amrubicinol and Adenocarcinoma

Amrubicin is a promising agent in the treatment of lung cancer, but predictive biomarkers have not yet been described. NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme known to metabolize amrubicinol, the active metabolite of amrubicin, to an inactive compound. We examined the relationship between NQO1 and amrubicinol cytotoxicity.. Gene and protein expression of NQO1, amrubicinol cytotoxicity, and C609T single-nucleotide polymorphism of NQO1 were evaluated in 29 lung cancer cell lines: 14 small cell lung cancer (SCLC) and 15 non-SCLC (NSCLC). The involvement of NQO1 in amrubicinol cytotoxicity was evaluated by small interfering RNA against NQO1.. A significant inverse relationship between both gene and protein expression of NQO1 and amrubicinol cytotoxicity was found in all cell lines. Treatment with NQO1 small interfering RNA increased amrubicinol cytotoxicity and decreased NQO1 expression in both NSCLC and SCLC cells. Furthermore, cell lines genotyped homozygous for the 609T allele showed significantly lower NQO1 protein expression and higher sensitivity for amrubicinol than those with the other genotypes in both NSCLC and SCLC cells.. NQO1 expression is one of the major determinants for amrubicinol cytotoxicity, and C609T single-nucleotide polymorphism of NQO1 could be a predictive biomarker for response to amrubicin treatment.

Topics: Adenocarcinoma; Anthracyclines; Biomarkers, Tumor; Blotting, Western; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; DNA, Neoplasm; Genotype; Humans; Lung Neoplasms; NAD(P)H Dehydrogenase (Quinone); Polymorphism, Single Nucleotide; Real-Time Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Small Cell Lung Carcinoma; Tumor Cells, Cultured

Amrubicin is a completely synthetic 9-aminoanthracycline agent for the treatment of lung cancer in Japan. The cytotoxicity of C-13 hydroxy metabolite, amrubicinol, is 10 to 100 times greater than that of amrubicin. The transporters responsible for the intracellular pharmacokinetics of amrubicin and amrubicinol remains unclear. This study was aimed to determine whether P-glycoprotein (P-gp) plays functional and preventive role in cellular accumulation and cytotoxicity of amrubicin and its active metabolite amrubicinol by in vitro transport and toxicity experiments. Cytotoxicity and intracellular accumulation of amrubicin and amrubicinol were evaluated by LLC-PK1 cells, MDR1 gene-transfected LLC-PK1 (L-MDR1) cells overexpressing P-gp, and human A549 lung adenocarcinoma cells. L-MDR1 cells showed 6- and 12-fold greater resistance to amrubicin and amrubicinol, respectively, than the parental LLC-PK1 cells. The intracellular accumulation of both drugs in L-MDR1 cells was significantly reduced compared to the LLC-PK1 cells. The basal-to-apical transepithelial transport of both drugs markedly exceeded, whereas the apical-to-basal transport of both drugs was significantly lower in L-MDR1 cells than LLC-PK1 cells. Cyclosporin A (CyA) restored the sensitivity, intracellular accumulation and transport activity for both drugs in L-MDR1 cells. In A549 cells, CyA significantly increased the intracellular accumulation and cytotoxicity of both drugs. These findings indicated that P-gp is responsible for cellular accumulation and cytotoxicity of both amrubicin and amrubicinol, therefore suggesting that the antitumor effect of amrubicin could be affected by the expression level of P-gp in lung cancer cells in chemotherapeutic treatments.

Topics: Adenocarcinoma; Animals; Anthracyclines; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Humans; LLC-PK1 Cells; Lung Neoplasms; Molecular Structure; Swine; Transfection

The effects of amrubicin (AMR) and its active metabolite, amrubicinol (AMROH), on the sensitivity of human lung adenocarcinoma A549 cells to ionizing radiation were investigated in vitro. Further, the kinetics of apoptosis and necrosis induction were also analyzed. The cytocidal effects of X-ray irradiation on A549 cells resulted in a low level of radiosensitivity with a D0 value of 12 Gy. The slopes of the survival curves in the exponential phase were plotted on semilogarithmic paper for radiation combined with AMR (2.5 microg/ml) and AMROH (0.02 microg/ml) treatment, and were shown to be approximately parallel to treatment with irradiation alone. The initial shoulder-shape portion of the survival curve for radiation alone, indicating the repair of sublethal damage, was reduced as compared to that for sequential combined treatment with AMR or AMROH. Sequential treatments with AMR or AMROH prior to ionizing radiation resulted in an additive radio-enhancement effect that reduced not only survival, but also the shoulder width. Fractionated irradiation with 2 Gy per fraction of A549 cells was carried out in vitro similar to that commonly performed in clinical radiotherapy and the radio-resistance of the cells was shown to be inhibited by AMR and AMROH. Similar to AMR and AMROH, adriamycin and etoposide (VP-16) are DNA topoisomerase II inhibitors. The effects of these 4 agents on cells that received X-ray irradiation were compared and all of the agents exhibited comparable radio-enhancement effects. The induction of apoptosis was investigated at 48 and 72 h after administration of AMROH, radiation or combined treatment, and apoptosis was not significantly induced after any of the treatments. We also examined the induction of necrosis, and found that the incidence of necrosis following combined treatment was approximately 2 times higher than that with either of the single treatments.

Topics: Adenocarcinoma; Anthracyclines; Apoptosis; Cell Line, Tumor; Humans; Kinetics; Lung Neoplasms; Necrosis; Radiation Tolerance; Radiation-Sensitizing Agents; Topoisomerase II Inhibitors; X-Rays

The effects of amrubicin (AMR) and its active metabolite, amrubicinol (AMROH), on the sensitivity of human lung adenocarcinoma A549 cell line to hyperthermia at 44 degrees C were investigated. The cell phase response as well as the kinetics of apoptosis and necrosis induction were also analyzed. The cytocidal effects of 44 degrees C hyperthermia on A549 cells exhibited low thermosensitivity with a T(0) value of 12 min. The slope of the survival curve in the exponential phase, described semilogarithmically, in 44 degrees C hyperthermia combined treatment with AMROH (0.02 microg/ml) was approximately parallel to 44 degrees C hyperthermia alone. The initial shoulder shape portion of the survival curve from 44 degrees C hyperthermia alone, indicating the repair of sublethal thermal damage (SLTDR), was reduced with the sequential combined treatment of AMR or AMROH. Sequential treatments with AMR or AMROH prior to 44 degrees C hyperthermia resulted in additive thermo-enhancement effect by reducing not only survival but was shoulder wide. Furthermore, like AMR and AMROH, adriamycin (ADM) and etoposide (VP-16) are DNA topoisomerase II inhibitors, and the effects of these 4 agents on 44 degrees C hyperthermia were compared. All 4 agents exhibited comparable thermo-enhancement effects. Using synchronized A549 cells, AMR or AMROH did not elicit cell phase responses, irrespective of the concentration. The induction of apoptosis was investigated at 48 and 72 h after AMROH treatment, 44 degrees C hyperthermia or the combined treatment, in which apoptosis was not significantly induced after any treatment. Furthermore, the incidence of necrosis was examined as well as apoptosis. The incidence of necrosis at 48 and 72 h after AMROH was 2.4 and 4.3%, respectively; after 44 degrees C hyperthermia was 3.3 and 4.0%, respectively; and after the combined treatment it was 10.7 and 8.7%, respectively. The necrosis induced after the combined treatment was circa 3 times higher than that in either of the single treatments.

Topics: Adenocarcinoma; Anthracyclines; Apoptosis; Cell Line, Tumor; Cell Survival; Doxorubicin; Drug Synergism; Etoposide; Hot Temperature; Humans; Lung Neoplasms; Necrosis; Time Factors