midostaurin and Lung-Neoplasms

PKC412 (N-benzoyl-staurosporine), an oral inhibitor of protein kinase C, is capable of cell cycle inhibition and is endowed with anti-angiogenic properties. This dose-finding phase I study was designed to establish the maximum tolerated dose (MTD) of PKC412 when combined with cisplatin-gemcitabine.. Escalating doses of PKC412 were given every day of a 4 week cycle with cisplatin 100 mg/m2 on day 2 and gemcitabine 1000 mg/m2 on days 1, 8 and 15 in patients with non-small-cell lung cancer. Dose escalation was based on a modified continuous reassessment method.. Twenty-three patients, assigned to four cohorts receiving PKC412 at a dose ranging from 25 to 150 mg/day were evaluable. Grade 3 diarrhea occurring in 3/4 patients at cycle 1 led us to define 150 mg/day as the MTD. The MTD based on multiple cycles was redefined as 100 mg/day, since prolonged grade 2-3 nausea/vomiting leading to treatment discontinuation occurred in 3/7 patients after repeated cycles. The next lower dose tested of 50 mg/day was therefore considered as the recommended dose for phase II trials. Among 33 cycles in eight patients, toxicity consisted of grade 1-2 diarrhea (12.5%) and asthenia (50%) with only one patient experiencing grade 3 headache at this dose level. A partial response was observed in three patients.. The results of the present study indicate that PKC412 at a dose of 50 mg/day can be safely added to cisplatin and gemcitabine in patients with advanced non-small-cell lung cancer.

Topics: Administration, Oral; Adult; Aged; Angiogenesis Inhibitors; Antineoplastic Combined Chemotherapy Protocols; Asthenia; Carcinoma, Non-Small-Cell Lung; Cisplatin; Deoxycytidine; Diarrhea; Enzyme Inhibitors; Female; Gemcitabine; Humans; Infusions, Intravenous; Lung Neoplasms; Male; Maximum Tolerated Dose; Middle Aged; Protein Kinase C; Staurosporine

Drug combinations are key to circumvent resistance mechanisms compromising response to single anti-cancer targeted therapies. The implementation of combinatorial approaches involving MEK1/2 or KRASG12C inhibitors in the context of KRAS-mutated lung cancers focuses fundamentally on targeting KRAS proximal activators or effectors. However, the antitumor effect is highly determined by compensatory mechanisms arising in defined cell types or tumor subgroups. A potential strategy to find drug combinations targeting a larger fraction of KRAS-mutated lung cancers may capitalize on the common, distal gene expression output elicited by oncogenic KRAS. By integrating a signature-driven drug repurposing approach with a pairwise pharmacological screen, here we show synergistic drug combinations consisting of multi-tyrosine kinase PKC inhibitors together with MEK1/2 or KRASG12C inhibitors. Such combinations elicit a cytotoxic response in both in vitro and in vivo models, which in part involves inhibition of the PKC inhibitor target AURKB. Proteome profiling links dysregulation of MYC expression to the effect of both PKC inhibitor-based drug combinations. Furthermore, MYC overexpression appears as a resistance mechanism to MEK1/2 and KRASG12C inhibitors. Our study provides a rational framework for selecting drugs entering combinatorial strategies and unveils MEK1/2- and KRASG12C-based therapies for lung cancer.

Topics: Cell Line, Tumor; Drug Combinations; Drug Repositioning; Humans; Lung Neoplasms; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras)

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; DNA Nucleotidyltransferases; Drug Discovery; Drug Resistance, Neoplasm; ErbB Receptors; Genes, Reporter; High-Throughput Screening Assays; Humans; Luciferases; Lung Neoplasms; Mutation; Phosphorylation; Protein Kinase Inhibitors; Reproducibility of Results; Small Molecule Libraries; Staurosporine

Lung cancer is associated with high prevalence and mortality, and despite significant successes with targeted drugs in genomically defined subsets of lung cancer and immunotherapy, the majority of patients currently does not benefit from these therapies. Through a targeted drug screen, we found the recently approved multi-kinase inhibitor midostaurin to have potent activity in several lung cancer cells independent of its intended target, PKC, or a specific genomic marker. To determine the underlying mechanism of action we applied a layered functional proteomics approach and a new data integration method. Using chemical proteomics, we identified multiple midostaurin kinase targets in these cells. Network-based integration of these targets with quantitative tyrosine and global phosphoproteomics data using protein-protein interactions from the STRING database suggested multiple targets are relevant for the mode of action of midostaurin. Subsequent functional validation using RNA interference and selective small molecule probes showed that simultaneous inhibition of TBK1, PDPK1 and AURKA was required to elicit midostaurin's cellular effects. Immunoblot analysis of downstream signaling nodes showed that combined inhibition of these targets altered PI3K/AKT and cell cycle signaling pathways that in part converged on PLK1. Furthermore, rational combination of midostaurin with the potent PLK1 inhibitor BI2536 elicited strong synergy. Our results demonstrate that combination of complementary functional proteomics approaches and subsequent network-based data integration can reveal novel insight into the complex mode of action of multi-kinase inhibitors, actionable targets for drug discovery and cancer vulnerabilities. Finally, we illustrate how this knowledge can be used for the rational design of synergistic drug combinations with high potential for clinical translation.

Topics: Aurora Kinase A; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Drug Discovery; Drug Synergism; Humans; Lung Neoplasms; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proteomics; Proto-Oncogene Proteins; RNA Interference; Signal Transduction; Staurosporine

Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cytokines; Drug Discovery; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Lung Neoplasms; Mice; Molecular Targeted Therapy; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proteomics; Xenograft Model Antitumor Assays

Lung cancer cells are sensitive to 5-aza-2'-deoxycytidine (decitabine) or midostaurin (PKC412), because decitabine restores the expression of methylation-silenced tumor suppressor genes, whereas PKC412 inhibits hyperactive kinase signaling, which is essential for cancer cell growth. Here, we demonstrated that resistance to decitabine (decitabine(R)) or PKC412 (PKC412(R)) eventually results from simultaneously remethylated DNA and reactivated kinase cascades. Indeed, both decitabine(R) and PKC412(R) displayed the up-regulation of DNA methyltransferase DNMT1 and tyrosine-protein kinase KIT, the enhanced phosphorylation of KIT and its downstream effectors, and the increased global and gene-specific DNA methylation with the down-regulation of tumor suppressor gene epithelial cadherin CDH1. Interestingly, decitabine(R) and PKC412(R) had higher capability of colony formation and wound healing than parental cells in vitro, which were attributed to the hyperactive DNMT1 or KIT, because inactivation of KIT or DNMT1 reciprocally blocked decitabine(R) or PKC412(R) cell proliferation. Further, DNMT1 knockdown sensitized PKC412(R) cells to PKC412; conversely, KIT depletion synergized with decitabine in eliminating decitabine(R). Importantly, when engrafted into nude mice, decitabine(R) and PKC412(R) had faster proliferation with stronger tumorigenicity that was caused by the reactivated KIT kinase signaling and further CDH1 silencing. These findings identify functional cross-talk between KIT and DNMT1 in the development of drug resistance, implying the reciprocal targeting of protein kinases and DNA methyltransferases as an essential strategy for durable responses in lung cancer.

Topics: Animals; Antigens, CD; Azacitidine; Cadherins; Cell Line, Tumor; Cell Proliferation; Decitabine; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Proto-Oncogene Proteins c-kit; Staurosporine

Approximately half of EGFR-mutant non-small cell lung cancer (NSCLC) patients treated with small-molecule EGFR kinase inhibitors develop drug resistance associated with the EGF receptor (EGFR) T790M "gatekeeper" substitution, prompting efforts to develop covalent EGFR inhibitors, which can effectively suppress EGFR T790M in preclinical models. However, these inhibitors have yet to prove clinically efficacious, and their toxicity in skin, reflecting activity against wild-type EGFR, may limit dosing required to effectively suppress EGFR T790M in vivo. While profiling sensitivity to various kinase inhibitors across a large cancer cell line panel, we identified indolocarbazole compounds, including a clinically well-tolerated FLT3 inhibitor, as potent and reversible inhibitors of EGFR T790M that spare wild-type EGFR. These findings show the use of broad cancer cell profiling of kinase inhibitor efficacy to identify unanticipated novel applications, and they identify indolocarbazole compounds as potentially effective EGFR inhibitors in the context of T790M-mediated drug resistance in NSCLC.. EGFR-mutant lung cancer patients who respond to currently used EGFR kinase inhibitors invariably develop drug resistance, which is associated with the EGFR T790M resistance mutation in about half these cases. We unexpectedly identified a class of reversible potent inhibitors of EGFR T790M that do not inhibit wild-type EGFR, revealing a promising therapeutic strategy to overcome T790M-associated drug-resistant lung cancers.

Topics: Acrylamides; Amino Acid Substitution; Animals; Area Under Curve; Base Sequence; Carbazoles; Cell Line, Tumor; Drug Resistance, Neoplasm; ErbB Receptors; Erlotinib Hydrochloride; Humans; Immunoblotting; Lung; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, Transgenic; Mutation; Protein Kinase Inhibitors; Pyrimidines; Quinazolines; RNA Interference; Staurosporine; Xenograft Model Antitumor Assays

To investigate the combination of conventional cytotoxic anticancer agents and a small molecule kinase inhibitor in preclinical models of non-small-cell lung cancer (NSCLC).. We compared the induction of apoptosis by DNA-damaging anticancer drugs and PKC412, a predominantly protein kinase C (PKC)-specific small molecule inhibitor, in six NSCLC cell lines of different histologic and genetic backgrounds. The outcome of various combinations and schedules of DNA-damaging agents and PKC412 was studied, and isobolograms were calculated. Conditional expression of pro-apoptotic BAK was applied to specifically target apoptotic signal transduction in combination with drug therapy.. Resistance of NSCLC cells to DNA damage-induced apoptosis was mainly determined at the mitochondrial step of the intrinsic pathway of caspase activation. PKC412 effectively inhibited the growth factor signal transduction, but failed to induce apoptosis in NSCLC cells resistant to DNA-damaging agents. Combining conventional anticancer drugs with PKC412 at different doses and schedules resulted in unpredictable outcomes, including synergistic, additive, and antagonistic interactions. In contrast, conditional expression of BAK reliably sensitized drug-resistant NSCLC cells to apoptosis induced by cytotoxic agents or PKC412.. Combining DNA-damaging anticancer drugs with a pharmacologic inhibitor of growth and survival factor signaling in NSCLC may result in unpredictable treatment outcomes. In contrast, targeting specific death effector mechanisms, such as apoptotic signal transduction, is a promising strategy to sensitize NSCLC to cytotoxic agents or kinase inhibitors.

Topics: Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; bcl-X Protein; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Dactinomycin; Dose-Response Relationship, Drug; Doxorubicin; Drug Antagonism; Drug Synergism; Etoposide; Humans; Immunoblotting; Lung Neoplasms; Paclitaxel; Protein Kinase C; Signal Transduction; Staurosporine; Time Factors

Clinical biomarker tests that aid in making treatment decisions will play an important role in achieving personalized medicine for cancer patients. Definitive evaluation of the clinical utility of these biomarkers requires conducting large randomized clinical trials (RCTs). Efficient RCT design is therefore crucial for timely introduction of these medical advances into clinical practice, and a variety of designs have been proposed for this purpose. To guide design and interpretation of RCTs evaluating biomarkers, we present an in-depth comparison of advantages and disadvantages of the commonly used designs. Key aspects of the discussion include efficiency comparisons and special interim monitoring issues that arise because of the complexity of these RCTs. Important ongoing and completed trials are used as examples. We conclude that, in most settings, randomized biomarker-stratified designs (ie, designs that use the biomarker to guide analysis but not treatment assignment) should be used to obtain a rigorous assessment of biomarker clinical utility.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Clinical Trials, Phase III as Topic; DNA-Binding Proteins; Endonucleases; Enzyme Activation; ErbB Receptors; Erlotinib Hydrochloride; fms-Like Tyrosine Kinase 3; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glutamates; Guanine; Humans; Leukemia, Myeloid, Acute; Lung Neoplasms; Mutation; Pemetrexed; Protein Kinase Inhibitors; Quinazolines; Randomized Controlled Trials as Topic; Research Design; Staurosporine

Non-small cell lung cancer cells (NSCLC) are more resistant to anticancer treatment as compared with other types of cancer cells. Recently (Hemström et al., Exp Cell Res 2005;305:200-13) we showed that apoptosis of U1810 NSCLC cells induced by the staurosporine analog PKC 412 correlated with inhibition of Akt and ERK1/2, suggesting the involvement of these kinases in cell survival. Here we investigated the contribution of the PI3-kinase/Akt and MEK/ERK pathways to survival of NSCLC cells. The two signaling pathways were studied by using different combinations of the PI3-kinase inhibitors LY-294002 and wortmannin, the Akt activator Ro 31-8220, the MEK inhibitor PD 98059 and PKC 412. PI3-kinase inhibitors induced apoptosis-like death in U1810 cells. H157 cells in general were relatively resistant to PI3 kinase/Akt inhibitors yet these compounds sensitized cells to the DNA-damaging drug VP-16, while Ro 31-8220 could not. PD 98059 only had a sensitizing effect on H157 cells when combined with PI3-kinase inhibition and VP-16. Morphological data indicated that LY-294002 and PKC 412 induced cell death at anaphase and metaphase, respectively, suggesting death by mitotic catastrophe. Analyzes of cells blocked in G2/M-phase by nocodazol revealed that LY-294002 increased, while PKC 412 decreased histone H3 phosphorylation, suggesting that LY-294002 allowed, while PKC 412 inhibited cells to leave M-phase. Flow cytometric analysis of cell cycle distribution demonstrated that LY-294002 allowed cells to leave G2/M phase, while PKC 412 inhibited cytokinesis, resulting in formation of multinucleated cells. These results indicate that sensitization of NSCLC cells by PI3-kinase inhibition involves interplay between cell cycle regulation, mitotic catastrophe and apoptosis.

Topics: Androstadienes; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Survival; Chromones; Enzyme Inhibitors; Etoposide; Flavonoids; Flow Cytometry; Humans; Indoles; Lung Neoplasms; MAP Kinase Kinase Kinases; Mitosis; Morpholines; Nocodazole; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Staurosporine; Wortmannin

Non-small cell lung carcinoma (NSCLC) is characterized by resistance to drug-induced apoptosis, which might explain the survival of lung cancer cells following treatment. Recently we have shown that the broad-range kinase inhibitor staurosporine (STS) reactivates the apoptotic machinery in U1810 NSCLC cells [Joseph et al., Oncogene 21 (2002) 65]. Lately, several STS analogs that are more specific in kinase inhibition have been suggested for tumor treatment. In this study the apoptosis-inducing ability of the STS analogs PKC 412 and Ro 31-8220 used alone or in combination with DNA-damaging agents in U1810 cells was investigated. In these cells Ro 31-8220 neither induced apoptosis when used alone, nor sensitized cells to etoposide treatment. PKC 412 as a single agent induced death of a small number of U1810 cells, whereas it efficiently triggered a dose- and time-dependent apoptosis in U1285 small cell lung carcinoma cells. In both cell types PKC 412 triggered release of mitochondrial proteins followed by caspase activation. However, concomitant activation of a caspase-independent pathway was essential to kill NSCLC cells. Importantly, PKC 412 was able to sensitize etoposide- and radiation-induced death of U1810 cells. The best sensitization was achieved when PKC 412 was administered 24 h after treatments. In U1810 cells, Ro 31-8220 decreased PMA-induced ERK phosphorylation as efficiently as PKC 412, indicating that the failure of Ro 31-8220 to induce apoptosis was not due to weaker inhibition of conventional and novel PKC isoforms. However, Ro 31-8220 increased the basal level of ERK and Akt phosphorylation in both cell lines, whereas Akt phosphorylation was suppressed in the U1810 cells, which might influence apoptosis. These results suggest that PKC 412 could be a useful tool in increasing the efficiency of therapy of NSCLC.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; DNA Damage; DNA, Neoplasm; Enzyme Inhibitors; Humans; Intracellular Membranes; Kinetics; Lung Neoplasms; Membrane Potentials; Mitochondria; Protein Kinase Inhibitors; Staurosporine

PKC412, a selective inhibitor of protein kinase C (PKC), is currently in clinical trials as an anti-tumor drug. In the present study, we investigated the anti-metastatic effect of PKC412 using an experimental metastatic mouse model intravenously injected with melanoma cells. One-hour exposure to various concentrations of PKC412 (0.5, 5 and 50 microM) dose-dependently reduced the lung-metastatic potential of highly metastatic B16-F10 and -BL6 mouse melanoma cells in syngeneic mice. Following the exposure, PKC activities in B16-F10 and -BL6 cells were significantly decreased, but growth curves were not influenced. To elucidate the mechanism of the anti-metastatic effect of PKC412, we examined the activity to invade the extracellular matrix and the platelet-aggregating activity of the melanoma cells incubated with PKC412 (0.5, 5 and 50 microM) for 1 hour. PKC412 significantly reduced both the invasive and platelet-aggregating activities. These results suggest that PKC412 shows an anti-metastatic function through the inhibition of the invasive and/or platelet-aggregating activities of melanoma cells. PKC412 is potentially a promising candidate for an anti-metastatic agent.

Topics: Animals; Cell Division; Enzyme Inhibitors; Female; Lung Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Neoplasm Transplantation; Platelet Aggregation; Protein Kinase C; Rabbits; Staurosporine; Tumor Cells, Cultured

The staurosporine analogues CGP 41251, UCN-01 and Ro 31-8220 are specific inhibitors of protein kinase C (PKC). CGP 41251 and UCN-01 exert anti-neoplastic activity against human tumours grown in rodents, and CGP 41251 reverses multidrug resistance. The hypothesis was tested that these agents can induce drug resistance and alter cellular levels of target kinases. Human-derived A549 lung carcinoma cells were exposed for 6 months to CGP 41251, UCN-01 or Ro 31-8220 at gradually increasing concentrations. Cells acquired resistance against these agents, 4.3-fold against CGP 41251 (A549/CGP cells), 4.0-fold against UCN-01 (A549/UCN cells) and 14-fold against Ro 31-8220 (A549/Ro cells). Cells were neither collaterally cross-resistant towards the PKC inhibitors nor resistant against the growth-inhibitory properties of 12-O-tetradecanoylphorbol-13-acetate. However, cross-resistance was observed in A549/CGP cells against staurosporine (13-fold) and in A549/Ro cells against doxorubicin (26-fold). All 3 cell types expressed multidrug resistance-associated protein, and A549/Ro cells expressed P-glycoprotein, as adjudged by Western blot analysis. Phorbol ester-stimulated PKC activity in these cells was decreased by between 57% and 96% compared to wild-type A549 cells. Levels of the PKC isoenzymes alpha and theta in all 3 resistant cell types and of PKC-epsilon in A549/UCN cells were concomitantly reduced. Cells regained drug sensitivity after culture in the absence of drug for 6 (A549/Ro cells), 5 (A549/CGP cells) and 1 (A549/UCN cells) months. Our results suggest the following features of this type of anti-signalling drug: (i) they can induce drug resistance, (ii) they may be potentially useful in combination because of the lack of cross-resistance between them and (iii) they can down-regulate PKC, which may have pharmacological or toxicological consequences.

Topics: Adenocarcinoma; Alkaloids; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Cell Division; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; Indoles; Lung Neoplasms; Multidrug Resistance-Associated Proteins; Protein Kinase C; Staurosporine; Tumor Cells, Cultured

CGP 41251 (4'-N-benzoyl staurosporine, CAS 120685-11-2) exerts increased selectivity for Ca(2+)- and phospholipid-dependent protein kinase C inhibition. In this study, the effects of CGP 41251 on cell cycle distribution and growth inhibition were examined in SBC3 human small cell lung cancer (SCLC) cell line. CGP 41251 caused the inhibition of cell proliferation and at 1.0 mumol/l showed almost complete effect. In early S phase synchronized SBC3 cells, CGP 41251 at 1.0 mumol/l did not inhibit an initial progression from early S to G2 phase, but it blocked a process from G2/M to G1 phase completely. After removal of nocodazole block, CGP 41251 at 1.0 mumol/l caused DNA re-replication and induction of polyploidy. In nude mice xenograft, CGP 41251 at a dose of 200 mg/kg showed statistically significant inhibition against this tumor with a T/C value of 21.4%. Histopathologically, expansion of central necrosis was observed by the administration of CGP 41251. These results in SBC3 cells indicated that CGP 41251 showed antitumor activity through the inhibition of cell cycle progression from G2/M to G1 phase, and through induction of cells with higher DNA content.

Topics: Animals; Apoptosis; Carcinoma, Small Cell; Cell Cycle; Cell Division; DNA, Neoplasm; Enzyme Inhibitors; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Protein Kinase C; Rats; Staurosporine; Transplantation, Heterologous

The antitumor effect of CGP41251 (4'-N-benzoyl staurosporine), a selective protein kinase C (PKC) inhibitor, was examined on two kinds of human non-small cell lung cancer (NSCLC) cell lines (adenocarcinoma: A549 and squamous cell carcinoma: NCI-H520). CGP41251 at 0.5 or 1.0 microM inhibited the proliferation of these tumor cell lines significantly; However, at 0.1 microM, it did not show any significant inhibition. Cell cycle analysis indicated that CGP41251 at 0.5 or 1.0 microM arrested the cell cycle progression at the G2/M phase up to 24 hr, but 0.1 microM did not. It seems that the antiproliferative action of CGP41251 against human NSCLC is related to G2/M accumulation. In NCI-H520, CGP41251 caused DNA re-replication without mitosis. In a nude mice xenograft, CGP41251 at a dose of 200 mg/kg showed antitumor activity against these cell lines. Histopathologically, expansion of central necrosis was observed, although no destruction of tumor nests was seen by CGP41251 administration. In both tumor tissues, the PKC activity of the particulate fraction was significantly decreased by CGP41251 treatment. From these results, it is thought that the antitumor activity of CGP41251 against human NSCLS is accompanied by the decrease of PKC activity in the particulate fraction. Moreover, the G2/M arrest of the cell cycle induced by CGP41251 might be important for the growth inhibitory action of this compound.

Topics: Adenocarcinoma; Analysis of Variance; Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Cycle; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Transplantation; Protein Kinase C; Staurosporine; Tumor Cells, Cultured

Inhibitors of protein kinase C (PKC) such as the staurosporine analogues UCN-01 and CGP 41251 possess antineoplastic properties, but the mechanism of their cytostatic action is not understood. We tested the hypothesis that the ability of these compounds to arrest growth is intrinsically linked with their propensity to inhibit PKC. Compounds with varying degrees of potency and specificity for PKC were investigated in A549 and MCF-7 carcinoma cells. When the log values of drug concentration which arrested cell growth by 50% (IC50) were plotted against the logs of the IC50 values for inhibition of cytosolic PKC activity, two groups of compound could be distinguished. The group which comprised the more potent inhibitors of enzyme activity (calphostin C, staurosporine and its analogues UCN-01, RO 31-8220, CGP 41251) were the stronger growth inhibitors, whereas the weaker enzyme inhibitors (trimethylsphingosine, miltefosine, NPC-15437, H-7, H-7I) affected proliferation less potently. GF 109203X was exceptional in that it inhibited PKC with an IC50 in the 10(-8) M range, yet was only weakly cytostatic. To substantiate the role of PKC in the growth inhibition caused by these agents, cells were depleted of PKC by incubation with bryostatin 1 (1 microM). The susceptibility of these enzyme-depleted cells towards growth arrest induced by staurosporine, RO 31-8220, UCN-01 or H-7 was studied. The drug concentrations which inhibited incorporation of [3H]thymidine into PKC-depleted A549 cells by 50% were slightly, but not significantly, lower than significantly, lower than those observed in control cells. These results suggest that PKC is unlikely to play a direct role in the arrest of the growth of A549 and MCF-7 cells mediated by these agents. Staurosporine is not only a strong inhibitor of PKC but also mimics activators of this enzyme in that it elicits the cellular redistribution of certain PKC isoenzymes. The ability of kinase inhibitors other than staurosporine to exert a similar effect was investigated. Calphostin C, H-7, H-7I, miltefosine, staurosporine, UCN-01, RO 31-8220, CGP 41251 or GF 109203X were incubated for 30 min with A549 cells in the absence or presence of the PKC activator 12-O-tetradecanoyl phorbol-13-acetate. The subcellular distribution of PKC-alpha-, -epsilon and -zeta was measured by Western blot analysis. None of the agents affected PKC-alpha or -zeta.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Alkaloids; Antineoplastic Agents; Breast Neoplasms; Cell Division; Cell Line; Dose-Response Relationship, Drug; Humans; Isoenzymes; Lung Neoplasms; Protein Kinase C; Staurosporine; Structure-Activity Relationship; Tumor Cells, Cultured