MiR-34a functions as a tumor suppressor in oral cancer through the inhibition of the Axl/Akt/GSK-3ß pathway.

Background/purpose: Oral cancer is a prevalent malignancy affecting men globally. This study aimed to investigate the regulatory role of miR-34a in oral cancer cells through the Axl/Akt/glycogen synthase kinase-3ß (GSK-3ß) pathway and its impact on cellular malignancy. Materials and methods: We examined the effects of miR-34a overexpression on the malignancy of oral cancer cells. Multiple oral cancer cell lines were assessed to determine the correlation between endogenous miR-34a and Axl levels. Transfection experiments with miR-34a were conducted to analyze its influence on Axl mRNA and protein expression. Luciferase reporter assays were performed to investigate miR-34a's modulation of Axl gene transcription. Manipulation of miR-34a expression was utilized to demonstrate its regulatory effects on oral cancer cells through the Axl/Akt/GSK-3ß pathway. Results: Overexpression of miR-34a significantly suppressed the malignancy of oral cancer cells. We observed an inverse correlation between endogenous miR-34a and Axl levels across multiple oral cancer cell lines. Transfection of miR-34a resulted in decreased Axl mRNA and protein expression, and luciferase reporter assays confirmed miR-34a-mediated modulation of Axl gene transcription. The study revealed regulatory effects of miR-34a on oral cancer cells through the Axl/Akt/GSK-3ß pathway, leading to alterations in downstream target genes involved in cellular proliferation and tumorigenesis. Conclusion: Our findings highlight the significance of the miR-34a/Axl/Akt/GSK-3ß signaling axis in modulating the malignancy of oral cancer cells. Targeting miR-34a may hold therapeutic potential in oral cancer treatment, as manipulating its expression can attenuate the aggressive behavior of oral cancer cells via the Axl/Akt/GSK-3ß pathway.

Drastic Synergy of Lovastatin and Antrodia camphorata Extract Combination against PC3 Androgen-Refractory Prostate Cancer Cells, Accompanied by AXL and Stemness Molecules Inhibition.

Prostate cancer (PC) is the second most frequently diagnosed cancer and the fifth leading cause of cancer-related death in males worldwide. Early-stage PC patients can benefit from surgical, radiation, and hormonal therapies; however, once the tumor transitions to an androgen-refractory state, the efficacy of treatments diminishes considerably. Recently, the exploration of natural products, particularly dietary phytochemicals, has intensified in response to addressing this prevailing medical challenge. In this study, we uncovered a synergistic effect from combinatorial treatment with lovastatin (an active component in red yeast rice) and Antrodia camphorata (AC, a folk mushroom) extract against PC3 human androgen-refractory PC cells. This combinatorial modality resulted in cell cycle arrest at the G0/G1 phase and induced apoptosis, accompanied by a marked reduction in molecules responsible for cellular proliferation (p-Rb/Rb, Cyclin A, Cyclin D1, and CDK1), aggressiveness (AXL, p-AKT, and survivin), and stemness (SIRT1, Notch1, and c-Myc). In contrast, treatment with either AC or lovastatin alone only exerted limited impacts on the cell cycle, apoptosis, and the aforementioned signaling molecules. Notably, significant reductions in canonical PC stemness markers (CD44 and CD133) were observed in lovastatin/AC-treated PC3 cells. Furthermore, lovastatin and AC have been individually examined for their anti-PC properties. Our findings elucidate a pioneering discovery in the synergistic combinatorial efficacy of AC and clinically viable concentrations of lovastatin on PC3 PC cells, offering novel insights into improving the therapeutic effects of dietary natural products for future strategic design of therapeutics against androgen-refractory prostate cancer.

AXL transcriptionally up-regulates TMEM14A expression to mediate cell proliferation in non-small-cell lung cancer cells.

In non-small cell lung cancer (NSCLC), the receptor tyrosine kinase AXL has been identified as a potent activator of tumor progression and resistance to therapies. However, the molecular mechanisms behind AXL-mediated oncogenesis remain elusive. Current study thus aimed to uncover potential downstream genes regulated by AXL in NSCLC. Through transcriptomic RNA sequencing of AXL-silenced NSCLC cells, TMEM14A was identified as a significantly up-regulated gene. Clinical evaluations using GEPIA2 revealed that TMEM14A mRNA expression was notably higher in lung adenocarcinoma (LUAD) tumor tissues compared to normal tissues. Further, significantly increased TMEM14A levels were associated with poorer overall survival in LUAD patients. Experimentally, silencing TMEM14A in NSCLC cells led to reduced cellular proliferation and ATP levels, highlighting a key role of TMEM14A in NSCLC progression. Moreover, our promoter analysis demonstrated that AXL-mediated regulation of TMEM14A transcription could involve binding of transcription factors STAT and NF-κB to 5'-promoter of TMEM14A. Collectively, current study unveils TMEM14A as a novel downstream target of AXL, suggesting its potential as a therapeutic target to counteract resistance in future NSCLC patients undergoing AXL-targeted therapies.

Intervention of AXL in EGFR Signaling via Phosphorylation and Stabilization of MIG6 in Non-Small Cell Lung Cancer.

About 80% of lung cancer patients are diagnosed with non-small cell lung cancer (NSCLC). EGFR mutation and overexpression are common in NSCLC, thus making EGFR signaling a key target for therapy. While EGFR kinase inhibitors (EGFR-TKIs) are widely used and efficacious in treatment, increases in resistance and tumor recurrence with alternative survival pathway activation, such as that of AXL and MET, occur frequently. AXL is one of the EMT (epithelial-mesenchymal transition) signature genes, and EMT morphological changes are also responsible for EGFR-TKI resistance. MIG6 is a negative regulator of ERBB signaling and has been reported to be positively correlated with EGFR-TKI resistance, and downregulation of MIG6 by miR-200 enhances EMT transition. While MIG6 and AXL are both correlated with EMT and EGFR signaling pathways, how AXL, MIG6 and EGFR interplay in lung cancer remains elusive. Correlations between AXL and MIG6 expression were analyzed using Oncomine or the CCLE. A luciferase reporter assay was used for determining MIG6 promoter activity. Ectopic overexpression, RNA interference, Western blot analysis, qRT-PCR, a proximity ligation assay and a coimmunoprecipitation assay were performed to analyze the effects of certain gene expressions on protein-protein interaction and to explore the underlying mechanisms. An in vitro kinase assay and LC-MS/MS were utilized to determine the phosphorylation sites of AXL. In this study, we demonstrate that MIG6 is a novel substrate of AXL and is stabilized upon phosphorylation at Y310 and Y394/395 by AXL. This study reveals a connection between MIG6 and AXL in lung cancer. AXL phosphorylates and stabilizes MIG6 protein, and in this way EGFR signaling may be modulated. This study may provide new insights into the EGFR regulatory network and may help to advance cancer treatment.

Periostin promotes ovarian cancer metastasis by enhancing M2 macrophages and cancer-associated fibroblasts via integrin-mediated NF-κB and TGF-ß2 signaling.

BACKGROUND: Ovarian cancer has the highest mortality among gynecological cancers due to late diagnosis and lack of effective targeted therapy. Although the study of interplay between cancer cells with their microenvironment is emerging, how ovarian cancer triggers signaling that coordinates with immune cells to promote metastasis is still elusive. METHODS: Microarray and bioinformatics analysis of low and highly invasive ovarian cancer cell lines were used to reveal periostin (POSTN), a matrix protein with multifunctions in cancer, with elevated expression in the highly invasive cells. Anchorage independent assay, Western blot, RNA interference, confocal analysis and neutralizing antibody treatment were performed to analyze the effects of POSTN on tumor promotion and to explore the underlying mechanism. Chemotaxis, flow cytometry and cytokine array analyses were undertaken to analyze the involvement of POSTN in cancer-associated fibroblast (CAF) and macrophage modulation. Correlations between POSTN expression levels and clinical characteristics were analyzed using the Oncomine, commercial ovarian cancer cDNA and China Medical University Hospital patient cohort. In vivo effect of POSTN on metastasis was studied using a mouse xenograft model. RESULTS: Expression of POSTN was found to be elevated in highly invasive ovarian cancer cells. We observed that POSTN was co-localized with integrin ß3 and integrin ß5, which was important for POSTN-mediated activation of ERK and NF-κB. Ectopic expression of POSTN enhanced whereas knockdown of POSTN decreased cancer cell migration and invasion in vitro, as well as tumor growth and metastasis in vivo. POSTN enhanced integrin/ERK/NF-κB signaling through an autocrine effect on cancer cells to produce macrophage attracting and mobilizing cytokines including MIP-1ß, MCP-1, TNFα and RANTES resulting in increased chemotaxis of THP-1 monocytes and their polarization to M2 macrophages in vitro. In agreement, tumors derived from POSTN-overexpressing SKOV3 harbored more tumor-associated macrophages than the control tumors. POSTN induced TGF-ß2 expression from ovarian cancer cells to promote activation of adipose-derived stromal cells to become CAF-like cells expressing alpha smooth muscle actin and fibroblast activation protein alpha. Consistently, increased CAFs were observed in POSTN overexpressing SKOV3 cells-derived metastatic tumors. In clinical relevance, we found that expression of POSTN was positively correlated with advanced-stage diseases and poor overall survival of patients. CONCLUSIONS: Our study revealed a POSTN-integrin-NF-κB-mediated signaling and its involvement in enhancing M2 macrophages and CAFs, which could potentially participate in promoting tumor growth. Our results suggest that POSTN could be a useful prognosis marker and potential therapeutic target.

Selenium Yeast and Fish Oil Combination Diminishes Cancer Stem Cell Traits and Reverses Cisplatin Resistance in A549 Sphere Cells.

Cisplatin is a prevalent chemotherapeutic agent used for non-small cell lung cancer (NSCLC) that is difficult to treat by targeted therapy, but the emergence of resistance severely limits its efficacy. Thus, an effective strategy to combat cisplatin resistance is required. This study demonstrated that, at clinically achievable concentrations, the combination of selenium yeast (Se-Y) and fish oil (FO) could synergistically induce the apoptosis of cancer stem cell (CSC)-like A549 NSCLC sphere cells, accompanied by a reversal of their resistance to cisplatin. Compared to parental A549 cells, sphere cells have higher cisplatin resistance and possess elevated CSC markers (CD133 and ABCG2), epithelial-mesenchymal transition markers (anexelekto (AXL), vimentin, and N-cadherin), and cytoprotective endoplasmic reticulum (ER) stress marker (glucose-regulated protein 78) and increased oncogenic drivers, such as yes-associated protein, transcriptional coactivator with PDZ-binding motif, ß-catenin, and cyclooxygenase-2. In contrast, the proapoptotic ER stress marker CCAAT/enhancer-binding protein homologous protein and AMP-activated protein kinase (AMPK) activity were reduced in sphere cells. The Se-Y and FO combination synergistically counteracted the above molecular features of A549 sphere cells and diminished their elevated CSC-like side population. AMPK inhibition by compound C restored the side population proportion diminished by this nutrient combination. The results suggest that the Se-Y and FO combination can potentially improve the outcome of cisplatin-treated NSCLC with phenotypes such as A549 cells.

MicroRNA-485-5p targets keratin 17 to regulate oral cancer stemness and chemoresistance via the integrin/FAK/Src/ERK/ß-catenin pathway.

BACKGROUND: The development of drug resistance in oral squamous cell carcinoma (OSCC) that frequently leads to recurrence and metastasis after initial treatment remains an unresolved challenge. Presence of cancer stem cells (CSCs) has been increasingly reported to be a critical contributing factor in drug resistance, tumor recurrence and metastasis. Thus, unveiling of mechanisms regulating CSCs and potential targets for developing their inhibitors will be instrumental for improving OSCC therapy. METHODS: siRNA, shRNA and miRNA that specifically target keratin 17 (KRT17) were used for modulation of gene expression and functional analyses. Sphere-formation and invasion/migration assays were utilized to assess cancer cell stemness and epithelial mesenchymal transition (EMT) properties, respectively. Duolink proximity ligation assay (PLA) was used to examine molecular proximity between KRT17 and plectin, which is a large protein that binds cytoskeleton components. Cell proliferation assay was employed to evaluate growth rates and viability of oral cancer cells treated with cisplatin, carboplatin or dasatinib. Xenograft mouse tumor model was used to evaluate the effect of KRT17- knockdown in OSCC cells on tumor growth and drug sensitization. RESULTS: Significantly elevated expression of KRT17 in highly invasive OSCC cell lines and advanced tumor specimens were observed and high KRT17 expression was correlated with poor overall survival. KRT17 gene silencing in OSCC cells attenuated their stemness properties including markedly reduced sphere forming ability and expression of stemness and EMT markers. We identified a novel signaling cascade orchestrated by KRT17 where its association with plectin resulted in activation of integrin ß4/α6, increased phosphorylation of FAK, Src and ERK, as well as stabilization and nuclear translocation of ß-catenin. The activation of this signaling cascade was correlated with enhanced OSCC cancer stemness and elevated expression of CD44 and epidermal growth factor receptor (EGFR). We identified and demonstrated KRT17 to be a direct target of miRNA-485-5p. Ectopic expression of miRNA-485-5p inhibited OSCC sphere formation and caused sensitization of cancer cells towards cisplatin and carboplatin, which could be significantly rescued by KRT17 overexpression. Dasatinib treatment that inhibited KRT17-mediated Src activation also resulted in OSCC drug sensitization. In OSCC xenograft mouse model, KRT17 knockdown significantly inhibited tumor growth, and combinatorial treatment with cisplatin elicited a greater tumor inhibitory effect. Consistently, markedly reduced levels of integrin ß4, active ß-catenin, CD44 and EGFR were observed in the tumors induced by KRT17 knockdown OSCC cells. CONCLUSIONS: A novel miRNA-485-5p/KRT17/integrin/FAK/Src/ERK/ß-catenin signaling pathway is unveiled to modulate OSCC cancer stemness and drug resistance to the common first-line chemotherapeutics. This provides a potential new therapeutic strategy to inhibit OSCC stem cells and counter chemoresistance.

LC3A-mediated autophagy regulates lung cancer cell plasticity.

ABBREVIATIONS: ATG14: autophagy related 14; CDH2: cadherin 2; ChIP-qPCR: chromatin immunoprecipitation quantitative polymerase chain reaction; CQ: chloroquine; ECAR: extracellular acidification rate; EMT: epithelial-mesenchymal transition; EPCAM: epithelial cell adhesion molecule; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; NDUFV2: NADH:ubiquinone oxidoreductase core subunit V2; OCR: oxygen consumption rate; ROS: reactive oxygen species; RT-qPCR: reverse-transcriptase quantitative polymerase chain reaction; SC: scrambled control; shRNA: short hairpin RNA; SNAI2: snail family transcriptional repressor 2; SOX2: SRY-box transcription factor 2; SQSTM1/p62: sequestosome 1; TGFB/TGF-ß: transforming growth factor beta; TOMM20: translocase of outer mitochondrial membrane 20; ZEB1: zinc finger E-box binding homeobox 1.

Astragalus Polysaccharide (PG2) Suppresses Macrophage Migration Inhibitory Factor and Aggressiveness of Lung Adenocarcinoma Cells.

Astragalus membranaceus is the most popular traditional Chinese medicine for managing vital energy deficiency. Its injectable polysaccharide PG2 has been used for relieving cancer-related fatigue, and PG2 has immune-modulatory and anti-inflammatory effects. In this study, we explored the effects of PG2 in lung adenocarcinoma A549 and CL1-2 cells and investigated its anticancer activity, and the results were validated in severe combined immunodeficiency (SCID) mice. Although PG2 did not inhibit the growth of these cells, it dose-dependently suppressed their migration and invasion, accompanied by reduced vimentin and AXL and induced epithelial cadherin (E-cadherin) expression. Regarding the underlying molecular mechanism, PG2 treatment reduced the macrophage migration inhibitory factor (MIF), an inflammatory cytokine that promotes the epithelial-mesenchymal transition and aggressiveness of cancer cells. Consistent with the previous finding that MIF regulates matrix metalloproteinase-13 (MMP-13) and AMP-activated protein kinase (AMPK), treatment with PG2 reduced MMP-13 and activated AMPK in A549 and CL1-2 cells in this study. In SCID mice injected with A549 cells through the tail vein, intraperitoneal injection with PG2 reduced lung and abdominal metastases in parallel with decreased immunohistochemical staining of AXL, vimentin, MMP-13, and MIF in the tumor. Collectively, data revealed a potential application of PG2 in integrative cancer treatment through the suppression of MIF in cancer cells and their aggressiveness.

Omega-3 Fatty Acid-Enriched Fish Oil and Selenium Combination Modulates Endoplasmic Reticulum Stress Response Elements and Reverses Acquired Gefitinib Resistance in HCC827 Lung Adenocarcinoma Cells.

Non-small cell lung cancer (NSCLC)-carrying specific epidermal growth factor receptor (EGFR) mutations can be effectively treated by a tyrosine kinase inhibitor such as gefitinib. However, the inevitable development of acquired resistance leads to the eventual failure of therapy. In this study, we show the combination effect of omega-3 fatty acid-enriched fish oil (FO) and selenium (Se) on reversing the acquired gefitinib-resistance of HCC827 NSCLC cells. The gefitinib-resistant subline HCC827GR possesses lowered proapoptotic CHOP (CCAAT/enhancer-binding protein homologous protein) and elevated cytoprotective GRP78 (glucose regulated protein of a 78 kDa molecular weight) endoplasmic reticulum (ER) stress response elements, and it has elevated ß-catenin and cyclooxygenase-2 (COX-2) levels. Combining FO and Se counteracts the above features of HCC827GR cells, accompanied by the suppression of their raised epithelial-to-mesenchymal transition (EMT) and cancer stem markers, such as vimentin, AXL, N-cadherin, CD133, CD44, and ABCG2. Accordingly, an FO and Se combination augments the gefitinib-mediated growth inhibition and apoptosis of HCC827GR cells, along with the enhanced activation of caspase -3, -9, and ER stress-related caspase-4. Intriguingly, gefitinib further increases the elevated ABCG2 and cancer stem-like side population in HCC827GR cells, which can also be diminished by the FO and Se combination. The results suggest the potential of combining FO and Se in relieving the acquired resistance of NSCLC patients to targeted therapy.

PTBP1-mediated regulation of AXL mRNA stability plays a role in lung tumorigenesis.

AXL is expressed in many types of cancer and promotes cancer cell survival, metastasis and drug resistance. Here, we focus on identifying modulators that regulate AXL at the mRNA level. We have previously observed that the AXL promoter activity is inversely correlated with the AXL expression levels, suggesting that post-transcriptional mechanisms exist that down-regulate the expression of AXL mRNA. Here we show that the RNA binding protein PTBP1 (polypyrimidine tract-binding protein) directly targets the 5'-UTR of AXL mRNA in vitro and in vivo. Moreover, we also demonstrate that PTBP1, but not PTBP2, inhibits the expression of AXL mRNA and the RNA recognition motif 1 (RRM1) of PTBP1 is crucial for this interaction. To clarify how PTBP1 regulates AXL expression at the mRNA level, we found that, while the transcription rate of AXL was not significantly different, PTBP1 decreased the stability of AXL mRNA. In addition, over-expression of AXL may counteract the PTBP1-mediated apoptosis. Knock-down of PTBP1 expression could enhance tumor growth in animal models. Finally, PTBP1 was found to be negatively correlated with AXL expression in lung tumor tissues in Oncomine datasets and in tissue micro-array (TMA) analysis. In conclusion, we have identified a molecular mechanism of AXL expression regulation by PTBP1 through controlling the AXL mRNA stability. These findings may represent new thoughts alternative to current approaches that directly inhibit AXL signaling and may eventually help to develop novel therapeutics to avoid cancer metastasis and drug resistance.

Epigenetic Modification and Differentiation Induction of Malignant Glioma Cells by Oligo-Fucoidan.

Malignant glioma (MG) is a poor prognostic brain tumor with inevitable recurrence after multimodality treatment. Searching for more effective treatment is urgently needed. Differentiation induction via epigenetic modification has been proposed as a potential anticancer strategy. Natural products are known as fruitful sources of epigenetic modifiers with wide safety margins. We thus explored the effects of oligo-fucoidan (OF) from brown seaweed on this notion in MG cells including Grade III U87MG cells and Grade IV glioblastoma multiforme (GBM)8401 cells and compared to the immortalized astrocyte SVGp12 cells. The results showed that OF markedly suppress the proliferation of MG cells and only slightly affected that of SVGp12 cells. OF inhibited the protein expressions of DNA methyltransferases 1, 3A and 3B (DNMT1, 3A and 3B) accompanied with obvious mRNA induction of differentiation markers (MBP, OLIG2, S100ß, GFAP, NeuN and MAP2) both in U87MG and GBM8401 cells. Accordingly, the methylation of p21, a DNMT3B target gene, was decreased by OF. In combination with the clinical DNMT inhibitor decitabine, OF could synergize the growth inhibition and MBP induction in U87MG cells. Appropriated clinical trials are warranted to evaluate this potential complementary approach for MG therapy after confirmation of the effects in vivo.

Protective Effect of Antrodia cinnamomea Extract against Irradiation-Induced Acute Hepatitis.

Radiotherapy for treatment of hepatocellular carcinoma causes severe side effects, including acute hepatitis and chronic fibrosis. Complementary and alternative medicine (CAM) has emerged as an important part of integrative medicine in the management of diseases. Antrodia cinnamomea (AC), a valuable medicinal fungus originally found only in Taiwan, has been shown to possess anti-oxidation, vaso-relaxtation, anti-inflammation, anti-hepatitis, and anti-cancer effects. In this paper we evaluate the protective effects of ethanol extract of Antrodia cinnamomea (ACE) against radiotoxicity both in normal liver cell line CL48 and in tumor-bearing mice. In CL48, ACE protects cells by eliminating irradiation-induced reactive oxygen species (ROS) through the induction of Nrf2 and the downstream redox system enzymes. The protective effect of ACE was also demonstrated in tumor-bearing mice by alleviating irradiation-induced acute hepatitis. ACE could also protect mice from CCl4-induced hepatitis. Since both radiation and CCl4 cause free radicals, these results indicate that ACE likely contains active components that protect normal liver cells from free radical attack and can potentially benefit hepatocellular carcinoma (HCC) patients during radiotherapy.

AXL phosphorylates and up-regulates TNS2 and its implications in IRS-1-associated metabolism in cancer cells.

BACKGROUND: TNS2 is a focal adhesions protein and a binding partner for many proteins, including the receptor tyrosine kinase Axl. Although TNS2 can bind with Axl, the details of their interactions have not been elucidated. TNS2 is involved in IRS-1 signaling pathway. In this study, we confirmed the relationship between TNS2 expression and the expression of Axl, IRS-1, PDK1 and Glut4 in pancreatic cancer patients. METHODS: The expression levels of TNS2, Axl, IRS-1, PDK1 and Glut4 in human cancer cells were measured by Western blot and/or IP-Western blot assays. Paired samples of pancreatic cancer and non-cancer tissues were obtained from 33 patients and were used to construct tissue microarrays. The expression levels of these markers in the tissue microarrays were measured by enzyme-linked Immunohistochemistry assay, and the relationships were analyzed by Pearson's chi-square test and two-tailed t-test analysis. RESULTS: We demonstrated for the first time that TNS2 is a phosphorylation substrate of Axl. Moreover, we found a positive relationship between TNS2 expression and the expression of Axl, IRS-1, PDK1 and Glut4 in pancreatic cancer patients. Based on these results, we suggest that Axl modulates glucose metabolism potentially through TNS2 and IRS-1. We hypothesize that there exists a novel mechanism whereby Axl binds to and phosphorylates TNS2, releasing TNS2 from interaction with IRS-1 and resulting in increased stability of IRS-1. The two key enzymes of aerobic glycolysis (Glut4 and PDK1) were found to be up-regulated by Axl/TNS2/IRS-1 cross-talk and may play a critical role in glucose metabolism of cancer cells. CONCLUSIONS: Our results revealed for the first time that Axl binds to and phosphorylates TNS2 and that Axl/TNS2/IRS-1 cross-talk may potentially play a critical role in glucose metabolism of cancer cells.

Opposite Regulation of CHOP and GRP78 and Synergistic Apoptosis Induction by Selenium Yeast and Fish Oil via AMPK Activation in Lung Adenocarcinoma Cells.

Selenium has been intensively studied for the use of cancer prevention and treatment. However, the clinical effects are still plausible. To enhance its efficacy, a combinational study of selenium yeast (SY) and fish oil (FO) was performed in A549, CL1-0, H1299, HCC827 lung adenocarcinoma (LADC) cells to investigate the enhancement in apoptosis induction and underlying mechanism. By sulforhodamine B staining, Western blot and flow cytometric assays, we found a synergism between SY and FO in growth inhibition and apoptosis induction of LADC cells. In contrast, the fetal lung fibroblast cells (MRC-5) were unsusceptible to this combination effect. FO synergized SY-induced apoptosis of A549 cells, accompanied with synergistic activation of AMP-activated protein kinase (AMPK) and reduction of Cyclooxygenase (COX)-2 and ß-catenin. Particularly, combining with FO not only enhanced the SY-elevated proapoptotic endoplasmic reticulum (ER) stress marker CCAAT/enhancer-binding protein homologous protein (CHOP), but also reduced the cytoprotective glucose regulated protein of molecular weight 78 kDa (GRP78). Consequently, the CHOP downstream targets such as phospho-JNK and death receptor 5 were also elevated, along with the cleavage of caspase-8, -3, and the ER stress-related caspase-4. Accordingly, inhibition of AMPK by compound C diminished the synergistic apoptosis induction, and elevated CHOP/GRP78 ratio by SY combined with FO. The AMPK-dependent synergism suggests the combination of SY and FO for chemoprevention and integrative treatment of LADC.

Human Urine Extract Cell Differentiation Agent 2 Protects PC12 Cells from Serum Deprivation-Induced Apoptosis Accompanied with Priming of Extracellular Signal-Regulated Kinase Activation and Differentiation Induction.

OBJECTIVE: To investigate the potential neuroprotective effect of human urine extract cell differentiation agent 2 (CDA-2) by the model of serum deprivation-induced apoptosis of PC12 cells and study the underlying molecular mechanisms. METHODS: Apoptosis of PC12 cells was induced by serum deprivation. CDA-2 at doses of 0.5-4 mg/mL was used to treat the serum-deprived PC12 cells. The cellular viability was measured by sulforhodamine B binding assay and the cell apoptosis was determined by flow cytometer. Western blot was used to analyze the expression of differentiation markers and activity of extracellular signal-regulated kinase (ERK). The cellular morphology was examined under an inverted microscope. RESULTS: CDA-2 inhibited apoptotic cell death of serum-deprived PC12 cells in a dose-dependent manner. Expression of low- and mid-sized neurofilaments was observed in serum-deprived PC12 cells treated with CDA-2 or nerve growth factor (NGF). However, CDA-2 did not induce proliferation of these cells like NGF. The morphology of CDA-2 treated cells was changed from rounded to neuron-like flat polygonal shape in contrast to the extensive neurite outgrowth induced by NGF. CDA-2 transiently induced the phosphorylation of ERK in serum deprived-PC12 cells and the expression of neurofilaments induced by CDA-2 was attenuated by mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059. CONCLUSIONS: Human urine extract CDA-2 showed a potential neuroprotective activity which may correlate with ERK activation and differentiation induction.

Active fraction (HS7) from Taiwanofungus camphoratus inhibits AKT-mTOR, ERK and STAT3 pathways and induces CDK inhibitors in CL1-0 human lung cancer cells.

BACKGROUND: The non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. In NSCLC, the oncogenic AKT-mTOR, ERK and STAT3 pathways are commonly dysregulated and have emerged as attractive targets for therapeutic developments. In a relatively limited subset of NSCLC, these pathways driven by mutant EGFR can be treated by the tyrosine kinase inhibitors (TKIs)-mediated targeted therapy. However, for the most NSCLC, more novel targeted agents are imperatively needed. Therefore, we investigated the inhibitory effects of the active fraction HS7 from Taiwanofungus camphoratus, a unique medicinal fungus in Taiwan, on these pathways in CL1-0 EGFR wild-type human NSCLC cells. METHODS: The active fraction HS7 was prepared by n-hexane extraction of T. camphoratus followed by silica gel chromatography. Its effects on the cell viabilities were determined by sulforhodamine B colorimetric assay. Flow cytometry was used to analyze cell-cycle regulation and apoptosis induction. The changes in cellular protein levels were examined by Western blot. RESULTS: The active fraction HS7 vigorously inhibits AKT-mTOR, ERK and STAT3 signaling pathways in CL1-0 cells. At dose of 25 µg/mL, these signaling pathways were almost completely inhibited by HS7, accompanied with induction of cyclin-dependent kinase inhibitors such as p15, p21 and p27. Accordingly, the AKT-mTOR downstream targets p-p70S6K and HIF-1α were also suppressed as well. At this dose, the cell proliferation was profoundly suppressed to 23.4% of control and apoptosis induction was observed. CONCLUSIONS: The active fraction HS7 from n-hexane extract of T. camphoratus exerts multi-targeting activity on the suppression of AKT-mTOR, ERK and STAT3 pathways and induction of p15, p21 and p27 in EGFR wild-type NSCLC cells. This multi-targeting activity of HS7 suggests its potential as an alternative medicine for the treatment of EGFR TKIs resistant NSCLC.

Induction of Forkhead Class box O3a and apoptosis by a standardized ginsenoside formulation, KG-135, is potentiated by autophagy blockade in A549 human lung cancer cells.

BACKGROUND: KG-135, a standardized formulation enriched with Rk1, Rg3, and Rg5 ginsenosides, has been shown to inhibit various types of cancer cells; however, the underlying mechanisms are not fully understood. In this study, we explored its effects in A549 human lung cancer cells to investigate the induction of Forkhead Class box O3a (FOXO3a) and autophagy. METHODS: Cell viability was determined by sulforhodamine B staining. Apoptosis and cell cycle distribution were analyzed using flow cytometry. The changes of protein levels were determined using Western blot analysis. Autophagy induction was monitored by the formation of acidic vesicular organelles stained with acridine orange. RESULTS: KG-135 effectively arrested the cells in G1 phase with limited apoptosis. Accordingly, a decrease of cyclin-dependent kinase-4, cyclin-dependent kinase-6, cyclin D1, and phospho-retinoblastoma protein, and an increase of p27 and p18 proteins were observed. Intriguingly, KG-135 increased the tumor suppressor FOXO3a and induced the accumulation of autophagy hallmark LC3-II and acidic vesicular organelles without an increase of the upstream marker Beclin-1. Unconventionally, the autophagy adaptor protein p62 (sequestosome 1) was increased rather than decreased. Blockade of autophagy by hydroxychloroquine dramatically potentiated KG-135-induced FOXO3a and its downstream (FasL) ligand accompanied by the cleavage of caspase-8. Meanwhile, the decrease of Bcl-2 and survivin, as well as the cleavage of caspase-9, were also drastically enhanced, resulting in massive apoptosis. CONCLUSION: Besides arresting the cells in G1 phase, KG-135 increased FOXO3a and induced an unconventional autophagy in A549 cells. Both the KG-135-activated extrinsic FOXO3a/FasL/caspase-8 and intrinsic caspase-9 apoptotic pathways were potentiated by blockade of autophagy. Combination of KG-135 and autophagy inhibitor may be a novel strategy as an integrative treatment for cancers.

Corrigendum to "Honokiol Eliminates Human Oral Cancer Stem-Like Cells Accompanied with Suppression of Wnt/ß-Catenin Signaling and Apoptosis Induction".

[This corrects the article DOI: 10.1155/2013/146136.].