Synergistic Effect Of Combined Treatment Research Articles

MP52-14 SYNERGISTIC EFFECTS OF COMBINATION OF DOCETAXEL AND SAHA ON AR SIGNALING IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2014MP52-14 SYNERGISTIC EFFECTS OF COMBINATION OF DOCETAXEL AND SAHA ON AR SIGNALING IN PROSTATE CANCER Kyung-Sik Han, Taekmin Kwon, In Gab Jeong, Dalsan You, Bumjin Lim, Donghyun Lee, Chunwoo Lee, Sungwoo Hong, Ha-Gyeong Kim, Jung Jin Hwang, Jun Hyuk Hong, Myung-Soo Choo, Tai Young Ahn, and Choung-Soo Kim Kyung-Sik HanKyung-Sik Han More articles by this author , Taekmin KwonTaekmin Kwon More articles by this author , In Gab JeongIn Gab Jeong More articles by this author , Dalsan YouDalsan You More articles by this author , Bumjin LimBumjin Lim More articles by this author , Donghyun LeeDonghyun Lee More articles by this author , Chunwoo LeeChunwoo Lee More articles by this author , Sungwoo HongSungwoo Hong More articles by this author , Ha-Gyeong KimHa-Gyeong Kim More articles by this author , Jung Jin HwangJung Jin Hwang More articles by this author , Jun Hyuk HongJun Hyuk Hong More articles by this author , Myung-Soo ChooMyung-Soo Choo More articles by this author , Tai Young AhnTai Young Ahn More articles by this author , and Choung-Soo KimChoung-Soo Kim More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1624AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Globally it is the sixth leading cause of cancer-related death in men, and it is now the first in the United Kingdom and second in the United States. At the early stage, androgen is required for cell growth and progression of prostate cancer. Therefore, androgen-deprivation therapy (ADT) is effective to patient with prostate cancer by inhibition of androgen synthesis. With time, however, most patients with prostate cancer eventually lose sensitivity to androgen withdrawal and progress to the development of hormone-refractory prostate cancer (HRPC) or castration-resistant prostate cancer (CRPC). Since the treatment for patient with CRPC is very limited, the novel therapeutic strategies and combination therapy are needed to overcome this resistance. Therefore, we examined whether histone deacetylase inhibitor (HDACI) might enhance the effects of docetaxel in CRPCs. Methods Hormone-independent CWR22Rv1 cells harboring androgen receptor (AR) were treated with docetaxel and/or HDACI, suberoylanilide hydroxamic acid (SAHA). Cell viability was measured with CellTiter-Glo¢ç reagent. AR signaling was analyzed by immunocytochemistry and Western blot of total cell lysates and nuclear fractionated proteins. Results The growth inhibitory effect of sequential treatment with docetaxel and SAHA was synergistic in CWR22Rv1 cells. Combination treatment decreased the levels of androgen receptor (AR), prostate specific antigen (PSA), and anti-apoptotic Bcl-2 proteins (Mcl-1, Bcl-xl) more efficiently, compared with docetaxel or SAHA alone. And combination treatment increased activation of caspase-3 remarkably, while docetaxel or SAHA alone increased it slightly. Moreover, combination treatment induced acetylation and bundling of tubulin, inhibiting nuclear accumulation of AR significantly. These synergistic effects of combination treatment were also observed in hormone-dependent LNCaP cells. Conclusions Docetaxel in combination with SAHA inhibited cell growth efficiently and induced apoptosis by repressing AR expression and translocation to nucleus, resulting in down regulation of AR signaling in hormone-independent CWR22Rv1 cells. These results suggest that combination treatment with docetaxel and SAHA can be a useful therapeutics for the treatment of CRPC patients. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e585 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Kyung-Sik Han More articles by this author Taekmin Kwon More articles by this author In Gab Jeong More articles by this author Dalsan You More articles by this author Bumjin Lim More articles by this author Donghyun Lee More articles by this author Chunwoo Lee More articles by this author Sungwoo Hong More articles by this author Ha-Gyeong Kim More articles by this author Jung Jin Hwang More articles by this author Jun Hyuk Hong More articles by this author Myung-Soo Choo More articles by this author Tai Young Ahn More articles by this author Choung-Soo Kim More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Combination of lycopene, quercetin and poloxamer 188 alleviates anxiety and depression in 3-nitropropionic acid-induced Huntington's disease in rats.

Aim:The present study investigates synergistic effect of combination treatment of lycopene, quercetin and poloxamer188 in 3-nitropropionic acid (3-NP)-induced Huntington’s disease (HD).Materials and Methods:Anxiety and depression were induced in male Wistar rat by intra-peritoneal administration of 3-NP (10 mg/kg) for 14 days. Body weight was assessed on day 1, 7 and 14, whereas locomotion, anxiety and depression were assessed at the end of the experiment.Results:Administration of 3-NP induces HD like symptoms and produced a significant decrease in body weight on day 7 (P < 0.01)and day 14 (P < 0.001), further decreased locomotion, time spent and number of entries in light area as well as increased immobility period were observed. The rats treated with lycopene and quercetin alone significantly restore the body weight and locomotion count as well as alleviate anxiety and depression. However, combination treatment of lycopene and quercetin with and without poloxamer 188 produced more significant effect on body weight compared to Huntington control rats, but no significant effect was found between the treated groups. However, significant increased on locomotion, time spent and number of entries in light area and decreased immobility period was observed in the combination treated groups when compared to single drug therapy.Conclusion:Combination treatment of lycopene and quercetin with and without poloxamer 188 in HD more effectively alleviate anxiety and depression than single drug therapy.

Alteration in the Balance of Prosurvival and Proapoptotic Signalling Pathways Leads to Sequence-Dependent Synergism Between Docetaxel and Sorafenib in Human Non-small Cell Lung Cancer Cell Lines

To examine the antiproliferative effect of the combination of docetaxel and sorafenib, applied to the representative non-small cell lung cancer cell line A549 cells either wild type or with acquired resistance to docetaxel (A549/D). The aim of this study is to evaluate the synergistic effect of combination treatment on cell growth inhibition and to elucidate the involved molecular mechanisms. A549 cells with acquired resistance to docetaxel were established by continuous exposure to docetaxel. We examined the effect of different combinatorial treatment on cell proliferation and cell cycle distribution. In addition, the effect of combinatorial treatments on proliferative and apoptotic signalling pathway were studied. Our results showed that the synergistic effect presented when A549 cells were treated with docetaxel followed by sorafenib or when A549/D cells were treated in reverse sequence. Furthermore, we suggested that synergistic effect in A549/D cells was caused by inhibiting P-gp function and altering in the balance of growth and apoptotic signalling pathways. Our data suggested a potential role of sorafenib in chemosensitizing docetaxel-resistant cancer cells. This study also provides molecular evidence for applying different therapeutic strategies for patients with different genetic and proteomic profile.

Pemetrexed downregulates ERCC1 expression and enhances cytotoxicity effected by resveratrol in human nonsmall cell lung cancer cells

The multitargeted antifolate pemetrexed has demonstrated certain clinical activities against nonsmall cell lung cancer (NSCLC). Resveratrol (3,5,4-trihydroxy-trans-stilbene) is a polyphenol found in grapes and other plants and has great potential as a preventative and therapeutic agent due to its anticarcinogenic activity. The efficacy of adding resveratrol to pemetrexed to prolong the survival of patients with NSCLC still remains unclear. The excision repair cross-complementation 1 (ERCC1) is a DNA repair gene coding 5' endonuclease in nucleotide excision repair and is overexpressed in chemo- or radioresistant carcinomas. In this study, resveratrol (10-50 μM) inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with resveratrol increased ERCC1 messenger RNA and protein levels in a MKK3/6-p38 MAPK signal activation-dependent manner. Furthermore, blocking p38 MAPK activation by SB202190 or knocking down ERCC1 expression by transfection with small interfering RNA of ERCC1 enhanced the cytotoxicity of resveratrol. Combining resveratrol with pemetrexed resulted in a synergistic cytotoxic effect, accompanied with the reduction of phospho-p38 MAPK and ERCC1 protein levels, and a DNA repair capacity. Expression of constitutively active MKK6 (MKK6E) or HA-p38 MAPK vectors significantly rescued the decreased p38 MAPK activity, and restored ERCC1 protein levels and cell survival in resveratrol and pemetrexed cotreated NSCLC cells. In this study, for the first time, we have demonstrated the synergistic effect of combined treatment with resveratrol and pemetrexed in human NSCLC cells through downregulation of the MKK3/6-p38 MAPK-ERCC1 signal, suggesting a potential benefit of combining resveratrol and pemetrexed to treat lung cancer in the future.

Synergistic effect of combined treatment with gamma-tocotrienol and statin on human malignant mesothelioma cells

The present study is the first to demonstrate the synergetic effect of statins (atorvastatin and simvastatin) and gamma-tocotrienol (γ-T3) on human malignant mesothelioma (MM). Statin+γ-T3 combinations induced greater cell growth inhibition more than each single treatment via inhibition of mevalonate pathway, a well-known target of both γ-T3 and statins. γ-T3 was necessary for endoplasmic reticulum stress markers CHOP and GRP78, whereas an intrinsic apoptotic marker, caspase 3 activation was induced only in the presence of statins. Overall, the combination of γ-T3 and statins could be useful for MM therapy and functions in a complementary style.

Treating a collagen scaffold with a low concentration of nicotine promoted angiogenesis and wound healing

BackgroundNicotine, one of the major pharmacologically active agents of cigarette smoke, has various effects on cell proliferation, and it has recently been reported to have angiogenic effects. In our previous study, we showed that the topical administration of nicotine at a low concentration accelerated wound healing. This study aimed to evaluate the efficacy of nicotine and synergistic effects of combination treatment with nicotine and basic fibroblast growth factor (bFGF) in a murine excisional wound model treated with artificial dermis. MethodsFull-thickness defects (8 mm in diameter) were created on the backs of mice, and artificial dermis was sutured to the defects. Phosphate-buffered saline (10 μL), nicotine (10−3, 10−4, or 10−5 M), bFGF (0.5 μg), and both bFGF and 10−4 M nicotine were topically administered to the artificial dermal tissue for 7 d. The mice were killed on day 14, and the wound area, neoepithelium length, and area of newly formed capillaries in the artificial dermis were evaluated. ResultsThe wound areas treated with 10−4 M nicotine, bFGF, or bFGF plus 10−4 M nicotine were significantly smaller than those in the control group. In these three groups, the neoepithelium in the bFGF plus 10−4 M nicotine group was significantly longer than that in the other groups. There was no significant difference between the neoepithelium lengths of the control and 10−5 M nicotine groups. The 10−3 M nicotine group displayed the least re-epithelization among the groups. ConclusionsIn this study, 10−4 M nicotine induced angiogenesis in, and accelerated the healing of, wounds treated with artificial dermis. bFGF and nicotine had synergistic effects, and the combined use of nicotine and bFGF is an effective wound healing method.

Reduction of Transplant Arteriosclerosis After Treatment With Mycophenolate Mofetil and Ganciclovir in a Mouse Aortic Allograft Model

Transplant arteriosclerosis is a major obstacle for long-term allograft survival in heart transplant. The aim of this study was to investigate potential synergistic effects of combined treatment with mycophenolate mofetil and ganciclovir on the development of transplant arteriosclerosis, presence of regulatory T cells, and expression of donor specific alloantibodies. Donor aortas from C57BL/6 (H2b) mice that were fully mismatched to the major histocompatibility complex were transplanted into CBA (H2k) mouse recipients. Groups of mice received mycophenolate mofetil (100 or 300 mg/kg, oral), ganciclovir (10 or 72 mg/kg, intraperitoneal), or a mycophenolate mofetil and ganciclovir combination. Grafts were analyzed by histology and morphometry on day 30 after transplant. Numbers of regulatory T cells and donor-specific alloantibodies were examined by fluorescence-activated cell sorting analysis of splenic tissue and peripheral blood. Mycophenolate mofetil (100 mg/kg) and ganciclovir (10 mg/kg and 72 mg/kg) did not show effects on transplant arteriosclerosis formation or alloantibody production. However, groups treated with mycophenolate mofetil (300 mg/kg) or a low- or high-dose mycophenolate mofetil and ganciclovir combination had significantly reduced transplant arteriosclerosis and alloantibody levels. Expression of regulatory T cells within the spleen was similar between all experimental groups and untreated controls. The combination of mycophenolate mofetil and ganciclovir significantly reduced the development of transplant arteriosclerosis in a mouse abdominal aortic allograft model. This effect may be a result of decreased alloantibody production.

Synergistic effects of combined treatment with simvastatin and exemestane on MCF-7 human breast cancer cells.

Breast cancer is associated with high levels of incidence, morbidity and mortality; therefore, the identification of effective chemopreventive strategies is crucial. It is important for clinicians to be able to identify the populations at risk who would benefit from chemoprevention, and the interventions that are effective and safe. The aim of the present study was to investigate the combined effects of simvastatin and exemestane on MCF-7 human breast cancer cells. The anti-proliferative effects of simvastatin and exemestane, alone and in combination, on the growth of MCF-7 human breast cancer cells were assessed by MTT assay. The synergism between the two drugs was determined in vitro using the combination index (CI) analysis. Cell cycle distribution and apoptosis were analyzed by flow cytometry, and alterations to the signaling pathway in MCF-7 cells were examined by immunoblotting following treatment with various regimens. The results of the MTT assay indicated that the combined treatment of simvastatin and exemestane significantly decreased the viability of MCF-7 estrogen receptor-positive (ER+) human breast cancer cells, as compared with those that were treated with the individual drugs (CI<1). In addition, coadministration of exemestane and simvastatin was shown to result in marked inhibition of tumor cell proliferation, significant cell cycle arrest at G0/G1 phase and induction of apoptosis, as compared with that of the control and individual drug-treated cells. Furthermore, the results of the present study indicated that these synergistic effects may be associated with the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein apoptotic pathway and the mitogen-activated protein kinase/mammalian target of rapamycin/p70S6 kinase growth pathway. The combination of exemestane and simvastatin generated synergistic effects on MCF-7 ER+ breast cancer cells, indicating that the combination of these drugs may be a potential therapeutic strategy for the treatment of hormone-dependent breast cancer. The combination of the two inhibitors markedly increased the efficacy, as compared with the single-agent treatment, suggesting that combination treatment could become a highly effective approach for breast cancer. The results of the present study suggested that this combination of drugs has therapeutic potential, and requires further mechanistic and biomarker investigations in clinical trials.

Synergistic Effects of Combination Treatment with Bortezomib and Doxorubicin in Human Neuroblastoma Cell Lines

Background: Neuroblastoma (NB) is the most common extracranial solid tumor in infants. Currently, the mainstay of NB chemotherapy is combination treatment with some traditional drugs, but these combination regimens are always inefficient. Methods: The aim of this study was to evaluate the inhibitory effect of a combination of doxorubicin and bortezomib, a novel anticancer drug and the first prote-asome inhibitor approved for the treatment of human malignant tumors, on the proliferation of two human NB cell lines, SK-N-SH and SH-SY5Y. The general mechanism underlying this combined effect was also investigated. Synergistic inhibitory effects on human NB cell proliferation were evaluated using the median-effect principle. The pro-apoptotic effects of these drugs were evaluated using double staining with annexin-V-FITC and propidium iodide. Results: Synergistic inhibitory effects on proliferation were observed when a combination of bortezomib and doxorubicin was applied to cultured NB cells. A similar synergistic effect on apoptosis was also observed when the two drugs were used concurrently, which suggested that the possible mechanism underlying the observed synergistic inhibitory effect might be related to apoptosis. Conclusion: The combination of bortezomib and doxorubicin appears to be a promising strategy to treat NB.

A Systems Biology Approach to Uncovering Pharmacological Synergy in Herbal Medicines with Applications to Cardiovascular Disease

Background. Clinical trials reveal that multiherb prescriptions of herbal medicine often exhibit pharmacological and therapeutic superiority in comparison to isolated single constituents. However, the synergistic mechanisms underlying this remain elusive. To address this question, a novel systems biology model integrating oral bioavailability and drug-likeness screening, target identification, and network pharmacology method has been constructed and applied to four clinically widely used herbs Radix Astragali Mongolici, Radix Puerariae Lobatae, Radix Ophiopogonis Japonici, and Radix Salviae Miltiorrhiza which exert synergistic effects of combined treatment of cardiovascular disease (CVD). Results. The results show that the structural properties of molecules in four herbs have substantial differences, and each herb can interact with significant target proteins related to CVD. Moreover, the bioactive ingredients from different herbs potentially act on the same molecular target (multiple-drug-one-target) and/or the functionally diverse targets but with potentially clinically relevant associations (multiple-drug-multiple-target-one-disease). From a molecular/systematic level, this explains why the herbs within a concoction could mutually enhance pharmacological synergy on a disease. Conclusions. The present work provides a new strategy not only for the understanding of pharmacological synergy in herbal medicine, but also for the rational discovery of potent drug/herb combinations that are individually subtherapeutic.

Synergistic Effects of Combined Disinfection Using Sanitizers and UV to Reduce the Levels of Staphylococcus aureus in Oyster Mushrooms

Synergistic effects of combined treatment of sanitizers, including ethanol, hydrogen peroxide (H2O2), sodium hypochlorite (NaOCl), and UV radiation on eliminating Staphylococcus aureus ATCC 35556 found in oyster mushrooms were evaluated. Various concentrations of ethanol (10, 30, 50, and 70%), H2O2 (100, 500, 1000, 1500, and 2000 ppm) and NaOCl (20, 60, 100, 150, and 200 ppm) were tested along with various UV exposure doses (6, 96, 216, 360, and 504 mWs/cm2). The combined sanitizers/UV treatments produced greater reductions in bacterial counts than either treatment alone. The synergistic values of combined ethanol/UV, H2O2/UV, and NaOCl/UV treatment with regards to the elimination of S. Aureus were 0.93-2.35, 0.07-0.93, and 0.06-0.89 log10 colony forming unit (CFU)/g, respectively. These results suggest that the significant synergistic benefit in eliminating food-borne pathogens on oyster mushroom results from combined sanitizer/ UV treatment.

Triptolide augments the effects of 5-lipoxygenase RNA interference in suppressing pancreatic tumor growth in a xenograft mouse model

Pancreatic cancer has one of the highest fatality rates of all cancers, and new strategies or reagents to tackle this disease are needed. Triptolide (TL) is able to potently inhibit the growth of pancreatic tumor cells in vitro. On the other hand, blockage of 5-LOX pathway might be useful for treatment of pancreatic cancer. In the current study, we tested the effects of 5-LOX RNA interference and TL individually or in combination in suppressing human pancreatic tumor growth in xenograft mouse model. 5-LOX short hairpin RNA (shRNA) vectors were developed and screened out for their efficacy in human pancreatic cancer cell line SW1990 in vitro. Their antitumor effects were also evaluated by measuring cell proliferation and apoptosis. An effective 5-LOX shRNA was given alone or in combination with TL to treat pancreatic tumor xenograft. Expression levels of 5-LOX and VEGF were measured with Western blotting and immunohistology. Knocking down 5-LOX gene suppressed cancer cell growth in vitro and intra-tumoral delivering of 5-LOX shRNA inhibited growth of transplanted tumor in vivo. TL treatment induced tumor suppression and greatly enhanced antitumor effects of 5-LOX shRNA in the mouse model. 5-LOX RNA interference or TL treatment suppresses VEGF expression in tumor tissue, and combined treatment further reduces its expression. Both treatments exerted antitumor effects in vivo, and combined use of the two approaches produced more powerful antitumor effects. Synergistic effects of combined treatment in VEGF expression may contribute to the mechanisms of the strong antitumor effects.

Therapeutic implications of the enhanced short and long-term cytotoxicity of radiation treatment followed by oncolytic Parvovirus H-1 infection in high-grade glioma cells

The prognosis of malignant brain tumors remains extremely bad in spite of moderate improvements of conventional treatments. A promising alternative approach is the use of oncolytic viruses. Strategies to improve viral toxicity include the combination of oncolytic viruses with standard therapies. Parvovirus H-1 (H-1PV) is an oncolytic virus with proven toxicity in glioma cells. Recently it has been demonstrated that the combination of ionizing radiation (IR) with H-1PV showed promising results. Previously irradiated glioma cells remained fully permissive for H-1PV induced cytotoxicity supporting the use of H-1PV for recurrent gliomas, which typically arise from irradiated cell clones. When glioma cells were infected with H-1PV shortly (24 h) after IR, cell killing improved and only the combination of both treatments lead to complete long-term tumor cell killing. The latter finding raises the question whether IR in combination with H-1PV exerts an additional therapeutic effect on highly resistant glioma stem cells. A likely translation into current clinical treatment protocols is to use stereotactic radiation of non-resectable recurrent gliomas followed by intratumoral injection of H-1PV to harvest the synergistic effects of combination treatment.

Tonic-clonic seizure following combined treatment of mycophenolate mofetil and acyclovir

Dear Editor, We have read about the anti-herpetic synergistic effect of combined treatment with mycophenolate mofetil (MMF) and acyclovir in patients with herpetic eye disease [1]. However, we encountered unexpected neuronal toxicity with this combination. A 55-year-old man was brought to the emergency department on the day of his discharge after he experienced a generalized tonic–clonic seizure. Two days before, he had undergone penetrating keratoplasty on his left eye. Fortunately, he had the seizure just in front of the hospital while he was on his way home. His vital signs were stable except mild hypertension; 157/82 mmHg. He had two additional generalized tonic–clonic seizures with loss of consciousness, as documented by a neurologist at the emergency department. The first documented seizure spontaneously subsided, but the second documented attack lasted for 1 min, and subsided after the neurologist ordered intravenous administration of lorazepam 5 mg. A computed tomography scan of the head showed no evidence of acute intracranial involvement. An electroencephalogram did not show epileptiform discharge in his brain. He denied history of seizure or general medical illness, except his eye problem. He had had recurrent herpetic keratitis in both eyes and subsequent opacity in his left eye. Three years before, he had undergone penetrating keratoplasty, but had experienced graft rejection after 6 months. One year before, he had undergone a second penetrating keratoplasty, but had experienced graft rejection again after 3 months. This operation was his third penetrating keratoplasty. Before the first penetrating keratoplasty, he had been treated with oral acyclovir (400 mg b.i.d.) for 2 months. After the first penetrating keratoplasty, he had continued with oral acyclovir and started an oral steroid (30 mg q.d.) for 1 month. After tapering of the oral steroid therapy, he continued oral acyclovir until rejection. After the second penetrating keratoplasty, he had been treated with oral acyclovir and an oral steroid, and the oral steroid was replaced with oral cyclosporine (100 mg b.i.d.). In the intervening period, he was treated with both oral steroid and cyclosporine with acyclovir. After the third penetrating keratoplasty, he had been treated with oral acyclovir (400 mg b.i.d.) and oral steroid (30mg q.d.), andMMF 1000mg (b.i.d.). On the second day after initiation of this combination, he had three sequential generalized tonic–clonic seizures with loss of consciousness. The patient had not experienced seizure attacks with any of the other previous combinations. After discontinuation ofMMF, he did not suffer anymore seizures, and did not require any anti-epileptic medication. However, he experienced graft rejection in his left eye again. In this patient, MMF or combination ofMMF and acyclovir or oral steroid must have caused the seizures, because he did not suffer from seizures before initiation or after discontinuation of MMF combined with oral acyclovir and steroid or even cyclosporine. The neurotoxicity of immunosuppressant drugs has been reported, especially in anti-metabolites [2] such as methotrexate [3]. No study has reported neurotoxicity of MMF [4, 5]; in fact there was a report of improvement of K. M. Lee :M. K. Kim :W. R. Wee : J.-h. Lee Department of Ophthalmology, Seoul National University College of Medicine, Seoul, South Korea

High-concentration glutaraldehyde fixation of bovine pericardium in organic solvent and post-fixation glycine treatment: in vitro material assessment and in vivo anticalcification effect☆

Glutaraldehdye (GA)-fixed xenografts are widely used in cardiovascular surgery. The objective of this study was to evaluate the anticalcification effect of glycine treatment and high-concentration GA fixation in organic solvent on GA-fixed bovine pericardium, and to evaluate the possible synergistic effect of combined treatment. Bovine pericardial tissues were divided into four groups according to the methods of treatment. Group 1 consisted of tissues fixed with 0.5% GA (control), group 2 fixed with 0.5% GA and post-treated with glycine, group 3 fixed with 2% GA in organic solvent (65% ethanol+5% octanol), and group 4 fixed with 2% GA in organic solvent and post-treated with glycine. The material characteristics of the treated tissues were assessed by amino acid analysis, thermal stability test, uniaxial mechanical test and light microscopy. The tissues were subcutaneously implanted into 4-week-old rats for 8 weeks, and the calcium contents of the explanted tissues were measured. Differently treated tissues resulted in no significant alterations in material characteristics and morphology as assessed by amino acid analysis, thermal stability test, uniaxial mechanical test, and light microscopy. Median calcium contents of groups 1, 2, 3, and 4 were 80.5 μg mg(-1), 1.0 μg mg(-1), 0.5 μg mg(-1) and 1.7 μg mg(-1), respectively. The calcium contents of groups 2, 3 and 4 were all significantly lower than that of group 1 (p < 0.05). Post-fixation treatment with glycine, high-concentration GA fixation in organic solvent and combined treatment of these all strongly prevented calcification of GA-fixed bovine pericardium in rat subcutaneous implantation model.

Intracellular Src Family Kinases Mediate PI3K-Akt Pathway Activation and Resistance to Lapatinib in HER2-Overexpressing Human Breast Cancer Cells.

Abstract We investigated mechanisms of resistance to the HER2 tyrosine kinase inhibitor (TKI) lapatinib (Lap) in breast cancer cells that overexpress HER2. Inhibition of phosphoinositide-3 kinase (PI3K)-Akt signaling downstream of HER2 has been proposed as required for the antitumor activity of HER2 inhibitors. Resistance to HER2 inhibitors has been associated with aberrant activation of the PI3K pathway. HER2-overexpressing breast cancer cells (BT-474, SKBR-3, HCC1954, MDA-MB-361, and SUM190) were made resistant to Lap by exposure to increasing concentrations of the drug. Lapatinib-resistant (LR) cells exhibited the same level of HER2 gene amplification as parental, sensitive cells as measured by fluorescent in situ hybridization. HER2 activity measured by Y1248 P-HER2 immunoblots was undetectable in LR cells. However, they consistently showed recovery of PI3K-Akt activity as demonstrated by phosphorylation of Akt at Ser473. We hypothesized that resistance to HER2 inhibitors results from reactivation of the PI3K-Akt axis through upregulation of alternate compensatory signaling pathways. To identify these putative escape pathways, we profiled the tyrosine (tyr) phosphoproteome using an immunoaffinity method of purification of tyr-phosphorylated peptides and identification by tandem liquid chromatography-mass spectrometry (LC-MS). Cell lysates from LR and sensitive cells were digested with trypsin and precipitated with a P-tyr antibody followed by tandem LC-MS. Peptide sequences were assigned from mass spectra using Myrimatch software and protein identities were determined by analysis with IDPicker. In this analysis, 820 spectra corresponding to 173 tyr-phosphorylated peptides were identified. Of these, 29 peptides were isolated more frequently or uniquely in LR cells based on spectral counts. To identify kinases that might be responsible for the unique phosphopeptides in the resistant cells, we used Kinase Enrichment Analysis with the 21 protein subtrates from these 29 peptides. This analysis suggested that Src or a Src family kinase (SFK) activity was enriched in LR cells. By western blot, we observed increased Y416 P-SFK in BT-474 and HCC1954 LR cells compared to drug-sensitive cells. The MS phosphotyrosine profiling identified Yes in BT-474 LR cells. This was confirmed by western analysis with Yes specific antibodies and qRT-PCR with SFK-specific primers. In HCC1954 cells, western analysis with Src and Lyn antibodies and qRT-PCR suggested that Lyn and/or Src are the active kinases. Treatment of LR cells with the small molecule Src inhibitors AZD0530 or dasatinib in combination with Lap resulted in inhibition of Y416 P-Src, partial inhibition of S473 P-Akt, and inhibition of cell growth. Treatment of HCC1954 sensitive cells with AZD0530 and Lap for 14 days partially abrogated the emergence of lapatinib-resistant colonies. These data suggest that combination of Src inhibitors with Lap will prevent or overcome therapeutic resistance to the HER2 TKI. Studies to determine 1) the mechanism whereby PI3K-Akt is activated by SFKs in the resistant cells, and 2) the synergistic effect of combination treatment with Src inhibitors and lapatinib against HER2-overexpressing xenografts are ongoing at this time. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 707.

Abstract PR-6: Src family kinases mediate PI3K-Akt pathway activation and resistance to lapatinib in HER2-overexpressing human breast cancer cells

Abstract We investigated mechanisms of resistance to the HER2 tyrosine kinase inhibitor (TKI) lapatinib (Lap) in breast cancer cells that overexpress HER2. Inhibition of phosphoinositide-3 kinase (PI3K)-Akt signaling downstream of HER2 has been proposed as required for the antitumor activity of HER2 inhibitors. Resistance to HER2 inhibitors has been associated with aberrant activation of the PI3K pathway. HER2-overexpressing breast cancer cells (BT-474, SKBR-3, HCC1954, MDA-MB-361, SUM190, and UACC893) were made resistant to Lap by exposure to increasing concentrations of the drug. Lapatinib-resistant (LR) cells exhibited the same level of HER2 gene amplification as parental, sensitive cells as measured by fluorescent in situ hybridization. HER2 activity measured by Y1248 P-HER2 immunoblots remained inhibited in LR cells. However, they consistently showed recovery of PI3K-Akt activity as demonstrated by phosphorylation of Akt at Ser473. We hypothesized that resistance to HER2 inhibitors results from reactivation of the PI3K-Akt axis through upregulation of alternate compensatory signaling pathways. To identify these putative escape pathways, we profiled the tyrosine (tyr) phosphoproteome using an immunoaffinity method of purification of tyr-phosphorylated peptides and identification by tandem liquid chromatography-mass spectrometry (LC-MS). Cell lysates from LR and sensitive cells were digested with trypsin and precipitated with a P-tyr antibody followed by tandem LC-MS. Peptide sequences were assigned from mass spectra using Myrimatch software and protein identities were determined by analysis with IDPicker. In this analysis, 820 spectra corresponding to 173 tyr-phosphorylated peptides were identified. Of these, 29 peptides were isolated more frequently or uniquely in LR cells based on spectral counts. To identify kinases that might be responsible for the unique phosphopeptides in the resistant cells, we used Kinase Enrichment Analysis with the 21 protein substrates from these 29 peptides. This analysis suggested that Src or a Src family kinase (SFK) activity was enriched in LR cells. By western blot, we observed increased Y416 P-SFK in four of the LR lines: BT-474, MDA-MB-361, HCC1954, and UACC893. The MS phosphotyrosine profiling identified Yes in BT-474 LR cells. This was confirmed by western analysis with Yes specific antibodies and qRT-PCR with SFK-specific primers. In HCC1954 cells, western analysis with Src and Lyn antibodies and qRT-PCR suggested that Lyn and/or Src are the active kinases, whereas Yes was the predominant SFK in MDA-MB-361 and UACC893 cells. Treatment of LR cells with the small molecule Src inhibitors AZD0530 or dasatinib in combination with Lap resulted in inhibition of Y416 P-Src, partial inhibition of S473 P-Akt, and inhibition of cell growth. Treatment of HCC1954 or MDA-MB-361 sensitive cells with Src inhibitors and Lap for 21 days partially abrogated the emergence of lapatinib-resistant colonies. These data suggest that combination of Src inhibitors with Lap will prevent or overcome therapeutic resistance to the HER2 TKI. Studies to determine 1) the mechanism whereby PI3K-Akt is activated by SFKs in the resistant cells, and 2) the synergistic effect of combination treatment with Src inhibitors and lapatinib against HER2-overexpressing xenografts are ongoing at this time. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):PR-6.

Synergistic immunostimulating activity of pidotimod and red ginseng acidic polysaccharide against cyclophosphamide-induced immunosuppression

We investigated the synergistic effect of combined treatment with red ginseng acidic polysaccharide (RGAP) from Panax ginseng C.A. Meyer and pidotimod in cyclophosphamide-treated mice. The combination of pidotimod and RGAP restored concanavalin A-induced splenic T cell proliferation and LPS-stimulated B cell proliferation significantly. The production of nitric oxide from peritoneal macrophages was increased by the combinations. NK cell activity was increased by RGAP alone or in combination with pidotimod. A synergistic increase in the level of serum IL-12 and interferongamm was observed when the combination of the two was used. RGAP alone or in combination with pidotimod modulated the level of serum C-reactive protein to a near-normal level. These results indicate that combinations of pidotimod and RGAP are synergistic and suggest that combination therapy using pidotimod and RGAP for improving immune activity may provide an additional benefit over the use of the two drugs by themselves.

M1823 Synergistic Intestinal Growth in Parenterally-Fed Rats Given Supplemental Enteral Nutrients (SEN) and a Low-Dose of GLP-2 Is Correlated with Greater Proglucagon and IGF-I Expression in Ileum

Background: Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent, proglucagon-derived gut hormone that stimulates intestinal adaptation through unknown pathways, possibly IGFI. Objective: To determine the mechanisms underlying the synergistic growth response to combination treatment with SEN and a low-dose of GLP-2 in parenterally-fed rats. Methods: Rats were randomized to 4 treatment groups and maintained with TPN for 7 days: total parenteral nutrition (TPN) alone, parenteral nutrition (PN)+SEN, TPN+GLP-2, PN+SEN+GLP-2 (combination treatment), n=9/group. The 2 main treatment effects are: ±GLP-2 (100 μg/kg body wt/day) and ± SEN (40% of energy needs, day4-6). Results: Single treatment with GLP-2 and combination treatment with SEN+GLP-2 induced significant enterocytes proliferation in ileum based on the number of BrdU labeled crypt cells (TPN alone=6±1, PN+SEN=5±0, TPN+GLP-2=10±1*, and PN+SEN+GLP-2=8±1*). Plasma concentrations of bioactive GLP-2 were significantly increased in TPN+GLP-2 compared to TPN alone and further increased by 22% in combination treatment suggesting a synergistic effect (SEN×GLP-2 interaction, p=0.0987). Combination treatment significantly decreased plasma dipeptidyl peptidase IV (DPPIV) activity compared to either treatment alone. There was no significant difference in plasma IGF-I level among groups. A synergistic effect of combination treatment to significantly increase proglucagon mRNA expression was noted in ileum whereas single treatment with either GLP-2 or SEN did not significantly alter proglucagon expression compared to TPN alone (TPN alone=0.04±0.02, PN+SEN=0.19±0.07, TPN+GLP-2= 0.17±0.09, and PN+SEN+GLP-2=0.33±0.14*; SEN×GLP-2 interaction, p=0.0072). Plasma concentrations of bioactive GLP-2 were positively correlated with ileal proglucagon mRNA expression in both +GLP-2 and -GLP-2 groups (p 0.44). PN+SEN did not alter IGF-I mRNA expression in jejunum and ileum compared to TPN alone whereas TPN+GLP-2 significantly increased IGF-I mRNA expression in ileum. Combination treatment significantly increased IGF-I mRNA expression in both jejunum and ileum. Jejunal and ileal IGF-I mRNA expressions were positively correlated with ileal proglucagon mRNA expression (p 0.42). Conclusions: Combination treatment with SEN+GLP-2 induced synergistic growth resulting in greater concentrations of bioactive GLP-2 in plasma and greater mucosal cellularity in ileum in parenterally-fed rats. These findings suggest that GLP2 provides a stimulus for proliferation of enterocytes that is associated with greater express of ileal proglucagon and IGF-I mRNA and a decrease in plasma DPPIV activity.

Comparison of independent and combined chronic metabolic effects of GIP and CB1 receptor blockade in high-fat fed mice

GIP receptor antagonism with (Pro3)GIP protects against obesity, insulin resistance, glucose intolerance and associated disturbances in mice fed high-fat diet. Furthermore, cannabinoid CB1 receptor antagonism with AM251 reduces appetite and body weight gain in mice. The present study has examined and compared the effects of chronic daily administrations of (Pro3)GIP (25 nmol/kg body weight), AM251 (6 mg/kg body weight) and a combination of both drugs in high-fat fed mice. Daily i.p. injection of (Pro3)GIP, AM251 or combined drug administration over 22 days significantly (P<0.05 to <0.01) decreased body weight compared with saline-treated controls. This was associated with a significant (P<0.05 to <0.01) reduction of food intake in mice treated with AM251. Plasma glucose levels and glucose tolerance were significantly (P<0.05) lowered by 22 days (Pro3)GIP, AM251 or combined drug treatment. These changes were accompanied by a significant (P<0.05) improvement of insulin sensitivity in all treatment groups. In contrast, AM251 lacked effects on glucose tolerance, metabolic response to feeding and insulin sensitivity in high-fat mice when administered acutely. These data indicate that chemical blockade of GIP- or CB1-receptor signaling using (Pro3)GIP or AM251, respectively provides an effective means of countering obesity and related abnormalities induced by consumption of high-fat energy-rich diet. AM251 lacks acute effects on glucose homeostasis and there was no evidence of a synergistic effect of combined treatment with (Pro3)GIP.