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Metastasis and drug resistance are the major causes of mortality in patients with non–small cell lung cancer (NSCLC). Several receptor tyrosine kinases (RTKs), including AXL, are involved in the progression of NSCLC. The AXL/MER/SKY subfamily is involved in cell adhesion, motility, angiogenesis, and signal transduction and may play a significant role in the invasiveness of cancer cells. Notably, no specific inhibitors of AXL have been described. A series of CL1 sublines with progressive invasiveness established from a patient with NSCLC has been identified that positively correlates with AXL expression and resistance to chemotherapeutic drugs. The ectopic overexpression of AXL results in elevated cell invasiveness and drug resistance. Nuclear factor-κB (NF-κB) signaling activity is associated with AXL expression and may play an important role in the enhancement of invasiveness and doxorubicin resistance, as shown by using the NF-κB inhibitor, sulfasalazine, and IκB dominant-negative transfectants. In the current study, sulfasalazine exerted a synergistic anticancer effect with doxorubicin and suppressed cancer cell invasiveness in parallel in CL1 sublines and various AXL-expressing cancer cell lines. Phosphorylation of AXL and other RTKs (ErbB2 and epidermal growth factor receptor) was abolished by sulfasalazine within 15 min, suggesting that the inhibition of NF-κB and the kinase activity of RTKs are involved in the pharmacologic effects of sulfasalazine. Our study suggests that AXL is involved in NSCLC metastasis and drug resistance and may therefore provide a molecular basis for RTK-targeted therapy using sulfasalazine to enhance the efficacy of chemotherapy in NSCLC. [Cancer Res 2007;67(8):3878–87]

Supplementary Figures 1-6 from Sulfasalazine Suppresses Drug Resistance and Invasiveness of Lung Adenocarcinoma Cells Expressing AXL

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, β-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.

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.

Cancer cell plasticity generates heterogeneous oncogenic subpopulations in tumors. How macroautophagy/autophagy, a catabolic system required for sustaining cell homeostasis, affects cancer cell plasticity, remains elusive. In this study, we report that MAP1LC3A/LC3A (microtubule associated protein 1 light chain 3 alpha), a key molecule in autophagy, is negatively associated with histological grade and distant metastasis of lung cancer. This is achieved in part, if not all, by maintaining the mitochondria and energy homeostasis to meet the proliferation demand of lung cancer cells driven by SOX2 (SRY-box transcription factor 2) signaling. Basal autophagy is preferentially active in SOX2-positive lung cancer cells with high-proliferative and low-invasive properties. The high-proliferative cancer cells exhibit higher oxygen consumption rate (OCR), elevated reactive oxygen species (ROS), and profound fragmented mitochondrial patterns compared to their high-invasive counterparts. SOX2 expression promotes LC3A expression and enhances proliferation but attenuates invasion in lung cancer cells. LC3A silencing enriches cells harboring low-proliferative and high-invasive features, concomitant with decreased OCR and ROS levels and reduced expression of SOX2. Our findings provide novel insights into how basal autophagy cross talks with SOX2 proliferation signaling to regulate mitochondrial metabolism and determines cancer cell plasticity with an impact on lung tumor progression. 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-β: transforming growth factor beta; TOMM20: translocase of outer mitochondrial membrane 20; ZEB1: zinc finger E-box binding homeobox 1

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 &beta, 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.

Background: Pancreatic adenocarcinoma (PDAC) is known for its deregulated TGFβ signal pathway. Therapeutic strategy targeting TGFβ is controversial due to the dual role of TGFβ with anti-proliferation in early phase and pro-metastatic in late phase of cancer progression. KLF10, an early response gene of TGFβ, positively feedback the antiproliferative effect in cancer. Recent studies revealed KLF10 expression were suppressed epigenetically in various cancers including PDAC. In contrary to TGFβ, KLF10 transcriptionally suppress Slug and Sirtuin 6 to prevent metastasis in advanced PDAC. The role of KLF10 in regulating tumorigenesis and stem cell phenotype is still unknown. Materials and Methods: From 105 patients of resectable PDAC, KLF10 expression level was evaluated to be reduced in 62% of tumor specimens. Low KLF10 expression correlated with larger tumor size and rapid loco-regional recurrence. In murine model of Kras mutation (KC mice), additional depletion of KLF10 induced 57% of PDAC compared to 0% at 18 to 24 wk of age. PDAC cells were genetically manipulated with KLF10mRNA silencing (PDACshKLF10) or inducible overexpression (PDACoeKLF10). Using limiting dilution assay of tumor growth, and orthotopic tumor implantation, we found higher tumorigenic ability of PDACshKlf10 compared to that of wild type. The stem cell phenotypes of PDACshKLF10 and PDACoeKLF10 were confirmed by sphere formation assay and FACS analysis of surface markers including CD24/CD44, ESA/c-Met, CD47 and CD133. Results: Notch signal pathway was found to be significantly enhanced from microarray analysis of wild type versus PDACshKLF10 cells. The increased Notch signal molecules were confirmed in RNA and protein level in genetically manipulated PDAC cells as well as clinical tissue specimens. Using Chip-PCR and luciferase promoter assay, KLF10 was demonstrated to bind to the promoter of Notch receptor 1, 3, and 4. DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), a Notch inhibitor, suppressed in vitro spheroid formation, cell surface expression of stem cell markers, and tumor growth of PDACshKLF10 in orthotopic murine model. We conclude that KLF10 modulates stem cell phenotype of PDAC by transcriptionally suppressing Notch signal pathway. Loss of KLF10 in PDAC patients leads to Notch signal activation, development of stem cell phenotype and tumor progression. Notch inhibition may reverse malignant growth of PDAC with reduced KLF10 expression. Conflict of interest statement: nothing to declare Citation Format: Yi-Chih Tsai, Su-Liang Chen, Shu-Ling Peng, Kuang-Hung Cheng, Shih-Sheng Jiang, Shuang-En Chuang, Ch'ang Hui-Ju. Krupple like factor 10 modulates stem cell phenotype of pancreatic adenocarcinoma via transcriptional regulation of Notch signal pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2001.

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. Electronic supplementary material The online version of this article (10.1186/s13020-017-0154-9) contains supplementary material, which is available to authorized users.

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.

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.

The AXL receptor tyrosine kinase is frequently overexpressed in cancers and is important in cancer invasion/metastasis and chemoresistance. Here, we demonstrate a regulatory feedback loop between AXL and microRNA (miRNA) at the post-transcriptional level. Both the GAS6-binding domain and the kinase domain of AXL, particularly the Y779 tyrosine phosphorylation site, are shown to be crucial for this autoregulation. To clarify the role of miRNAs in this regulation loop, approaches using bioinformatics and molecular techniques were applied, revealing that miR-34a may target the 3′ UTR of AXL mRNA to inhibit AXL expression. Interestingly and importantly, AXL overexpression may induce miR-34a expression by activating the transcription factor ELK1 via the JNK signaling pathway. In addition, ectopic overexpression of ELK1 promotes apoptosis through, in part, down-regulation of AXL. Therefore, we propose that AXL is autoregulated by miR-34a in a feedback loop; this may provide a novel opportunity for developing AXL-targeted anticancer therapies.

MicroRNAs (miRNAs) play important roles in various biologic processes, and aberrant miRNA expression is one of the mechanisms of tumorigenesis. Previous studies indicate that oncogenic HPV infection interrupts the expression of tumor-suppressive miR-34a and contributes to the development of cervical cancer. In addition, the traditional Chinese medicine Gynostemma pentaphyllum (Thunb.) Makino may exert its pharmacologic effects through modifying the gut microbiota by regulating hepatic miR-34a expression. MiR-449 miRs are members of the tumor suppressor miR-34 family and are evolutionarily conserved. In this study, we demonstrated that, in lung cancer cells, ectopic expression of miR-449 family, including miR-449a, miR-449b, and miR-449c, decreased the levels of several G1 phase proteins including cyclin D1, cyclin D3, CDK4, and CDK6 via directly targeting their mRNA’s 3’-UTR. We also observed a differential induction of apoptosis vs. senescence by the miR-449 cluster. While miR-449a and miR-449c induced senescence, miR-449b induced apoptosis and sensitized cancer cells to apoptotic stimuli, such as serum deprivation. Interestingly, among this cluster miRNAs, only miR-449a could induce significant senescence-associated secretory phenotype (SASP), e.g., expression of IL-8, IL-6, IL-1b, and TNF-alpha. Moreover, we also found that miR-449a increased NF-kB phosphorylation at S276, but not phosphorylation site of S536, and promoted the expression of IL-8 mRNA. In this regard, the gut microbiota has been indicated to induce proinflammatory cytokines such as IL-8 to influence the outcomes of cancer patients. In addition to MRX34 (a miR-34a mimic) used in clinical trials, miR-449 family may also potentially become a new therapeutic candidate for NSCLC and other solid tumors’ treatment. Citation Format: Chun-Yu Cho, Shih-Ying Chung, Shuang-En Chuang. Differential regulation of apoptosis and senescence by the miR-449 family microRNAs and its implications in tumor microenvironments of NSCLC [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A29.

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. Electronic supplementary material The online version of this article (10.1186/s12929-018-0465-x) contains supplementary material, which is available to authorized users.

Clinicopathological characteristics of the 33 pancreatic adenocarcinoma patients. The H-score data are shown as mean ± SD. (PDF 55 kb)

Expression of TNS2 (TENC1) in the Oncomine reference database. (PDF 148Â kb)

The original gel image of Fig. 3a. To demonstrate the binding of TNS2 to Axl, HEK293T cells were transfected with TNS2 or variant mutants of Axl and lysed. TNS2 was co-immunoprecipitated (co-IP) by Myc and Axl. K567R: Kinase-dead Axl (KD-Axl). (PDF 132Â kb)

Total TNS2 and total AXL expression in pancreatic cancer cells. Both proteins were detected by IHC in 33 PDAC. Representative micrographs show the histopathologic features and the expressions of TNS2, Axl, IRS-1, and GLUT4 in tumor and the adjacent normal tissues by immunohistochemical analysis. (PDF 164Â kb)

The endogous expression of TNS2, p-Axl and Axl in five pancreatic cancer cell lines. Densityometric quantitative analyses of the TNS2 expression and pAxl/Axl ratio was performed in bar graphs. (PDF 117Â kb)

Accumulating evidence has revealed that fucoidan exhibits anti-tumor activities by arresting cell cycle and inducing apoptosis in many types of cancer cells including hepatocellular carcinoma (HCC). Exploring its effect on microRNA expression, we found that fucoidan markedly upregulated miR-29b of human HCC cells. The induction of miR-29b was accompanied with suppression of its downstream target DNMT3B in a dose-dependent manner. The reduction of luciferase activity of DNMT3B 3′-UTR reporter by fucoidan was as markedly as that by miR-29b mimic, indicating that fucoidan induced miR-29b to suppress DNMT3B. Accordingly, the mRNA and protein levels of MTSS1 (metastasis suppressor 1), a target silenced by DNMT3B, were increased after fucoidan treatment. Furthermore, fucoidan also down-regulated TGF-β receptor and Smad signaling of HCC cells. All these effects leaded to the inhibition of EMT (increased E-cadherin and decreased N-cadherin) and prevention of extracellular matrix degradation (increased TIMP-1 and decreased MMP2, 9), by which the invasion activity of HCC cells was diminished. Our results demonstrate the profound effect of fucoidan not only on the regulation of miR-29b-DNMT3B-MTSS1 axis but also on the inhibition of TGF-β signaling in HCC cells, suggesting the potential of using fucoidan as integrative therapeutics against invasion and metastasis of HCC.

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumour. The neoplasms are difficult to resect entirely because of their highly infiltration property and leading to the tumour edge is unclear. Gliadel wafer has been used as an intracerebral drug delivery system to eliminate the residual tumour. However, because of its local low concentration and short diffusion distance, patient survival improves non-significantly. Axl is an essential regulator in cancer metastasis and patient survival. In this study, we developed a controlled-release polyanhydride polymer loading a novel small molecule, n-butylidenephthalide (BP), which is not only increasing local drug concentration and extending its diffusion distance but also reducing tumour invasion, mediated by reducing Axl expression. First, we determined that BP inhibited the expression of Axl in a dose- and time-dependent manner and reduced the migratory and invasive capabilities of GBM cells. In addition, BP downregulated matrix metalloproteinase activity, which is involved in cancer cell invasion. Furthermore, we demonstrated that BP regulated Axl via the extracellular signal-regulated kinases pathway. Epithelial-to-mesenchymal transition (EMT) is related to epithelial cells in the invasive migratory mesenchymal cells that underlie cancer progression; we demonstrated that BP reduced the expression of EMT-related genes. Furthermore, we used the overexpression of Axl in GBM cells to prove that Axl is a crucial target in the inhibition of GBM EMT, migration and invasion. In an in vivo study, we demonstrated that BP inhibited tumour growth and suppressed Axl expression in a dose-dependent manner according to a subcutaneous tumour model. Most importantly, in an intracranial tumour model with BP wafer in situ treatment, we demonstrated that the BP wafer not only significantly increased the survival rate but also decreased Axl expression, and inhibited tumour invasion. These results contribute to the development of a BP wafer for a novel therapeutic strategy for treating GBM invasion and increasing survival in clinical subjects.

Honokiol, an active compound of Magnolia officinalis, exerted many anticancer effects on various types of cancer cells. We explored its effects on the elimination of cancer stem-like side population (SP) cells in human oral squamous cell carcinoma SAS cells. The sorted SP cells possessed much higher expression of stemness genes, such as ABCG2, ABCC5, EpCAM, OCT-4, CD133, CD44, and β-catenin, and more clonogenicity as compared with the Non-SP cells. After 48 h of treatment, honokiol dose dependently reduced the proportion of SP from 2.53% to 0.09%. Apoptosis of honokiol-treated SP cells was evidenced by increased annexin V staining and cleaved caspase-3 as well as decreased Survivin and Bcl-2. Mechanistically, honokiol inhibited the CD44 and Wnt/β-catenin signaling of SP cells. The Wnt signaling transducers such as β-catenin and TCF-4 were decreased in honokiol-treated SP cells, while the β-catenin degradation promoting kinase GSK-3α/β was increased. Consistently, the protein levels of β-catenin downstream targets such as c-Myc and Cyclin D1 were also downregulated. Furthermore, the β-catenin-related EMT markers such as Slug and Snail were markedly suppressed by honokiol. Our findings indicate honokiol may be able to eliminate oral cancer stem cells through apoptosis induction, suppression of Wnt/β-catenin signaling, and inhibition of EMT.

Additional file 2. Minimum standards of reporting checklist-R.

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.

Honokiol, an active compound ofMagnolia officinalis, exerted many anticancer effects on various types of cancer cells. We explored its effects on the elimination of cancer stem-like side population (SP) cells in human oral squamous cell carcinoma SAS cells. The sorted SP cells possessed much higher expression of stemness genes, such asABCG2,ABCC5,EpCAM,OCT-4,CD133,CD44, andβ-catenin, and more clonogenicity as compared with the Non-SP cells. After 48 h of treatment, honokiol dose dependently reduced the proportion of SP from 2.53% to 0.09%. Apoptosis of honokiol-treated SP cells was evidenced by increased annexin V staining and cleaved caspase-3 as well as decreased Survivin and Bcl-2. Mechanistically, honokiol inhibited the CD44 and Wnt/β-catenin signaling of SP cells. The Wnt signaling transducers such asβ-catenin and TCF-4 were decreased in honokiol-treated SP cells, while theβ-catenin degradation promoting kinase GSK-3α/βwas increased. Consistently, the protein levels ofβ-catenin downstream targets such asc-MycandCyclin D1were also downregulated. Furthermore, theβ-catenin-related EMT markers such as Slug and Snail were markedly suppressed by honokiol. Our findings indicate honokiol may be able to eliminate oral cancer stem cells through apoptosis induction, suppression of Wnt/β-catenin signaling, and inhibition of EMT.

The Chinese herbal mixture, Tien-Hsien Liquid (THL), has been proven to suppress the growth and invasiveness of cancer cells and is currently regarded as a complementary medicine for the treatment of cancer. Our previous study using acute promyelocytic leukemia cells uncovered its effect on the downregulation of DNA methyltransferase 1 (DNMT1) which is often overexpressed in cancer cells resulting in the repression of tumor suppressors via hypermethylation. Herein, we explored the effects of THL in MCF-7 breast cancer cells that also demonstrate elevated DNMT1. The results show that THL dose-dependently downregulated DNMT1 accompanied by the induction of tumor suppressors such as p21 and p15. THL arrested cell cycle in G2/M phase and decreased the protein levels of cyclin A, cyclin B1, phospho-pRb, and AKT. DNMT1 inhibition was previously reported to exert a radiosensitizing effect in cancer cells through the repression of DNA repair. We found that THL enhanced radiation-induced clonogenic cell death in MCF-7 cells and decreased the level of DNA double-strand break repair protein, Rad51. Our observations may be the result of DNMT1 downregulation. Due to the fact that DNMT1 inhibition is now a mainstream strategy for anticancer therapy, further clinical trials of THL to confirm its clinical efficacy are warranted.

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.

Momordica charantiahas been found to exhibit anticancer activity, in addition to its well-known therapeutic functions. We have demonstrated that the leaf extract ofMomordica charantia(MCME) induces apoptosis in several human cancer cells through caspase- and mitochondria-dependent pathways. In this study, a different susceptibility to MCME was found in human lung adenocarcinoma CL1 cells with different metastatic ability, leading to the significant difference of cell viability and invasiveness between MCME-treated CL1-0 and CL1-5 cells. MCME was found to upregulate the expression of Wnt-2 and affect the migratory and invasive ability of CL1 cells through suppressed MMP-2 and MMP-9 enzymatic activities. We proposed that MCME mediates inhibition against migration of CL1 cells by reducing the expression and activation of Src and FAK to decrease the expression of downstream Akt,β-catenin, and MMPs.

There are increasing pieces of evidence suggesting that the recurrence of cancer may result from a small subpopulation of cancer stem cells, which are resistant to the conventional chemotherapy and radiotherapy. We investigated the effects of Chinese herbal mixtureTien-Hsien Liquid(THL) on the cancer stem-like side population (SP) cells isolated from human hepatoma cells. After sorting and subsequent culture, the SP cells from Huh7 hepatoma cells appear to have higher clonogenicity and mRNA expressions of stemness genes such asSMO, ABCG2, CD133,β-catenin,andOct-4than those of non-SP cells. At dose of 2 mg/mL, THL reduced the proportion of SP cells in HepG2, Hep3B, and Huh7 cells from 1.33% to 0.49%, 1.55% to 0.43%, and 1.69% to 0.27%, respectively. The viability and colony formation of Huh7 SP cells were effectively suppressed by THL dose-dependently, accompanied with the inhibition of stemness genes, e.g.,ABCG2, CD133,andSMO. The tumorigenicity of THL-treated Huh7 SP cells in NOD/SCID mice was also diminished. Moreover, combination with THL could synergize the effect of doxorubicin against Huh7 SP cells. Our data indicate that THL may act as a cancer stem cell targeting therapeutics and be regarded as complementary and integrative medicine in the treatment of hepatoma.

Aberrant activation of Wnt/β-catenin signaling plays an important role in the development of colon cancer. HS7 is an active fraction extracted fromTaiwanofungus camphoratus, which had been widely used as complementary medicine for Taiwan cancer patients in the past decades. In this study, we demonstrated the effects of HS7 on the growth inhibition, apoptosis induction, and Wnt/β-catenin signaling suppression in human colon cancer cells. HS7 significantly inhibited proliferation of HT29, HCT116, and SW480 colon cancer cells in a dose- and time-dependent manner. The apoptosis induction was evidenced by DNA fragmentation and subG1 accumulation, which was associated with increased Bax/Bcl-2 ratio, activation of caspase-3 and cleavage of PARP. By using Tcf-dependent luciferase activity assay, HS7 was found to inhibit theβ-catenin/Tcf transcriptional activities. In addition, HS7 strongly suppressed the binding of Tcf complexes to its DNA-binding site shown in electrophoretic mobility shift assay. This inhibition was further confirmed by the decreased protein levels of Tcf-4 andβ-catenin. Theβ-catenin/Tcf downstream target genes, such assurvivin,c-myc,cyclin D1,MMP7, andMT1-MMPinvolved in apoptosis, invasion, and angiogenesis were also diminished as well. These results indicate thatTaiwanofungus camphoratusmay provide a benefit as integrative medicine for the treatment of colon cancer.

Tien-HsienLiquid (THL) is a Chinese herbal mixture that has been used worldwide as complementary treatment for cancer patients in the past decade. Recently, THL has been shown to induce apoptosis in various types of solid tumor cellsin vitro. However, the underlying molecular mechanisms have not yet been well elucidated. In this study, we explored the effects of THL on acute promyelocytic leukemia (APL) NB4 cells, which could be effectively treated by some traditional Chinese remedies containing arsenic trioxide. The results showed THL could induce G2/M arrest and apoptosis in NB4 cells. Accordingly, the decrease of cyclin A and B1 were observed in THL-treated cells. The THL-induced apoptosis was accompanied with caspase-3 activation and decrease of PML-RARαfusion protein. Moreover, DNA methyltransferase 1 and oncogenic signaling pathways such as Akt/mTOR, Stat3 and ERK were also down-regulated by THL. By using ethyl acetate extraction and silica gel chromatography, an active fraction of THL named as EAS5 was isolated. At about 0.5–1% of the dose of THL, EAS5 appeared to have most of THL-induced multiple molecular targeting effects in NB4 cells. Based on the findings of these multi-targeting effects, THL might be regarding as a complementary and alternative therapeutic agent for refractory APL.

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 &beta, 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.

Troglitazone (TGZ) is a synthetic thiazolidinedione drug belonging to a group of potent peroxisome proliferator-activated receptor gamma (PPAR gamma) agonists known to inhibit proliferation, alter cell cycle regulation, and induce apoptosis in various cancer cell types. TGZ is an oral anti-type II diabetes drug that can reverse insulin resistance. For more then 100 yr, aspirin, a nonselective cyclooxygenase (COX) inhibitor, has been successfully used as an anti-inflammatory drug. Recently, Aspirin (ASA) and some other nonsteroidal anti-inflammatory drugs (NSAIDs) have drawn much attention for their protective effects against colon cancer and cardiovascular disease; it has been observed that ASA's anti-tumor effect can be attributed to inhibition of cell cycle progression, induction of apoptosis, and inhibition of angiogenesis. In this report we demonstrate for the first time that, when administered in combination, TGZ and ASA can produce a strong synergistic effect in growth inhibition and G(1) arrest in lung cancer CL1-0 and A549 cells. Examination by colony formation assay revealed an even more profound synergy. In Western blot, combined TGZ and ASA also could downregulate Cdk2, E2F-1, cyclin B1, cyclin D3 protein, and the ratio of phospho-Rb/Rb. Importantly, apoptosis was synergistically induced by the combination treatment, as evidenced by caspase-3 activation and PARP cleavage. The involvement of PI3K/Akt inhibition and p27 upregulation, as well as hypophosphorylation of Rac1 at ser71, were demonstrated. Taken together, these results suggest that clinically achievable concentrations of TGZ and ASA used in combination may produce a strong anticancer synergy that warrants further investigation for its clinical applications.

Carbonic anhydrase III (CAIII) is distinguished from the other members of the CA family by low carbon dioxide hydratase activity, resistance to the CA inhibitor acetazolamide, and a predominant expression in the liver of males. In this report the effects of CAIII expression on liver cancer cells invasiveness were explored. Overexpression of CAIII in the HCC cell line SK-Hep1 resulted in increased anchorage-independent growth and invasiveness. And siRNA-mediated silencing of CAIII expression decreased the invasive ability of SK-Hep1 cells. Furthermore, CAIII transfectants showed elevated focal adhesion kinase (FAK) and Src activity. Silencing of FAK expression in CAIII transfectants led to suppression of HCC cell invasion. More importantly, the CAIII transfectants acidified the culture medium at an accelerated speed than the control cells did. Taken together, these data suggest that the CAIII-promoted invasive ability of HCC cells may probably be mediated through, at least in part, the FAK signaling pathway via intracellular and/or extracellular acidification. © 2008 Wiley-Liss, Inc.

By using a novel profiling analysis of protein tyrosine kinases differentially expressed in the sensitive and refractory leukemia from the same patients we found that AXL was upregulated in drug-resistant leukemia. Furthermore, AXL could be induced by chemotherapy drugs in the acute myeloid leukemia U937 cells and this induction was dependent on the CCWGG methylation status of the AXL promoter. In U937 cells ectopically overexpressing AXL, addition of exogenous Gas6 induced AXL phosphorylation and activation of the Akt and ERK1/2 survival pathways. The Gas6-AXL activation pathway of drug resistance was associated with increased expression of Bcl-2 and Twist. These results show that upregulation of AXL by chemotherapy might induce drug resistance in acute myeloid leukemia in the presence of Gas6 stimulation.

Peroxisome proliferator-activated receptor (PPAR) gamma agonists are used clinically for treating diabetes mellitus and cancer. 2-Methyl-2[(1-{3-phenyl-7-propylbenzol[d]isoxazol-6-yl}oxy)propyl]-1H-4-indolyl) oxy]propanoic acid (BPR1H0101) is a novel synthetic indole-based compound, discovered through research to identify new PPARgamma agonists, and it acts as a dual agonist for PPARgamma and PPARalpha. Isobologram analysis demonstrated that BPR1H0101 is capable of antagonistic interaction with the topoisomerase (topo) II poison, VP16. A study of its mechanism showed that BPR1H0101 could inhibit the catalytic activity of topo II in vitro, but did not produce detectable topo II-mediated DNA strand breaks in human oral cancer KB cells. Furthermore, BPR1H0101 could inhibit VP16-induced topo II-mediated DNA cleavage and ataxia-telangiectasia-mutated phosphorylation in KB cells. The results suggest that BPR1H0101 can interfere with the topo II reaction by inhibiting catalytic activity before the formation of the intermediate cleavable complex; consequently, it can impede VP16-induced topo II-mediated DNA cleavage and cell death. This is the first identified PPARalpha/gamma agonist that can serve as a topo II catalytic inhibitor, to interfere with VP16-induced cell death. The result might have relevance to the clinical use of the PPARalpha/gamma agonist in combination chemotherapy.

We have previously shown that six propolins, A-F, could be isolated from Taiwanese propolis (TP) and that they exerted a broad spectrum of biological activities. Recently, we isolated a seventh compound, propolin G. Its chemical structure has been identified by NMR and fast atom bombardment-mass spectrometry spectra and was found to be identical to a known compound, nymphaeol C. We used high-performance liquid chromatography to determine the relative contents of propolins C, D, F, and G in TP collected in various seasons and regions and found them to be relatively higher in TPs collected from May to July than from September to October. In our present study, we were interested in the various biological activities of TP extract as well as in propolin G as a pure compound. We found that propolin G could efficiently induce apoptosis in brain cancer cell lines (glioma and glioblastoma). The apoptosis might have been through a mitochondrial- and caspase-dependent pathway. This result demonstrated that the TP collection season was more an important factor than the geographical region. Propolis has been suggested to possess a potent antioxidant activity. We further evaluated the antioxidant property of propolin G using DPPH (1,2-diphenyl-2-picryhydrazyl). Our results indicate that propolin G does possess free radical scavenging activity. We also evaluated the neuroprotective action of propolin G, TP, and BP (Brazilian propolis) extracts against oxidative stress in rat primary cortical neurons. Our data demonstrate that propolin G and TP extracts have a marked neuroprotective effect that is greater than BP extract. In conclusion, the isolation and characterization of propolin G from TP have demonstrated for the first time that this compound is a potent inducer of apoptosis in brain cancer cells and that this compound and TP extract exhibit a protective effect against oxidative stress in rat cortical neurons.

Connective tissue growth factor (CTGF) expression is elevated in advanced stages of breast cancer, but the regulatory role of CTGF in invasive breast cancer cell phenotypes is unclear. Presently, overexpression of CTGF in MCF-7 cells (MCF-7/CTGF cells) enhanced cellular migratory ability and spindle-like morphological alterations, as evidenced by actin polymerization and focal-adhesion-complex aggregation. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) impaired cellular migration and promoted a change to an epithelial-like morphology. A neutralizing antibody against integrin alphavbeta3 significantly attenuated CTGF-mediated ERK1/2 activation and cellular migration, indicating that the integrin-alphavbeta3-ERK1/2 signaling pathway is crucial in mediating CTGF function. Moreover, the cDNA microarray analysis revealed CTGF-mediated regulation of the prometastatic gene S100A4. Transfection of MCF-7/CTGF cells with AS-S100A4 reversed the CTGF-induced cellular migratory ability, whereas overexpression of S100A4 in MDA231/AS cells restored their high migratory ability. Genetic and pharmacological manipulations suggested that the CTGF-mediated S100A4 upregulation was dependent on ERK1/2 activation, with expression levels of CTGF and S100A4 being closely correlated with human breast tumors. We conclude that CTGF plays a crucial role in migratory/invasive processes in human breast cancer by a mechanism involving activation of the integrin-alphavbeta3-ERK1/2-S100A4 pathway.

The infection of T cells by Epstein-Barr virus (EBV) may result in hemophagocytic syndrome (HPS) through enhanced cytokine secretion, particularly tumor necrosis factor-α (TNF-α), by EBV latent membrane protein-1 (LMP-1). One bewildering observation of HPS patients is relapsing disease or progression to T-cell lymphoma. This finding raises the question whether EBV LMP-1-expressing T cells may survive and proliferate in the cytokine milieu of HPS. To explore this possibility, we tested the sensitivity of LMP-1-expressing T cells to apoptosis in the presence of TNF-α. LMP-1 up-regulated TNF-α through TRAF2,5 and nuclear factor-κB pathway in T cells. The LMP-1-expressing T cells then became resistant to TNF-α-induced apoptosis. Interestingly, the expression of TNFR1 was remarkably down-regulated by LMP-1 in T cells. Furthermore, the TNF-α/TNFR1 downstream death signal TNFR1-associated death domain protein was constitutively recruited by LMP-1, and the activities of apoptotic caspases 3, 8, and 9 were suppressed. Reconstitution of TNFR1 successfully reversed the TNF-α-induced apoptotic cascades. Therefore, EBV LMP-1 not only activates T cells to proliferate but also confers resistance to TNF-α-mediated apoptosis via down-regulation of TNFR1 in the cytokine milieu of HPS. This finding provides a potential mechanism to explain the disease persistence or progression to T-cell lymphoma in HPS patients.

Background Eliminating cancer stem cells (CSCs) has been suggested for prevention of tumor recurrence and metastasis. Honokiol, an active compound of Magnolia officinalis, had been proposed to be a potential candidate drug for cancer treatment. We explored its effects on the elimination of oral CSCs both in vitro and in vivo. Methods By using the Hoechst side population (SP) technique, CSCs-like SP cells were isolated from human oral squamous cell carcinoma (OSCC) cell lines, SAS and OECM-1. Effects of honokiol on the apoptosis and signaling pathways of SP-derived spheres were examined by Annexin V/Propidium iodide staining and Western blotting, respectively. The in vivo effectiveness was examined by xenograft mouse model and immunohistochemical tissue staining. Results The SP cells possessed higher stemness marker expression (ABCG2, Ep-CAM, Oct-4 and Nestin), clonogenicity, sphere formation capacity as well as tumorigenicity when compared to the parental cells. Treatment of these SP-derived spheres with honokiol resulted in apoptosis induction via Bax/Bcl-2 and caspase-3-dependent pathway. This apoptosis induction was associated with marked suppression of JAK2/STAT3, Akt and Erk signaling pathways in honokiol-treated SAS spheres. Consistent with its effect on JAK2/STAT3 suppression, honokiol also markedly inhibited IL-6-mediated migration of SAS cells. Accordingly, honokiol dose-dependently inhibited the growth of SAS SP xenograft and markedly reduced the immunohistochemical staining of PCNA and endothelial marker CD31 in the xenograft tumor. Conclusions Honokiol suppressed the sphere formation and xenograft growth of oral CSC-like cells in association with apoptosis induction and inhibition of survival/proliferation signaling pathways as well as angiogenesis. These results suggest its potential as an integrative medicine for combating oral cancer through targeting on CSCs. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2265-6) contains supplementary material, which is available to authorized users.