ABSTRACT
OBJECTIVES: To investigate the molecular machinery of docetaxel (Taxotere)-initiated death signaling on prostate cancer cell lines LNCaP and PC-3. Taxotere is a member of the taxane family of chemotherapeutic agents. It has been shown to disrupt microtubule dynamics causing mitotic arrest, which leads to cell death. Taxotere has demonstrated induction of cell death in LNCaP and PC-3 cells. However, the pathways by which apoptosis occurs differ in each cell line. METHODS: The prostate cancer cell lines, LNCaP and PC-3, were treated with 40 nM Taxotere for various lengths of time (0.5 to 24 hours). Western blot analysis was used for protein analysis. RESULTS: LNCaP cells demonstrated caspase-3 and caspase-7 cleavage, and PC-3 cells demonstrated only caspase-8 and BH3-interacting domain death agonist cleavage. Only LNCaP cells were observed to express clusterin expression; PC-3 cells expressed a novel apoptosis inhibitor, survivin. CONCLUSIONS: In this study, we demonstrated two distinctly different Taxotere-induced apoptotic pathways in LNCaP and PC-3 cells that may be of clinical importance when treating prostate cancer.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Microtubule-Associated Proteins , Paclitaxel/analogs & derivatives , Paclitaxel/pharmacology , Prostatic Neoplasms/pathology , Taxoids , Blotting, Western , Caspases/drug effects , Caspases/metabolism , Clusterin , Docetaxel , Glycoproteins/drug effects , Glycoproteins/metabolism , Humans , Inhibitor of Apoptosis Proteins , Male , Molecular Chaperones/drug effects , Molecular Chaperones/metabolism , Neoplasm Proteins/drug effects , Neoplasm Proteins/metabolism , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Proteins/drug effects , Proteins/metabolism , Survivin , Tumor Cells, CulturedABSTRACT
BACKGROUND: Estramustine in combination with other chemotherapeutic agents has demonstrated synergy in hormone-refractory prostate cancer. Docetaxel has demonstrated antineoplastic activity in a variety of chemotherapeutic-unresponsive tumors. We evaluated the effects of estramustine and docetaxel in preclinical models of prostate cancer. METHODS: Cell viability of PC-3 and MAT-LyLu (MLL) cells were assessed 48 hr after drug treatment. For in vivo studies, each flank of five animals in six groups was injected with 1 x 10(6) MLL cells: control, estramustine, docetaxel (low- and high-dose), and low- and high-dose docetaxel with estramustine. Animals were treated on days 4 and 11, and sacrificed on day 14. RESULTS: The IC(50) value for docetaxel was 2 nM in the PC-3 cells and 40 nM in the MLL cells. The addition of 100 nM of estramustine did not alter the IC(50) value for PC-3 cells. In the MLL cells, however, the IC(50) value was lowered to 15 nM. In vivo, low-dose docetaxel with estramustine demonstrated antineoplastic activity similar to that of high-dose docetaxel alone, suggesting additive activity between the drugs. CONCLUSIONS: These results demonstrate that when used in combination, docetaxel and estramustine can be more effective at lower dosages than when the individual drugs are used alone.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Paclitaxel/analogs & derivatives , Prostatic Neoplasms/drug therapy , Taxoids , Animals , Docetaxel , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Drug Synergism , Estramustine/administration & dosage , Humans , Inhibitory Concentration 50 , Male , Microtubules/drug effects , Neoplasm Transplantation , Neoplasms, Hormone-Dependent/drug therapy , Paclitaxel/administration & dosage , Rats , Tumor Cells, Cultured/drug effectsABSTRACT
BACKGROUND: Gemcitabine has demonstrated clinical activity against several common cancers. Our studies examine the ability of gemcitabine, both alone and in combination with other chemotherapeutic agents, to inhibit the in vitro and in vivo growth of several prostate cancer cell lines. MATERIALS AND METHODS: Cultures of LNCaP, PC-3 or MLL cells were exposed to either gemcitabine or other appropriate agents for specified amounts of time. Cells were lysed and nuclei counted utilizing a Coulter Counter. For in vivo experiments, animals were injected with 1 x 10(5) MLL cells subcutaneously into the right flank. Animals were treated as indicated for 14 days. Tumors were then excised, weighed and measured. RESULTS: In both human (PC-3 and LNCaP) and rat prostate (MLL) cancer cell lines our studies demonstrated gemcitabine had a strong effect in vitro, with an IC50 of approximately 500 nM in the human lines and 10 nM in MLL cells. In vivo, studies using the Dunning prostate cancer model in Copenhagen rats resulted in a dose response inhibition of tumor growth, with an 80% decrease in tumor size in rats treated with gemcitabine at 10 mg/kg. CONCLUSIONS: Our results demonstrated the potent activity of gemcitabine against prostate cancer in the Dunning rat model and suggest the addition of paclitaxel may not aid in this activity.
Subject(s)
Antimetabolites, Antineoplastic/toxicity , Antineoplastic Agents/toxicity , Deoxycytidine/analogs & derivatives , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Animals , Antimetabolites, Antineoplastic/therapeutic use , Antineoplastic Agents/therapeutic use , Carboplatin/toxicity , Cell Division/drug effects , Cell Survival/drug effects , Deoxycytidine/therapeutic use , Deoxycytidine/toxicity , Drug Synergism , Estramustine/toxicity , Etoposide/toxicity , Humans , Male , Paclitaxel/toxicity , Rats , Tumor Cells, Cultured , GemcitabineABSTRACT
Prostate cancer patients experiencing a relapse in disease often express high serum tumor necrosis factor-alpha (TNF-alpha) levels. Many androgen-insensitive prostate cancer cells are TNF-alpha insensitive because of the expression of antiapoptotic genes as part of the nuclear factor-kappaB (NF-kappaB) family of transcription factors. NF-kappaB stimulates gene transcription when expressed in the nucleus; however, in resting cells, this nuclear import is prevented by association with the cytoplasmic inhibitor IkappaBalpha. This cytoplasmic retention of NF-kappaB is uncoupled by many extracellular signals including low levels of TNF-alpha. During normal cell activation, nuclear translocation of NF-kappaB is preceded by phosphorylation and degradation of IkappaBalpha. When phosphorylation is blocked, IkappaBalpha remains intact, thereby blocking NF-kappaB translocation to the nucleus and subsequent activation of antiapoptotic genes that cause TNF-alpha insensitivity. We tested whether a "super-repressor" of NF-kappaB activity could be transfected into prostate cancer cells and make them TNF-alpha sensitive. PC-3 and LNCaP cells were stimulated with TNF-alpha (10 ng/ml) for 24 h in the presence or absence of the IkappaBalpha "super-repressor" (p6R-IkappaB(S32A + S36A)). NF-kappaB activity was measured by electrophoretic mobility shift assay and the steady state levels of the cytoplasmic IkappaBalpha protein were measured by Western blot. Secretory IL-6 and IL-6 mRNA were measured by ELISA. p6R-IkappaB(S32A + S36A) blocked the stimulation of NF-kappaB activity by TNF-alpha in prostate cancer cells. It also subsequently decreased IL-6 production by TNF-alpha. We conclude that these data demonstrate that inhibition of NF-kappaB selectively sensitizes previously insensitive prostate cancer cells to TNF-alpha.
Subject(s)
Apoptosis/drug effects , DNA-Binding Proteins/metabolism , I-kappa B Proteins , NF-kappa B/antagonists & inhibitors , Prostatic Neoplasms/drug therapy , Tumor Necrosis Factor-alpha/pharmacology , Amino Acid Sequence , Biological Transport , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Humans , Interleukin-6/genetics , Male , Mutation , NF-KappaB Inhibitor alpha , NF-kappa B/metabolism , Prostatic Neoplasms/pathology , Tumor Cells, CulturedABSTRACT
The nuclear matrix is the site of many nuclear functions including transcription, replication, formation of chromatin loops, and control of DNA supercoiling. It contains various structural and functional components that represent targets for antineoplastic agents. Antimetabolites and topoisomerase II inhibitors interact specifically with matrix-associated enzymes, DNA primase, and DNA topoisomerase II, respectively. Alkylating agents and ionizing radiation interact with nuclear matrix proteins and matrix-associated DNA. Many nuclear functions, including multidrug resistance, and others which lead to cell death, have been shown to be compromised when these anticancer agents interact with the nuclear matrix.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Nucleus/drug effects , Neoplasms/drug therapy , Nuclear Matrix/drug effects , Cell Nucleus/genetics , Cell Nucleus/ultrastructure , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type II/genetics , Humans , Neoplasms/genetics , Neoplasms/pathology , Nuclear Matrix/genetics , Nuclear Matrix/ultrastructureABSTRACT
"Poly-plat", SSP, and SAP are second generation analogs of cisplatin (CDDP) with higher efficacy and potency. In order to understand the mechanism of action of these compounds, isolated murine peritoneal macrophages were treated with "poly-plat", SSP, or SAP (5 micrograms/ml) for 2 h. Treated macrophages demonstrated an increase in the number of lysosomes, but only "poly-plat" and SSP treated macrophages were stimulated to form the cytoplasmic extensions so very characteristic of cisplatin after 2 h and 24 h post-treatment. SAP showed cytoplasmic extensions only after 24 h post-treatment, and demonstrated a back to the normal discoid form when viewed at 24 h post-treatment. When drug treated macrophages were co-incubated with S180 tumor cells, cytoplasmic extensions of the macrophages developed contacts, and cytoplasmic continuity with the tumor cells, and a subsequent transfer of lysosomes from macrophage to tumor cell was observed after only 2 h of co-incubation. After 24 h of co-incubation, lysis of S180 cells was achieved. Analysis of the tissue culture supernatants collected from "poly-plat", SSP, and SAP treated macrophages demonstrated the enhanced activity of interleukin-1 alpha of over 400 pg/ml after 2 h post-treatment, compared to only 300 pg/ml with cisplatin 24 h post-treatment. However, only SSP demonstrated an increase in TNF-alpha activity (2000 pg/ml) after 2 h post-treatment, which is comparable to that of cisplatin. Based on our observations we propose that "poly-plat", SSP, and SAP activate various cytolytic factors of the immune system better, than cisplatin.
Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Macrophage Activation/drug effects , Macrophages, Peritoneal/drug effects , Organoplatinum Compounds/pharmacology , Animals , Cells, Cultured , Cisplatin/analogs & derivatives , Cytoplasm/drug effects , Cytoplasm/physiology , Interleukin-1/biosynthesis , Kinetics , Lysosomes/drug effects , Lysosomes/ultrastructure , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/physiology , Mice , Molecular Structure , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase Type II , Nitrites/metabolism , Structure-Activity Relationship , Time FactorsABSTRACT
Swiss Webster mice treated with bolus injections of 'poly-plat' (10 mg/kg) show increased macrophage activation after 2 and 12 days when isolated in cultures. Such macrophages demonstrate an increase in the number of lysosomes and cytoplasmic extension formation with enhanced cytokine (interleukin-1alpha) activity. In addition, peripheral blood smears demonstrated an increase in lymphocytes and monocytes compared to cisplatin-treated animals. These results show that 'poly-plat' activates the immune system more effectively than cisplatin both in vitro and in vivo.
Subject(s)
Antineoplastic Agents/pharmacology , Macrophages, Peritoneal/drug effects , Organoplatinum Compounds/pharmacology , Animals , Cell Adhesion , Interleukin-1/metabolism , Leukocyte Count/drug effects , Macrophage Activation/drug effects , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/ultrastructure , Mice , Nitric Oxide/biosynthesis , Tumor Cells, Cultured/drug effectsABSTRACT
Cisplatin and paclitaxel are potent antineoplastic agents. Their distinctly different mechanisms of action have prompted laboratory and clinical research into their use in combination therapies. Murine peritoneal macrophages treated with cisplatin and paclitaxel in combination elicit an increase in their number of lysosomes. Drug-treated macrophages, when co-incubated with sarcoma 180 cells, establish cytoplasmic contact and transfer lysosomes into tumor cells causing tumor cell lysis. In addition, analysis of tissue culture supernatants show increased levels of interleukin-1alpha and tumor necrosis factor-alpha. Our study shows that cisplatin and paclitaxel in combination enhance elements of the immune system with greater efficacy and potency than when used alone.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Cytotoxicity, Immunologic/drug effects , Macrophage Activation , Macrophages, Peritoneal/drug effects , Paclitaxel/pharmacology , Animals , Drug Combinations , Interleukin-1/metabolism , Lysosomes , Macrophages, Peritoneal/immunology , Mice , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolismABSTRACT
'Poly-plat', SSP and SAP are second generation analogs of cisplatin which have been shown to activate murine peritoneal macrophages in vivo and in vitro. Murine peritoneal macrophages treated with 'poly-plat', SSP or SAP (5 microg/mg) for 2 h are stimulated to form cytoplasmic extensions. Drug-treated macrophages also elicit an increase in the number of lysosomes. In addition, analysis of tissue culture supernatants shows increased levels of interleukin-1alpha and tumor necrosis factor-alpha. These results show that 'poly-plat', SSP and SAP enhance the immune system with greater efficacy and potency than cisplatin.