Strategic tillage achieves lower carbon footprints with higher carbon accumulation and grain yield in a wheat-maize cropping system.

Continuous single tillage has the potential to increase greenhouse gas (GHG) emissions and decrease the accumulation of soil organic carbon (SOC), thus increasing carbon footprints (CFs). However, in a wheat-maize cropping system, limited information was available about the effects of strategic tillage on CFs. Thus, a four-year field experiment was conducted, including continuous rotary tillage (RT), continuous no-till (NT), RT + subsoiling (RS), and NT + subsoiling (NS), to investigate the effects of NS (strategic tillage) on the unit area and unit yield. The results showed that CO2 emission was the highest contributor to CFs (73.92%) in a winter wheat-summer maize cropping system, following the order of NS < NT < RS < RT. The direct N2O emissions from fertilizers and residues were 4.43-4.51 t CO2-eq ha-1 yr-1 during the wheat and maize seasons, and indirect N2O emissions from irrigation and fertilizer inputs had a proportion of >80% from total agricultural inputs. The differences in SOC storage significantly affected the CFs. Although the NS treatment increased the amount of GHG emissions from the residues returned and consumption of diesel, the enhancement of SOC storage by deeper SOC increased. Thus, lower area-scaled CFs were observed in the NS treatment. Furthermore, a higher grain yield and an annual change of SOC storage compared with other treatments were observed under the NS system, which helped to reduce the CFs. The yield-scaled CFs followed the order of RT > RS > NT > NS when considering the changes in SOC storage. Therefore, the NS treatment resulted in a higher grain yield and SOC sequestration with lower CFs, and thus, it could be recommended as the best tillage method to achieve sustainable production and environmental balance in a wheat-maize cropping system.

[Effect of Fuzheng Kang'ai Recipe Combined Gefitinib on Lung Cancer A549 Cells and Its Mecha- nism Research].

Objective To observe the effect of Fuzheng Kang'ai Recipe (FKR) combined ge- fitinib on the proliferation and apoptosis of lung cancer A549 cells , and to study its potential synergistic mechanish with gefitinib. Methods The effects of FKR (0. 211, 0. 316, 0. 474, 0. 711, 1. 067, 1. 600, 2. 400, 3. 600 mg/mL) combined gefitinib (3. 95, 5. 92, 8. 18, 13. 33, 20. 00, 30. 00, 45. 00, 67. 50 µmol/ L) on the proliferation of A549 cells were detected by MTT assay. The apoptosis of A549 cells in the control group (complete culture medium) , FKR (1. 6 mg/mL) , gefitinib (45 µmol/L) , and FKR plus gefitinib (1. 6 mg/mL +45 µmol/L) were detected by flow cytometry (FCM). Their expressions of epidermal growth factor receptor (EGFR) , phosphorylating epidermal growth factor receptor ( p-EGFR) , enhancer of zeste homolog 2 (EZH2), peroxisome proliferator-activated receptor-γ ( PPAR-γ) , and P53 protein in A549 cells were detected by Western blot. Results Both FKR and gefitinib could inhibit the proliferation of A549 cells. The apoptotic rate was 12. 6% ±4. 5% in the FKR combined gefitinib group, obviously higher than that of the FKR group (4. 6% ± 0. 7%) and the gefitinib group (7. 8% ± 2. 7%) , showing statistical difference (P <0. 05). Compared with the control group, the expressions of p-EGFR and EZH2 were sig- nificantly down-regulated (P <0. 05) , the expressions of PPAR-γ and P53 protein were up-regulated in the FKR combined gefitinib group (P <0. 05); the expression of EZH2 was down-regulated in the gefitinib group and the FKR group (P <0. 05) ; the expression of PPAR-y was up-regulated in the FKR group (P < 0. 05). Compared with the gefitinib group, the expression of p-EGFR was down-regulated, and the expression of PPAR-γ was up-regulated in the FKR combined gefitinib group (both P <0. 05). Compared with the FKR group, the expression of p-EGFR was down-regulated in the FKR combined gefitinib group (P < 0. 05). Conclusions Combination of FKR and gefitinib could significantly inhibit the proliferation and growth of A549 cells,and induce cell apoptosis. Its potential synergistic mechanism of anti-tumor activities might be associated with down-regulating mRNA expressions of p-EGFR and EZH2, and up-regulating protein expressions of PPAR-y and P53.

Dietary compound isoliquiritigenin targets GRP78 to chemosensitize breast cancer stem cells via ß-catenin/ABCG2 signaling.

Accumulating evidence suggests that ß-catenin signaling in breast cancer stem cells (CSCs) is closely correlated to chemoresistance and adenosine triphosphate (ATP)-binding cassette subfamily G2 (ABCG2) expression. Targeting the aberrant ß-catenin signaling in CSCs has become a promising strategy to improve chemosensitivity in cancer treatment. In a pilot screening study, we found that the natural compound isoliquiritigenin (ISL) blocked ß-catenin transcription activity with the highest inhibition ratio. Here, we investigated the chemosensitizing effects of ISL on breast CSCs and the underlying mechanisms regulating the ß-catenin pathway. ISL could have synergistic effects with chemotherapeutic drugs to inhibit breast cancer cell proliferation and colony formation. In addition, ISL could significantly limit the side population and CSC ratios in breast cancer cells, accompanied by inhibited self-renewal and multidifferentiation abilities. A mechanistic study revealed that ISL could inhibit ß-catenin/ABCG2 signaling by activating the proteasome degradation pathway. The drug affinity responsive target stability strategy further identified GRP78 as the direct target of ISL. Subsequent molecular docking analysis and functional studies demonstrated that ISL could dock into the ATP domain of GRP78 and thereby inhibit its ATPase activity, resulting in its dissociation from ß-catenin. An in vivo study also suggested that ISL could chemosensitize breast CSCs via the GRP78/ß-catenin/ABCG2 pathway, with little toxicity in normal tissues and mammary stem cells. Taken together, the data from this study not only suggest ISL as a natural candidate to enhance breast CSC chemosensitivity but also highlight the significance of GRP78 in mediating cancer drug resistance and ß-catenin signaling in CSCs.

Dietary compound isoliquiritigenin inhibits breast cancer neoangiogenesis via VEGF/VEGFR-2 signaling pathway.

Angiogenesis is crucial for cancer initiation, development and metastasis. Identifying natural botanicals targeting angiogenesis has been paid much attention for drug discovery in recent years, with the advantage of increased safety. Isoliquiritigenin (ISL) is a dietary chalcone-type flavonoid with various anti-cancer activities. However, little is known about the anti-angiogenic activity of isoliquiritigenin and its underlying mechanisms. Herein, we found that ISL significantly inhibited the VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) at non-toxic concentration. A series of angiogenesis processes including tube formation, invasion and migration abilities of HUVECs were also interrupted by ISL in vitro. Furthermore, ISL suppressed sprout formation from VEGF-treated aortic rings in an ex-vivo model. Molecular mechanisms study demonstrated that ISL could significantly inhibit VEGF expression in breast cancer cells via promoting HIF-1α (Hypoxia inducible factor-1α) proteasome degradation and directly interacted with VEGFR-2 to block its kinase activity. In vivo studies further showed that ISL administration could inhibit breast cancer growth and neoangiogenesis accompanying with suppressed VEGF/VEGFR-2 signaling, elevated apoptosis ratio and little toxicity effects. Molecular docking simulation indicated that ISL could stably form hydrogen bonds and aromatic interactions within the ATP-binding region of VEGFR-2. Taken together, our study shed light on the potential application of ISL as a novel natural inhibitor for cancer angiogenesis via the VEGF/VEGFR-2 pathway. Future studies of ISL for chemoprevention or chemosensitization against breast cancer are thus warranted.

Bioactivity-guided identification and cell signaling technology to delineate the lactate dehydrogenase A inhibition effects of Spatholobus suberectus on breast cancer.

Aerobic glycolysis is an important feature of cancer cells. In recent years, lactate dehydrogenase A (LDH-A) is emerging as a novel therapeutic target for cancer treatment. Seeking LDH-A inhibitors from natural resources has been paid much attention for drug discovery. Spatholobus suberectus (SS) is a common herbal medicine used in China for treating blood-stasis related diseases such as cancer. This study aims to explore the potential medicinal application of SS for LDH-A inhibition on breast cancer and to determine its bioactive compounds. We found that SS manifested apoptosis-inducing, cell cycle arresting and anti-LDH-A activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cell. Oral herbal extracts (1 g/kg/d) administration attenuated tumor growth and LDH-A expression in both breast cancer xenografts. Bioactivity-guided fractionation finally identified epigallocatechin as a key compound in SS inhibiting LDH-A activity. Further studies revealed that LDH-A plays a critical role in mediating the apoptosis-induction effects of epigallocatechin. The inhibited LDH-A activities by epigallocatechin is attributed to disassociation of Hsp90 from HIF-1α and subsequent accelerated HIF-1α proteasome degradation. In vivo study also demonstrated that epigallocatechin could significantly inhibit breast cancer growth, HIF-1α/LDH-A expression and trigger apoptosis without bringing toxic effects. The preclinical study thus suggests that the potential medicinal application of SS for inhibiting cancer LDH-A activity and the possibility to consider epigallocatechin as a lead compound to develop LDH-A inhibitors. Future studies of SS for chemoprevention or chemosensitization against breast cancer are thus warranted.

alpha5beta1-integrin expression is essential for tumor progression in experimental lung cancer.

The matrix glycoprotein, fibronectin, stimulates the proliferation of non-small cell lung carcinoma in vitro through α5ß1 integrin receptor-mediated signals. However, the true role of fibronectin and its receptor in lung carcinogenesis in vivo remains unclear. To test this, we generated mouse Lewis lung carcinoma cells stably transfected with short hairpin RNA shRNA targeting the α5 integrin subunit. These cells were characterized and tested in proliferation, cell adhesion, migration, and soft agar colony formation assays in vitro. In addition, their growth and metastatic potential was tested in vivo in a murine model of lung cancer. We found that transfected Lewis lung carcinoma cells showed decreased expression of the α5 gene, which was associated with decreased adhesion to fibronectin and reduced cell migration, proliferation, and colony formation when compared with control cells and cells stably transfected with α2 integrin subunit in vitro. C57BL/6 mice injected with α5-silenced cells showed lower burden of implanted tumors, and a dramatic decrease in lung metastases resulting in higher survival as compared with mice injected with wild-type or α2 integrin-silenced cells. These observations reveal that recognition of host- and/or tumor-derived fibronectin via α5ß1 is important for tumor growth both in vitro and in vivo, and unveil α5ß1 as a potential target for the development of anti-lung cancer therapies.

Nicotine stimulates PPARbeta/delta expression in human lung carcinoma cells through activation of PI3K/mTOR and suppression of AP-2alpha.

We previously showed that nicotine stimulates non-small cell lung carcinoma (NSCLC) cell proliferation through nicotinic acetylcholine receptor (nAChR)-mediated signals. Activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) has also been shown to induce NSCLC cell growth. Here, we explore the potential link between nicotine and PPARbeta/delta and report that nicotine increases the expression of PPARbeta/delta protein; this effect was blocked by an alpha7 nAChR antagonist (alpha-bungarotoxin), by alpha7 nAChR short interfering RNA, and by inhibitors of phosphatidylinositol 3-kinase (PI3K; wortmannin and LY294002) and mammalian target of rapamycin (mTOR; rapamycin). In contrast, this effect was enhanced by PUN282987, an alpha7 nAChR agonist. Silencing of PPARbeta/delta attenuated the stimulatory effect of nicotine on cell growth, which was overcome by transfection of an exogenous PPARbeta/delta expression vector. Of note, nicotine induced complex formation between alpha7 nAChR and PPARbeta/delta protein and increased PPARbeta/delta gene promoter activity through inhibition of AP-2alpha as shown by reduced AP-2alpha binding using electrophoretic gel mobility shift and chromatin immunoprecipitation assays. In addition, silencing of Sp1 attenuated the effect of nicotine on PPARbeta/delta. Collectively, our results show that nicotine increases PPARbeta/delta gene expression through alpha7 nAChR-mediated activation of PI3K/mTOR signals that inhibit AP-2alpha protein expression and DNA binding activity to the PPARbeta/delta gene promoter. Sp1 seems to modulate this process. This study unveils a novel mechanism by which nicotine promotes human lung carcinoma cell growth.

PPARbeta/delta agonist increases the expression of PGE2 receptor subtype EP4 in human lung carcinoma cells.

Lung carcinoma remains one of the most common malignant tumors in the world despite recent advancements in the development of new chemotherapeutic agents for its treatment. Therefore, novel approaches for drug target discovery play an important role in the effort to help extend its dismal 5-year survival rate (<15%). Many mechanisms contribute to oncogenic transformation in carcinoma cells in the lung and recent evidence indicates that the overproduction of prostaglandin E(2) (PGE(2)), and the prostag-landin E(2) receptor subtype, EP4, promote the growth and progression of human nonsmall cell lung carcinoma (NSCLC), the most common lung carcinoma. Peroxisome proliferator-activated receptor beta/ delta (PPARbeta/delta), one of the nuclear hormone ligand-dependent transcription factors, has recently been reported to be involved in tumorigeniCity. We have shown that NSCLC cells express PPARbeta/delta protein and that treatment with a selective PPARbeta/delta agonist, GW501516, stimulated the expression of EP4 and induced NSCLC cell proliferation. In addition, this PPARbeta/delta agonist also induced EP4 promoter activity through the binding of C/EBP to the NF-IL6 site in the EP4 promoter. Therefore, PPARbeta/delta activation represents a novel molecular mechanism for regulating human cancer cell growth.

Fish oil inhibits human lung carcinoma cell growth by suppressing integrin-linked kinase.

We previously showed that synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) ligands inhibit non-small cell lung carcinoma (NSCLC) cell growth through multiple signaling pathways. Here, we show that dietary compounds, such as fish oil (which contains certain kinds of fatty acids like omega3 and omega6 polyunsaturated fatty acids), also inhibit NSCLC cell growth by affecting PPARgamma and by inhibiting the expression of integrin-linked kinase (ILK). Exogenous expression of ILK overcame, whereas silencing ILK enhanced the inhibitory effect of fish oil on cell growth. The inhibitor of p38 mitogen-activated protein kinase, SB239023, abrogated the inhibitory effect of fish oil on ILK expression, whereas the inhibitor of extracellular signal-regulated kinase, PD98059, had no effect. Transient transfection experiments showed that fish oil reduced ILK promoter activity, and this effect was abolished by AP-2alpha small interfering RNA and SB239023 and by deletion of a specific portion of the ILK gene promoter. Western blot analysis and gel mobility shift assay showed that fish oil significantly induced AP-2alpha protein expression and AP-2 DNA-binding activity in the ILK gene promoter and that this was dependent on PPARgamma activation. Blockade of AP-2alpha abrogated the effect of fish oil on ILK expression and on cell growth, whereas exogenous expression of AP-2alpha enhanced cell growth in the setting of fish oil exposure. Taken together, these findings show that fish oil inhibits ILK expression through activation of PPARgamma-mediated and p38 mitogen-activated protein kinase-mediated induction of AP-2alpha. In turn, this leads to inhibition of NSCLC cell proliferation. This study unveils a novel mechanism by which fish oil inhibits human lung cancer cell growth.

Prostaglandin E2 stimulates human lung carcinoma cell growth through induction of integrin-linked kinase: the involvement of EP4 and Sp1.

Cyclooxygenase-2-derived prostaglandin E(2) (PGE(2)) stimulates tumor cell growth and progression. However, the mechanisms by which PGE(2) increases tumor growth remain incompletely understood. In studies performed in non-small cell lung carcinoma (NSCLC) cells, we found that PGE(2) stimulates the expression of integrin-linked kinase (ILK). ILK small interfering RNA (siRNA) inhibited the mitogenic effects of PGE(2). In view of its perceived importance, we turned our attention to the mechanisms involved in PGE(2)-induced ILK expression and found that this effect was blocked by an antagonist of the PGE(2) receptor subtype EP4 and by EP4 siRNA. Furthermore, we showed that PGE(2) induction of ILK was associated with phosphorylation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt, which were abrogated by ILK siRNA. Transient transfection, gel mobility shift assays, and chromatin immunoprecipitation experiments showed that PGE(2) induced ILK promoter activity and increased Sp1, although it had no effect on nuclear factor-kappaB and AP-2 DNA-binding activity. Blockade of Sp1 abrogated the effect of PGE(2) on expression of ILK and promoter activity and on cell growth. In summary, our observations show that PGE(2) increases NSCLC cell growth through increased ILK expression, which is dependent on EP4 signaling and on induction of Sp1 protein and Sp1 DNA-binding activity in the ILK promoter. These studies suggest a novel molecular mechanism by which PGE(2) stimulates NSCLC cell growth and unveils a new molecular target for the development of therapies against NSCLC.

Rosiglitazone inhibits alpha4 nicotinic acetylcholine receptor expression in human lung carcinoma cells through peroxisome proliferator-activated receptor gamma-independent signals.

We and others have shown previously that nicotine, a major component of tobacco, stimulates non-small cell lung carcinoma (NSCLC) proliferation through nicotinic acetylcholine receptor (nAChR)-mediated signals. Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to inhibit NSCLC cell growth, but the exact mechanisms responsible for this effect remain incompletely defined. Herein, we show that nicotine induces NSCLC cell proliferation in part through alpha4 nAChR, prompting us to explore the effects of rosiglitazone, a synthetic PPARgamma ligand, on the expression of this receptor. Rosiglitazone inhibited the expression of alpha4 nAChR, but this effect was through a PPARgamma-independent pathway, because GW9662, an antagonist of PPARgamma, and the transfection of cells with PPARgamma small interfering RNA failed to abolish the response. The inhibitory effect of rosiglitazone on alpha4 nAChR expression was accompanied by phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 and down-regulation of Akt phosphorylation. These signals mediated the inhibitory effects of rosiglitazone on alpha4 nAChR expression because chemical inhibitors prevented the effect. Rosiglitazone was also found to stimulate p53, a tumor suppressor known to mediate some of the effects of nicotine. Interestingly, p53 up-regulation was needed for rosiglitazone-induced inhibition of alpha4 nAChR. Thus, rosiglitazone inhibits alpha4 nAChR expression in NSCLC cells through activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, which triggers induction of p53. Finally, like others, we found that nicotine stimulated the expression of alpha4 nAChR. This process was also inhibited by rosiglitazone through similar pathways.

Activation of peroxisome proliferator-activated receptor beta/delta induces lung cancer growth via peroxisome proliferator-activated receptor coactivator gamma-1alpha.

We previously demonstrated that a selective agonist of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta), GW501516, stimulated human non-small cell lung carcinoma (NSCLC) growth, partly through inhibition of phosphatase and tensin homolog deleted on chromosome 10 expression. Here, we show that GW501516 also decreases the phosphorylation of AMP-activated protein kinase alpha (AMPKalpha), a major regulator of energy metabolism. This was mediated through specific activation of PPARbeta/delta, as a PPARbeta/delta small interfering RNA inhibited the effect. However, AMPKalpha did not mediate the growth-promoting effects of GW501516, as silencing of AMPKalpha did not inhibit GW501516-induced cell proliferation. Instead, we found that GW501516 stimulated peroxisome proliferator-activated receptor coactivator gamma (PGC)-1alpha, which activated the phosphatidylinositol 3 kinase (PI3-K)/Akt mitogenic pathway. An inhibitor of PI3-K, LY294002, had no effect on PGC-1alpha, consistent with PGC-1alpha being upstream of PI3-K/Akt. Of note, an activator of AMPKalpha, 5-amino-4-imidazole carboxamide riboside, inhibited the growth-promoting effects of GW501516, suggesting that although AMPKalpha is not responsible for the mitogenic effects of GW501516, its activation can oppose these events. This study unveils a novel mechanism by which GW501516 and activation of PPARbeta/delta stimulate human lung carcinoma cell proliferation, and suggests that activation of AMPKalpha may oppose this effect.

PPARbeta/delta agonist stimulates human lung carcinoma cell growth through inhibition of PTEN expression: the involvement of PI3K and NF-kappaB signals.

Recent studies suggest that activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) promotes cancer cell survival. We previously demonstrated that a selective PPARbeta/delta agonist, GW501516, stimulated human non-small cell lung carcinoma (NSCLC) cell growth. Here, we explore the mechanisms responsible for this effect. We show that GW501516 decreased phosphate and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor known to decrease cell growth and induce apoptosis. Activation of PPARbeta/delta and phosphatidylinositol 3-kinase (PI3K)/Akt signaling was associated with inhibition of PTEN. GW501516 increased NF-kappaB DNA binding activity and p65 protein expression through activation of PPARbeta/delta and PI3K/Akt signals and enhanced the physical interactions between PPARbeta/delta and p65 protein. Conversely, inhibition of PI3K and silencing of p65 by small RNA interference (siRNA) blocked the effect of GW501516 on PTEN expression and on NSCLC cell proliferation. GW501516 also inhibited IKBalpha protein expression. Silencing of IKBalpha enhanced the effect of GW501516 on PTEN protein expression and on cell proliferation. It also augmented the GW501516-induced complex formation of PPARbeta/delta and p65 proteins. Overexpression of PTEN suppressed NSCLC cell growth and eliminated the effect of GW501516 on phosphorylation of Akt. Together, our observations suggest that GW501516 induces the proliferation of NSCLC cells by inhibiting the expression of PTEN through activation of PPARbeta/delta, which stimulates PI3K/Akt and NF-kappaB signaling. Overexpression of PTEN overcomes this effect and unveils PPARbeta/delta and PTEN as potential therapeutic targets in NSCLC.

Stimulation of lung carcinoma cell growth by fibronectin-integrin signalling.

Throughout many countries, lung cancer will kill more people this year than malignancies related to breast, prostate, colon, liver, kidney and melanoma combined. Despite recent advances in understanding the molecular biology of lung carcinoma and the introduction of multiple new chemotherapeutic agents for its treatment, its dismal five-year survival rate (<15%) has not changed substantially. The lack of advancement in this area reflects the limited knowledge available concerning the factors that promote oncogenic transformation and proliferation of carcinoma cells in the lung. Malignant transformation plays a key role in tumor growth and invasion; however, other factors such as the surrounding stroma, local growth factors, vascularity, and systemic hormones are important contributors as well. We believe that the composition of the lung extracellular matrix is also important due to its ability to affect malignant cell behavior in vitro. The matrix glycoprotein fibronectin, for example, is highly expressed in chronic lung disorders where most lung carcinomas are identified. This document reviews information that implicates fibronectin in the stimulation of lung carcinoma cell growth. Data available to date indicate that by binding to specific integrin receptors expressed on the surface of tumor cells, fibronectin stimulates intracellular signals implicated in the pathobiology of lung carcinogenesis and lung tumor chemoresistance including mitogen-activated protein kinases, GTPases, and the PI3-kinase/Akt/mTOR pathway. Thus, integrin-mediated signals triggered by fibronectin in tumor cells represent promising targets for the development of novel anti-cancer strategies.

Rosiglitazone, an Agonist of PPARgamma, Inhibits Non-Small Cell Carcinoma Cell Proliferation In Part through Activation of Tumor Sclerosis Complex-2.

PPARgamma ligands inhibit the proliferation of non-small cell lung carcinoma (NSCLC) cells in vitro. The mechanisms responsible for this effect remain incompletely elucidated, but PPARgamma ligands appear to inhibit the mammalian target of rapamycin (mTOR) pathway. We set out to test the hypothesis that PPARgamma ligands activate tuberous sclerosis complex-2 (TSC2), a tumor suppressor gene that inhibits mTOR signaling. We found that the PPARgamma ligand rosiglitazone stimulated the phosphorylation of TSC2 at serine-1254, but not threonine-1462. However, an antagonist of PPARgamma and PPARgamma siRNA did not inhibit these effects. Rosiglitazone also increased the phosphorylation of p38 MAPK, but inhibitors of p38 MAPK and its downstream signal MK2 had no effect on rosiglitazone-induced activation of TSC2. Activation of TSC2 resulted in downregulation of phosphorylated p70S6K, a downstream target of mTOR. A TSC2 siRNA induced p70S6K phosphorylation at baseline and inhibited p70S6K downregulation by rosiglitazone. When compared to a control siRNA in a thymidine incorporation assay, the TSC2 siRNA reduced the growth inhibitory effect of rosiglitazone by fifty percent. These observations suggest that rosiglitazone inhibits NSCLC growth partially through phosphorylation of TSC2 via PPARgamma-independent pathways.

Nicotine stimulates human lung cancer cell growth by inducing fibronectin expression.

The mechanisms by which tobacco promotes lung cancer remain incompletely understood. Herein, we report that nicotine, a major component of tobacco, promotes the proliferation of cultured non-small cell lung carcinoma (NSCLC) cells; this effect was most noticeable at 5 days. However, nicotine had no effect on apoptosis of NSCLC cells. In experiments designed to unveil the mechanisms for this effect, we found that nicotine also stimulated mRNA and protein expression of fibronectin. Fibronectin is a matrix glycoprotein that regulates important cellular processes (e.g., adhesion, proliferation, and differentiation) and is highly expressed in tobacco-related lung disorders. Of note, reagents against the integrin alpha5beta1 (antibodies, RGD peptides, alpha5 shRNA) blocked the mitogenic effects of nicotine. Thus, nicotine stimulated NSCLC cell proliferation indirectly via fibronectin induction. We then focused on the mechanisms responsible for nicotine-induced fibronectin expression in NSCLC cells and found that nicotine stimulated the surface expression of alpha7 nicotinic acetylcholine receptor (alpha7 nAChR), and that alpha-bungarotoxin, an inhibitor of alpha7 nAChR, abolished the nicotine-induced fibronectin response. The fibronectin-inducing effects of nicotine were associated with activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3-K)/mammalian target of rapamycin (mTOR) signaling pathways, and were abrogated by inhibitors of ERK (PD98059), PI3-K (LY294002), and mTOR (rapamycin), but not by inhibitors of protein kinase (PK)C (calphostin C) and PKA (H89). These observations suggest that nicotine stimulates NSCLC proliferation through induction of fibronectin, and that these events are mediated through nAChR-mediated signals that include ERK and PI3-K/mTOR pathways. This work highlights the role of fibronectin and alpha5beta1 integrins as potential targets for anti-lung cancer therapies.

Peroxisome proliferator-activated receptor gamma: a novel target for cancer therapeutics?

Peroxisome proliferator-activated receptors are ligand-activated intracellular transcription factors that have been implicated in important biological processes such as inflammation, tissue remodeling and atherosclerosis. Emerging information also implicates peroxisome proliferator-activated receptors in oncogenesis. Peroxisome proliferator-activated receptor gamma, the best studied of the peroxisome proliferator-activated receptors, modulates the proliferation and apoptosis of many cancer cell types, and it is expressed in many human tumors including lung, breast, colon, prostate and bladder. Natural and synthetic agents capable of activating peroxisome proliferator-activated receptor gamma have been found to inhibit cancer cell growth in vitro and in animal models. These agents, however, are not specific and both peroxisome proliferator-activated receptor gamma-dependent and peroxisome proliferator-activated receptor gamma-independent pathways involved in their effects have been identified. Together, these studies, coupled with a few clinical trials, suggest that peroxisome proliferator-activated receptor gamma is a novel target for the development of new and effective anticancer therapies.

Extracellular matrix fibronectin increases prostaglandin E2 receptor subtype EP4 in lung carcinoma cells through multiple signaling pathways: the role of AP-2.

We have previously demonstrated that fibronectin (Fn) stimulates the proliferation of non-small cell lung carcinoma (NSCLC) cell growth through the induction of cyclooxygenase-2 (COX-2) and prostaglandin E2 secretion. Here, we demonstrate that NSCLC cells express mRNA and protein for the prostaglandin E2 receptor EP4 and that Fn enhances its stimulatory effect by inducing the expression of EP4, but not of EP1, EP2, and EP3 receptor subtypes. The effect of Fn on EP4 was inhibited by an antibody against alpha5beta1 integrin and by inhibitors of phosphoinositide 3-kinase (wortmannin) and extracellular signal-regulated kinase (PD98095), but not by inhibitors of protein kinase C (calphostin C), of protein kinase A (H-89), or of mammalian target of rapamycin (rapamycin). A COX-2 small interfering RNA was also inhibitory. Fn significantly increased AP-2 binding activity in the promoter of the EP4 gene, and AP-2 antisense oligonucleotides blocked Fn-induced EP4 expression. Using full-length and mutated EP4 promoter constructs, we found that Fn stimulation of EP4 gene expression was inhibited when one AP-2 site (-1000 bp) was mutated. Fn induced nuclear AP-2alpha protein expression through multiple signaling pathways. Our results indicate that Fn-induced NSCLC cell proliferation is mediated through EP4. Furthermore, they show that Fn induces EP4 expression through the activation of alpha5beta1-dependent signals that include induction of extracellular signal-regulated kinase and phosphoinositide 3-kinase pathways as well as expression of COX-2. These events lead to activation of the transcription factor AP-2alpha, which interacts with specific regions in the EP4 gene promoter, leading to transcription of the EP4 gene.

Peroxisome proliferator-activated receptors in lung cancer.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. Their discovery in the 1990s provided insights into the cellular mechanisms involved in the control of energy homeostasis; the regulation of cell differentiation, proliferation, and apoptosis; and the modulation of important biological and pathological processes related to inflammation, among others. Since then, PPARs have become an exciting therapeutic target for several diseases. PPARs are expressed by many tumors including lung carcinoma cells, and their function has been linked to the process of carcinogenesis in lung. Consequently, intense research is being conducted in this area with the hope of discovering new PPAR-related therapeutic targets for the treatment of lung cancer. This review summarizes the research being conducted in this area and focuses on the mechanisms by which PPARs are believed to affect lung tumor cell biology.

Fibronectin increases matrix metalloproteinase 9 expression through activation of c-Fos via extracellular-regulated kinase and phosphatidylinositol 3-kinase pathways in human lung carcinoma cells.

Enhanced expression of matrix metalloproteinase-9 (MMP-9) is associated with human lung tumor invasion and/or metastasis. We have demonstrated that fibronectin (FN), a matrix glycoprotein, stimulates human non-small cell lung carcinoma (NSCLC) cell proliferation. The current study examines the effect of FN on MMP-9 expression in NSCLC cells. We show that FN increases MMP-9 protein, mRNA expression, and gelatinolytic activity in NSCLC cells. The integrin alpha5beta1 mediated the effects of FN because alpha5 small interfering RNA blocked FN-stimulated MMP-9 protein expression, and also abrogated FN-induced phosphorylation of ERK and phosphatidylinositol 3-kinase (PI3K) signals. The inhibitor of ERK, PD98095, and of PI3K, wortmannin, but not that of protein kinase A, H89, of Rho kinase, Y-27632, of mTOR, rapamycin, or of JNK, SP600125, prevented FN-induced MMP-9 gelatinolytic activity and gene expression. FN enhanced MMP-9 gene promoter activity; however, there was no response to FN in DNA constructs with an AP-1 site mutation. FN increased AP-1 DNA binding activity, and this was abrogated by cyclic AMP response element decoy oligonucleotides, which also diminished FN-induced MMP-9 promoter activity. FN increased the expression of the AP-1 subunit c-Fos protein, but not in the presence of PD98095 and wortmannin. The AP-1 inhibitor, nordihydroguaiaretic acid, and a c-Fos small interfering RNA eliminated the effect of FN on MMP-9 expression. This study indicates that FN, by binding to the integrin alpha5beta1 receptor, stimulates the expression of MMP-9 through increased AP-1/DNA binding and c-Fos protein expression via ERK and PI3K signaling pathways. The data unveils a novel mechanism by which FN could promote NSCLC cell invasion and metastasis.