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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.

Leukemia is one of the major diseases causing cancer-related deaths in the young population, and its cure rate is unsatisfying with side effects on patients. Fluorouracil (5-FU) is currently used as an anticancer drug for leukemia patients. Casticin, a natural polymethoxyflavone, exerts anticancer activity against many human cancer cell lines in vitro, but no other reports show 5-FU combined with casticin increased the mouse leukemia cell apoptosis in vitro. Herein, the antileukemia activity of 5-FU combined with casticin in WEHI-3 mouse leukemia cells was investigated in vitro. Treatment of two-drug combination had a higher decrease in cell viability and a higher increase in apoptotic cell death, the level of DNA condensation, and the length of comet tail than that of 5-FU or casticin treatment alone in WEHI-3 cells. In addition, the two-drug combination has a greater production rate of reactive oxygen species but a lower level of Ca2+ release and mitochondrial membrane potential (ΔΨm ) than that of 5-FU alone. Combined drugs also induced higher caspase-3 and caspase-8 activities than that of casticin alone and higher caspase-9 activity than that of 5-FU or casticin alone at 48 hours treatment. Furthermore, 5-FU combined with casticin has a higher expression of Cu/Zn superoxide dismutase (SOD [Cu/Zn]) and lower catalase than that of 5-FU or casticin treatment alone. The combined treatment has higher levels of Bax, Endo G, and cytochrome C of proapoptotic proteins than that of casticin alone and induced lower levels of B-cell lymphoma 2 (BCL-2) and BCL-X of antiapoptotic proteins than that of 5-FU or casticin only. Furthermore, the combined treatment had a higher expression of cleaved poly (ADP-ribose) polymerase (PARP) than that of casticin only. Based on these findings, we may suggest that 5-FU combined with casticin treatment increased apoptotic cell death in WEHI-3 mouse leukemia cells that may undergo mitochondria and caspases signaling pathways in vitro.

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.

Bauhinia championii (Benth.) is one of the commonly used herbs in Taiwan. The stem of this plant has been used to treat epigastria pain and rheumatoid arthritis. However, the antitumor activities of this herb have never been reported. This study aims to investigate the mechanism of anticancer activity of the extracts from B. championii (BC). BC was fractionated with a series of organic solvents, including n-hexane (H), ethyl acetate (EA), 1-butanol (B), and water (W). We first investigated the effects of BC-H, BC-EA, BC-B and BC-W partitioned fraction on cell viability. In HCT 116 colon cancer cell lines, BC-EA showed the highest inhibition of cell viability and changed the morphology of cells. With dose- and time-dependent manners, BC-EA inhibited the proliferation of HCT 116 cells by inducing apoptosis and G0/G1phase arrest of cell cycle. To determine the underlying mechanisms, down-regulated CDK2, Cyclin D, and Cyclin E and up-regulated p16, p21, and p53 may account for the cell cycle arrest, while the apoptotic effect of BC-EA may attribute to increased intracellular Ca[Formula: see text], loss of mitochondria membrane potential ([Formula: see text]), increase of Bax, Bak, puma, and AIF, and decrease of Bcl-2. Furthermore, the inactivation of Ras signaling pathway by BC-EA also contributed to its apoptotic effect on HCT 116. Our study demonstrates that BC-EA not only inhibits cell growth but also induces apoptosis through inhibiting Ras signal pathway and increasing p53 expression levels. We suggest that BC-EA may be a new dietary supplement and a useful tool to search for therapeutic candidates against colon cancer.

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.

A previous study presented that glycyrrhizic acid as the hepatoprotective agent inhibits total parenteral nutrition-associated acute liver injury in rats. However, the anticancer effect and mechanism of glycyrrhizic acid in human hepatocellular carcinoma (HCC) is ambiguous. The purpose of the present study was to investigate the effect of glycyrrhizic acid on apoptosis dysregulation and metastatic potential in HCC in vitro and in vivo. Both SK-Hep1 and Hep3B cells were treated with different concentrations of glycyrrhizic acid for 24 or 48[Formula: see text]h. SK-Hep1/luc2 tumor-bearing mice were treated with vehicle or glycyrrhizic acid (50[Formula: see text]mg/kg/day by intraperitoneal injection) for 7 days. Tumor cells growth, apoptotic, and metastatic signaling transduction were evaluated by using MTT assay, digital caliper, bioluminescence imaging (BLI), flow cytometry, western blotting assay, and immunohistochemistry (IHC) staining. The results demonstrated glycyrrhizic acid significantly inhibits tumor cell growth, cell invasion, and expression of AKT (Ser473), extracellular-signal-regulated kinase (ERK), epidermal growth factor receptor (EGFR) phosphorylation, anti-apoptotic and metastatic proteins in HCC in vitro and in vivo. Glycyrrhizic acid also significantly triggered apoptosis and extrinsic/intrinsic apoptotic signaling transduction. In addition, PD98059 (ERK inhibitor) and LY294002 (AKT inhibitor) obviously reduced cell invasion and expression of metastasis-associated proteins. Taken together, these results indicated that glycyrrhizic acid induces apoptosis through extrinsic/intrinsic apoptotic signaling pathways and diminishes EGFR/AKT/ERK-modulated metastatic potential in HCC in vitro and in vivo.

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 most frequently occurring liver malignancy in Asia. Glycyrrhizic acid is known to reduce the risk of HCC formation in patients with chronic hepatitis C. To identify whether glycyrrhizic acid may play a role in anti-HCC therapy as an adjuvant is important. However, the inhibitory effect of glycyrrhizic acid on cell cycle progression in HCC cells and the mechanism of such have not been fully elucidated. This study used the comet assay, cell cycle analysis, immunofluorescence staining, the TUNEL assay, and Western blotting to identify the anti-HCC role of glycyrrhizic acid. Glycyrrhizic acid may induce DNA damage, apoptosis, activation of ATM, and expression of p21, and p27 in HCC cells. In addition, glycyrrhizic acid may also induce G

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.

Cardamonin, a monomeric alkaloid, is isolated from Alpinia conchigera Griff and other natural plants. Recently, it has been focused on its anticancer activities, and no information showing its immune effects on leukemia mice was reported. In this study, we investigated the immune effects of cardamonin on WEHI-3 cell-generated leukemia mice. Forty BALB/c mice were randomly divided into four groups: Group I mice were normal animals and groups II-IV were leukemia. Group II mice, as a positive control, were administered with normal diet, and group III and IV mice were treated with 1 and 5 mg/kg of cardamonin, respectively, by intraperitoneal injection every 2 days for 14 days. The population of white blood cells, macrophage phagocytosis, and the proliferations of T and B cells were analyzed by flow cytometry. Another forty mice were also separated randomly into four groups for the determination of survival rate. Results showed that cardamonin did not affect body weight. Cardamonin decreased CD3, CD11b, and Mac-3 cell populations but increased CD19 number. Cardamonin enhanced phagocytic abilities of macrophages from the peripheral blood mononuclear cells of leukemia mice. Furthermore, cardamonin at 1 mg/kg treatment improved the survival rate of leukemia mice in vivo. Therefore, cardamonin could be applied for a leukemia therapeutic reagent at a defined dose.

Although hepatitis B and/or hepatitis C virus were recognized as major risk factor for the development of hepatocellular carcinoma (HCC), certain occupational, environmental, and lifestyle factors also play key roles in HCC tumorigenesis. Moreover, in molecular signaling route, extracellular signal-regulated kinase (ERK)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling was found to be overexpressed and linked to poor prognosis in HCC. Thus, to identify possible nature compound that can suppress ERK/NF-κB may be benefit to HCC patient. Magnolol, a natural compound derived from herbal plant Magnolia officinalis, has been recognized as a liver protection and antitumor reagent. However, whether magnolol-inhibited HCC progression correlates with disruption of ERK/NF-κB signaling is remained unclear. In this studies, we performed SK-Hep1/luc2 HCC bearing animal model to investigate the anticancer efficacy and mechanism of magnolol on tumor progression. Tumor size and tumor growth rate were dramatically suppressed after treatment of magnolol. In addition, expression of phospho-ERK (p-ERK), NF-κB p65 (Ser536), and tumor progression-associated proteins, such as matrix metallopeptidase 9 (MMP-9), vascular endothelial growth factor (VEGF), X-linked inhibitor of apoptosis protein (XIAP), and CyclinD1 were all significantly decreased by magnolol. Most important, major extrinsic and intrinsic apoptosis signaling factors, including active caspase-8 and caspase-9 were both enhanced by magnolol. This study indicated that apoptosis induction through extrinsic/intrinsic pathways and blockage of ERK/NF-κB activation were associated with magnolol-inhibited tumor progression in HCC in vivo.

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.

The anticancer activity of cationic antimicrobial peptides (AMPs) has become more interesting because some AMPs have selective recognition against cancer cells. However, their antitumor properties and underlying mechanisms in cancer cells have not been clearly understood. In this study, we evaluated the effects of KT2 (lysine/tryptophan-rich AMP) on the cellular uptake and internalization mechanism, cell viability, surface charge of the cell membrane, membrane integrity, apoptotic cell death, and autophagy in human HCT 116 colon cancer cells. We found that KT2 interacted with the cell membrane of HCT 116 cells and was internalized into HCT 116 cells via clathrin-mediated and caveolae-mediated endocytosis mechanisms. The interaction of KT2 with cells caused cell membrane structure change, elevated membrane permeability, and KT2 also affected the lipid component. The results of atomic force microscopy showed cellular membrane defects of KT2-treated cells. The internalized KT2 induced nuclear condensation and apoptotic cell death. It elevated the apoptotic factor levels including those of cytochrome c and apoptosis-inducing factor. Furthermore, KT2 inhibited autophagy by the suppression of autophagy-related 5, autophagy-related 7, autophagy-related 16 like 1, and Beclin-1 proteins. In conclusion, these results revealed the cytotoxicity of cationic KT2 against HCT 116 cells and may help to clarify the interactions between cationic AMPs and cancer cells.

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.

The aim of this study was to investigate the effects of bufalin on human nasopharyngeal carcinoma NPC-TW 076 cells in vitro. Bufalin is a cardiotonic steroid and a key active ingredient of the Chinese medicine ChanSu. The extracts of Chansu are used for various cancer treatments in China. In the present study, bufalin induced cell morphological changes, decreased total cell viability and induced G2/M phase arrest of cell cycle in NPC-TW 076 cells. Results also indicated that bufalin induced chromatin condensation (cell apoptosis) and DNA damage by DAPI staining and comet assay, respectively. The induced apoptotic cell death was further confirmed by annexin-V/PI staining assay. In addition, bufalin also increased ROS and Ca[Formula: see text] production and decreased the levels of [Formula: see text]. Furthermore, the alterations of ROS, ER stress and apoptosis associated protein expressions were investigated by Western blotting. Results demonstrated that bufalin increased the expressions of ROS associated proteins, including SOD (Cu/Zn), SOD2 (Mn) and GST but decreased that of catalase. Bufalin increased ER stress associated proteins (GRP78, IRE-1[Formula: see text], IRE-1[Formula: see text], caspase-4, ATF-6[Formula: see text], Calpain 1, and GADD153). Bufalin increased the pro-apoptotic proteins Bax, and apoptotic associated proteins (cytochrome c, caspase-3, -8 and -9, AIF and Endo G) but reduced anti-apoptotic protein Bcl-2 in NPC-TW 076 cells. Furthermore, bufalin elevated the expressions of TRAIL-pathway associated proteins (TRAIL, DR4, DR5, and FADD). Based on these findings, we suggest bufalin induced apoptotic cell death via caspase-dependent, mitochondria-dependent and TRAIL pathways in human nasopharyngeal carcinoma NPC-TW 076 cells.

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.

Glioblastoma is the most common primary malignant tumor of the central nervous system, with an annual incidence of 5.26 per 100000 people. The clinical outcome of standard therapy and the survival rate remain poor; therefore, there is an unmet need for a new strategy to treat this lethal disease. Although amentoflavone was known to have anticancer potential in various types of cancers, its antiglioblastoma ability and mechanism remain unrecognized. We demonstrated that amentoflavone may suppress glioblastoma invasion and migration by transwell assay. Moreover, we established NF-[Formula: see text]B reporter gene system and used that for verifying NF-[Formula: see text]B inhibition efficacy of amentoflavone on in vitro and in vivo studies. Here, we indicated that amentoflavone not only diminished NF-[Formula: see text]B activation, but also reduced NF-[Formula: see text]B-mediated downstream oncogenes expression, such as MMP-2, MMP-9, XIAP, cyclinD1 and VEGF, which was elucidated by Western blot and immunohistochemistry (IHC). Tumor growth inhibition and NF-[Formula: see text]B reduction was found in the amentoflavone treatment group, which was revealed by the glioblastoma-bearing animal model. In this study, we also used ERK inhibitor and NF-[Formula: see text]B inhibitor (QNZ) to confirm whether the beneficial result of amentoflavone on glioblastoma was mainly regulated by blockage of ERK/NF-[Formula: see text]B signaling. In summary, ERK/NF-[Formula: see text]B signaling pathway has a role in the inhibition of tumor growth by amentoflavone in glioblastoma.

Casticin, derived from Fructus Viticis, has anticancer properties in many human cancer cells, however, there is no report to show that casticin promotes immune responses and affects the survival rate of leukemia mice in vivo. The aim of this study is to evaluate the effects of casticin on immune responses and the survival rate of WEHI-3 cells generated in leukemia mice in vivo. Animals were divided into six groups: normal control mice, leukemia control mice, mice treated with ATRA (all-trans retinoic acid), and casticin (0.1, 0.2, and 0.4[Formula: see text]mg/kg) treated mice. All animals were treated for 14 days and then measured for body weights, total survival rate, cell markers, the weights of liver and spleen, phagocytosis of spleen cells, NK cell activities and cell proliferation. Results show that casticin did not affect animal appearances, however, it increased body weights and decreased the weights of liver at 0.2[Formula: see text]mg/kg and 0.4[Formula: see text]mg/kg treatment. Casticin also decreased spleen weight at 0.2[Formula: see text]mg/kg and 0.4[Formula: see text]mg/kg treatment, increased CD3 at 0.1, 0.2 and 0.4[Formula: see text]mg/kg doses and increased CD19 at 0.2[Formula: see text]mg/kg treatment but decreased CD11b and Mac-3 at 0.1, 0.2 and 0.4[Formula: see text]mg/kg treatment. Casticin (0.1, 0.2 and 0.4[Formula: see text]mg/kg) increased macrophage phagocytosis from PBMC (peripheral blood mononuclear cell) and peritoneal cavity. Furthermore, casticin increased NK cells’ cytotoxic activity and promoted T cell proliferation at 0.1–0.4[Formula: see text]mg/kg treatment with or without concanavalin A (Con A) stimulation, but only increased B cell proliferation at 0.1 mg/kg treatment. Based on these observations, casticin could be used as promoted immune responses in leukemia mice in vivo.

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.

Fisetin, a naturally occurring flavonoid, is found in common fruits and vegetables and has been shown to induce cytotoxic effects in many human cancer cell lines. No information has shown that fisetin induced cell cycle arrest and apoptosis in mouse leukemia WEHI-3 cells. We found that fisetin decreased total viable cells through G0/G1 phase arrest and induced sub-G1 phase (apoptosis). We have confirmed fisetin induced cell apoptosis by the formation of DNA fragmentation and induction of apoptotic cell death. Results indicated that fisetin induced intracellular Ca[Formula: see text] increase but decreased the ROS production and the levels of [Formula: see text]m in WEHI-3 cells. Fisetin increased the activities of caspase-3, -8 and -9. Cells were pre-treated with inhibitors of caspase-3, -8 and -9 and then treated with fisetin and results showed increased viable cell number when compared to fisetin treated only. Fisetin reduced expressions of cdc25a but increased p-p53, Chk1, p21 and p27 that may lead to G0/G1 phase arrest. Fisetin inhibited anti-apoptotic protein Bcl-2 and Bcl-xL and increased pro-apoptotic protein Bax and Bak. Furthermore, fisetin increased the protein expression of cytochrome c and AIF. Fisetin decreased cell number through G0/G1 phase arrest via the inhibition of cdc25c and induction of apoptosis through caspase-dependent and mitochondria-dependent pathways. Therefore, fisetin may be useful as a potential therapeutic agent for leukemia.

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.