Effects of CCN6 overexpression on the cell motility and activation of p38/bone morphogenetic protein signaling pathways in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancer types that is highly resistant to conventional treatments, such as chemotherapy and radiotherapy. As the demand for more effective therapeutics for PDAC treatment increases, various approaches have been studied to develop novel targets. The cellular communication network (CCN) family is a matricellular protein that modulates various cellular functions, including cell adhesion, proliferation, migration, and invasiveness. Despite this, little is known about the role of CCN6 in PDAC. The current study investigated the role of CCN6 in PDAC by generating CCN6-overexpressing PANC-1 cells (PANC-1-CCN6) by infecting lentivirus particles containing CCN6. PANC-1-CCN6 induces cell viability and tumorigenesis than PANC-1 cells with empty vector (control). The PANC-1-CCN6 formed more colonies, and the size of spheroids increased compared to the control. The upregulation of CCN6 enhances the expression of bone morphogenetic proteins (BMPs) genes and the upregulation of p38 mitogen-activated protein kinases (MAPKs). In PANC-1-CCN6 cells, the levels of N-cadherin, VEGF, and Snail expression were higher than the control, while E-cadherin expression was lower, which is associated with upregulation of epithelial-to-mesenchymal transition (EMT). Consistent with the changes in EMT-related proteins in PANC-1-CCN6, the migratory ability and invasiveness were enhanced in PANC-1-CCN6. Xenografted PANC-1-CCN6 in immunocompromised mice exhibited accelerated tumor growth than the control group. In immunohistochemistry (IHC), the PANC-1-CCN6 xenografted tumor showed an increased positive area of PCNA and Ki-67 than the control. These results suggest that CCN6 plays a tumorigenic role and induces the metastatic potential by the p38 MAPK and BMPs signaling pathways. Although the role of CCN6 has been introduced as an antitumor factor, there was evidence of CCN6 acting to cause tumorigenesis and invasion in PANC-1.

Divergent roles of the regulatory subunits of class IA PI3K.

The regulatory subunit of phosphatidylinositol 3-kinase (PI3K), known as p85, is a critical component in the insulin signaling pathway. Extensive research has shed light on the diverse roles played by the two isoforms of p85, namely p85α and p85ß. The gene pik3r1 encodes p85α and its variants, p55α and p50α, while pik3r2 encodes p85ß. These isoforms exhibit various activities depending on tissue types, nutrient availability, and cellular stoichiometry. Whole-body or liver-specific deletion of pik3r1 have shown to display increased insulin sensitivity and improved glucose homeostasis; however, skeletal muscle-specific deletion of p85α does not exhibit any significant effects on glucose homeostasis. On the other hand, whole-body deletion of pik3r2 shows improved insulin sensitivity with no significant impact on glucose tolerance. Meanwhile, liver-specific double knockout of pik3r1 and pik3r2 leads to reduced insulin sensitivity and glucose tolerance. In the context of obesity, upregulation of hepatic p85α or p85ß has been shown to improve glucose homeostasis. However, hepatic overexpression of p85α in the absence of p50α and p55α results in increased insulin resistance in obese mice. p85α and p85ß have distinctive roles in cancer development. p85α acts as a tumor suppressor, but p85ß promotes tumor progression. In the immune system, p85α facilitates B cell development, while p85ß regulates T cell differentiation and maturation. This review provides a comprehensive overview of the distinct functions attributed to p85α and p85ß, highlighting their significance in various physiological processes, including insulin signaling, cancer development, and immune system regulation.

Overexpression of KiSS1 Induces the Proliferation of Hepatocarcinoma and Increases Metastatic Potential by Increasing Migratory Ability and Angiogenic Capacity.

Liver cancer has a high prevalence, with majority of the cases presenting as hepatocellular carcinoma (HCC). The prognosis of metastatic HCC has hardly improved over the past decade, highlighting the necessity for liver cancer research. Studies have reported the ability of the KiSS1 gene to inhibit the growth or metastasis of liver cancer, but contradictory research results are also emerging. We, therefore, sought to investigate the effects of KiSS1 on growth and migration in human HCC cells. HepG2 human HCC cells were infected with lentivirus particles containing KiSS1. The overexpression of KiSS1 resulted in an increased proliferation rate of HCC cells. Quantitative polymerase chain reaction and immunoblotting revealed increased Akt activity, and downregulation of the G1/S phase cell cycle inhibitors. A significant increase in tumor spheroid formation with upregulation of ß-catenin and CD133 was also observed. KiSS1 overexpression promoted the migratory, invasive ability, and metastatic capacity of the hepatocarcinoma cell line, and these effects were associated with changes in the expressions of epithelial mesenchymal transition (EMT)-related genes such as E-cadherin, N-cadherin, and slug. KiSS1 overexpression also resulted in dramatically increased tumor growth in the xenograft mouse model, and upregulation of proliferating cell nuclear antigen (PCNA) and Ki-67 in the HCC tumors. Furthermore, KiSS1 increased the angiogenic capacity by upregulation of the vascular endothelial growth factor A (VEGF-A) and CD31. Based on these observations, we infer that KiSS1 not only induces HCC proliferation, but also increases the metastatic potential by increasing the migratory ability and angiogenic capacity.

Next-Generation Antisense Oligonucleotide of TGF-ß2 Enhances T Cell-Mediated Anticancer Efficacy of Anti-PD-1 Therapy in a Humanized Mouse Model of Immune-Excluded Melanoma.

Anti-programmed death-1 (PD-1) immunotherapy is one of the most promising therapeutic interventions for treating various tumors, including lung cancer, bladder cancer, and melanoma. However, only a subset of patients responds to anti-PD-1 therapy due to complicated immune regulation in tumors and the evolution of resistance. In the current study, we investigate the potential of a novel transforming growth factor-beta2 (TGF-ß2) antisense oligonucleotide (ngTASO), as a combination therapy with an anti-PD-1 antibody in melanoma. This study was conducted in a melanoma-bearing human immune system mouse model that recapitulates immune-excluded phenotypes. We observed that the TGF-ß2 blockade by ngTASO in combination with PD-1 inhibition downregulated the tumor intrinsic ß-catenin, facilitated the infiltration of CD8+ cytotoxic lymphocytes (CTLs) in the tumor, and finally, enhanced the antitumor immune potentials and tumor growth delays. Blockade of TGF-ß2 combined with PD-1 inhibition also resulted in downregulating the ratio of regulatory T cells to CTLs in the peripheral blood and tumor, resulting in increased granzyme B expression. In addition, co-treatment of ngTASO and anti-PD-1 augmented the PD-L1 expression in tumors, which is associated with an improved response to anti-PD-1 immunotherapy. These results indicate that the combination of ngTASO and anti-PD-1 exerts an enhanced T cell-mediated antitumor immune potential. Hence, co-inhibition of TGF-ß2 and PD-1 is a potentially promising immunotherapeutic strategy for immune-excluded melanoma.

The role of KiSS1 gene on the growth and migration of prostate cancer and the underlying molecular mechanisms.

Metastatic prostate cancers have a high mortality rate. KiSS1 was originally identified as a metastasis suppressor gene in metastatic melanoma and breast cancer, but its role in prostate cancer has been contradictory. This study was therefore undertaken to investigate the effects of KiSS1 overexpression on the growth and migration of human metastatic prostate cancer cells. We first tested the effect of KiSS1 overexpression on the growth and migration of DU145 human metastatic prostate cancer cells in vitro. DU145 cells were infected with the culture medium of 293T cells, which produce lentivirus particles containing KiSS1. A 2.5-fold increase in proliferation of KiSS1-overexpressing cancer cells was observed, and these cells formed tumor spheroids about 3 times larger than the vector control group. qPCR and immunoblotting revealed the association between increased cell growth and regulation of the PI3K/Akt and cell cycle genes, and also that increases in ß-catenin and CD133 contribute to tumor aggregation. KiSS1 overexpression resulted in upregulation of the ß-arrestin1/2 and Raf-MEK-ERK-NF-κB pathways via KiSS1R. Moreover, the migration and invasion of KiSS1-overexpressing cells were determined to be faster than the control group, along with 1.6-fold increased metastatic colonization of the KiSS1-overexpressing cancer cells. These were associated to the regulation of EMT gene expressions, such as E-cadherin and N-cadherin, and the upregulation of MMP9. In a xenograft mouse model inoculated with DU145 cells infected GFP or KiSS1 via a lentiviral vector, KiSS1 statistically significantly increased the tumor growth, with upregulation of PCNA and Ki-67 in the tumor tissues. In addition, KiSS1 increased the angiogenic capacity by upregulating VEGF-A and CD31, both in vitro and in vivo. Taken together, our results indicate that KiSS1 not only induces prostate cancer proliferation, but also promotes metastasis by increasing the migration, invasion, and angiogenesis of malignant cells.

Establishment of a platform for measuring mitochondrial oxygen consumption rate for cardiac mitochondrial toxicity.

The heart has an abundance of mitochondria since cardiac muscles require copious amounts of energy for providing continuous blood through the circulatory system, thereby implying that myocardial function is largely reliant on mitochondrial energy. Thus, cardiomyocytes are susceptible to mitochondrial dysfunction and are likely targets of mitochondrial toxic drugs. Various methods have been developed to evaluate mitochondrial toxicity by evaluating toxicological mechanisms, but an optimized and standardized assay for cardiomyocytes remains unmet. We have therefore attempted to standardize the evaluation system for determining cardiac mitochondrial toxicity, using AC16 human and H9C2 rat cardiomyocytes. Three clinically administered drugs (acetaminophen, amiodarone, and valproic acid) and two anticancer drugs (doxorubicin and tamoxifen) which are reported to have mitochondrial effects, were applied in this study. The oxygen consumption rate (OCR), which directly reflects mitochondrial function, and changes in mRNA levels of mitochondrial respiratory complex I to complex V, were analyzed. Our results reveal that exposure to all five drugs results in a concentration-dependent decrease in the basal and maximal levels of OCR in AC16 cells and H9C2 cells. In particular, marked reduction in the OCR was observed after treatment with doxorubicin. The reduction in OCR after exposure to mitochondrial toxic drugs was found to be associated with reduced mRNA expression in the mitochondrial respiratory complexes, suggesting that the cardiac mitochondrial toxicity of drugs is majorly due to dysfunction of mitochondrial respiration. Based on the results of this study, we established and standardized a protocol to measure OCR in cardiomyocytes. We expect that this standardized evaluation system for mitochondrial toxicity can be applied as basic data for establishing a screening platform to evaluate cardiac mitochondrial toxicity of drugs, during the developmental stage of new drug discovery.

A fungicide, fenhexamid, is involved in the migration and angiogenesis in breast cancer cells expressing estrogen receptors.

Fenhexamid (Fen) is used to eradicate gray mold of fruits and vegetables leading to greater detection of its residual concentration in wine than other fungicides. Here, we further investigated the malign influence of Fen on the migration and angiogenesis via regulation of the estrogen receptor (ER) and phosphoinositide 3-kinase (PI3K) pathways in breast cancer models. ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells were exposed to 17ß-estradiol (E2, 10-9 M), Fen (10-5 M and 10-7 M), ICI 182,780 (ICI; an ER antagonist, 10-8 M) or/and Pictilisib (Pic; a PI3K inhibitor, 10-7 M), and subsequently subjected to migration assay, live cell motility monitoring, trans-chamber assay, immunofluorescence, angiogenesis assay, tumor spheroid formation, and Western blot analysis. In MCF-7 cells, E2 and Fen induced cell migration by regulating the cell migration-related proteins. Although expressions of N-cadherin and Vimentin remained unchanged E2 and Fen induced the decrease of E-cadherin and Occludin in the immunofluorescence assay and Western blot analysis. In addition, Fen increased vessel formation in HUVEC cells. Furthermore, Fen treatment induced the formation of larger and denser tumor spheroids in MCF-7 cells. Western blot further confirmed the increased expressions of vascular endothelial growth factor (VEGF) and sex-determining region Y-box 2 (SOX2) after exposure to Fen. We conclude that Fen plays an important role as an endocrine-disrupting chemical in breast cancer migration and metastasis through the regulation of ER and PI3K signaling pathways.

Epithelial-Mesenchymal Transition-Inducing Factors Involved in the Progression of Lung Cancers.

Although there have been advances in cancer therapy and surgical improvement, lung cancer has the lowest survival rate (19%) at all stages. This is because most patients are diagnosed with concurrent metastasis, which occurs due to numerous related reasons. Especially, lung cancer is one of the most common and malignant cancers in the world. Although there are advanced therapeutic strategies, lung cancer remains one of the main causes of cancer death. Recent work has proposed that epithelialmesenchymal transition (EMT) is the main cause of metastasis in most cases of human cancers including lung cancer. EMT involves the conversion of epithelial cells, wherein the cells lose their epithelial abilities and become mesenchymal cells involved in embryonic development, such as gastrulation and neural crest formation. In addition, recent research has indicated that EMT contributes to altering the cancer cells into cancer stem cells (CSCs). Although EMT is important in the developmental stages, this process also activates lung cancer progression, including complicated and diverse signaling pathways. Despite the numerous investigations on signaling pathways involved in the progression of lung cancer, this malignancy is considered critical for treatment. EMT in lung cancer involves many transcription factors and inducers, for example, Snail, TWIST, and ZEB are the master regulators of EMT. EMT-related factors and signaling pathways are involved in the progression of lung cancer, proposing new approaches to lung cancer therapy. In the current review, we highlight the signaling pathways implicated in lung cancer and elucidate the correlation of these pathways, indicating new insights to treat lung cancer and other malignancies.

Targeting transforming growth factor-ß2 by antisense oligodeoxynucleotide accelerates T cell-mediated tumor rejection in a humanized mouse model of triple-negative breast cancer.

Transforming growth factor-beta (TGF-ß) pathway mediates suppression of antitumor immunity and is associated with poor prognosis in triple-negative breast cancer (TNBC). In this study, we generated a humanized animal model by transplanting human peripheral blood mononuclear cells into immunodeficient mice followed by inoculation of MDA-MB-231 cells and subsequently analyzed the role of TGF-ß2 in the interaction between human T cells and human tumor cells. Following reconstitution of the human immune system, inhibition of TGF-ß signaling by TGF-ß2 antisense oligodeoxynucleotide (TASO) resulted in accelerated tumor growth inhibition. TGF-ß2 inhibition also resulted in downregulation of peripheral Foxp3 + regulatory T cells (Treg), whereas no effect was seen in the expression of CD8 + cytotoxic T cells. Analysis of the TASO-treated mice serum revealed elevated levels of human IFN-γ and reduced levels of human IL-10 and TGF-ß2. Moreover, TGF-ß2 inhibition resulted in increased CD8 + T cell infiltration, whereas the reduced infiltration of Tregs into the tumor partly resulted from decreased expression of CCL22. Decreased intratumoral Tregs facilitated the activation of cytotoxic T cells, associated with increased granzyme B expression. These results indicate that TASO potentiated T cell-mediated antitumor immunity, and it is proposed that TGF-ß2 may be a promising target in the immunotherapeutic strategy of TNBC.

Gallic Acid Hindered Lung Cancer Progression by Inducing Cell Cycle Arrest and Apoptosis in A549 Lung Cancer Cells via PI3K/Akt Pathway.

This study elucidates the anti-cancer potential of gallic acid (GA) as a promising therapeutic agent that exerts its effect by regulating the PI3K/Akt pathway. To prove our research rationale, we used diverse experimental methods such as cell viability assay, colony formation assay, tumor spheroid formation assay, cell cycle analysis, TUNEL assay, Western blot analysis, xenograft mouse model and histological analysis. Treatment with GA inhibited cell proliferation in dose-dependent manner as measured by cell viability assay at 48 h. GA and cisplatin (CDDP) also inhibited colony formation and tumor spheroid formation. In addition, GA and CDDP induced apoptosis, as determined by the distribution of early and late apoptotic cells and DNA fragmentation. Western blot analysis revealed that inhibition of the PI3K/Akt pathway induced upregulation of p53 (tumor suppressor protein), which in turn regulated cell cycle related proteins such as p21, p27, Cyclin D1 and E1, and intrinsic apoptotic proteins such as Bax, Bcl-2 and cleaved caspase-3. The anti-cancer effect of GA was further confirmed in an in vivo mouse model. Intraperitoneal injection with GA for 4 weeks in an A549-derived tumor xenograft model reduced the size of tumor mass. Injection of them downregulated the expression of proliferating cell nuclear antigen and p-Akt, but upregulated the expression of cleaved caspase-3 in tumor tissues. Taken together, these results indicated that GA hindered lung cancer progression by inducing cell cycle arrest and apoptosis, suggesting that GA would be a potential therapeutic agent against non-small cell lung cancer.

Potential Roles of Iridoid Glycosides and Their Underlying Mechanisms against Diverse Cancer Growth and Metastasis: Do They Have an Inhibitory Effect on Cancer Progression?

Iridoids are glycosides found in plants, having inherent roles in defending them against infection by viruses and microorganisms, and in the rapid repair of damaged areas. The emerging roles of iridoid glycosides on pharmacological properties have aroused the curiosity of many researchers, and studies undertaken indicate that iridoid glycosides exert inhibitory effects in numerous cancers. This review focuses on the roles and the potential mechanism of iridoid glycosides at each stage of cancer development such as proliferation, epithelial mesenchymal transition (EMT), migration, invasion and angiogenesis. Overall, the reviewed literature indicates that iridoid glycosides inhibit cancer growth by inducing cell cycle arrest or by regulating apoptosis-related signaling pathways. In addition, iridoid glycosides suppress the expression and activity of matrix metalloproteinases (MMPs), resulting in reduced cancer cell migration and invasiveness. The antiangiogenic mechanism of iridoid glycosides was found to be closely related to the transcriptional regulation of pro-angiogenic factors, i.e., vascular endothelial growth factors (VEGFs) and cluster of differentiation 31 (CD31). Taken together, these results indicate the therapeutic potential of iridoid glycosides to alleviate or prevent rapid cancer progression and metastasis.

Effects of anticancer drugs on the cardiac mitochondrial toxicity and their underlying mechanisms for novel cardiac protective strategies.

Mitochondria are organelles that play a pivotal role in the production of energy in cells, and vital to the maintenance of cellular homeostasis due to the regulation of many biochemical processes. The heart contains a lot of mitochondria because those muscles require a lot of energy to keep supplying blood through the circulatory system, implying that the energy generated from mitochondria is highly dependent. Thus, cardiomyocytes are sensitive to mitochondrial dysfunction and are likely to be targeted by mitochondrial toxic drugs. It has been reported that some anticancer drugs caused unwanted toxicity to mitochondria. Mitochondrial dysfunction is related to aging and the onset of many diseases, such as obesity, diabetes, cancer, cardiovascular and neurodegenerative diseases. Mitochondrial toxic mechanisms can be mainly explained concerning reactive oxygen species (ROS)/redox status, calcium homeostasis, and endoplasmic reticulum stress (ER) stress signaling. The toxic mechanisms of many anticancer drugs have been revealed, but more studying and understanding of the mechanisms of drug-induced mitochondrial toxicity is required to develop mitochondrial toxicity screening system as well as novel cardioprotective strategies for the prevention of cardiac disorders of drugs. This review focuses on the cardiac mitochondrial toxicity of commonly used anticancer drugs, i.e., doxorubicin, mitoxantrone, cisplatin, arsenic trioxide, and cyclophosphamide, and their possible chemopreventive agents that can prevent or alleviate cardiac mitochondrial toxicity.

Blockade of transforming growth factor ß2 by anti-sense oligonucleotide improves immunotherapeutic potential of IL-2 against melanoma in a humanized mouse model.

BACKGROUND AIMS: IL-2 is a potent cytokine that activates natural killer cells and CD8+ cytotoxic T lymphocytes (CTLs) and has been approved for the treatment of metastatic renal cell carcinoma and metastatic melanoma. However, the medical use of IL-2 is restricted because of its narrow therapeutic window and potential side effects, including the expansion of regulatory T cells (Tregs). METHODS: In this study, the authors investigated the complementary effects of transforming growth factor-ß2 (TGF-ß2) anti-sense oligodeoxynucleotide (TASO) on the immunotherapeutic potential of IL-2 in a melanoma-bearing humanized mouse model. RESULTS: The authors observed that the combination of TASO and IL-2 facilitated infiltration of CTLs into the tumor, thereby potentiating the tumor killing function of CTLs associated with increased granzyme B expression. In addition, TASO attenuated the increase in Tregs by IL-2 in the peripheral blood and spleen and also inhibited infiltration of Tregs into the tumor, which was partly due to decreased CCL22. Alteration of T-cell constituents at the periphery by TGF-ß2 inhibition combined with IL-2 might be associated with the synergistic augmentation of serum pro-inflammatory cytokines (such as interferon Î³ and tumor necrosis factor α) and decreased ratio of Tregs to CTLs in tumor tissues, which consequently results in significant inhibition of tumor growth CONCLUSIONS: These results indicate that the application of TASO improves IL-2-mediated anti-tumor immunity, thus implying that blockade of TGF-ß2 in combination with IL-2 may be a promising immunotherapeutic strategy for melanoma.

Fenhexamid induces cancer growth and survival via estrogen receptor-dependent and PI3K-dependent pathways in breast cancer models.

Fenhexamid (Fen), a fungicide used to treat gray mold of fruits and vegetables, is reported to function as an endocrine disrupting chemical via the estrogen receptors (ER), despite low-toxicity of the pesticide. In this study, we elucidated that the disrupting effects of Fen are exerted via the ER and phosphatidylinositol 3-kinase (PI3K) pathways in breast cancer models. The WST assay, live cell monitoring, cell cycle analysis, colony formation assay, apoptotic analysis by JC-1 dyeing, and Western blot analysis were applied in ER positive MCF-7 and ER negative MDA-MB-231 breast cancer cells, after exposure to 17ß-estradiol (E2), Fen, ICI 182,780 (ICI; an ER antagonist) and/or Pictilisib (Pic; a PI3K inhibitor). Exposure to E2 and Fen induced the cell growth and survival ability of MCF-7 cells by increasing the S-phase cells and regulating the cell cycle-related proteins (Cyclin D1 and E1, p21 and p27). In addition, E2 and Fen treatment resulted in elevated levels of the survival-related proteins (Survivin and PCNA), and inhibited apoptosis by increasing the mitochondrial membrane potential and regulating the apoptosis-related proteins (BAX, BCL-2, and Caspase-9). These changes were reversed to the same level as the control group when exposed to their respective inhibitors, thereby indicating that the changes are exerted via the ER and PI3K pathways. In particular, co-treatment with these inhibitors induced greater inhibition than single treatment. Conversely, no alterations were observed in the ER-negative MDA-MB-231 breast cancer cells. Taken together, these results indicate that Fen promotes the growth of breast cancer cells via the ER and/or PI3K pathways, similar to the E2 mechanism. Although a relatively safe pesticide, Fen possibly exerts its influence as an endocrine disrupting chemical in ER-positive breast cancer cells via the ER and PI3K pathways.

Inhalation exposure by cigarette smoke: Effects on the progression of bleomycin- and lipopolysaccharide-induced lung injuries in rat models.

The toxic substances of cigarette smoke (CS) induce inflammatory responses in the lung by recruiting inflammatory cells. In this study, we investigated the effects of CS on the progression of lung disease in bleomycin (BLM) and lipopolysaccharide (LPS)-induced lung injury rat models. Briefly, rats were exposed to CS via inhalation (nose-only) for 28 consecutive days, for 4 h per day. Using an automatic video instillator, rats were administered a single dose of 2.5 mg/kg BLM (day 1) or 0.5 mg/kg LPS (day 26), prepared in 50 µL phosphate-buffered saline (PBS) solution. Examination of the bronchoalveolar lavage fluid (BALF) revealed that the number of neutrophils increased in a concentration-dependent manner of CS. Exposure to CS also enhanced the expression of cytokines, i.e., CCL2 (MCP-1), CCL3 (MIP-1α), CXCL2 (CINC3), CXCL10 (IP-10), TNF-α, IFN-γ, IL-2, IL-4 in the BALF of the vehicle (VC) and BLM groups in a concentration-dependent manner. In particular, the expressions of CCL2, CXCL10 and TNF-α were remarkably upregulated in the BLM + CS 300 treatment as compared to VC, while there were no differences in these cytokine levels in the serum following CS exposure. Exposure to CS resulted in compacted alveolar spaces and macrophage aggregation in the lung tissues following BLM and LPS treatments. Compared to VC, pulmonary fibrosis and chronic inflammation of bronchioloalveoli were observed in the BLM + CS treatment and inflammatory cell infiltration of bronchioloalveoli was observed in the LPS + CS treatment in a concentration-dependent manner by CS. The expression levels of CCL2 and IFN-γ in the lung tissues were increased similar to the levels obtained in BALF, in a concentration-dependent manner by CS. Taken together, these results indicate that repeated exposure to CS may exacerbate the lung injury initially caused by BLM and LPS.

Inhibitory effects of cigarette smoke extracts on neural differentiation of mouse embryonic stem cells.

Maternal smoking during the perinatal period is linked to adverse neonatal outcomes such as low birth weight and birth defects. Numerous studies have shown that cigarette smoke or nicotine exposure has a widespread effect on fetal nerve development. However, there exists a lack of understanding of what specific changes occur at the cellular level on persistent exposure to cigarette smoke during the differentiation of embryonic stem cells (ESCs) into neural cells. We previously investigated the effects of cigarette smoke extract (CSE) and its major component, nicotine, on the neural differentiation of mouse embryonic stem cells (mESCs). Differentiation of mESCs into neural progenitor cells (NPCs) or neural crest cells (NCCs) was induced with chemically defined media, and the cells were continuously exposed to CSE or nicotine during neural differentiation and development. Disturbed balance of the pluripotency state was observed in the NPCs, with consequent inhibition of neurite outgrowth and glial fibrillary acidic protein (Gfap) expression. These inhibitions correlated with the altered expression of proteins involved in the Notch-1 signaling pathways. The migration ability of NCCs was significantly decreased by CSE or nicotine exposure, which was associated with reduced protein expression of migration-related proteins. Taken together, we concluded that CSE and nicotine inhibit differentiation of mESCs into NPCs or NCCs, and may disrupt functional development of neural cells. These results imply that cigarette smoking during the perinatal period potentially inhibits neural differentiation and development of ESCs cells, leading to neonatal abnormal brain development and behavioral abnormalities.

Effects of cigarette smoke components on myocardial differentiation of mouse embryonic stem cells.

The heart is the first organ formed in the developing fetus, and abnormal development of the heart is a major cause of fetal death. The adverse effects of cigarette smoke on the heart have been well established, but it is not well understood how cigarette smoke components regulate signaling molecules and cardiac specific functions during the early differentiation stage of the embryonic heart. In this study, we identified changes in the size of mouse embryoid bodies (mEBs) in response to treatment with cigarette smoke extract (CSE) via regulation of HDAC2, p53, p21, and cyclin D1 protein expression, which are cardiac differentiation and cell-cycle markers, respectively. In addition, exposure of mouse embryonic stem cells (mESCs) to cigarette smoke components inhibited myocardial differentiation and development through the expression of HDAC1, HDAC2, GATA4, NKX2-5, TBX5, HAND1, and Troponin I. Long-term exposure studies showed that CSE and nicotine may delay the development of mouse cardiomyocytes from mESCs and inhibit the contractibility, which is a fundamental function of the heart. Taken together, these findings suggest that cigarette smoke components, including nicotine, may affect abnormal myocardial differentiation and development.

Apoptotic effects of cigarette smoke extracts on mouse embryonic stem cells via oxidative stress.

Previous studies have reported that cigarette smoke and cigarette smoke extract (CSE) have negative effects on embryonic development. However, no studies have investigated the mechanism through which CSE affects the cellular signaling pathway leading to apoptosis and oxidative stress in embryonic cells, or how the two pathways are cross-linked. Thus, we studied the effects of CSE on apoptosis and oxidative stress in mouse embryonic stem cells (mESCs). Specifically, we measured changes in cell viability in response to CSEs (3R4F and two domestic cigarettes CSE 1 and 2) using a water soluble tetrazolium (WST) assay and a neutral red uptake (NRU) assay, which revealed that cell viability decreased in a concentration-dependent manner. Western blot analysis revealed that the expression of cyclin D1 and cyclin E1 was decreased and that of p21 and p27 was increased by CSE. Additionally, the number of terminal deoxynucleotidyl transferase (TUNEL)-stained cells was increased by CSE, while the levels of Bax and Caspase-3 increased and Bcl-2 decreased. Moreover, a 2',7'-dichlorofluorescin diacetate (DCF-DA) assay and reactive oxygen species (ROS)-Glo H2 O2 assay confirmed that ROS were generated in response to CSE and that they were associated with up-regulated Keaf-1 and CHOP. Overall, the results revealed that cigarette smoke extract (CSE) inhibited cell proliferation by regulating cell cycle-related protein expression and increased oxidative stress by regulating the expression of Kelch-like ECH-associated protein 1 (Keap-1) and CCAAT/enhancer-binding protein homologous protein (CHOP), resulting in apoptosis in mESCs.

Effects of cigarette smoke extracts on apoptosis and oxidative stress in two models of ovarian cancer in vitro.

Cigarette smoke contains thousands of harmful components, many of which exert toxic effects on reproductive organs, including the ovaries, and their development. In this study, we investigated the effects of cigarette smoke extract (CSE) on cell proliferation, apoptosis and oxidative stress in the SKOV3 and OVCAR3 ovarian cancer cell lines. A water soluble tetrazolium (WST) assay revealed that the cell proliferation of both ovarian cells was significantly inhibited by three types of CSE in a concentration-dependent manner. Western blot analysis showed that the inhibition of cell proliferation was related to regulation of cell cycle-related protein expression. To determine the effects of cigarette smoke on the death of ovarian cells, we conducted a TUNEL assay, which revealed a time-dependent increase in TUNEL-stained cells in response to CSE exposure. Moreover, the protein expression of the Bcl-2 family signaling pathway observed upon Western blot analysis were consistent with the results of the TUNEL assay. We also examined the reactive oxygen species (ROS) generation in two ovarian cell lines using DCF-DA (5(6)-Carboxy-2',7'-dichlorofluorescein diacetate) assay and ROS-Glo™ H2O2 assay to investigate the oxidative stress caused by CSE. Both ROS detection assays demonstrated an increase in the amount of ROS by CSE in ovarian cells, which was consistent with the results observed for the Keap1-Nrf2 pathway upon Western blot analysis. Taken together, these results suggest that CSE may induce cell proliferation inhibition and oxidative stress in ovarian cells.