Bafilomycin induces the p21-mediated growth inhibition of cancer cells under hypoxic conditions by expressing hypoxia-inducible factor-1alpha

Bafilomycin A1, a macrolide antibiotic isolated from Streptomyces species, has been used as an inhibitor of vacuolar H(+) ATPase (V-ATPase). Bafilomycin has been also evaluated as a potential anticancer agent because it inhibits cell proliferation and tumor growth. Although these anticancer effects of bafilomycin are considered to be attributable to the intracellular acidosis by V-ATPase inhibition, the exact mechanism remains unclear. In the present study, we tested the possibility that bafilomycin targets a tumor-promoting factor, hypoxia-inducible factor-1alpha (HIF-1alpha). Bafilomycin A1 and its analog, concanamycin A, were found to up-regulate HIF-1alpha in eight human cancer cell-lines, and this effect is attributed to inhibited degradation of HIF-1alpha protein. Furthermore, the HIF-1alpha induction by bafilomycin was augmented by hypoxia, which caused a robust induction of p21 and cell cycle arrest in cancer cells. The cell cycle inhibition was shown only in cancer cells expressing both HIF-1alpha and p21. In HIF-1alpha(+/+) or HIF-1alpha(-/-) fibrosarcomas grafted in nude mice, bafilomycin showed the HIF-1alpha-dependent anticancer effect. Based on these results, the exorbitant expression of HIF-1alpha is likely to contribute to the anticancer action of bafilomycin.     

New anticancer strategies targeting HIF-1

Hypoxia-inducible factor-1 (HIF-1), which is present at high levels in human tumors, plays crucial roles in tumor promotion by up-regulating its target genes, which are involved in anaerobic energy metabolism, angiogenesis, cell survival, cell invasion, and drug resistance. Therefore, it is apparent that the inhibition of HIF-1 activity may be a strategy for treating cancer. Recently, many efforts to develop new HIF-1-targeting agents have been made by both academic and pharmaceutical industry laboratories. The future success of these efforts will be a new class of HIF-1-targeting anticancer agents, which would improve the prognoses of many cancer patients. This review focuses on the potential of HIF-1 as a target molecule for anticancer therapy, and on possible strategies to inhibit HIF-1 activity. In addition, we introduce YC-1 as a new anti-HIF-1, anticancer agent. Although YC-1 was originally developed as a potential therapeutic agent for thrombosis and hypertension, recent studies demonstrated that YC-1 suppressed HIF-1 activity and vascular endothelial growth factor expression in cancer cells. Moreover, it halted tumor growth in immunodeficient mice without serious toxicity during the treatment period. Thus, we propose that YC-1 is a good lead compound for the development of new anti-HIF-1, anticancer agents.     

A quassinoid 6alpha-tigloyloxychaparrinone inhibits hypoxia-inducible factor-1 pathway by inhibition of eukaryotic translation initiation factor 4E phosphorylation

Hypoxia-inducible factor-1 (HIF-1) is the central mediator of cellular responses to low oxygen and vital to many aspects of cancer biology. In a search for HIF-1 inhibitors, we identified a quassinoid 6alpha-tigloyloxychaparrinone (TCN) as an inhibitor of HIF-1 activation from Ailantus altissima. We here demonstrated the effect of TCN on HIF-1 activation induced by hypoxia or CoCl(2). TCN showed the potent inhibitory activity against HIF-1 activation induced by hypoxia in various human cancer cell lines. This compound markedly decreased the hypoxia-induced accumulation of HIF-1alpha protein dose-dependently, whereas it did not affect the expressions of HIF-1beta and topoisomerase-I. Furthermore, TCN prevented hypoxia-induced expression of HIF-1 target genes for vascular endothelial growth factor (VEGF) and erythropoietin. Further analysis revealed that TCN strongly inhibited HIF-1alpha protein synthesis, without affecting the expression level of HIF-1alpha mRNA or degradation of HIF-1alpha protein. Moreover, the levels of phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2), mitogen-activated protein (MAP) kinase-interacting protein kinase-1 (MNK1) and eukaryotic initiation factor 4E (eIF4E) were significantly suppressed by the treatment of TCN, without changing the total levels of these proteins. Our data suggested that TCN may exhibit anticancer activity by inhibiting HIF-1alpha translation through the inhibition of eIF4E phosphorylation pathway and thus provide a novel mechanism for the anticancer activity of quassinoids. TCN could be a new HIF-1-targeted anticancer agent and be effective on mammalian target of rapamycin (mTOR)-targeted cancer therapy, in which mTOR inhibition increases eIF4E phosphorylation.     

Perillyl alcohol efficiently scavenges activity of cellular ROS and inhibits the translational expression of hypoxia-inducible factor-1α via mTOR/4E-BP1 signaling pathways

Perillyl alcohol (POH) is a dietary monoterpene present in a variety of plants with a pure or mixed form, and it is one of the very few natural substances with anticancer activity. However, the mechanism by which POH unleashes its anticancer activity in tumor cells remains unclear. We here demonstrated the effect of POH on hypoxia-inducible factor-1α (HIF-1α) activation. POH showed the potent inhibitory activity against HIF-1 activation induced by hypoxia in various human cancer cell lines and efficient scavenging activity of cellular Reactive oxygen species (ROS) by hypoxia in tumor cells. Further analysis revealed that POH inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. Moreover, we found that suppression of HIF-1α accumulation by POH correlated with strong de-phosphorylation of mammalian target of rapamycin (mTOR) and eIF4E binding protein-1 (4E-BP1), and eukaryotic initiation factor 4E (eIF4E). These results showed that POH inhibited HIF-1α protein synthesis through the inhibition of mTOR/4E-BP1 signaling pathways. Furthermore, POH increased the expression of p53, p21, induced cell cycle arrest in the G1 phase as well as decreased cyclin D1, c-Myc, and Skp2 expression. In vivo studies further confirmed the inhibitory effect of POH on the expression of HIF-1α proteins, leading to a decrease growth of HCT116 cells in a xenograft tumor model. There results show that POH is an effective inhibitor of HIF-1 and provide new perspectives in to the mechanism of its anticancer activity.     

Baffled by bafilomycin: an anticancer agent that induces hypoxia-inducible factor-1alpha expression

In an article presented in this issue of Molecular Pharmacology, Lim et al. (p. 1856) investigate the anticancer effect of bafilomycin, an inhibitor of the vacuolar ATPase. The authors report that bafilomycin inhibits cell cycle progression and tumor growth by inducing the expression of hypoxia-inducible factor (HIF) 1alpha and the cyclin-dependent kinase inhibitor p21(CIP1), a surprising result because HIF-1alpha overexpression is associated with tumor growth and angiogenesis in preclinical models and with increased patient mortality in clinical studies. However, the authors demonstrate that bafilomycin-induced HIF-1alpha expression leads to increased CIP1 gene expression but does not lead to increased expression of other HIF-1-regulated genes that promote tumor progression.     

Role of HIF signaling on tumorigenesis in response to chronic low-dose arsenic administration.

Trivalent inorganic arsenic (arsenite, arsenic trioxide, As(III)) is a primary contaminant of groundwater supplies worldwide. As(III), marketed as trisenox, is also an FDA-approved agent to treat cancer It has been previously shown by our laboratory that As(III) administered at doses lower than a therapeutic anticancer dose results in an increase in tumor formation and blood vessel density of tumors. In this work it was found that chronic administration of As(III) approaching the EPA action level of 10 ppb, given in the drinking water of mice 5 weeks prior to B16-F10 melanoma implantation, increased the growth rate of primary tumors and the number of metastases to the lung. Further, levels of arsenic in the tumor and lung were found to be much greater than those in the blood and similar to pro-angiogenic As(III) doses. Levels of hypoxia inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) surrounding the blood vessels in the tumors of the As(III)-treated mice were also found to be increased. Exposure of isolated B16-F10 tumor cells to chronic (3 or 7 day) but not acute (4 h) low-dose As(III) was found to increase HIF-1alpha expression and secretion of VEGF. Finally, coadministration of an inhibitor of HIF (YC-1) or a VEGFR-2 kinase inhibitor (SU5416) was found to antagonize the pro-angiogenic effects of low-dose As(III). Together, these results suggest that chronic exposure to low-dose As(III) could stimulate growth of tumors through a HIF-dependent stimulation of angiogenesis.     

Berberine inhibits HIF-1alpha expression via enhanced proteolysis

We have studied the antiangiogenic property of berberine. We showed that berberine could directly inhibit in vitro human umbilical vein endothelial cell (HUVEC) tube formation and migration. In addition, to determine whether berberine could influence the cross-talk between the gastric adenocarcinoma cell line SC-M1 and vascular endothelial cells, we performed modified confrontation culture experiments and showed that berberine (7.5 microM, 16 h) could inhibit the capacity of hypoxic SC-M1 cells to stimulate HUVEC migration. These results demonstrated berberine's antiangiogenic property and its clinical potential as an inhibitor of tumor angiogenesis. Parallel Western blot analyses revealed that berberine prevented hypoxic SC-M1 cultures from expressing vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1alpha, two key factors in mediating tumor angiogenesis. However, overexpression of HIF-1alpha in SC-M1 cells dramatically reversed the inhibitory effect of berberine on SC-M1-induced in vitro HUVEC migration. These data indicated that HIF-1alpha repression is a critical step in the inhibitory effect of berberine on tumor-induced angiogenesis. Northern blot analyses plus pulse-chase assays revealed that berberine did not down-regulate HIF-1alpha mRNA but destabilized HIF-1alpha protein. We found that berberine-induced HIF-1alpha degradation was blocked by a 26S proteasome inhibitor. Moreover, immunoprecipitation and Western blot analyses showed that berberine increased the lysine-acetylated HIF-1alpha in hypoxic SC-M1 cultures. These data indicated that a proteasomal proteolytic pathway and lysine acetylation were involved in berberine-triggered HIF-1alpha degradation. In conclusion, our data provided molecular evidence to support berberine as a potent antiangiogenic agent in cancer therapy.     

Inhibition of IkappaB kinase-nuclear factor-kappaB signaling pathway by 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24), a novel monoketone analog of curcumin

The nuclear factor-kappaB (NF-kappaB) signaling pathway has been targeted for therapeutic applications in a variety of human diseases, includuing cancer. Many naturally occurring substances, including curcumin, have been investigated for their actions on the NF-kappaB pathway because of their significant therapeutic potential and safety profile. A synthetic monoketone compound termed 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24) was developed from curcumin and exhibited potent anticancer activity. Here, we report a mechanism by which EF24 potently suppresses the NF-kappaB signaling pathway through direct action on IkappaB kinase (IKK). We demonstrate that 1) EF24 induces death of lung, breast, ovarian, and cervical cancer cells, with a potency about 10 times higher than that of curcumin; 2) EF24 rapidly blocks the nuclear translocation of NF-kappaB, with an IC(50) value of 1.3 microM compared with curcumin, with an IC(50) value of 13 microM; 3) EF24 effectively inhibits tumor necrosis factor (TNF)-alpha-induced IkappaB phosphorylation and degradation, suggesting a role of this compound in targeting IKK; and 4) EF24 indeed directly inhibits the catalytic activity of IKK in an in vitro-reconstituted system. Our study identifies IKK as an effective target for EF24 and provides a molecular explanation for a superior activity of EF24 over curcumin. The effective inhibition of TNF-alpha-induced NF-kappaB signaling by EF24 extends the therapeutic application of EF24 to other NF-kappaB-dependent diseases, including inflammatory diseases such as rheumatoid arthritis.     

Curcumin-induced antitumor effects on triple-negative breast cancer patient-derived xenograft tumor mice through inhibiting salt-induced kinase-3 protein

This study demonstrated for the first time that curcumin effectively inhibits the growth of triple-negative breast cancer (TNBC) tumors by inhibiting the expression of salt-induced kinase-3 (SIK3) protein in patient-derived xenografted tumor mice (TNBC-PDX). For TNBC patients, chemotherapy is the only option for postoperative adjuvant treatment. In this study, we detected the SIK3 mRNA expression in paired-breast cancer tissues by qPCR analysis. The results revealed that SIK3 mRNA expression was significantly higher in tumor tissues when compared to the normal adjacent tissues (73.25 times, n = 183). Thus, it is proposed for the first time that the antitumor effect induced by curcumin by targeting SIK3 can be used as a novel strategy for the therapy of TNBC tumors. In vitro mechanism studies have shown that curcumin (>25 μM) inhibits the SIK3-mediated cyclin D upregulation, thereby inhibiting the G1/S cell cycle and arresting TNBC (MDA-MB-231) cancer cell growth. The SIK3 overexpression was associated with increased mesenchymal markers (i.e., Vimentin, α-SMA, MMP3, and Twist) during epithelial–mesenchymal transition (EMT). Our results demonstrated that curcumin inhibits the SIK3-mediated EMT, effectively attenuating the tumor migration. For clinical indications, dietary nutrients (such as curcumin) as an adjuvant to chemotherapy should be helpful to TNBC patients because the current trend is to shrink the tumor with preoperative chemotherapy and then perform surgery. In addition, from the perspective of chemoprevention, curcumin has excellent clinical application value.     

Hypoxia and succinate antagonize 2-deoxyglucose effects on glioblastoma

Glioblastoma multiforme (GBM) are highly proliferative brain tumors characterized by a hypoxic microenvironment which controls GBM stem cell maintenance. Tumor hypoxia promotes also elevated glycolytic rate; thus, limiting glucose metabolism is a potential approach to inhibit tumor growth. Here we investigate the effects mediated by 2-deoxyglucose (2-DG), a glucose analogue, on primary GBM-derived cells maintained under hypoxia. Our results indicate that hypoxia protects GBM cells from the apoptotic effect elicited by 2-DG, which raises succinate dehydrogenase activity thus promoting succinate level decrease. As a consequence hypoxia inducible factor-1α (HIF-1α) degradation occurs and this induces GBM cells to acquire a neuronal committed phenotype. By adding succinate these effects are reverted, as succinate stabilizes HIF-1α and increases GBM stem cell fraction particularly under hypoxia, thus preserving the tumor stem cell niche.2-DG inhibits anaerobic glycolysis altering GBM cell phenotype by forcing tumor cells into mitochondrial metabolism and by inducing differentiation.