CYB5R3 functions as a tumor suppressor by inducing ER stress-mediated apoptosis in lung cancer cells via the PERK-ATF4 and IRE1α-JNK pathways.

Cytochrome b5 reductase 3 (CYB5R3) is involved in various cellular metabolic processes, including fatty acid synthesis and drug metabolism. However, the role of CYB5R3 in cancer development remains poorly understood. Here, we show that CYB5R3 expression is downregulated in human lung cancer cell lines and tissues. Adenoviral overexpression of CYB5R3 suppresses lung cancer cell growth in vitro and in vivo. However, CYB5R3 deficiency promotes tumorigenesis and metastasis in mouse models. Transcriptome analysis revealed that apoptosis- and endoplasmic reticulum (ER) stress-related genes are upregulated in CYB5R3-overexpressing lung cancer cells. Metabolomic analysis revealed that CYB5R3 overexpression increased the production of nicotinamide adenine dinucleotide (NAD+) and oxidized glutathione (GSSG). Ectopic CYB5R3 is mainly localized in the ER, where CYB5R3-dependent ER stress signaling is induced via activation of protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme 1 alpha (IRE1α). Moreover, NAD+ activates poly (ADP-ribose) polymerase16 (PARP16), an ER-resident protein, to promote ADP-ribosylation of PERK and IRE1α and induce ER stress. In addition, CYB5R3 induces the generation of reactive oxygen species and caspase-9-dependent intrinsic cell death. Our findings highlight the importance of CYB5R3 as a tumor suppressor for the development of CYB5R3-based therapeutics for lung cancer.

Automatic classification of 3D positional relationship between mandibular third molar and inferior alveolar canal using a distance-aware network.

The purpose of this study was to automatically classify the three-dimensional (3D) positional relationship between an impacted mandibular third molar (M3) and the inferior alveolar canal (MC) using a distance-aware network in cone-beam CT (CBCT) images. We developed a network consisting of cascaded stages of segmentation and classification for the buccal-lingual relationship between the M3 and the MC. The M3 and the MC were simultaneously segmented using Dense121 U-Net in the segmentation stage, and their buccal-lingual relationship was automatically classified using a 3D distance-aware network with the multichannel inputs of the original CBCT image and the signed distance map (SDM) generated from the segmentation in the classification stage. The Dense121 U-Net achieved the highest average precision of 0.87, 0.96, and 0.94 in the segmentation of the M3, the MC, and both together, respectively. The 3D distance-aware classification network of the Dense121 U-Net with the input of both the CBCT image and the SDM showed the highest performance of accuracy, sensitivity, specificity, and area under the receiver operating characteristic curve, each of which had a value of 1.00. The SDM generated from the segmentation mask significantly contributed to increasing the accuracy of the classification network. The proposed distance-aware network demonstrated high accuracy in the automatic classification of the 3D positional relationship between the M3 and the MC by learning anatomical and geometrical information from the CBCT images.

Amelioration of alcohol­induced gastric mucosa damage by oral administration of food­polydeoxyribonucleotides.

Gastritis refers to inflammation caused by injury to the gastric epithelium, which is usually due to excessive alcohol consumption and prolonged use of nonsteroidal anti­inflammatory drugs. Millions of individuals worldwide suffer from this disease. However, the lack of safe and promising treatments makes it urgent to explore and develop leads from natural resources. Therefore, food as medicine may be the best approach for the treatment of these disorders. The present study described the protective effects of food­polydeoxyribonucleotides (f­PDRNs) in a rat model of gastric mucosal injury induced by HCl­EtOH. Administration of f­PDRN was performed with low­PRF002 (26 mg/kg/day), medium­PRF002 (52 mg/kg/day) and high­PRF002 (78 mg/kg/day) on the day of autopsy. The site of damage to the mucous membrane was also analysed. In addition, an increase in gastric juice pH, total acidity of gastric juice and decrease in gastric juice secretion were confirmed, and gastric juice secretion­related factors corresponding to the administration of f­PDRN were analysed. Administration of f­PDRN reduced the mRNA expression of histamine H2 receptor, muscarinic acetylcholine receptor M3, cholecystokinin 2 receptor and H+/K+ ATPase related to gastric acid secretion and downregulation of histamine, myeloperoxidase and cyclic adenosine monophosphate. In addition, it was histologically confirmed that the loss of epithelial cells and the distortion of the mucosa were recovered in the group in which f­PDRN was administered compared to the model group with gastric mucosa damage. In summary, the present study suggested that f­PDRN has therapeutic potential and may have beneficial effects if taken regularly as a food supplement.

Mistletoe Extract Targets the STAT3-FOXM1 Pathway to Induce Apoptosis and Inhibits Metastasis in Breast Cancer Cells.

Mistletoe extracts (Viscum album L.) have been widely used as complementary and alternative medicines for the treatment of cancer, and their cytotoxic effects have been reported on various types of cancer. However, the molecular targets of mistletoe extracts have not been well studied. Herein, we investigated molecules associated with the in vitro and in vivo anticancer effects of mistletoe extract using 4T1 murine breast cancer cells. Mistletoe extract induced apoptosis and inhibited the signal transducer and activator of transcription3 (STAT3) phosphorylation. This inhibition was accompanied by the downregulations of forkhead box M1 (FOXM1) and the DNA repair proteins, RAD51 and survivin. Mistletoe extract simultaneously increased the expression of the DNA damage marker proteins, phosphorylated H2A histone family member X (H2A.X), and phosphorylated p38. Furthermore, mistletoe extract effectively suppressed tumor growth in 4T1 tumor-bearing BALB/c mice. In addition to tumor growth inhibition, mistletoe extract inhibited lung metastasis in the tumor-bearing mice and cell invasiveness by downregulating the expressions of matrix metalloproteinases (MMPs), urokinase-type plasminogen activator (uPA), uPA receptor, and markers of epithelial-mesenchymal transition (snail and fibronectin). Taken together, our results suggest that mistletoe extract targets the STAT3-FOXM1 pathway for its cytotoxic effects, and that mistletoe extracts might be useful for the treatment of patients with cancers highly expressing the STAT3-FOXM1 pathway.

DDIAS promotes STAT3 activation by preventing STAT3 recruitment to PTPRM in lung cancer cells.

DNA damage-induced apoptosis suppressor (DDIAS) regulates cancer cell survival. Here we investigated the involvement of DDIAS in IL-6-mediated signaling to understand the mechanism underlying the role of DDIAS in lung cancer malignancy. We showed that DDIAS promotes tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3), which is constitutively activated in malignant cancers. Interestingly, siRNA protein tyrosine phosphatase (PTP) library screening revealed protein tyrosine phosphatase receptor mu (PTPRM) as a novel STAT3 PTP. PTPRM knockdown rescued the DDIAS-knockdown-mediated decrease in STAT3 Y705 phosphorylation in the presence of IL-6. However, PTPRM overexpression decreased STAT3 Y705 phosphorylation. Moreover, endogenous PTPRM interacted with endogenous STAT3 for dephosphorylation at Y705 following IL-6 treatment. As expected, PTPRM bound to wild-type STAT3 but not the STAT3 Y705F mutant. PTPRM dephosphorylated STAT3 in the absence of DDIAS, suggesting that DDIAS hampers PTPRM/STAT3 interaction. In fact, DDIAS bound to the STAT3 transactivation domain (TAD), which competes with PTPRM to recruit STAT3 for dephosphorylation. Thus we show that DDIAS prevents PTPRM/STAT3 binding and blocks STAT3 Y705 dephosphorylation, thereby sustaining STAT3 activation in lung cancer. DDIAS expression strongly correlates with STAT3 phosphorylation in human lung cancer cell lines and tissues. Thus DDIAS may be considered as a potential biomarker and therapeutic target in malignant lung cancer cells with aberrant STAT3 activation.

Metformin selectively targets 4T1 tumorspheres and enhances the antitumor effects of doxorubicin by downregulating the AKT and STAT3 signaling pathways.

Recent studies have reported that metformin (Met), the first-line medication for the treatment of type 2 diabetes, exhibited anticancer and chemoprotective effects in diverse cancer cells. In this study, we investigated the effects of Met on the drug-resistance of 4T1 murine breast cancer tumorspheres (TS) and the mechanism responsible for its drug-resistance. 4T1 TS exhibited accumulations of cells at the G0/G1 phase compared with cells in monolayer culture, which suggested the majority of cells in TS were quiescent. Furthermore, it was identified that activations of the signal transducer and activator of transcription 3 (STAT3) and protein kinase B (AKT) signaling pathways in 4T1 TS conferred drug-resistance to doxorubicin (Dox) and lapatinib (Lapa). However, Met selectively targeted TS rather than cells in monolayer culture and increased the cytotoxic effect of Dox on TS by inhibiting activations of the STAT3 and AKT signaling pathways. These observations suggested that inhibitions of STAT3 and AKT underlie the selective cytotoxic effects of Met on TS. In addition, Met exhibited synergistic antitumor effects with Dox on 4T1 tumor-bearing BALB/c mice. Our findings suggest that combinations of Met and cytotoxic anticancer drugs may offer an advantage for treating drug-resistant breast cancer.

Arsenic May Act as a Pro-Metastatic Carcinogen Through Promoting Tumor Cell-Induced Platelet Aggregation.

Arsenic-associated carcinogenesis and related mortality are a major public health concern worldwide; however, the underlying mechanism of action remains unclear. Here, we demonstrated that arsenic promotes tumor metastasis by stimulating tumor cell-platelet aggregation (TCPA), which can ultimately increase cancer-related mortality. In freshly isolated human platelets in vitro, arsenic potentiated TCPA prompted by diverse cancer cell lines, which was attributable to increased platelet reactivity to TCPA with respect to thrombin generation and P-selectin, GPIIb/IIIa expression. Consistently, the co-existence of platelets and arsenic significantly enhanced tumor cell adhesion, extravasation and invasion along with increased metastasis-related markers like metallo-matrix proteinase-2 and -9 in vitro, which was attenuated by platelet activation blockers. Importantly, the exposure to arsenic-contaminated drinking water (2 ppm, 3 weeks) in mice in vivo significantly increased the metastasis of intravenously injected melanoma cells into lung. Furthermore, the exposure to arsenic-contaminated drinking water significantly reduced the survival of melanoma cell-injected mice, which was attenuated by the pretreatment of platelet-activation blockers; aspirin and eptifibatide. All these results provide an important clue to understand the mechanism underlying arsenic-associated cancer mortality and its prevention.

Anti-metastatic potential of a proton beam is regulated by p38 MAPK/c-Fos signaling pathway in TPA-treated HepG2 human hepatocellular carcinoma.

A proton beam therapy is considered the next generation radio-therapeutic tool for liver cancer treatments. However, its effect on metastasis has not been fully elucidated. Herein, we assessed the effects of a proton beam on the metastatic potential in HepG2, Hep3B and SK-Hep1 hepatocellular carcinomas. The result showed that a proton beam suppressed TPA-induced cell migration and invasion in HepG2 cells, but not in Hep3B and SK-Hep1 cells. In addition, matrix metalloproteinase-9 (MMP-9) activity and mRNA expressions were reversed by proton beam irradiation in TPA-treated HepG2 cells only. Furthermore, a proton beam suppressed TPA-induced gene expressions of urokinase plasminogen activator (uPA), uPA receptor (uPAR), Snail-1 and vascular endothelial growth factor (VEGF) in HepG2 cells in a dose-dependent manner. Moreover, we found that proton beam irradiation restrained p38 MAPK phosphorylation and c-Fos expression. Therefore, the result demonstrates that the anti-metastatic effects of a proton beam in TPA-treated HepG2 cells are associated with the inhibition of MMP-9 activity and the down-regulations of MMP-9, uPA, uPAR, Snail-1 and VEGF gene expression via the p38 MAPK/c-Fos signaling pathway. Taken together, this investigation suggests that the establishment of a customized proton beam therapeutic strategy for each liver cancer type is necessary to improve therapeutic efficiency.

The prevention of TNF-α/IFN-γ mixture-induced inflammation in human keratinocyte and atopic dermatitis-like skin lesions in Nc/Nga mice by mineral-balanced deep sea water.

Atopic dermatitis (AD) is a chronic inflammatory skin disease caused by environmental and chemical allergens. Despite the complexity of its pathogenesis, many investigations have shown that substances having anti-inflammatory activities alleviated the pathology of AD. Here, we evaluated the effects of mineral-balanced deep sea water (DSW) on AD-like skin damage in both in vitro and in vivo. The results showed that mineral-balanced DSW regressed inflammatory chemokines, such as macrophage-derived chemokine (MDC), thymus- and activation-regulated chemokine (TARC) and regulated on activation, normal T-cell expressed and secreted (RANTES), and cytokines, interleukin (IL)-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA expression in HaCaT immortal human keratinocyte treated with tumor necrosis factor (TNF)-α/ interferon (IFN)-γ mixture. Furthermore, increased cyclooxygenase (COX)-2 protein expressions were also reversed, filaggrin gene expression was enhanced and decreased involucrin transcriptions was recovered by mineral-balanced DSW in TNF-α/IFN-γ mixture-treated HaCaT human keratinocyte. Moreover, we revealed that the inhibitory effects of mineral-balanced DSW were mediated with the suppression of signal transducer and activator of transcription (STAT) 1 phosphorylation. In animal experiments, we showed that hardness 2000 of mineral-balanced DSW decreased the serum levels of IgE, IL-4, and histamine, and alleviated the severity score and numbers of scratching in dinitrochlorobezene (DNCB)-treated Nc/Nga mice. Furthermore, increased epidermal thickness and mast cell infiltration by DNCB treatment were reversed by the application of hardness 2000 mineral-balanced DSW. Taken together, the present investigation indicates that mineral-balanced DSW is a potent substance with anti-atopic dermatitis activity.

Refined Deep-Sea Water Suppresses Inflammatory Responses via the MAPK/AP-1 and NF-κB Signaling Pathway in LPS-Treated RAW 264.7 Macrophage Cells.

Atopic dermatitis (AD) is a type of inflammatory skin disease caused by genetics, immune system dysfunction, and environmental stresses. It is, however, still considered to be a refractory disease. Macrophages are inflammatory immune cells that infiltrate the skin and induce inflammation. We investigated the effect of refined deep-sea water (RDSW) on lipopolysaccharide (LPS)-induced inflammatory response in RAW 264.7 macrophage cells. The results showed that RDSW suppressed the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. Furthermore, nitric oxide, a product of iNOS, and prostaglandin (PG) D2 and PGE2, products of COX-2, were significantly inhibited by RDSW in a hardness-dependent manner. Moreover, we found that RDSW reversed the release of histamines and regressed the mRNA expressions and production of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and IL-10, and vascular endothelial growth factor, in a hardness-dependent manner. We also found that the suppressive effect of RDSW on LPS-induced inflammatory responses was regulated by the inhibition of NF-κB nuclear translocation, and ERK 1/2 and JNK 1/2 mediated the suppression of c-Jun and c-Fos expressions. In conclusion, the present investigation suggests the possibility that RDSW may be used to treat and/or prevent inflammatory diseases, including AD.

The inhibitory effects of deep-sea water on doxorubicin­induced epithelial-mesenchymal transition.

It has been revealed that the induction of epithelial­mesenchymal transition (EMT) is associated with drug resistance, leading to tumor recurrence and metastasis. Recent studies have shown that chemotherapeutic agents, besides their therapeutic effects, can induce EMT and enhance invasive and metastatic properties of tumor cells. Previously, we revealed that deep-sea water (DSW) exhibited antimetastatic effects in several human cancer cell lines. In the present study, we investigated the effects of DSW on doxorubicin-induced EMT in MCF-7 human breast cancer cells. When treated with doxorubicin, MCF-7 cells displayed characteristics of EMT, such as, mesenchymal markers (vimentin and fibronectin) and EMT-related transcription factors (Slug and Snail-1) in their RNA expression. However, DSW efficiently inhibited doxorubicin-induced EMT, revealing the decreased expression of vimentin, fibronectin, Slug and Snail-1. Moreover, treatment of MCF-7 cells with DSW significantly suppressed their increased migratory ability by doxorubicin as determined by wound-healing assay. We further demonstrated that the inhibitory effects of DSW on doxorubicin-induced EMT appeared to be mediated by inhibition of the ERK1/2, p38 MAPK and PI3K/AKT signaling pathways. Collectively, our data revealed that DSW has the potential to abolish undesired side-effects of doxorubicin by targeting EMT.

Deep sea water improves hypercholesterolemia and hepatic lipid accumulation through the regulation of hepatic lipid metabolic gene expression.

A high­fat diet or high­cholesterol diet (HCD) is a major cause of metabolic diseases, including obesity and diabetes; vascular diseases, including hypertension, stroke and arteriosclerosis; and liver diseases, including hepatic steatosis and cirrhosis. The present study aimed to evaluate the effects of deep sea water (DSW) on rats fed a HCD. DSW decreased HCD­induced increases in total cholesterol and low­density lipoprotein (LDL) cholesterol in the blood, and recovered high­density lipoprotein cholesterol. In addition, DSW decreased levels of liver injury markers, which were increased in response to HCD, including glutamate­oxaloacetate transaminase, glutamate­pyruvate transferase and alkaline phosphatase. Lower lipid droplet levels were observed in the livers of rats fed a HCD and treated with DSW at a hardness of 1,500, as compared with those in the HCD only group. Semi­quantitative reverse transcription­polymerase chain reaction (RT­PCR) revealed that mRNA expression levels of fatty acid synthase and sterol regulatory element binding protein­1c (SREBP­1c) in rats fed a HCD with DSW were lower compared with the HCD only group. Furthermore, quantitative RT­PCR revealed that DSW enhanced LDL receptor (LDLR) mRNA expression in a hardness­dependent manner. Combined, the results of the present study indicated that DSW may reduce HCD­induced increases in blood and liver lipid levels, indicating that DSW may protect against hypercholesterolemia and non­alcoholic hepatic steatosis. In addition, the present study demonstrated that DSW­induced downregulation of lipids in the blood and hepatic lipid accumulation was mediated by enhancement of LDLR expression and suppression of fatty acid synthase and SREBP­1c.

The anti-metastatic effects of the phytoestrogen arctigenin on human breast cancer cell lines regardless of the status of ER expression.

Arctigenin is a plant lignan extracted from Arctium lappa that has been shown to have estrogenic properties. In spite of the health benefits of phytoestrogens reducing the risk of osteoporosis, heart disease, and menopausal symptoms, its benefits against the risk of breast cancer have not been fully elucidated. Thus, we investigated the effects of arctigenin on metastasis of breast cancer using both estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cell lines to see if the effects are dependent on the status of ER expression. In ER-positive MCF-7 cells, arctigenin efficiently inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell migration and invasion. The activity of crucial metastatic protease matrix metalloprotease (MMP)-9 in gelatin zymography was also efficiently decreased by arctigenin, as well as its mRNA expression. Notably, arctigenin exhibited similar anti-metastatic effects even in ER-negative MDA-MB-231 cells, suggesting that the anti-metastatic effects of arctigenin were not exerted via the ER. The upstream signaling pathways involved in the regulation of MMP-9 and urokinase plasminogen activator (uPA) were analyzed using western blotting. The activation of Akt, NF-κB and MAPK (ERK 1/2 and JNK 1/2) was found to be inhibited. Taken together, these data suggest that arctigenin confers anti-metastatic effects by inhibiting MMP-9 and uPA via the Akt, NF-κB and MAPK signaling pathways on breast cancer, regardless of ER expression. Therefore, we propose that the intake of arctigenin could be an effective supplement for breast cancer patients.

Suppressive effects of a proton beam on tumor growth and lung metastasis through the inhibition of metastatic gene expression in 4T1 orthotopic breast cancer model.

A proton beam is a next generation tool to treat intractable cancer. Although the therapeutic effects of a proton beam are well known, the effect on tumor metastasis is not fully described. Here, we investigated the effects of a proton beam on metastasis in highly invasive 4T1 murine breast cancer cells and their orthotopic breast cancer model. Cells were irradiated with 2, 4, 8 or 16 Gy proton beam, and changes in cell proliferation, survival, and migration were observed by MTT, colony forming and wound healing assays. 4T1 breast cancer cell-implanted BALB/c mice were established and the animals were randomly divided into 4 groups when tumor size reached 200 mm3. Breast tumors were selectively irradiated with 10, 20 or 30 Gy proton beam. Breast tumor sizes were measured twice a week, and breast tumor and lung tissues were pathologically observed. Metastasis-regulating gene expression was assessed with quantitative RT-PCR. A proton beam dose-dependently decreased cell proliferation, survival and migration in 4T1 murine breast cancer cells. Also, growth of breast tumors in the 4T1 orthotopic breast cancer model was significantly suppressed by proton beam irradiation without significant change of body weight. Furthermore, fewer tumor nodules metastasized from breast tumor into lung in mice irradiated with 30 Gy proton beam, but not with 10 and 20 Gy, than in control. We observed correspondingly lower expression levels of urokinase plasminogen activator (uPA), uPA receptor, cyclooxygenase (COX)-2, and vascular endothelial growth factor (VEGF), which are important factors in cancer metastasis, in breast tumor irradiated with 30 Gy proton beam. Proton beam irradiation did not affect expressions of matrix metalloproteinase (MMP)-9 and MMP-2. Taken together, the data suggest that, although proton beam therapy is an effective tool for breast cancer treatment, a suitable dose is necessary to prevent metastasis-linked relapse and poor prognosis.

Crosstalk between Wnt signaling and Phorbol ester-mediated PKC signaling in MCF-7 human breast cancer cells.

Although many studies have implicated the crosstalk between the Wnt and PKC signaling pathways in tumor initiation and progression, the molecular roles of PKC isoforms in the Wnt signaling pathway remain poorly understood. In this study, we explored the contribution of PKC isoforms to canonical and noncanonical Wnt signaling pathway in mediating cell migration and an epithelial-mesenchymal transition (EMT). When MCF-7 cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for up to 3 weeks, the effect of TPA on Wnt signaling pathway was dramatically different depending on the exposure time. The short term exposure (3 days) of MCF-7 cells to TPA exhibited significant induction of Wnt5a expression, along with the enhanced expression of PKC-α, to promote cell migration, which suggested that activation of noncanonical Wnt signaling pathway is associated with PKC-α. However, the chronic exposure (3 weeks) of cells to TPA completely suppressed Wnt5a expression and the expression of PKC-η and PKC-δ, whereas the expression of Wnt3a and PKC-θ were up-regulated to activate the canonical Wnt signaling pathway. Moreover, the loss of epithelial markers, including E-cadherin and GATA-3, suggested that chronic exposure of TPA stimulates EMT. Taken together, our data suggest that PKC-θ positively regulates the canonical Wnt signaling pathway, and that PKC-η and PKC-δ negatively modulate this signaling pathway.

Lapatinib enhances the cytotoxic effects of doxorubicin in MCF-7 tumorspheres by inhibiting the drug efflux function of ABC transporters.

Increasing evidences indicate that cancer stem cells are resistant to chemotherapy due to their cell quiescence and the expression of ATP-binding cassette (ABC) transporters. In this study, we utilized tumorsphere cultures to seek better strategies to overcome chemoresistance since tumorsphere cultures have been used widely for the enrichment of cancer stem cells. We found that tumorspheres generated from MCF-7 human breast cancer cells exhibited high proportions of quiescent cells and expressed MDR-1 at elevated levels, leading to resistance to 5-fluorouracil, paclitaxel, and doxorubicin. Because the expression of EGFR/HER2 was increased in MCF-7 tumorspheres, we assessed the combinational effect of the dual ErbB1/ErbB2 inhibitor, lapatinib, with doxorubicin in tumorspheres. The results showed that inhibition of EGFR/HER2 signaling by lapatinib sensitized MCF-7 tumorspheres to doxorubicin by inhibiting the expression of the ABC transporters, MDR-1 and BCRP, and thus, enhancing the intracellular accumulation of doxorubicin. These findings suggest that combinations of lapatinib and cytotoxic anticancer drugs may offer an advantage for treating the drug-resistant cancers.

Lapatinib sensitizes quiescent MDA-MB-231 breast cancer cells to doxorubicin by inhibiting the expression of multidrug resistance-associated protein-1.

The quiescent state plays an important role in tumor recurrence because it protects cancer cells from chemotherapy. Previously, we optimized tumorsphere cultures for in vitro screening methods for targeting quiescent cell population since the majority of cells in tumorspheres are quiescent. In this study, we analyzed efficacies of current chemotherapeutics in tumorsphere assays to seek better strategies for eradicating quiescent cell population. Tumorspheres generated from MDA-MB-231 cells exhibited accumulations of cells in the G0/G1 phase as compared with cells in monolayer culture, suggesting that sphere formation contributes to an increase of quiescent cells. As a result of a decreased doxorubicin uptake, MDA-MB-231 tumorspheres exhibited chemoresistance to both doxorubicin and paclitaxel. Since we found that the enhanced EGFR signaling is characteristics of MDA-MB-231 tumorspheres, the combination effects of chemotherapy with lapatinib, a dual ErbB1/ErbB2 inhibitor, were accessed in tumorsphere assays. Western blot analysis revealed that lapatinib inhibited the phosphorylation of EGFR, AKT and p38 in doxorubicin-treated tumorspheres. The inhibition of EGFR signaling by the treatment with lapatinib suppressed the expression of multidrug resistance-associated protein-1 (MRP-1), leading to increased cytotoxicity of doxorubicin to tumorspheres. Furthermore, blockade of the PI3K/AKT and p38 MAPK signaling pathways resulted in a remarkable decrease in the expression of MRP-1 in doxorubicin-treated tumorspheres. These results demonstrate that lapatinib sensitizes quiescent MDA-MB-231 breast cancer cells to doxorubicin by inhibiting doxorubicin-induced MRP-1 expression via PI3K/AKT and p38 MAPK signaling pathways. Thus, this study suggests that treatment with lapatinib in combination with anti-mitotic drugs maybe a useful approach to improve clinical response by eradicating the quiescent cancer cell population.

Metastatic potential in MDA-MB-231 human breast cancer cells is inhibited by proton beam irradiation via the Akt/nuclear factor-κB signaling pathway.

A previous study has revealed that proton beam irradiation affects cell migration in MDA-MB-231 human breast cancer cells. Cyclooxygenase-2 (COX-2) and matrix metalloproteinase­9 (MMP-9) are highly expressed in various cancers, such as colon, lung and breast cancer, and enhance cell migration and metastasis in vitro and in vivo. In the present study, the effects of proton beam irradiation on COX-2 and MMP-9 expression levels in MDA-MB­231 human breast cancer cells were investigated, along with the signaling pathway involved in the proton beam irradiation­mediated antimetastatic effect. The results revealed that 12-O-tetradecanoylphorbol-13­acetate­induced increases in COX-2 and MMP-9 expression levels were reversed by proton beam irradiation in a dose-dependent manner. In addition, proton beam irradiation inhibited phosphorylation of protein kinase B (also known as Akt) and nuclear factor-κB (NF-κB), which are activated by phosphoinositide 3-kinase (PI3K) stimulation. MMP-9 and COX-2 expression levels are regulated by PI3K/Akt and/or protein kinase C/mitogen-activated protein kinase signaling pathways that enhance NF-κB and activator protein-1 transcriptional activities. Therefore, the results suggest that proton beam irradiation inhibited the cancer cell growth and metastasis associated with COX-2 and MMP-9 expression in MDA-MB­231 human breast cancer cells, and that the antimetastatic effect of proton beam irradiation is achieved by the suppression of NF-κB phosphorylation via inhibition of Akt activation.

Mineral-enriched deep-sea water inhibits the metastatic potential of human breast cancer cell lines.

Recently, the scientific community has begun to establish the health benefits of deep-sea water (DSW) due to its enrichment in nutrients and minerals. In this study, we investigated the effects of deep-sea water (DSW) on the metastatic potential of two human breast cancer cell lines exhibiting highly different phenotypes. MDA-MB-231 cells exhibit invasive/metastatic tumor features with rapid migration ability and high endogenous expression of TGF-ß and Wnt5a. DSW treatment significantly inhibits their migratory ability in a wound-healing assay. This inhibitory effect of DSW appears to be mediated through TGF-ß and Wnt5a signaling, resulting in attenuated expression of CD44. We further investigated the preventive effect of DSW on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced invasive/metastatic tumor features in non-invasive MCF-7 cells. Similar to the inhibitory effects shown in MDA-MB-231 cells, we observed that DSW treatment resulted in the inhibition of TPA-induced migration and MMP-9 activity with a concomitant decrease in mRNA levels of MMP-9, TGF-ß, Wnt5a and Wnt3a. Taken together, our data show that DSW has inhibitory effects on breast cancer invasion/metastasis, suggesting that DSW has some promise in improving cancer survival by preventing tumor metastasis.

Genetic modification of primate amniotic fluid-derived stem cells produces pancreatic progenitor cells in vitro.

Insulin therapy for type 1 diabetes does not prevent serious long-term complications including vascular disease, neuropathy, retinopathy and renal failure. Stem cells, including amniotic fluid-derived stem (AFS) cells - highly expansive, multipotent and nontumorigenic cells - could serve as an appropriate stem cell source for ß-cell differentiation. In the current study we tested whether nonhuman primate (nhp)AFS cells ectopically expressing key pancreatic transcription factors were capable of differentiating into a ß-cell-like cell phenotype in vitro. nhpAFS cells were obtained from Cynomolgus monkey amniotic fluid by immunomagnetic selection for a CD117 (c-kit)-positive population. RT-PCR for endodermal and pancreatic lineage-specific markers was performed on AFS cells after adenovirally transduced expression of PDX1, NGN3 and MAFA. Expression of MAFA was sufficient to induce insulin mRNA expression in nhpAFS cell lines, whereas a combination of MAFA, PDX1 and NGN3 further induced insulin expression, and also induced the expression of other important endocrine cell genes such as glucagon, NEUROD1, NKX2.2, ISL1 and PCSK2. Higher induction of these and other important pancreatic genes was achieved by growing the triply infected AFS cells in media supplemented with a combination of B27, betacellulin and nicotinamide, as well as culturing the cells on extracellular matrix-coated plates. The expression of pancreatic genes such as NEUROD1, glucagon and insulin progressively decreased with the decline of adenovirally expressed PDX1, NGN3 and MAFA. Together, these experiments suggest that forced expression of pancreatic transcription factors in primate AFS cells induces them towards the pancreatic lineage.