Inhibition of primary colon carcinoma growth and liver metastasis by the A3 adenosine receptor agonist CF101

Adenosine is a purine nucleoside that acts as a regulatory molecule by binding to specific G-protein-coupled A1, A(2A), A(2B), and A3 cell surface receptors. We have recently demonstrated that adenosine inhibits tumour cell growth and concomitantly stimulates bone marrow cell proliferation via activation of the A3 adenosine receptor (A3AR). In the present study, we show that a synthetic agonist to the A3AR, CF101, at the low nanomolar concentration range, inhibits HCT-116 human colon carcinoma cell growth. This effect was reversed by the selective A3AR antagonist MRS1523, demonstrating the specificity of the response. CF101 (given orally) was efficacious in inhibiting the development of primary tumours in xenograft and syngeneic models in which mice were inoculated subcutaneously with human HCT-116 or murine CT-26 colon carcinoma cells, respectively. Moreover, CF101 suppressed (50%, P<0.01) colon cancer liver metastases in syngeneic mice inoculated to the spleen with CT-26 cells. The mechanism of action entailed upregulation of interleukin-12 production in the CF101-treated groups and potentiation of NK cell activity. In the HCT-116 xenograft model in which a combined therapy of CF101 and 5-fluorouracyl (5-FU) was examined, an additive antitumour effect was demonstrated. Moreover, CF101 prevented the 5-FU-induced myelotoxicity, resulting in normal values of white blood cell and neutrophil counts. We conclude that the A3AR agonist CF101, a small orally bioavailable molecule, exerts systemic anticancer, antimetastatic, and myeloprotective effects in colon carcinoma-bearing mice, and may serve as an adjuvant treatment to enhance the chemotherapeutic index and prevent myelotoxicity.     

Violacein synergistically increases 5-fluorouracil cytotoxicity, induces apoptosis and inhibits Akt-mediated signal transduction in human colorectal cancer cells

Despite recent additions to the armory of chemotherapeutic agents for colorectal cancer (CRC) treatment, the results of chemotherapy remain unsatisfactory. 5-Fluorouracil (5-FU) still represents the cornerstone of treatment and resistance to its actions is a major obstacle to successful chemotherapy. Therefore, new active agents in CRC and agents that increase the chemosensitivity of cancer cells to 5-FU are still urgently required. Violacein, a pigment isolated from Chromobacterium violaceum in the Amazon river, has a diverse spectrum of biological activities, and represents a novel cytotoxic drug with known antileukemic properties. To assess the suitability of violacein as a chemotherapeutic agent in CRC its cytotoxic effects were evaluated both as a single agent and in combination with 5-FU. Its underlying mechanisms of action were further investigated by studying its effects on the cell cycle, apoptosis and cell survival pathways [phosphatidylinositol-3-kinase/Akt, p44/42 mitogen activated protein kinase and nuclear factor kappaB (NF-kappaB)] in colon cancer cell lines. Violacein inhibits the growth of all four colon cancer cell lines tested. It induces apoptosis, and potentiates the cytotoxic effect of 5-FU in a poorly differentiated microsatellite unstable cell line (HCT116). Violacein causes cell cycle block at G(1), upregulates p53, p27 and p21 levels and decreases the expression of cyclin D1. Violacein leads to dephosphorylation of retinoblastoma protein and activation of caspases and a pancaspase inhibitor abrogates its biological activity. Our data provide evidence that violacein acts through the inhibition of Akt phosphorylation with subsequent activation of the apoptotic pathway and downregulation of NF-kappaB signaling. This leads to the increase in chemosensitivity to 5-FU in HCT116 colon cancer cells. Taken together, our findings suggest that violacein will be active in the treatment of colorectal tumors and offers new prospects for overcoming 5-FU resistance.     

Chemosensitization by STI571 targeting the platelet-derived growth factor/platelet-derived growth factor receptor-signaling pathway in the tumor progression and angiogenesis of gastric carcinoma

BACKGROUND: Autocrine and paracrine growth mediated by the platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR)-signaling pathway plays an important role in the progression of solid tumors. The authors assessed the effect of STI571 on the tumor growth of gastric carcinoma in combination with 5-fluorouracil (5-FU) or paclitaxel targeting the PDGF/PDGFR-signaling pathway. METHODS: In MKN-45 gastric carcinoma cells, the cytotoxic effect was evaluated by 3-(4,5 dimethiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and the in vivo antitumor effect was evaluated in a nude mouse xenograft. Both STI571 and an antitumor drug were administered intraperitoneally. Gene expression was assessed by Western blot analysis and immunohistochemical staining. Apoptotic cell death was evaluated by the terminal deoxyuridine triphosphate-biotin nick-end labeling assay, and tumor angiogenesis was evaluated by microvessel density analysis. RESULTS: Treatment with STI571 alone was not effective in vitro, as assessed by a 50% inhibitory concentration value of 24.3 microM. Combination treatment with STI571 and 5-FU or paclitaxel enhanced the cytotoxic effect somewhat when the concentration of STI571 was increased to 10 microM. Combination treatment with STI571 and 5-FU or paclitaxel enhanced the antitumor effect of the antitumor drug significantly in vivo. The enhanced antitumor effect was associated with increased apoptotic cell death and inhibition of tumor angiogenesis. Treatment with STI571 down-regulated the expression of PDGF-BB and PDGFR-beta in tumor cells and decreased the production of phosphorylated PDGFR-beta and phosphorylated Akt. Furthermore, treatment with STI571 inhibited the expression of PDGFR-beta in stromal cells. CONCLUSIONS: STI571 was an effective chemosensitizer of antitumor drugs, such as 5-FU and paclitaxel for gastric carcinoma, targeting the PDGF/PDGFR-signaling pathway of tumor cells and stromal cells in disease progression and angiogenesis.     

Knockdown of focal adhesion kinase reverses colon carcinoma multicellular resistance

Chemotherapy resistance in solid tumors is broad and encompasses diverse unrelated drugs. Three-dimensional multicellular spheroids (MCSs) are a good model for studying in vitro drug resistance. In the current study, we investigated the role of focal adhesion kinase (FAK) in 5-fluorouracil (5-FU) chemoresistance in colon carcinoma MCS culture cells. The expression of FAK was inhibited significantly by specific small hairpin RNA targeting FAK. The suppression of FAK expression did not affect the growth of spheroid cells. However, silencing of FAK combined with 5-FU treatment significantly decreased the 50% inhibitory concentration (IC₅₀) of 5-FU and markedly increased the population of apoptosis cells, which was associated with the reduction of the levels of Akt and nuclear factor–kappa B (NF-κB). Moreover, knockdown of FAK could inhibit tumor growth and increase the sensitivity of the tumor to 5-FU in the nude mouse xenograft. These results indicate that while not affecting cellular proliferation in the absence of 5-FU, RNA interference targeting FAK potentiated 5-FU-induced cytotoxicity in vitro and in vivo , and partially reversed multicellular resistance, which may contribute to its chemosensitizing effect through efficiently suppressing Akt/NF-κB activity. ( Cancer Sci 2009; 100: 1708–1713)     

Liposomal prednisolone phosphate potentiates the antitumor activity of liposomal 5-fluorouracil in C26 murine colon carcinoma in vivo

The antitumor efficacy of 5-fluorouracil (5-FU) in advanced colorectal cancer (CRC) is hindered not only by the low therapeutic index, but also by tumor cell resistance to this cytotoxic drug. Therefore, to enhance the 5-FU antitumor activity, the present research used a novel tumor-targeted therapy based on the co-administration of 5-FU encapsulated in long-circulating liposomes (LCL-5-FU) together with liposomal prednisolone phosphate (LCL-PLP), a formulation with known anti-angiogenic actions on C26 murine colon carcinoma cells. Thus, we assessed the in vivo effects of the combined liposomal drug therapy on C26 carcinoma growth as well as on the production of molecular markers with key roles in tumor development such as angiogenic, inflammatory, and oxidative stress molecules. To get further insight into the polarization state of tumor microenvironment after the treatment, we determined the IL-10/IL-12p70 ratio in tumors. Our results showed that combined liposomal drug therapy inhibited almost totally tumor growth and was superior as antitumor activity to both single liposomal drug therapies tested. The antitumor efficacy of the combined therapy was mainly related to the anti-angiogenic and anti-inflammatory actions on C26 carcinoma milieu, being favored by its controlling effect on intratumor oxidative stress and the skewing of polarization of tumor microenvironmental cells toward their antineoplastic phenotypes. Thus, our study unveils a promising treatment strategy for CRC that should be furthermore considered.     

Role of NF-kappaB and Akt/PI3K in the resistance of pancreatic carcinoma cell lines against gemcitabine-induced cell death

Pancreatic cancer is resistant to almost all cytotoxic drugs. Currently, gemcitabine appears to be the only clinically active drug but, because of pre-existing or acquired chemoresistance of most of the tumor cells, it failed to significantly improve the outcome of pancreatic carcinoma patients. The current study examined the relevance of nuclear factor kappaB (NF-kappaB) and PI3K/Akt in the resistance of five pancreatic carcinoma cell lines towards gemcitabine. Treatment for 24 h with gemcitabine (0.04-20 micro M) led to a strong induction of apoptosis in PT45-P1 and T3M4 cells but not in BxPc-3, Capan-1 and PancTu-1 cells. These resistant cell lines exhibited a high basal NF-kappaB activity in contrast to the sensitive cell lines. Furthermore, gemcitabine showed a dose-dependent induction of NF-kappaB. At a dose of 0.04 micro M, gemcitabine still induced apoptosis in the sensitive cell lines, but did not induce NF-kappaB. In addition, NF-kappaB inhibition by MG132, sulfasalazine or the IkappaBalpha super-repressor strongly diminished the resistance against gemcitabine (0.04-20 micro M). In contrast to this obvious correlation between basal NF-kappaB activity and gemcitabine resistance, PI3K/Akt seems not to be involved in gemcitabine resistance of these cell lines. Neither did the basal Akt activity correlate with the sensitivity towards gemcitabine treatment, nor did the inhibition of PI3K/Akt by LY294002 alter gemcitabine-induced apoptosis. These results indicate that constitutive NF-kappaB activity confers resistance against gemcitabine and that modulation of this activity by pharmacological or genetic approaches may have therapeutical potential when combined with gemcitabine in the treatment of pancreatic carcinoma.     

Xenografts in pharmacologically immunosuppressed mice as a model to test the chemotherapeutic sensitivity of human tumors

A human tumor xenograft model using pharmacologically immunosuppressed mice was assessed for its suitability to test preclinically the sensitivity of colorectal carcinomas, bone sarcomas and melanomas against anticancer agents. Besides ionizing radiation, 14 cytotoxic drugs including 5-fluorouracil (5-FU), dimethylmyleran (DMM), cytosine arabinoside, cyclophosphamide, melphalan, BCNU, mitomycin C, adriamycin, bleomycin, etoposide, vinblastine, cisplatin, procarbazine and DTIC were assayed. Ionizing radiation, 5-FU and DMM were also applied at lethal doses followed by bone-marrow rescue heavy therapy. Four colon carcinomas responded poorly to most of the agents but one tumor displayed marked sensitivity to BCNU. Lethal doses of radiation, 5-FU and DMM could also show considerable activity. High sensitivity was shown by a Ewing sarcoma to DMM and cyclophosphamide and by an osteosarcoma to the latter drug. No strong effects were seen against melanomas. Lethal doses of DMM induced the best regression of one colon carcinoma. In general, the superiority of heavy therapy for solid human tumors compared to maximally tolerated doses was demonstrated. Individual carcinomas of the same type displayed different drug sensitivity.     

Chemosensitization and radiosensitization of human lung and colon cancers by antimitotic agent, ABT-751, in athymic murine xenograft models of subcutaneous tumor growth

Purpose

ABT-751 is an orally active antimitotic agent that is currently in Phase II clinical trials. This agent binds to the colchicine site on ß-tubulin and inhibits polymerization of microtubules. This disruption of microtubule dynamics leads to a block in the cell cycle at the G₂/M phase, and promotes apoptosis. ABT-751, as a single agent, has antitumor activity against a series of xenograft models including non-small cell lung cancer (NSCLC) and colon cancer. The current studies were conducted to determine whether ABT-751 enhances antitumor activity of standard cytotoxic therapies currently in clinical use.

Methods

Efficacy of ABT-751, in combination with cisplatin, 5-FU, and radiation, was evaluated in the Calu-6 NSCLC, HT-29 colon, and HCT-116 colon carcinoma xenograft models, respectively. Tumor-bearing athymic mice were treated with ABT-751 orally once a day at 75 or 100 mg/kg/day on a 5-days-on, 5-days-off schedule for two cycles.

Results

Efficacy of ABT-751 at 100 mg/kg/day was tested in combination with cisplatin at its maximum tolerable dose (MTD) (10 mg/kg/day, i.p. x1) in Calu-6 tumor-bearing athymic mice. The percent treated/control (%T/C) tumor volume ratios on day 38 were 35, 37, and 6, and the percent tumor growth delay (%TGD) values were 71, 65, and 188 for cisplatin, ABT-751 and the combination groups, respectively. HT-29 colon tumors were used to test ABT-751 in combination with an MTD of 5-FU, 30 mg/kg/day, i.p., q.d. x5. The %T/C ratios on day 38 were 22, 28, and 5 and the %TGD values were 75, 75, and 150 for 5-FU, ABT-751, and the combination groups, respectively. Treatment of HCT-116 colon carcinoma tumors with ABT-751, concurrent with the radiation treatment, was able to both enhance radiation-induced tumor regression, and delay the time to recurrence and progression. Growth curves allowed calculation of enhancement of radiation-induced growth delay (defined as the additional time required for a treated tumor to reach four times its original size) of 2, 9, and 12 days, for ABT-751 alone, radiation alone, and the combination, respectively.

Conclusion

Collectively, these studies demonstrate that ABT-751 enhanced efficacy of standard cytotoxic therapies in a variety of tumor xenograft models, and that enhancement was at least additive in all systems.     

PUMA mediates the combinational therapy of 5-FU and NVP-BEZ235 in colon cancer

Colon cancer is the third most common cancer in humans which has a high mortality rate, and 5-Fluorouracil (5-FU) is one of the most widely used drugs in colon cancer therapy. However, acquired chemoresistance is becoming the major challenges for patients, and the molecular mechanism underlying the development of 5-FU resistance is still poorly understood. In this study, a newly designed therapy in combination with 5-FU and NVP-BEZ235 in colon cancer cells (HCT-116 and RKO) was established, to investigate the mechanism of 5-FU resistance and optimize drug therapy to improve outcome for patients. Our results show 5-FU induced cell apoptosis through p53/PUMA pathway, with aberrant Akt activation, which may well explain the mechanism of 5-FU resistance. NVP-BEZ235 effectively up-regulated PUMA expression, mainly through inactivation of PI3K/Akt and activation of FOXO3a, leading to cell apoptosis even in the p53^−/− HCT-116 cells. Combination treatment of 5-FU and NVP-BEZ235 further increased cell apoptosis in a PUMA/Bax dependent manner. Moreover, significantly enhanced anti-tumor effects were observed in combination treatment in vivo. Together, these results demonstrated that the combination treatment of 5-FU and NVP-BEZ235 caused PUMA-dependent tumor suppression both in vitro and in vivo, which may promise a more effective strategy for colon cancer therapy.     

Effect of Z-360, a novel orally active CCK-2/gastrin receptor antagonist on tumor growth in human pancreatic adenocarcinoma cell lines in vivo and mode of action determinations in vitro

Purpose Gastrin is known to enhance the growth of pancreatic carcinoma via the cholecystokinin (CCK)-2/gastrin receptor. We investigated the anti-tumor effect of Z-360 (calcium bis [(R)-(−)-3-[3-{5-cyclohexyl-1-(3,3-dimethyl-2-oxo-butyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepin-3-yl}ureido]benzoate]), a novel orally active CCK-2 receptor antagonist alone or combined with the chemotherapeutic agent, gemcitabine in human pancreatic adenocarcinoma cell lines. Results Z-360 potently inhibited specific binding of [³H]CCK-8 to the human CCK-2 receptor, with a K _i value of 0.47 nmol/l, and showed antagonistic activity for this receptor. The anti-tumor effect of Z-360 alone or combined with gemcitabine was assessed using subcutaneous xenografts of MiaPaCa2 and PANC-1 and an orthotopic xenograft model (PANC-1). Oral administration of Z-360 significantly inhibited the growth of MiaPaCa2 (41.7% inhibition at 100 mg/kg, P < 0.01). Combined administration of Z-360 and gemcitabine significantly inhibited subcutaneous PANC-1 tumor growth compared with either agent alone (27.1% inhibition compared to effect with gemcitabine, P < 0.05), and significantly prolonged survival compared with the vehicle control (median survival of 49 days in vehicle compared to 57 days in the combination group, P < 0.05). In vitro studies showed that Z-360 significantly inhibited gastrin-induced proliferation of human CCK-2 receptor-expressing cells, and also significantly reduced gastrin-induced PKB/Akt phosphorylation to the level of untreated controls. Conclusion In the present study, we have shown that Z-360 combined with gemcitabine can inhibit pancreatic tumor growth and prolong survival in a pancreatic carcinoma xenograft model, on a possible mode of action being the inhibition of gastrin-induced PKB/Akt phosphorylation through blockade of the CCK-2 receptor. Our results suggest that Z-360 may be a useful adjunct to gemcitabine for the treatment of pancreatic carcinoma and a therapeutic option for patients with advanced pancreatic cancer.     

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.     

Multi-chemothermoimmunotherapy for human colon adenocarcinoma in vitro

Effective adjunctive therapies for colorectal carcinoma are clearly needed. We evaluated the cytotoxic responses in vitro of human colon carcinoma cell lines to combined modalities: 5-fluorouracil/leucovorin (5-FU/LV), carboplatin (CP), tumor necrosis factor (TNF) and hyperthermia (HTX). Cytotoxicity was evaluated in a cell proliferation assay using crystal violet staining. 5-FU/LV was administered 2–3 days before TNF and CP, followed 1 h later by HTX. These cell lines were relatively resistant to HTX alone (42°C for 2 h), but were heterogeneous in their responses to various doses of the other single agents. This heterogeneity was also evident for combined modalities: the geneity was also evident for combined modalities: the HCT-15 cell line exhibited significant supra-additivity for selected doses of CP, TNF and 5-FU/LV, which was further enhanced by hyperthermia. In contrast, the HT-29 cell line did not demonstrate a strong pattern for supra-additivity, whereas the DLD-1 cell line had an intermediate response. Thus, our results suggest one approach to develop effective and dose-sparing multimodality therapeutic regimens for colon adenocarcinoma.     

三氧化二砷联合5-氟尿嘧啶抗裸鼠人结肠癌移植瘤的作用及其机制的研究

目的探讨三氧化二砷（As2O3）联合5-氟尿嘧啶（5-FU）对裸鼠人结肠癌移植瘤的抗肿瘤作用、作用机制以及药物毒性.方法建立裸鼠人结肠癌模型,随机分为生理盐水组、As2O3组、5-FU组和As2O3联合5-FU组,腹腔分别注射生理盐水、As2O3、5-FU和As2O3 ＋5-FU,观察各组抑瘤作用和裸鼠一般状态的变化,并对体内标本行光镜、电镜、原位末端标记（TUNEL）、免疫组化和血常规检测.结果与单药作用相比,As2O3联合5-FU可显著抑制结肠癌移植瘤的增长,CDI值〈1,并显著增强诱导瘤细胞凋亡作用、调控凋亡相关基因（Bcl-2、Bax、Fas和FasL）表达.联合用药并未增加裸鼠肝、肾和造血系统的毒性.结论 As2O3与5-FU联合应用对结肠癌移植瘤有协同抗肿瘤作用,其机制可能与增强诱导癌细胞凋亡、细胞周期特异性药物与细胞周期调控剂联合增效以及调控凋亡相关基因表达有关;同时联合用药对于荷瘤裸鼠的肝、肾和造血系统无明显毒性.     

Cyclin A correlates with the sensitivity of human cancer cells to cytotoxic effects of 5-FU

We investigated the role of cyclin A in the cytotoxic effect of 5-fluorouracil (5-FU) on cancer cell lines. Experiments were performed using gastric cancer chemosensitive NUGC3, and chemoresistant NUGC3/5FU/L established by repeated exposure to 5-FU. 5-FU inhibited cell growth of NUGC3 in a dose-dependent manner. Low concentrations of 5-FU did not inhibit cell growth of NUGC3/5FU/L, while high concentrations slightly inhibited cell growth. Examination of the cell cycle pattern of NUGC3 cells showed accumulation at S-phase at 10 micro M and at G1-S-phase at 100 micro M of 5-FU. Cell cycle pattern of NUGC3/5FU/L cells did not change 5-FU concentrations. 5-FU increased cyclin A mRNA level in NUGC3 cells but not NUGC3/5FU/L cells. In the presence of 100 micro M 5-FU, cyclin A protein level increased 2.6-fold in NUGC3 and 1.47-fold in NUGC3/5FU/L. 5-FU dose-dependently increased the percentage of cyclin A-positive NUGC3 cells, but not NUGC3/5FU/L cells. The percentage of cyclin A-positive cells in other 5-FU sensitive esophageal, colon and gastric cancer cell lines (T.Tn, LOVO, DLD-1, MKN-7), increased in the presence of 1 and 10 micro M 5-FU, while cyclin A-positive cells in 5-FU resitant hepatocellular and colon carcinoma cell lines (HCC50 and C-1), did not increase with the same treatment. Our results indicate that the cytotoxic effects of 5-FU in human cancer cell lines correlate with cyclin A and it may be used as a predictive factor for chemotherapy response.     

5-Fluorouracil plus leucovorin as adjuvant treatment of an experimental liver tumor in rats

Recent experimental and clinical work has shown that leucovorin potentiates the cytotoxic effect of 5-fluorouracil (5-FU). To investigate the adjuvant role for this combination, 5-FU, 30 mg/kg, combined with leucovorin, 15 mg/kg, was administered intraperitoneally on 3 consecutive days beginning on the same day as tumor cell inoculation in the liver. Tumor take and tumor volume were registered on day 14, followed by recording of survival time. The results of the combined treatment were compared with treatment with 5-FU alone. Leucovorin in combination with 5-FU, but not 5-FU alone, significantly reduced the tumor take compared with untreated animals (p less than 0.01). The combination also resulted in smaller tumors compared with untreated animals (p less than 0.001) or with animals given 5-FU alone (p less than 0.01). The present study supports the use of leucovorin in combination with 5-FU as adjuvant treatment of patients with primary colorectal cancer.     

Inhibition of ILK in PTEN-mutant human glioblastomas inhibits PKB/Akt activation, induces apoptosis, and delays tumor growth

The tumor suppressor gene phosphatase and tensin homologue (PTEN) regulates the phosphatidylinositol-3'-kinase (PI3K) signaling pathway and has been shown to correlate with poor prognosis in high-grade astrocytomas when mutational inactivation or loss of the PTEN gene occurs. PTEN mutation leads to constitutive activation of protein kinase B (PKB)/Akt with phosphorylation at the PKB/Akt sites Thr-308 and Ser-473. Integrin-linked kinase (ILK) has been shown to regulate PKB/Akt activity with the loss of PTEN in prostate cancer. We now demonstrate that ILK activity regulates PKB/Akt activity in glioblastoma cells. The activity of ILK is constitutively elevated in a serum-independent manner in PTEN mutant cells, and transfection of wild-type PTEN under the control of an inducible promoter into mutant PTEN cells inhibits ILK activity. Transfection of ILK antisense (ILKAS) or exposure to a small-molecule ILK inhibitor suppresses the constitutive phosphorylation of PKB/Akt on Ser-473 in PTEN-mutant glioblastoma cell lines. In addition, the delivery of ILKAS to PTEN-negative glioblastoma cells resulted in apoptosis. Rag-2M mice bearing established ( approximately 100 mg) human U87MG glioblastoma tumors, treated QD x 5 for 3 consecutive weeks with ILKAS (i.p. 5 mg/kg), exhibited stable disease with < or =7% increase in tumor volume over the 3-week course of treatment. In contrast, animals treated with an oligonucleotide control or saline exhibited a >100% increase in tumor volume over the same time period. Our initial results indicate that therapeutic strategies targeting ILK may be beneficial in the treatment of glioblastomas.     

The histone deacetylase inhibitor PXD101 increases the efficacy of irinotecan in in vitro and in vivo colon cancer models

Purpose

Histone deacetylase inhibitors (HDACIs), such as PXD101 and suberoylanilide hydroxamic acid, inhibit proliferation and stimulate apoptosis of tumor cells. The enhanced effectiveness of chemotherapy or radiotherapy when combined with HDACIs has been observed in several cancers. In this study, we investigated the antitumor effect of PXD101 combined with irinotecan in colon cancer.

Methods

HCT116 and HT29 colon cancer cells for cell viability assay were treated with PXD101 and/or SN-38, the active form of irinotecan. Antitumor effects of HCT116 and HT29 xenografts treated with these combinations were evaluated. [¹⁸F]FLT-PET was used to detect early responses to PXD101 and irinotecan in colon cancer.

Results

PXD101 and SN38 possessed dose-dependent antiproliferative activity against HCT116 and HT29 cells and exerted a synergistic effect when used in combination. In xenografted mice, PXD101 in combination with irinotecan dramatically inhibited tumor growth without causing additive toxicity. Apoptotic effects on xenograft tumors were greater with combined treatment than with irinotecan alone. [¹⁸F]FLT-PET imaging revealed a 64% decrease in [¹⁸F]FLT uptake in tumors of HCT116 xenograft-bearing mice treated with a combination of PXD101 and irinotecan, indicating a decrease in thymidine kinase 1 (TK1) activity. These results were supported by Western blot analyses showing a decrease in tumor thymidine kinase 1 protein levels, suggesting that [¹⁸F]FLT-PET can be used to non-invasively detect early responses to these agents.

Conclusions

These data show that PXD101 increases the cytotoxic activity of irinotecan in in vitro and in vivo colon cancer models and suggest these agent combinations should be explored in the treatment of colon cancer.     

Astragalus saponins induce growth inhibition and apoptosis in human colon cancer cells and tumor xenograft

Astragalus memebranaceus is used as immunomodulating agent in treating immunodeficiency diseases and to alleviate the adverse effects of chemotherapeutic drugs. In recent years, it has been proposed that Astragalus may possess anti-tumorigenic potential in certain cancer cell types. In this study, the anti-carcinogenic effects of Astragalus saponin extract were investigated in HT-29 human colon cancer cells and tumor xenograft. Our findings have shown that Astragalus saponins (AST) inhibit cell proliferation through accumulation in S phase and G2/M arrest, with concomitant suppression of p21 expression and inhibition of cyclin-dependent kinase activity. Besides, AST promotes apoptosis in HT-29 cells through caspase 3 activation and poly(ADP-ribose) polymerase cleavage, which is indicated by DNA fragmentation and nuclear chromatin condensation. Nevertheless, we also demonstrate the anti-tumorigenic effects of AST in vivo, of which the reduction of tumor volume as well as pro-apoptotic and anti-proliferative effects in HT-29 nude mice xenograft are comparable with that produced by the conventional chemotherapeutic drug 5-fluorouracil (5-FU). In addition, the side effects (body weight drop and mortality) associated with the drug combo 5-FU and oxaliplatin are not induced by AST. These results indicate that AST could be an effective chemotherapeutic agent in colon cancer treatment, which might also be used as an adjuvant in combination with other orthodox chemotherapeutic drugs to reduce the side effects of the latter compounds.