Tuangporn Suthiphongchai

Prolactin directly enhances bone turnover by raising osteoblast-expressed receptor activator of nuclear factor κB ligand/osteoprotegerin ratio

Hyperprolactinemia leads to high bone turnover as a result of enhanced bone formation and resorption. Although its osteopenic effect has long been explained as hyperprolactinemia-induced hypogonadism, identified prolactin (PRL) receptors in osteoblasts suggested a possible direct action of PRL on bone. In the present study, we found that hyperprolactinemia induced by anterior pituitary transplantation (AP), with or without ovariectomy (Ovx), had no detectable effect on bone mineral density and content measured by dual-energy X-ray absorptiometry (DXA). However, histomorphometric studies revealed increases in the osteoblast and osteoclast surfaces in the AP rats, but a decrease in the osteoblast surface in the AP+Ovx rats. The resorptive activity was predominant since bone volume and trabecular number were decreased, and the trabecular separation was increased in both groups. Estrogen supplement (E2) fully reversed the effect of estrogen depletion in the Ovx but not in the AP+Ovx rats. In contrast to the typical Ovx rats, bone formation and resorption became uncoupled in the AP+Ovx rats. Therefore, hyperprolactinemia was likely to have some estrogen-independent and/or direct actions on bone turnover. Osteoblast-expressed PRL receptor transcripts and proteins shown in the present study confirmed our hypothesis. Furthermore, we demonstrated that the osteoblast-like cells, MG-63, directly exposed to PRL exhibited lower expression of alkaline phosphatase and osteocalcin mRNA, and a decrease in alkaline phosphatase activity. The ratios of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) proteins were increased, indicating an increase in the osteoclastic bone resorption. The present data thus demonstrated that hyperprolactinemia could act directly on bone to stimulate bone turnover, with more influence on bone resorption than formation. PRL enhanced bone resorption in part by increasing RANKL and decreasing OPG expressions by osteoblasts.

Vanillin suppresses metastatic potential of human cancer cells through PI3K inhibition and decreases angiogenesis in vivo.

Vanillin, a food flavoring agent, has been shown to suppress cancer cell migration and metastasis in a mouse model, but its mechanism of action is unknown. In this report, we have examined the antimetastatic potential of vanillin and its structurally related compounds, vanillic acid, vanillyl alcohol, and apocynin on hepatocyte growth factor (HGF)-induced migration of human lung cancer cells by the Transwell assay. Vanillin and apocynin could inhibit cell migration, and both compounds selectively inhibited Akt phosphorylation of HGF signaling, without affecting phosphorylation of Met and Erk. Vanillin and apocynin could inhibit the enzymatic activity of phosphoinositide 3-kinase (PI3K), as revealed by an in vitro lipid kinase assay, suggesting that inhibition of PI3K activity was a mechanism underlying the inhibitory effect on cancer cell migration, and the presence of an aldehyde or ketone group in the vanillin structure was important for this inhibition. Vanillin and apocynin also inhibited angiogenesis, determined by the chick chorioallantoic membrane assay.

Prolactin decreases the expression ratio of receptor activator of nuclear factor κB ligand/osteoprotegerin in human fetal osteoblast cells

Prolactin (PRL) enhanced bone remodeling leading to net bone loss in adult and net bone gain in young animals. Studies in PRL‐exposed osteoblasts derived from adult humans revealed an increase in the expression ratio of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG), thus supporting the previous finding of PRL‐induced bone loss in adults. This study thus investigated the effects of PRL on the osteoblast functions and the RANKL/OPG ratio in human fetal osteoblast (hFOB) cells which strongly expressed PRL receptors. After 48 h incubation, PRL increased osteocalcin expression, but had no effect on cell proliferation. However, the alkaline phosphatase activity was decreased in a dose—response manner within 24 h. The effect of PRL on alkaline phosphatase was abolished by LY294002, a phosphoinositide 3‐kinase (PI3K) inhibitor. PRL also decreased the RANKL/OPG ratio by downregulating RANKL and upregulating OPG expression, implicating a reduction in the osteoblast signal for osteoclastic bone resorption. It could be concluded that, unlike the osteoblasts derived from adult humans, PRL‐exposed hFOB cells exhibited indices suggestive of bone gain, which could explain the in vivo findings in young rats. The signal transduction of PRL in osteoblasts involved the PI3K pathway.

High level of urokinase plasminogen activator contributes to cholangiocarcinoma invasion and metastasis

AIM To investigate the role of urokinase plasminogen activator (uPA) in cholangiocarcinoma (CCA) invasion and its correlation with clinicopathological parameters. METHODS uPA expression in CCA tissue was determined by immunohistochemistry. The level of uPA from two CCA cell lines (HuCCA-1 and KKU-M213) and a non-cancer immortalized cholangiocyte cell line (H69) was monitored by plasminogen-gelatin zymography and western blotting, whereas that of plasminogen activator inhibitor type 1 (PAI-1) protein and uPA receptor (uPAR) mRNA was monitored by western blotting and quantitative real-time reverse transcriptase polymerase chain reaction, respectively. Two independent methods were employed to suppress uPA function: a synthetic uPA inhibitor (B428) and silencing of uPA gene expression using siRNA. In vitro invasion of the uPA-disrupted cells was assessed by Matrigel-coated Transwell assay. RESULTS The immunohistochemical study showed that 75.3% (131/174) of CCA tissues expressed uPA. High uPA expression was correlated with lymphatic invasion and metastasis of CCA patients. Plasminogen-gelatin zymography of the conditioned media and cell-surface eluates showed that both CCA cell lines, but not H69, expressed both secreted and membrane-bound forms of uPA. Although the two CCA cell lines, HuCCA-1 and KKU-M213, expressed a relatively high level of uPA and uPAR, the latter exhibited a much lower degree of in vitro invasiveness, correlating with a high expression of PAI-1 in the latter, but not in the former. Suppressing uPA function with a specific uPA inhibitor, B428, or with siRNA against uPA reduced in vitro invasiveness of KKU-M213 cells, demonstrating the requirement for uPA in the invasiveness of CCA cells. Therefore, our in vivo and in vitro studies suggest that uPA is an important requirement for the invasion process of CCA. CONCLUSION uPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro, suggesting its potential as a therapeutic target.

Involvement of ERK1/2 in invasiveness and metastatic development of rat prostatic adenocarcinoma.

Extracellular signal-regulated kinase (ERK) activation has been implicated in cell motility and invasion. In this study, we demonstrated that the steady-state levels of activated ERK1/2 correlated with the degree of invasiveness and metastatic potential of three Dunning cancer cell lines, originating from the same parental tumor. Inhibition of mitogen-activated protein kinase kinase 1 (MEK1), an upstream regulator of ERK1/2, with PD98059 resulted in a dose-dependent reduction of invasiveness with different IC50 values in the three Dunning cell lines. These results suggest that ERK is, at least in part, responsible for regulating invasiveness and may underlie the differences in the metastatic ability of the cell lines.

Transcriptome analysis of mammary tissues reveals complex patterns of transporter gene expression during pregnancy and lactation

As a complex Ca2+‐rich fluid mixture of water, casein, lactose and several ions, milk secretion requires a number of unknown transporters, which can be identified by a genome‐wide microarray study in mammary tissues of lactating animals. Ca2+ was reported to be secreted across mammary epithelial cells through the transcellular pathway, presumably involving TRPC (canonical transient receptor potential) channels. In the present study, we have used quantitative real‐time PCR to demonstrate that the human mammary cell line MCF‐7, as well as rat mammary tissues from pregnant and lactating rats, expressed TRPC1, TRPC5 and TRPC6. Expression of TRPC1, TRPC5 and TRPC7 were markedly up‐regulated, whereas that of TRPC3 and TRPC4 was down‐regulated in the early lactating period. To further identify other transporter genes affected by lactation, a highly sensitive Illumina microarray featuring Bead Array technology was performed on RNA samples from mammary tissues of lactating rats. We found that, of the 384 transcripts changed during lactation, 31 transcripts were involved in the transport of water and electrolytes, such as Ca2+, Na+, K+, Cl−, I−, Fe2+, sulfate and phosphate. The present study, therefore, provides information for further investigation of the mechanism of lactation‐induced transport adaptation in mammary epithelial cells.

Vanadium-dependent bromoperoxidases from Gracilaria algae

Red algae from the Gulf of Thailand were examined for haloperoxidatic activity. Six species, Gracilaria changii, G. edulis, G. firma, G. fisheri, G. salicornia, and G. tenuistipitata, showed bromoperoxidatic activity. Duplicate polyacrylamide electrophoretic gels showed enzyme activity patterns developed by phenol red staining for bromoperoxidatic activity and by 3,3′-diaminobenzidine staining for peroxidatic activity. All algae gave isoenzymic bromoperoxidatic activity bands and peroxidatic activity bands, but there were peroxidatic and bromoperoxidatic activity bands that did not correspond. The bromoperoxidatic activity of the crude enzyme extracts as well as previously dialyzed enzyme solutions was enhanced significantly by incubation with vanadium pentoxide. The three purified bromoperoxidases from G. fisheri contained vanadium, and their relative activities corresponded to the ratio of vanadium to enzyme. In addition, they were not inhibited by H2O2. These data confirm that the enzymes are vanadium bromoperoxidases.

Suppression of prometastatic phenotype of highly metastatic androgen-independent rat prostate cancer MLL cell line by PI3K inhibitor LY294002.

Upregulation of the PI3K pathway has often been reported in androgen-independent prostate cancer and is implicated in cancer cell survival and proliferation in the absence of androgen. Inhibition of PI3K by LY294002 suppressed cell invasion and motility of the highly metastatic androgen-independent Dunning rat prostate cancer MLL cell line with similar IC50 values and inhibition profile. Moreover, LY294002 attenuated expression of urokinase plasminogen activator (uPA) without any significant effect on that of matrix metalloproteinase 2. These results indicated that the role of PI3K in MLL cell invasion is by regulating cell motility and uPA expression.

Blocking ERK1/2 signaling impairs TGF-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells

BackgroundTransforming growth factor-β (TGF-β) plays a paradoxical role in cancer: it suppresses proliferation at early stages but promotes metastasis at late stages. This cytokine is upregulated in cholangiocarcinoma and is implicated in cholangiocarcinoma invasion and metastasis. Here we investigated the roles of non-Smad pathway (ERK1/2) and Smad in TGF-β tumor promoting and suppressing activities in intrahepatic cholangiocarcinoma (ICC) cells.MethodsTGF-β1 effects on proliferation, invasion and migration of ICC cells, KKU-M213 and/or HuCCA-1, were investigated using MTT, colony formation, in vitro Transwell and wound healing assays. Levels of mRNAs and proteins/phospho-proteins were measured by quantitative (q)RT-PCR and Western blotting respectively. E-cadherin localization was examined by immunofluorescence and secreted MMP-9 activity was assayed by gelatin zymography. The role of ERK1/2 signaling was evaluated by treating cells with TGF-β1 in combination with MEK1/2 inhibitor U0126, and that of Smad2/3 and Slug using siSmad2/3- and siSlug-transfected cells.Resultsh-TGF-β1 enhanced KKU-M213 cell invasion and migration and induced epithelial-mesenchymal transition as shown by an increase in vimentin, Slug and secreted MMP-9 levels and by a change in E-cadherin localization from membrane to cytosol, while retaining the cytokine’s ability to attenuate cell proliferation. h-TGF-β1 stimulated Smad2/3 and ERK1/2 phosphorylation, and the MEK1/2 inhibitor U0126 attenuated TGF-β1-induced KKU-M213 cell invasion and MMP-9 production but moderately enhanced the cytokine growth inhibitory activity. The latter effect was more noticeable in HuCCA-1 cells, which resisted TGF-β-anti-proliferative activity. Smad2/3 knock-down suppressed TGF-β1 ability to induce ERK1/2 phosphorylation, Slug expression and cell invasion, whereas Slug knock-down suppressed cell invasion and vimentin expression but marginally affected ERK1/2 activation and MMP-9 secretion. These results indicate that TGF-β1 activated ERK1/2 through Smad2/3 but not Slug pathway, and that ERK1/2 enhanced TGF-β1 tumor promoting but repressed its tumor suppressing functions.ConclusionsInhibiting ERK1/2 activation attenuates TGF-β1 tumor promoting effect (invasion) but retains its tumor suppressing role, thereby highlighting the importance of ERK1/2 in resolving the TGF-β paradox switch.

Abstract A24: High expression of ErbB2 contributes to cholangiocarcinoma cell invasion and proliferation through AKT/p70S6K

Cholangiocarcinoma (CCA), an adenocarcinoma of the biliary tract, is an incurable and highly lethal cancer. Its incidence has been increasing worldwide during to the past three decades and all cases have almost the same mortality rate. Due to the lack of a specific tumor marker and its silent symptom, this tumor is difficult to diagnose and the majority of patients present at the late stage (stage III or IV) of the disease progression. From 70% to 80% of CCA patients are inoperable and are generally treated by chemotherapy and radiation, which unfortunately have no significant impact for long-term survival. Thus, new diagnostic/prognosis markers and targeted therapies for CCA need to be investigated. Several lines of evidences have implicated the role of ErbB2 in aggressive forms of CCA. High expression of ErbB2 has been found in a variety of non-cancerous biliary proliferative diseases, such as in hepatolithiasis and primary sclerosing cholangitis, both of which are risk factors associated with CCA. Immunohistochemical data have demonstrated that 20% to 30% of tumor specimens from CCA patients show moderate to strong immunostaining for ErbB2 and most of them confer poor clinical outcomes (high metastasis and short survival rate). Moreover, about 30% of transgenic mice constitutively expressing wide type Neu develop gallbladder cancer and intrahepatic CCA within 8 months. Although many reports have indicated the involvement of ErbB2 in cholangiocarcinogenesis and CCA progression, the mechanism of aberrant ErbB2 expression in promoting CCA progression remains unclear. In this study, the role of ErbB2 in governing the malignant phenotype (invasion and proliferation) of CCA was investigated by suppressing ErbB2 function in three human CCA cell lines expressing different levels of ErbB2. Level of endogenous ErbB2 expression in three CCA cell lines, namely HuCCA-1, KKU-100 and KKU-M213, was determined by real time RT-PCR. Two ErbB2 inhibitory methods, a small molecule ErbB2 kinase inhibitor (AG825) and siRNA, were used to disrupt ErbB2 function in the cell lines. CCA cell invasion, motility and proliferation under ErbB2-disrupted conditions were detected using Transwell and MTT assays. In addition, ErbB2 downstream effectors were investigated by western blot analysis. Suppression of ErbB2 activity using a specific kinase inhibitor (AG825) reduced invasion, motility, and proliferation of all three CCA cell lines. The ability of this drug to inhibit neoplastic properties (invasion, motility and proliferation) increased concomitantly with the level of ErbB2 expression. Similarly, knockdown of ErbB2 level by siRNA inhibited cell invasion and proliferation of KKU-M213, a high ErbB2-expressing cell, better than those of the lower ErbB2-expressing cells, HuCCA-1 and KKU-100. Thus, both inhibitory methods indicated that there is more ErbB2-dependency for malignancy of high ErbB2-expressing cell, KKU-M213, than that of low ErbB2-expressing ones. In addition, interrupting ErbB2 activity decreased phosphorylation of AKT and p70S6K, but not ERK1/2, in the high ErbB2 expressing CCA cell line. Our data indicated that high ErbB2 expression enhances CCA invasion, motility and proliferation via AKT/p70S6K pathway, indicating the possibility of targeting these molecules for cancer therapy. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A24.