Linghua Meng

A Series of α-Heterocyclic Carboxaldehyde Thiosemicarbazones Inhibit Topoisomerase IIα Catalytic Activity

A series of novel thiosemicarbazone derivatives bearing condensed heterocyclic carboxaldehyde moieties were designed and synthesized. Among them, TSC24 exhibited broad antiproliferative activity in a panel of human tumor cells and suppressed tumor growth in mice. The mechanism research revealed that TSC24 was not only an iron chelator but also a topoisomerase IIalpha catalytic inhibitor. Its inhibition on topoisomerase IIalpha was due to direct interaction with the ATPase domain of topoisomerase IIalpha which led to the block of ATP hydrolysis. Molecular docking predicted that TSC24 might bind at the ATP binding site, which was confirmed by the competitive inhibition assay. These results about the mechanisms involved in the anticancer activities of thiosemicarbazones will aid in the rational design of novel topoisomerase II-targeted drugs and will provide insights into the discovery and development of novel cancer therapeutics based on the dual activity to chelate iron and to inhibit the catalytic activity of topoisomerase IIalpha.

Gambogic acid inhibits the catalytic activity of human topoisomerase IIα by binding to its ATPase domain

This study is intended to characterize the cellular target of gambogic acid (GA), a natural product isolated from the gamboge resin of Garcinia hurburyi tree, which possesses potent in vitro and in vivo antitumor activities. The antiproliferative activity of GA was further confirmed here in a panel of human tumor cells and multidrug-resistant cells. We found that GA significantly inhibited the catalytic activity of topoisomerase (Topo) II and, to a comparatively less extent, of Topo I, without trapping and stabilizing covalent topoisomerase-DNA cleavage complexes. Down-regulation of Topo IIα but not Topo I and Topo IIβ, reduced GA-induced apoptosis and the phosphorylation of c-Jun, and restored cell proliferation upon GA treatment. Moreover, GA antagonized etoposide-induced DNA damage and abrogated the antiproliferative activity of etoposide, whereas it did not affect camptothecin-induced DNA damage. By dissecting the actions of GA on the individual steps of Topo IIα catalytic cycle, we found that GA inhibited DNA cleavage and ATP hydrolysis. Moreover, GA directly bound to the ATPase domain of Topo IIα, and may share common binding sites with ATP. The results reported here show that GA exerts its antiproliferative effect by inhibiting the catalytic activity Topo IIα. They also indicate that GA inhibits Topo IIα-mediated DNA cleavage and modulate the activity of Topo II poisons, which provide rationale for further clinical evaluation of GA. [Mol Cancer Ther 2007;6(9):2429–40]

Dihydroartemisinin induces apoptosis in HL-60 leukemia cells dependent of iron and p38 mitogen-activated protein kinase activation but independent of reactive oxygen species

Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is one of the most effective anti-malarial analogs of artemisinin. In the current study, we found that DHA inhibited the proliferation of a panel of tumor cells originated from different tissue types. DHA effectively induced apoptosis in human promyelocytic leukemia HL-60 cells, which was accompanied with mitochondrial dysfunction and caspases activation. Further studies indicated that DHA-induced apoptosis was iron-dependent. Though DHA slightly elicited superoxide anion, these reactive oxygen species (ROS) contribute little to DHA-induced apoptosis in HL-60 cells. Moreover, DHA time-dependently activated mitogen-activeted protein kinases (MAPKs) and specific inhibition of p38 MAPK, but not c-Jun-NH2-terminal kinase (JNK) or extracellular signal-regulated kinase (ERK), abolished DHA-induced apoptosis, indicating that activation of p38 MAPK is required for DHA-induced apoptosis in HL-60 cells. Altogether, our data uncover that DHA induces apoptosis is dependent of iron and p38 MAPK activation but not ROS in HL-60 cells.

Salvicine, a novel DNA topoisomerase II inhibitor, exerting its effects by trapping enzyme-DNA cleavage complexes

Salvicine, a structurally modified diterpenoid quinone derived from Salvia prionitis, is a novel anticancer drug candidate. The compound has significant in vitro and in vivo activity against malignant tumor cells and xenografts, especially some human solid tumor models. This anticancer activity of salvicine is associated with its ability to induce tumor cell apoptosis. Salvicine was also found to have a profound cytotoxic effect on multidrug-resistant (MDR) cell lines by down-regulating the expression of MDR-1 mRNA of MDR cells. Salvicine acted as a topoisomerase II (Topo II) poison through its marked enhancement effect on Topo II-mediated DNA double-strand breaks as observed in the DNA cleavage assay. Strong inhibitory activity of salvicine against Topo II was observed in a kDNA decatenation assay, with an approximate IC(50) value of 3 microM. A similar result was obtained by a Topo II-mediated supercoiled DNA relaxation assay. In contrast, no inhibitory activity was observed against the catalytic activity of Topo I. When the effects of salvicine on individual steps of the catalytic cycle of Topo II were dissected, it was found that the mechanism by which salvicine inactivates Topo II is different from that by other anti-Topo II agents. Salvicine greatly promoted Topo II-DNA binding and inhibited pre- and post-strand Topo II-mediated DNA religation without interference with the forward cleavage steps. In addition, salvicine was not a DNA intercalative agent, as demonstrated by DNA unwinding assays. The results of this study indicate that the inhibitory activity of salvicine against Topo II was derived from its ability to stabilize DNA strand breaks through interactions with the enzyme alone or with the DNA-enzyme complex. It is therefore postulated that salvicine acts on Topo by trapping the DNA-Topo II complex, which in turn produces anticancer effects.

The anti-cancer activity of dihydroartemisinin is associated with induction of iron-dependent endoplasmic reticulum stress in colorectal carcinoma HCT116 cells

SummaryDihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is among the artemisinin derivatives possessing potent anti-malarial and anti-cancer activities. In the present study, we found that DHA displayed significant anti-proliferative activity in human colorectal carcinoma HCT116 cells, which may be attributed to its induction of G1 phase arrest and apoptosis. To further elucidate the mechanism of action of DHA, a proteomic study employed two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed. Glucose-regulated protein 78 (GRP78), which is related with endoplasmic reticulum stress (ER stress), was identified to be significantly up-regulated after DHA treatment. Further study demonstrated that DHA enhanced expression of GRP78 as well as growth arrest and DNA-damage-inducible gene 153 (GADD153, another ER stress-associated molecule) at both mRNA and protein levels. DHA treatment also led to accumulation of GADD153 in cell nucleus. Moreover, pretreatment of HCT116 cells with the iron chelator deferoxamine mesylate salt (DFO) abrogated induction of GRP78 and GADD153 upon DHA treatment, indicating iron is required for DHA-induced ER stress. This result is consistent with the fact that the anti-proliferative activity of DHA is also mediated by iron. We thus suggest the unbalance of redox may result in DHA-induced ER stress, which may contribute, at least in part, to its anti-cancer activity.

Inhibition of chemokine (CXC motif) ligand 12/chemokine (CXC motif) receptor 4 axis (CXCL12/CXCR4)-mediated cell migration by targeting mammalian target of rapamycin (mTOR) pathway in human gastric carcinoma cells.

Background: The cross-talk between CXCL12/CXCR4 axis and PI3K/mTOR pathway in migration of gastric carcinoma cells is unknown. Results: p110β provided a conduit for CXCL12-stimulated signaling and targeting PI3K/mTOR blocked CXCL12-activated cell motility. Conclusion: Targeting PI3K/mTOR pathway inhibited CXCL12-mediated cell migration. Significance: Drugs targeting mTOR pathway may be used for the therapy of metastatic gastric cancer expressing high levels of CXCL12. CXCL12/CXCR4 plays an important role in metastasis of gastric carcinoma. Rapamycin has been reported to inhibit migration of gastric cancer cells. However, the role of mTOR pathway in CXCL12/CXCR4-mediated cell migration and the potential of drugs targeting PI3K/mTOR pathway remains unelucidated. We found that CXCL12 activated PI3K/Akt/mTOR pathway in MKN-45 cells. Stimulating CHO-K1 cells expressing pEGFP-C1-Grp1-PH fusion protein with CXCL12 resulted in generation of phosphatidylinositol (3,4,5)-triphosphate, which provided direct evidence of activating PI3K by CXCL12. Down-regulation of p110β by siRNA but not p110α blocked phosphorylation of Akt and S6K1 induced by CXCL12. Consistently, p110β-specific inhibitor blocked the CXCL12-activated PI3K/Akt/mTOR pathway. Moreover, CXCR4 immunoprecipitated by anti-p110β antibody increased after CXCL12 stimulation and Gi protein inhibitor pertussis toxin abrogated CXCL12-induced activation of PI3K. Further studies demonstrated that inhibitors targeting the PI3K/mTOR pathway significantly blocked the chemotactic responses of MKN-45 cells triggered by CXCL12, which might be attributed primarily to inhibition of mTORC1 and related to prevention of F-actin reorganization as well as down-regulation of active RhoA, Rac1, and Cdc42. Furthermore, rapamycin inhibited the secretion of CXCL12 and the expression of CXCR4, which might form a positive feedback loop to further abolish upstream signaling leading to cell migration. Finally, we found cells expressing high levels of cxcl12 were sensitive to rapamycin in its activity inhibiting migration as well as proliferation. In summary, we found that the mTOR pathway played an important role in CXCL12/CXCR4-mediated cell migration and proposed that drugs targeting the mTOR pathway may be used for the therapy of metastatic gastric cancer expressing high levels of cxcl12.

S9, a Novel Anticancer Agent, Exerts Its Anti-Proliferative Activity by Interfering with Both PI3K-Akt-mTOR Signaling and Microtubule Cytoskeleton

Background Deregulation of the phosphatidylinositol 3-kinases (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway plays a central role in tumor formation and progression, providing validated targets for cancer therapy. S9, a hybrid of α-methylene-γ-lactone and 2-phenyl indole compound, possessed potent activity against this pathway. Methodology/Principal Findings Effects of S9 on PI3K-Akt-mTOR pathway were determined by Western blot, immunofluorescence staining and in vitro kinas assay. The interactions between tubulin and S9 were investigated by polymerization assay, CD, and SPR assay. The potential binding modes between S9 and PI3K, mTOR or tubulin were analyzed by molecular modeling. Anti-tumor activity of S9 was evaluated in tumor cells and in nude mice bearing human cancer xenografts. S9 abrogated EGF-activated PI3K-Akt-mTOR signaling cascade and Akt translocation to cellular membrane in human tumor cells. S9 possessed inhibitory activity against both PI3K and mTOR with little effect on other tested 30 kinases. S9 also completely impeded hyper-phosphorylation of Akt as a feedback of inhibition of mTOR by rapamycin. S9 unexpectedly arrested cells in M phase other than G1 phase, which was distinct from compounds targeting PI3K-Akt-mTOR pathway. Further study revealed that S9 inhibited tubulin polymerization via binding to colchicine-binding site of tubulin and resulted in microtubule disturbance. Molecular modeling indicated that S9 could potentially bind to the kinase domains of PI3K p110α subunit and mTOR, and shared similar hydrophobic interactions with colchicines in the complex with tubulin. Moreover, S9 induced rapid apoptosis in tumor cell, which might reflect a synergistic cooperation between blockade of both PI3-Akt-mTOR signaling and tubulin cytoskeleton. Finally, S9 displayed potent antiproliferative activity in a panel of tumor cells originated from different tissue types including drug-resistant cells and in nude mice bearing human tumor xenografts. Conclusions/Significance Taken together, S9 targets both PI3K-Akt-mTOR signaling and microtubule cytoskeleton, which combinatorially contributes its antitumor activity and provides new clues for anticancer drug design and development.

Crystal Structures of PI3Kα Complexed with PI103 and Its Derivatives: New Directions for Inhibitors Design

The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays important roles in cell proliferation, growth, and survival. Hyperactivated PI3K is frequently found in a wide variety of human cancers, validating it as a promising target for cancer therapy. We determined the crystal structure of the human PI3Kα-PI103 complex to unravel molecular interactions. Based on the structure, substitution at the R1 position of the phenol portion of PI103 was demonstrated to improve binding affinity via forming a new H-bond with Lys802 at the bottom of the ATP catalytic site. Interestingly, the crystal structure of the PI3Kα-9d complex revealed that the flexibility of Lys802 can also induce additional space at the catalytic site for further modification. Thus, these crystal structures provide a molecular basis for the strong and specific interactions and demonstrate the important role of Lys802 in the design of novel PI3Kα inhibitors.

Toward rapamycin analog (rapalog)-based precision cancer therapy.

Rapamycin and its analogs (rapalogs) are the first generation of mTOR inhibitors, which have the same molecular scaffold, but different physiochemical properties. Rapalogs are being tested in a wide spectrum of human tumors as both monotherapy and a component of combination therapy. Among them, temsirolimus and everolimus have been approved for the treatment of breast and renal cancer. However, objective response rates with rapalogs in clinical trials are modest and variable. Identification of biomarkers predicting response to rapalogs, and discovery of drug combinations with improved efficacy and tolerated toxicity are critical to moving this class of targeted therapeutics forward. This review focuses on the aberrations in the PI3K/mTOR pathway in human tumor cells or tissues as predictive biomarkers for rapalog efficacy. Recent results of combinational therapy using rapalogs and other anticancer drugs are documented. With the rapid development of next-generation genomic sequencing and precision medicine, rapalogs will provide greater benefits to cancer patients.

New microtubule-inhibiting anticancer agents

Importance of this field: Microtubule-inhibiting drugs are among the most commonly prescribed agents in the combat against cancer, though the clinical use of these drugs is limited by acquired resistance, risk of hypersensitivity reactions and intolerable toxicity. With progress in our understanding of cytoskeleton structure and its related signaling pathways, a number of new microtubule-inhibiting agents with diversified structures and modes of action haveemerged. What the reader will gain: This review mainly describes new microtubule-targeting anticancer agents that have been discovered (especially over the past 2 years), with emphasis on their diversity of structures and distinct modes ofaction. Areas covered in this review: Data were obtained exclusively from public sources, including journals and scientific meeting abstracts, up to September 2009. Take home message: A number of new agents have been discovered, and some have entered clinical trials. Even though most of these agents stabilize or destabilize tubulin via binding on the recognized tubulin binding sites, a few compounds bind to tubulin on undefined sites or interrupt microtubule in diverse ways. Moreover, some agents target microtubule indirectly such that they alter the post-translational modification of tubulin. Further investigation into their mechanism of action and evaluation of their anticancer efficacy will help to develop novel regimens that are superior to existingapproaches.