Enhancement of antitumor activity of mitomycin C in vitro and in vivo by UCN-01, a selective inhibitor of protein kinase C

UCN-01 (7-hydroxy-staurosporine) is a potent and selective inhibitor of protein kinase C (PKC), one of several protein kinases examined. UCN-01 itself was shown to exhibit antitumor activity in vitro and in vivo in oncogene-activated human and murine tumor cell lines. Since the mechanism(s) of action of UCN-01 is thought to be different from those of alkylating agents, including mitomycin C (MMC), we tested the combined effect of UCN-01 with MMC on human epidermoid carcinoma A431 cells. UCN-01 potentiated the antiproliferative activity of MMC and yet it did not affect the growth of the cells in vitro. However, other nonselective protein kinase inhibitors, such as staurosporine, K-252a, KT6124 (a derivative of K-252a) and H7, did not enhance the activity of MMC. Isobologram analysis revealed that the interaction of UCN-01 with MMC was synergistic in its antiproliferative activity. A DNA histogram of A431 cells treated with both UCN-01 and MMC showed a block in the cell cycle at the G₁/S phase. However, a histogram of cells treated with UCN-01 or MMC alone showed a G₁ or a G₂M block, respectively. The combined effect of UCN-01 with MMC was further examined in vivo in xenografted A431 cells in nude mice. The combination of both drugs in a single i.v. injection exhibited greater antitumor activity than MMC and UCN-01 alone (P<0.01). This synergistic antitumor effect was also confirmed in two other solid tumor cell lines, i.e. human xenografted colon carcinoma Co-3 and murine sarcoma 180. The same was observed in the i.v.-inoculated P388 leukemia model, in which we saw an increased lifespan of mice when UCN-01 was combined with MMC. These results suggests the feasibility of using UCN-01 in clinical oncology, especially in combination with alkylating agents such as MMC. In addition, this combination therapy might be a novel chemotherapeutic approach to MMC-insensitive tumors in clinical trials.     

Antitumor activity of UCN-01, a selective inhibitor of protein kinase C, in murine and human tumor models

Antitumor activity of UCN-01 (7-hydroxy staurosporine), a selective inhibitor of Ca2+- and phospholipid-dependent protein kinase C, was examined in comparison with staurosporine, a nonselective inhibitor of protein kinases, on human and murine tumor cell lines which have some aberrations in cellular signal transduction. UCN-01 inhibited the growth of five tumor cell lines about 9 to 90 times less potently than staurosporine in vitro. UCN-01 showed an in vivo antitumor effect against three human tumor xenografts [epidermoid carcinoma A431 (c-erbB-1 overexpression), fibrosarcoma HT1080 (N-ras activation), and acute myeloid leukemia HL-60 (N-ras activation)], giving a minimum treated/control ratio of 0.40 (P less than 0.01), 0.17 (P less than 0.01), and 0.61 (P less than 0.05), respectively. UCN-01 also exhibited significant antitumor activity against two murine tumor models (fibrosarcoma, K-BALB and M-MSV-BALB), which activated the v-ras and v-mos oncogenes, showing a minimum treated/control ratio of 0.27 (P less than 0.01) and 0.21 (P less than 0.01). Staurosporine did not show significant antitumor activity against any of these five tumors. UCN-01 inhibited the down-modulation of epidermal growth factor receptor caused by phorbol 12-myristate 13-acetate in A431 cells at a near 50% inhibitory concentration for cell growth. These results imply that UCN-01 is a promising antitumor agent which has a novel mechanism(s) of action.     

Effects of staurosporine, K 252a and other structurally related protein kinase inhibitors on shape and locomotion of Walker carcinosarcoma cells.

The structure/activity relationship of the protein kinase inhibitors, staurosporine and K 252a and their analogues on motility of Walker carcinosarcoma cells has been studied in vitro. Staurosporine and K 252a, similar to phorbol myristate acetate (PMA) and diacylglycerols, suppress cell polarity and locomotor activity of Walker carcinosarcoma cells. Staurosporine inhibits spontaneous and colchicine-induced front-tail polarity (ID50 of about 6.0 x 10(-8) M) as well as spontaneous and colchicine-stimulated locomotion at 10(-7) M. K 252a suppresses cell polarity (ID50 of about 4.5 x 10(-6) M) and inhibits spontaneous and colchicine-stimulated locomotion at 10(-5) M, but suppression of locomotor activity is not complete in the presence of colchicine. CGP 41251, a staurosporine derivative with a much higher specificity for protein kinase C (PKC) than staurosporine, induces a dose-dependent increase in the proportion of polarised cells, and stimulates cell locomotion. Two K252a analogues, KT 5720 and KT 5822, which act preferentially on cyclic nucleotide-dependent protein kinases, and CGP 42700, an inactive staurosporine analogue, had no effect on cell polarity and locomotion. The findings suggest that protein kinase inhibitors acting preferentially on PKC may be of interest in pharmacological regulation of tumour cell locomotion.     

Selective downregulation of EGF receptor and downstream MAPK pathway in human cancer cell lines by active components partially purified from the seeds of Livistona chinensis R. Brown

Deregulation of protein kinase-mediated signaling events is one of the major causes to malignant transformation. In this work, we tried to purify protein kinase inhibitory activity and antitumor activity from ethanol extracts of the seeds of Livistona chinensis R. Brown (LC), a traditional herb used for the treatment of nasopharyngeal carcinoma (NPC). Both activities were found to be co-purified in various chromatography steps, and a highly purified fraction, LC-X, was obtained and its biological effects were characterized further. LC-X inhibited the activities of various protein kinases in vitro, including PAK2, PKA, PKC, GSK-3alpha, CK2, mitogen-activated protein kinase (MAPK), and JNK1, with IC(50) between approximately 1 and 40microg/ml. The proliferation of two NPC (NPC-TW02 and -TW04) and one breast cancer (MCF-7) cell lines, but not the epidermoid (A431) and cervical (HeLa) carcinoma cell lines, were significantly blocked by LC-X at the dose of >50microg/ml. Cell cycle arrested at G(2)/M phase and apoptosis were detected in NPC-TW02 cells treated with LC-X for 24h. Further studies revealed that epidermal growth factor (EGF)-induced activation of epidermal growth factor receptor (EGFR) and MAPK could be potently inhibited by LC-X in both NPC-TW02 and A431cells in a dose-dependent manner. More interestingly, the level of EGFR protein detected by Western blot decreased drastically in LC-X-treated A431 and NPC-TW02 cells in a dose- and time-dependent fashion. Further analysis of the plasma membrane and cytosolic fractions from LC-X-treated and untreated A431 cells showed that a 170kDa protein selectively disappeared from the plasma membrane of LC-X-treated cells. The protein was identified as EGFR by MALDI-TOF mass spectrometry, indicating EGFR as a selective target for LC-X. Moreover, the electrophoretic mobility of purified EGFR in SDS-PAGE was altered dramatically post LC-X treatment, suggesting that LC-X may chemically modify EGFR. In conclusion, the active components with both antitumor and protein kinases inhibitor activities were highly purified from LC, which can inhibit the EGF signaling events mainly through EGFR modification. Blockage of the functions of EGFR may account for the antitumor activity of these active components.     

Cytotoxicity against human tumor cells mediated by the conjugate of anti-epidermal growth factor receptor monoclonal antibody to recombinant ricin A chain

We have produced monoclonal antibodies against the epidermal growth factor (EGF) receptor which bind to the receptor with high affinity, compete with EGF for binding, block EGF-induced tyrosine kinase activity, and activate internalization and down-regulation of the receptor. These antibodies are cytostatic against cultured A431 cells at concentrations of 5-20 nM. In addition, they prevent the growth of A431 tumor xenografts in athymic mice. In the present experiments, we have attempted to improve the antitumor activity of monoclonal antibody 528 IgG2a against the EGF receptor by linking it to recombinant ricin A chain (rRA). The immunoconjugate (528 IgG-rRA) showed a potent cytotoxic effect on A431 cells in vitro. At a concentration of 10 pM, it inhibited the proliferation of cultured A431 cells by 50% and also inhibited protein synthesis in these cells by 50%. Proliferation was prevented and cell death occurred at 528 IgG-rRA concentrations of 60 pM or greater. Recombinant free ricin A chain was far less toxic. The cytotoxic effect of the immunoconjugate was neutralized by 528 IgG at concentrations 100-fold higher than 528 IgG-rRA. When the cytotoxic effect of 528 IgG-rRA was compared among several human cell lines expressing different numbers of EGF receptors, the capacity to inhibit both proliferation and protein synthesis generally correlated with the number of EGF receptors on the plasma membranes of these cells. Since 528 IgG-rRA is a very potent immunotoxin against A431 cells in culture, we designed experiments to test its in vivo antitumor activity against A431 xenografts in athymic mice. To measure the clearance of 528 IgG-rRA, 50 micrograms of immunotoxin were injected i.p. into athymic mice, blood was collected from the animals at regular intervals, and the level of immunotoxin in the serum was assayed by protein synthesis inhibition in cultured A431 cells. The blood level of active immunoconjugate reached a maximum 6 h after i.p. injection. The half-life of the absorption phase was 2.2 h, the half-life for elimination was 9.2 h, and blood levels which could be potentially cytotoxic were maintained for 48-72 h. We investigated a number of immunotoxin treatment schedules, including every other day for 4 days, based on these data. The results demonstrate that, while 528 IgG-rRA has higher in vivo antitumor activity than 528 IgG against A431 cell xenografts, this is accompanied by toxicity against the murine host.     

Antitumoral activity of a xanthate compound. I. Cytotoxicity studies with neoplastic cell lines in vitro

Xanthate derivatives were shown previously to display antitumor activity against transformed fibroblasts and lymphoma cells in combination with monocarboxylic acids [1]. Various malignant cell lines of human origin were treated in vitro to explore the range of antitumoral activity of the compounds. The combination of tricyclodecan-9-yl-xanthogenate (D 609) with undecanoic acid (C11) exerted dose dependent cytotoxic and antiproliferative effects on cell lines both from solid tumors (glioblastomas, colon-carcinomas) and hematological diseases (lymphomas, CML/BC). Additionally, the combination of D 609/C11 was able to kill both methotrexate- and adriamycin-resistant L 1210 and S 180 cells, indicating that there is no cross-resistance for these drugs and D 609/C11 in vitro.     

A new derivative of camptothecin, irinotecan hydrochloride (cpt-11) induces programmed cell-death in leukemia-lymphoma cell-lines

Irinotecan Hydrochloride (CPT-11) and 7-ethyl-10-hydroxycamptothecin (SN-38), which are both topoisomerase I inhibitors with potent antitumor effects in vivo and in vitro, were tested for the induction of programmed cell death (PCD) in leukemia/lymphoma cell lines. When the human T-cell leukemia cell line HUT-102 and the human promyelocytic leukemia cell line HL-60 cells were exposed to CPT-11, PCD characterized by a DNA fragmentation ladder of 180-200 bp in agarose gel electrophoresis and loss of cell viability was induced. The PCD inducing activity of SN-38, an active metabolite of CPT-11, was much more powerful than that of CPT-11. Besides inducing PCD in HUT-102 and HL-60 cells, SN-38 also induced PCD in the human erythroblast leukemia cell line K-562, which was resistant to CPT-11. Induction of PCD by SN-38 and CPT-11 was dose- and time-dependent. PCD in HUT-102 cells induced by SN-38 was prevented neither by aurintricarboxylic acid (ATA), an endonuclease inhibitor, as determine by DNA electrophoretic profiles and the number of viable cells, nor by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). The present data suggest that the topoisomerase I inhibitors, SN-38 and CPT-11 exert antitumor activity through induction of PCD in involved cells, at least in part. The PCD-inducing activity of the topoisomerase II inhibitor VP-16 was also tested in the above three cell lines and compared with CPT-11 and SN-38.     

In vitro evaluation of the new anticancer agents KT6149, MX-2, SM5887, menogaril, and liblomycin using cisplatin- or adriamycin-resistant human cancer cell lines

A new model to predict antitumor activity of new analogues was developed, and the cross-resistance against cisplatin (CDDP) and Adriamycin (ADM) was examined. A preclinical evaluation of various new analogues using this new model was performed. The antitumor activities of KT6149, MX-2 (KRN8602), SM5887, menogaril (TUT-7), and liblomycin (NK313) were evaluated against four non-small cell lung cancer cell lines, PC-7, -9, -13, and -14; two small cell lung cancer cell lines, H69 and N231; four CDDP-resistant cell lines, PC-7/1.0, PC-9/0.5, PC-14/1.5, and H69/0.4; a human myelogenous leukemia cell line, K562; and its ADM-resistant subline, K562/ADM by clonogenic assay. The relative antitumor activities of these new analogues were compared with those of parental agents, mitomycin C, ADM, bleomycin, and several anticancer drugs, CDDP, daunomycin, vindesine, and etoposide. KT6149 was more active than mitomycin C against all lung cancer cell lines and the human myelogenous leukemia cell line. Menogaril showed greater activity than ADM, and MX-2 showed activity similar to ADM. However, the antitumor activity of SM5887 was lower than that of ADM. SM5887 and menogaril showed cross-resistance to K562/ADM. Nevertheless, the antitumor activity against K562/ADM of MX-2 was similar to that of the parental cell lines. The activity of liblomycin was similar to that of bleomycin. Thus, KT6149 appears to be the best analogue for use in a clinical trial against lung cancer. MX-2 was active even against ADM-resistant cancer cells. The values of relative resistance to CDDP or ADM were 4.7, 8.1, 7.5, 20.0, and 13.6 for PC-7/1.0, PC-9/0.5, PC-14/1.5, H69/0.4, and K562/ADM, respectively. CDDP-resistant cell lines showed no cross-resistance with other drugs in this study. K562/ADM showed cross-resistance against daunomycin, etoposide, and vindesine. In contrast, mitomycin C and bleomycin had nearly equal activity against K562 and K562/ADM. However, K562/ADM was 2.4-fold more sensitive to CDDP than its parental cell line, K562 (P less than 0.001). These results suggested that the mechanism of CDDP resistance is different from that of multidrug resistance.     

Reversal by two dihydropyridine compounds of resistance to multiple anticancer agents in mouse P388 leukemia in vivo and in vitro

We investigated whether two representative 1,4-dihydropyridine derivatives, NK-250 and NK-252, could potentiate the antitumor activity of multiple anticancer agents including vincristine (VCR), vinblastine, vindesine and actinomycin D in drug-resistant tumor cells and their parental drug-sensitive tumor cells. NK-250 and NK-252 at 5-10 microM almost completely reversed VCR resistance in cultured VCR-resistant P388/VCR cells derived from the mouse drug-sensitive P388/S leukemia cell line and also potentiated the cytocidal activity of VCR in drug-sensitive P388/S cells. NK-250 and NK-252 at 1-10 microM inhibited the photoaffinity labeling by [3H]azidopine of the cell-surface 170,000-molecular-weight P-glycoprotein. In chemotherapeutic experiments with leukemia-bearing mice, NK-250 or NK-252 was orally administered in combination with different drugs of the MDR phenotype administered intraperitoneally. The antitumor activity of the various combinations was found to be augmented in mice bearing P388/S- and P388/VCR-leukemia. Among the combinations examined, the combination of NK-250 and VCR was the most effective. These two 1,4-dihydropyridines, NK-250 and NK-252, are unique compounds because they were effective not only in circumventing the drug resistance, but also in potentiating the action of antitumor drugs against drug-sensitive tumors.     

Anti-mesothelin immunotoxin SS1P in combination with gemcitabine results in increased activity against mesothelin-expressing tumor xenografts.

PURPOSE: To determine the antitumor activity of the anti-mesothelin immunotoxin SS1P in combination with gemcitabine against mesothelin-expressing tumor xenografts. EXPERIMENTAL DESIGN: The in vitro activity of SS1P in combination with gemcitabine against the mesothelin-expressing cell line A431/K5 was evaluated using cytotoxicity and apoptosis assays. The antitumor activity of this combination was evaluated in nude mice bearing A431/K5 tumor xenografts. Tumor-bearing mice were treated with different doses and schedules of gemcitabine alone, SS1P alone (0.2 mg/kg i.v. every other day x three doses), or with both agents together, and tumor volumes were measured over time. RESULTS: In vitro studies failed to show the synergy of SS1P plus gemcitabine against the mesothelin-expressing A431/K5 cells. In contrast, in the in vivo setting, there was a marked synergy when SS1P was combined with gemcitabine for the treatment of mesothelin-expressing tumor xenografts. This synergy was present using different doses and schedules of gemcitabine administration. In mice treated with fractionated doses of gemcitabine in combination with SS1P, complete tumor regression was observed in all mice and was long-lasting in 60% of the animals. Also, this antitumor activity was specific to SS1P because HA22, an immunotoxin targeting CD22 not expressed on A431/K5 cells, did not increase the efficacy of gemcitabine. CONCLUSIONS: SS1P in combination with gemcitabine results in marked antitumor activity against mesothelin-expressing tumors. This combination could be potentially useful for the treatment of human cancers that express mesothelin and are responsive to gemcitabine therapy.     

DH334, a beta-carboline anti-cancer drug, inhibits the CDK activity of budding yeast

The beta-carboline alkaloids present in medicinal plants, such as Peganum harmala and Eurycoma longifolia, have recently drawn attention due to their antitumor activities. Further mechanistic studies indicate that beta-carboline derivatives inhibit DNA topoisomerases and interfere with DNA synthesis. Moreover, some beta-carboline compounds are specific inhibitors of cyclin dependent kinases (CDKs). In this study we used budding yeast as a model system to investigate the antitumor mechanism of beta-carboline drugs. We found that DH334, a beta-carboline derivative, inhibits the growth of budding yeast. Strikingly, deletion of SIC1, which encodes the budding yeast CDK inhibitor, results in resistance to DH334. In contrast, yeast cells defective for Sic1 degradation exhibit morepronounced sensitivity to DH334. The presence of DH334 causes accumulation of yeast cells in G(1) phase, indicating that DH334 blocks cell cycle initiation. We further demonstrated that DH334 inhibits CDK activity as indicated by the decreased phosphorylation of a CDK substrate. All these data suggest that the inhibition of CDK contributes to the toxicity of beta-carboline derivatives to budding yeast. DH334 also inhibits the kinase activity of Cdk2/CyclinA in vitro. Therefore, we speculate that the antitumor activity of beta-carboline drugs could be attributable to their inhibition of CDK.     

Mechanism of antitumor activity in mice for anti-epidermal growth factor receptor monoclonal antibodies with different isotypes

In previous studies of monoclonal antibodies (mAbs) against the receptor for epidermal growth factor (EGF) both 528 IgG2a and 225 IgG1 were shown to inhibit growth of A431 cell xenografts in athymic mice. The antitumor activities of the two mAbs were similar and, although they differ in their isotypes, they share many properties. The two mAbs bind to EGF receptors with identical affinities, compete with EGF for binding to EGF receptors, down regulate the receptors identically, block EGF-induced activation of tyrosine protein kinase activity to a comparable degree, and block EGF-induced changes in the proliferation of cultured cells. These similarities in physiological effects permit a direct comparison of the mechanisms of action mAbs of the IgG1 and IgG2a isotypes. We examined in vitro cytotoxicity against A431 cells, using 528 IgG2a and 225 IgG1 mAbs. 528 IgG2a, but not 225 IgG1, demonstrated partial complement-mediated cytotoxicity by the 51Cr release assay and by growth inhibition of cultured A431 cells. 528 IgG2a, but not 225 IgG1, was cytotoxic to A431 cells in the presence of activated peritoneal macrophages, as demonstrated by release of incorporated [3H]thymidine. Neither mAb showed any significant cytotoxicity to A431 cells in the presence of nonadherent spleen cells which contain K-cells. The results of in vitro cytotoxicity experiments suggested that the antitumor activity of 528 IgG2a, but not 225 IgG1, could be mediated by macrophages. This was verified by in vivo experiments in which s.c. tumor cell inocula containing activated macrophages showed enhancement of antitumor effects in animals treated i.p. twice weekly for 3 weeks with suboptimal doses of 528 IgG2a. This enhancement was not observed when 225 IgG1 was used with the same procedure. The results of these experiments suggest that immune mechanisms involving activated macrophages or complement could contribute to the antitumor activity of anti-EGF receptor mAb with the IgG2a isotype, but not with the IgG1 isotype. This observation confirms the findings of others who examined the antitumor activity of IgG2a mAbs in other model systems. IgG1 mAb 225, and possibly IgG2a mAb 528, may prevent growth of human tumor xenografts by altering the physiological functions of the EGF receptor rather than by immune mechanisms.     

Effect of UCN-01, a selective inhibitor of protein kinase C,on the cell-cycle distribution of human epidermoid carcinoma,A431 cells

UCN-01 (7-hydroxy-staurosporine), a selective inhibitor of protein kinase C (PKC), was shown to exhibit antitumor activity in murine and human tumor cell lines in vitro and in vivo. On the other hand, staurosporine, a nonselective protein kinase inhibitor, was not shown to exert antitumor activity in vivo despite its potent antiproliferative activity in vitro. To compare the modes of action of UCN-01 and staurosporine in vitro, the effects of both drugs on the cell cycle progression of human epidermoid carcinoma A431 cells were examined by flow cytometry using propidium iodide (PI) staining. At 50% growth inhibitory concentrations, both UCN-01 and staurosporine induced G1 phase accumulation in the cell cycle. At 80% growth inhibitory concentrations, UCN-01 also induced preferential G1 phase accumulation, but staurosporine mostly induced G2M phase accumulation. Staurosporine also induced higher DNA ploidy when the cells were exposed to the drug for more than one generation time of A431 cells. An analysis of cell kinetics by 5-bromo-2-deoxyuridine incorporation versus DNA content confirmed that the G1 phase block by UCN-01 and the G1 and G2M phase block by staurosporine at the respective doses, as was the case for PI staining. Additionally, DNA synthesis of the cells, which was determined by the uptake of³H-TdR, was not suppressed at least 8 h after the treatment with UCN-01. These results suggested that UCN-01 could affect the G1 phase of cell cycle in A431 cells in quite different manners from staurosporine. The G1 phase block induced by UCN-01 might be important for the growth inhibitory activity of UCN-01 against A431 cells in vitro and in vivo.Abbreviations used PKC Ca²⁺ and phospholipid-dependent serine/threonine kinase - PBS phosphate buffered saline - PI propidium iodide - FITC fluorescein isothiocyanate - cPKC conventional PKC including - PKC and - nPKC neo PKC including PKC , , (L) and - IC₅₀ concentration required for 50% growth inhibition - IPA phorbol 12-myristate 13-acetate     

Fusion tyrosine kinases: a result and cause of genomic instability

Reciprocal chromosomal translocations may arise as a result of unfaithful repair of spontaneous DNA double-strand breaks, most probably induced by oxidative stress, radiation, genotoxic chemicals and/or replication stress. Genes encoding tyrosine kinases are targeted by these mechanisms resulting in the generation of chimera genes encoding fusion tyrosine kinases (FTKs). FTKs display transforming activity owing to their constitutive kinase activity causing deregulated proliferation, apoptosis, differentiation and adhesion. Moreover, FTKs are able to facilitate DNA repair, prolong activation of G(2)/M and S cell cycle checkpoints, and elevate expression of antiapoptotic protein Bcl-X(L), making malignant cells less responsive to antitumor treatment. FTKs may also stimulate the generation of reactive oxygen species and enhance spontaneous DNA damage in tumor cells. Unfortunately, FTKs compromise the fidelity of DNA repair mechanisms, which contribute to the accumulation of additional genetic abnormalities leading to the resistance to inhibitors such as imatinib mesylate and malignant progression of the disease.     

靶向表皮生长因子受体的寡肽与力达霉素强化融合蛋白的构建及其抗肿瘤活性

背景与目的：表皮生长因子受体（epidermal growth factor receptor,EGFR）在多种肿瘤细胞表面异常表达。力达霉素是从链霉菌中分离得到的具有强抗肿瘤作用的烯二炔类抗生素。本研究的目的是制备一种特异性靶向EGFR的寡肽配体与力达霉素的强化融合蛋白（Ec—LDP-AE）,并初步探讨其抗肿瘤活性。方法：制备融合蛋白（Ec-LDP）,并分离纯化,用高效液相色谱法检测Ec-LDP的纯度,ELISA、流式细胞技术以及细胞免疫荧光方法分析纯化的Ec-LDP蛋白对不同肿瘤细胞的免疫结合活性。将纯化的Ec-LDP蛋白与力达霉素发色团进行组装,构建Ec-LDP-AE,用高效液相色谱法检测350nm处的吸收峰。MTY法检测Ec—LDP-AE的体外抗肿瘤活性。结果：成功构建并表达了Ec-LDP蛋白,目的蛋白分泌至大肠杆菌周质腔中,产量为每升发酵液18mg活性蛋白,其纯度为95．3％。ELISA和细胞流式检测结果均显示Ec-LDP蛋白对EGFR高表达的肿瘤细胞系A431和MCF-7都有很强的免疫结合活性．而对不表达EGFR的NIH3T3细胞则无结合活性。免疫荧光实验也证实Ec—LDP蛋白可与A431细胞膜受体结合。Ec-LDP-AE蛋白在350nm波长处出现特定吸收峰,表明其构建成功。MTT法检测结果显示Ee-LDP—AE对体外培养的肿瘤细胞有强烈的杀伤作用,对MCF-7和A431细胞的IC。值分别为3．06×10^11mol/L和9．38×10^-13mol／L。结论：本研究制备的强化融合蛋白Ec—LDP—AE对表皮生长因子受体有靶向作用,并且保留了力达霉素的强烈细胞毒作用。     

K252a induces cell cycle arrest and apoptosis by inhibiting Cdc2 and Cdc25c.

The indole carbazole K252a has been shown in previous studies to inhibit the platelet-derived growth factor signal transduction pathway in gliomas. Because K252a has nonspecific effects on protein kinase function, we studied its effect on cyclin-dependent kinases (CDK) and cell cycle blockade in glioma cells. K252a blocked T98G cells at the G1/S and G2/M checkpoints. Consistent with cell cycle arrest, K252a was shown to hypophosphorylate Rb, upregulate p21, and decrease Cdc2 and Cdc25c activity. Finally, cell cycle arrest in T98G cells resulted in apoptosis as determined by cell morphology and DNA laddering. K252a is a useful tool for studying the effects of CDK inhibition and cell cycle blockade in tumor cells.     

In vivo antitumor activity of choline kinase inhibitors: a novel target for anticancer drug discovery.

Transformation by some oncogenes is associated with increased activity of choline kinase (ChoK), resulting in elevated constitutive levels of phosphorylcholine, a proposed second messenger required for DNA synthesis induced by growth factors. Here we describe the characterization of ChoK inhibitors with antiproliferative properties against human tumor-derived cell lines. The new molecules were tolerated in mice at doses that showed in vivo antitumor activity against human tumor xenografts derived from HT-29 and A431 cell lines implanted s.c. in nude mice. This first generation of inhibitors provides in vivo evidence that blockade of phosphorylcholine production is a valid strategy for the development of new anticancer agents, opening a new avenue for the development of antitumor drugs with a novel mechanism of action.     

CR8, a potent and selective, roscovitine-derived inhibitor of cyclin-dependent kinases

Among the ten pharmacological inhibitors of cyclin-dependent kinases (CDKs) currently in clinical trials, the purine roscovitine (CYC202, Seliciclib) is undergoing phase 2 trials against non-small-cell lung and nasopharyngeal cancers. An extensive medicinal chemistry study, designed to generate more potent analogues of roscovitine, led to the identification of an optimal substitution at the N6 position (compound CR8). An extensive selectivity study (108 kinases) highlights the exquisite selectivity of CR8 for CDK1/2/3/5/7/9. CR8 was 2- to 4-fold more potent than (R)-roscovitine at inhibiting these kinases. Cocrystal structures of (R)-CR8 and (R)-roscovitine with pCDK2/cyclin A showed that both inhibitors adopt essentially identical positions. The cellular effects of CR8 and (R)-roscovitine were investigated in human neuroblastoma SH-SY5Y cells. CR8 inhibited the phosphorylation of CDK1 and 9 substrates, with a 25-50 times higher potency compared to (R)-roscovitine. CR8 was consistently more potent than (R)-roscovitine at inducing apoptotic cell death parameters: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reduction (40-fold), lactate dehydrogenase release (35-fold), caspases activation (68-fold) and poly-(ADP-ribose)polymerase cleavage (50-fold). This improved cell death-inducing activity of CR8 over (R)-roscovitine was observed in 25 different cell lines. Altogether these results show that second-generation analogues of (R)-roscovitine can be designed with improved antitumor potential.     

Tumor growth suppression by alpha-eleostearic acid, a linolenic acid isomer with a conjugated triene system, via lipid peroxidation

We have previously shown that conjugated linolenic acids (CLnA) prepared by alkaline isomerization have a stronger antitumor effect than conjugated linoleic acids (CLA). In this study we have compared the suppressive effect on tumor growth of alpha-eleostearic acid (alpha-ESA, 9Z11E13E-18:3) with those of the CLA isomers 9Z11E-CLA and 10E12Z-CLA, using nude mice into which DLD-1 human colon cancer cells were transplanted. The results showed that alpha-ESA, which is a CLnA that can be prepared from natural sources in bulk, had a stronger antitumor effect than CLA. DNA fragmentation was enhanced and lipid peroxidation was increased in tumor tissues of the alpha-ESA-fed mice, which suggested that alpha-ESA induced apoptosis via lipid peroxidation. Furthermore, treatment of DLD-1 cells with alpha-ESA, 9Z11E-CLA and 10E12Z-CLA confirmed that alpha-ESA had a stronger antitumor effect than CLA in cultured cell lines. The induction of apoptosis by alpha-ESA was consistent with enhanced DNA fragmentation, increased caspase activity and increased expression of caspase mRNA following alpha-ESA treatment. Addition of alpha-tocopherol, an antioxidant, suppressed oxidative stress and apoptosis, suggesting that these effects were associated with lipid peroxidation.     

紫草素及其衍生物的抗肿瘤作用

紫草为传统的中药,紫草的化学 成份复杂,具有广泛的药理作用,抗肿瘤 作用是其中之一。天然紫草素类化合物 由于抗瘤作用弱,仍未用于临床。为了减 少它们的毒副反应和增强抗肿瘤作用,人 们半合成了许多紫草素类衍生物。实验 证明,天然紫草素类化合物及其衍生物具 有不同程度的细胞毒作用和抗肿瘤作用。 初步的作用机制研究表明,紫草素类化合 物及其衍生物能诱导肿瘤细胞凋亡,激活 促细胞分裂原激活蛋白激酶(mitogen acti vatedproteinkinase,MAPK),抑制蛋白酪 氨酸激酶和DNA拓扑异构酶I的活性,从 而影响肿瘤细胞的代谢、增殖、分化、信号 传递、基因表达等过程,阻碍肿瘤细胞的 生长。