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Background/Aim: Lung cancer notably contributes to tumor-associated mortality worldwide, and standard chemotherapy is used for lung cancer patients. However, its therapeutic efficacy remains unsatisfactory. This study aimed to evaluate the effects and molecular mechanisms of sorafenib and bufalin combination therapy on lung cancer cells in vitro. Materials and Methods: NCI-H292 cells were treated with sorafenib, bufalin, and sorafenib in combination with bufalin. Cell viability, ROS production, Ca(2+) release, and mitochondrial membrane potential were examined by flow cytometric assay. Annexin V/PI staining and chromatin condensation were examined by the apoptosis assays. Finally the molecular mechanism of apoptosis-associated protein expression was investigated by western blotting. Results: NCI-H292 cells treated with sorafenib in combination with bufalin showed significantly decreased viability, enhanced cellular apoptosis, and DNA condensation when compared to that with sorafenib or bufalin alone. Moreover, the combination treatment exhibited higher reactive oxygen species (ROS) production and lower mitochondrial membrane potential (ΔΨm). The combined treatment resulted in higher expression of SOD but lower catalase compared to sorafenib treatment alone. Compared to sorafenib or bufalin treatment alone, the combination treatment resulted in lower Bcl-2 expression but higher Bax, Bad, APAF-1, caspase-3, and caspase-9. Conclusion: Sorafenib in combination with bufalin shows more potent cytotoxic effects and cell apoptosis than sorafenib or bufalin treatment alone in NCI-H292 cells. The combined treatment significantly enhanced apoptotic cell death in NCI-H292 lung cancer cells by activating ROS-, mitochondria-, and caspase-signaling pathways in vitro.

Background/aim Demethoxycurcumin (DMC), one of the derivatives of curcumin, has been shown to induce apoptotic cell death in many human cancer cell lines. However, there is no available information on whether DMC inhibits metastatic activity in human glioblastoma cancer cells in vitro. Materials and methods DMC at 1.0-3.0 μM significantly decreased the proliferation of GBM 8401 cells; thus, we used 2.0 μM for further investigation regarding anti-metastatic activity in human glioblastoma GBM 8401 cells. Results The internalized amount of DMC has reached the highest level in GBM 8401 cells after 3 h treatment. Wound healing assay was used to determine cell mobility and results indicated that DMC suppressed cell movement of GBM 8401 cells. The transwell chamber assays were used for measuring cell migration and invasion and results indicated that DMC suppressed cell migration and invasion in GBM 8401 cells. Gelatin zymography assay was used to examine gelatinolytic activity (MMP-2) in conditioned media of GBM 8401 cells treated by DMC and results demonstrated that DMC significantly reduced MMP-2 activity. Western blotting showed that DMC reduced the levels of p-EGFR(Tyr1068), GRB2, Sos1, p-Raf, MEK, p-ERK1/2, PI3K, p-Akt/PKBα(Thr308), p-PDK1, NF-κB, TIMP-1, MMP-9, MMP-2, GSK3α/β, β-catenin, N-cadherin, and vimentin, but it elevated Ras and E-cadherin at 24 h treatment. Conclusion DMC inhibited cancer cell migration and invasion through inhibition of PI3K/Akt and NF-κB signaling pathways in GBM 8401 cells. We suggest that DMC may be used as a novel anti-metastasis agent for the treatment of human glioblastoma cancer in the future.

Background/aim KT2 is a lysine/tryptophan-rich peptide modified from Crocodylus siamensis Leucrocin I. In this study, we examined the cell toxicity, cellular uptake, anti-migration and anti-invasion activities of KT2 in A375.S2 human melanoma cells. Materials and methods A375.S2 cells were treated with KT2 peptide and then we performed MTT assay, study of cellular uptake by a confocal microscope, wound healing assay, transwell migration/invasion assay, and evaluation of the expression of metastasis-associated proteins. Results KT2 can be internalized through the plasma membrane and can slightly alter cell morphology, decrease the percentage of viable cells and inhibit cell migration and invasion of A375.S2 cells in a dose-dependent manner. This peptide suppressed MMP-2 activity, as measured by gelatine zymography assay. The protein level of MMP-2 was decreased by KT2. KT2 also down-regulated metastasis pathway-related molecules, including FAK, RhoA, ROCK1, GRB2, SOS-1, p-JNK, p-c-Jun, PI3K, p-AKT (Thr308), p-AKT (Ser473), p-p38, MMP-9, NF-kB, and uPA. Conclusion These results indicate that KT2 inhibits the migration and invasion of human melanoma cells by decreasing MMP-2 and MMP-9 expression through inhibition of FAK, uPA, MAPK, PI3K/AKT NF-kB, and RhoA-ROCK signalling pathways. These findings suggest that KT2 deserves further investigation as an anti-metastatic agent for human melanoma.

Background/aim Quetiapine, an atypical antipsychotic, has been encountered as a potential protective agent to suppress various types of tumor growth. However, the inhibitory mechanism of quetiapine in hepatocellular carcinoma (HCC) still remains unclear. The purpose of present study was to investigate the inhibitory mechanism of quetiapine on cell survival and invasion in HCC. Materials and methods Changes of apoptotic signaling, migration/invasion ability, and signaling transduction involved in cell survival and invasion were evaluated with flow cytometry, migration/invasion, and western blot assays. Results Quetiapine inhibited cell proliferation and migration/invasion in SK-Hep1 and Hep3B cells. Quetiapine induced extrinsic and intrinsic apoptotic pathways. Activation of extracellular signal-regulated kinases (ERK), protein kinase B (AKT), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), expression of anti-apoptotic, and metastasis-associated proteins were decreased by quetiapine. Conclusion The apoptosis induction, the decreased expression of ERK/AKT-mediated anti-apoptotic and the metastasis-associated proteins were associated with quetiapine-inhibited cell survival and invasion in HCC in vitro.

Background/Aim: Radiation (RT) induced ERK/NF-ĸB in hepatocellular carcinoma (HCC) has been reported in our previous works; it weakens the toxicity of RT or triggers a radioresistance effect. Thus, combining RT with a suitable NF-ĸB inhibitor may sensitize HCC to RT. Magnolol, a bioactive compound, was known to have anti-inflammatory and anti-tumor functions. Here, we aimed to investigate whether magnolol may enhance anti-HCC efficacy of RT in vivo. Materials and Methods: We established a Hep3B bearing mouse to evaluate the efficacy of the combination treatment of magnolol and RT. Results: Most significantly, tumor volume and tumor weight inhibition was found in the combination group. Tumor immunohistochemistry staining also illustrated the suppression of RT-induced ERK/NF-ĸB-related proteins expression by magnolol. In addition, intrinsic apoptosis-related proteins, such as caspase-3 and -9, were markedly increased in the combination group. Conclusion: Magnolol may effectively enhance anti-HCC ability of RT by downregulating the expression of ERK/NF-ĸB-related proteins and increasing the expression of apoptosis-related proteins.

Background/aim Hepatocellular carcinoma (HCC) is a primary malignancy of the liver and the third leading cause of cancer death worldwide. Although multiple chemotherapies options are available for HCC, chemo-induced toxicity is inevitable during clinical treatment. Therefore, identifying possible adjuvant agents with both liver-protective and antitumor effects is critical. Herbal medicines have chemopreventive and anti-HCC effect, such as Juzen taiho-to and Sho-saiko-to. Astragaloside IV is a compound extracted from the Chinese medical herb Astragalus membranaceus (Fisch.) Bge. with liver protection potential. However, whether astragaloside IV may also possess tumor-inhibitory capability and its underlying mechanism is remaining unknown. Materials and methods Viability analysis, cell-cycle analysis, apoptosis analysis, western blotting analysis and invasion trans-well assay were performed to identify tumor-inhibitory potential of astragaloside IV on HCC cells (SK-Hep1 and Hep3B cells). Results We found that astragaloside IV may induce cytotoxicity and extrinsic/intrinsic apoptosis effect, but also trigger G1 arrest in HCC cells. The expression of anti-apoptotic proteins of HCC were all reduced by astragaloside IV. Additionally, astragaloside IV also suppressed HCC cell invasion ability. Conclusion Astragaloside IV effectively suppressed HCC cell proliferation, invasion and anti-apoptosis in vitro.

Hepatocellular carcinoma (HCC) is the primary tumor of the liver and the fourth leading cause of cancer-related death. Recently, several studies indicated the anti-tumor potential of antipsychotic medicine. Quetiapine, an atypical antipsychotic, is used to treat schizophrenia, bipolar disorder, and major depressive disorder since 1997. However, whether quetiapine may show potential to suppress HCC progression and its underlying mechanism is persisting unclear. Quetiapine has been shown to induce apoptosis and inhibit invasion ability in HCC in vitro. Here, we established two different HCC (Hep3B, SK-Hep1) bearing animals to identify the treatment efficacy of quetiapine. Tumor growth, signaling transduction, and normal tissue pathology after quetiapine treatment were validated by caliper, bioluminescence image, immunohistochemistry (IHC), and hematoxylin and eosin staining, respectively. Quetiapine suppressed HCC progression in a dose-dependent manner. Extracellular signal-regulated kinases (ERKs) and Nuclear factor-κB (NF-κB) mediated downstream proteins, such as myeloid leukemia cell differentiation protein (MCL-1), cellular FLICE-inhibitory protein (C-FLIP), X-linked inhibitor of apoptosis protein (XIAP), Cyclin-D1, matrix metallopeptidase 9 (MMP-9), vascular endothelial growth factor-A (VEGF-A) and indoleamine 2,3-dioxygenase (IDO) which involved in proliferation, survival, angiogenesis, invasion and anti-tumor immunity were all decreased by quetiapine. In addition, extrinsic/intrinsic caspase-dependent and caspase-independent pathways, including cleaved caspase-3, -8, and - 9 were increased by quetiapine. In sum, the tumor inhibition that results from quetiapine may associate with ERK and NF-κB inactivation.

Chronic inflammation induces autoimmune disorders and chronic diseases. Several natural products activate nuclear factor erythroid 2-related factor 2 (Nrf2) signaling, attenuating inflammatory responses. Ergosta-7,9(11),22-trien-3β-ol (EK100) isolated from Cordyceps militaris showed anti-inflammatory and antioxidative activity, but those mechanisms are still unclear. This study is the first to investigate EK100 on antioxidant Nrf2 relative genes expression in LPS-stimulated macrophage-like cell lines. The results showed that EK100 reduced IL-6 (interleukin-6) and tumor necrosis factor-α production. EK100 also attenuated a mitogen-activated protein kinase/activator protein-1 (MAPK/AP-1) pathway and interleukin-6/Janus kinase/signal transducer and activator of transcription (IL-6/JAK/STAT) pathway in LPS-stimulated cells. Toll-like receptor 4 (TLR4) inhibitor CLI-095 and MAPK inhibitors can synergize the anti-inflammatory response of EK100 in LPS-stimulated cells. Moreover, EK100 activated Nrf2/HO-1 (heme oxygenase-1) signaling in LPS-stimulated murine macrophage-like RAW 264.7 cells, murine microglial BV2 cells, and human monocytic leukemia THP-1 cells. However, Nrf2 small interfering RNA (Nrf2 siRNA) reversed EK100-induced antioxidative proteins expressions. In conclusion, EK100 showed anti-inflammatory responses via activating the antioxidative Nrf2/HO-1 signaling and inhibiting TLR4 related MAPK/AP-1 induced IL-6/JAK/STAT pathways in the LPS-stimulated cells in vitro. The results suggest EK100 acts as a novel antioxidant with multiple therapeutic targets that can potentially be developed to treat chronic inflammation-related diseases.

Cantharidin (CTD), a sesquiterpenoid bioactive substance, has been reported to exhibit anticancer activity against various types of cancer cells. The aim of the present study was to investigate the apoptosis effects and the underlying mechanisms of CTD on osteosarcoma U-2 OS cells. Results showed that CTD induced cell morphologic changes, reduced total viable cells, induced DNA damage, and G2/M phase arrest. CTD increased the production of reactive oxygen species and Ca2+, and elevated the activities of caspase-3 and −9, but decreased the level of mitochondrial membrane potential. Furthermore, CTD increased the ROS- and ER stress-associated protein expressions and increased the levels of pro-apoptosis-associated proteins, but decreased that of anti-apoptosis-associated proteins. Based on these observations, we suggested that CTD decreased cell number through G2/M phase arrest and the induction of cell apoptosis in U-2 OS cells and CTD could be a potential candidate for osteosarcoma treatments.

Lupeol, one of the common components from the fruits and natural foods, has been reported to exert antitumor activities in many human cancer cell lines; however, its effects on osteosarcoma cell metastasis were not elucidated. In the present study, lupeol at 10–25 μM induced cell morphological changes and decreased total viable cell number in U-2 OS cells. Lupeol (5–15 μM) suppressed cell mobility, migration, and invasion by wound healing and transwell chamber assays, respectively. Lupeol inhibited the activities of MMP-2 and −9 in U-2 OS cells by gelatin zymography assay. Lupeol significantly decreased PI3K, pAKT, β-catenin, and increased GSK3β. Furthermore, lupeol decreased the expressions of Ras, p-Raf-1, p-p38, and β-catenin. Lupeol also decreased uPA, MMP-2, MMP-9, and N-cadherin but increased VE-cadherin in U-2 OS cells. Based on these observations, we suggest that lupeol can be used in anti-metastasis of human osteosarcoma cells in the future.

Background/aim Casticin shows anti-cancer effects in many types of cancer. However, there is no information regarding its role in DNA damage in human bladder cancer. The aim of this study was to investigate the effects of casticin on TSGH-8301 cells in vitro. Materials and methods Viability of cells was assayed by flow cytometry. DNA damage was assayed by DAPI staining, comet assay, and gel electrophoresis. Protein levels were examined by western blotting and confocal laser microscopy. Results Casticin decreased viability of cells and induced DNA damage. Furthermore, casticin decreased expression of p-ATM, p-ATR, MDC1 and MGMT levels after 48 h of treatment, however, it increased p-ATR and MGMT levels after 12 h. In contrast, casticin increased the levels of p-p53, p-H2A.X, and PARP after 48 h of treatment. As shown by confocal microscopy, casticin affected the translocation of DNA-PKcs and p-p53 to the nucleus of TSGH-8301 cells. Conclusion Casticin decreased viability of human bladder cancer cells through DNA damage.

The aim of the present study is to investigate anticancer effect and mechanism of regorafenib in bladder cancer in vitro and in vivo. Human bladder cancer TSGH 8301 cells were treated with regorafenib, NF‐κB, AKT, or mitogen‐activated protein kinase (MAPK) inhibitors for different time. The changes of cell viability, NF‐κB activation, apoptotic signaling transduction, and expression of tumor progression‐associated proteins were evaluated with MTT, NF‐κB reporter gene assay, flow cytometry, and Western blotting assay. TSGH 8301 tumor bearing mice were established and treated with vehicle (140 μL of 0.1% DMSO) or regorafenib (10 mg/kg/day by gavage) for 15 days. The changes of tumor volume, body weight, NF‐κB activation, MAPK activation, and tumor progression‐associated proteins (MMP‐9, XIAP, VEGF, and Cyclin‐D1) after regorafenib treatment were evaluated with digital caliper, digital weight, and ex vivo Western blotting assay. Our results demonstrated NF‐κB activation and protein levels of MMP‐9, XIAP, VEGF, and Cyclin‐D1 were significantly reduced by NF‐κB (QNZ), ERK (PD98059), and P38 (SB203580) inhibitors. Regorafenib also significantly induced extrinsic and intrinsic apoptotic signaling transduction in bladder cancer in vitro. In addition, regorafenib significantly inhibited tumor growth, NF‐κB, p38, ERK activation and expression of tumor progression‐associated proteins in bladder cancer in vitro and in vivo. Taken together, these results proved that regorafenib not only induced apoptosis through extrinsic and intrinsic pathways and but suppressed MAPK/ NF‐κB‐modulated tumor progression in bladder cancer.

Background/aim Muscle-invasive bladder cancer (MIBC) has long been recognized as a difficult to treat cancer type, thus a new treatment strategy is needed. The major purpose of the present study was to verify the anticancer effect of hyperforin and the mechanism through which it affects tumor cell growth and invasion in bladder cancer in vitro. Materials and methods Bladder cancer TSGH-8301 cells were treated with different concentrations of hyperforin for different durations of time. The changes in cell viability, production of calcium and reactive oxygen species (ROS), and anti-apoptotic signaling were evaluated using MTT assay, flow cytometry, and western blot analysis. The effect of hyperforin on the expression of nuclear factor-kappaB (NF-ĸB) p65 (Ser276), tumor progression-associated proteins, as well as on cell invasion was investigated using western blotting and cell invasion assay, respectively. Results Hyperforin significantly induces apoptosis, extrinsic/intrinsic apoptotic signaling, accumulation of cytosol ROS, and calcium signalling. Hyperforin also significantly diminishes the expression of NF-ĸB p65 (Ser276), anti-apoptotic and tumor progression-associated proteins, as well as the cell invasion ability of TSGH-8301 cells. Conclusion Our findings demonstrate that hyperforin triggers apoptosis depending on extrinsic/intrinsic pathways and suppresses NF-ĸB-mediated cell survival as well as the invasive properties of bladder cancer in vitro.

Osteoarthritis (OA) commonly affects the synovial joint and is characterized by degradation of articular cartilage, which is largely mobilized by increased matrix metalloproteinase (MMP) activity. Soya-cerebroside, an extract of Cordyceps militaris, inhibits inflammatory cytokine production and monocyte migration in human synovial fibroblasts, although its effects are uncertain on MMP expression in chondrocytes and cartilage degradation. In this study, soya-cerebroside antagonized interleukin 1 beta (IL-1β)-induced MMP-1 production in chondrocytes, without cytotoxic effects. Functional genomic data confirm high levels of MMP-1 expression in OA tissue compared with normal tissue. Soya-cerebroside reduced MMP-1 expression via the focal adhesion kinase (FAK), mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase (ERK) and AP-1 signalling pathways. In addition, soya-cerebroside suppressed IL-1β-promoted MMP-1 expression and cartilage degradation in animal models. Our report is the first to reveal that soya-cerebroside reduces MMP-1 production in chondrocytes and protects cartilage degradation through the FAK, MEK, ERK, and AP-1 signalling cascade.

Background/aim Evidence has indicated that fisetin induces cytotoxic effects in human cancer cell lines, including the inhibition of cell migration and invasion, however, the exact molecular mechanism of action of fisetin in human osteosarcoma cells remains unclear. Materials and methods The anti-metastatic mechanisms of fisetin in human osteosarcoma U-2 OS cells were investigated in vitro. Results Fisetin reduced the viability of cells at different concentrations (2.5, 5 and 10 μM) as measured by flow cytometric assay. Fisetin suppressed cell mobility, migration and invasion of U-2 OS cells, as shown by wound healing assay and transwell filter chambers, respectively. The gelatin zymography assay showed that fisetin inhibited MMP-2 activity in U-2 OS cells. Results from western blotting indicated that fisetin reduced the levels of pEGFR, SOS-1, GRB2, Ras, PKC, p-ERK1/2, p-JNK, p-p-38, VEGF, FAK, RhoA, PI3K, p-AKT, NF-ĸB, uPA, MMP-7, MMP-9, and MMP-13, but increased GSK3β and E-cadherin in U-2 OS cells after 48 h of treatment. Conclusion Fisetin can be used in the future, as a target for the treatment of metastasis of human osteosarcoma cells.

Plumbagin, a naturally occurring naphthoquinone component isolated from the root of Plumbago zeylanica, reduces angiogenesis in human umbilical vein endothelial cells; whether plumbagin also has anti-angiogenic activity in human endothelial progenitor cells (EPCs) has remained unclear. Importantly, the recruitment of bone marrow-derived EPCs promotes physiological and pathological neovascularization. In this study, plumbagin inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation of human EPCs, without cytotoxic effects. We also found that plumbagin inhibited angiogenesis via the phospholipase C (PLC), Akt, extracellular-signal-regulated kinase (ERK), nuclear factor (NF)-κB and hypoxia-inducible factor (HIF)-1 signaling pathways. In an EPC Matrigel plug assay, plumbagin significantly diminished microvessel formation and EPC-specific marker expression. Our report is the first to reveal that plumbagin reduces EPC-related angiogenesis both in vitro and in vivo. Further evaluations of plumbagin are warranted, to determine its antitumor activity and other angiogenesis-related disorders.

The recruitment of bone marrow-derived endothelial progenitor cells (EPCs) facilitates physiological and pathological processes involved in new blood vessel synthesis. Glucocerebroside, an extract of Cordyceps militaris, inhibits inflammatory cytokine production and monocyte migration, although its anti-angiogenic properties in human EPCs has remained largely unknown up until now. We describe how glucocerebroside reduces migration as well as tube formation induced by vascular endothelial growth factor (VEGF) stimulation in human EPCs, without affecting cell viability. This inhibitory effect was achieved through the focal adhesion kinase (FAK)/c-Src pathways. We also found that glucocerebroside reduced VEGF-promoted upregulation of the transcription factor Runx2 in the EPCs. The in vivo chick embryo chorioallantoic membrane model demonstrated that glucocerebroside reduces new vessel formation. Our investigation is the first to show that glucocerebroside reduces angiogenesis in human EPCs and to describe the underlying mechanisms. Further investigations are needed to examine the effects of glucocerebroside in other angiogenesis-related disorders.

Gastric cancer has a poor prognosis; once cancer has metastasized, it can easily lead to patient death. Melittin is one of the major components extracted from the bee venom. It has been shown that melittin emerges antitumor activities against many human cancer cell lines. Our results indicated that melittin at 0.2-0.5 µm significantly reduced total cell viability in human gastric cancer AGS cells. At low concentrations (0.05-0.15 µm), melittin displayed antimetastasis effects and inhibited cell adhesion and colony formation. Besides, it inhibited cell motility and suppressed cell migration and invasion. Melittin inhibited the activities of MMP-2 and MMP-9 and the integrity of cell membrane in AGS cells. Furthermore, Western blotting results showed that melittin decreased the protein expressions of Wnt/BMP and MMP-2 signaling pathways. Based on these observations, melittin inhibited cell migration and invasion of AGS cells through multiple signaling pathways. It may be used to treat metastasized gastric cancers in the future.

Ergosta-7, 9 (11), 22-trien-3β-ol (EK100) was isolated from Cordyceps militaris, which has been used as a traditional anti-inflammatory medicine. EK100 has been reported to attenuate inflammatory diseases, but its anti-inflammatory mechanism is still unclear. We were the first to investigate the effect of EK100 on the Toll-like receptor 4 (TLR4)/nuclear factor of the κ light chain enhancer of B cells (NF-κB) signaling in the lipopolysaccharide (LPS)-stimulated RAW264.7 cells and the green fluorescent protein (GFP)-labeled NF-κB reporter gene of Drosophila. EK100 suppressed the release of the cytokine and attenuated the mRNA and protein expression of pro-inflammatory mediators. EK100 inhibited the inhibitor kappa B (IκB)/NF-κB signaling pathway. EK100 also inhibited phosphatidylinositol-3-kinase (PI3K)/Protein kinase B (Akt) signal transduction. Moreover, EK100 interfered with LPS docking to the LPS-binding protein (LBP), transferred to the cluster of differentiation 14 (CD14), and bonded to TLR4/myeloid differentiation-2 (MD-2) co-receptors. Compared with the TLR4 antagonist, resatorvid (CLI-095), and dexamethasone (Dexa), EK100 suppressed the TLR4/AKT signaling pathway. In addition, we also confirmed that EK100 attenuated the GFP-labeled NF-κB reporter gene expression in Drosophila. In summary, EK100 might alter LPS docking to LBP, CD14, and TLR4/MD-2 co-receptors, and then it suppresses the TLR4/NF-κB inflammatory pathway in LPS-stimulated RAW264.7 cells and Drosophila.

Background/aim Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with poor prognosis. Lenvatinib is a multi-kinase inhibitor that has the potential to suppress tumor progression. Our previous study suggested that lenvatinib induces cytotoxicity and apoptosis in CL-1-5-F4 cells in vitro. However, whether lenvatinib suppresses NSCLC progression in vivo remains unclear. Materials and methods Tumor growth inhibition and normal tissue toxicity evaluation following lenvatinib treatment were performed on CL-1-5-F4-bearing mice. Results Tumor growth calculated by caliper and living cell intensity decreased by lenvatinib treatment as analysed by bioluminescence imaging. Phosphorylation of AKT, NF-κB, and NF-κB downstream proteins involved in tumor progression were reduced by lenvatinib in the tumor tissue. No pathological changes were found in the liver, kidney, and spleen after lenvatinib treatment. Conclusion Induction of apoptosis and suppression of AKT/NF-κB were associated with lenvatinib-induced inhibition of the progression of NSCLC in vivo.

Demethoxycurcumin (DMC), one of the derivatives of curcumin, has been shown to induce apoptotic cell death in many human cancer cell lines. However, there is no available information on whether DMC inhibits metastatic activity in human glioblastoma cancer cells in vitro.DMC at 1.0-3.0 μM significantly decreased the proliferation of GBM 8401 cells; thus, we used 2.0 μM for further investigation regarding anti-metastatic activity in human glioblastoma GBM 8401 cells.The internalized amount of DMC has reached the highest level in GBM 8401 cells after 3 h treatment. Wound healing assay was used to determine cell mobility and results indicated that DMC suppressed cell movement of GBM 8401 cells. The transwell chamber assays were used for measuring cell migration and invasion and results indicated that DMC suppressed cell migration and invasion in GBM 8401 cells. Gelatin zymography assay was used to examine gelatinolytic activity (MMP-2) in conditioned media of GBM 8401 cells treated by DMC and results demonstrated that DMC significantly reduced MMP-2 activity. Western blotting showed that DMC reduced the levels of p-EGFRDMC inhibited cancer cell migration and invasion through inhibition of PI3K/Akt and NF-κB signaling pathways in GBM 8401 cells. We suggest that DMC may be used as a novel anti-metastasis agent for the treatment of human glioblastoma cancer in the future.

Background/aim Non-small cell lung cancer (NSCLC) is a serious disease and the leading cause of death globally. Overexpression of protein kinase B/nuclear factor-kappa B (NF-κB) signaling transduction of NSCLC cells was recognized as a potential therapeutic target. Lenvatinib is a multiple kinase inhibitor against vascular endothelial growth factor receptor family. However, whether lenvatinib may affect AKT/NF-κB in NSCLC remains unknown. Materials and methods MTT assay, NF-κB reporter gene assay, flow cytometry, tranwell migration/invasion analysis and western blotting were used to identify the alteration of cell viability, NF-κB activation, apoptosis effect, migration/invasion potential and AKT/NF-κB related protein expression, respectively, in CL-1-5-F4 cells after lenvatinib treatment. Results The cell viability and NF-κB activity were suppressed by lenvatinib. Extrinsic and intrinsic apoptosis were activated by lenvatinib. Additionally, the metastatic potential of CL-1-5-F4 cells was also suppressed by lenvatinib. Conclusion Altogether, lenvatinib induced extrinsic/intrinsic apoptosis and suppressed migration/invasion ability of NSCLC cells that was associated with AKT/NF-κB signaling inactivation.

Background Although both chemotherapy and radiotherapy (RT) can sufficiently maintain tumor suppression of colorectal cancer (CRC), these treatments may trigger the expression of nuclear factor kappa B (NF-κB) and compromise patients' survival. Regorafenib suppresses NF-κB activity in various tumor types. However, whether regorafenib may act as a suitable radiosensitizer to enhance therapeutic efficacy of RT remains unknown. Materials and methods Here, we established a CRC-bearing animal model to investigate the therapeutic efficacy of regorafenib in combination with RT, through measurement of tumor growth, body weight, whole-body computed tomography (CT) scan and immunohisto-chemistry staining. Results Smallest tumor size and weight were found in the combination treatment group. In addition, RT-induced up-regulation of NF-κB and downstream proteins were diminished by regorafenib. Moreover, the body weight and liver pathology in the treated group were similar to those of the non-treated control group. Conclusion Regorafenib may enhance the anti-CRC efficacy of RT.

Background Diabetic patients are highly vulnerable to hypoxic injury, which is associated with hypoxia induced BNIP3 expression that subsequently activate apoptosis. Our previous research show that Tetramethylpyrazine (TMP), a food flavoring agent, represses the hypoxia induced BNIP3 expression attenuate myocardial apoptosis. In this study, we evaluate the effect of TMP to provide protection against hypoxia aggravated high-glucose associated cellular apoptosis. Methods The cytoprotective effect of TMP against high glucose induced cellular damages was determined on embryo derived H9c2 cardiomyoblast cells that were subjected to 5% hypoxia for 24 h and subjected to different duration of 33 mM high glucose challenge. Further, the involvement of HIF-1α and BNIP3 in cellular damage and the mechanism of protection of TMP were determined by overexpression and silencing HIF-1α and BNIP3 protein expression. Results The results show that hypoxic effects on cell viability aggravates with high glucose challenge and this augmentative effect is mediated through BNIP3 in H9c2 cardiomyoblast cells. However, TMP administration effectively reversed the augmented HIF-1α levels and BNIP3 elevation. TMP improved the survival of H9c2 cells and effectively suppressed apoptosis in H9c2 cells. Further comparison on the effects of TMP on H9c2 cells challenged with high glucose and those challenged with hypoxia show that TMP precisely regulated the hypoxic intensified apoptotic effects in high-glucose condition. Conclusion The results clearly show that flavoring agent-TMP attenuates cytotoxicity amplified by hypoxia challenge in high glucose condition by destabilizing HIF-1α.

Background/aim Etomidate, an intravenous anesthetic, has been shown to have anticancer effects, including induction of cell-cycle arrest and apoptosis. However, to our knowledge, there are no reports about the anti-metastasis effects of etomidate on A549 human lung adenocarcinoma cells. Materials and methods The cell viability, cell adhesion, gelatin zymography assay, transwell migration and invasion assay, and western blotting analysis were used to investigate the effects of etomidate on A549 cells. Results In our study, etomidate showed low cytotoxicity, inhibited cell adhesion, and suppressed the migration and invasion in A549 cells. The activity of matrix metallopeptidase 2 (MMP2) was reduced by 48 h treatment of etomidate. Results of western blotting analysis indicated that etomidate down-regulated the expression of protein kinase C, MMP7, MMP1, MMP9, and p-p-38, but up-regulated that of RAS, phosphoinositide 3-kinase, and phosphor-extracellular-signal related kinase after 24 and 48 h treatment, in A549 cells. Conclusion Etomidate suppressed the migration and invasion of lung adenocarcinoma A549 cells via inhibiting the expression of MMP1, MMP2, MMP7 and MMP9, and provides potential therapeutic targets for lung cancer treatment.

Background/aim Maslinic acid (MA), a pentacyclic triterpene extracted from wax-like coatings of olives, has been shown to reduce cancer cell number through induction of autophagy and apoptosis in many human cancer cells including human leukemia HL-60 cells. In the present study, we investigated whether or not MA affects immune responses in a leukemia mouse model. Materials and methods WEHI-3 cells were intraperitonealIy (i.p.) injected into normal BALB/c mice to develop leukemia. Mice were then treated by i.p. injection with MA at different doses (0, 8, 16 and 32 mg/kg) for 2 weeks. After treatment, all animals were weighed and blood, liver and spleen tissues were weighed. Blood or spleen both were used for determination of cell markers or phagocytosis, natural killer (NK) cell activities and T- and B-cell proliferation, respectively, by using a flow cytometric assay. Results MA did not significantly affect body, liver, and spleen weights. However, MA increased markers of T-cells (at 16 mg/kg treatment) and monocytes (at 32 mg/kg treatment), but reduced B-cell markers (at 8 mg/kg treatment); MA did not significantly affect cell marker of macrophages. Furthermore, MA increased phagocytosis by macrophages from peripheral blood mononuclear cells and peritoneal cavity at 32 mg/kg treatment and increased NK cell activity at target cell:splenocyte ratio of 25:1 but did not affect B- and T-cell proliferation. Conclusion MA increased immune responses by enhancing macrophage phagocytosis and NK cell activities in leukemic mice.

Background/aim Bisdemethoxycurcumin (BDMC) exhibits biological activities including anticancer and anti-metastasis in human cancer cell lines, but there is no available information to show whether BDMC suppresses cell migration and invasion of human cervical cancer cells. Materials and methods Wound-healing, migration, invasion, zymography, and western blotting assays were used to investigate the effects of BDMC on HeLa cells in vitro. Results BDMC reduced the total viable cell number in a dose-dependent manner. The wound-healing assay show BDMC suppressed the movement of HeLa cells. Furthermore, the trans-well chamber assays showed that BDMC suppressed the cell migration and invasion. Gelatin zymograph assay showed that BDMC did not inhibit matrix metalloproteinase-2 (MMP-2) and -9 activities in vitro. However, western blotting assay showed that BDMC significantly reduced protein levels of growth factor receptor-bound protein 2 (GRB2), Ras homolog gene family, member A (Rho A), urokinase-type plasminogen activator (uPA), RAS, MMP-2, and N-cadherin but increased those of phosphor-extracellular-signal related kinase (p-ERK1/2), E-cadherin and nuclear factor-ĸB (NF-ĸB) in HeLa cells. Confocal laser microscopy assay was used to further confirm BDMC increased NF-ĸB when compared to controls. Conclusion BDMC may have potential as a novel anti-metastasis agent for the treatment of human cervical cancer.

Tetrandrine (TET) exhibits biological activities, including anticancer activity. In Chinese medicine, TET has been used to treat hypertensive and arrhythmic conditions and has been demonstrated to induce cytotoxic effects on human cancer cell lines. However, to the best of the author's knowledge, no previous studies have revealed that TET affects cell metastasis in SW620 human colon cancer cells. The present study demonstrated that TET decreased the cell number and inhibited cell adhesion and mobility of SW620 cells. Furthermore, a wound healing assay was performed to demonstrate that TET suppressed cell movement, and Transwell chamber assays were used to reveal that TET suppressed the cell migration and invasion of SW620 cells. Western blotting demonstrated that TET significantly reduced protein expression levels of SOS Ras/Rac guanine nucleotide exchange factor 1, phosphatidylinositol 3-kinase, growth factor receptor bound protein 2, phosphorylated (p)-c Jun N-terminal kinase 1/2, p-p38, p38, 14-3-3, Rho A, β-catenin, nuclear factor-κB p65, signal transducer and activator of transcription-1 and cyclooxygenase-2, in comparison with untreated SW620 cells. Overall, the results of the present study suggested that TET may be used as a novel anti-metastasis agent for the treatment of human colon cancer in the future.

Background/aim Bufalin, bufadienolide present in Chan Su, has been shown to induce cancer cell apoptosis in many human cancer cells, including human leukemia cells, but its effects on immune responses are unknown. Materials and methods This study investigated whether bufalin affected immune responses of mice with WEHI-3 cell-generated leukemia in vivo. BALB/c mice were intraperitoneally injected with WEHI-3 cells to develop leukemia and then were treated with oral treatment with bufalin at different doses (0, 0.1, 0.2 and 0.4 mg/kg) for 2 weeks. At the end of treatment, all mice were weighted and blood was collected; liver and spleen tissues were collected for cell marker, phagocytosis, natural killer (NK) cell activity and T- and B-cell proliferation measurements by using flow cytometric assays. Results When compared with the leukemia control group, bufalin increased the body weight, but reduced liver and spleen weights, and reduced CD3, CD16 and Mac-3 cell markers at 0.4 mg/kg treatment and increased CD11b marker at 0.1 and 0.2 mg/kg treatment. Furthermore, bufalin at 0.4 mg/kg increased phagocytosis by macrophages isolated from peripheral blood mononuclear cells and at 0.1 mg/kg by those from the peritoneal cavity. Bufalin (0.2 and 0.4 mg/kg) increased NK cell cytotoxic activity at effector:target ratio of 50:1. Bufalin increased B-cell proliferation at 0.1 and 0.2 mg/kg treatment but only increased T-cell proliferation at 0.1 mg/kg. Bufalin increased glutamate oxaloacetate transaminase level at all dose treatments, increased glutamic pyruvic transaminase level only at 0.1 mg/kg treatment, but reduced the level of lactate dehydrogenase at all dose levels in mice with WEHI-3 cell-induced leukemia in vivo. Conclusion Bufalin increased immune responses by enhancing phagocytosis in mice with leukemia mice.

Background/aim Many antimicrobial peptides have been shown to have anticancer activity against human cancer cell lines. Cationic KT2 peptide, derived from white blood cell extract of Crocodylus siamensis has antibacterial activity and antitumor activity against human cervical cancer cells, but there are no data on the effect of KT2 peptide on tumor growth in vivo. The anticancer activity of KT2 peptide on human colon cancer xenografts was investigated in nude mice. Materials and methods Tumors in nude mice (BALB/c -nu/nu mice) were induced by subcutaneous injection with HCT116 cells. Twelve days after cancer cell xenograft, mice were treated by intratumoral injection with phosphate-buffered saline or KT2 peptide (25 and 50 mg/kg) once every 2 days for a total of four times and mice were sacrificed at 2 days after the last treatment. Results KT2 peptide treatment did not lead to significant difference in mouse body weight among groups, but reduced both tumor volume and weight of colon cancer xenografts. Moreover, KT2 peptide increased the expression of apoptotic proteins, such as BCL2-associated X (BAX), cleaved caspase-3, and poly (ADP-ribose) polymerase and reduced that of BCL2 apoptosis regulator in xenograft tumors. Conclusion This finding suggests that KT2 peptide may inhibit tumor growth via apoptosis induction in this mouse model and supports the antitumor ability of KT2 peptide.