Alopecia is a psychologically devastating complication of chemotherapy for which there is currently no effective therapy. PTH-CBD is a collagen-targeted parathyroid hormone analog that has shown promise as a therapy for alopecia disorders. To compare the efficacy of prophylactic versus therapeutic administration of PTH-CBD in chemotherapy-induced alopecia using a mouse model that mimics the cyclic chemotherapy dosing used clinically. C57BL/6J mice were treated with a single subcutaneous injection of PTH-CBD (320 mcg/kg) or vehicle control before or after hair loss developing from three courses of cyclophosphamide chemotherapy (50–150 mg/kg/week). Mice receiving chemotherapy alone developed hair loss and depigmentation over 6–12 months. Mice pretreated with PTH-CBD did not develop these changes and maintained a normal-appearing coat. Mice treated with PTH-CBD after development of hair loss showed a partial recovery. Observations of hair loss were confirmed quantitatively by gray scale analysis. Histological examination showed that in mice receiving chemotherapy alone, there were small, dystrophic hair follicles mostly in the catagen phase. Mice receiving PTH-CBD before chemotherapy showed a mix of normal-appearing telogen and anagen hair follicles with no evidence of dystrophy. Mice receiving PTH-CBD therapy after chemotherapy showed intermediate histological features. PTH-CBD was effective in both the prevention and the treatment of chemotherapy-induced alopecia in mice, but pretreatment appears to result in a better cosmetic outcome. PTH-CBD shows promise as an agent in the prevention of this complication of chemotherapy and improving the quality of life for cancer patients.
anagen VI; chemotherapy-induced alopecia; cyclophosphamide; dystrophy; PTH-CBD
We have previously prepared a prokaryotic expressed TNF-α mutant which exhibited a higher anti-tumor activity and a lower systemic toxicity compared with that of wild type TNF-α in both syngeneic murine tumor models and human tumor xenografts models. For its clinical use as an anti-tumor agent, we evaluate repeated dose toxicity, anaphylaxis, genetic toxicity, pharmacokinetic and metabolism in different animals according to the criteria for biological Investigational New Drug (IND) application. It was found to be safe at a dose of 4×106 IU/kg/day for 60 days after administration in rhesus monkeys but the TNF-α antibody level and liver toxicity needed to be monitored. No systemic anaphylaxis or genetic toxicity were found and the pharmacokinetic characteristics of the rmhTNF-α were suited for clinical use. Over 96.3% of rmhTNF-α could be reclaimed from the urine and feces in 24 hours after administration, which indicated the main excretion route. The results proved the characteristics of this rmhTNF-α satisfied clinical trial requirements. The related positive clinical trial results will be reported in future. This study of novel rmhTNF-α is of considerable importance, not only given the proven usefulness of TNF-α local application therapies under ILP (Isolated Limp Perfusion) and IHP (Isolated Hepatic Perfusion) conditions for selected indications, but also implicated for systemic application of TNF-α.
Tumor necrosis factor-alpha; Mutation; Cancer therapy; Safety evaluation; Systemic toxicity; Pharmacokinetic
Botanically derived natural products have recently become an attractive source of new chemotherapeutic agents. To explore active anti-colorectal cancer compounds, we performed phytochemical studies on Alkanna tinctoria and isolated eight quinone compounds. Using different spectrum methods, compounds were identified as alkannin (1), acetylalkannin (2), angelylalkannin (3), 5-methoxyangenylalkannin (4), dimethylacryl alkannin (5), arnebifuranone (6), alkanfuranol (7), and alkandiol (8). Compounds 4, 7, and 8 are novel compounds. The structures of the three novel compounds were elucidated based on extensive spectroscopic evidence including high-resolution mass spectrometry and NMR spectra. The antiproliferative effects of these eight compounds on HCT-116 and SW-480 human colorectal cancer cells were determined by the MTS method. Cell cycle and apoptosis were determined using flow cytometry. Enzymatic activities of caspases were determined by colorimetric assay, and interactions of compound 4 and caspase 9 were explored by docking analysis. Among the eight compounds, alkannin (1), angelylalkannin (3), and 5-methoxyangenylalkannin (4) showed strong antiproliferative effects, while compound 4 showed the most potent effects. Compound 4 arrested cancer cells in the S and G2/M phases, and significantly induced cell apoptosis. The apoptotic effects of compound 4 were supported by caspase assay and docking analysis. The structural functional relationship assay suggested that to increase anticancer potential, future modifications on alkannin (1) should focus on the hydroxyl groups at C-5 and C-8.
Alkanna tinctoria; naphthoquinones; alkannin derivatives; novel compounds; antiproliferation; cell cycle; apoptosis; caspase 9; docking analysis; colorectal cancer
Background and Aims
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Mito-carboxy proxyl (Mito-CP), a lipophilic cationic nitroxide, accumulates in the mitochondria due to the large negative transmembrane potential. Studies have shown that these agents act by disrupting the energy producing mechanism, inducing mitochondrial-mediated apoptosis, and also enhancing the action of other chemotherapeutic agents in cancer cells. We hypothesized that the combination of Mito-CP and glycolysis inhibitor, 2-deoxyglucose (2-DG), would synergistically inhibit HCC in vitro.
HepG2 cells and primary hepatocytes were treated with various combinations of Mito-CP and 2-DG. Cell cytotoxicity was measured using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, and adenosine triphosphate (ATP) bioluminescence assay. In addition, caspase 3/7 enzymatic activity was examined after treatment.
Mito-CP and 2-DG induced synergistic cytotoxicity in HepG2 cells in a dose- and time-dependent fashion, while primary cells remained viable and unaffected after treatment. The intracellular ATP levels of HepG2 cells were suppressed within 6 h of combination treatment, whereas primary cells maintained higher levels of ATP. Dose-dependent increases of caspase 3/7 activity occurred in HepG2 cells with time dependent manner, demonstrating the initiation of cell death via apoptotic pathway.
These findings indicate that a combination of Mito-CP and 2-DG effectively inhibit HCC growth in vitro. The increase in caspase 3/7 activity supports the occurrence of 2-DG and Mito-CP induced apoptotic death in HCC. Inability of the compounds to induce cytotoxicity or suppress the production of ATP in primary hepatocytes provides selective and synergistic approach for treatment of HCC.
HCC; apoptosis; Warburg effect, mitochondrial target, glycolysis inhibitor; combination therapy
There is a need for efficacious therapies for metastatic castration-resistant prostate cancer (mCRPC) after disease progression on docetaxel. The SRC tyrosine kinase and its related family members may be important drivers of prostate cancer and can be inhibited by dasatinib. mCRPC patients, after one previous chemotherapy, started dasatinib at 70mg twice daily, amended to 100mg daily. The primary endpoint was the disease control (DC) rate, defined as complete response (CR), partial response (PR), or stable disease (SD) in prostate specific antigen (PSA), RECIST, bone scan, and FACT-P score. Up to 41 patients were to be accrued (two-stage design, 21+20) to rule out a null-hypothesized effect of 5 versus 20% (α=0.05, β=0.1). Secondary endpoints included progression-free survival, toxicity, and pharmacokinetic and pharmacodynamic correlatives. Of 38 patients, 27 were evaluable for response or toxicity. The median duration of therapy was 55 days (6–284). Five patients showed DC after 8 weeks of therapy (18.5% DC, 95% CI: 6.3–38.1%). One PR (3.7% response rate, 95% CI: 0.1–19.0%) was observed in a patient treated for 284 days. Twelve patients (43%) discontinued treatment for toxicity. Dasatinib induced a decrease in phytohemagglutinin-stimulated CSF2, CD40L, GZMB, and IL-2 mRNAs in blood cells, indicating target engagement. Decreases in plasma IL-6 and bone alkaline phosphatase, and in urinary N-telopeptide, were associated with DC. Dasatinib has definite but limited activity in advanced mCRPC, and was poorly tolerated. The observation of a patient with prolonged, objective, clinically significant benefit warrants molecular profiling to select the appropriate patient population.
dasatinib; prostate cancer; SRC inhibition
Inhibition of either vascular endothelial growth factor receptor (VEGFR) or mammalian target of rapamycin (mTOR) signaling improves outcomes in patients with several advanced solid tumors. We conducted a phase I trial of temsirolimus with pazopanib to investigate the feasibility of simultaneous “vertical inhibition” of VEGFR and mTOR pathways.
Patients with advanced solid tumors, no prior pazopanib or mTOR inhibitor, good performance status and acceptable end-organ function were eligible. In a typical 3 + 3 escalation design starting at temsirolimus 15 mg by intravenous (IV) infusion weekly and pazopanib 400 mg orally daily, we defined dose-limiting toxicity (DLT) as attributable grade 3 or higher non-hematologic adverse events in the first 28-day cycle and the maximum tolerable dose as the maximum dose level at which less than 2 patients experienced DLT.
At the initial dose level, 2 patients had 4 DLTs (anorexia, fatigue, hyponatremia, hypophosphatemia). After reduction to temsirolimus 10 mg IV weekly and pazopanib 200 mg orally daily, 1 of 3 patients had DLT (fatigue) and the first patient in the subsequent expansion had dose-limiting hypophosphatemia. Attributable grade 3 or higher adverse events in more than one patient included leukopenia, neutropenia, fatigue, and hypophosphatemia. Tumor reduction not meeting RECIST criteria for partial response was the best response in 4 of 7 evaluable patients.
The combination of temsirolimus and pazopanib was not feasible at clinically meaningful doses in this population due to constitutional and electrolyte disturbances.
Pazopanib; Temsirolimus; Vertical Pathway Inhibition; Renal Cell Carcinoma; Phase I
Ovarian cancer is the fifth leading cause of cancer deaths for women in America. With no known carcinogens or manageable risk factors, targeted prevention is currently unavailable. Angioprevention is a nonspecific strategy to limit the growth of solid tumors and is especially suitable for ovarian cancers. In search of angiopreventive agents, we examined chaetoglobosin K (ChK), a natural cytochalasan compound from the fungus Diplodia macrospora. We found that ChK significantly inhibits cell viability at concentrations as low as 0.5 μmol/l for A2780/CP70 ovarian cancer cells and 1.0 μmol/l for OVCAR-3 cells. ChK also significantly inhibits the secretion of key angiogenesis mediators, including Akt (which is also known as protein kinase B), hypoxia-inducible factor 1α (HIF-1α), and vascular epithelial growth factor (VEGF) by ovarian cancer cells. More importantly, ChK inhibits in-vitro and in-vivo angiogenesis induced by ovarian cancer cells and reduces the migratory capability of human umbilical vein endothelial cells. Through transfection of HIF-1α plasmids in luciferase assays, we found that ChK executes its VEGF inhibition by mediating the downregulation of HIF-1α. Furthermore, chromatin immunoprecipitation assays using the HIF-1α antibody revealed that ChK inhibits the interaction of HIF-1α with the VEGF promoter. Through transfection of Akt plasmids, we found that inhibition of HIF-1α by ChK occurs through downregulation of Akt. To our knowledge, this is the first report about the potential angioprevention of ChK. Our data suggest that this natural fungal bioactive compound effectively inhibits angiogenesis through downregulation of VEGF-binding HIF-1α and could be an effective agent for cancer treatment.
Akt; angiogenesis; chaetoglobosin K; hypoxia-inducible factor; ovarian cancer cells; vascular epithelial growth factor
Targeting the molecular pathways associated with carcinogenesis remains the greatest opportunity to reduce treatment-related morbidity and mortality. Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as CD147, is a cell surface molecule known to promote tumor growth and angiogenesis in preclinical studies of head and neck carcinoma making it an excellent therapeutic target. To evaluate the feasibility of anti-EMMPRIN therapy, an ex-vivo human head and neck cancer model was established using specimens obtained at the time of surgery (n=22). Tumor slices were exposed to varying concentrations of anti-EMMPRIN monoclonal antibody and cetuximab for comparison purposes. Cetuximab is the only monoclonal antibody currently approved for the treatment of head and neck carcinoma. After treatment, tumor slices were assessed by immunohistochemistry and western blot analysis for apoptosis (TUNEL) and EMMPRIN expression. Of the tumor specimens 33% showed a significant reduction in mean ATP levels after treatment with cetuximab compared with untreated controls, whereas 58% of the patients responded to anti-EMMPRIN therapy (P<0.05). Samples, which showed reactivity to anti-EMMPRIN, also had greater EMMPRIN expression based on immunohistochemistry staining (49%) when compared with nonresponders (25%, P=0.06). In addition, TUNEL analysis showed a larger number of cells undergoing apoptosis in antibody-treated tumor slices (77%) compared with controls (30%, P<0.001) with activation of apoptotic proteins, caspase 3 and caspase 8. This study shows the potential of anti-EMMPRIN to inhibit proliferation and promote apoptosis and suggests its future role in the targeted treatment of head and neck carcinoma.
anti-EMMPRIN antibody; CNTO3899; ex vivo; head and neck squamous cell carcinoma; tissue slices
Neuroblastoma (NB), a childhood neoplasm arising from neural crest cells, is characterized by a diversity of clinical behaviors ranging from spontaneous remission to rapid tumor progression and death. In addition to genetic abnormalities, recent studies have indicated that epigenetic aberrations also contribute to NB pathogenesis. However, the epigenetic mechanisms underlying the pathogenesis of NB are largely unknown.
Inhibition of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) was evaluated through the measurement of H3K9Me2 levels. Cell proliferation was examined by the use of cell counting in human NB cell lines (LA1-55n, IMR-5 and NMB). The RNA expression of EHMT2, MYCN, and p21 was measured by real-time PCR. The expression of PCNA, MYCN, p53, cyclinD1, H3, H3K27M2, and H3K9Me2 was examined by Western blot analysis. In vitro invasion and the effects of the EHMT2 inhibitor (BIX-01294) were assessed in the Transwell chamber assay. Caspase-3 and -8 activities were measured by a Caspase-Glo assay kit. The level of global DNA methylation was measured by liquid chromatography-mass spectroscopy.
BIX-01294, a specific inhibitor of EHMT2 (a key enzyme for histone H3 dimethylation at lysine-9), specifically decreases global H3K9Me2 level but not H3K27Me2. The inhibition of EHMT2 decreased proliferation of NB cells and induced apoptosis by increasing caspase 8/caspase 3 activity. BIX-01294 inhibited NB cell mobility and invasion. This was accompanied with a decreased expression of the MYCN oncogene. Inhibition of EHMT2 enhanced a doxorubicin induced inhibitory effect on cell proliferation. Finally EHMT2 inhibition modulated global DNA methylation levels in NB cells.
Our results demonstrate that histone lysine methylation is involved in cell proliferation, apoptosis, cell invasion, and global DNA methylation in human NB cells. Further understanding of this mechanism may provide insight into the pathogenesis of NB progression and lead to novel treatment strategies.
EMHT2; Histone lysine methyltransferase; Cell proliferation; Global DNA methylation; Caspase; Phenotype; Neuroblastoma
A pediatric study has established a maximum tolerated dose (MTD) for temsirolimus (Tem) of more than 150 mg/m2 IV/week. A phase I trial was conducted to establish the MTD for Tem in combination with valproic acid (VPA) in children and adolescents with refractory solid tumors. Secondary aims included expression of mTOR markers on archival tumor tissue; Tem pharmacokinetics (PK); assessment of histone acetylation (HA); and tumor response.
Patients were treated with VPA (5mg/kg PO TID) with a target serum level of 75–100 mcg/mL. Tem was started at an initial dose of 60 mg/m2/week. PK and HA measurements were performed weeks 1 and 5.
Two of the first 3 subjects experienced dose-limiting toxicity (DLT) (grade 3 mucositis). Tem at 35 mg/m2/week was found to be tolerable. Peak Tem concentrations were higher in all subjects compared to those in previously published reports of single agent Tem. Increases in HA correlated with VPA levels. All tumor samples expressed mTORC1 and mTORC2. An objective response was seen in one patient (melanoma); transient stable disease was seen in 4 other patients (spinal cord ependymoma, alveolar soft part sarcoma; medullary thyroid carcinoma; hepatocellular carcinoma).
The MTD of Tem when administered with VPA is considerably lower than when used as a single agent, with mucositis the major DLT. The combination merits further study and may have activity in melanoma. Attention to drug-drug interactions will be important in future multi-agent trials including Tem.
Temsirolimus; Valproic; Pediatrics; Solid Tumors; Phase I
Tumor cells are characterized by their high rate of glycolysis and clotrimazole has been shown to disrupt the glycolysis pathway thereby arresting the cells in the G1 cell cycle phase. Herein, we present data to support our hypothesis that clotrimazole arrests tumor cells in a radiosensitizing, late G1 phase. The effects of clotrimazole were studied using the glioblastoma cell line, U-87 MG. Flow cytometry was used to analyze cell cycle redistribution and induction of apoptosis. Immunoblots were probed to characterize a late G1 cell cycle arrest. Nuclear and cytoplasmic fractions were collected to follow the clotrimazole-induced translocation of hexokinase II. Clonogenic assays were designed to determine the radiosensitizing effect by clotrimazole. Our studies have shown a dose-dependent and time-dependent clotrimazole arrest in a late G1 cell cycle phase. Concurrent with the late G1 arrest, we observed an overexpression of p27Kip along with a decreased expression of p21Cip, cyclin-dependent kinase 1, cyclin-dependent kinase 4, and cyclin D. Clotrimazole induced the translocation of mitochondrial-bound hexokinase II to the cytoplasm and the release of cytochrome c into the cytoplasm. Clotrimazole-induced apoptosis was enhanced when combined with radiation. Clotrimazole was shown to sensitize tumor cells to radiation when the cells were irradiated for 18 h post-clotrimazole treatment. The disruption of the glycolysis pathway by clotrimazole leads to cell cycle arrest of U-87 MG cells in the radiosensitizing late G1 phase. The use of clotrimazole as a radiosensitizing agent for cancer treatment is novel and may have broad therapeutic applications.
antitumor; clotrimazole; glioblastoma; hexokinase II; radiosensitizing
Src and αV integrins are both important for tumor growth and angiogenesis. They are interconnected and responsible for important features of the tumor phenotype including invasiveness, metastasis, angiogenesis and resistance to apoptosis. This study examines whether combinational inhibition of both integrin and Src pathways would exhibit greater anti-angiogenesis and anti-tumor effects than either pathway alone.
Using in vitro cell culture systems, the activity of CNTO95 (Intetumumab), an αV integrin inhibitor, and dasatinib, a Src inhibitor, on proliferation, adhesion and migration was evaluated in colon cancer cell lines, HCT-116 and RKO, as well as HUVEC cells. The effects of CNTO95 and dasatinib on the activation of Src and integrin pathway signal transduction were also measured by western blotting.
The combination of CNTO95 plus dasatinib inhibited adhesion, migration and paxillin phosphorylation in both HCT-116 and RKO cells. CNTO95 and dasatinib also led to increased apoptosis of HCT-116 cells; however, similar effects were not observed in RKO cells. In addition, dual treatment of CNTO95 and dasatinib produced enhanced effects on HUVEC cell proliferation, invasion and paxillin phosphorylation.
Our results suggest that concurrent inhibition of both the integrin and src pathways elicit more pronounced anti-angiogenic and anti-tumor effects than with either pathway being inhibited alone.
integrins; src; paxillin; FAK; colorectal cancer
Protein kinase C iota (PKCι) is overexpressed in non-small cell lung (NSCLC), ovarian and pancreatic cancers where it plays a critical role in oncogenesis. The gold compound aurothiomalate (ATM) has been shown to inhibit PKCι signaling and exhibits potent anti-tumor activity in preclinical models. We sought to determine the maximum tolerated dose (MTD) of ATM.
We conducted a phase I dose escalation trial of ATM in patients with NSCLC, ovarian or pancreatic cancer. Patients received ATM IM weekly for three cycles (cycle duration 4 weeks) at 25 mg, 50 mg or 75 mg in a 3+3 design. The dose was not escalated for individual patients. Blood samples were analyzed for elemental gold levels. Patients were evaluated every four weeks for toxicity and every eight weeks for response.
Fifteen patients were enrolled in this study. Six patients were treated at 25 mg, 7 patients at 50 mg, and 2 at 75 mg. There was 1 dose limiting toxicity at 25 mg (hypokalemia), one at 50 mg (urinary tract infection), and none at 75 mg. There were 3 grade 3 hematologic toxicities. The recommended MTD of ATM is 50 mg. Patients received treatment for a median of 2 cycles (range 1-3). There appeared to be a dose-related accumulation of steady-state plasma concentrations of gold consistent with linear pharmacokinetics.
In summary, this phase I study was successful in identifying ATM 50 mg IM weekly as the MTD. Future clinical investigations targeting PKCι are currently in progress.
protein kinase C iota; aurothiomalate; non-small cell lung cancer; ovarian cancer; pancreatic cancer
Carcinoids are neuroendocrine malignancies characterized by their over production of various bioactive hormones that lead to the carcinoid syndrome. We have previously shown that AKT serves as a key regulator of growth and phenotypic expression of tumor markers in carcinoids by genetic depletion of AKT expression. However, no small molecule inhibitor of AKT kinase activity has been developed until recently. MK-2206, a novel allosteric inhibitor of AKT, is currently undergoing clinical trials for treatment of solid tumors. In this study, we explored the effect of MK-2206 on carcinoid cell proliferation and bioactive hormone production in vitro in two carcinoid cell lines-pancreatic carcinoid BON and bronchopulmonary H727. Treatment with MK-2206 effectively suppressed AKT phosphorylation at serine 473 and significantly reduced cell proliferation in a dose dependent manner. Most importantly, MK-2206 treatment resulted in a significant reduction of ASCL1, CgA, and NSE expression, collectively recognized as markers of neuroendocrine tumor malignancy. Furthermore, MK-2206 treated cells exhibited an increase in levels of cleaved PARP and cleaved caspase-3, with a concomitant reduction in levels of Mcl-1 and XIAP, suggesting that the anti-proliferative effect of MK-2206 occurs through the induction of apoptosis. In conclusion, MK-2206 alters neuroendocrine phenotype and suppresses carcinoid tumor growth, suggesting that this drug may be beneficial for patients with carcinoid syndrome. These studies merit further clinical investigation.
carcinoids; Akt pathway; chromogranin A; neuroendocrine markers; ASCL1
Large (>6 µm) rigid microparticles (MPs) become passively entrapped within the lungs following intravenous injection making them an attractive and highly efficient alternative to inhalation for pulmonary delivery. In the current studies, PEGylated 6 μm polystyrene MPs with multiple copies of the norvaline (Nva) α-amino acid prodrug of camptothecin (CPT) were prepared. Surface morphology was characterized using a scanning electron microscope (SEM). CPT was released from the CPT-Nva-MPs over 24 hours in rat plasma at 37°C. In vivo CPT plasma concentrations were low (~1 ng/mL or less) and constant over a period of 4 days after a single intravenous injection of CPT-Nva-MPs as compared to high but short-lived systemic exposures after an IV injection of free CPT. This suggests that sustained local CPT concentrations were achieved in the lung after administration of the MP delivery system. Anti-cancer efficacy was evaluated in an orthotopic lung cancer animal model and compared to a bolus injection of CPT. Animals receiving either free CPT (2 mg/kg) or CPT-Nva-MPs (0.22 mg/kg CPT, 100 mg/kg MPs) were found to have statistically significant smaller areas of lung cancer (P<0.05, P<0.01, respectively) than untreated animals. In addition, 40% of the animals receiving CPT-Nva-MPs were found to be free of cancer. The CPT dose using targeted MPs was ten fold lower than after IV injection of free CPT but was more effective in reducing the amount of cancerous areas. In conclusion, CPT-Nva-MPs were able to achieve effective local lung and low systemic CPT concentrations at a dose that was ten times lower than systemically administered CPT resulting in a significant improvement in anticancer efficacy in an orthotopic rat model of lung cancer.
Lung cancer; microparticles; passive pulmonary targeting; camptothecin
Epigenetic aberrations and a CpG island methylator phenotype are associated with poor outcome in children with neuroblastoma (NB). Previously, we demonstrated that valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, has antitumor effects in an NB xenograft model. However, the underlying antitumor molecular mechanisms are largely unknown. In this study, we investigated the role of HDAC in cell proliferation, cell cycle progression, gene expression patterns, and epigenome in neuroblastoma. Cell proliferation, cell cycle progression, caspase activity, RNA and protein expression, quantitative methylation, and global DNA methylation were examined in NBL-W-N and LA1-55n NB cell lines. Our studies demonstrated that Inhibition of HDAC decreased NB proliferation and induced G1 growth arrest. Expression patterns of cancer-related genes were modulated by VPA. THBS1, CASP8, SPARC, CDKN1A, HIC1, CDKN1B, and HIN1 expression was upregulated, and MYCN and TIG1 were downregulated. HDAC inhibition decreased methylation levels of THBS1 and RASSF1A promoters. Inhibition of HDAC increased acetylation of histone 4 and global DNA methylation levels. Our studies demonstrated that inhibition of HDAC blocked cell proliferation and cell cycle progression in relation to alteration of cancer related genes, increasing global DNA methylation and decreasing methylation of tumor suppressor genes. Further studies investigating the anti-tumor effects of VPA in NB are warranted.
Histone deacetylase; histone acetylation; cell proliferation; cell cycle; valproic acid; neuroblastoma; DNA methylation
Breast cancer mortality is primarily due to the occurrence of metastatic disease. We have identified a novel potential therapeutic agent derived from an edible root of the plant Colocasia esculenta, commonly known as taro, that has demonstrable activity in a preclinical model of metastatic breast cancer and that should have minimal toxicity. We have shown for the first time that a water-soluble extract of taro (TE) potently inhibits lung colonizing ability as well as spontaneous metastasis from mammary gland-implanted tumors, in a murine model of highly metastatic ER, PR and Her-2/neu negative breast cancer. TE modestly inhibits proliferation of some, but not all, breast and prostate cancer cell lines. Morphologic changes including cell rounding were observed. Tumor cell migration was completely blocked by TE. TE treatment also inhibited prostaglandin E2 (PGE2) synthesis and downregulated cyclooxygenase (COX) 1 and 2 mRNA expression. We purified the active compound(s) to near homogeneity with antimetastatic activity comparable to stock TE. The active compound with a native size of approximately 25 kD contains two fragments of nearly equal size. The N-terminal amino acid sequencing of both fragments reveals that the active compound is highly related to three taro proteins; 12 kD storage protein, tarin and lectin. All are similar in terms of amino acid sequence, post-translational processing and all contain a carbohydrate-binding domain. This is the first report describing a compound(s) derived from taro, that potently and specifically inhibits tumor metastasis.
Taro; Breast cancer; Antimetastatic activity; Tumor; Cancer therapy
Peptides are receiving increased attention as therapeutic agents, due to their high binding specificity and versatility to be modified as targeting or carrier molecules. Particularly, peptides with anti-angiogenic activity are of high interest due to their applicability to a wide range of cancers. In this study we investigate the biological activity of two novel antiangiogenic peptides in pre-clinical glioma models. One peptide SP2000 is derived from collagen IV and the other peptide SP3019 belongs to the CXC family. We previously characterized the capacity of SP2000 and SP3019 to inhibit multiple biological endpoints linked to angiogenesis in human endothelial cells in several assays. Here we report additional studies using endothelial cells and focus on the activity of these peptides against human glioma cell growth, migration and adhesion in vitro and growth as tumor xenografts in vivo. We found that SP2000 completely inhibits migration of the glioma cells at 50 μM and SP3019 produced 50% inhibition at 100 μM. Their relative anti-adhesion activities were similar with SP2000 and SP3019 generating 50% adhesion inhibition at 4.9 ± 0.82 μM and 21.3 ± 5.92 μM respectively. In vivo glioma growth inhibition was 63 % for SP2000 and 76% for SP3019 after 2 weeks of administration at daily doses of 10mg/kg and 20 mg/kg, respectively. The direct activity of these peptides against glioma cells in conjunction with their anti-angiogenic activities warrants their further development as either stand-alone agents or in combination with standard cytotoxic or emerging targeted therapies in malignant brain tumors.
Angiogenesis; cancer therapy; endothelial cell; glioblastoma; proliferation; migration; adhesion
An in vitro 72 hour assay using median effect analysis and curve shift analysis was employed to evaluate the utility of potentially clinically useful combinations of agents for synergism or antagonism. Six human breast cancer cell lines both receptor rich and receptor poor were studied. Panobinostat (LBH-589), a pan histone deacetylase inhibitor with a multitude of biological effects, exhibits time dependent synergistic effects in breast cancer cell lines with docetaxel, doxorubicin, or gemcitabine in clinically relevant concentrations. Survivin expression was markedly down regulated in the presence of panobinostat with gemcitabine. Bortezomib, a proteasome inhibitor, markedly enhanced the cytotoxic effects of panobinostat combined with gemcitabine. Panobinostat did not demonstrate universal enhancement of cytotoxic drugs, and therefore synergy was dependent upon the second agent selected. No synergy was noted with anti Her2 agents in Her2 over expressing cell lines. Metformin combined with panobinostat demonstrated no synergy in this test system. These effects were confirmed by apoptosis assay and caspase-3 production. A positive drug interaction was identified. The triplet of panobinostat with either doxorubicin /carboplatin or gemcitabine / carboplatin was especially potent in all cell lines. As all these agents are clinically available, further studies of the potent combinations are warranted.
synergism; breast cancer; therapy; survivin; combination; in vitro
The objective of this study was to evaluate extracelluar matrix metalloproteinase (EMMPRIN) as a novel target in orthotopic pancreatic-cancer murine models. MIA PaCa-2 human pancreatic tumor cells were implanted in groups 1 and 3-7, while MIA PaCa-2 EMMPRIN knockdown cells were implanted in group 2. Dosing with anti-EMMPRIN antibody started immediately after implantation for groups 1-3 (residual tumor model) and at 21 days after cell implantation for groups 4-7 (established tumor model). Groups 3, 5, and 7 were treated with anti-EMMRPIN antibody (0.2-1.0 mg) twice weekly for 2-3 weeks, while the other groups served as the control. In residual tumor model, tumor growth of anti-EMMPRIN treated group was successfully arrested for 21 days (15±4 mm3), significantly lower than that of EMMPRIN knockdown group (80±15 mm3; p=0.001) or control group (240±41 mm3; p<0.001). In established tumor model, anti-EMMPRIN therapy lowered tumor-volume increase about 40% compared with control regardless of dose amount. Ki67-expressed cell densities of group 5 was 939±150 mm−2, significantly lower than that of group 4 (1709±145 mm−2; p=0.006). Microvessel density of group 5 (30±6 mm−2) was also significantly lower than that of group 4 (53±5 mm−2; p=0.014), while the microvessel size of group 5 (191±22 μm2) was significantly larger than that of group 4 (113±26 μm2; p=0.049). These data show the high potential of anti-EMMPRIN therapy for pancreatic cancer, and support its clinical translation.
EMMPRIN; Targeted therapy; Pancreatic cancer
Ovarian cancer is the most lethal gynecological malignancy among US women. Paclitaxel/carboplatin is the current drug therapy used to treat ovarian cancer, but most women develop drug resistance and recurrence of the disease, necessitating alternative strategies for treatment. A possible molecular target for cancer therapy is glycogen synthase kinase 3β (GSK3β), a downstream kinase in the Wnt signaling pathway that is overexpressed in serous ovarian cancer. Novel maleimide-based GSK3β inhibitors (GSK3βi) were synthesized, selected, and tested in vitro using SKOV3 and OVCA432 serous ovarian cancer cell lines. From a panel of 10 inhibitors, the GSK3βi 9ING41 was found to be the most effective in vitro. 9ING41 induced apoptosis as indicated by 4′6-diamidino-2-phenylindole (DAPI) positive nuclear condensation, poly (ADP-ribose) polymerase (PARP) cleavage, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The mechanism for apoptosis was through caspase-3 cleavage. GSK3βi upregulated phosphorylation of the inhibitory serine residue of GSK3β in the OVCA432 and SKOV3 cell lines as well as inhibited phosphorylation of the downstream target glycogen synthase. An in vivo xenograft study using SKOV3 cells demonstrated that tumor progression was hindered by 9ING41 in vivo. The maximum tolerated dose for 9ING41 was greater than 500 mg/kg in rats. Pharmacokinetic analysis showed 9ING41 to have a bioavailability of 4.5% and was well distributed in tissues. Therefore, GSK3β inhibitors alone or in combination with existing drugs may hinder growth of serous ovarian cancers.
Ovarian cancer; Wnt; GSK3beta; xenograft; drug discovery
Mutations/deletions of the tumor suppressor phosphatase and tensin homolog PTEN, results in PI3K/Akt pathway hyperactivation and potentially alters oncogenic responses to targeted receptor tyrosine kinase inhibitors. We previously showed that hepatocyte growth factor (HGF):c-Met pathway inhibition decreases tumor growth and oncogenic signaling responses in PTEN-null/Met+ gliomas. Here we utilize two tet-on PTENwt-inducible glioma cell lines and xenograft models to examine the influence of PTEN on oncogenic signaling responses to HGF:c-Met pathway inhibitors. Reconstitution of PTEN inhibited Akt by >80% and inhibited cell growth by ~70–75 % in both cell lines in vitro. C-Met inhibition alone inhibited in vitro cell growth by ~80–85 % and the magnitude of growth inhibition was not altered by combining PTEN reconstitution with c-Met inhibition. Combining PTEN reconstitution with Met inhibition arrested a higher percentage of cells in G1/G0 phase of the cell cycle when compared to either PTEN reconstitution or c-Met inhibition alone. Both PTEN reconstitution alone and inhibiting autocrine HGF:c-Met signaling alone, using anti-HGF mAb, robustly inhibited the growth of subcutaneous and intracranial glioma xenografts. Combining anti-HGF therapy with PTEN reconstitution did not significantly alter the magnitude of xenograft growth inhibition. Semi-quantitative immunohistopathological analyses revealed that the inhibition of glioma xenograft angiogenesis and cell proliferation by anti-HGF mAb was greatest in conjunction with PTEN reconstitution. In contrast, xenograft cell apoptosis was greatest in response to anti-HGF therapy alone and PTEN reconstitution abrogated the apoptotic response to anti-HGF therapy. These results provide new insights into how PTEN modulates glioma responses to the inhibition of HGF:c-Met signaling and possibly other receptor tyrosine kinase pathways.
hepatocyte growth factor; Akt; xenograft; apoptosis; angiogenesis
Gap junctions are intercellular channels connecting adjacent cells, allowing cells to transport small molecules. The loss of gap junctional intercellular communication (GJIC) is one of the important hallmarks of cancer. Restoration of GJIC is related to the reduction of tumorigenesis and increase in drug sensitivity. Previous reports have shown that PQ1, a quinoline derivative, increases GJIC in T47D breast cancer cells, and subsequently attenuates xenograft breast tumor growth. Combinational treatment of PQ1 and tamoxifen can lower the effective dose of tamoxifen in cancer cells. In this study, the effects of PQ1 were examined in normal C57BL/6J mice, evaluating the distribution, toxicity, and adverse effects. The distribution of PQ1 was quantified by high-performance liquid chromatography and mass spectrometry. The expressions of survivin, caspase-8, cleaved caspase-3, aryl hydrocarbon receptor (AhR), and gap junction protein, connexin 43 (Cx43), were assessed using western blot analysis. Our results showed that PQ1 was absorbed and distributed to vital organs within 1 h and the level of PQ1 decreased after 24 h. Furthermore, PQ1 increased the expression of survivin, but decreased the expression of caspase-8 and caspase-3 activity. Interestingly, the expression of AhR increased in the presence of PQ1, suggesting that PQ1 may be involved in the AhR-mediated response. Previously, PQ1 caused an increase in Cx43 expression in breast cancer cells; however, PQ1 induced a decrease in Cx43 in normal tissues. Hemotoxylin and eosin staining of the tissues showed no histological change between the treated and the untreated organs. Our studies indicate that the administration of PQ1 by an oral gavage can be achieved with low toxicity to normal vital organs.
adverse effect; anti-breast cancer agent; distribution; gap junction; PQ1; toxicity