Dynamic contrast-enhanced magnetic resonance imaging of sunitinib-induced vascular changes to schedule chemotherapy in renal cell carcinoma xenograft tumors

In an attempt to develop better therapeutic approaches for metastatic renal cell carcinoma (RCC), the combination of the antiangiogenic drug sunitinib with gemcitabine was studied. Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), we have previously determined that a sunitinib dosage of 20 mg/kg per day increased kidney tumor perfusion and decreased vascular permeability in a preclinical murine RCC model. This sunitinib dosage causing regularization of tumor vessels was selected to improve delivery of gemcitabine to the tumor. DCE-MRI was used to monitor regularization of vasculature with sunitinib in kidney tumors to schedule gemcitabine. We established an effective and nontoxic schedule of sunitinib combined with gemcitabine consisting of pretreatment with sunitinib for 3 days followed by four treatments of gemcitabine at 20 mg/kg given 3 days apart while continuing daily sunitinib treatment. This treatment caused significant tumor growth inhibition resulting in small residual tumor nodules exhibiting giant tumor cells with degenerative changes, which were observed both in kidney tumors and in spontaneous lung metastases, suggesting a systemic antitumor response. The combined therapy caused a significant increase in mouse survival. DCE-MRI monitoring of vascular changes induced by sunitinib, gemcitabine, and both combined showed increased tumor perfusion and decreased vascular permeability in kidney tumors. These findings, confirmed histologically by thinning of tumor blood vessels, suggest that both sunitinib and gemcitabine exert antiangiogenic effects in addition to cytotoxic antitumor activity. These studies show that DCE-MRI can be used to select the dose and schedule of antiangiogenic drugs to schedule chemotherapy and improve its efficacy.     

Altered testicular microsomal steroidogenic enzyme activities in rats with lifetime exposure to soy isoflavones

Androgen production in the testis is carried out by the Leydig cells, which convert cholesterol into androgens. Previously, isoflavones have been shown to affect serum androgen levels and steroidogenic enzyme activities. In this study, the effects of lifelong exposure to dietary soy isoflavones on testicular microsomal steroidogenic enzyme activities were examined in the rat. F1 male rats were obtained from a multi-generational study where the parental generation was fed diets containing alcohol-washed soy protein supplemented with increasing amounts of Novasoy, a commercially available isoflavone supplement. A control group was maintained on a soy-free casein protein-based diet (AIN93G). The diets were designed to approximate human consumption levels and ranged from 0 to 1046.6 mg isoflavones/kg pelleted feed, encompassing exposures representative of North American and Asian diets as well as infant fed soy-based formula. Activities of testicular 3β-hydroxysteroid dehydrogenase (3β-HSD), P450c17 (CYP17), 17β-hydroxysteroid dehydrogenase (17β-HSD) were assayed on post natal day (PND) 28, 70, 120, 240 and 360 while 5-reducatase was assayed on PND 28. At PND 28, 3β-HSD activity was elevated by approximately 50% in rats receiving 1046.6 mg total isoflavones/kg feed compared to those on the casein only diet. A similar increase in activity was observed for CYP17 in rats receiving 235.6 mg total isoflavones/kg feed, a level representative of infant exposure through formula, compared to those receiving 0 mg isoflavones from the casein diet. These results demonstrate that rats fed a mixture of dietary soy isoflavones showed significantly altered enzyme activity profiles during development at PND 28 as a result of early exposure to isoflavones at levels obtainable by humans.     

Enhanced bioavailability of soy isoflavones by complexation with beta-cyclodextrin in rats

In order to improve the solubility and bioavailability of a soy isoflavone extract (IFE), inclusion complexes (IFE-beta-CD) of the isoflavone extract with beta-cyclodextrin (beta-CD) were prepared and studied for their solubility and bioavailability. The aqueous solubility of the complexes of IFE with beta-CD (2.0 mg/ml) was about 26 times that of IFE itself (0.076 mg/ml). The same dosages of IFE and IFE-beta-CD were orally administered to SD rats (Sprague-Dawley) on an isoflavone glycoside (IFG) basis (daidzin, genistin and glycitin), and the plasma concentrations of daidzein, genistein and glycitein were measured over time to estimate the average AUC (area under the plasma concentration versus time curve) of the isoflavones. After the oral administration, the AUC values for daidzein, genistein and glycitein were 340, 11 and 28 microg x min/ml, respectively. In contrast, the respective AUC values after the administration of IFE-beta-CD were 430, 20 and 48 microg x min/ml. The bioavailability of daidzein in IFE-beta-CD was increased to 126% by the formation of inclusion complexes with beta-CD, compared with that in IFE. Furthermore, the bioavailability of genistein and glycitein in IFE-beta-CD formulation was significantly higher by up to 180% and 170%, respectively, compared with that of IFE p=0.008 and p=0.028, respectively). These results show that the absorption of IFE could be improved by the complexation of IFE with beta-CD (IFE-beta-CD).     

Multiple complexation of cyclodextrin with soy isoflavones present in an enriched fraction

In the present study we evaluated the complexation of daidzein/genistein/glycitein, present in an isoflavone enriched fraction (IEF), with β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin (HPβCD). Based on the increased solubility and higher complexation efficiency, IEF and HPβCD solid complexes were prepared by kneading, freeze-drying, co-evaporation, spray-drying and microwave. The solid complexes were characterized using Fourier transformed-infrared spectroscopy, differential scanning calorimetry, X-ray diffraction, scanning electron microscopy, and nuclear magnetic resonance spectroscopy, and the isoflavone content and solubility were determined by liquid chromatography. The results suggest that the isoflavones daidzein, genistein and glycitein may be externally associated to HPβCD as well as that isoflavones/HPβCD inclusion complexes are formed through the insertion of B-ring into the cyclodextrin cavity. Except for the freeze-dried IEF/HPβCD solid complex, all complexes showed similar content and solubility. In conclusion, the three isoflavones showed to be able to simultaneously complex with HPβCD.     

Berberine inhibits human tongue squamous carcinoma cancer tumor growth in a murine xenograft model

Our primary studies showed that berberine induced apoptosis in human tongue cancer SCC-4 cells in vitro. But there is no report to show berberine inhibited SCC-4 cancer cells in vivo on a murine xenograft animal model. SCC-4 tumor cells were implanted into mice and groups of mice were treated with vehicle, berberine (10 mg/kg of body weight) and doxorubicin (4 mg/kg of body weight). The tested agents were injected once per four days intraperitoneally (i.p.), with treatment starting 4 weeks prior to cells inoculation. Treatment with 4 mg/kg of doxorubicin or with 10 mg/kg of berberine resulted in a reduction in tumor incidence. Tumor size in xenograft mice treated with 10 mg/kg berberine was significantly smaller than that in the control group. Our findings indicated that berbeirne inhibits tumor growth in a xenograft animal model. Therefore, berberine may represent a tongue cancer preventive agent and can be used in clinic.     

Adequate but not supplemental folic acid combined with soy isoflavones during early life improves bone health at adulthood in male mice

Previous investigations from our laboratory have demonstrated that neonatal exposure to soy isoflavones (ISO) improves bone outcomes in CD-1 mice at adulthood with greater benefits in females than males. This study determined whether early-life exposure to supplemental folic acid (FA) — that may enhance DNA methylation of target genes — in combination with ISO provides greater benefits to male bone development than ISO alone. CD-1 dams were randomized to a low (0 mg/kg diet), adequate (2 mg/kg diet) or supplemental (8 mg/kg diet) level of FA during pregnancy and lactation. Offspring received corn oil or ISO (7 mg/kg of body weight per day) from postnatal day 1–10. From weaning, males were fed adequate FA and studied to age 4 months. Offspring exposed to adequate FA+ISO had multiple benefits to bone health: higher (P<.05) bone mineral density (BMD) and greater (P<.05) resistance to fracture at the femur and lumbar spine than mice exposed to adequate FA alone. Exposure to supplemental FA+ISO resulted in higher (P<.05) serum osteoprotegerin (OPG), and a higher ratio of OPG to receptor activator for nuclear factor κβ ligand (RANKL) but did not result in greater BMD or strength at the femur or lumbar spine than supplemental FA alone. In conclusion, early-life exposure to adequate FA+ISO provided functional benefits to male bone development, while improvements induced by supplemental FA+ISO were limited to a higher level of serum OPG. Mechanistic studies are needed to better understand how FA and ISO improve bone development in male offspring.     

Pharmacokinetics of isoflavones,daidzein and genistein,after ingestion of soy beverage compared with soy extract capsules in postmenopausal Thai women

Background  

Isoflavones from soybeans may provide some beneficial impacts on postmenopausal health. The purpose of this study was to compare the pharmacokinetics and bioavailability of plasma isoflavones (daidzein and genistein) after a single dose of orally administered soy beverage and soy extract capsules in postmenopausal Thai women.     

The inhibitory effects of Endostar combined with chemotherapy on human esophageal squamous cell carcinoma xenograft in mice

The purpose of this study was to investigate the efficacy of Endostar combined with chemotherapy on human esophageal squamous cell carcinoma Eca-109 in mice. The tumor xenograft models were established and randomly assigned to 4 groups: control group, Endostar group (1.5?mg/kg?day, once daily for 3?weeks), chemotherapy group (Paclitaxel 10?mg/kg?day, Cisplatin 5?mg/kg?day, for 1, 7, 14, 21?days), and chemotherapy?+?Endostar group (combination). The length and width of tumor were measured and the tumor volumes (cm³) were calculated every other day. Three weeks later, the mice were executed, and the tumor tissues were collected to weigh and analyse the histopathology and proliferation for tumor xenograft. The results demonstrated that the tumor volumes and weight in chemotherapy?+?Endostar group were significantly lower than that in other three groups. Meanwhile, the cell proliferation of tumor xenograft in combined treatment group was significantly lower than that in other three groups. It was concluded that Endostar combined with chemotherapy could obviously enhance the inhibitory effect on esophageal squamous cell carcinoma Eca-109 in mice.     

Soy protein with or without isoflavones, soy germ and soy germ extract, and daidzein lessen plasma cholesterol levels in golden Syrian hamsters

Dietary isolated soy protein (ISP, containing approximately equal amounts of daidzein and genistein), ethanol-extracted ISP (ISP (-)), soygerm or soygerm extract (containing large amounts of daidzein and glycitein and little genistein) and the isoflavone, daidzein, were hypothesized to lessen plasma cholesterol in comparison with casein. Sixty male and 60 female golden Syrian hamsters (6-8 weeks of age) were randomly assigned to six treatments fed for 10 weeks. Four of the experimental diets (ISP, daidzein, soygerm, and soygerm extract) contained 1.3 mmol total isoflavones/kg. The ISP (-) diet contained 0.013 mmol isoflavone/kg, whereas the casein diet contained no isoflavones. Hamsters fed ISP, ISP (-), daidzein, soygerm, and soygerm extract had significantly less plasma total cholesterol (by 16%-28%), less non-HDL cholesterol (by 15%-50%) and less non-HDL/HDL cholesterol ratios compared with hamsters fed casein (P < 0.01). For male hamsters, there were no differences among treatments in plasma HDL concentrations. Female hamsters fed ISP (-) had significantly greater HDL levels (P < 0.01) than females fed casein or daidzein. Triglyceride concentration was significantly less in hamsters fed ISP (-) compared with the casein-fed females. Because soy protein with or without isoflavones, soygerm and soygerm extract, and daidzein lessened plasma cholesterol to an approximately equal extent, soy protein alone, varying mixtures of isoflavones, and other extractable components of soy are responsible for cholesterol-lessening effects of soy foods, mainly due to their effects to lessen LDL cholesterol.     

Combined effects of radiotherapy and endostatin gene therapy in melanoma tumor model

PEgr–Endostatin–EGFP plasmid was constructed to investigate its expression properties induced by ionizing irradiation and the effect of pEgr–Endostatin–EGFP gene-radiotherapy on melanoma tumor-bearing mice. The pEgr–Endostatin–EGFP plasmid was transfected into B16 cell line with liposome. The expression property of endostatin was investigated by RT-PCR and that of EGFP was detected by flow cytometry. Tumor-bearing mice were treated by the plasmid injection and 2 Gy X-irradiation of three fractions. Tumor growth was observed for 18 days after treatment. Change of tumor capillary formation was measured with histochemistry assay at the end of the experiment. The expression of GFP in B16 melanoma cells was detected after X-irradiation with 0.05–20 Gy. Time-course studies showed that the expression of GFP in B16 cells reached its peak at 8 h after irradiation with 2 Gy. The injection of pEgr–Endostatin–EGFP recombinant plasmid into the implanted B16 melanoma in C57BL/6J mice followed by local X-irradiation could significantly inhibit tumor growth with inhibition of intratumor micro-vessel density. The inhibitory effect of pEgr–Endostatin–EGFP gene-radiotherapy on the growth of B16 melanoma is correlated with the marked decrease of intratumoral vascularization. The present data point to the potential of an anti-angiogenic approach in gene-radiotherapy of cancer.     

Factorial designs combined with the steepest ascent method to optimize serum-free media for CHO cells

A serum free medium for recombinant CHO NTHU 108 cell growth and fusion protein (CD20 linked to a human IgG-Fc gamma4 fragment) synthesis were systematically developed using factorial designs combined with the steepest ascent method. Experimental results indicate that the optimal composition of serum replacement for specific fusion protein production was 1% SITE (selenium, insulin, transferrin, ethanolamine), 0.3 g/L yeast extract, and 0.09% linoleic acid-BSA. Cell growth and fusion protein production of the adapted CHO NTHU 108 cultured in Iscove's modified Dulbecco's medium supplemented with these serum substitutes were comparable to those in the Ex-Cell 301 commercial serum-free medium. These serum substitutes can also promote CHO cell growth and fusion protein production in nine kinds of commercial media. The low protein content of the developed medium facilitates downstream processing and product purification.     

Simultaneous in vivo optical quantification of key metabolic and vascular endpoints reveals tumor metabolic diversity in murine breast tumor models

Therapeutically exploiting vascular and metabolic endpoints becomes critical to translational cancer studies because altered vascularity and deregulated metabolism are two important cancer hallmarks. The metabolic and vascular phenotypes of three sibling breast tumor lines with different metastatic potential are investigated in vivo with a newly developed quantitative spectroscopy system. All tumor lines have different metabolic and vascular characteristics compared to normal tissues, and there are strong positive correlations between metabolic (glucose uptake and mitochondrial membrane potential) and vascular (oxygen saturations and hemoglobin concentrations) parameters for metastatic (4T1) tumors but not for micrometastatic (4T07) and nonmetastatic (67NR) tumors. A longitudinal study shows that both vascular and metabolic endpoints of 4T1 tumors increased up to a specific tumor size threshold beyond which these parameters decreased. The synchronous changes between metabolic and vascular parameters, along with the strong positive correlations between these endpoints suggest that 4T1 tumors rely on strong oxidative phosphorylation in addition to glycolysis. This study illustrates the great potential of our optical technique to provide valuable dynamic information about the interplay between the metabolic and vascular status of tumors, with important implications for translational cancer investigations.      

Antitumor effects of residual tumor after cryoablation: the combined effect of residual tumor and a protein-bound polysaccharide on multiple liver metastases in a murine model

Cryoablation is a low-invasive surgical treatment for malignant tumors. It may induce an immunological response leading to the eradication of distant metastases or alternatively it might promote the growth of residual tumors. In this paper we confirm the occurrence of both phenomena and we describe the preventive effect of a protein-bound polysaccharide preparation. Metastatic liver tumors were produced in BALB/c mice by the intrasplenic inoculation of colon 26 cells and cryoablation was carried out using liquid nitrogen (-170 degrees C) applied by a contact method. The value of combining cryoablation with administration of the polysaccharide preparation in the prevention of growth of residual tumors was investigated. It was shown that the number of metastatic liver nodules and the size of the primary tumor at the site of inoculation in the spleen were significantly lower when the volume that was frozen was small. The production by splenocytes of the tumor necrosis factor TNF-alpha, interferon INF-gamma, and the interleukins IL-4 and IL-10 increased significantly after freezing and thawing of the tumor tissue. The polysaccharide treatment significantly reduced the production of IL-4 and IL-10 following cryoablation; the production of TNF-alpha and INF-gamma was slightly promoted; the natural killer and cytotoxic T-cell activities of splenocytes were slightly enhanced. It was concluded that the polysaccharide preparation was beneficial by suppressing IL-4 and IL-10 production and might inhibit the growth of residual tumor that is sometimes induced by large-volume cryoablation.     

Antitumor effects of IL-6 on murine liver tumor cells in vivo

IL-6 is a pleiotropic cytokine that is capable of modulating the diverse functions of hepatocytes such as acute phase responses and inflammation in the liver. To learn its antitumor effects in vivo, the cDNA of IL-6 was transfected into murine liver cells, TIB cells. IL-6-transfected TIB cells (TIB73-IL-6 or TIB75-IL-6) produced much higher levels of IL-6 compared with vector-transfected TIB cells (TIB73-vec or TIB75-vec). To investigate the effects of IL-6 on TIB tumor growth in vivo, IL-6-transfected TIB cells or vector-transfected TIB cells were injected subcutaneously into syngeneic mice. Vector-transfected TIB cells grew rapidly 3 weeks after injection, but IL-6-transfected TIB cells did not grow at all for up to 6 weeks. Pathologically, IL-6-transfected TIB cells demonstrated a severe necrosis and apoptotic pattern. Taken together, these results indicate that IL-6 functions as a growth inhibiting factor in vivo, and another biological role of IL-6 in the liver is suggested.     

The response of murine stem spermatogonia to radiation combined with 3-aminobenzamide

The influence of 3-aminobenzamide (3-AB) on the radiation response of the stem spermatogonia of the CBA mouse has been investigated. Doses of 3-AB from 66 to 450 mg/kg, administered 1 h before irradiation, significantly enhanced stem-cell killing. Enhancement was observed when 3-AB (450 mg/kg) was given up to 5 h before, but not if administered after, irradiation. When radiation was delivered at a lower dose rate (5 cGy/min compared to 180 cGy/min) significant dose sparing was achieved for radiation alone. Pretreatment with 3-AB resulted in slightly less enhancement at the low dose rate than at the high. Split-dose studies (9 Gy total dose) with radiation alone resulted in a recovery ratio of 1.4-1.5. Administration of 3-AB before the first dose resulted in a similar recovery ratio, but if given immediately after the first dose the ratio was smaller. Pretreatment of mice with the radiosensitizer RSU-1069 indicated that at least some of the stem cells were radiobiologically hypoxic. We suggest therefore that the enhancement of spermatogonial stem-cell killing by 3-AB is not entirely due to inhibition of repair processes but may also involve modification of the oxygen status of the testis.     

The B-Raf status of tumor cells may be a significant determinant of both antitumor and anti-angiogenic effects of pazopanib in xenograft tumor models

Pazopanib is an FDA approved Vascular Endothelial Growth Factor Receptor inhibitor. We previously reported that it also inhibits tumor cell B-Raf activity in an experimental brain metastatic setting. Here, we determine the effects of different B-Raf genotypes on pazopanib efficacy, in terms of primary tumor growth and anti-angiogenesis. A panel of seven human breast cancer and melanoma cell lines harboring different mutations in the Ras-Raf pathway was implanted orthotopically in mice, and tumor growth, ERK1/2, MEK1/2 and AKT activation, and blood vessel density and permeability were analyzed. Pazopanib was significantly inhibitory to xenografts expressing either exon 11 mutations of B-Raf, or HER2 activated wild type B-Raf; no significant inhibition of a xenograft expressing the common V600E B-Raf mutation was observed. Decreased pMEK staining in the responsive tumors confirmed that B-Raf was targeted by pazopanib. Interestingly, pazopanib inhibition of tumor cell B-Raf also correlated with its anti-angiogenic activity, as quantified by vessel density and area. In conclusion, using pazopanib, tumor B-Raf status was identified as a significant determinant of both tumor growth and angiogenesis.     

Small-sample inference for incomplete longitudinal data with truncation and censoring in tumor xenograft models

In cancer drug development, demonstrating activity in xenograft models, where mice are grafted with human cancer cells, is an important step in bringing a promising compound to humans. A key outcome variable is the tumor volume measured in a given period of time for groups of mice given different doses of a single or combination anticancer regimen. However, a mouse may die before the end of a study or may be sacrificed when its tumor volume quadruples, and its tumor may be suppressed for some time and then grow back. Thus, incomplete repeated measurements arise. The incompleteness or missingness is also caused by drastic tumor shrinkage (<0.01 cm3) or random truncation. Because of the small sample sizes in these models, asymptotic inferences are usually not appropriate. We propose two parametric test procedures based on the EM algorithm and the Bayesian method to compare treatment effects among different groups while accounting for informative censoring. A real xenograft study on a new antitumor agent, temozolomide, combined with irinotecan is analyzed using the proposed methods.     

Hormonal therapy combined with radiotherapy in locally advanced prostate cancer

At present radiation therapy and radical prostatectomy are considered to be the treatment of choice for clinical T1-T2 prostate cancer. In a more advanced stage of the disease (T3) 10-year overall survival is observed in approximately 40% of patients treated with conventional radiotherapy. So far only a few methods for improving the efficacy of radiotherapy have been introduced. One of them is a three-dimensional conformal radiotherapy with 3 dimensional treatment planning. These novel methods make it possible to escalate the dose to the target and protect healthy tissue at the same time. The optimal volume of irradiation, total dose, fraction dose, techniques of radiotherapy, and the end points used during the follow-up are open to debate. In recent years a few clinical trials involving hormonal therapy and radiotherapy have been carried out. The most important of these are: RTOG 8307, RTOG 8610, RTOG 9202, and EORTC 22863.In the RTOG 8307 trial the comparison of outcomes of a combined treatment with a matched-control group of patients treated by radiotherapy alone has shown that adding hormonal therapy to radiotherapy resulted in a better outcome. Another trials RTOG 8531 and RTOG 8610 produced benefit due to the implementation of hormonal therapy in radiotherapy. The EORTC trial No. 22863 showed improvement in the 5-year overall survival when hormonal therapy after the completion of radiotherapy was continued for 3 years in the investigational arm. The RTOG 9202 study indicated benefit obtained from 2 years of adjuvant hormonal therapy.The results of these trials have had a substantial impact on the management of locally advanced prostate cancer, but there are still questions that have to be answered. There is no doubt that hormonal therapy is an important component of the management of locally advanced prostate cancer. Still the optimal combination of drugs and the timing of such treatment remains controversial. Considering the potential side effects of a combined treatment on the quality of life of patients and care costs, additional properly designed randomised trials are needed to identify the subgroup of patients who will obtain the greatest benefit. Currently, it can be concluded that in the group of patients with a high risk of relapse by adding hormonal therapy to radiotherapy the outcome of treatment in patients with prostate cancer has improved.