Candidate Anticancer Agent Research Articles

Tumor cell-specific bioluminescence platform to identify stroma-induced changes to anticancer drug activity

Conventional anti-cancer drug screening is typically performed in the absence of accessory cells of the tumor microenvironment, which can profoundly alter anti-tumor drug activity. To address this major limitation, we developed the tumor cell-specific in vitro bioluminescence imaging (CS-BLI) assay. Tumor cells (e.g. myeloma, leukemia and solid tumors) stably expressing luciferase are co-cultured with non-malignant accessory cells (e.g. stromal cells) for selective quantification of tumor cell viability, in presence vs. absence of stromal cells or drug treatment. CS-BLI is high-throughput scalable and identifies stroma-induced chemoresistance in diverse malignancies, including imatinib-resistance in leukemic cells. A stromal-induced signature in tumor cells correlates with adverse clinical prognosis and includes signatures for activated Akt, Ras, NF-κB, HIF-1α, myc, hTERT, and IRF4; signatures for biological aggressiveness and for self-renewal. Unlike conventional screening, CS-BLI can also identify agents with increased activity against tumor cells interacting with stroma. One such compound, reversine, exhibits more potent activity in an orthotopic model of diffuse myeloma bone lesions than in conventional subcutaneous xenografts. Use of CS-BLI, therefore, enables refined screening of candidate anti-cancer agents to enrich preclinical pipelines with potential therapeutics that overcome stroma-mediated drug resistance and can act in a synthetic lethal manner in the context of tumor-stromal interactions.

Anticancer effect of Lycium barbarum polysaccharides on colon cancer cells involves G0/G1 phase arrest

Colorectal cancer is one of the most common cancers worldwide. The anticancer effect of Wolfberry (Lycium barbarum) polysaccharide (LBP) on colon cancer cells is largely unknown. To investigate the growth effect of LBP on human colon cancer cell and its possible mechanisms, human colon cancer SW480 and Caco-2 cells were treated with 100-1,000 mg/l LBP for 1-8 days. Cell growth was measured by MTT assay and crystal violet assay. Distribution of the cell cycle was analyzed by flow cytometry. Western blotting was used to indicate changes in the level of cyclins and cyclin-dependent kinases (CDKs). LBP treatment inhibited both colon cancer cell lines in a dose-dependent manner. At concentrations from 400 to 1,000 mg/l, LBP significantly inhibited the growth of SW480 cells (400 mg/l, P < 0.01; 800 and 1,000 mg/l, P < 0.001); while at concentrations from 200 to 1,000 mg/l, LBP significantly inhibited the growth of Caco-2 cells (200 mg/l, P < 0.05; 400-1,000 mg/l, P < 0.001). Crystal violet assay showed that LBP had a long-term anti-proliferative effect. More importantly, cells were arrested at the G0/G1 phase. The changes in cell-cycle-associated protein, cyclins, and CDKs were consistent with the changes in cell-cycle distribution. This is one of the first studies to focus on LBP-induced interruption of the cell cycle in human colon carcinoma cells. The results suggest that LBP is a candidate anticancer agent.

Impact of Standardized Rhus verniciflua Stokes Extract as Complementary Therapy on Metastatic Colorectal Cancer: A Korean Single-Center Experience

To investigate the clinical feasibility of the standardized Rhus verniciflua Stokes (RVS) extract for the metastatic colorectal cancer (mCRC), experimentally proven to have anticancer activities. From July 2006 to November 2007, patients with conventional chemotherapy refractory mCRC were checked. After fulfilling inclusion/exclusion criteria, 36 patients were eligible for the final analysis. Overall survival and adverse events of patients treated with RVS in the aftercare period were determined. On October 21, 2008, a total of 26 patients died while the remaining 10 patients were alive with evidence of disease. The median RVS administration period was 2.7 months (95% confidence interval, 1.9-3.5). The median overall survival for the entire population was 10.9 months (95% confidence interval, 5.6-16.1) and 1-year survival rate was 44.4%, which is compatible with external controls. By survival analysis using Cox proportional hazards model, the performance status and the prior chemotherapy regimen number significantly affected overall survival. Adverse reactions to the RVS treatment were mostly mild and self-limiting. Complementary treatment with the standardized RVS extract might be beneficial for patients with mCRC, since it positively affected overall survival without significant side effects. This study suggests that RVS could be a natural anticancer agent candidate for the treatment of colorectal adenocarcinoma.

Quantification of Hormesis in Anticancer-Agent Dose-Responses

Quantitative features of dose responses were analyzed for 2,189 candidate anticancer agents in 13 strains of yeast (Saccharomyces cerevisiae). The agents represent a diverse class of chemical compounds including mustards, other alkylating agents, and antimetabolites, inter alia. Previous analyses have shown that the responses below the toxic threshold were stimulatory and poorly predicted by a threshold dose-response model, while better explained by a hormetic dose-response model. We determined the quantitative features of the hormetic concentration-responses (n = 4,548) using previously published entry and evaluative criteria. The quantitative features that are described are: (1) the width of the concentration range showing stimulation above 10% of the control (mean of 5-fold), (2) the maximum stimulation of the concentration-responses (mean of 27% above the control), and (3) the width from the maximum stimulation to the toxicological threshold (mean of 3.7-fold). These results show that 52.5% of the 2,189 chemicals evaluated display hormetic concentration-responses in at least one of the 13 yeast strains. Many chemicals showed hormesis in multiple strains, and 24 agents showed hormesis in all 13 strains. The data are compared to previously reported quantitative features of hormesis based on published literature.

Barbigerone, a Natural Isoflavone, Induces Apoptosis in Murine Lung-Cancer Cells via the Mitochondrial Apoptotic Pathway

Barbigerone is a naturally occurring isoflavone with antioxidant activity. In present study, we investigated the antitumor activity of barbigerone against murine lung cancer cells LL/2 and the possible mechanism in vitro. Our results showed that barbigerone inhibited LL/2 cells proliferation in a concentration- and time-dependent manner and caused apoptotic death of LL/2 cells. Barbigerone-induced apoptosis was characterized by enhanced mitochondrial cytochrome c release, activation of caspase-3,-9, but not caspase-8. Exposure of LL/2 cells to barbigerone resulted in upregulation of Bcl-2-associated protein (Bax) and down-regulation of Bcl-2. In addition, proliferation inhibitory effect of barbigerone was associated with decreased level of phosphorylated p42/44 mitogen-activated protein kinase (p42/44 MAPK) and phosphorylated Akt. Moreover, barbigerone exhibit less toxicity to non-cancer cells than tumor cells. In conclusion, our results indicated that barbigerone can inhibit murine lung cancer cell proliferation by inducing apoptosis via mitochondrial apoptotic pathway and by decreasing phosphorylated p42/44 MAPK and Akt. Its potential to be a candidate of anti-cancer agent is worth being further investigated.

Anti-Gout Agent Allopurinol Exerts Cytotoxicity to Human Hormone-Refractory Prostate Cancer Cells in Combination with Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand

Allopurinol has been used for the treatment of gout and conditions associated with hyperuricemia for several decades. We explored the potential of allopurinol on cancer treatment. Allopurinol did not expose cytotoxicity as a single treatment in human hormone refractory prostate cancer cell lines, PC-3 and DU145. However, allopurinol drastically induced apoptosis of PC-3 and DU145 in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is a promising candidate for anticancer agent but its efficacy is limited by the existence of resistant cancer cells. We examined the underlying mechanism by which allopurinol overcomes the resistance of prostate cancer cells to TRAIL. Allopurinol up-regulated the expression of a proapoptotic TRAIL receptor, death receptor 5 (DR5). Allopurinol increased DR5 protein, mRNA, and promoter activity. Using DR5 small interfering RNA (siRNA), we showed that allopurinol-mediated DR5 up-regulation contributed to the enhancement of TRAIL effect by allopurinol. Furthermore, we examined the mechanism of allopurinol-mediated DR5 up-regulation. DR5 promoter activity induced by allopurinol was diminished by a mutation of a CAAT/enhancer binding protein homologous protein (CHOP)-binding site. In addition, allopurinol also increased CHOP expression, suggesting that allopurinol induced DR5 expression via CHOP. Allopurinol possesses the activity of a xanthine oxidase (XO) inhibitor. We used XO siRNA instead of allopurinol. XO siRNA also up-regulated DR5 and CHOP expression and sensitized the prostate cancer cells to TRAIL-induced apoptosis. Here, we show the novel potential of allopurinol in cancer treatment and indicate that the combination of allopurinol with TRAIL is effective strategy to expand the TRAIL-mediated cancer therapy.

Inactivation of JAK/STAT Cell Signaling by SK-7041, a Novel HDAC Inhibitor, Effectively Inhibits Growth of Acute Myeloid Leukemia Cells

Activation of the JAK/STAT pathway appears common in AML, occurring in up to 70% of AML patients. Therefore, JAK/STAT signal inhibitors are promising as candidate anti-cancer agents in AML. Recently, we reported that SK-7041, an HDAC inhibitor, inhibited the growth of AML cells via activation of caspase-3 and down-regulation of cyclin D1. These findings lead us to further examine whether SK-7041 inhibits the growth of KG1 AML cells via inactivation of JAK/STAT signals. Multi-immunoblotting technique (Kinetworks™ analysis) showed that expression of p-STAT-3, p-STAT-5, and p-Erk was down-regulated in KG1 cells treated with SK-7041. These results were confirmed by individual western blot analysis. In addition, IL-6-induced activation of STAT-3 and Erk was inhibited by treatment of SK-7041. Combined treatment of SK-7041 and JAK inhibitor (AG490) showed additive anti-leukemic effect as evidenced by caspase-3 activation, down-regulation of cyclin D1 (cMYC), and inhibition of phosphorylation of STATs (−1, −3). These results suggest that HDAC inhibitor, SK-7041, inhibited AML cell growth via inactivation of JAK/STATs pathway.

FLT3, CD32, PU.1, ERG, uPAR, and TAP2 Are Strongly Associated with the Progression of Chronic Myeloid Leukemia and Combination of Small Interference RNA of FLT3 and STI571 Synergistically Induced Apoptosis of K562 Cells

To characterize molecular mechanisms by which transition from chronic phase to blast crisis in chronic myeloid leukemia (CML) for developing novel therapeutic targets, we analyzed gene-expression profiles of leukemic cells from 12 patients in chronic phase and 9 patients in blast crisis using a 8.7K cDNA chip. We identified 89 genes that were up-regulated as well as 54 genes that were down-regulated in blast crisis of CML. The expression profile included oncogenes, tumor suppression genes, and human genes encoding proteins involved in transcription, signal transduction, metabolism, cell growth, differentiation, apoptosis and immune functions. 18 genes were selected among the up-regulated group for analysis using real-time PCR. Real-time PCR data indicated that the expression of FLT3 (p < 0.001), CD32 (p < 0.001), ERG (p < 0.001), uPAR (p < 0.001), MAD (p < 0.001) and TAP2 (p < 0.001) showed statistically significant difference between chronic phase and blast crisis. For further analysis, we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human FLT3. These small interfering RNA constructs significantly inhibited FLT3 expression at mRNA and protein levels in K562 cells. After treating both the FLT3 knockdown cells and control cells (FLT3 wild type) with STI571, MTT assay and the expression patterns of apoptosis related genes (PARP, caspase-3, Bax) were examined. MTT assay and caspase-3 activity assay showed that silencing of the gene for FLT3 significantly reduced cell viability and ultimately facilitated the induction of apoptotic cell death by STI571. These findings uncovered evidence of a complex signaling network operating down-stream of FLT3 that actively contributes to tumor progression. Thus, RNA interference-directed targeting of FLT3 can be a potential candidate anticancer agent in association with STI571 against chronic myeloid leukemia.

Antitumoral and antiangiogenic activity of Synadenium umbellatum Pax

Aim of the study Synadenium umbellatum Pax (SU), a plant used in the Midwestern region of Brazil, was tested for its antitumor and antiangiogenic activities in vitro, using K-562 and Ehrlich ascites tumor (EAT) cells, and in vivo, using the EAT-bearing model. Materials and methods The viability of tumor cells was evaluated by MTT and trypan blue exclusion assays, after incubation with the ethanolic extract of SU (EESU) (0.15–20 mg/mL) or equivalent concentrations of its partitioned fractions (chloroformic, hexanic, and methanolic). In vivo studies were performed in EAT-bearing mice treated intraperitoneally with 5, 10, and 25 mg/kg of the EESU or equivalent doses of the fractions for 10 days. The methotrexate (1.5 mg/kg), for 10 days, was used as control. Results SU and fractions, except the methanolic, decreased the viability of the cells in a concentration-dependent manner. In vivo results showed a significant dose-dependent antitumoral efficacy of SU against EAT growth. The best results in prolonging life span were produced by 25 mg/kg of EESU. In these animals, the levels of vascular endothelial growth factor were markedly decreased after the treatment. Conclusions The data presented herein could open interesting perspectives for further research of SU as a candidate anticancer agent.

Increased therapeutic potential of an experimental anti-mitotic inhibitor SB715992 by genistein in PC-3 human prostate cancer cell line.

BackgroundKinesin spindle proteins (KSP) are motor proteins that play an essential role in mitotic spindle formation. HsEg5, a KSP, is responsible for the formation of the bipolar spindle, which is critical for proper cell division during mitosis. The function of HsEg5 provides a novel target for the manipulation of the cell cycle and the induction of apoptosis. SB715992, an experimental KSP inhibitor, has been shown to perturb bipolar spindle formation, thus making it an excellent candidate for anti-cancer agent. Our major objective was a) to investigate the cell growth inhibitory effects of SB715992 on PC-3 human prostate cancer cell line, b) to investigate whether the growth inhibitory effects of SB715992 could be enhanced when combined with genistein, a naturally occurring isoflavone and, c) to determine gene expression profile to establish molecular mechanism of action of SB715992.MethodsPC-3 cells were treated with varying concentration of SB715992, 30 μM of genistein, and SB715992 plus 30 μM of genistein. After treatments, PC-3 cells were assayed for cell proliferation, induction of apoptosis, and alteration in gene and protein expression using cell inhibition assay, apoptosis assay, microarray analysis, real-time RT-PCR, and Western Blot analysis.ResultsSB715992 inhibited cell proliferation and induced apoptosis in PC-3 cells. SB715992 was found to regulate the expression of genes related to the control of cell proliferation, cell cycle, cell signaling pathways, and apoptosis. In addition, our results showed that combination treatment with SB715992 and genistein caused significantly greater cell growth inhibition and induction of apoptosis compared to the effects of either agent alone.ConclusionOur results clearly show that SB715992 is a potent anti-tumor agent whose therapeutic effects could be enhanced by genistein. Hence, we believe that SB715992 could be a novel agent for the treatment of prostate cancer with greater success when combined with a non-toxic natural agent like genistein.

Biological Evaluation and Structure—Activity Relationships of Bis‐(3‐aryl‐3‐oxo‐propyl)methylamine Hydrochlorides and 4‐Aryl‐3‐arylcarbonyl‐1‐methyl‐4‐piperidinol Hydrochlorides as Potential Cytotoxic Agents and Their Alkylating Ability Towards Cellular Glutathione in Human Leukemic T Cells.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Alkylphosphocholine-induced glioma cell death is BCL-X(L)-sensitive, caspase-independent and characterized by massive cytoplasmic vacuole formation.

Alkylphosphocholines (APC) are candidate anticancer agents. We here report that APC induce the formation of large vacuoles and typical features of apoptosis in human glioma cell lines, but not in immortalized astrocytes. APC promote caspase activation, poly(ADP-ribose)-polymerase (PARP) processing and cytochrome c release from mitochondria. Adenoviral X-linked inhibitor of apoptosis (XIAP) gene transfer, or exposure to the caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoro-methylketone zVAD-fmk, blocks caspase-7 and PARP processing, but not cell death, whereas BCL-X(L) blocks not only caspase-7 and PARP processing but also cell death. APC induce changes in Delta Psi m in sensitive glioma cells, but not in resistant astrocytes. The changes in Delta Psi m are unaffected by crm-A (cowpox serpin-cytokine response modifier protein A), XIAP or zVAD-fmk, but blocked by BCL-X(L), and are thus a strong predictor of cell death in response to APC. Free radicals are induced, but not responsible for cell death. APC thus induce a characteristic morphological, BCL-X(L)-sensitive, apparently caspase-independent cell death involving mitochondrial alterations selectively in neoplastic astrocytic cells.

N-/C-terminal deleted mutant of human endostatin efficiently acts as an anti-angiogenic and anti-tumorigenic agent

Non-collagenous (NC-1) fragments at the C-terminus of basement membrane collagen IV, XV and XIII have been implicated as negative regulators of angiogenesis. Endostatin is an endogenous carboxyl-terminal fragment of collagen XVIII/NC-1, suppressing endothelial cell proliferation, migration in vitro and tumor growth in mouse models. Endostatin can bind zinc through N- and C-terminal residues. Here we demonstrate that N-/C-terminal deleted derivative of human endostatin, H5, effectively inhibited the proliferation of human umbilical vein endothelial cells (HUVECs). Also, tube formation in vitro was comparably inhibited either by H5 or endostatin. The anti-angiogenic and anti-tumorigenic activities of H5 and endostatin were confirmed by an in vivo assay using chick chorioallantoic membrane (CAM) and mouse models, respectively. Treatment of 30 ng of H5 inhibited the capillary formation in CAM by 50%. In addition, H5 exhibited more potent anti-tumor activity than wild-type endostatin in in vivo mouse metastasis assay. These results indicate that the N-/C-terminal deletion mutant would be a strong candidate of anti-cancer agent against spontaneous tumor development and growth.

Potentiated inhibition of Escherichia coli by certain combinations of agents.

Much useful information has been obtained on the modes of action of chemotherapeutic agents, including tumor-inhibiting agents, by inhibition analysis in bacterial systems. In a number of cases the mode of action appears to be the same in bacterial and mammalian systems. Therefore, it is not unreasonable to test agents or combinations of agents against bacteria to serve as a guide for tests in mammals. In this laboratory, candidate anticancer agents are tested singly and in combinations as growth inhibitors for bacteria prior to testing against neoplasms in animals. This report presents some of the data that have been obtained for combinations of agents in tests with bacteria. Methods. To screen for potentiating growth-inhibitory activity, a simple test method was devised using Escherichia coli (ATCC No. 9637) as the test organism. The use of this organism grown in a simple, chemically defined glucose-salts medium is ideal for biochemical studies of this kind because the observed action of specific inhibitors or antimetabolites is not obscured by the presence in the medium of the chemically complex metabolites required in similar studies on more fastidious organisms. Duplicate paper discs saturated with known concentrations of a candidate compound were placed on the surfaces of minimal agar cultures of E. coli in a control plate and in a test plate. The control plate contained no test compound, whereas the test plate contained a subinhibitory concentration of the second member of the pair of candidate compounds being tested for potentiation. If the diameter of the zone of inhibition around the paper disc on the test plate was greater than 2 times that on the control plate, then the combination was considered worthy of further study. Of approximately one thousand pairs of compounds tested, approximately one hundred pairs gave evidence of significant additive or potentiated inhibition.