PCTAIRE1-knockdown sensitizes cancer cells to TNF family cytokines.

While PCTAIRE1/PCTK1/Cdk16 is overexpressed in malignant cells and is crucial in tumorigenesis, its function in apoptosis remains unclear. Here we investigated the role of PCTAIRE1 in apoptosis, especially in the extrinsic cell death pathway. Gene-knockdown of PCTAIRE1 sensitized prostate cancer PPC1 and Du145 cells, and breast cancer MDA-MB-468 cells to TNF-family cytokines, including TNF-related apoptosis-inducing ligand (TRAIL). Meanwhile, PCTAIRE1-knockdown did not sensitize non-malignant cells, including diploid fibroblasts IMR-90 and the immortalized prostate epithelial cell line 267B1. PCTAIRE1-knockdown did not up-regulate death receptor expression on the cell surface or affect caspase-8, FADD and FLIP expression levels. PCTAIRE1-knockdown did promote caspase-8 cleavage and RIPK1 degradation, while RIPK1 mRNA knockdown sensitized PPC1 cells to TNF-family cytokines. Furthermore, the kinase inhibitor SNS-032, which inhibits PCTAIRE1 kinase activity, sensitized PPC1 cells to TRAIL-induced apoptosis. Together these results suggest that PCTAIRE1 contributes to the resistance of cancer cell lines to apoptosis induced by TNF-family cytokines, which implies that PCTAIRE1 inhibitors could have synergistic effects with TNF-family cytokines for cytodestruction of cancer cells.

ARTS and Siah collaborate in a pathway for XIAP degradation.

ARTS (apoptosis-related protein in the TGF-ß signaling pathway) is a mitochondrial protein that binds XIAP (X-linked inhibitor of apoptosis protein) upon entering the cytosol, thus promoting cell death. Expression of ARTS is lost in some malignancies. Here, we show that ARTS binds to XIAP at BIR1, a domain distinct from the caspase-binding sites. Furthermore, ARTS interacts with the E3 ligase Siah-1 (seven in absentia homolog 1) to induce ubiquitination and degradation of XIAP. Cells lacking either Siah or ARTS contain higher steady-state levels of XIAP. Thus, ARTS serves as an adaptor to bridge Siah-1 to XIAP, targeting it for destruction.

Inhibition of protein kinase C-driven nuclear factor-kappaB activation: synthesis, structure-activity relationship, and pharmacological profiling of pathway specific benzimidazole probe molecules.

A unique series of biologically active chemical probes that selectively inhibit NF-kappaB activation induced by protein kinase C (PKC) pathway activators have been identified through a cell-based phenotypic reporter gene assay. These 2-aminobenzimidazoles represent initial chemical tools to be used in gaining further understanding on the cellular mechanisms driven by B and T cell antigen receptors. Starting from the founding member of this chemical series 1a (notated in PubChem as CID-2858522), we report the chemical synthesis, SAR studies, and pharmacological profiling of this pathway-selective inhibitor of NF-kappaB activation.

Chemical biology strategy reveals pathway-selective inhibitor of NF-kappaB activation induced by protein kinase C.

Dysregulation of NF-kappaB activity contributes to many autoimmune and inflammatory diseases. At least nine pathways for NF-kappaB activation have been identified, most of which converge on the IkappaB kinases (IKKs). Although IKKs represent logical targets for potential drug discovery, chemical inhibitors of IKKs suppress all known NF-kappaB activation pathways and thus lack the selectivity required for safe use. A unique NF-kappaB activation pathway is initiated by protein kinase C (PKC) that is stimulated by antigen receptors and many growth factor receptors. Using a cell-based high-throughput screening (HTS) assay and chemical biology strategy, we identified a 2-aminobenzimidazole compound, CID-2858522, which selectively inhibits the NF-kappaB pathway induced by PKC, operating downstream of PKC but upstream of IKKbeta, without inhibiting other NF-kappaB activation pathways. In human B cells stimulated through surface immunoglobulin, CID-2858522 inhibited NF-kappaB DNA-binding activity and expression of endogenous NF-kappaB-dependent target gene, TRAF1. Altogether, as a selective chemical inhibitor of the NF-kappaB pathway induced by PKC, CID-2858522 serves as a powerful research tool and may reveal new paths toward therapeutically useful NF-kappaB inhibitors.

Selective benzimidazole inhibitors of the antigen receptor-mediated NF-kappaB activation pathway.

Dysregulated antigen receptor-mediated NF-kappaB activation can contribute to development of autoimmunity, chronic inflammation, and malignancy. A chemical biology screening strategy has identified a substituted benzimidazole that selectively inhibits antigen receptor-mediated NF-kappaB activation without blocking other NF-kappaB activation pathways. A library of analogs was synthesized and the structure-activity relationship and metabolic stability for the series is presented.

Luteolin, a flavonoid with potential for cancer prevention and therapy.

Luteolin, 3',4',5,7-tetrahydroxyflavone, is a common flavonoid that exists in many types of plants including fruits, vegetables, and medicinal herbs. Plants rich in luteolin have been used in Chinese traditional medicine for treating various diseases such as hypertension, inflammatory disorders, and cancer. Having multiple biological effects such as anti-inflammation, anti-allergy and anticancer, luteolin functions as either an antioxidant or a pro-oxidant biochemically. The biological effects of luteolin could be functionally related to each other. For instance, the anti-inflammatory activity may be linked to its anticancer property. Luteolin's anticancer property is associated with the induction of apoptosis, and inhibition of cell proliferation, metastasis and angiogenesis. Furthermore, luteolin sensitizes cancer cells to therapeutic-induced cytotoxicity through suppressing cell survival pathways such as phosphatidylinositol 3'-kinase (PI3K)/Akt, nuclear factor kappa B (NF-kappaB), and X-linked inhibitor of apoptosis protein (XIAP), and stimulating apoptosis pathways including those that induce the tumor suppressor p53. These observations suggest that luteolin could be an anticancer agent for various cancers. Furthermore, recent epidemiological studies have attributed a cancer prevention property to luteolin. In this review, we summarize the progress of recent research on luteolin, with a particular focus on its anticancer role and molecular mechanisms underlying this property of luteolin.

Apoptotic activity and mechanism of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic-acid and related synthetic triterpenoids in prostate cancer.

Synthetic triterpenoids 2-cyano-3, 12-dioxooleana-1, 9-(11)-dien-28-oic acid (CDDO) and CDDO-Me (CDDO-methyl ester) have entered clinical trials for cancer. We determined that CDDO analogues at submicromolar concentrations induce apoptosis of cultured prostate cancer cell lines, LNCaP, ALVA31, Du145, PC3, and PPC1, with lethal dose 50% approximately 1 micromol/L for CDDO-Me and an imidazole analogue (CDDO-Im). These compounds induced apoptosis of prostate cancer cells as characterized by cleavage of caspase-3, caspase-7, caspase-8, caspase-9, caspase-10, BID, and poly(ADP)ribose polymerase and by dependence on caspase activity. Moreover, triterpenoid-induced cell death was abolished by caspase-8-targeting small interfering (si) RNA. To explore the mechanism(s) involved in caspase-8 activation, we examined cell surface expression of death receptor (DR)4 and DR5 after triterpenoid treatment. Cell surface DR4 and DR5 expression was significantly up-regulated by CDDO or CDDO-Im but not by CDDO-Me. DR4 and DR5 knockdown with siRNA significantly inhibited apoptosis induced by CDDO and CDDO-Im but had no effect on CDDO-Me-induced killing, suggesting that CDDO and CDDO-Im induce apoptosis by a different mechanism than CDDO-Me. In addition to activating the caspase-8-dependent extrinsic apoptosis pathway, we observed that Bcl-X(L) overexpression inhibited triterpenoid-mediated killing of prostate cancer cell line Du145, suggesting that the intrinsic pathway (via mitochondria) also participates in triterpenoid-mediated killing. In vivo antitumor activity of CDDO-Me was shown using a Du145 tumor xenograft model in nude rats. Altogether, these findings suggest CDDO and related synthetic triterpenoids should be further evaluated as potential novel therapeutics for hormone refractory prostate cancers.

Luteolin sensitizes the anticancer effect of cisplatin via c-Jun NH2-terminal kinase-mediated p53 phosphorylation and stabilization.

Luteolin is an important flavonoid with a potential anticancer effect. In this study, we examined the molecular mechanisms involved in the sensitization effect of luteolin on cancer cell killing induced by cisplatin, an important cancer chemotherapeutic agent. First, we provided evidence that the sensitization effect of luteolin on cisplatin-induced apoptosis is p53 dependent, as such effect is only found in p53 wild-type cancer cells but not in p53 mutant cancer cells. Moreover, knockdown of p53 by small interfering RNA made p53 wild-type cancer cells resistant to luteolin and cisplatin. Second, we observed a significant increase of p53 protein level in luteolin-treated cancer cells without increase of p53 mRNA level, indicating the possible effect of luteolin on p53 posttranscriptional regulation. Third, we identified the critical role of c-Jun NH(2)-terminal kinase (JNK) in regulation of p53 protein stability: luteolin activates JNK, and JNK then stabilizes p53 via phosphorylation, leading to reduced ubiquitination and proteasomal degradation. Finally, by using an in vivo nude mice xenograft model, we confirmed that luteolin enhanced the cancer therapeutic activity of cisplatin via p53 stabilization and accumulation. In summary, data from this study reveal a novel molecular mechanism involved in the anticancer effect of luteolin and support its potential clinical application as a chemosensitizer in cancer therapy.

Protein kinase C inhibition and x-linked inhibitor of apoptosis protein degradation contribute to the sensitization effect of luteolin on tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in cancer cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an important member of the TNF superfamily with great potential in cancer therapy. Luteolin is a dietary flavonoid commonly found in some medicinal plants. Here we found that pretreatment with a noncytotoxic concentration of luteolin significantly sensitized TRAIL-induced apoptosis in both TRAIL-sensitive (HeLa) and TRAIL-resistant cancer cells (CNE1, HT29, and HepG2). Such sensitization is achieved through enhanced caspase-8 activation and caspase-3 maturation. Further, the protein level of X-linked inhibitor of apoptosis protein (XIAP) was markedly reduced in cells treated with luteolin and TRAIL, and ectopic expression of XIAP protected against cell death induced by luteolin and TRAIL, showing that luteolin sensitizes TRAIL-induced apoptosis through down-regulation of XIAP. In search of the molecular mechanism responsible for XIAP down-regulation, we found that luteolin and TRAIL promoted XIAP ubiquitination and proteasomal degradation. Next, we showed that protein kinase C (PKC) activation prevented cell death induced by luteolin and TRAIL via suppression of XIAP down-regulation. Moreover, luteolin inhibited PKC activity, and bisindolylmaleimide I, a general PKC inhibitor, simulated luteolin in sensitizing TRAIL-induced apoptosis. Taken together, these results present a novel anticancer effect of luteolin and support its potential application in cancer therapy in combination with TRAIL. In addition, our data reveal a new function of PKC in cell death: PKC activation stabilizes XIAP and thus suppresses TRAIL-induced apoptosis.

Luteolin sensitizes tumor necrosis factor-alpha-induced apoptosis in human tumor cells.

Tumor necrosis factor-alpha (TNFalpha) activates both cell death and cell survival pathways, which render most cancer cells resistant to its cytotoxicity. In this study, we found that pretreatment with luteolin, a plant flavonoid, greatly sensitized TNFalpha-induced apoptotic cell death in a number of human cancer cell lines; including colorectal cancer COLO205, HCT116 cells and cervical cancer HeLa cells. In the search of the molecular mechanisms responsible for the sensitization effect of luteolin, we discovered that luteolin inhibited TNFalpha-induced activation of nuclear transcription factor-kappa B (NF-kappaB), the main survival factor in TNFalpha signaling. As a result, luteolin suppressed the expression of NF-kappaB-targeted antiapoptotic genes, including A20 and cellular inhibitor of apoptosis protein-1 (c-IAP1). The role of A20 and c-IAP1 was further confirmed by ectopic expression of these two genes, which significantly protected cell death induced by luteolin followed by TNFalpha. In addition, inhibition of NF-kappaB by luteolin led to augmentation and prolongation of c-Jun N-terminal kinase (JNK) activation induced by TNFalpha. Suppression of JNK activation, either by a synthetic JNK inhibitor (SP600125) or by overexpression of the dominant negative forms of JNK kinase 1 (JNKK1) and JNK kinase 2 (JNKK2), conferred significant protection against apoptotic cell death induced by luteolin and TNFalpha, suggesting that NF-kappaB and JNK are closely associated with the sensitization effect of luteolin. Data from this study reveal a novel function of luteolin and enhance the value of luteolin as an anticancer agent.