Molecular Mechanisms Of Antitumor Activity Research Articles

Synthesis, Pharmacological Properties, and Potential Molecular Mechanisms of Antitumor Activity of Betulin and Its Derivatives in Gastrointestinal Cancers.

Gastrointestinal (GI) cancers are an increasingly common type of malignancy, caused by the unhealthy lifestyles of people worldwide. Limited methods of treatment have prompted the search for new compounds with antitumor activity, in which betulin (BE) is leading the way. BE as a compound is classified as a pentacyclic triterpene of the lupane type, having three highly reactive moieties in its structure. Its mechanism of action is based on the inhibition of key components of signaling pathways associated with proliferation, migration, interleukins, and others. BE also has a number of biological properties, i.e., anti-inflammatory, hepatoprotective, neuroprotective, as well as antitumor. Due to its poor bioavailability, betulin is subjected to chemical modifications, obtaining derivatives with proven enhanced pharmacological and pharmacokinetic properties as a result. The method of synthesis and substituents significantly influence the effect on cells and GI cancers. Moreover, the cytotoxic effect is highly dependent on the derivative as well as the individual cell line. The aim of this study is to review the methods of synthesis of BE and its derivatives, as well as its pharmacological properties and molecular mechanisms of action in colorectal cancer, hepatocellular carcinoma, gastric cancer, and esophageal cancer neoplasms.

Purification, Structural Characterization, and Antitumor Activity of a Polysaccharide from Perilla Seeds.

A previous study found that a crude Perilla seed polysaccharide (PFSP) fraction exhibited obviously antitumor activity; however, the structural characterization and antitumor properties of this polysaccharide remain unclear. In this study, the PFSP was extracted and purified via combined column chromatography, and the structure of a single polysaccharide fraction was characterized by methylation, IC, GC-MS, NMR, and AFM. The results demonstrated that the efficient antitumor polysaccharide fraction PFSP-2-1 was screened from PFSP with a relative molecular weight of 8.81 × 106 Da. The primary structure of the PFSP main chain was →1)-Araf-(5→, →1,3)-Galp-(6→, →1)-Galp-(6→, →1,3)-Araf-(5→ and →1)-Xylp-(4→, and that of the side chains was →1)-Arap, →1,3)-Galp-(6→, →1)-Araf and →1)-Glcp-(4→, →1)-Galp-(3→ and →1)-Glcp, leading to a three-dimensional helical structure. CCK-8 experiments revealed that PFSP-2-1 significantly inhibited the growth of human hepatocellular carcinoma cells in vitro (p < 0.05), and its inhibitory effect positively correlation with the concentration of PFSP-2-1, and when the concentration of PFSP-2-1 was 1600 µg/mL, it showed the highest inhabitation rate on three hepatocellular carcinoma cells (HepG-2, Hep3b, and SK-Hep-1), for which the survival rates of HepG-2, Hep3b, and SK-Hep-1 were 53.34%, 70.33%, and 71.06%. This study clearly elucidated the structure and antitumor activity of PFSP-2-1, which lays a theoretical foundation for revealing the molecular mechanism of antitumor activity of Perilla seed polysaccharides and provides an important theoretical basis for the development of high-value Perilla.

Active Tumor-Targeting Nano-formulations Containing Simvastatin and Doxorubicin Inhibit Melanoma Growth and Angiogenesis.

Primary melanoma aggressiveness is determined by rapid selection and growth of cellular clones resistant to conventional treatments, resulting in metastasis and recurrence. In addition, a reprogrammed tumor-immune microenvironment supports melanoma progression and response to therapy. There is an urgent need to develop selective and specific drug delivery strategies for modulating the interaction between cancer cells and immune cells within the tumor microenvironment. This study proposes a novel combination therapy consisting of sequential administration of simvastatin incorporated in IL-13-functionalized long-circulating liposomes (IL-13-LCL-SIM) and doxorubicin encapsulated into PEG-coated extracellular vesicles (PEG-EV-DOX) to selectively target both tumor-associated macrophages and melanoma cells. To this end, IL-13 was conjugated to LCL-SIM which was obtained via the lipid film hydration method. EVs enriched from melanoma cells were passively loaded with doxorubicin. The cellular uptake of rhodamine-tagged nano-particles and the antiproliferative potential of the treatments by using the ELISA BrdU-colorimetric immunoassay were investigated in vitro. Subsequently, the therapeutic agents were administered i.v in B16.F10 melanoma-bearing mice, and tumor size was monitored during treatment. The molecular mechanisms of antitumor activity were investigated using angiogenic and inflammatory protein arrays and western blot analysis of invasion (HIF-1) and apoptosis markers (Bcl-xL and Bax). Quantification of oxidative stress marker malondialdehyde (MDA) was determined by HPLC. Immunohistochemical staining of angiogenic markers CD31 and VEGF and of pan-macrophage marker F4/80 was performed to validate our findings. The in vitro data showed that IL-13-functionalized LCL were preferentially taken up by tumor-associated macrophages and indicated that sequential administration of IL-13-LCL-SIM and PEG-EV-DOX had the strongest antiproliferative effect on tumor cells co-cultured with tumor-associated macrophages (TAMs). Accordingly, strong inhibition of tumor growth in the group treated with the sequential combination therapy was reported in vivo. Our data suggested that the antitumor action of the combined treatment was exerted through strong inhibition of several pro-angiogenic factors (VEGF, bFGF, and CD31) and oxidative stress-induced upregulation of pro-apoptotic protein Bax. This novel drug delivery strategy based on combined active targeting of both cancer cells and immune cells was able to induce a potent antitumor effect by disruption of the reciprocal interactions between TAMs and melanoma cells.

Molecular mechanisms of antitumor activity of 3-(4-chlorobenyzl)-5-isopropyl-5-phenylhydantoin in human colon cancer HCT-116 cells

Molecular mechanisms of antitumor activity of 3-(4-chlorobenyzl)-5-isopropyl-5-phenylhydantoin in human colon cancer HCT-116 cells

Multiple functions of reversine on the biological characteristics of sheep fibroblasts

Previous reports have demonstrated that Reversine can reverse differentiation of lineage-committed cells to mesenchymal stem cells and suppress tumors growth. However, the molecular mechanisms of antitumor activity and promoting cellular dedifferentiation for reversine have not yet been clearly elucidated. In the present study, it was demonstrated that reversine of 5 μM could induce multinucleated cells through cytokinesis failure rather than just arrested in G2 or M phase. Moreover, reversine reversed the differentiation of sheep fibroblasts into MSC-like style, and notably increased the expression of pluripotent marker genes Oct4 and MSCs-related surface antigens. The fibroblasts treated with reversine could transdifferentiate into all three germ layers cells in vitro. Most importantly, the induced β-like cells and hepatocytes had similar metabolic functions with normal cells in vivo. In addition, reversine promoted fibroblasts autophagy, ROS accumulation, mitochondrial dysfunction and cell apoptosis via the mitochondria mediated intrinsic pathway. The results of high-throughput RNA sequencing showed that most differentially expressed genes (DEGs) involved in Mismatch repair, Nucleotide excision repair and Base excision repair were significantly up-regulated in reversine treated fibroblasts, which means that high concentration of reversine will cause DNA damage and activate the DNA repair mechanism. In summary, reversine can increase the plasticity of sheep fibroblasts and suppress cell growth via the mitochondria mediated intrinsic pathway.

Xanthomicrol Exerts Antiangiogenic and Antitumor Effects in a Mouse Melanoma (B16F10) Allograft Model.

Xanthomicrol, a trimethoxylated hydroxyflavone, is the main active component of Dracocephalum kotschyi Boiss leaf extract. Preliminary in vitro studies identified this compound as a potential antiangiogenic and anticancer agent. This study aimed to evaluate in vivo anticancer effect of xanthomicrol and investigate its molecular mechanism of action in a mouse melanoma (B16F10) model. Effect of xanthomicrol on B16F10 melanoma cell viability was determined using the MTT assay. For in vivo experiments, C57BL/6 mice were inoculated subcutaneously with B16F10 cells. After five days, once daily administration of xanthomicrol, thalidomide, or vehicle was commenced and continued for 21 consecutive days. On the 26th day, blood samples and tumor biopsies were taken for subsequent molecular analysis. Xanthomicrol showed inhibitory effect on viability of B16F10 melanoma cells (IC50 value: 3.433 μg/ml). Initial tumor growth, tumor volume and weight, and angiogenesis were significantly decreased in xanthomicrol-treated animals compared with those in vehicle group. Protein expression of phosphorylated Akt, mRNA expressions of HIF-1α and VEGF in tumor tissues, and serum VEGF were significantly decreased in xanthomicrol-treated animals compared with vehicle-treated animals. Thus, xanthomicrol inhibited cancer cell growth both in vitro and in vivo. This effect, at least in part, was exerted by interfering with PI3K/Akt signaling pathway and inhibiting VEGF secretion by tumor cells. Further studies are required to elucidate the exact molecular mechanisms of antitumor activity of xanthomicrol.

Molecular Mechanisms Involved in the Antitumor Activity of Isolated Lectins from Marine Organisms: A Systematic Review.

Tumor cells may present several molecular alterations that favor their malignancy, among which there is the expression of tumor-related antigens, such as truncated T-glycans, Thomsen-nouvelle, sialyl-Lewis X and sialyl Tn, which may help in the diagnosis and treatment using specific target molecules. Lectins are ubiquitous proteins capable of interacting with specific carbohydrates. Lectins isolated from marine organisms have important characteristics such as low immunogenicity and can bind to complex glycans compared to plant lectins. This work evaluated, through a systematic review, the molecular mechanisms of antitumor activity of lectins isolated from marine organisms. The Pubmed, Lilacs, Science Direct, Wiley and Scopus databases were reviewed using the descriptors: marine lectin and cancer. Articles in English, published between January 2008 and December 2018, which proposed the molecular mechanisms of anticancer activity of lectins from marine organisms were eligible for the study. 17 articles were eligible. The lectins showed promising performance against cancer cells, presenting specific cytotoxicity for some types of malignant cells. The articles presented several lectins specific to different carbohydrates, modulating: pro and anti-apoptotic proteins, transcription factor E2F-1, via mitogen-activated protein kinase. In addition, exogenous lectin expression in cancer cells has been shown to be a promising way to treat cancer. This review showed the various studies that described the molecular mechanisms caused by marine lectins with antineoplastic potential. This knowledge is relevant for the development and use of the next generations of lectins isolated from marine organisms, supporting their potential in cancer treatment.

Molecular Mechanisms of Antitumor Activity of PAMAM Dendrimer Conjugates with Anticancer Drugs and a Monoclonal Antibody.

Taxanes are considered fundamental drugs in the treatment of breast cancer, but despite the similarities, docetaxel (doc) and paclitaxel (ptx) work differently. For this reason, it is interesting to identify mechanisms of antitumor activity of PAMAM dendrimer conjugates that carry docetaxel or paclitaxel and monoclonal antibody trastuzumab, specifically targeted to cells which overexpressed HER-2. For this purpose, the impact on the level of reactive oxygen species, the mitochondrial membrane potential, cell cycle distribution and the activity of caspases-3/7, -8 and -9 of PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates was determined and compared with free docetaxel and paclitaxel toward HER-2-positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines. Moreover, apoptosis and necrosis were studied using flow cytometry and confocal microscopy, respectively. Our studies show the complexity of the potential mechanism of cytotoxic action of PAMAM-drug-trastuzumab conjugates that should be sought as a resultant of oxidative stress, mitochondrial activation of the caspase cascade and the HER-2 receptor blockade.

Blocking of methionine aminopeptidase-2 by TNP-470 induces apoptosis and increases chemosensitivity of cholangiocarcinoma.

Resistance of cancer cells to chemotherapeutic drugs is a major pitfall of the failure of chemotherapy treatment for cholangiocarcinoma (CCA). A new therapeutic strategy that can improve treatment efficacy is mandatory for CCA patients. Our previous findings demonstrated the overexpression of methionine aminopeptidase-2 (MetAP2) in CCA patients. In addition, supplementation of TNP-470, a MetAP2 inhibitor, significantly inhibited the growth and metastatic activities of CCA cell lines. However, the molecular mechanism of antitumor activity of TNP-470 and the synergistic antitumor activity of TNP-470 combined with chemotherapeutic drugs are still unknown. The aim of this study is to evaluate the molecular mechanism of anticancer activity and the potential use of TNP-470 as a chemosensitizing agent in CCA cell lines. Cell cycle and apoptosis of CCA cell lines were evaluated using flow cytometry with propidium iodide staining. Expression of apoptosis regulatory proteins was measured by Western blotting. The chemosensitizing effect of TNP-470 was determined using combination index. TNP-470 inhibited the growth of CCA cells via induction of apoptosis through activation of the p38-phosphorylation and up- and down-regulation of Bax and Bcl-xL, respectively. Furthermore, TNP-470 significantly enhanced the antitumor activity of 5-fluorouracil, cisplatin, doxorubicin, and gemcitabine. The present results show that TNP-470 could be a potential therapeutic or adjuvant agent for CCA.

Solanine induced apoptosis and increased chemosensitivity to Adriamycin in T-cell acute lymphoblastic leukemia cells.

Solanine is an alkaloid and is the main extract of the traditional Chinese herb, Solanum nigrum Linn. It has been reported that Solanine has anti-inflammatory and antitumor properties. The present study aimed to investigate the antitumor effect of Solanine in Jurkat cells and demonstrate the molecular mechanism of antitumor activity of Solanine. A Cell Counting Kit-8 assay demonstrated that Solanine inhibited the proliferation of Jurkat cells in a dose-and time-dependent manner. Cell apoptosis was measured by flow cytometry. Flow cytometry revealed that Solanine induced apoptosis in a dose-dependent manner in Jurkat cells. Reverse transcription-quantitative polymerase chain reaction demonstrated that Solanine modulated the mRNA levels of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax). Additionally, Bcl-2 and Bax expression was measured using western blot analysis. Western blot analysis revealed a significant increase in the expression of Bax and decrease in the expression of Bcl-2. Solanine increased the chemosensitivity of Jurkat cells to Adriamycin. In summary, the present results indicated that the antitumor activity of Solanine was associated with inhibition of cell proliferation, induction of apoptosis and increasing cytotoxicity of Adriamycin. Therefore, Solanine may have potential as a novel agent for the treatment of acute lymphocytic leukemia.

Molecular mechanisms of antitumor activity of the polymeric form of niclosamide with respect to human colorectal cancer cells

Using poly(lactic-co-glycolic) acid a polymeric form of niclosamide (PFN) has been developed and its antitumor activity against human colorectal cancer cell lines SW837, Caco-2, COLO 320 HSR has been investigated in comparison with free niclosamide. PFN was shown to be more cytotoxic against cancer cells and less cytotoxic against normal cells (human embryonic lung fibroblasts) as compared to niclosamide. Free niclosamide and PFN share a common mechanism of the cytotoxic action on tumor cells, which is associated with mitochondrial damage (evaluated as a decrease in rhodamine 123 accumulation), and increased levels of reactive oxygen species, particularly mitochondrial superoxide anion, causing oxidative damage of intracellular targets. The action of niclosamide and PFN was accompanied by G0/G1 cell cycle arrest.

N-myc downstream-regulated gene 1 promotes oxaliplatin-triggered apoptosis in colorectal cancer cells via enhancing the ubiquitination of Bcl-2.

N-myc downstream-regulated gene1 (NDRG1) has been identified as a potent tumor suppressor gene. The molecular mechanisms of anti-tumor activity of NDRG1 involve its suppressive effects on a variety of tumorigenic signaling pathways. The purpose of this study was to investigate the role of NDRG1 in the apoptosis of colorectal cancer (CRC) cells. We first collected the clinical data of locally advanced rectal cancer (LARC) patients receiving oxaliplatin-based neoadjuvant chemotherapy in our medical center. Correlation analysis revealed that NDRG1 positively associated with the downstaging rates and prognosis of patients. Then, the effects of over-expression and depletion of NDRG1 gene on apoptosis of colorectal cancer were tested in vitro and in vivo. NDRG1 over-expression promoted apoptosis in colorectal cancer cells whereas depletion of NDRG1 resulted in resistance to oxaliplatin treatment. Furthermore, we observed that Bcl-2, a major anti-apoptotic protein, was regulated by NDRG1 at post-transcriptional level. By binding Protein kinase Cα (PKCα), a classical regulating factor of Bcl-2, NDRG1 enhanced the ubiquitination and degradation of Bcl-2, thus promoting apoptosis in CRC cells. In addition, NDRG1 inhibited tumor growth and promoted apoptosis in mouse xenograft model. In conclusion, NDRG1 promotes oxaliplatin-triggered apoptosis in colorectal cancer. Therefore, colorectal cancer patients can be stratified by the expression level of NDRG1. NDRG1-positive patients may benefit from oxaliplatin-containing chemotherapy regimens whereas those with negative NDRG1 expression should avoid the usage of this cytotoxic drug.

Molecular mechanisms of antitumor activity of niclosamide

The review summarizes literature data on the antitumor activity of niclosamide and its molecular mechanisms. Earlier niclosamide was used for treatment of intestinal parasitic infections. Recent screening of various drugs and chemical compounds, aimed at identifying putative agents with high antitumor activity, revealed that it possesses such activity. Niclosamide not only inhibits such signaling pathways as Wnt/β-cate-nin, mTORC1, Stat3, NF-κB and Notch, but also damages tumor cell mitochondria, inhibits proliferation and induces apoptosis. The effectiveness of niclosamide has been demonstrated in vitro experiments as well as in vivo models using xenotransplantation of human tumors to immunodeficient mice. Niclosamide was active not only against tumor cells but also cancer stem cells. Normal cells are resistant to niclosamide. The accumulated experimental data suggest that niclosamide is a promising pharmacological agent for the treatment of various types of cancer.

Melatonin inhibits the Migration of Colon Cancer RKO cells by Down-regulating Myosin Light Chain Kinase Expression through Cross-talk with p38 MAPK

Melatonin, which is mainly produced by the pineal gland, has a good inhibitory effect on cell growth of multiple cancer types. However, the underlying molecular mechanisms of anti-tumor activity for colon cancer have not been fully elucidated. In this study, we investigated the effects of melatonin on migration in human colon cancer RKO cells and the potential molecular mechanisms. The viability of RKO cells was investigated by MTT assay after treatment with melatonin, SB203580 (p38 inhibitor) and phorbol 12-myristate 13-acetate (PMA, MAPK activator) alone or in combination for 48h. The effects of melatonin, and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, and PMA on the migration of RKO cells were analyzed by in vitro scratch-wound assay. The relative mRNA levels of MLCK was assessed by real-time quantitative RT-PCR. Western blotting analysis was performed to examine the expression of MLCK, phosphorylation of myosin light chain (pMLC) and p38 (pp38). The proliferation and migration of human colon cancer RKO cells were inhibited significantly after treatment with melatonin. The expression levels of MLCK and phosphorylation of MLC of RKO cells were reduced, and real-time quantitative RT-PCR showed that melatonin had significant effects on suppressing the expression of MLCK. Furthermore, the phosphorylation level of p38, which showed the same trend, was also reduced when cells were treated by melatonin. In addition, ML-7 (25umol/l) could down-regulate the phosphorylation of p38. Melatonin could inhibit the proliferation and migration of RKO cells, and further experiments confirmed that p38 MAPK plays an important role in regulating melatonin-induced migration inhibition through down-regulating the expression and activity of MLCK.

GS-2, a pyrazolo[1,5-a]indole derivative with inhibitory activity of topoisomerases, exerts its potent cytotoxic activity by ROS generation

Pyrazolo[1,5-a]indole derivatives, a new type of topoisomerase (topo) inhibitor, demonstrate a broad spectrum of antitumor activities. However, the mechanism underlying the induced cytotoxicity remains unclear. In this study, we investigated whether GS-2, one of the derivatives, altered the levels of ROS in breast cancer MDA-231 cells and whether these ROS contributed to the observed antitumoral activity. Our data revealed that GS-2 caused a time- and dose-dependent elevation of intracellular ROS level in MDA-231 cells. GS-2 subsequently elicited notable inhibition on the expression of topos, DNA damage, activation of caspase-3, -9. The loss of mitochondrial membrane potential (MMP) was observed during the induction. The addition of N-acetyl cysteine (NAC, a well-known antioxidant) could effectively attenuate the GS-2-induced ROS enhancement and subsequent apoptosis. NAC attenuated the induced inhibition on expression of topos, indicating that topos might be the target of GS-2-induced ROS. The finding of the induced ROS provides new evidence for the molecular mechanisms of antitumor activity of pyrazolo[1,5-a]indole derivatives.

Molecular Mechanisms of Antitumor Activity of the Selective Inhibitor of Nuclear Export (SINE) CRM1 Antagonist KPT-185 in Mantle Cell Lymphoma.

Abstract 2438CRM1, a member of the importin b super family of nuclear transport receptors, functions as a major nuclear export factor by shuttling transcription factors including p53, p21, I-kB, and FOXO3a from nucleus to cytoplasm, thereby preventing their activity. CRM1 is also involved in the transport of rRNA and a certain subset of mRNAs including Cyclin D1. Upregulated CRM1 expression has been reported to correlate with poor prognosis in various hematopoietic malignancies. MCL is a subtype of B-cell lymphoma which is frequently resistant to standard chemotherapy. The t(11,14)(q13;32) translocation of MCL juxtaposes the cyclin D1 gene, and constitutively overexpressed cyclin D1 is believed to be associated with oncogenesis. Additional genetic events such as mutation/overexpression of TP53 have been reported as adverse prognostic indicators. TP53 mutations are rare in typical MCL, although about 30% of aggressive blastoid MCL have mt-TP53. Because of the multiple signaling pathways that are dysregulated in MCL, a novel strategy aimed at restoring multiple anti-oncogenetic pathways, especially targeting p53-independent signaling pathways, is of considerable interest.In this study, we investigated the antitumor effects and molecular mechanisms of the SINE CRM1 antagonist KPT-185 (Karyopharm Therapeutics) in 4 MCL cells with known TP53 mutation status (wt-TP53: JVM2, Z138; mt-TP53: MINO, Jeko-1).Treatment with KPT-185 resulted in reduction of cell proliferation in a concentration-dependent manner without significant differences between wt- and mt-TP53 cells (IC50 at 72hrs by trypan blue exclusion method; 35nM for Z138, 92 nM for JVM2, 96 nM for MINO, 103 nM for Jeko-1). KPT-185 exhibited limited pro- apoptotic activity in the tested MCL cells except Z138 (ED50 at 48hrs by Annexin V positivity; 62 nM for Z138, 910 nM for JVM2, 665 nM for MINO, 618 nM for Jeko-1).We then investigated KPT-185-induced TP53 target gene expression changes (24 genes) by TaqMan low density arrays (TLDA) (Applied Biosystems). In wt-TP53 JVM2 and Z138 cells, KPT-185 (100nM for Z138, MINO, and 500nM for JVM2, Jeko-1) upregulated classical p53 targets such as p21 and MDM2 mRNA (>2.0 fold), while there was no increase in mt-TP53 MINO and Jeko-1 cells. Of note, in both wt- and mt-TP53 cells, KPT-185 upregulated gene expression of PUMA which is a target of FOXO3a, p73 and p53 (3.3 fold for JVM2, 2.5 fold for Z138, 3.3 fold for MINO, 4.8 fold for Jeko-1).Recently, CRM1 has been reported to positively modulate the nuclear export of Cyclin D1 mRNA in a eIF4E-dependent manner. We therefore examined Cyclin D1 protein levels by western blot analysis, and observed significantly high baseline expression of Cyclin D1 in Z-138 cells which are highly sensitive to KPT-185, as compared to less sensitive MCL cells. KPT-185 treatment decreased Cyclin D1 expression in a dose-dependent manner (50nM and 100nM) after 12hrs of treatment accompanied by p21 induction and decreased p-Rb.These findings demonstrate that KPT-185 successfully inhibits CRM1 activity in MCL resulting in inhibition of Cyclin D1 and cell proliferation, and in the p-53-independent upregulation of pro-apoptotic PUMA. In conclusion, CRM1 inhibition by KPT-185 results in cell growth inhibition and in moderate cell death in a TP-53 independent manner. Results also suggest that the sensitivity to KPT-185 in MCL may be dependent on Cyclin D1 expression. Therefore, KPT-185may be an effective agent for the treatment of MCL. Disclosures:Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment.

Abstract C192: Molecular mechanisms of antitumor activity of the combination of E-3810 and paclitaxel in MDA-MB-231 triple-negative breast xenograft.

Abstract E-3810 is a small molecule inhibitor of VEGFR-1, -2 and -3 and FGFR-1 tyrosine kinases with IC50 &amp;lt;30nM, currently in clinical Phase 1. We have previously shown that in vivo E-3810 as a single agent (s.a.) has potent antitumor activity and strong anti-angiogenic effects (Cancer Res. 2011 Feb 15;71(4):1396–405) and that, when combined with paclitaxel (PTX) in triple negative breast cancer MDA-MB-231 xenograft, E-3810 induces complete regressions with no major safety issues; the E-3810/PTX combination was more active than brivanib/PTX and sunitinib/PTX. PK studies suggested that the increased antitumor activity of the combination was not due to an increase in PTX tumor levels ((Poster #2574, AACR, April 2011, Orlando, FL). In vitro we observed no synergy in the cytotoxic activity of E-3810 and PTX on MDA-MB-231 cells, suggesting that host mediated mechanisms cause the tumor cures in vivo. MDA-MB-231 tumor-bearing mice (n = 4; tumor weight approximately 400 mg) were treated orally with E-3810 (20mg/kg), sunitinb (40mg/kg), brivanib (100 mg/kg) or vehicle for 10 days; on day 7 (4 hours from the anti-angiogenic drug), PTX (20 mg/kg) was administered i.v. and tumor and plasma samples were collected 24 and 72 hours thereafter to evaluate early and late biological pharmacodinamic effects on tumor cells and tumor vasculature. Immuno-histochemical staining of tumor cells for the proliferation marker Ki-67 showed that the positive population was around 30%, with no major differences in the experimental groups both at 24 and 72 hours. A 2- to 20-fold increase over controls in the percentage of cellular necrosis, detected after staining with hematossilin/eosin, was observed in all groups except for PTX s.a., with the highest increase in the E-3810 s.a and the E-3810/PTX groups; a similar trend was observed in the caspase 3/7 activity, indicative of apoptosis. PTX treatment caused no modification in CD31 count compared to controls, whereas a % decrease, ranging from 63 to 90, was seen with E-3810, sunitinb and brivanib s.a and PTX combinations. There was a decreasing trend vs. vehicle (significant at 72 hours) in the collagen IV immunoreactivity only for the E-3810/PTX combination. We assessed, by commercially available ELISA kits, the plasma concentrations of soluble factors including mVEGF, mFGF, msVEGFR and msCollagen IV. Plasma mVEGF values were increased only in E-3810 s.a. and E-3810/PTX treated mice. There was a statistically significant decrease in levels of msVEGFR in mice treated with PTX, E-3810 and brivanib alone and in combination with PTX compared to control mice, while no changes were observed in mice treated with sunitinb alone and in combination with PTX. No change versus controls in the levels of mFGF was found in any of the experimental groups. An increase in plasma levels of soluble collagen IV was observed in mice treated with E-3810 alone or in combination with PTX; an opposite trend was found in mice treated with sunitinb, PTX and their combination whereas levels were unaffected by brivanib treatment as s.a and in combination. Our data suggest that the synergistic activity of the combination E-3810/PTX observed in this model relies on complex and dynamic interactions between tumor cells and host factors and that further studies are warranted to fully understand the relevant mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C192.

Paclitaxel Induces Apoptosis Through Activation of Nuclear Protein Kinase C-δ and Subsequent Activation of Golgi Associated Cdk1 in Human Hormone Refractory Prostate Cancer

Emerging evidence shows that the translocation of apoptosis related factors on cellular organelles, such as mitochondria, endoplasmic reticulum, Golgi apparatus and nucleus, has a crucial role in the apoptotic process. We characterized the effect of paclitaxel (Sigma®) on Golgi involved apoptosis in human hormone refractory prostate cancer. FACScan™ flow cytometric analysis was used to determine cell cycle distribution and the subG1 (apoptosis) population. Protein expression and localization were detected by Western blot, confocal microscopic examination and the sucrose gradient separation technique. Paclitaxel induced Golgi apparatus disassembly and interaction between Golgi complexes and mitochondria. Discontinuous sucrose gradient fractionation was used to determine and collect Golgi containing fractions. Data revealed that paclitaxel induced an increase of Cdk1 activity and DR5 expression on the Golgi complex that was associated with increased cleavage of caspase-8, a DR5 downstream factor, and caspase-3 into catalytically active fragments. Data were validated by confocal immunofluorescence microscopy. Golgi associated effects were inhibited by the Cdk1 inhibitor roscovitine (Sigma), suggesting a critical role for Golgi-Cdk1. Also, paclitaxel caused an increase of nuclear but not of Golgi associated PKC-δ activity. The selective PKC-δ inhibitor rottlerin (Sigma) completely inhibited the increase of Golgi-Cdk1 activity, suggesting that nuclear PKC-δ served as an upstream regulator of Golgi-Cdk1. Data suggest that paclitaxel induces nuclear translocation and activation of PKC-δ, which in turn causes Golgi-Cdk1 activation, leading to Golgi associated DR5 up-regulation, and caspase-8 and 3 activation. Golgi mediated signaling cascades facilitate mitochondria involved apoptotic pathways and at least partly explain the anticancer activity of paclitaxel action.

A review of lenalidomide in combination with dexamethasone for the treatment of multiple myeloma

Lenalidomide (also known as Revlimid((R)), CC-5013) is an immunomodulatory derivative of thalidomide and has more potent anti-tumor and anti-inflammatory effects than thalidomide. The molecular mechanisms of anti-tumor activity of lenalidomide have been extensively studied in multiple myeloma (MM) both preclinical models and in clinical trials. Lenalidomide: directly triggers growth arrest and/or apoptosis of drug resistant MM cells; inhibits binding of MM cells to bone marrow (BM) extracellular matrix proteins and stromal cells; modulates cytokine secretion and inhibits angiogenesis in the BM milieu; and augments host anti-tumor immunity. Lenalidomide achieved responses in patients with relapsed refractory MM. Moreover, lenalidomide with dexamethasone (Dex) demonstrates more potent anti-MM activities than Dex both in vitro and in randomized phase III clinical trials. Specifically, the combination improved overall and extent of response, as well as prolonged time to progression and overall survival, resulting in FDA approval of lenalidomide with Dex for therapy MM relapsing after prior therapy.

Antitumor Effects of the Proteasome Inhibitor Bortezomib in Medullary and Anaplastic Thyroid Carcinoma Cells in Vitro

The ubiquitin-proteasome pathway is a major pathway for degradation of intracellular proteins. Proteasome inhibitors constitute a novel class of antitumor agents with preclinical and clinical evidence of activity against hematological malignancies and solid tumors. The proteasome inhibitor bortezomib (PS-341, Velcade) has been approved by the Food and Drug Administration for the treatment of multiple myeloma and is being studied intensely in several other malignancies. Its mechanism of action is complex but appears to include the inhibition of inhibitory-kappaB degradation, which leads to inactivation of the transcriptional factor nuclear factor-kappaB (NF-kappaB). NF-kappaB has been implicated in the pathophysiology of the most aggressive forms of thyroid carcinoma, i.e. medullary and anaplastic. We evaluated the effect of bortezomib on a panel of thyroid carcinoma cell lines, originating from papillary, follicular, anaplastic, and medullary carcinomas. Bortezomib induced apoptosis in medullary and anaplastic cell lines with IC(50) values well within the range of clinically achievable concentrations and much lower than respective IC(50) values for other solid malignancies. Bortezomib inhibited NF-kappaB activity; increased p53, p21, and jun expression; and induced caspase-dependent apoptosis. Sensitivity of thyroid carcinoma cells to bortezomib was partially decreased by overexpression of Bcl-2 or treatment with IGF-I, whereas the combination of bortezomib with chemotherapy (doxorubicin) was synergistic. These data provide both insights into the molecular mechanisms of antitumor activity of proteasome inhibitors and the rationale for future clinical trials of bortezomib, alone or in combination with conventional chemotherapy, to improve patient outcome in medullary and anaplastic thyroid carcinomas.