive breast cancer cells could be targeted for selective photodynamic killing of tumor cell with antiestrogen-porphyrin conjugates by combining the over-expression of ER in hormone-sensitive breast cancer cells and tumor-retention property of porphyrin photosensitizers. In this study we describe that a tamoxifen (TAM)-pyropheophorbide conjugate that specifically binds to ER alpha, caused selective cell-kill in MCF-7 breast cancer cells upon light exposure. Therefore, it is a potential candidate for ER-targeted photodynamic therapy of cancers (PDT) of tissues and organs that respond to estrogens/antiestrogens.


Assuntos
Antineoplásicos Hormonais , Neoplasias da Mama , Linhagem Celular Tumoral/efeitos dos fármacos , Clorofila/análogos & derivados , Fotoquimioterapia/métodos , Porfirinas/farmacologia , Porfirinas/uso terapêutico , Tamoxifeno/análogos & derivados , Antineoplásicos Hormonais/química , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Sobrevivência Celular , Clorofila/química , Clorofila/farmacologia , Clorofila/uso terapêutico , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Estrutura Molecular , Porfirinas/química , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Tamoxifeno/química , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico
10.
J Cell Biochem ; 99(3): 966-77, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16741968

RESUMO

We hypothesized that over-expression of estrogen receptor (ER) in hormone-sensitive breast cancer could be harnessed synergistically with the tumor-migrating effect of porphyrins to selectively deliver estrogen-porphyrin conjugates into breast tumor cells, and preferentially kill the tumor cells upon exposure to red light. In the present work we synthesized four (4) conjugates of C17-alpha-alkynylestradiol and chlorin e6-dimethyl ester with varying tether lengths, and showed that all these conjugates specifically bound to recombinant ER alpha. In a cellular uptake assay with ER-positive MCF-7 and ER-negative MDA-MB 231 human breast cancer cell-lines, we observed that one such conjugate (E17-POR, XIV) was selectively taken up in a dose-dependent and saturable manner by MCF-7 cells, but not by MDA-MB 231 cells. Furthermore, MCF-7 cells, but not MDA-MB 231 cells, were selectively and efficiently killed by exposure to red light after incubation with E17-POR. Therefore, the combination approach, including drug and process modalities has the potential to be applied clinically for hormone-sensitive cancers in organs where ER is significantly expressed. This could potentially be carried out either as monotherapy involving a photo-induced selective destruction of tumor cells and/or adjuvant therapy in post-surgical treatment for the destruction of residual cancer cells in tissues surrounding the tumor.


Assuntos
Antineoplásicos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Estradiol/análogos & derivados , Receptor alfa de Estrogênio/metabolismo , Estrogênios , Fotoquimioterapia , Porfirinas , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular , Estradiol/química , Estradiol/metabolismo , Estrogênios/química , Estrogênios/metabolismo , Feminino , Humanos , Luz , Estrutura Molecular , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/metabolismo , Porfirinas/química , Porfirinas/metabolismo
11.
Gynecol Oncol ; 103(1): 261-70, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16624391

RESUMO

OBJECTIVE: Epithelial ovarian cancer has the highest mortality rate among gynecologic cancers. Chemotherapy is an essential component of its treatment. While isothiocyanates are known to possess chemopreventive effects against various cancers, yet little is known about their chemotherapeutic potential in ovarian cancer (OC). In the present study, we examined the antiproliferative and apoptotic effect of phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate on OVCAR-3 cells. METHODS: Cytotoxic activity of PEITC on OVCAR-3 cells was determined using cell proliferation, apoptosis (DNA fragmentation and TUNEL assay) and caspase-activation studies. The role of PARP-1, Bax, and Bcl-2 in apoptosis was analyzed by Western blotting. Activation of JNK1/2, p38, Akt, ERK1/2, and c-Myc was examined by immunoblotting. Specific inhibitors of caspases, JNK1/2, p38, and MEK were used to corroborate these data. RESULTS: PEITC was cytotoxic to OVCAR-3 cells, and inhibited proliferation in a dose-dependent fashion (IC(50) = 23.2 microM). PEITC induced apoptosis by activating caspase-3 and -9, without capsase-8 activation. Anti-apoptotic Bcl-2 levels were suppressed while pro-apoptotic Bax levels were enhanced. PEITC suppressed activation of Akt, ERK1/2, and the expression of transcription factor c-Myc, while simultaneously activating pro-apoptotic p38 and JNK1/2. Specific inhibitors of caspase-3 and -9, JNK1/2, and p38 reversed the cytotoxic effect of PEITC. CONCLUSIONS: These findings demonstrate that PEITC exhibits cytotoxicity towards OVCAR-3 cells and induces apoptosis via caspase-9 and -3 pathways. PEITC inhibits Akt, ERK1/2 survival signaling, and c-Myc while simultaneously activating pro-apoptotic p38 and JNK1/2. Systematic preclinical and clinical trials with PEITC in ovarian cancer are indicated.


Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Isotiocianatos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Apoptose/fisiologia , Caspase 3 , Caspase 9 , Inibidores de Caspase , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
Gynecol Oncol ; 103(1): 176-80, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16564074

RESUMO

OBJECTIVES: Total parenteral nutrition (TPN) for terminal ovarian cancer patients remains controversial. In this study, we compared survival from time of terminal intestinal obstruction (TIO) diagnosis in patients who received TPN versus those who did not. METHODS: A historical cohort of 55 patients with stage IIIC/IV epithelial ovarian cancer hospitalized for TIO between 1994 and 2002 was studied. All patients were previously treated with paclitaxel/platinum following cytoreductive surgery. Exposure was administration of TPN after TIO. The primary outcome was survival from TIO diagnosis to death. Number of chemotherapy cycles completed after TIO diagnosis, major complications of TPN, and demographics were measured. Survival analysis was performed using Kaplan-Meier methods. RESULTS: The median survival from time of TIO diagnosis was 72 days (range 16-485) for patients receiving TPN and 41.0 days (range 4-133) for those not receiving TPN (P = 0.05), but no difference in survival was observed when adjusting for chemotherapy. Overall survival [median 23 (range 6-67) vs. 35 months (range 8-67), P = 0.03] was shorter for the TPN group. Demographic data were similar in both groups. Patients receiving TPN after obstruction were more likely to undergo concurrent chemotherapy (64 vs. 26%, P = 0.004). One major TPN-related complication was found. CONCLUSIONS: Ovarian cancer patients with TIO receiving TPN had a median survival benefit of 4 weeks. This benefit decreased when patients were treated with concurrent chemotherapy. Issues of cost, quality of life, and human values need to be investigated to assess the full impact of TPN in this patient population.


Assuntos
Obstrução Intestinal/terapia , Neoplasias Ovarianas/terapia , Nutrição Parenteral Total , Estudos de Coortes , Células Epiteliais/patologia , Feminino , Humanos , Obstrução Intestinal/complicações , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/complicações , Neoplasias Ovarianas/patologia , Estudos Retrospectivos , Taxa de Sobrevida , Assistência Terminal
13.
Gynecol Oncol ; 100(1): 116-27, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16203029

RESUMO

OBJECTIVES: Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Deprivation of iron (Fe), an essential micro-nutrient, by chelation is known to inhibit proliferation of several human cancers but its potential in ovarian cancer treatment remains unknown. We have evaluated the anti-proliferative activities of iron chelators, deferoxamine (DFO), and diethylenetriamine pentaacetic acid (DTPA), in human and rat ovarian cancer cells. METHODS: The effect of DFO and DTPA on CaOV-3 (human) and NUTU-19 (rat) ovarian cancer cells was determined by cell proliferation and apoptosis assays (Hoechst staining, DNA fragmentation, and caspase activation), cell cycle analysis, and Fe supplementation studies. RESULTS: DFO and DTPA were cytotoxic to ovarian cancer cells in a dose- and time-dependent manner. DFO inhibited proliferation of NUTU-19 and CaOV-3 cells (IC(50) at 45 and 280 microM, respectively), while DTPA inhibited proliferation of only NUTU-19 cells (IC(50) at 50 microM), at 48 h. DNA synthesis was inhibited in CaOV-3 cells by DFO (>90% at 200 microM) and in NUTU-19 by both DFO and DTPA (>90% at 50 microM). Fe supplementation effectively reversed the cytotoxic effects of DFO and DTPA. Cell cycle analysis showed a G0/G1- and S-phase block with increased apoptosis. DNA fragmentation analysis confirmed apoptosis. Increase in caspase-3, -8, and -9 activities ( approximately 2.4-fold) was associated with apoptosis. CONCLUSIONS: Our studies show that Fe chelators suppress ovarian cancer growth by inhibiting proliferation and inducing apoptosis. Therefore, Fe chelators can be potentially developed as novel therapeutic agents to treat ovarian cancer.


Assuntos
Apoptose/efeitos dos fármacos , Desferroxamina/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Ácido Pentético/farmacologia , Animais , Caspases/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Inibidores do Crescimento/farmacologia , Humanos , Ferro/metabolismo , Quelantes de Ferro/farmacologia , Neoplasias Ovarianas/metabolismo , Ratos
14.
Angiogenesis ; 8(4): 349-60, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16400520

RESUMO

Angiogenesis is a complex process involving coordinated steps of endothelial cell activation, proliferation, migration, tube formation and capillary sprouting with participation of intracellular signaling pathways. Regulation of angiogenesis carries tremendous potential for cancer therapy. Our earlier studies showed that vitamin D-binding protein-macrophage activating factor (DBP-maf) acts as a potent anti-angiogenic factor and inhibits tumor growth in vivo. The goal of this investigation was to understand the effect of DBP-maf on human endothelial cell (HEC) and the mechanism of angiogenesis inhibition. DBP-maf inhibited human endothelial cell (HEC) proliferation by inhibiting DNA synthesis (IC(50) = 7.8 +/- 0.15 microg/ml). DBP-maf significantly induced S- and G0/G1-phase arrest in HEC in 72 h. DBP-maf potently blocked VEGF-induced migration, tube-formation of HEC in a dose dependent manner. In addition, DBP-maf inhibited growth factor-induced microvessel sprouting in rat aortic ring assay. Moreover, DBP-maf inhibited VEGF signaling by decreasing VEGF-mediated phosphorylation of VEGFR-2 and ERK1/2, a downstream target of VEGF signaling cascade. However, Akt activation was not affected. These studies collectively demonstrate that DBP-maf inhibits angiogenesis by blocking critical steps such as HEC proliferation, migration, tube formation and microvessel sprouting. DBP-maf exerts its effect by inhibiting VEGR-2 and ERK1/2 signaling cascades. Understanding the cellular and molecular mechanisms of anti-endothelial activity of DBP-maf will allow us to develop it as an angiogenesis targeting novel drug for tumor therapy.


Assuntos
Regulação para Baixo/fisiologia , Endotélio Vascular/metabolismo , Inibidores do Crescimento/fisiologia , Fatores Ativadores de Macrófagos/fisiologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Proteína de Ligação a Vitamina D/fisiologia , Inibição de Migração Celular/fisiologia , Proliferação de Células , Células Cultivadas , Replicação do DNA/fisiologia , Células Endoteliais/metabolismo , Fase G1/fisiologia , Humanos , Microvasos/crescimento & desenvolvimento , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neovascularização Patológica/fisiopatologia , Fosforilação , Fase S/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia
15.
Clin Cancer Res ; 10(23): 8018-27, 2004 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-15585637

RESUMO

The 25-hydroxyvitamin D(3) (25-OH-D(3)) is a nontoxic and low-affinity vitamin D receptor (VDR)-binding metabolic precursor of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. We hypothesized that covalent attachment of a 25-OH-D(3) analog to the hormone-binding pocket of VDR might convert the latter into transcriptionally active holo-form, making 25-OH-D(3) biologically active. Furthermore, it might be possible to translate the nontoxic nature of 25-OH-D(3) into its analog. We showed earlier that 25-hydroxyvitamin D(3)-3-bromoacetate (25-OH-D(3)-3-BE) alkylated the hormone-binding pocket of VDR. In this communication we describe that 10(-6) mol/L of 25-OH-D(3)-3-BE inhibited the growth of keratinocytes, LNCaP, and LAPC-4 androgen-sensitive and PC-3 and DU145 androgen-refractory prostate cancer cells, and PZ-HPV-7 immortalized normal prostate cells with similar or stronger efficacy as 1,25(OH)(2)D(3). But its effect was strongest in LNCaP, PC-3, LAPC-4, and DU145 cells. Furthermore, 25-OH-D(3)-3-BE was toxic to these prostate cancer cells and caused these cells to undergo apoptosis as shown by DNA-fragmentation and caspase-activation assays. In a reporter assay with COS-7 cells, transfected with a 1alpha,25-dihydroxyvitamin D(3)-24-hydroxylase (24-OHase)-construct and VDR-expression vector, 25-OH-D(3)-3-BE induced 24-OHase promoter activity. In a "pull down assay" with PC-3 cells, 25-OH-D(3)-3-BE induced strong interaction between VDR and general transcription factors, retinoid X receptor, and GRIP-1. Collectively, these results strongly suggested that the cellular effects of 25-OH-D(3)-3-BE were manifested via 1,25(OH)(2)D(3)/VDR signaling pathway. A toxicity study in CD-1 mice showed that 166 microg/kg of 25-OH-D(3)-3-BE did not raise serum-calcium beyond vehicle control. Collectively, these results strongly suggested that 25-OH-D(3)-3-BE has a strong potential as a therapeutic agent for androgen-sensitive and androgen-refractory prostate cancer.


Assuntos
Apoptose/efeitos dos fármacos , Calcitriol/análogos & derivados , Calcitriol/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Animais , Células COS , Proteínas de Transporte/metabolismo , Caspases/metabolismo , Cloranfenicol O-Acetiltransferase , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Masculino , Camundongos , Neoplasias Hormônio-Dependentes/patologia , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas , Próstata/citologia , Próstata/efeitos dos fármacos , Neoplasias da Próstata/patologia , Receptores de Calcitriol/metabolismo , Receptores X de Retinoides/metabolismo , Timidina/metabolismo , Células Tumorais Cultivadas
16.
J Steroid Biochem Mol Biol ; 89-90(1-5): 427-31, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15225814

RESUMO

BACKGROUND: Calcitriol [1,25-(OH)(2)D(3)] is a strong anti-proliferative agent both in vitro and in vivo. Earlier studies have established that calcitriol inhibits the growth factor-stimulated proliferation of endothelial cells (EC) and angiogenesis. However, the lethal calcemic side effects of calcitriol prohibit its use as a therapeutic agent. Several analogs of vitamin D have been developed to minimize these calcemic side effects. 1,25-dihydroxy-3-epi-vitamin D(3) (3-epiD(3)), a naturally formed vitamin D metabolite is one such analog. OBJECTIVE: To demonstrate that 3-epiD(3), a calcitriol analog, inhibits endothelial cell proliferation and induces apoptosis. RESULTS: Treatment of EC with 3-epiD(3) showed 60% inhibition (P < 0.006) of proliferation. Cell viability assays corroborated these results. Pro-apoptotic caspase-3 activity was increased fourfold (P < 0.01) in 3-epiD(3)-treated cells over controls. 3-epiD(3) induced apoptosis in EC as shown by genomic DNA fragmentation. Cell cycle analysis of 3-epiD(3)-treated EC revealed a G0/G1 arrest. CONCLUSIONS: 3-epiD(3), a low-calcemic, natural analog of calcitriol, inhibits EC proliferation by causing a G0/G1 arrest and induces apoptosis more effectively than 1,25-(OH)(2)D(3). These results suggest that 3-epiD(3) is a potent inhibitor of EC growth.


Assuntos
Colecalciferol/análogos & derivados , Colecalciferol/farmacologia , Endotélio Vascular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/citologia , Ativação Enzimática , Humanos
17.
J Cell Biochem ; 90(1): 87-96, 2003 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-12938159

RESUMO

Vitamin D-binding protein-macrophage-activating factor (DBP-maf) is derived from serum vitamin D binding protein (DBP) by selective deglycosylation during inflammation. In the present study, we investigated the effect of DBP-maf on RAW 264.7 macrophages and the underlying intracellular signal transduction pathways. DBP-maf increased proapoptotic caspase-3, -8, and -9 activities and induced apoptosis in RAW 264.7 cells. However, DBP, the precursor to DBP-maf did not induce apoptosis in these cells. Cell cycle analysis of DBP-maf-treated RAW 264.7 cells revealed growth arrest with accumulation of cells in sub-G(0)/G(1) phase. We also investigated the role of mitogen-activated protein kinase (MAPK) pathways in the DBP-maf-induced apoptosis of RAW264.7 cells. DBP-maf increased the phosphorylation of p38 and JNK1/2, while it decreased the ERK1/2 phosphorylation. Treatment with the p38 MAPK inhibitor, SB202190, attenuated DBP-maf-induced apoptosis. PD98059, a MEK specific inhibitor, did not show a significant inhibition of apoptosis induced by DBP-maf. Taken together, these results suggest that the p38 MAPK pathway plays a crucial role in DBP-maf-mediated apoptosis of macrophages. Our studies indicate that, during inflammation DBP-maf may function positively by causing death of the macrophages when activated macrophages are no longer needed at the site of inflammation. In summary, we report for the first time that DBP-maf induces apoptosis in macrophages via p38 and JNK1/2 pathway.


Assuntos
Apoptose/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Fatores Ativadores de Macrófagos/metabolismo , Macrófagos/metabolismo , Proteína de Ligação a Vitamina D/metabolismo , Animais , Ciclo Celular/fisiologia , Linhagem Celular , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Humanos , Macrófagos/citologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Explosão Respiratória
18.
J Cell Biochem ; 89(5): 909-16, 2003 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12874825

RESUMO

In this report we describe that 1,25(OH)(2)D(3)-3-BE, a VDR-affinity labeling analog of 1,25(OH)(2)D(3), showed strong and dose-dependent growth-inhibitory effect in several epithelial cells, i.e., keratinocytes (primary cells), MCF-7 breast cancer, PC-3, and LNCaP prostate cancer and PZ-HPV-7 immortalized normal prostate cell-lines. Furthermore, 10(-6) M of 1,25(OH)(2)D(3)-3-BE induced apoptosis specifically in LNCaP and PC-3 cells; and the effect was much less pronounced at lower doses. We also showed that the effect (of 1,25(OH)(2)D(3)-3-BE) was not due to probable degradation (hydrolysis) of 1,25(OH)(2)D(3)-3-BE or random interaction of this molecule with cellular proteins. Tissue- or cell-specific action of 1,25(OH)(2)D(3) and its mimics is not common due to the ubiquitous nature of VDR. Furthermore, variable effects of 1,25(OH)(2)D(3) and its analogs in various cell-lines potentially limits their application as anticancer agents. We showed that 1,25(OH)(2)D(3)-3-BE displayed similar growth-inhibitory and cytotoxic activities towards androgen sensitive LNCaP and androgen-independent PC-3 cell-lines. Therefore, these results raise the possibility that 1,25(OH)(2)D(3)-3-BE or similar VDR-cross linking analogs of 1,25(OH)(2)D(3) might be considered for further development as potential candidates for prostate cancer.


Assuntos
Calcitriol/análogos & derivados , Calcitriol/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Marcadores de Afinidade/química , Marcadores de Afinidade/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Calcitriol/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Citometria de Fluxo , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Masculino , Azul de Metileno/química , Próstata/citologia , Próstata/efeitos dos fármacos , Neoplasias da Próstata/patologia , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , Timidina/metabolismo
19.
Neoplasia ; 5(1): 32-40, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12659668

RESUMO

We have isolated a selectively deglycosylated form of vitamin D binding protein (DBP-maf) generated from systemically available DBP by a human pancreatic cancer cell line. DBP-maf is antiproliferative for endothelial cells and antiangiogenic in the chorioallantoic membrane assay. DBP-maf administered daily was able to potently inhibit the growth of human pancreatic cancer in immune compromised mice (T/C=0.09). At higher doses, DBP-maf caused tumor regression. Histological examination revealed that treated tumors had a higher number of infiltrating macrophages as well as reduced microvessel density, and increased levels of apoptosis relative to untreated tumors. Taken together, these data suggest that DBP-maf is an antiangiogenic molecule that can act directly on endothelium as well as stimulate macrophages to attack both the endothelial and tumor cell compartment of a growing malignancy.


Assuntos
Fatores Ativadores de Macrófagos/farmacologia , Neovascularização Patológica/prevenção & controle , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/patologia , Proteína de Ligação a Vitamina D/farmacologia , Alantoide/citologia , Alantoide/efeitos dos fármacos , Animais , Bovinos , Divisão Celular/efeitos dos fármacos , Embrião de Galinha/metabolismo , Córion/citologia , Córion/efeitos dos fármacos , Meios de Cultivo Condicionados , Endotélio Vascular/metabolismo , Humanos , Imunidade Celular , Ativação de Macrófagos , Macrófagos , Masculino , Camundongos , Camundongos SCID , Neuraminidase/metabolismo , Neoplasias Pancreáticas/metabolismo , Taxa de Sobrevida , Células Tumorais Cultivadas/transplante , beta-Galactosidase/metabolismo
20.
Bioorg Med Chem Lett ; 13(2): 213-6, 2003 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-12482426

RESUMO

Synthesis of an affinity alkylating non steroidal mimic of 1alpha,25-dihydroxyvitamin D(3) and its radiolabeled counterpart is presented. We also report the affinity labeling of the VDR-ligand binding domain (VDR-LBD) with this analogue.


Assuntos
Alquilantes/síntese química , Alquilantes/farmacologia , Receptores de Calcitriol/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Marcadores de Afinidade , Calcitriol/química , Estrogênios/química , Estrogênios/farmacologia , Indicadores e Reagentes , Mimetismo Molecular , Receptores de Estrogênio/efeitos dos fármacos
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