The synergistic interaction of MEK and PI3K inhibitors is modulated by mTOR inhibition

Background:

Combined targeting of MAPK and PI3K signalling pathways may be necessary for optimal therapeutic activity in cancer. This study evaluated the MEK inhibitors AZD6244 and PD0325901, alone and in combination with the dual mTOR/PI3K inhibitor NVP-BEZ235 or the PI3K inhibitor GDC-0941, in three colorectal cancer cell lines.

Methods:

Growth inhibition, survival and signal transduction were measured using the Sulforhodamine B assay, clonogenicity and western blotting, respectively, in HCT116, HT29 and DLD1 cell lines.

Results:

All MEK/PI3K inhibitor combinations exhibited marked synergistic growth inhibition; however, GDC-0941 displayed greater synergy in combination with either MEK inhibitor. NVP-BEZ235 exhibited stronger inhibition of 4EBP1 phosphorylation, and similar inhibition of S6 and AKT phosphorylation, compared with GDC-0941. Both PD0325901 and AZD6244 inhibited ERK phosphorylation, and with MEK/PI3K inhibitor combinations inhibition of S6 phosphorylation was increased. The reduced synergy exhibited by NVP-BEZ235 in combination with MEK inhibitors, compared with GDC-0941, may be due to inhibition of mTOR, and the addition of the mTORC1/2 inhibitor KU0063794 compromised the synergy of GDC-0941:PD0325901 combinations.

Conclusion:

These studies confirm that dual targeting of PI3K and MEK can induce synergistic growth inhibition; however, the combination of specific PI3K inhibitors, rather than dual mTOR/PI3K inhibitors, with MEK inhibitors results in greater synergy.     

Retention of amphotericin-B therapeutic efficacy at half doses by synergistic activation of phagocytes.

Amphotericin B (AMB) is a mainstay in the treatment of serious systemic fungal infections, such as those occurring prevalently in immuno-compromised patients treated with immunosuppressive agents or affected by Acquired Immunodeficiency Syndrome (AIDS). However AMB is an extremely toxic agent whose therapeutical utilization is often accompanied by acute side effects and chronic impairment of renal function. It is here reported that the preactivation of polymorphonucleated cells (PMN) in vivo, by a new immunomodulatory agent (PCF 39:N alpha-5[1,6,dihydro-(6-oxo-9 purinyl) pentoxycarbonyl]-L-Arginine) allows marked reduction of the AMB doses with full retention of therapeutic efficacy. This was observed in an experimental fungal infection induced in mice by intravenous inoculation of Candida albicans.     

Molecular basis for the synergistic interaction of adriamycin with the formaldehyde-releasing prodrug pivaloyloxymethyl butyrate (AN-9).

The interaction of Adriamycin and pivaloyloxymethyl butyrate (AN-9) was investigated in IMR-32 neuroblastoma and MCF-7 breast adenocarcinoma cells. Adriamycin is a widely used anticancer drug, whereas AN-9 is an anticancer agent presently undergoing Phase II clinical trials. The anticancer activity of AN-9 has been attributed to its ability to act as a butyric acid prodrug, although it also releases formaldehyde and pivalic acid. Adriamycin and AN-9 in combination display synergy when exposed simultaneously to cells or when AN-9 treatment is up to 18 h after Adriamycin administration. However, the reverse order of addition results in antagonism. These interactions have been established using cell viability assays and classical isobologram analysis. To understand the molecular basis of this synergy, the relative levels of Adriamycin-DNA adducts were determined using various treatment combinations. Levels of Adriamycin-DNA adducts were enhanced when treatment combinations known to be synergistic were used and were diminished using those treatments known to be antagonistic. The relative timing of the addition of Adriamycin and AN-9 was critical, with a 20-fold enhancement of Adriamycin-DNA adducts occurring when AN-9 was administered 2 h after the exposure of cells to Adriamycin. The enhanced levels of these adducts and the accompanying decreased cell viability were directly related to the esterase-dependent release of formaldehyde from AN-9, providing evidence for the formaldehyde-mediated activation of Adriamycin.     

Modulation of the antineoplastic efficacy of mitomycin C by dicoumarol in vivo

Summary Dicoumarol (DIC) modulates the intracellular metabolism of mitomycin C (MC) in vitro, increasing the toxicity of MC to hypoxic EMT6 cells and decreasing its toxicity to aerobic cells. The present experiments asessed whether DIC could be used to increase the therapeutic ratio attainable in vivo when MC was used as an adjunct to radiotherapy. Experiments with transplanted EMT6 tumors in mice showed that DIC increased the toxicity of MC to hypoxic tumor cells and increased the antineoplastic efficacy of regimens combining MC with radiation. DIC did not increase the hematologic toxicity of MC, and pretreatment with DIC plus MC did not augment radiation-induced skin reactions. The increase in antineoplastic effect was therefore obtained without a concomitant increase in normal tissue toxicities, and therapeutic gain was obtained.This research was supported by research grants PDT-145 from the American Cancer Society (SR) and CA-43659 from the National Cancer Institute (ACS, SRK)     

Carcinogen specificity in the activation of transforming genes by direct-acting alkylating agents

DNAs from rat nasal and mouse skin carcinomas and fibrosarcomasinduced by the alkylating agents methylmethane sulfonate (MMS),ß-propiolactone (BPL), and dimethyl-carbamyl chloride(DMCC) were tested for their ability to transform NIH3T3 cellsby DNA transfection. Each of eight MMS-induced rat nasal carcinomasand two of five BPL-induced mouse skin tumors were positivein the transfection assay while all of four fibrosarcomas andsix carcinomas induced by DMCC were negative. Anchorage independentgrowth, tumorigenicity in nude mice, and secondary transfectionconfirmed the transformed phenotype of the positive transfectants.The transfectants from MMS-induced tumor DNAs did not containrestriction fragments homologous to rat H-, K- or N-ras oncogenesalthough exogenous (rat) tumor-derived DNA sequences were detectedin transfectant genomes by Southern analysis. In contrast aBPL-induced mouse skin tumor showed evidence of containing activatedH-ras. These results suggest specificity among causal carcinogensfor activation of transforming genes in experimental tumors.     

Persistence of cytogenetic damage induced by alkylating antineoplastic drug phopurinum in human lymphocytes in vivo and in vitro

Sister chromatid exchanges (SCEs) and structural chromosome aberrations (CAs) induced by cytostatic drug phopurinum in vivo and in vitro were studied in human lymphocytes. Phopurinum was found to cause a significant increase of CAs in lymphocytes of patients undergoing cytostatic therapy. Increased CA rates, however, declined rapidly after the cessation of treatment. This decline observed in vivo is in agreement with experimental results obtained in vitro, where it is found that the induction of SCEs and CAs occur only during the 1st cell cycle after pulse-treatment as G1 stage with phopurinum. Thus, phopurinum induced short-lived DNA damage both experimentally and in vivo.     

The efficacy of the anthracycline prodrug daunorubicin-GA3 in human ovarian cancer xenografts.

The prodrug N-[4-(daunorubicin-N-carbonyl-oxymethyl)phenyl] O-beta-glucuronyl carbamate (DNR-GA3) was synthesized for specific activation by human beta-glucuronidase, released in necrotic areas of tumour lesions. In vitro, DNR-GA3 was 18 times less toxic than daunorubicin (DNR) and the prodrug was completely activated to the parent drug by human beta-glucuronidase. The maximum tolerated dose of DNR-GA3 in nude mice bearing s.c. human ovarian cancer xenografts was 6-10 times higher than that of DNR. The prodrug was cleared more rapidly from the circulation (elimination t1/2 = 20 min) than the parent drug (elimination t1/2 = 720 min). The anti-tumour effects of DNR-GA3 and DNR were investigated in four different human ovarian cancer xenografts OVCAR-3, FMa, A2780 and MRI-H-207 at a mean tumour size between 100 and 200 mm3. In three out of four of these tumour lines, the prodrug given i.v. at the maximum tolerated dose ranging from 150 to 250 mg kg(-1) resulted in a maximum tumour growth inhibition from 82% to 95%. The standard treatment with DNR at a dose of 8 mg kg(-1) given i.v. weekly x 2 resulted only in a maximum tumour growth inhibition from 40% to 47%. Tumour line FMa did not respond to DNR, nor to DNR-GA3. Treatment with DNR-GA3 was also given to mice with larger tumours that would contain more necrosis (mean size 300-950 mm3). The specific growth delay by DNR-GA3 was extended from 2.1 to 4.4 in OVCAR-3 xenografts and from 4.4 to 6.0 in MRI-H-207 xenografts. Our data indicate that DNR-GA3 is more effective than DNR and may be especially of use for treatment of tumours with areas of necrosis.     

Interactions of the UVRABC endonuclease in vivo and in vitro with DNA damage produced by antineoplastic anthracyclines

The anthracycline antineoplastic agents Adriamycin and N-trifluoroacetyl-Adriamycin-14-valerate were assayed in vivo and in vitro for ability to produce DNA lesions recognized by the UVRABC endonuclease, a DNA repair enzyme of Escherichia coli which recognizes large, bulky lesions in DNA. We found that, while both drugs produce DNA lesions, only the lesions produced by Adriamycin were toxic. Hence, anthracycline antineoplastic activity may be related to production of large, bulky lesions in DNA, while toxicity may correlate with toxicity measured in a simple E. coli DNA repair mutant test system.     

Mutagenicity of dibromodulcitol (DBD), an alkylating anticancer drug) and its mono- and bifunctional conversion products studied by the Salmonella/microsome assay

Dibromodulcitol (DBD) and one of its most important bifunctionaltransformation products, dianhydrogalactitol (DAG) with similarcytostatic effect, were tested by the Salmonella/microsome assayon strains TA 1535, TA 1538, TA 98 and TA 100 using the plateincorporation technique. Both drugs were direct mutagens instrains TA 1535 and TA 100 and non-mutagenic in other strains.Their mutagenic effect was not influenced by S-9 mix from ratliver. Mutagenicity of DAG was very limited because of its markedtoxicity. The other monofunctional alkylating derivatives, i.e.,1-bromo-3, 6-anhydrodulcitol and 1,2-epoxy-3,6-anhydrodulcitolwere highly mutagenic in strains TA 1535 and TA 100 with andwithout S-9 mix despite having no anticancer effect. Anticanceractivity was exerted only by the bifunctional alkylating hexitols(DBD, DAG) which showed moderate mutagenic activity comparedto the monofunctional derivatives. No correlation could be establishedbetween the mutagenic and anticancer effect of the four structurallyrelated hexitols. Mutagenicity of urine and bile from rats aftera single administration of the maximum tolerated (450 mg/kg)dose of DBD was also examined, and the hexitol components ofthe same urine sample were identified by t.l.c. DBD and itsmutagenic transformation products were excreted in urine andnot through the bile. The mutagenic effect of DBD observed cannotbe attributed exclusively to DBD itself, because the parentmolecule, like other alkylating agents, easily undergoes spontaneousdecomposition under in vitro and in vivo conditions to releaseboth bi- and monofunctional alkylating solvolysis products andthese highly reactive derivatives may play a role in this effect.No significant difference in the relative mutagenicity was detectedbetween DBD and cyclophosphamide, used as a reference substance.     

Quantitation of the synergistic interaction of edatrexate and cisplatin in vitro

Summary Cytotoxicity studies combining edatrexate (EDX) and cisplatin (Cis-Pt) were carried out in HL-60 cells in vitro as a retrospective analysis of the same combination in animal models and as a prospective study of this combination for future clinical trials. For purposes of comparison, parallel studies were carried out using methotrexate (MTX) and Cis-Pt. Dose-effect relationships were analyzed by the median-effect principle and the combination index-isobologram technique. EDX was the most cytotoxic agent of the three examined. The doses effective in 50% inhibition of the cell proliferation (ED₅₀ values) for EDX, MTX, and Cis-Pt were 0.001, 0.0043, and 1.08 m, respectively. Synergism occurred at effect levels corresponding to greater than 65% inhibition of cell growth by EDX+Cis-Pt, with an increase in synergism being observed at high doses. By contrast, MTX+Cis-Pt exhibited moderate synergism, with a decrease in synergism being noted at high doses. Preceding one drug by another for 4 h during the 48-h incubation period did not result in synergism greater than that produced by simultaneous exposure to both drugs for both pairs of combinations. Due to the synergism arising from these combinations, the ED₉₀ values can be reduced by as many as 52 and 7.3 times for Cis-Pt and EDX, respectively, as compared with only 4.0 and 1.9 times for Cis-Pt and MTX, respectively. The calculation of these drug interactions was carried out automatically with the use of computer software and was also illustrated by a sample calculation performed without computer simulation.Abbreviations MTX methotrexate - EDX edatrexate, 10-ethyl-10-deaza-aminopterin - Cis-Pt cisplatin cis-diamminedichloroplatinum(II) - CI combination index - DRI dose-reduction index This work was supported in part by NCI grant CA18 856 and by the Elsa U. Pardee Foundation     

Plasma clearance of an antibody--enzyme conjugate in ADEPT by monoclonal anti-enzyme: its effect on prodrug activation in vivo.

The effect of anti-enzyme antibody clearance on prodrug turnover in antibody-directed enzyme prodrug therapy (ADEPT) has been studied. Mice bearing LS174T xenografts were given localising carboxypeptidase G2 (CPG)2 conjugate (AEC) and 19 h later galactosylated anti-CPG2 antibody (SB43-GAL). In regimen I prodrug was injected 5 h after SB43-GAL as previously described. In regimen 2 and 3 a shortened and extended clearance time was used in which prodrug was administered 0.5 h or 53 h after SB43-GAL respectively. Regimen 1 resulted in similar tumour and normal tissue levels of active drug to those of the control in which prodrug was given 72 h after AEC. SB43-GAL therefore accelerated clearance of enzyme allowing early administration of prodrug. In regimen 2, very high active drug levels were found in the liver, showing removal of AEC from the blood followed by reactivation of enzyme and extensive and rapid prodrug turnover. Active drug levels in tumour and blood reached similar peak levels to those of the control. Regimen 3 resulted in lower active drug levels in tissues, consistent with degradation and excretion of enzyme. Regimen 3 also produced the best tumour to normal ratios for active drug. Residual prodrug in tumour was unaffected by SB43-GAL, showing the advantage of galactosylation in minimising inactivation of CPG2 in tumour. By contrast, residual prodrug in blood persisted for longer when SB43-GAL was used. Circulatory clearance of enzyme with SB43-GAL allows prodrug to be administered expediently with reduced toxicity and with the prospect of increasing the dosage.     

The efficacy of Herceptin therapies is influenced by the expression of other erbB receptors, their ligands and the activation of downstream signalling proteins

ErbB2 and EGFR are attractive oncology therapeutic targets as their overexpression in tumors predicts a poorer clinical outcome in a variety of epithelial malignancies. However, clinical results with therapeutic compounds targeting these receptors have been mixed. Therefore, there is a need for improved predictive biomarkers for these targeted therapeutics. In this study we analysed tissue microarrays of patients treated with combination chemotherapy and Herceptin for expression or phosphorylation of signalling proteins associated with erbB receptors to identify protein biomarkers that are predictive of breast cancer patient response. A comparison of expression or phosphorylation of these markers with patient outcome revealed that response to Herceptin depended not only on expression levels of erbB2 but also on expression of EGFR, expression of erbB ligands, expression of other receptors and phosphorylation of downstream proteins. Elucidating the biological effects of EGFR/erbB2 targeted therapeutics will enable patient tumor profiling to identify likely responders and the determination of biologically effective doses that allows chronic administration of these agents in order to maximise efficacy.     

The bioactivation of CB 1954 and its use as a prodrug in antibody-directed enzyme prodrug therapy (ADEPT)

Walker cellsin vivo orin vitro are exceptionally sensitive to the monofunctional alkylating agent CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The basis of the sensitivity is that CB 1954 forms DNA interstrand crosslinks in Walker cells but not in insensitive cells. Crosslink formation is due to the aerobic reduction of CB 1954 to form 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide by the enzyme DT diaphorase. The 4-hydroxylamine can not crosslink DNA directly but requires further activation by a non-enzymatic reaction with a thioester (such as acetyl coenzyme A). As predicted from their measured DT diaphorase activities, a number of rat hepatoma and hepatocyte cell lines are also sensitive to CB 1954. However, no CB 1954-sensitive tumours or cell lines of human origin have been found. This is because the rate of reduction of CB 1954 by the human form of DT diaphorase is much lower than that of the Walker enzyme (ratio of k_cat= 6.4). To overcome this intrinsic resistance of human cells towards CB 1954 a number of strategies have been developed. First, analogues have been developed that are more rapidly reduced by the human form of CB 1954. Second, the cytotoxicity of CB 1954 can be potentiated by reduced pyridinium compounds. Third, a CB 1954 activating enzyme can be targeted to human tumours by conjugating it to an antibody (ADEPT). A nitroreductase enzyme has been isolated fromE. coli that can bioactivate CB 1954 much more rapidly than Walker DT diaphorase and is very suitable for ADEPT. Thus CB 1954 may have a role in the therapy of human tumours.     

Evaluation of the efficacy of potential antineoplastic drugs on tumour metastasis by a computer-assisted image analysis

Computerised image analysis, performed on histological sections of (C57BL6/N) mouse lungs that had been intravenously (i.v.) injected with B16-F10 melanoma cells was used to develop a novel method to quantify the efficacy of potential antineoplastic drugs. This procedure allowed the evaluation of the rate of inhibition of growth and the anti-invasive capability of new molecules, thus resulting in more accurate data than that obtained from common macroscopical counting of surface metastatic foci. Several morphological parameters can be measured by this method: the percentage of tissue area occupied by metastases, which accounts for tumour implantation into the organ; the growth index, related to the size of the metastases, and the invasion index, related to the frequency of foci. These morphometric data were found to be correlated to the levels of lung hydroxyproline and transglutaminase activity, well known markers of tumour invasion and cell differentiation, respectively. The main objective of this computerised procedure was to evaluate how the tumour cell is affected in the host by the drug under investigation. The use of the method is exemplified by an analysis of the antitumour activity of some methylxanthines.     

不同前路减压术治疗脊髓型颈椎病的疗效分析

目的 观察不同术式前路减压手术效果,为脊髓型颈椎病的前路手术治疗提供术式选择依据.方法 回顾性分析2001年7月至2009年6月笔者施行多种前路减压融合内固定术治疗并有完整随访资料的106例脊髓型颈椎病病例,男73例,女33例;单间隙25例,双间隙71例,三间隙10例.术前术后按JOA评分标准及SF-36生活质量评分评估患者的手术疗效.结果 本组病例根据压迫情况不同而采用不同前路术式均获得良好效果,前路直接减压彻底、效果好.术后随访7个月至9年,平均3.9年,1年平均改善率86.26%,有效率100%,优良率89.62%.结论 对于压迫主要来自前方的脊髓型颈椎病,根据颈髓受压情况选择合适的前路减压术,可以直接切除脊髓前方致压物,减压效果明显.