Manumycin抗人肝癌细胞HepG2裸鼠移植瘤的作用及其对肿瘤血管生成的影响

背景与目的:法尼基转移酶抑制剂manumycin体内外抗胰腺癌、结肠癌和退行性甲状腺癌的研究已见报道。我们先前的体外研究发现,manumycin能抑制人肝癌HepG2细胞的增殖通路和生存通路。本研究拟探讨manumycin在裸小鼠体内的抗肝癌作用及其对肿瘤的血管生成的影响。方法:建立人肝癌HepG2细胞裸鼠移植瘤模型,接种后第8天按肿瘤大小随机分为5组:生理盐水对照组(20ml/kg)、阳性药对照组(CTX,25mg/kg)、0.1%Me2SO阴性对照组(20ml/kg)、manumycin低剂量组(2.5mg/kg)和高剂量组(5mg/kg)。测量各组肿瘤体积观察manumycin的体内抗瘤作用。采用免疫组化方法观察移植瘤的微血管密度(MVD),应用Westernblot法检测VEGF和b鄄FGF的蛋白水平变化,RT鄄PCR法检测VEGFmRNA的水平。结果:manumycin低剂量组和高剂量组的平均肿瘤体积比(V/V0)分别为0.68±0.09和0.59±0.04(n=5),与0.1%Me2SO阴性对照组(V/V0为1.38±0.21)相比,manumycin处理组肿瘤体积明显缩小(P<0.01)。与对照组相比较,manumycin处理组肿瘤组织MVD明显降低(P<0.01)。manumycin能明显抑制HepG2细胞及HepG2细胞裸鼠移植瘤VEGF蛋白的表达,而对b鄄FGF蛋白的表达影响不大。manumycin也明显抑制了HepG2细胞裸小鼠移植瘤VEGFmRNA的表达。结论:manumycin能明显抑制人肝癌HepG2细胞裸小鼠移植瘤的生长。下调VEGF表达,抑制肿瘤的血管生成可能是manumycin抗肿瘤作用的重要机理之一。     

Modified radiosensitivity of pancreatic cancer xenografts by farnesyl protein transferase inhibitor and MEK inhibitor

We investigated the effects of the farnesyl transferase inhibitor (FTI) manumycin and the MEK inhibitor PD98059 on growth of human pancreatic cancer, with mutant (SUIT2) or wild-type (BxPC-3) K-ras, xenografted into nude mice. Tumor growth was not reduced by either of the agents at a dose of 3 mg/kg without irradiation. Growth of SUIT2 irradiated at 15 Gy or 30 Gy was reduced by manumycin and PD98059: at 15 Gy, tumor volume doubling time (TVDT) increased from 18.6+/-3.8 to 36.3+/-14.2 days with PD98059 (p<0.05); at 30 Gy, TVDT increased from 32.8+/-6.8 to 70.5+/-10.5 days and 70.7+/-1.5 days, respectively. Manumycin tended to reduce growth of BxPC-3, but the difference in TVDT was not statistically significant. PD98059 significantly increased the TVDT of BxPC-3 at 30 Gy from 34.4+/-18 to 62.6+/-9.8 at 30 Gy. The present results suggest that Ras signaling pathways are potential targets for manipulation of radiosensitivity, and that induction of an alternative pathway may enhance radiosensitivity of pancreatic cancer.     

Manumycin inhibits ras signal transduction pathway and induces apoptosis in COLO320-DM human colon tumour cells

The aim of the present study was to assess the cytotoxicity of manumycin, a specific inhibitor of farnesyl:protein transferase, as well as its effects on protein isoprenylation and kinase-dependent signal transduction in COLO320-DM human colon adenocarcinoma which harbours a wild-type K-ras gene. Immunoblot analysis of isolated cell membranes and total cellular lysates of COLO320-DM cells demonstrated that manumycin dose-dependently reduced p21 ras farnesylation with a 50% inhibitory concentration (IC50) of 2.51 +/- 0.11 microM and 2.68 +/- 0.20 microM, respectively, while the geranylgeranylation of p21 rhoA and p21rap1 was not affected. Manumycin dose-dependently inhibited (IC50 = 2.40 +/- 0.67 microM) the phosphorylation of the mitogen-activated protein kinase/extracellular-regulated kinase 2 (p42MAPK/ERK2), the main cytoplasmic effector of p21ras, as well as COLO320-DM cell growth (IC50 = 3.58 +/- 0.27 microM) without affecting the biosynthesis of cholesterol. Mevalonic acid (MVA, 100 microM), a substrate of the isoprenoid synthesis, was unable to protect COLO320-DM cells from manumycin cytotoxicity. Finally, manumycin 1-25 microM for 24-72 h induced oligonucleosomal fragmentation in a dose- and time-dependent manner and MVA did not protect COLO320-DM cells from undergoing DNA cleavage. The present findings indicate that the inhibition of p21ras processing and signal transduction by manumycin is associated with marked inhibition of cell proliferation and apoptosis in colon cancer cells and the effect on cell growth does not require the presence of a mutated ras gene for maximal expression of chemotherapeutic activity.     

Manunycin对人肝癌HepG2细胞的生长抑制作用与Ras通路的关系

背景与目的:目前肝癌药物治疗的临床疗效还不尽人意。在肝癌的发生发展过程中,Ras起着重要的作用。Ras必须在法尼基转移酶的作用下,经过法尼基化修饰才具有生物活性。我们观察了法尼基转移酶抑制剂Manumycin对人肝癌HepG2细胞株生长的抑制作用,并探讨其对Ras通路的影响。方法:应用氚标胸腺嘧啶脱氧核苷掺入实验观察法尼基转移酶抑制剂Manumycin对人肝癌HepG2细胞株生长的抑制作用,Westernblot技术检测Manumycin对HepG2细胞Ras通路相关蛋白,包括pan-Ras、N-Ras、membrane-pan-Ras、ERK1/2、p-ERK1/2、AKT、p-AKT及MKP-1表达的影响。结果:Manumycin(5、10、20、40、80μmol/L)处理24h对肝癌HepG2细胞株生长具有明显的抑制作用,并呈浓度依赖性,其IC50为(17.1±2.6)μmol/L。同时Westernblot分析显示,Manumycin对肝癌HepG2细胞pan-Ras蛋白和N-Ras蛋白及细胞膜pan-Ras蛋白表达均有抑制作用,且对细胞膜pan-Ras蛋白的抑制作用更明显。进一步研究发现Manumycin对Ras蛋白下游ERK1/2和AKT活性也有抑制作用,表现为ERK1/2和AKT磷酸化水平降低。Manumycin对AKT活性的影响与PI3K抑制剂Wortmannin作用相似,两者联用可加强对AKT的抑制。此外,Manumycin诱导MKP-1的表达具有浓度依赖性。结论:Manumycin抑制肝癌HepG2细胞株     

Combined therapy of xenografts of human pancreatic carcinomas with rTNF-alpha and mitomycin C

Based on previously published experimental studies in nude mice indicating that rTNF-alpha as well as mitomycin C are able to induce significant growth inhibition of xenotransplants of gastrointestinal and pancreatic carcinomas we have investigated the antitumor effects of rTNF-alpha (0.8 mg/kg daily for 21 days) and mitomycin C (2.4 mg/kg once weekly, day 1, 7, 14) as single drugs and in combination (0.8 + 2.4) in nude mice bearing xenografts of 4 human pancreatic carcinomas and 1 colorectal cancer. Serum CA 19-9 was measured additionally to tumor growth rate. The results demonstrate that combined treatment is more effective compared to rTNF-alpha and mitomycin C alone. Combined therapy resulted in a significant inhibition of tumor growth in 3 of 5 xenografts and in decrease of tumor volume to less than 50% of the initial values in 2 of 5 tumors. The results support the concept that combinations of cytokines with cytostatics might be of value for treatment of gastrointestinal and pancreatic cancer in vivo.     

Characterization of the antitumor effects of the selective farnesyl protein transferase inhibitor R115777 in vivo and in vitro

R115777 [(B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)-methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone] is a potent and selective inhibitor of farnesyl protein transferase with significant antitumor effects in vivo subsequent to oral administration in mice. In vitro, using isolated human farnesyl protein transferase, R115777 competitively inhibited the farnesylation of lamin B and K-RasB peptide substrates, with IC50s of 0.86 nM and 7.9 nM, respectively. In a panel of 53 human tumor cell lines tested for growth inhibition, approximately 75% were found to be sensitive to R115777. The majority of sensitive cell lines had a wild-type ras gene. Tumor cell lines bearing H-ras or N-ras mutations were among the most sensitive of the cell lines tested, with responses observed at nanomolar concentrations of R115777. Tumor cell lines bearing mutant K-ras genes required higher concentrations for inhibition of cell growth, with 50% of the cell lines resistant to R115777 up to concentrations of 500 nM. Inhibition of H-Ras, N-Ras, and lamin B protein processing was observed at concentrations of R115777 that inhibited cell proliferation. However, inhibition of K-RasB protein-processing could not be detected. Oral administration b.i.d. of R115777 to nude mice bearing s.c. tumors at doses ranging from 6.25-100 mg/kg inhibited the growth of tumors bearing mutant H-ras, mutant K-ras, and wild-type ras genes. Histological evaluations revealed heterogeneity in tumor responses to R115777. In LoVo human colon tumors, treatment with R115777 produced a prominent antiangiogenic response. In CAPAN-2 human pancreatic tumors, an antiproilferative response predominated, whereas in C32 human melanoma, marked induction of apoptosis was observed. The heterogeneity of histological changes associated with antitumor effects suggested that R115777, and possibly farnesyl protein transferase inhibitors as a class, alter processes of transformation related to tumor-host interactions in addition to inhibiting tumor-cell proliferation.     

Survival efficacy of adjuvant cytosine-analogue CS-682 in a fluorescent orthotopic model of human pancreatic cancer

Adjuvant treatment with the cytosine analogue 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-N(4)-palmitoylcytosine (CS-682) results in a highly significant increase in survival in the aggressive orthotopic MIA-PaCa-2 human pancreatic cancer mouse model. Seven days after implantation, mice were randomized into eight groups, depending on whether they were to be treated by tumor resection, 5 weeks of CS-682 chemotherapy at 40-60 mg/kg once daily, or both. Throughout the course of treatment, noninvasive optical whole-body imaging based on brilliant red fluorescent protein expression of the tumor permitted visualization and quantification of primary, metastatic, and recurrent disease. Total tumor burden negatively correlated with survival. Untreated mice died of disseminated disease with a median survival of 26 days. Surgical resection alone conferred a small but significant survival advantage (median survival, 28 days, P = 0.03). Primary CS-682 treatment at all doses also significantly prolonged survival compared with untreated animals (P < 0.05) and was more effective than surgery alone at doses of 50 and 60 mg/kg (median survival, 34 days, P = 0.045, and 38.5 days, P = 0.03, respectively). Maximal survival (median, 48 days, with 30% of animals surviving longer than 60 days) was achieved by adjuvant CS-682 (50 mg/kg), given after surgical resection of the primary pancreatic tumor (P = 0.004 compared with surgery alone). The results demonstrate that adjuvant oral administration of CS-682 for pancreatic cancer is highly effective with acceptable toxicity, suggesting its potential for cure of this disease in appropriate combinations.     

Manumycin and gliotoxin derivative KT7595 block Ras farnesylation and cell growth but do not disturb lamin farnesylation and localization in human tumour cells.

Recently, many inhibitors of farnesyl protein transferase (FPTase) have been identified. Some of them interrupt cell growth in addition to Ras and nuclear lamin processing of Ras-transformed cells. We have tested the effect of the FPTase inhibitors manumycin, an analogue of farnesyl diphosphate, and KT7595, a gliotoxin derivative, on Ras farnesylation, DNA synthesis and the anchorage-dependent and -independent growth of human colon carcinoma (LoVo), hepatoma (Mahlavu and PLC/PRF/5) and gastric carcinoma (KATO III). Both drugs severely inhibited DNA synthesis, cellular proliferation and Ras farnesylation in LoVo and moderately reduced them in Mahlavu and PLC/PRF/5 but not in KATO III. Complete sequencing of ras genes, however, revealed that LoVo and KATO III have activated Ki-ras and activated N-ras, respectively, whereas Mahlavu and PLC/PRF/5 have no activated ras. We next checked whether the inhibition of the cellular proliferation is due to the blocking of nuclear lamin function. Neither drug disturbed lamin farnesylation and localization, as demonstrated using metabolic labelling, immunoblotting and indirect immunofluorescence. These results indicate that manumycin and KT7595 can inhibit Ras farnesylation and cell growth without disturbing the farnesylation and localization of the lamins on human tumour cell lines.     

The farnesyl protein transferase inhibitor SCH66336 synergizes with taxanes in vitro and enhances their antitumor activity in vivo

Purpose: SCH66336 is an orally active, farnesyl protein transferase inhibitor. SCH66336 inhibits ras farnesylation in tumor cells and suppresses tumor growth in human xenograft and transgenic mouse cancer models in vivo. The taxanes, paclitaxel (Taxol) and docetaxel (Taxotere) block cell mitosis by enhancing polymerization of tubulin monomers into stabilized microtubule bundles, resulting in apoptosis. We hypothesized that anticancer combination therapy with SCH66336 and taxanes would be more efficacious than single drug therapy. Methods: We tested the efficacy of SCH66336 and taxanes when used in combination against tumor cell proliferation in vitro, against NCI-H460 human lung tumor xenografts in nude mice, and against mammary tumors in wap-ras transgenic mice. Results: SCH66336 synergized with paclitaxel in 10 out of 11 tumor cells lines originating from breast, colon, lung, ovary, prostate, and pancreas. SCH66336 also synergized with docetaxel in four out of five cell lines tested. In the NCI-H460 lung cancer xenograft model, oral SCH66336 (20 mg/kg twice daily for 14 days) and intraperitoneal paclitaxel (5 mg/kg once daily for 4 days) caused a tumor growth inhibition of 56% by day 7 and 65% by day 14 compared to paclitaxel alone. Male transgenic mice of the wap-ras/F substrain [FVB/N-TgN(WapHRAS)69LlnYSJL] spontaneously develop mammary tumors at 6–9 weeks of age which have been previously shown to be resistant to paclitaxel. Paclitaxel resistance was confirmed in the present study, while SCH66336 inhibited growth of these tumors. Most importantly, SCH66336 was able to sensitize wap-ras/F mammary tumors to paclitaxel chemotherapy. Conclusion: Clinical investigation of combination therapy using SCH66336 and taxanes in cancer patients is warranted. Further, SCH66336 may be useful for sensitizing paclitaxel-resistant tumors to taxane treatment. Received: 30 November 1999 / Accepted: 10 May 2000     

Inhibition of cell growth of human hepatoma cell line (HepG2) by a farnesyl protein transferase inhibitor: A preferential suppression of ras farnesylation

So far, treatment with anti-cancer agents has failed to achieve satisfactory results in hepatocellular carcinoma. In the process of hepatocarcinogenesis, ras has been shown to play a role, ras requires a farnesyl moiety for activation. It has been found that UCFI-C (manumycin), an antibiotic, inhibits farnesyl protein transferase, an enzyme that catalyzes farnesylation. Therefore, we investigated the effects of UCFI-C on cell growth, prenylation of cellular proteins including ras and Rap I, MAP kinase activity, activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and synthesis of cholesterol in a ras-activated human hepatoma cell line, Hep G2. Treatment with varying concentrations of UCFI-C (10–30 μM) for 24 and 72 hr resulted in a time· and dose-dependent inhibition of cell numbers. ³H-Thymidine incorporation was also inhibited in a dose-dependent manner, with 50% inhibition after 44 hr being observed at a concentration of 17 μM. UCFI-C dose-dependently inhibited ras farnesylation and MAP kinase activity, but did not decrease Rap I geranylgeranylation or prenylation of 21- to 26-kDa proteins. Neither the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase nor cholesterol synthesis were inhibited. These results suggest that UCFI-C antagonizes the growth of Hep G2 via the suppression of ras farnesylation and could be a lead for the development of new anti-cancer agents blocking the function of oncogenic ras associated with human cancer, including hepatocellular carcinoma. © 1996 Wiley-Liss, Inc.     

UCN-01 (7-hydoxystaurosporine) inhibits in vivo growth of human cancer cells through selective perturbation of G1 phase checkpoint machinery.

Mechanisms underlying tumor sensitivity to the antitumor agent UCN-01 (7-hydroxystaurosporine) were examined in the nude mouse model using three human tumor xenografts, two pancreatic cancers (PAN-3-JCK and CRL 1420) and a breast cancer (MX-1). UCN-01 antitumor activity was evaluated in terms of relative tumor weights in treated and untreated mice bearing the tumor xenografts. The activity of cyclin-dependent kinase 2 (CDK2), levels of p21 and p27 proteins, pRb status and cell cycle were evaluated. Induction of p21 and apoptosis were also assessed immunohistochemically in CRL 1420. UCN-01 was administered intraperitoneally at a dose of either 5 or 10 mg / kg daily for 5 days followed by a further 5 injections after an interval of 2 days. UCN-01 significantly suppressed the growth of both pancreatic cancers, but was ineffective against MX-1. p21 protein expression was markedly induced in the UCN-01-sensitive pancreatic carcinoma xenografts at both doses, but p21 induction was only evident in the UCN-01-resistant MX-1 at 10 mg / kg. MX-1 exhibited CDK2 activity that was 6-fold higher than that of pancreatic cancer strains, which may explain the resistance of MX-1 to UCN-01 despite the induction of p21 at the dose of 10 mg / kg. The UCN-01-sensitive tumors exhibited G1 arrest and increased levels of apoptosis, changes not observed in resistant MX-1. In conclusion, it appears that a determining factor of in vivo UCN-01 sensitivity involves the balance of CDK2 kinase activity and p21 protein induction, resulting in augmented pRb phosphorylation, G1 cell cycle arrest and apoptosis.     

Antiangiogenic treatment with the three thrombospondin-1 type 1 repeats recombinant protein in an orthotopic human pancreatic cancer model.

PURPOSE: This study investigates the antiangiogenesis and antitumor efficacy of a recombinant protein composed of the three type 1 repeats (3TSR) of thrombospondin-1 in an orthotopic human pancreatic cancer model and provides useful preclinical data for pancreatic cancer treatment. EXPERIMENTAL DESIGN: Human pancreatic cancer cells (AsPC-1) were injected into the pancreas of severe combined immunodeficient mice. The animals were treated with 3TSR (3 mg per kg per day) or PBS for 3 weeks. Subsequently, the effects of 3TSR on tumor growth, microvessel density, cancer cell proliferation, apoptosis, and endothelial cell apoptosis were analyzed. The in vitro effects of 3TSR on human pancreatic cancer cells were also studied. RESULTS: 3TSR treatment significantly reduced angiogenesis and tumor growth of orthotopic pancreatic cancer. 3TSR-treated mice had a 69% reduction in tumor volume (316.6 +/- 79.3 versus 1,012.2 +/- 364.5 mm(3); P = 0.0001), and a significant increase in tumor necrotic area. After 3TSR treatment, both the vessel number and average microvessel size were significantly decreased, and microvessel density was decreased from 8.0% to 3.7% (P < 0.0001). The apoptotic rate of tumoral endothelial cells in 3TSR-treated tumors increased to 14.7% comparing to 4.2% in control tumors (P < 0.0001). 3TSR showed no direct effects on pancreatic cancer cell proliferation or apoptosis either in vivo or in vitro. CONCLUSION: 3TSR, a domain of a natural occurring angiogenesis inhibitor, showed potent therapeutic effect in pancreatic cancer by inhibiting tumor angiogenesis and may prove to be a promising agent for clinical pancreatic cancer treatment.     

Inhibition of protein farnesylation enhances the chemotherapeutic efficacy of the novel geranylgeranyltransferase inhibitor BAL9611 in human colon cancer cells

Proteins belonging to the ras superfamily are involved in cell proliferation of normal and neoplastic tissues. To be biologically active, they require post-translational isoprenylation by farnesyl-transferase and geranylgeranyl-transferase. Enzyme inhibition by drugs may thus represent a promising approach to the treatment of cancer. Therefore, the combined effect of BAL9611, a novel inhibitor of geranylgeranylation, and manumycin, a farnesyl-transferase inhibitor, was evaluated on the SW620 human colon cancer cell line which harbours a mutated K-ras gene. BAL9611 and manumycin dose-dependently inhibited SW620 cell growth with 50% inhibitory concentration (IC(50)) of 0.47 +/- 0.03 and 5.24 +/- 1.41 microM (mean +/- SE), respectively. The isobologram analysis performed at the IC(50)level revealed that the combined treatment was highly synergistic with respect to cell growth inhibition. BAL9611 and manumycin were able to inhibit the geranylgeranylation of p21rhoA and farnesylation of p21ras; both drugs inhibited p42ERK2/MAPK phosphorylation, but their combination was more effective than either drug alone. Moreover, the enhanced inhibition of cell growth in vitro by the BAL9611-manumycin combination was also observed in vivo in CD nu/nu female mice xenografted with SW620 tumours. Finally, both drugs were able to induce cell death by apoptosis in vitro and in vivo, as demonstrated by perinuclear chromatin condensation, cytoplasm budding and nuclear fragmentation, and interoligonucleosomal DNA digestion. In conclusion, the inhibition of protein farnesylation enhances the chemotherapeutic effect of BAL9611 in vitro and in vivo in a synergistic fashion, as a result of the impairment of post-translational isoprenylation of proteins and phosphorylation of p42ERK2/MAPK, whose activation is associated with post-translational geranylgeranylation and farnesylation of p21rhoA and p21ras.     

Manumycin enhances the cytotoxic effect of paclitaxel on anaplastic thyroid carcinoma cells

Despite the current multimodal approach to treatment of anaplastic thyroid cancer (ATC), the prognosis for patients with the disease is poor. New effective therapy for ATC is desperately needed. Thus, we investigated the effects of manumycin (a farnesyl:protein transferase inhibitor), alone and in combination with other drugs frequently used to treat ATC, in six human ATC cell lines: ARO, C643, DRO, Hth-74, KAT-4, and KAT-18. By means of a formazan dye-based spectrophotometric assay of cell viability and light microscopy, manumycin was shown to decrease the number of viable cells in all six of the cell lines though to a lesser degree in DRO and C643 cells than in ARO, Hth-74, KAT-4, and KAT-18 cells. In combination, manumycin enhanced the effect of paclitaxel in all six of the cell lines. The mechanism of cell death was investigated by measuring caspase-3 activity, immunoblotting with anti-poly-(ADP-ribose)polymerase (PARP) antibody and electrophoresis of DNA. After an 18-h incubation, manumycin plus paclitaxel caused enhanced activation of caspase-3 activity, cleavage of PARP into Mr 89,000 and 28,000 fragments, and internucleosomal fragmentation of DNA (all of which are characteristic of apoptotic cell death). In contrast, neither manumycin alone, paclitaxel alone, doxorubicin alone, nor doxorubicin plus manumycin produced significant specific cleavage of PARP and internucleosomal DNA fragmentation after 18 h of incubation. The in vivo effect and toxicity of combined manumycin and paclitaxel treatments were evaluated in a nude mouse xenograft model using ARO and KAT-4 cells. Drugs were injected i.p. on days 1 and 3 of a 7-day cycle for three cycles. Both manumycin (7.5 mg/kg/dose) and paclitaxel (20 mg/kg/dose) had significant inhibitory effects on tumor growth. Combined manumycin and paclitaxel treatments seemed as effective as manumycin against ARO cells and more effective than either manumycin or paclitaxel alone against KAT-4 cells. No significant morbidity or mortality was caused by the treatments. In conclusion, manumycin can inhibit the growth of ATC both in vitro and in vivo. Manumycin plus paclitaxel has enhanced cytotoxic effects and increased apoptotic cell death in ATC cells in vitro compared with either drug by itself. The combination of manumycin and paclitaxel is also effective in vivo with no significant toxicity observed. The lack of synergy observed in this in vivo experiment may be due to a ceiling effect, and further experimentation is warranted to ascertain the optimal way to combine these two agents for maximal therapeutic effects.     

UCN-01 (7-Hydoxystaurosporine) Inhibits in vivo Growth of Human Cancer Cells through Selective Perturbation of G1 Phase Checkpoint Machinery

Mechanisms underlying tumor sensitivity to the antitumor agent UCN-01 (7-hydroxystaurosporine) were examined in the nude mouse model using three human tumor xenografts, two pancreatic cancers (PAN-3-JCK and CRL 1420) and a breast cancer (MX-1). UCN-01 antitumor activity was evaluated in terms of relative tumor weights in treated and untreated mice bearing the tumor xenografts. The activity of cyclin-dependent kinase 2 (CDK2), levels of p21 and p27 proteins, pRb status and cell cycle were evaluated. Induction of p21 and apoptosis were also assessed immuno-histochemically in CRL 1420. UCN-01 was administered intraperitoneally at a dose of either 5 or 10 mg/kg daily for 5 days followed by a further 5 injections after an interval of 2 days. UCN-01 significantly suppressed the growth of both pancreatic cancers, but was ineffective against MX-1. p21 protein expression was markedly induced in the UCN-01-sensitive pancreatic carcinoma xenografts at both doses, but p21 induction was only evident in the UCN-01-resistant MX-1 at 10 mg/kg. MX-1 exhibited CDK2 activity that was 6-fold higher than that of pancreatic cancer strains, which may explain the resistance of MX-1 to UCN-01 despite the induction of p21 at the dose of 10 mg/kg. The UCN-01-sensitive tumors exhibited G1 arrest and increased levels of apoptosis, changes not observed in resistant MX-1. In conclusion, it appears that a determining factor of in vivo UCN-01 sensitivity involves the balance of CDK2 kinase activity and p21 protein induction, resulting in augmented pRb phosphorylation, G1 cell cycle arrest and apoptosis.     

Virulizin-2gamma, a novel immunotherapeutic agent, in treatment of human pancreatic cancer xenografts

Virulizin-2gamma, a novel biological response modifier extracted from bovine bile, has shown in clinical studies a significant antitumor activity against pancreatic cancer. We report here the results of preclinical evaluation of Virulizin-2gamma in treatment of human pancreatic cancer xenograft in nude mice. In this in vivo study, 14 daily bolus intraperitoneal administrations of Virulizin-2gamma (0.2 ml/mouse/day) significantly inhibited the growth of BxPC-3 human pancreatic tumor xenografts, with T/C value of 60%. Virulizin-2gamma also potentiated the antitumor activity of gemcitabine (120 mg/kg x4, q3d) in this tumor model. The combination therapy resulted in T/C values of 26% compared to gemcitabine alone (T/C 33%). In addition, based on body weight observation, no signs of toxicity related to treatment with Virulizin-2gamma, either as a single agent or when combined with gemcitabine were found. Virulizin-2gamma was well tolerated by the mice. The data from in vitro studies revealed that Virulizin-2gamma did not have direct cytotoxicity against cultured tumor cell lines, indicating that the in vivo antitumor activity is likely due to its immunomodulating effects on host immune cells. These preclinical results supported the safety and efficacy observed in clinical studies and indicated that Virulizin-2gamma is a promising immunotherapeutic agent for treatment of pancreatic cancer and worthy of further clinical evaluation.     

Radiosensitizing effects of the prenyltransferase inhibitor AZD3409 against RAS mutated cell lines

Mutations at the H-, N- and K-ras loci are among the most frequent genetic alterations in human cancers. In this study, we have investigated the effect of AZD3409, a novel, peptidomimetic prenyltransferase inhibitor (PTI), on the radiosensitivity of cells with mutated ras alleles. AZD3409, developed by AstraZeneca, inhibits both farnesyl- and geranylgeranyl transferase in cell free systems. AZD3409 inhibits the growth of a variety of human cancer cell lines, including cells that express mutant alleles of either K- or H- ras and was well tolerated when administered orally to healthy volunteers in a phase I clinical trial. We have previously shown that PTI can radiosensitize human and rodent cancer cell lines that express activated RAS. Here we assessed the ability of AZD3409 to radiosensitize human cancer cell lines in vivo and in vitro and the activation state of RAS proteins in treated cells. Once daily oral administration of AZD3409 to nude mice bearing PSN-1 and MiaPaCa-2 human pancreatic cancer xenografts expressing mutant K-ras was well tolerated and resulted in a supra-additive reduction in clonogenic cell survival after irradiation. Similarly, AZD3409 reduced clonogenic survival in cells that express either mutant K- or H- ras in vitro. We next examined the effect of AZD3409 on the processing and activation of K- and H-RAS. AZD3409-mediated radiosensitization, both in vivo and in vitro, correlates with a decrease in H-RAS processing without detectable effect on K-RAS processing. RAS activation assays show that the decreased H-RAS processing is accompanied by decreased H-RAS activation in cell lines with mutations in either K- or H-ras. However, no decrease in K-RAS activation was detected. Thus, radiosensitization of human cancer cells that express mutated K-RAS occurred under conditions where AZD3409 inihibits the activation of farneyslated H-RAS, but did not inhibit K-RAS activation.     

Wortmannin inhibits pkb/akt phosphorylation and promotes gemcitabine antitumor activity in orthotopic human pancreatic cancer xenografts in immunodeficient mice.

Pancreatic cancer is resistant to almost all classes of cytotoxic agents. Gemcitabine seems to be the current drug of choice. We have recently reported that inhibition of the phosphatidylinositide 3-kinase-protein kinase B (PKB/Akt) cell survival pathway by wortmannin enhances gemcitabine-induced apoptosis in cultured human pancreatic cancer cells (1). The present study investigated the effects of wortmannin on orthotopic human pancreatic cancer xenografts implanted in severe combined immunodeficient mice. Animals were given single i.v. bolus injections of 0.175, 0.35, or 0.7 mg/kg of wortmannin and killed at 0.5, 1, 2, or 4 h after treatment. Phosphorylated PKB/Akt levels in tumor tissues were measured by fluorescence image analysis. Wortmannin was found to inhibit PKB/Akt phosphorylation in a time- and dose-dependent manner, reaching a plateau at 4 h and at 0.7 mg/kg. The levels of phosphorylated PKB/Akt were maximally decreased by approximately 50% relative to the vehicle control. Subsequently, the extent of apoptosis in tumors treated with gemcitabine or wortmannin alone or in combination was determined using terminal deoxynucleotidyl transferase-mediated nick end labeling assay and computerized image analysis. Orthotopic tumors exposed to 80 mg/kg gemcitabine for 48 h and then 0.7 mg/kg wortmannin for 4 h showed a 5-fold increase (P = 0.002) in apoptosis compared with those treated with each agent alone and with the vehicle control. The combination treatment also significantly (P < 0.001) inhibited tumor growth. Taken together, our findings support the potential of phosphatidylinositide 3-kinase inhibitors as adjuncts to conventional chemotherapy in the treatment of pancreatic cancer.     

Preclinical antitumor activity and pharmacodynamic studies with the farnesyl protein transferase inhibitor R115777 in human breast cancer.

Antitumor and pharmacodynamic studies were performed in MCF-7 human breast cancer cells and companion xenografts with the farnesyl protein transferase inhibitor, R115777, presently undergoing Phase II clinical trials, including in breast cancer. R115777 inhibited growth of MCF-7 cells in vitro with an IC(50) of 0.31 +/- 0.25 microM. Exposure of MCF-7 cells to increasing concentrations of R115777 for 24 h resulted in the inhibition of protein farnesylation, as indicated by the appearance of prelamin A at concentrations >1 microM. After continuous exposure to 2 microM R115777, prelamin A levels peaked at 2 h post drug exposure and remained high for up to 72 h. R115777 administered p.o. twice daily for 10 consecutive days to mice bearing established s.c. MCF-7 xenografts induced tumor inhibition at a dose of 25 mg/kg [percentage of treated versus control (% T/C) = 63% at day 21]. Greater inhibition was observed at doses of 50 mg/kg (% T/C at day 21 = 38%) or 100 mg/kg (% T/C at day 21 = 43%). The antitumor effect appeared to be mainly cytostatic with little evidence of tumor shrinkage to less than the starting volume. Tumor response correlated with an increase in the appearance of prelamin A, but no changes in the prenylation of lamin B, heat shock protein 40, or N-Ras were detectable. In addition, significant increases in apoptotic index and p21(WAF1/CIP1) expression were observed, concomitant with a decrease in proliferation as measured by Ki-67 staining. An increase in prelamin A was also observed in peripheral blood lymphocytes in a breast cancer patient who responded to R115777. These data show that R115777 possesses preclinical antitumor activity against human breast cancer and that the appearance of prelamin A may provide a sensitive and convenient pharmacodynamic marker of inhibition of prenylation and/or response.