The HDAC inhibitor GCJ-490A suppresses c-Met expression through IKKα and overcomes gefitinib resistance in non-small cell lung cancer

Objective: The novel compound GCJ-490A has been discovered as a pan-histone deacetylase(HDAC) inhibitor that exerts potent inhibitory activity against HDAC1, HDAC3, and HDAC6. Because of the important roles of HDACs in lung cancer development and the high distribution of GCJ-490A in lung tissue, we explored the anti-tumor potency of GCJ-490A against non-small cell lung cancer(NSCLC) in vitro and in vivo in this study.Methods: The in vitro effects of GCJ-490A alone or combined with the EGFR inhib...     

THE BIOLOGICAL CHARACTERISTICS OF ADENOVIRUS-MEDIATED IL-18 GENE-MODIFIED MURINE COLORECTAL ADENOCARCINOMA CELL IN VIVO AND IN VITRO

Interleukin 18 (IL-18) has multiple biological activities, such as promoting the generation of Th1 cytokines and GM-CSF, activating NK cells and CTL, which contributes to its anti-tumor activity.[1(5] So IL-18 might have promising application in the immunotherapy and gene therapy of cancer. To confirm the anti-tumor activity of IL-18, we constructed the recombinant adenovirus encoding IL-18 gene, observed preliminarily the biological characteristics of IL-18-modified murine colorectal …     

A phase Ⅰ study of Hemay022, an irreversible dual EGFR/HER2 tyrosine kinase inhibitor in Chinese patients with HER2-positive advanced breast cancer

Objective: Hemay022 is a novel small-molecule and an irreversible tyrosine kinase inhibitor with the target of epidermal growth factor receptor(EGFR)/human epidermal growth factor receptor 2(HER2), which demonstrated anti-tumor activity in preclinical studies. This first-in-human study evaluated the safety, pharmacokinetics,tolerability and preliminary anti-tumor activity of Hemay022 in HER2-positive advanced breast cancer patients.Methods: Heavily pretreated patients with HER2-positive advanced...     

Two-Dimensional （Polyacrylamide） Gel Electrophoresis Analysis of Apoptosis Induced by Harringtonine in K562 Cells

OBJECTIVE The anti-tumor drug, harringtonine (HT), has been extensively used with satisfactory results in the treatment of acute or chronic myeloid leukemia. Previous studies have shown that the anti-tumor activity of the drug is related to induced apoptosis of tumor cells, but the molecular mechanism still remains unclear. The main purpose of this research was to analyze the protein profiles formed during HT-induced apoptosis in K562 cells and to screen the apoptotic-related proteins.METHODS Annexin V and Pi double staining was used in combination with flow cytometry to examine the early and the late stages of HT-induced apoptosis in K562 cells. In addition two-dimensional gel electrophoresis and computer-assisted image analysis were employed to separate and compare the HT-induced apoptotic proteins of the K562 cells and the controls.RESULTS When a concentration of 10 μg/ml HT was used to treat K562 cells,the percentage of the early-apoptotic cells (Annexin V /PI-) was found to be 28.3% and 18.1% at 5 and 24 h, respectively (P＜0.01), while the rate of lateapoptotic cells (Annexin V /PI ) was at a level of 9.1% and 20.2%,respectively (P＜0.01). Matching analysis of the proteome among the control group and the early- and late-apoptotic groups showed 1,300 ± 50 protein spots which were identified in the control K562 cells with a matching rate of 88.3 ± 2.0 % for the protein spots in the two treated groups. Ten protein spots showed overt and steady changes in both quality and quantity in the cells of the late-apoptotic group (P＜0.01), among which the level of expression for eight of the ten protein spots was up-regulated after apoptosis, one was down-regulated and one was merely expressed as in the control cells.CONCLUSION The proteins with differential expression might be important proteins involved in the process of apoptosis in K562 cells induced by HT.     

Activity of Angiogenesis Inhibitors in Metastatic Epithelioid Hemangioendothelioma: A Case Report

This report describes a patient with metastatic epithelioid hemangioendothelioma treated with bevacizumab and nanoparticle albumin-bound paclitaxel.The treatment was well tolerated and led to the stabilization of an aggressive variant of the disease. This case report is the first one that describes the activity of the combination of chemotherapy and bevacizumab in epithelioid hemangioendothelioma.Literature describing the activity of bevacizumab and other agents(thalidomide,lenalidomide,and interferon) believed to possess anti-angiogenic activities is also reviewed.     

Anti-tumor Activities of Novel Estrogen Compound 17aα-D-Homo-Ethynylestradiol-3-Acetate

Objective:To study the anti-tumor activities of novel estrogen compound 17a α-D-homo-ethvnvlestradiol-3-acetate in vitro and in vivo. Methods:In vitro anti-tumor activity was assayed in adenoma cells A549 and human liver cancer cells Bel-7402 using MTT method,and half-inhibitory concentration (IC50)were observed. In vivo the pulmonary adenoma LA795 cells was selected and the conventional assay method of anti-tumor activity was employed.5,7.5,10 mg/kg of 17a α-D-homo-ethynylestradiol-3-acetate was administered by i.P., and tumor-inhibitory rate, thymus and spleen indexes,bone marrow cells(BMC)were observed. Results:IC50 of 17a α-D-homo-ethynylestradiol-3-acetate in vitro for A549 and Bel-7402 cells were 12.28 μg/ml and 17.79 μg/ml, respectively.In vivo the highest tumor-inhibitory rates for LA795 was 60.0%(P＜0.01).The drug had hardly any side-effect in spleen indexes,thymus indexes,and BMC compared with control mice. Nevertheless,compared with the positive control drug cyclophosphamide(CY),thymus and spleen indexes,BMC showed obvious diffefences(P＜0.01). Conclusion:17a α-D-homo-ethynylestradiol-3-acetate has obvious anti-tumor activities in vitro and in vivo with low side-effect, thus worth further investigation.     

Anti-tumor Activities of Novel Estrogen Compound 17a α-D-Homo-Ethynylestradiol-3-Acetate

Objective： To study the anti-tumor activities of novel estrogen compound 17a α-D-homo-ethynylestradiol-3-acetate in vitro and in vivo. Methods： In vitro anti-tumor activity was assayed in adenoma cells A549 and human liver cancer cells Bel-7402 using MTT method, and half-inhibitory concentration （IC50） were observed. In vivo the pulmonary adenoma LA795 cells was selected and the conventional assay method of anti-tumor activity was employed. 5, 7.5, 10 mg/kg of 17a α-D-homo-ethynylestradiol-3-acetate was administered by i.p., and tumor-inhibitory rate, thymus and spleen indexes, bone marrow cells （BMC） were observed. Results： IC50 of 17a α-D-homo-ethynylestradiol-3-acetate in vitro for A549 and Bel-7402 cells were 12.28 μg/ml and 17.79 μg/ml, respectively. In vivo the highest tumor-inhibitory rates for LA795 was 60.0% （P〈0.01）. The drug had hardly any side-effect in spleen indexes, thymus indexes, and BMC compared with control mice. Nevertheless, compared with the positive control drug cyclophosphamide （CY）, thymus and spleen indexes, BMC showed obvious differences （P〈0.01）. Conclusion： 17a α-D-homo-ethynylestradiol-3-acetate has obvious anti-tumor activities in vitro and in vivo with low side-effect, thus worth further investigation.     

ANTI-HUMAN LUNG GIANT CELL CANCER (PG) EFFECT OF HUMAN LAK CELLS IN VITRO AND IN NUDE MICE

Human LAK cells were prepared by culturing normal human peripheral blood mononuclear cells (PBMC) with or without rIL-2 and assayed for T cell surface markers as well as anti-tumor activity against PC in vitro and in nude mice. Although the percentages of T3, T4, and T8 positive cells in rIL-2-activated cells did not differ significantly from those of control cells in vitro, the former showed stronger cytotoxicity than control cells to PG tumor cells in vitro. In vivo, LAK cells completely inhibited the growth of PG tumor in nude mice, whereas PBMC control cells were to be of no effect. The anti-tumor effect of human LAK cells in nude mice may offer a useful model to study the role of human LAK cells against human tumor in vivo.     

Telomerase activity during 7,12-dimethylbenz [a] anthracene-induced hamster buccal pouch carcinogenesis

Telomerase is a ribonucleoprotein complex intimately involved in cell immortalization and carcinogenesis. Increasing evidence shows that telomerase activity has been detected in human tumor cells but not in normal somatic tissues.[1] Thus, anhypothesis has been proposed that the activation of telomerase activity is essential for cells to overcome senescence and also may result in the immortality and malignant progression of a cancer.[2] Recently, comparatively low telomerase activity has been …     

Studies on the pharmacodynamics and pharmacokinetics of paclitaxel(Zisu®)

Pharmacological studies demonstrated that paclitaxel (Zisu®) was very active in the inhibition of the growth of human cancer cell panel including KB cells, HCT-8, A2780, andMCF-7 cells. The IC₅₀ was as low as 0.0019, 0.0019, 0.0036 and 0.01 µ g/ml respectively. Experimental therapeutic studies indicated that paclitaxel(Zisu®) significantly inhibited the growth of melanoma B-16, Walker carcinomsarcoma and heterotransplanted human ovarian cancer in nude mice. Biochemical pharmacological studies showed that paclitaxel (Zisu®) could accelerate microtubule assembly and inhibit its deassembly; population in G₁ was decreased while the cell population in G₂+M phase was increased significantly. In addition, a polyploid cell population appeared. Pharmacokinetic studies demonstrated that the $t_{{1 \mathord{\left/ {\vphantom {1 {2\alpha }}} \right. \kern-0em} {2\alpha }}} $ was 0.12 h and $t_{{1 \mathord{\left/ {\vphantom {1 {2\beta }}} \right. \kern-0em} {2\beta }}} $ was 5.02 h when it was injected intravenously at a dose of 5 mg/kg in rats. The AUC, Vc and CLs were 11.82(µg.h)/ml, 0.50L/kg and 0.42L(h.kg) respectively.     

组蛋白去乙酰化酶抑制剂SAHA与紫杉醇联用诱导宫颈癌HeLa细胞凋亡的实验

目的 探讨组蛋白乙酰化抑制剂SAHA联合紫杉醇体外对宫颈癌HeLa细胞增殖抑制效果及其机制。方法 分别设置空白对照组SAHA（10 μmol/L）组、紫杉醇（10 nmol/L）组、SAHA（10 μmol/L）+紫杉醇（10 nmol/L）联合组,采用MTT法检测各组HeLa细胞的抑制率,采用流式细胞术检测HeLa细胞凋亡情况和细胞周期。结果 MTT结果显示经SAHA组、紫杉醇组和联合组分别处理24、48 h后,与SAHA和紫杉醇组比较,联合组能够显著抑制HeLa细胞的增殖,差异有统计学意义,且二者具有叠加作用（Q=0.861, Q=1.25）; HeLa细胞的凋亡实验结果显示联合组的凋亡率分别高于紫杉醇组、SAHA组;HeLa细胞周期实验发现：联合组处理后的HeLa细胞主要处于G0/G1期,提示SAHA与紫杉醇联合使用能够抑制HeLa细胞有丝分裂过程中的DNA合成和复制。结论 SAHA与紫衫醇联用能够抑制宫颈癌HeLa细胞增殖,增强诱导HeLa细胞凋亡的能力,阻滞细胞周期,最终增强抗肿瘤的能力。     

Bradykinin antagonist dimer, CU201, inhibits the growth of human lung cancer cell lines in vitro and in vivo and produces synergistic growth inhibition in combination with other antitumor agents.

Small cell lung cancers (SCLCs), many non-SCLCs, and other cancers have neuroendocrine features, including paracrineand autocrine growth stimulation by various neuropeptides. Interference with this pathway is an attractive target for novel therapies. We developed a novel bradykinin antagonist dimer, CU201 (B9870), that acts as a "biased agonist" for neuropeptides by blocking G(alphaq) signaling and activating G(alpha12,13) signaling. CU201 induced apoptosis and complete growth inhibition in various lung cancer and other cancer cell lines. CU201 was 10-fold more potent than substance P derivatives and was stable in serum for >7 days. In this study, we evaluated the ability of CU201 to produce additive or synergistic growth inhibition in combination with various antitumor agents used in lung cancer therapy. We found that CU201 produced additive or synergistic growth inhibition when combined with doxorubicin, etoposide, cisplatin, vinorelbine, and paclitaxel for SCLC lines and with paclitaxel and ZD1839, an epidermal growth factor receptor tyrosine kinase inhibitor, for non-SCLC cell lines. Pharmacokinetic parameters associated with the i.v. administration of CU201 were evaluated in normal mice, and the effects of CU201 on the growth of human lung cancer xenografts were evaluated in athymic nude mice. In CD2F1 mice given an i.v. bolus infusion of 5 mg/kg, the c(max) was 5773 ng/ml (5 microM), and the decay was biexponential. When fitted to a two-compartment model, the t(1/2alpha) was 14.4 min, and the t(1/2beta) was 44.3 h, indicating a long terminal half-life consistent with the prolonged in vitro effects. CU201 inhibited the growth of human lung cancers in athymic nude mice by the intratumoral, s.c., and i.p. routes at a dose of 5 mg/kg/day. This dose is >10-fold less than the dose of substance P derivatives used to inhibit SCLC xenografts in nude mice. We conclude that CU201 should undergo further preclinical toxicology studies in its development as a novel targeted therapy for the treatment of lung cancers with neuroendocrine features. These studies are in progress through the NCI RAID mechanism.     

紫杉醇对不同p53基因型肺癌细胞作用的研究

目的:观察紫杉醇对3种不同p53基因型肺癌细胞的作用。方法:以不同浓度紫杉醇(0.1、1.0、10、100、1 000nmol/L)分别作用肺癌细胞A549、H322、H1299不同时间(4、12、24、48、96 h),实验同时设阴性对照组(不加紫杉醇)和空白对照组(只加细胞培养液)。采用MTT、流式细胞术、Western blotting检测细胞的生长及细胞周期、凋亡、乙酰化微管蛋白、p53蛋白等指标变化。结果:紫杉醇对3株肺癌细胞的生长抑制作用均随着作用时间的延长而增强(P0.05);不同浓度紫杉醇对3株细胞生长的影响也存在差异(P0.05)。3株细胞中A549细胞半数抑制浓度相对较低,但3株细胞间对紫杉醇敏感性的差异无统计学意义(P0.05)。紫杉醇10 nmol/L作用时,3株细胞G2/M期变化趋势基本一致,1 000 nmol/L作用时,A549细胞G2/M期比例与作用时间呈正相关(P0.05),H322和H1299的G2/M细胞于24 h达高峰,作用后期H1299出现异常多倍体;同浓度紫杉醇作用的各株肺癌细胞的凋亡呈时间依赖性,但1 000 nmol/L组诱导的细胞凋亡比10 nmol/L组更晚出现。浓度依赖性实验中,10 nmol/L诱导凋亡比例最高;紫杉醇浓度≤100 nmol/L时,G2/M期比例随浓度增加而增高(P0.05);紫杉醇浓度≥100 nmol/L时,各肺癌细胞株G2/M期比例间的差异无统计学意义。凋亡和G2/M期阻滞无相关性(P0.05)。紫杉醇作用后,A549细胞p53蛋白呈浓度和时间依赖性增加,H322细胞p53蛋白无明显变化。3株细胞乙酰化微管蛋白均较对照组显著上升(P0.05)。结论:紫杉醇对不同p53基因型肺癌细胞生长的抑制作用呈时间依赖性。紫杉醇可增加野生型p53蛋白表达,诱导p53依赖和非p53依赖的两种凋亡途径,p53基因型影响了高浓度紫杉醇对肺癌细胞的周期阻滞作用,但对紫杉醇化疗敏感性无显著影响。     

Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin increases chemosensitivity of CaSki cells to paclitaxel

Paclitaxel, a potent anti-neoplastic agent, has been found to be effective against several tumours, including cervical cancer. However, the exact mechanism underlying the cytotoxic effects of pacitaxel, especially in the survival-signalling pathway, is poorly understood. The aim of this study was to investigate the molecular pathway of the cytotoxic effect of paclitaxel in human cervical cancer cell lines. Four human cervical cancer cell lines were treated for 24 h with various concentration of paclitaxel, and the sensitivity was analysed by an MTT assay. The cell cycle progression and sub-G1 population were analysed by flow cytometry. Apoptosis was further measured by DNA fragmentation and microscope examination. The protein expression was determined by Western blot analysis. Our results showed that HeLa cells demonstrated the highest sensitivity to paclitaxel, whereas CaSki cells showed the lowest. In cervical cancer cells, paclitaxel induced apoptosis through an intrinsic pathway with prior G2/M arrest. In addition, we showed that paclitaxel downregulated the phosphorylation of Akt in both HeLa and CaSki cells. Interestingly, in CaSki cells, which were more suggestive of a resistant phenotype, paclitaxel induced the activation of mTOR as a downstream target of Akt. Pre-treatment with rapamycin inhibited activation of mTOR signalling and significantly enhanced the sensitivity of CaSki cells to paclitaxel by increasing apoptotic cell death. This effect was mediated, at least partly, through caspase activation. Overall, paclitaxel exerts its anti-tumour effects on cervical cancer cells by inducing apoptosis through intrinsic pathway, and rapamycin targeted to mTOR can sensitise paclitaxel-resistant cervical cancer cells.     

Clinical pharmacokinetic and in vitro combination studies of nolatrexed dihydrochloride (AG337, Thymitaq) and paclitaxel

A clinical study of nolatrexed dihydrochloride (AG337, Thymitaq) in combination with paclitaxel was performed. The aims were to optimize the schedule of administration and determine any pharmacokinetic (PK) interactions between the two drugs. In vitro combination studies were performed to assist with schedule optimization. Three patients were entered on each of three different schedules of administration of the two drugs: (1) paclitaxel 0-3 h, nolatrexed 24-144 h; (2) nolatrexed 0-120 h, paclitaxel 48-51 h; (3) nolatrexed 0-120 h, paclitaxel 126-129 h. Paclitaxel was administered at a dose of 80 mg m(-2) over 3 h and nolatrexed at a dose of 500 mg m(-2) day(-1) as a 120-h continuous intravenous infusion. Plasma concentrations of both drugs were determined by high performance liquid chromatography. In vitro growth inhibition studies using corresponding schedules were performed using two head and neck cancer cell lines. In both HNX14C and HNX22B cell lines, synergistic growth inhibition was observed on schedule 2, whereas schedules 1 and 3 demonstrated antagonistic effects. In the clinical study, there was no effect of schedule on the pharmacokinetics of nolatrexed. However, patients on schedules 1 and 3 had a higher clearance of paclitaxel (322-520 ml min(-1) m(-2)) than those on schedule 2 (165-238 ml min(-1) m(-2)). Peak plasma concentrations (1.66-1.93 vs. 0.86-1.32 microM) and areas under the curve (392-565 vs. 180-291 microM min(-1)) of paclitaxel were correspondingly higher on schedule 2. The pharmacokinetic interaction was confirmed by studies with human liver microsomes, nolatrexed being an inhibitor of the major routes of metabolism of paclitaxel. Toxicity was not schedule-dependent. Nolatrexed and paclitaxel may be safely given together when administered sequentially at the doses used in this study. Studies in vitro suggest some synergy, however, due to a pharmacokinetic interaction, paclitaxel doses should be reduced when administered during nolatrexed infusion.     

GW843682X增加肺癌A549细胞对紫杉醇化疗敏感性的体外研究

[目的]研究GW843682X对肺癌A549细胞化疗敏感性的影响。[方法]培养肺癌A549细胞,均等浓度接种于6孔板,分四组：1空白对照组;2紫杉醇处理组：48μg/ml紫杉醇处理48h;3GW843682X处理组：0.5μmol/L GW843682X处理48h;4联合用药组：48μg/ml紫杉醇联合0.5μmol/L GW843682X处理48h。倒置显微镜下观察细胞形态的变化。流式细胞仪检测细胞凋亡率的变化。RT-PCR检测plk1在mRNA水平的变化。Western blot检测plk1在蛋白质水平的变化。[结果]紫杉醇或GW843682X均能够抑制A549细胞的增殖,倒置显微镜下发现细胞形态变化明显,联合用药组变化更明显。流式细胞仪检测结果发现各组细胞凋亡率分别是2.5%,9.4%,5.8%和22.1%。RT-PCR结果显示紫杉醇或GW843682X均能下调plk1mRNA表达水平,联合用药组下调最明显。Western blot结果显示紫杉醇或GW843682X抑制了plk1蛋白表达水平,联合用药组下调最明显。[结论]GW843682X能够通过抑制plk1的表达增加肺癌A549细胞对紫杉醇的化疗敏感性。     

紫杉醇对p53缺失型肺癌细胞株H1299的生长抑制作用

背景与目的:评价紫杉醇对p53缺失型肺癌细胞株H1299的生长抑制作用.材料与方法:分别以体外药物敏感试验(Mir法)、流式细胞术观察紫杉醇不同作用浓度、不同作用时间处理H1299细胞后,其生长抑制情况以及细胞周期、细胞凋亡等指标的变化,并以荧光显微镜观察细胞核形态的改变.结果:紫杉醇对H1299细胞毒性呈时间依赖性(P＜0.05);在紫杉醇1 nmol/L以上各浓度组,随着紫杉醇浓度的增加,H1299细胞的存活率降低,与对照组间差异具有统计学意义(P＜0.05);各实验组H1299细胞的生长被阻滞于Gz/M期,在0.1～1 000 nmol/L浓度范围内呈浓度依赖性(P＜0.05),其细胞凋亡比例在0.1～10 nmol/L浓度范围内随浓度的增加而增高(P＜0.05);在时间依赖性试验中,10 nmol/L作用时,G2/M期的阻滞于12 h达高峰;1 000 nmol/L作用时于24 h达高峰,延长1 000 nmol/L作用时间,H1299出现异常多倍体现象;凋亡发生呈时间依赖性,但高浓度诱导的凋亡较低浓度出现的时间晚.紫杉醇诱导的凋亡和G2/M期阻滞无相关性(P＞0.05).荧光显微镜下可见细胞凋亡与细胞死亡共存现象.结论:紫杉醇可以同时诱导H1299细胞凋亡和死亡,对其生长抑制作用呈时间和浓度依赖性.     

小檗胺对胃癌细胞增殖、凋亡的影响及其分子机制

目的:探讨小檗胺对胃癌细胞（AGS和SGC-7901）增殖、凋亡的影响并探讨其分子机制。方法:利用MTT 实验检测胃癌细胞的增殖,利用集落形成实验检测胃癌细胞的生长, 流式细胞技术检测胃癌细胞的凋亡率以及细胞周期, Western blot检测凋亡相关蛋白的表达水平。结果:小檗胺（0~64 μg/ml）能够剂量依赖性以及时间依赖性的抑制 AGS和SGC-7901细胞的增殖 （P＜0.05）。集落形成实验结果显示小檗胺(32 μg/ml)能够抑制AGS和SGC-7901细胞的生长 （P＜0.05）。流式细胞实验显示,小檗胺(32 μg/ml)同时能够促进AGS和SGC-7901细胞的凋亡 （P＜0.05）,增殖G0/G1期细胞比例,降低S期细胞比例 （P＜0.05）。Western blot实验结果显示小檗胺(32 μg/ml)能够增加AGS和SGC-7901细胞的cleaved caspase-3,cleaved caspase-9以及Bax的蛋白水平,同时降低Bcl-2的蛋白水平 （P＜0.05）。结论:小檗胺能够抑制胃癌细胞的增殖,其机制可能与改变细胞周期以及促进细胞凋亡有关。     

The in vivo effect of bryostatin-1 on paclitaxel-induced tumor growth, mitotic entry, and blood flow.

Pretreatment of tumor cells with the protein kinase C (PKC) inhibitor bryostatin-1 enhances the cytotoxicity of most chemotherapeutic agents. However, in the case of paclitaxel, this effect has been shown in vitro to be best achieved when bryostatin-1 follows (rather than precedes) paclitaxel treatment. With combination trials of bryostatin-1 and paclitaxel planned for clinical trials and with only in vitro data available regarding drug sequence, we elected to undertake an in vivo study evaluating the effect of sequential bryostatin-1 and paclitaxel in a tumor-bearing mouse model and to correlate this effect to cell cycle events, tumor metabolism, and tumor blood flow. At the maximum tolerated i.p. dose, bryostatin-1 at 80 microg/kg resulted in a small but significant increase in tumor doubling time (4.2 +/- 0.3 days) compared with control tumors (3.0 +/- 0.3 days; P < 0.01). Mice treated with i.v. paclitaxel, administered at a dose of 12 mg/kg every 12 h for three doses, weekly for 3 weeks, had a tumor doubling time of 23.4 +/- 1.7 days. Mice pretreated with i.p. bryostatin-1 (80 microg/kg) followed 12 h later by i.v. paclitaxel (12 mg/kg every 12h for three doses) weekly for 3 weeks had a tumor doubling time of 9.7 +/- 1.1 days. This was significantly less (P < .001) than paclitaxel alone, which indicated an inhibitory effect by bryostatin-1 on paclitaxel therapy. In comparison, tumor-bearing mice that were treated with the same dose but with the sequence of paclitaxel followed by bryostatin-1 had a tumor doubling time of 29.6 +/- 0.6 days. This was significantly greater than the tumor doubling times for any condition tested (P < 0.01), demonstrating the sequence dependence of this combination. The efficacy of paclitaxel is dependent on mitotic entry, a step that requires activation of p34cdc2 kinase activity. Treatment with paclitaxel in vivo increased p34 cdc2 kinase activity in the mouse mammary tumors, whereas administration of bryostatin-1 before paclitaxel prevented the p34cdc2 kinase activation by paclitaxel. This was further evaluated in vitro by flow cytometry in MKN-74 human gastric cancer cells. As determined by MPM-2 labeling, which identifies cells in mitosis, pretreatment with bryostatin-1 prevented paclitaxel-treated cells from entering mitosis. Bryostatin-1 has been reported to induce changes in muscle metabolism and to decrease muscle blood flow. These events could impact on the interaction of bryostatin-1 with paclitaxel. Using proton-decoupled phosphorus nuclear magnetic resonance (31P-NMR) spectroscopy in vivo, bryostatin-1 at 80 micro1g/kg induced a decrease in both intratumoral pH and high-energy phosphates. In vivo perfusion studies, using dynamic enhanced NMR imaging with gadolinium diethylenetriamine pentaacetic acid, also demonstrated decreased tumor blood flow. These studies suggest that the inhibition of tumor response to paclitaxel by bryostatin-1 is multifactorial and includes such diverse factors as inhibition of cell entry into mitosis, a decrease in pH and energy metabolism, and a decrease in tumor blood flow. These results indicate that, as this combination enters Phase I clinical trials, the sequence of paclitaxel followed by bryostatin-1 will be critical in the clinical trial design.