2-2-bis(hydroxymethyl)-1-azabicyclo(2-2-2-)octan-3-one and Lung-Neoplasms

The metabolic inhibitor 3-bromopyruvate (3-BrPA) is a promising anti-cancer alkylating agent, shown to inhibit growth of some colorectal carcinoma with KRAS mutation. Recently, we demonstrated increased resistance to 3-BrPA in wt p53 tumor cells compared to those with p53 silencing or mutation. Since hypoxic microenvironments select for tumor cells with diminished therapeutic response, we investigated whether hypoxia unequally increases resistance to 3-BrPA in wt p53 MelJuso melanoma harbouring (Q61L)-mutant NRAS and wt BRAF, C8161 melanoma with (G12D)-mutant KRAS (G464E)-mutant BRAF, and A549 lung carcinoma with a KRAS (G12S)-mutation. Since hypoxia increases the toxicity of the p53 activator, Prima-1 against breast cancer cells irrespective of their p53 status, we also investigated whether Prima-1 reversed hypoxic resistance to 3-BrPA.. In contrast to the high susceptibility of hypoxic mutant NRAS MelJuso cells to 3-BrPA or Prima-1, KRAS mutant C8161 and A549 cells revealed hypoxic resistance to 3-BrPA counteracted by Prima-1. In A549 cells, Prima-1 increased p21CDKN1mRNA, and reciprocally inhibited mRNA expression of the SLC2A1-GLUT1 glucose transporter-1 and ALDH1A1, gene linked to detoxification and stem cell properties. 3-BrPA lowered CAIX and VEGF mRNA expression. Death from joint Prima-1 and 3-BrPA treatment in KRAS mutant A549 and C8161 cells seemed mediated by potentiating oxidative stress, since it was antagonized by the anti-oxidant and glutathione precursor N-acetylcysteine.. This report is the first to show that Prima-1 kills hypoxic wt p53 KRAS-mutant cells resistant to 3-BrPA, partly by decreasing GLUT-1 expression and exacerbating pro-oxidant stress.

Topics: A549 Cells; Acetylcysteine; Aza Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Hypoxia; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Drug Resistance, Neoplasm; Free Radical Scavengers; Gene Expression Regulation, Neoplastic; Glucose Transporter Type 1; GTP Phosphohydrolases; Humans; Lung Neoplasms; Melanoma; Membrane Proteins; Mutation; Proto-Oncogene Proteins p21(ras); Pyruvates; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Protein p53

Derivatives of PRIMA-1 compound, 8a and 8b have been shown to increase cytotoxicity in lung cancer cells through sphingomyelinase pathways in IR and 8a or 8b co-treated lung cancer cells. The goal of the present study was to further elaborate the molecular mechanism of 8a- or 8b-treated lung cancer cells in order to understand their potential as anti-cancer drugs.. Biochemical assays, western blot, flow cytometry and gene array analyses were employed to distinguish these mechanisms.. Herein we demonstrated that 8a and 8b cause apoptosis with S-phase arrest in lung cancer cells by activating neutral sphingomyelinase with ceramide production. 8a induces expression of TNF family genes while 8b induces p53-mediated apoptosis genes. Protein analysis shows an increased expression in caspase 8, bcl-2, bax, caspase 9 and cytochrome c.. PRIMA-1 derivatives provoke cytotoxicity in lung cancer cells mainly through the neutral sphingomyelinase-dependent apoptosis pathway.

Topics: Antineoplastic Agents; Apoptosis; Aza Compounds; Bridged Bicyclo Compounds, Heterocyclic; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Ceramides; Humans; Lung Neoplasms; S Phase; Sphingomyelin Phosphodiesterase; Tumor Suppressor Protein p53

Lung cancer is the leading cause of cancer deaths worldwide. Expression of the p53 tumor suppressor protein is frequently altered in tobacco-associated lung cancers. We studied chemopreventive effects of p53-modulating agents, namely, CP-31398 and Prima-1, on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung adenoma and adenocarcinoma formation in female A/J mice. Seven-week-old mice were treated with a single dose of NNK (10 µmol/mouse) by intraperitoneal injection and, 3 weeks later, were randomized to mice fed a control diet or experimental diets containing 50 or 100 ppm CP-31398 or 150 or 300 ppm Prima-1 for either 17 weeks (10 mice/group) or 34 weeks (15 mice/group) to assess the efficacy against lung adenoma and adenocarcinoma. Dietary feeding of 50 or 100 ppm CP-31398 significantly suppressed (P < .0001) lung adenocarcinoma by 64% and 73%, respectively, after 17 weeks and by 47% and 56%, respectively, after 34 weeks. Similarly, 150 or 300 ppm Prima-1 significantly suppressed (P < .0001) lung adenocarcinoma formation by 56% and 62%, respectively, after 17 weeks and 39% and 56%, respectively, after 34 weeks. Importantly, these results suggest that both p53 modulators cause a delay in the progression of adenoma to adenocarcinoma. Immunohistochemical analysis of lung tumors from mice exposed to p53-modulating agents showed a significantly reduced tumor cell proliferation and increased accumulation of wild-type p53 in the nucleus. An increase in p21- and apoptotic-positive cells was also observed in lung tumors of mice exposed to p53-modulating agents. These results support a chemopreventive role of p53-modulating agents in tobacco carcinogen-induced lung adenocarcinoma formation.

Topics: Adenocarcinoma; Animals; Anticarcinogenic Agents; Apoptosis; Aza Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Transformation, Neoplastic; Chemoprevention; Cyclin-Dependent Kinase Inhibitor p21; Female; Lung Neoplasms; Mice; Mice, Inbred A; Nicotiana; Nitrosamines; Pyrimidines; Random Allocation; Tumor Suppressor Protein p53

p53-dependent apoptosis is important for the efficacy of cancer treatment, and tumors carrying mutant p53 are often resistant to chemotherapy. Non-small cell lung cancer (NSCLC) cells generally exhibit resistance to apoptosis following treatment with many cytotoxic drugs. The new molecule PRIMA-1 appears to kill human tumor cells by restoring the transcriptional activity to mutated p53. We investigated the induction of apoptosis in response to this drug in three NSCLC cell lines carrying different p53 proteins: A549 (p53wt), LX1 (p53R273H), and SKMes1 (p53R280K). PRIMA-1 alone did not trigger apoptosis but significantly reduced cell viability. However, in combination with adriamycin, PRIMA-1 strengthen the adriamycin-induced apoptosis in A549 and LX1. Interestingly, even in SKMes1 cells, the combined treatment triggered a strong PARP cleavage without DNA fragmentation. Our data suggest that in NSCLC cells, PRIMA-1 may induce cell death through pathways other than apoptosis but may synergize with adriamycin to trigger an apoptotic response.

Topics: Apoptosis Regulatory Proteins; Aza Compounds; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; DNA Fragmentation; Doxorubicin; Drug Screening Assays, Antitumor; Drug Synergism; Humans; Lung Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53; Ultraviolet Rays

We have synthesized and tested novel quinuclidinone analogs to assay the effects on H1299 lung cancer cell lines alone or with gamma-radiation. We have found two series of quinuclidinone analogs that act as anti-cancer agents. Of these, four interesting analogs significantly decreased cell viability in H1299 lung cancer cell lines. Two derivatives decreased cell proliferation in a dose-dependent fashion alone or in the presence of gamma-radiation. Radiosensitization increased when derivative treatment preceded radiation treatment for both derivatives. These preliminary studies show an evidence for both additive and synergistic cytotoxicity for treatment of lung cancer by these novel quinuclidinone analogs.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Humans; Lung Neoplasms; Molecular Structure; Quinuclidines; Structure-Activity Relationship; Tumor Suppressor Protein p53

Mutant p53-carrying tumors are often more resistant to chemotherapeutical drugs. We demonstrate here that the mutant p53-reactivating compound PRIMA-1(MET) acts synergistically with several chemotherapeutic drugs to inhibit tumor cell growth. Combined treatment with cisplatin and PRIMA-1(MET) resulted in a synergistic induction of tumor cell apoptosis and inhibition of human tumor xenograft growth in vivo in SCID mice. The induction of mutant p53 levels by chemotherapeutic drugs is likely to increase the sensitivity of tumor cells to PRIMA-1(MET). Thus, the combination of PRIMA-1(MET) with currently used chemotherapeutic drugs may represent a novel and more efficient therapeutic strategy for treatment of mutant p53-carrying tumors.

Topics: Adenocarcinoma; Animals; Apoptosis; Aza Compounds; Bone Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Non-Small-Cell Lung; Cisplatin; Drug Interactions; Drug Resistance, Neoplasm; Genes, p53; Humans; Lung Neoplasms; Mice; Mice, SCID; Mutation; Osteosarcoma; Quinuclidines; Transplantation, Heterologous; Tumor Cells, Cultured

PRIMA-1 (p53 reactivation and induction of massive apoptosis) is a chemical compound that was originally identified as a selective mutant p53-dependent growth suppressor by screening a library of low-molecular-weight compounds. However, its mechanism of action is unknown. In this study, we examined toxicity of PRIMA-1 to three premalignant human colorectal adenoma cell lines (RG/C2, BR/C1, and AA/C1) and four colorectal carcinoma cell lines (DLD-1, SW480, LOVO, and HCT116) and its mechanism of action. It selectively induced apoptosis only in the mutant p53 premalignant and malignant colon cell lines, but was not toxic to the wild-type p53 premalignant and malignant colon cell lines. Using stable transfectants of temperature-sensitive p53 mutant Ala(143) in null p53 H1299 lung cancer cells, we found that PRIMA-1 induced significantly more apoptosis in cells with mutant p53 conformation (37 degrees C) than the wild-type p53 conformation (32.5 degrees C). Cell cycle analysis indicated that its inhibition of cell growth was correlated with induction of G(2) arrest. Western blot analysis showed PRIMA-1 increased p21 and GADD45 expression selectively in the mutant p53 cells. However, Fas, Bcl-2 family proteins, and caspases were not involved in PRIMA-1-induced cell death. The c-Jun-NH(2)-kinase (JNK) inhibitor SP 600125, but not p38 mitogen-activated protein kinase inhibitor SB 203580 or extracellular signal-regulated kinase inhibitor PD 98059, blocked PRIMA-1-induced apoptosis. Transfection with a dominant-negative phosphorylation mutant JNK, but not a dominant-negative p38 or wild-type JNK, inhibited PRIMA-1-induced cell death, suggesting that the JNK pathway plays an important role in PRIMA-1-induced apoptosis. PRIMA-1 is a highly selective small molecule toxic to p53 mutant cells and may serve as a prototype for the development of new p53-targeting agents for therapy of premalignant and malignant cells.

Topics: Apoptosis; Aza Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cell Transformation, Neoplastic; Colonic Neoplasms; Humans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Neoplasms; Precancerous Conditions; Protein Kinase Inhibitors; RNA, Messenger; Signal Transduction; Temperature; Tumor Suppressor Protein p53