Effect of colicin E3 on leukemia cells P 388 in vitro

167

149

The molecular chaperone heat shock protein 90 (Hsp90) is required for the stabilization and conformational maturation of various oncogenic proteins in cancer. The loading of protein kinases to Hsp90 is actively mediated by the cochaperone Cdc37. The crucial role of the Hsp90-Cdc37 complex has made it an exciting target for cancer treatment. In this study, we characterize Hsp90 and Cdc37 interaction and drug disruption using a reconstituted protein system. The GST pull-down assay and ELISA assay show that Cdc37 binds to ADP-bound/nucleotidefree Hsp90 but not ATP-bound Hsp90. Celastrol disrupts Hsp90-Cdc37 complex formation, whereas the classical Hsp90 inhibitors (e.g. geldanamycin) have no effect. Celastrol inhibits Hsp90 ATPase activity without blocking ATP binding. Proteolytic fingerprinting indicates celastrol binds to Hsp90 C-terminal domain to protect it from trypsin digestion. These data suggest that celastrol may represent a new class of Hsp90 inhibitor by modifying Hsp90 C terminus to allosterically regulate its chaperone activity and disrupt Hsp90-Cdc37 complex.Heat shock protein 90 (Hsp90) 2 is a highly abundant and essential molecular chaperone in eukaryotic cells, accounting for as much as 1-2% of the cytosolic protein even under nonstressed conditions (1). Hsp90 protects cells not only through correcting the misfolded proteins under stress conditions, but also plays a key role under normal conditions in regulating the stability, maturation, and activation of a wide range of client substrates, including kinases, hormone receptors, and transcription factors (2). There is strong evidence that Hsp90 plays an important role in disease states, particularly in cancer. Hsp90 is expressed 2-10-fold higher in cancer cells compared with their normal counterparts, implying its crucial role in tumor cell growth or survival (3). The largest subset of Hsp90 clients is the protein kinase, many of which are mutated and/or overexpressed signaling proteins in cancers (4 -6). Furthermore, cancer cells are significantly more sensitive to Hsp90 inhibition than non-transformed cells (7). Therefore, Hsp90 has emerged as a promising target for cancer treatment.The crystal structure reveals that Hsp90 consists of three highly conserved domains: an N-terminal ATP-binding domain (25 kDa), a middle domain (35 kDa), and a C-terminal dimerization domain (12 kDa) (8 -10). Hsp90 exists as a homodimer (11). The N-terminal domain contains a specific ATP-binding pocket, which has been well characterized (9, 12). The middle domain is highly charged, and its major role is to distinguish various types of client proteins and adjust the molecular chaperone for proper substrate activation (13). The C-terminal domain strengthens the weak association between the two N-terminal domains of the Hsp90 dimer (10). A second ATP-binding site is located in the C terminus, which does not exhibit ATPase activity (14).Hsp90 chaperone function depends on the conformational changes driven by its ATPase activity (15). Numerous Hsp90 inhibitors, ranging from...

Section: Discussionmentioning

confidence: 80%

Characterization of Celastrol to Inhibit Hsp90 and Cdc37 Interaction

Zhang

et al. 2009

167

149

“…As shown in Fig. 5B, it is apparent that WA with an IC 50 of 250 nM is the major and most active NFB-inhibiting compound in WS, whereas both WdA (50 M) and 12DW (90 M) required 200 -400-fold higher concentrations to elicit similar NFB repression. Furthermore NFB reporter gene assays confirmed that essentially all NFB-repressing activity is concentrated in the WA peak fraction as no repression could be observed in pre-or postpeak fractions as prepared by semipreparative HPLC (supplemental Fig.…”

Section: Ws Induces the Phosphorylation Of Ikk␤ Through The Mek/erkmentioning

confidence: 85%

“…5B). With respect to structure function analysis, important roles have been attributed to the double bond (C-2ϭC-3) configuration and the C-26 lactone moiety for its biological activities (12,50), but all tested WS-derived reference compounds share these features. Thus, the presence of a C-5-C-6 epoxide and the absence of a C-20 hydroxyl seem critical for optimal activity.…”

Section: Ws Induces the Phosphorylation Of Ikk␤ Through The Mek/erkmentioning

confidence: 99%

Withaferin A Strongly Elicits IκB Kinase β Hyperphosphorylation Concomitant with Potent Inhibition of Its Kinase Activity

Kaileh

Berghe

Heyerick

et al. 2007

281

196

The transcription factor NFB plays a critical role in normal and pathophysiological immune responses. Therefore, NFB and the signaling pathways that regulate its activation have become a major focus of drug development programs. Withania somnifera (WS) is a medicinal plant that is widely used in Palestine for the treatment of various inflammatory disorders. In this study we show that the leave extract of WS, as well as its major constituent withaferin A (WA), potently inhibits NFB activation by preventing the tumor necrosis factor-induced activation of IB kinase ␤ via a thioalkylation-sensitive redox mechanism, whereas other WS-derived steroidal lactones, such as withanolide A and 12-deoxywithastramonolide, are far less effective. To our knowledge, this is the first communication of IB kinase ␤ inhibition by a plant-derived inhibitor, coinciding with MEK1/ ERK-dependent Ser-181 hyperphosphorylation. This prevents IB phosphorylation and degradation, which subsequently blocks NFB translocation, NFB/DNA binding, and gene transcription. Taken together, our results indicate that pure WA or WA-enriched WS extracts can be considered as a novel class of NFB inhibitors, which hold promise as novel anti-inflammatory agents for treatment of various inflammatory disorders and/or cancer.

“…Several potential mechanisms have been implicated in the inhibition of tumorigenesis by withanolides. First, withanolides induce growth arrest and apoptosis in cancer cells (4,5). Second, withanolides can inhibit angiogenesis by suppressing endothelial cell proliferation (6).…”

mentioning

confidence: 99%

Tubocapsanolide A Inhibits Transforming Growth Factor-β-activating Kinase 1 to Suppress NF-κB-induced CCR7

Pan

Chang

et al. 2009

Withanolides are steroidal lactones that were originally isolated from Withania somnifera, one of the most important herbs used as a traditional remedy for several illnesses in Asian countries (1). These compounds are biologically active and have been shown to inhibit the enzymatic activity of cyclooxygenase-2 and suppress inflammation (2). In addition, recent studies demonstrate that withanolides exhibit anti-cancer effect on human lung, colon, and breast cancer cells in vitro and exert immunopotentiating activity in vivo (3, 4). Several potential mechanisms have been implicated in the inhibition of tumorigenesis by withanolides. First, withanolides induce growth arrest and apoptosis in cancer cells (4, 5). Second, withanolides can inhibit angiogenesis by suppressing endothelial cell proliferation (6). Third, withanolides can reduce cancer cell invasion and metastasis (7). These data suggest that withanolides may be developed as a novel class of anti-cancer drugs.Lymph node invasion by cancer cells is an important step for tumor metastasis and is frequently correlated with early recurrence and poor prognosis. However, the underlying mechanism by which cancer cells metastasize into peripheral lymphatic capillaries is poorly defined. Recent studies indicate that the interaction between chemokine CCL21 and its cognate receptor CCR7 may play an important role in this process (8, 9). The hypothesis suggests that lymphatic endothelial cells (LECs) 2 express and release chemotactic factors such as CCL19 and CCL21 to direct cancer cells with high expression of chemokine receptor CCR7 to grow and migrate toward lymphatic capillaries (10 -12). Indeed, many highly metastatic cancer cells express large amount of CCR7 receptor (13,14), and CCR7 expression is associated with lymph node metastasis in breast, lung, gastric, esophageal cancer, and melanoma (15-19). It is possible that inhibition of CCR7 expression or block of CCL21-CCR7 interaction may cause reduction of lymph node invasion and tumor metastasis. However, the control of CCR7 gene transcription in cancer cells is largely unclear, and no natural products have been shown to regulate CCR7 expression.We have isolated a new bioactive withanolide tubocapsanolide A (Tubo A) from Tubocapsicum anomalum (20). Tubo A exhibited cytotoxic activity on various types of human cancer cells (20). In addition, our recent results demonstrated that Tubo A suppressed the transcription of Skp2 oncogene and up-regulated cyclin-dependent kinase inhibitory proteins p27