Pharmacological targeting of Axin2 suppresses cell growth and metastasis in colorectal cancer.

BACKGROUND AND PURPOSE: The scaffold molecule Axin2 is constitutively activated in colorectal cancer (CRC) and functions as a potent promoter of CRC behaviour. Pharmacological targeting of Axin2 may therefore exert a therapeutic effect in patients with CRC. Here, we discovered a potent small-molecule inhibitor of Axin2, based on the mechanism by which Axin2 is regulated post-translationally, and investigated its antitumour effects. EXPERIMENTAL APPROACH: Compound discovery and its inhibitory action on Axin2 protein were revealed by microscale thermophoresis, in vitro kinase assay, quantitative kinetic assay, immunoblotting/immunoprecipitation, RT-qPCR and cycloheximide pulse-chase assay. Compound antitumour effects and the underlying mechanisms were evaluated in multiple cell-based assays and mouse models. KEY RESULTS: We discovered that glycogen synthase kinase 3ß (GSK3ß) phosphorylates Axin2 at two consensus motifs and coupled Axin2 phosphorylation to its ubiquitination (mediated by the E3 ligase ß-Trcp2) and proteasomal degradation. The binding of Axin2 to GSK3ß in CRC cells is faint, which enables most of the Axin2 protein to maintain an unphosphorylated status and thereby permits the cells to preserve high levels of Axin2. Importantly, we identified a small-molecule compound CW85319 that enhances Axin2's interaction with GSK3ß via forming a high affinity for Axin2. Treatment of CRC cells with CW85319 enhanced Axin2 binding with GSK3ß, thereby promoting Axin2 phosphorylation, subsequent ubiquitination, and degradation. Furthermore, we demonstrated that CW85319 efficiently suppressed Axin2-driven CRC growth and metastasis, without eliciting side toxicity. CONCLUSIONS AND IMPLICATIONS: These findings suggest that pharmacological targeting of Axin2 by CW85319 may provide therapeutic benefits against certain human cancers, especially CRC.

Structural Optimization and Improving Antitumor Potential of Moreollic Acid from Gamboge.

Moreollic acid, a caged-tetraprenylated xanthone from Gamboge, has been indicated as a potent antitumor molecule. In the present study, a series of moreollic acid derivatives with novel structures were designed and synthesized, and their antitumor activities were determined in multifarious cell lines. The preliminary screening results showed that all synthesized compounds selectively inhibited human colon cancer cell proliferation. TH12-10, with an IC50 of 0.83, 1.10, and 0.79 µM against HCT116, DLD1, and SW620, respectively, was selected for further antitumor mechanism studies. Results revealed that TH12-10 effectively inhibited cell proliferation by blocking cell-cycle progression from G1 to S. Besides, the apparent structure-activity relationships of target compounds were discussed. To summarize, a series of moreollic acid derivatives were discovered to possess satisfactory antitumor potentials. Among them, TH12-10 displays the highest antitumor activities against human colon cancer cells, in which the IC50 values in DLD1 and SW620 are lower than that of 5-fluorouracil.

Omeprazole suppresses aggressive cancer growth and metastasis in mice through promoting Snail degradation.

Omeprazole is a proton pump inhibitor that has recently been reported to exhibit anticancer activity against several types of cancer. However, the anticancer mechanisms of omeprazole remain elusive. Snail is an oncogenic zinc finger transcription factor; aberrant activation of Snail is associated with the occurrence and progression of cancer. In this study, we investigated whether Snail acted as a direct anticancer target of omeprazole. We showed that omeprazole displayed a high binding-affinity to recombinant Snail protein (Kd = 0.076 mM), suggesting that omeprazole directly and physically binds to the Snail protein. We further revealed that omeprazole disrupted CREB-binding protein (CBP)/p300-mediated Snail acetylation and then promoted Snail degradation through the ubiquitin-proteasome pathway in HCT116 cells. Omeprazole treatment markedly suppressed Snail-driven epithelial-mesenchymal transition (EMT) in aggressive HCT116, SUM159, and 4T1 cancer cells in vitro and reduced EMT-associated tumor invasion and metastasis in cancer cell xenograft models. Omeprazole also inhibited tumor growth by limiting Snail-dependent cell cycle progression. Overall, this study, for the first time, identifies Snail as a target of omeprazole and reveals a novel mechanism underlying the therapeutic effects of omeprazole against cancer. This study strongly suggests that omeprazole may be an excellent auxiliary drug for treating patients with malignant tumors.

The antibiotic drug trimethoprim suppresses tumour growth and metastasis via targeting Snail.

BACKGROUND AND PURPOSE: The zinc finger transcription factor Snail is aberrantly activated in many human cancers and strongly associated with poor prognosis. As a transcription factor, Snail has been traditionally considered an 'undruggable' target. Here, we identified a potent small-molecule inhibitor of Snail, namely trimethoprim, and investigated its potential antitumour effects and the underlying mechanisms. EXPERIMENTAL APPROACH: The inhibitory action of trimethoprim on Snail protein and the related molecular mechanisms were revealed by molecular docking, biolayer interferometry, immunoblotting, immunoprecipitation, qRT-PCR, pull-down and cycloheximide pulse-chase assays. The anti-proliferative and anti-metastatic effects of trimethoprim via targeting Snail were tested in multiple cell-based assays and animal models. KEY RESULTS: This study identified trimethoprim, an antimicrobial drug, as a potent antitumour agent via targeting Snail. Molecular modelling analysis predicted that trimethoprim directly binds to the arginine-174 pocket of Snail protein. We further discovered that trimethoprim strongly interrupts the interaction of Snail with CREB-binding protein (CBP)/p300, which consequently suppresses Snail acetylation and promotes Snail degradation through the ubiquitin-proteasome pathway. Furthermore, trimethoprim sufficiently inhibited the proliferation, epithelial-mesenchymal transition (EMT) and migration of cancer cells in vitro via specifically targeting Snail. More importantly, trimethoprim effectively reduced Snail-driven tumour growth and metastasis to vital organs such as lung, bone and liver. CONCLUSIONS AND IMPLICATIONS: These findings indicate, for the first time, that trimethoprim suppresses tumour growth and metastasis via targeting Snail. This study provides insights for a better understanding of the anticancer effects of trimethoprim and offers a potential anticancer therapeutic agent for clinical treatment.

A potent CBP/p300-Snail interaction inhibitor suppresses tumor growth and metastasis in wild-type p53-expressing cancer.

The zinc finger transcription factor Snail is aberrantly activated in many human cancers and associated with poor prognosis. Therefore, targeting Snail is expected to exert therapeutic benefit in patients with cancer. However, Snail has traditionally been considered "undruggable," and no effective pharmacological inhibitors have been identified. Here, we found a small-molecule compound CYD19 that forms a high-affinity interaction with the evolutionarily conserved arginine-174 pocket of Snail protein. In aggressive cancer cells, CYD19 binds to Snail and thus disrupts Snail's interaction with CREB-binding protein (CBP)/p300, which consequently impairs CBP/p300-mediated Snail acetylation and then promotes its degradation through the ubiquitin-proteasome pathway. Moreover, CYD19 restores Snail-dependent repression of wild-type p53, thus reducing tumor growth and survival in vitro and in vivo. In addition, CYD19 reverses Snail-mediated epithelial-mesenchymal transition (EMT) and impairs EMT-associated tumor invasion and metastasis. Our findings demonstrate that pharmacologically targeting Snail by CYD19 may exert potent therapeutic effects in patients with cancer.

Inactivation of endothelial ZEB1 impedes tumor progression and sensitizes tumors to conventional therapies.

Current antiangiogenic therapy is limited by its cytostatic property, scarce drug delivery to the tumor, and side toxicity. To address these limitations, we unveiled the role of ZEB1, a tumor endothelium-enriched zinc-finger transcription factor, during tumor progression. We discovered that the patients who had lung adenocarcinomas with high ZEB1 expression in tumor endothelium had increased prevalence of metastases and markedly reduced overall survival after the diagnosis of lung cancer. Endothelial ZEB1 deletion in tumor-bearing mice diminished tumor angiogenesis while eliciting persistent tumor vascular normalization by epigenetically repressing TGF-ß signaling. This consequently led to improved blood and oxygen perfusion, enhanced chemotherapy delivery and immune effector cell infiltration, and reduced tumor growth and metastasis. Moreover, targeting vascular ZEB1 remarkably potentiated the anticancer activity of nontoxic low-dose cisplatin. Treatment with low-dose anti-programmed cell death protein 1 (anti-PD-1) antibody elicited tumor regression and markedly extended survival in ZEB1-deleted mice, conferring long-term protective anticancer immunity. Collectively, we demonstrated that inactivation of endothelial ZEB1 may offer alternative opportunities for cancer therapy with minimal side effects. Targeting endothelium-derived ZEB1 in combination with conventional chemotherapy or immune checkpoint blockade therapy may yield a potent and superior anticancer effect.

Endothelial ZEB1 promotes angiogenesis-dependent bone formation and reverses osteoporosis.

Recent interest in the control of bone metabolism has focused on a specialized subset of CD31hiendomucinhi vessels, which are reported to couple angiogenesis with osteogenesis. However, the underlying mechanisms that link these processes together remain largely undefined. Here we show that the zinc-finger transcription factor ZEB1 is predominantly expressed in CD31hiendomucinhi endothelium in human and mouse bone. Endothelial cell-specific deletion of ZEB1 in mice impairs CD31hiendomucinhi vessel formation in the bone, resulting in reduced osteogenesis. Mechanistically, ZEB1 deletion reduces histone acetylation on Dll4 and Notch1 promoters, thereby epigenetically suppressing Notch signaling, a critical pathway that controls bone angiogenesis and osteogenesis. ZEB1 expression in skeletal endothelium declines in osteoporotic mice and humans. Administration of Zeb1-packaged liposomes in osteoporotic mice restores impaired Notch activity in skeletal endothelium, thereby promoting angiogenesis-dependent osteogenesis and ameliorating bone loss. Pharmacological reversal of the low ZEB1/Notch signaling may exert therapeutic benefit in osteoporotic patients by promoting angiogenesis-dependent bone formation.

A ZEB1/p53 signaling axis in stromal fibroblasts promotes mammary epithelial tumours.

Accumulating evidence indicates that the zinc-finger transcription factor ZEB1 is predominantly expressed in the stroma of several tumours. However, the role of stromal ZEB1 in tumour progression remains unexplored. In this study, while interrogating human databases, we uncover a remarkable decrease in relapse-free survival of breast cancer patients expressing high ZEB1 levels in the stroma. Using a mouse model of breast cancer, we show that ZEB1 inactivation in stromal fibroblasts suppresses tumour initiation, progression and metastasis. We associate this with reduced extracellular matrix remodeling, immune cell infiltration and decreased angiogenesis. ZEB1 deletion in stromal fibroblasts increases acetylation, expression and recruitment of p53 to FGF2/7, VEGF and IL6 promoters, thereby reducing their production and secretion into the surrounding stroma. Importantly, p53 ablation in ZEB1 stroma-deleted mammary tumours sufficiently recovers the impaired cancer growth and progression. Our findings identify the ZEB1/p53 axis as a stroma-specific signaling pathway that promotes mammary epithelial tumours.

Discovery of a natural small-molecule compound that suppresses tumor EMT, stemness and metastasis by inhibiting TGFß/BMP signaling in triple-negative breast cancer.

BACKGROUND: The transforming growth factor ß (TGFß) and bone morphogenetic protein (BMP) signaling pathways are both constitutively activated in triple-negative breast cancer (TNBC). We are interested in isolating the naturally-derived small-molecule inhibitor that could simultaneously targeting TGFß/BMP pathways and further studying its anti-proliferative/-metastatic effects as well as the underlying mechanisms in multiple tumor models. METHODS: Multiple in vitro cell-based assays are used to examine the compound's inhibitory efficacy on TNBC cell growth, stemness, epithelial-mesenchymal transition (EMT), invasion and migration by targeting TGFß/BMP signaling pathways. Transgenic breast cancer mouse model (MMTV-PyMT), subcutaneous xenograft and bone metastasis models are used to examine ZL170's effects on TNBC growth and metastasis potentials in vivo. RESULTS: ZL170 dose-dependently inhibits cell proliferation, EMT, stemness, invasion and migration in vitro via specifically targeting canonical TGFß/BMP-SMADs pathways in TNBC cells. The compound significantly hinders osteolytic bone metastasis and xenograft tumor growth without inflicting toxicity on vital organs of tumor-bearing nude mice. ZL170 strongly inhibits primary tumor growth and lung metastases in MMTV-PyMT transgenic mice. ZL170-treated tumors exhibit impaired TGFß/BMP signaling pathways in both epithelial and stromal compartments, thereby creating a suppressive tumor microenvironment characterized by reduced extracellular matrix deposition and decreased infiltration of stromal cells. CONCLUSIONS: ZL170 inhibits tumor EMT, stemness and metastasis and could be further developed as a potent anti-metastatic agent used in combination with cytotoxic drugs for treatment of TNBC and other advanced metastatic cancers.

LW106, a novel indoleamine 2,3-dioxygenase 1 inhibitor, suppresses tumour progression by limiting stroma-immune crosstalk and cancer stem cell enrichment in tumour micro-environment.

BACKGROUND AND PURPOSE: Indoleamine 2,3-dioxygenase 1 (IDO1) is emerging as an important new therapeutic target for treatment of malignant tumours characterized by dysregulated tryptophan metabolism. However, the antitumour efficacy of existing small-molecule inhibitors of IDO1 is still unsatisfactory and the underlying mechanism remains largely undefined. Hence, we discovered a novel potent small-molecule inhibitor of IDO1, LW106, and studied its antitumour effects and the underlying mechanisms in two tumour models. EXPERIMENTAL APPROACH: C57BL6 mice, athymic nude mice or Ido1-/- mice were inoculated with IDO1-expressing and -nonexpressing tumour cells and treated with vehicle, epacadostat or increasing doses of LW106. Xenografted tumours, plasma, spleens and other vital organs were harvested and subjected to kynurenine/tryptophan measurement and flow cytometric, histological and immunohistochemical analyses. KEY RESULTS: LW106 dose-dependently inhibited the outgrowth of xenografted tumours that were inoculated in C57BL6 mice but not nude mice or Ido1-/- mice, showing a stronger antitumour efficacy than epacadostat, an existing IDO1 inhibitor. LW106 substantially elevated intratumoural infiltration of proliferative Teff cells, while reducing recruitment of proliferative Treg cells and non-haematopoietic stromal cells such as endothelial cells and cancer-associated fibroblasts. LW106 treatment resulted in a reduced subpopulation of cancer stem cells (CSCs) in xenografted tumours in which fewer proliferative/invasive tumour cells and more apoptotic tumour cells were observed. CONCLUSIONS AND IMPLICATIONS: LW106 inhibits tumour outgrowth by limiting stroma-immune crosstalk and CSC enrichment in the tumour micro-environment. LW106 has potential as a immunotherapeutic agent for use in combination with immune checkpoint inhibitors and (or) chemotherapeutic drugs for cancer treatment.

Snail1-dependent p53 repression regulates expansion and activity of tumour-initiating cells in breast cancer.

The zinc-finger transcription factor Snail1 is inappropriately expressed in breast cancer and associated with poor prognosis. While interrogating human databases, we uncovered marked decreases in relapse-free survival of breast cancer patients expressing high Snail1 levels in tandem with wild-type, but not mutant, p53. Using a Snail1 conditional knockout model of mouse breast cancer that maintains wild-type p53, we find that Snail1 plays an essential role in tumour progression by controlling the expansion and activity of tumour-initiating cells in preneoplastic glands and established tumours, whereas it is not required for normal mammary development. Growth and survival of preneoplastic as well as neoplastic mammary epithelial cells is dependent on the formation of a Snail1/HDAC1/p53 tri-molecular complex that deacetylates active p53, thereby promoting its proteasomal degradation. Our findings identify Snail1 as a molecular bypass that suppresses the anti-proliferative and pro-apoptotic effects exerted by wild-type p53 in breast cancer.

Wogonin inhibits multiple myeloma-stimulated angiogenesis via c-Myc/VHL/HIF-1α signaling axis.

Angiogenesis is associated with the progression of multiple myeloma (MM). Wogonin is an active mono-flavonoid with remarkable antitumor activity. However, its impact on MM-stimulated angiogenesis remains largely unknown. Here, we demonstrated that wogonin decreased expression and secretion of pro-angiogenic factors in MM cells via c-Myc/HIF-1α signaling axis, reducing MM-stimulated angiogenesis and MM cell proliferation in vivo. Overexpression of c-Myc in MM cells disrupted the balance between VHL SUMOylation and ubiquitination, and thus inhibited proteasome-mediated HIF-1α degradation. Impaired function of VHL ubiquitination complex in c-Myc-overexpressing cells was fully reversed by wogonin treatment via increasing HIF-1α-VHL interaction and promoting HIF-1α degradation. Collectively, our in vitro and in vivo studies reveal for the first time that wogonin represses MM-stimulated angiogenesis and tumor progression via c-Myc/VHL/HIF-1α signaling axis.

A Snail1/Notch1 signalling axis controls embryonic vascular development.

Notch1-Delta-like 4 (Dll4) signalling controls vascular development by regulating endothelial cell (EC) targets that modulate vessel wall remodelling and arterial-venous specification. The molecular effectors that modulate Notch signalling during vascular development remain largely undefined. Here we demonstrate that the transcriptional repressor, Snail1, acts as a VEGF-induced regulator of Notch1 signalling and Dll4 expression. EC-specific Snail1 loss-of-function conditional knockout mice die in utero with defects in vessel wall remodelling in association with losses in mural cell investment and disruptions in arterial-venous specification. Snail1 loss-of-function conditional knockout embryos further display upregulated Notch1 signalling and Dll4 expression that is partially reversed by inhibiting γ-secretase activity in vivo with Dll4 identified as a direct target of Snail1-mediated transcriptional repression. These results document a Snail1-Dll4/Notch1 axis that controls embryonic vascular development.

Canonical Wnt signaling regulates Slug activity and links epithelial-mesenchymal transition with epigenetic Breast Cancer 1, Early Onset (BRCA1) repression.

Slug (Snail2) plays critical roles in regulating the epithelial-mesenchymal transition (EMT) programs operative during development and disease. However, the means by which Slug activity is controlled remain unclear. Herein we identify an unrecognized canonical Wnt/GSK3ß/ß-Trcp1 axis that controls Slug activity. In the absence of Wnt signaling, Slug is phosphorylated by GSK3ß and subsequently undergoes ß-Trcp1-dependent ubiquitination and proteosomal degradation. Alternatively, in the presence of canonical Wnt ligands, GSK3ß kinase activity is inhibited, nuclear Slug levels increase, and EMT programs are initiated. Consistent with recent studies describing correlative associations in basal-like breast cancers between Wnt signaling, increased Slug levels, and reduced expression of the tumor suppressor Breast Cancer 1, Early Onset (BRCA1), further studies demonstrate that Slug-as well as Snail-directly represses BRCA1 expression by recruiting the chromatin-demethylase, LSD1, and binding to a series of E-boxes located within the BRCA1 promoter. Consonant with these findings, nuclear Slug and Snail expression are increased in association with BRCA1 repression in a cohort of triple-negative breast cancer patients. Together, these findings establish unique functional links between canonical Wnt signaling, Slug expression, EMT, and BRCA1 regulation.

Canonical Wnt suppressor, Axin2, promotes colon carcinoma oncogenic activity.

Aberrant activation of canonical Wingless-type MMTV integration site family (Wnt) signaling is pathognomonic of colorectal cancers (CRC) harboring functional mutations in either adenomatous polyposis coli or ß-catenin. Coincident with Wnt cascade activation, CRCs also up-regulate the expression of Wnt pathway feedback inhibitors, particularly the putative tumor suppressor, Axin2. Because Axin2 serves as a negative regulator of canonical Wnt signaling in normal cells, recent attention has focused on the utility of increasing Axin2 levels in CRCs as a means to slow tumor progression. However, rather than functioning as a tumor suppressor, we demonstrate that Axin2 acts as a potent promoter of carcinoma behavior by up-regulating the activity of the transcriptional repressor, Snail1, inducing a functional epithelial-mesenchymal transition (EMT) program and driving metastatic activity. Silencing Axin2 expression decreases Snail1 activity, reverses EMT, and inhibits CRC invasive and metastatic activities in concert with global effects on the Wnt-regulated cancer cell transcriptome. The further identification of Axin2 and nuclear Snail1 proteins at the invasive front of human CRCs supports a revised model wherein Axin2 acts as a potent tumor promoter in vivo.

Plk1 phosphorylation of TRF1 is essential for its binding to telomeres.

In a search for Polo-like kinase 1 (Plk1) interaction proteins, we have identified TRF1 (telomeric repeat binding factor 1) as a potential Plk1 target. In this communication we report further characterization of the interaction. We show that Plk1 associates with TRF1, and Plk1 phosphorylates TRF1 at Ser-435 in vivo. Moreover, Cdk1, serving as a priming kinase, phosphorylates TRF1 to generate a docking site for Plk1 toward TRF1. In the presence of nocodazole, ectopic expression of wild type TRF1 but not TRF1 with alanine mutation in the Plk1 phosphorylation site induces apoptosis in cells containing short telomeres but not in cells containing long telomeres. Unexpectedly, down-regulation of TRF1 by RNA interference affects cell proliferation and results in obvious apoptosis in cells with short telomeres but not in cells with long telomeres. Importantly, we observe that telomeric DNA binding ability of TRF1 is cell cycle-regulated and reaches a peak during mitosis. Upon phosphorylation by Plk1 in vivo and in vitro, the ability of TRF1 to bind telomeric DNA is dramatically increased. These results demonstrate that Plk1 interacts with and phosphorylates TRF1 and suggest that Plk1-mediated phosphorylation is involved in both TRF1 overexpression-induced apoptosis and its telomeric DNA binding ability.

Role for Plk1 phosphorylation of Hbo1 in regulation of replication licensing.

In a search for Polo-like kinase 1 (Plk1)-interacting proteins using a yeast two-hybrid system, we have identified histone acetyltransferase binding to the origin recognition complex 1 (Hbo1) as a potential Plk1 target. Here, we show that the interaction between Plk1 and Hbo1 is mitosis-specific and that Plk1 phosphorylates Hbo1 on Ser-57 in vitro and in vivo. During mitosis, Cdk1 phosphorylates Hbo1 on Thr-85/88, creating a docking site for Plk1 to be recruited. Significantly, the overexpression of Hbo1 mutated at the Plk1 phosphorylation site (S57A) leads to cell-cycle arrest in the G1/S phase, inhibition of chromatin loading of the minichromosome maintenance (Mcm) complex, and a reduced DNA replication rate. Similarly, Hbo1 depletion results in decreased DNA replication and a failure of Mcm complex binding to chromatin, both of which can be partially rescued by the ectopic expression of WT Hbo1 but not Hbo1-S57A. These results suggest that Plk1 phosphorylation of Hbo1 may be required for prereplicative complex (pre-RC) formation and DNA replication licensing.

Involvement of dopamine system in regulation of Na+,K+-ATPase in the striatum upon activation of opioid receptors by morphine.

The striatum is believed to be a crucial brain region associated with drug reward. Adaptive alteration of neurochemistry in this area might be one potential mechanism underlying drug dependence. It has been proposed that the dysfunction of Na+,K+-ATPase function is involved in morphine tolerance and dependence. The present study, therefore, was undertaken to study the adaptation of the striatal Na+,K+-ATPase activity in response to morphine treatment. The results demonstrated that in vivo short-term morphine treatment stimulated Na+,K+-ATPase activity in a dose-dependent manner. This action could be significantly inhibited by D2-like dopamine receptor antagonist S(-)-3-chloro-5-ethyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-hydroxy-2-methoxybenzamine (eticlopride). Contrary to shortterm morphine treatment, long-term morphine treatment significantly suppressed Na+,K+-ATPase activity. This effect could be significantly inhibited by D(1)-like dopamine receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH 23390). However, both short-term and long-term morphine treatment-induced changes in Na+,K+-ATPase activity could be reversed by opioid receptor antagonist naltrexone. It was further found that cAMP-dependent protein kinase (PKA) was crucially involved in regulating Na+,K+-ATPase activity by morphine. Different regulation of the phosphorylation levels of the alpha3 subunit of Na+,K+-ATPase by PKA was related to the distinct modulations of Na+,K+-ATPase by short-term and long-term morphine treatment. Short-term morphine treatment inhibited PKA activity and then decreased the phosphorylation of Na+,K+-ATPase, leading to increase in enzyme activity. These effects were sensitive to eticlopride or naltrexone. Conversely, long-term morphine treatment stimulated PKA activity and then increased the phosphorylation of Na+,K+-ATPase, leading to the reduction of enzyme activity. These effects were sensitive to SCH 23390 or naltrexone. These findings demonstrate that dopamine receptors are involved in regulation of Na+,K+-ATPase activity after activation of opioid receptors by morphine.

Involvement of cAMP/cAMP-dependent protein kinase signaling pathway in regulation of Na+,K+-ATPase upon activation of opioid receptors by morphine.

The depolarization of neurons induced by impairment of Na+,K+-ATPase activity after long-term opiate treatment has been shown to involve the development of opioid dependence. However, the mechanisms underlying changes in Na+,K+-ATPase activity after opioid treatment are unclear. The best-established molecular adaptation to long-term opioid exposure is up-regulation of the cAMP/cAMP-dependent protein kinase (PKA) signaling pathway; this study, therefore, was undertaken to investigate the role of up-regulation of cAMP/PKA signaling pathway in alteration of the mouse hippocampal Na+,K+-ATPase activity. The results demonstrated that short-term morphine treatment dose dependently stimulated Na+,K+-ATPase activity. This action could be significantly suppressed by adenylyl cyclase activator 7beta-acetoxy-8,13-epoxy-1alpha,6beta,9alpha-trihydroxylabd-14-en-11-one (forskolin), or the cAMP analog dibutyryl-cAMP. Contrary to short-term morphine treatment, long-term treatment significantly inhibited Na+,K+-ATPase activity. Moreover, an additional decrease in Na+,K+-ATPase activity was observed by naloxone precipitation. The effects of both short- and long-term morphine treatment on Na+,K+-ATPase activity were naltrexone-reversible. The regulation of Na+,K+-ATPase activity by morphine was inversely correlated with intracellular cAMP accumulation. N-[2-(4-Bromocinnamylamino)ethyl]-5-isoquinoline (H89), a specific PKA inhibitor, mimicked the stimulatory effect of short-term morphine but antagonized the inhibitory effect of long-term morphine treatment on Na+,K+-ATPase activity. However, okadaic acid, a protein phosphatase inhibitor, suppressed short-term morphine stimulation but potentiated long-term morphine inhibition of Na+,K+-ATPase activity. The regulation of Na+,K+-ATPase activity by morphine treatment seemed to associate with the alteration in phosphorylation level but not to be relevant to the change in abundance of Na+,K+-ATPase. These findings strongly demonstrate that cAMP/PKA signaling pathway involves regulation of Na+,K+-ATPase activity after activation of opioid receptors.

Gambogic acid inhibits proliferation of human lung carcinoma SPC-A1 cells in vivo and in vitro and represses telomerase activity and telomerase reverse transcriptase mRNA expression in the cells.

We determined the in vivo and in vitro antitumor activities of gambogic acid (GA) and one of the possible mechanisms for its inhibitory activities. In vivo antitumor activity of GA was evaluated by the relative tumor growth ratio (T/C) in nude mice, and in vitro inhibition of SPC-A1 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and trypan blue exclusion assay. Telomere repeats amplification protocol (TRAP)-polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) and RT-PCR were used to quantitatively detect telomerase activity and the expression of human telomerase reverse transcriptase (hTERT) mRNA, respectively. Results from our in vivo study showed that transplantable tumor growth remained suppressed for up to 21 d with minimal animal weight loss in nude mice treated with gambogic acid (i.v.). Proliferation of SPC-A1 cells cultured in vitro was significantly inhibited (p<0.01), showing time-dependent and dose-dependent inhibition. Telomerase activity and hTERT mRNA expression were both decreased significantly, when cells were exposed to gambogic acid for 24, 48 and 72 h (for 24 h p<0.05, and for 48, 72 h, p<0.01). These results suggeste that gambogic acid could inhibit the growth of SPC-A1 cells and its tumor xenografts, and when treated with gambogic acid for a period of time, telomerase activity and expression of hTERT mRNA in the tumor cells were both inhibited significantly. It is safe, at least in part, to conclude that the down-regulating telomerase activity of GA by modifying partly the expression of hTERT mRNA in SPC-A1 cells may be one possible mechanism for the inhibitory activity of GA in the cells.