Inhibition of phosphatidylinositol 3-kinase sensitizes ovarian cancer cells to carboplatin and allows adjunct chemotherapy treatment

Signal transduction pathways associated with cancer progression and chemotherapeutic resistance are being investigated as molecular targets for chemotherapy. The phosphatidylinositol 3-kinase (PI3K) pathway has been found to be frequently amplified and have increased activity in ovarian cancer. The current study investigates the efficacy of an antagonist of PI3K, LY294002, in inhibiting ovarian cancer cell growth and survival both in vitro and in vivo. The hypothesis tested is that inhibition of PI3K signaling makes ovarian cancer cells susceptible to the effects of platinum-based chemotherapy. Observations show that LY294002 is an effective inhibitor of ovarian cancer cell growth and survival in vitro. Inhibition of PI3K/Akt signaling increased the sensitivity of ovarian cell cultures to the cytotoxic effects of carboplatin. The combined treatment of LY294002 and carboplatin was needed to optimally promote cellular apoptosis and decrease ovarian cancer cell survival in vitro. To extend these observations, a model involving in vivo i.p. growth of human ovarian tumors in a nude mouse was used. LY294002 in combination with carboplatin was more effective in inhibiting ovarian cancer cell xenograft growth than either agent alone. The results of this study suggest that the combined treatment of carboplatin and LY294002 can effectively decrease ovarian tumor progression and support the use of a PI3K inhibitor (e.g., LY294002) as an adjunct platinum-based drug therapy for treatment of ovarian cancer.     

The mechanism of methylselenocysteine and docetaxel synergistic activity in prostate cancer cells

The study was designed to evaluate the combination treatment of methylselenocysteine (MSeC) and docetaxel and to delineate the underlying mechanism associated with observed in vitro synergy between MSeC and docetaxel in prostate cancer cells. Cells were treated with different concentrations and schedules (concurrent or sequential) of MSeC and docetaxel alone or in combination. Cell growth/death was assessed with sulforhodamine B assay, trypan blue assay, and time-lapse video. Loewe synergism/antagonism model was used to determine whether the combination effect was additive, synergistic, or antagonistic. Apoptosis and caspase-3 activity were evaluated with cell death ELISA assay and caspase activity assay, respectively. Synergy between MSeC and docetaxel was further assessed in the presence and absence of z-VAD-fmk, a pan-caspase inhibitor. Effect of MSeC and docetaxel alone or in combination on the cellular expression of the antiapoptotic protein survivin was measured with Western blot analyses. Pretreatment with MSeC was crucial to enhance docetaxel antitumor activity. The enhanced antitumor activity of the sequential combination treatment of MSeC and docetaxel (MSeC/docetaxel) was highly synergistic. Apoptosis increased after MSeC/docetaxel, compared with each drug alone or concurrent treatment. Pretreatment with z-VAD-fmk converted the synergy into antagonism, suggesting that the synergy is caspase-dependent apoptosis. The survivin level was down-regulated following MSeC/docetaxel treatment when compared with each drug alone. In conclusion, pretreatment with MSeC was essential to markedly sensitize cells to docetaxel. The synergy between MSeC and docetaxel in C2G prostate cancer cells is associated with increased level of caspase-dependent apoptosis and decreased level of survivin.     

Prevention of rifampicin resistance in Acinetobacter baumannii in an experimental pneumonia murine model, using rifampicin associated with imipenem or sulbactam

OBJECTIVES: To examine the development of rifampicin resistance in multidrug-resistant Acinetobacter baumannii exposed to rifampicin and the prevention of the appearance of rifampicin-resistant mutants when rifampicin is used in association with imipenem or sulbactam. METHODS: A clinical strain of multidrug-resistant A. baumannii was used to examine the frequency of resistance to rifampicin in vivo, in a pneumonia model in immunocompetent C57BL/6 mice. The in vitro and in vivo prevention of the development of resistance to rifampicin was analysed using rifampicin alone or in association with imipenem or sulbactam, in time-kill studies and in the experimental murine pneumonia, respectively. RESULTS: Rifampicin-resistant mutants were found at 48 and 72 h, both in vitro and in vivo, when rifampicin was used alone, with the MIC increasing from 4 to > or =128 mg/L. The in vivo frequency of rifampicin-resistant mutants was 3 x 10(-6). On the contrary, no resistant mutants appeared after 72 h, in vitro or in vivo, when rifampicin was employed in association with imipenem or sulbactam. After six daily passages in rifampicin-free agar plates the resistant mutants maintained the high resistance to rifampicin (> or =128 mg/L). CONCLUSIONS: These results suggest that rifampicin must not be used alone in the treatment of infections caused by multidrug-resistant A. baumannii. In these cases, rifampicin may be used in combination with imipenem or sulbactam, which prevent the development of resistance to rifampicin.     

In vitro and in vivo activities of E-101 solution against Acinetobacter baumannii isolates from U.S. military personnel

We evaluated the in vitro and in vivo activity of a novel topical myeloperoxidase-mediated antimicrobial, E-101 solution, against 5 multidrug-resistant Acinetobacter baumannii isolates recovered from wounded American soldiers. Time-kill studies demonstrated rapid bactericidal activity against all A. baumannii strains tested in the presence of 3% blood. The in vitro bactericidal activity of E-101 solution against A. baumannii strains was confirmed in a full-thickness excision rat model. Additional in vivo studies appear warranted.     

In vitro activity of newer β-lactam agents in combination with amikacin against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Serratia marcescens

The in vitro activity of cefoperazone, ceftazidime, ceftizoxime, moxalactam, and N-forminidoyl thienamycin was evaluated alone and in combination with amikacin to assess possible synergistic activity against isolates of amikacin-resistant and multidrug-resistant and susceptible to amikacin (one isolate was also resistant to amikacin). The checkerboard agar dilution method was used. Ceftazidime and thienamycin followed by moxalactam and cefoperazone were the most active agents versus the alone and in combination testing. Ceftazidime, moxalactam, and thienamycin showed the greatest activity against and all agents except cefoperazone were active against . The finding of synergy or partial synergy in combination testing was found in the majority with all three genera, including levels below the breakpoint for both amikacin and the β-lactam agents. This wide in vitro activity indicates that clinical evaluation of these agents in treatment of multidrug-resistant infections is warranted.     

In vitro activity of newer beta-lactam agents in combination with amikacin against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Serratia marcescens

The in vitro activity of cefoperazone, ceftazidime, ceftizoxime, moxalactam, and N-formimidoyl thienamycin was evaluated alone and in combination with amikacin to assess possible synergistic activity against isolates of amikacin-resistant Pseudomonas aeruginosa and multidrug-resistant Serratia marcescens and Klebsiella pneumoniae susceptible to amikacin (one S. marcescens isolate was also resistant to amikacin). The checkerboard agar dilution method was used. Ceftazidime and thienamycin followed by moxalactam and cefoperazone were the most active agents versus the P. aeruginosa alone and in combination testing. Ceftazidime, moxalactam, and thienamycin showed the greatest activity against S. marcescens, and all agents except cefoperazone were active against K. pneumoniae. The finding of synergy or partial synergy in combination testing was found in the majority with all three genera, including levels below the breakpoint for both amikacin and the beta-lactam agents. This wide in vitro activity indicates that clinical evaluation of these agents in treatment of multidrug-resistant infections is warranted.     

Exisulind-induced apoptosis in a non-small cell lung cancer orthotopic lung tumor model augments docetaxel treatment and contributes to increased survival

We reported previously a significant increase in survival of nude rats harboring orthotopic A549 human non-small cell lung cancer tumors after treatment with a combination of exisulind (Sulindac Sulfone) and docetaxel (D. C. Chan, Clin. Cancer Res., 8: 904-912, 2002). The purpose of the current study was to determine the biochemical mechanisms responsible for the increased survival by an analysis of the effects of both drugs on A549 orthotopic lung tumors and A549 cells in culture. Orthotopic A549 rat lung tissue sections from drug-treated rats and A549 cell culture responses to exisulind and docetaxel were compared using multiple apoptosis and proliferation analyses [i.e., terminal deoxynucleotidyl transferase-mediated nick end labeling, active caspase 3, the caspase cleavage products cytokeratin 18 and p85 poly(ADP-ribose) polymerase, and Ki-67]. Immunohistochemistry was used to determine cyclic GMP (cGMP) phosphodiesterase (PDE) expression in tumors. The cGMP PDE composition of cultured A549 cells was resolved by DEAE-Trisacryl M chromatography and the pharmacological sensitivity to exisulind, and additional known PDE inhibitors were determined by enzyme activity assays. Exisulind inhibited A549 cell cGMP hydrolysis and induced apoptosis of A549 cells grown in culture. PDE5 and 1 cGMP PDE gene family isoforms identified in cultured cells were highly expressed in orthotopic tumors. The in vivo apoptosis rates within the orthotopic tumors increased 7-8-fold in animals treated with the combination of exisulind and docetaxel. Exisulind increased the in vivo apoptosis rates as a single agent. Docetaxel, but not exisulind, decreased proliferative rates within the tumors. The data indicate that exisulind-induced apoptosis contributed significantly to the increased survival in rats treated with exisulind/docetaxel. The mechanism of exisulind-induced apoptosis involves inhibition of cGMP PDEs, and these results are consistent with a cGMP-regulated apoptosis pathway.     

艾素对原发和复发乳腺癌的体外药物敏感性研究

目的了解艾素（多西他赛，docetaxel）和其它化疗药物对原发和复发乳腺癌原代细胞的体外肿瘤杀伤作用。方法取得不同患者的乳腺癌组织标本，采用胶原酶消化法获取乳腺癌原代细胞，应用原代培养和MTT法检测各种化疗药物在体外对乳腺癌原代细胞的杀伤率。结果各种化疔药物对大部分原发乳腺癌患者的体外肿瘤杀伤作用无明显差异（P〉0．05），但对于小部分原发和全部的复发乳腺癌患者，艾索和其它化疗药物的体外肿瘤杀伤作用有明显差异（P〈0．01）。结论对于伴有原发多药耐药的乳腺癌和复发的乳腺癌患者，艾素仍然具有较高的有效率，可以作为一线治疗用药。     

Flavopiridol enhances the effect of docetaxel in vitro and in vivo in human gastric cancer cells

Gastric cancer is one of the leading causes of cancer death throughout the world. It is a disease in desperate need of new therapeutic approaches. Docetaxel, a semisynthetic taxane, has shown potent activity against a broad range of solid tumors. However, in gastric cancer, response rates to docetaxel remain only approximately 20%. In these studies we show that flavopiridol, a cyclin-dependent kinase inhibitor, potentiates docetaxel-induced apoptosis 3-fold in MKN-74 human gastric cells. This effect is sequence dependent, such that flavopiridol must follow docetaxel to induce this effect. Docetaxel induces transient arrest in the M phase of the cell cycle. Cells exit mitosis in a specific time window without cytokinesis with a decrease in cyclin B1/cdc-2 kinase activity and MPM-2 labeling. Flavopiridol treatment of docetaxel-treated cells enhances the exit from mitosis with a more rapid decrease in mitotic markers including MPM-2 labeling and cyclin B1/cdc2 kinase activity. In contrast, pretreatment with flavopiridol prevents cells from entering mitosis by inhibiting cyclin B1/cdc-2 kinase activity, thus antagonizing the docetaxel effect. The testing of this combination against MKN-74 xenografts confirms the sequence dependency. Treatment of MKN-74 tumor-bearing xenografts with docetaxel at a dose of 10 mg/kg followed 3-7 h later by flavopiridol at a dose of 2.5 mg/kg resulted in a 1-18% decrease in tumor volume. In contrast, treatment with docetaxel alone at this same dose resulted in a 394% increase in tumor volume. When flavopiridol was given immediately after docetaxel, the effect was not statistically different from that of docetaxel alone. The reverse combination of flavopiridol followed 7 h later by docetaxel was similar to treatment with docetaxel alone. Flavopiridol alone had no effect in this tumor model. Thus, flavopiridol, when combined with docetaxel in a sequence-specific manner, may provide a completely new therapeutic approach in the treatment of gastric cancer.     

Drug evaluation: CP-751871, a human antibody against type I insulin-like growth factor receptor for the potential treatment of cancer

Several phase II trials of CP-751871 are currently underway, including a phase Ib/II [corrected] trial of CP-751871 in combination with paclitaxel and carboplatin in patients with advanced NSCLC, a phase II trial of CP-751871 in combination with docetaxel and prednisone in patients with hormone-refractory prostate cancer, and a phase II trial of CP-751871 in combination with exemestane in hormone receptor positive advanced breast cancer [corrected]     

Smoothened antagonists reverse taxane resistance in ovarian cancer

The hedgehog pathway has been implicated in the formation and maintenance of a variety of malignancies, including ovarian cancer; however, it is unknown whether hedgehog signaling is involved in ovarian cancer chemoresistance. The goal of this study was to determine the effects of antagonizing the hedgehog receptor, Smoothened (Smo), on chemotherapy response in ovarian cancer. Expression of hedgehog pathway members was assessed in three pairs of parental and chemotherapy-resistant ovarian cancer cell lines (A2780ip2/A2780cp20, SKOV3ip1/SKOV3TRip2, HeyA8/HeyA8MDR) using quantitative PCR and Western blot analysis. Cell lines were exposed to increasing concentrations of two different Smo antagonists (cyclopamine, LDE225) alone and in combination with carboplatin or paclitaxel. Selective knockdown of Smo, Gli1, or Gli2 was achieved using siRNA constructs. Cell viability was assessed by MTT assay. A2780cp20 and SKOV3TRip2 orthotopic xenografts were treated with vehicle, LDE225, paclitaxel, or combination therapy. Chemoresistant cell lines showed higher expression (>2-fold, P < 0.05) of hedgehog signaling components compared with their respective parental lines. Smo antagonists sensitized chemotherapy-resistant cell lines to paclitaxel, but not to carboplatin. LDE225 treatment also increased sensitivity of ALDH-positive cells to paclitaxel. A2780cp20 and SKOV3TRip2 xenografts treated with combined LDE225 and paclitaxel had significantly less tumor burden than those treated with vehicle or either agent alone. Increased taxane sensitivity seems to be mediated by a decrease in P-glycoprotein (MDR1) expression. Selective knockdown of Smo, Gli1, or Gli2 all increased taxane sensitivity. Smo antagonists reverse taxane resistance in chemoresistant ovarian cancer models, suggesting combined anti-hedgehog and chemotherapies could provide a useful therapeutic strategy for ovarian cancer.     

The Combination of Sulfamethoxazole, Trimethoprim, and Isoniazid or Rifampin Is Bactericidal and Prevents the Emergence of Drug Resistance in Mycobacterium tuberculosis

The challenges of developing new drugs to treat tuberculosis (TB) are indicated by the relatively small number of candidates entering clinical trials in the past decade. To overcome these issues, we reexamined two FDA-approved antibacterial drugs, sulfamethoxazole (SMX) and trimethoprim (TMP), for use in TB treatment. SMX and TMP inhibit folic acid biosynthesis and are used in combination to treat infections of the respiratory, urinary, and gastrointestinal tracts. The MICs of SMX and TMP, alone and in combination, were determined for drug-susceptible, multidrug-resistant (MDR), and extensively drug-resistant Mycobacterium tuberculosis strains. While TMP alone was not effective against M. tuberculosis, the combination of TMP and SMX was bacteriostatic against M. tuberculosis. Surprisingly, the combination of SMX and TMP was also active against a subset of MDR M. tuberculosis strains. Treatment of M. tuberculosis with TMP-SMX and a first-line anti-TB drug, either isoniazid or rifampin, was bactericidal, demonstrating that the combination of TMP and SMX with isoniazid or rifampin was not antagonistic. Moreover, the addition of SMX-TMP in combination with either isoniazid or rifampin also prevented the emergence of drug resistance in vitro. In conclusion, this study further illustrates the opportunity to reevaluate the activity of TMP-SMX in vivo to prevent the emergence of drug-resistant M. tuberculosis.     

Histone deacetylase inhibitors and paclitaxel cause synergistic effects on apoptosis and microtubule stabilization in papillary serous endometrial cancer cells

The use of histone deacetylase (HDAC) inhibitors has shown promise for a variety of malignancies. In this investigation, we define the activity of this class of inhibitors in combination with traditional cytotoxic chemotherapy in endometrial cancer cells. Significant reductions in growth were observed in Ark2 and KLE endometrial cancer cells following treatment with paclitaxel, doxorubicin, carboplatin, or the HDAC inhibitor trichostatin A (TSA). However, only combined treatment with TSA/paclitaxel caused synergistic inhibition of cell growth. This combination also resulted in significant changes in cell morphology. Using cell cycle analysis, nuclear staining, and Western blot analysis for poly(ADP-ribose) polymerase and caspase-9 degradation products, TSA/paclitaxel showed the most dramatic activation of the apoptotic cascade. These effects were also observed when the HDAC inhibitors HDAC inhibitor-1 or oxamflatin were substituted for TSA. The anticancer properties of paclitaxel are known to result in part from inhibition of microtubule depolymerization, which results in apoptosis. We show that TSA administration also stabilizes microtubules via alpha-tubulin acetylation. Furthermore, using Western blot and immunohistochemical analysis, treatment with TSA/paclitaxel led to a significant increase in acetylated tubulin and microtubule stabilization. These effects were confirmed in a mouse xenograft model. Moreover, TSA/paclitaxel resulted in a 50% reduction in tumor weight compared with either agent alone. This study provides in vivo evidence of nonhistone protein acetylation as one possible mechanism by which HDAC inhibitors reduce cancer growth. The TSA/paclitaxel combination seems to hold promise for the treatment of serous endometrial carcinoma and other malignancies with limited sensitivity to paclitaxel.     

Genotoxic action of cycloplatam,a new platinum antitumor drug,on mammalian cells in vivo and in vitro

Cycloplatam (CP), a new antitumor platinum compound of the second generation, was first synthesized in Russia. This drug shows less toxicity in vivo than the widely used cisplatin and carboplatin and a higher antitumor activity than carboplatin. CP is not nephrotoxic with respect to the platinum-based antitumor agents. CP is actually studied in clinical phase II trials in Russia and Armenia. Promising results were obtained in patients with lung, ovary, and prostate cancers. The aim of this work was to study the micronuclei (MN) inducing effect of CP in vivo in murine bone marrow cells and in human lymphocytes in vitro. The mutagenic activity of CP in bone marrow of mice was significantly lower than that of cisplatin at equitoxic doses. In human lymphocytes CP induced a dose-dependent increase of MN, beginning at a very low dose (0.1 micro M). Our results showed that CP is much less toxic and MN inducing in vivo in murine bone marrow than cisplatin. In vitro data evidenced that CP is more toxic and genotoxic to human cells than cisplatin. The evidence of a MN-induced activity of an antitumor drug suggests a potential risk for long-time survivors.     

Paclitaxel does not cause central nervous adverse effects: a prospective vigilance-controlled EEG mapping study in ovarian cancer patients

BACKGROUND: The objective of this study was to evaluate potential adverse effects of paclitaxel-containing chemotherapy on the central nervous system in women with ovarian cancer. METHODS: Twenty-eight women with histologically confirmed epithelial ovarian carcinoma and treated with combination chemotherapy consisting of paclitaxel and carboplatin were entered into the study. Patients were tested with vigilance-controlled EEG before and after chemotherapy. Additionally, a battery of neuropsychologic tests before, after 3 cycles and at the end of the chemotherapy were performed. RESULTS: Twenty of the 28 patients responded to chemotherapy (71%). Eleven patients (39%) developed peripheral neurotoxicity. A reduced vigilance in patients with ovarian cancer prior to chemotherapy compared to healthy controls was observed (clearly demonstrated by an attenuated total power, a decrease in fast alpha and slow beta activity, an increase in delta and theta activity as well as a deceleration of the centroid of the total power spectrum). On the other hand, an improved vigilance in patients with ovarian cancer treated with paclitaxel/carboplatin was observed (demonstrated by an increase in total power and an increase in beta power, being more pronounced in the right than in the left hemisphere, between pre- and post-treatment). The results of EEG mapping concurred with those of psychometric tests. The test results did not deteriorate during chemotherapy. A statistically significant improvement in the alphabetical crossout test from the first to the third measurement was observed, indicating an improvement in short-term attention, concentration and the constancy of work during chemotherapy. CONCLUSIONS: Combination chemotherapy consisting of paclitaxel and carboplatin does not cause encephalopathy, but a trend towards an improved vigilance and an improvement of short-term attention and concentration was observed in patients with ovarian cancer after chemotherapy.