Yi Ping Huang

Benzyl isothiocyanate (BITC) induces G 2/M phase arrest and apoptosis in human melanoma A375.S2 cells through reactive oxygen species (ROS) and both mitochondria-dependent and death receptor-mediated multiple signaling pathways

Benzyl isothiocyanates (BITC), a member of the isothiocyanate (ITC) family, inhibits cell growth and induces apoptosis in many types of human cancer cell lines. The present study investigated mechanisms underlying BITC-induced apoptosis in A375.S2 human melanoma cancer cells. To observe cell morphological changes and viability, flow cytometric assays, cell counting, and a contrast-phase microscopic examination were carried out in A375.S2 cells after BITC treatment. Cell cycle distribution and apoptosis were assessed with the analysis of cell cycle by flow cytometric assays, DAPI staining, propidium iodide (PI), and annexin V staining. Apoptosis-associated factors such as reactive oxygen species (ROS) formation, loss of mitochondrial membrane potential (ΔΨ(m)), intracellular Ca(2+) release, and caspase-3 activity were evaluated by flow cytometric assays. Abundance of cell cycle and apoptosis associated proteins was determined by Western blotting. AIF and Endo G expression was examined by confocal laser microscope. Results indicated that (1) BITC significantly reduced cell number and induced cell morphological changes in a dose-dependent manner in A375.S2 cells; (2) BITC induced arrest in cell cycle progression at G(2)/M phase through cyclin A, CDK1, CDC25C/Wee1-mediated pathways; (3) BITC induced apoptosis and increased sub-G(1) population; and (4) BITC promoted the production of ROS and Ca(2+) and loss of ΔΨ(m) and caspase-3 activity. Furthermore, BITC induced the down-regulation of Bcl-2 expression and induced up-regulation of Bax in A375.S2 cells. Moreover, BITC-induced cell death was decreased after pretreatment with N-acetyl-l-cysteine (NAC, a ROS scavenger) in A375.S2 cells. In conclusion, the results showed that BITC promoted the induction of G(2)/M phase arrest and apoptosis in A375.S2 human melanoma cells through ER stress- and mitochondria-dependent and death receptor-mediated multiple signaling pathways. These data suggest that BITC has potential as an agent for the treatment of melanoma.

Ellagic acid induces apoptosis in TSGH8301 human bladder cancer cells through the endoplasmic reticulum stress- and mitochondria-dependent signaling pathways.

To investigate the effects of ellagic acid on the growth inhibition of TSGH8301 human bladder cancer cells in vitro, cells were incubated with various doses of ellagic acid for different time periods. The phase‐contrast microscope was used for examining and photographing the morphological changes in TSGH8301 cells. Flow cytometric assay was used to measure the percentage of viable cells, cell cycle distribution, apoptotic cells, ROS, mitochondrial membrane potential (ΔΨm), Ca2+, caspase‐9 and ‐3 activities in TSGH8301 cells after exposure to ellagic acid. Western blotting was used to examine the changes of cell cycle and apoptosis associated proteins levels. Results indicated that ellagic acid induced morphological changes, decreased the percentage of viable cells through the induction of G0/G1 phase arrest and apoptosis, and also showed that ellagic acid promoted ROS and Ca2+ productions and decreased the level of ΔΨm and promoted activities of caspase‐9 and ‐3. The induction of apoptosis also confirmed by annexin V staining, comet assay, DAPI staining and DNA gel electrophoresis showed that ellagic acid induced apoptosis and DNA damage in TSGH8301 cells. Western blotting assay showed that ellagic acid promoted p21, p53 and decreased CDC2 and WEE1 for leading to G0/G1 phase arrest and promoting BAD expression, AIF and Endo G, cytochrome c, caspase‐9 and ‐3 for leading to apoptosis in TSGH8301 cells. On the basis of these observations, we suggest that ellagic acid induced cytotoxic effects for causing a decrease in the percentage of viable cells via G0/G1 phase arrest and induction of apoptosis in TSGH8301 cells.

Proteomics-based identification of haptoglobin as a novel plasma biomarker in oral squamous cell carcinoma

BACKGROUND Identification of tumor biomarkers to assist early diagnosis and monitoring of disease progression may potentially decrease the mortality and morbidity associated with oral cancer. METHODS A mouse model with oral squamous cell carcinoma (OSCC) induced by 4-nitroquinoline 1-oxide (4-NQO)/arecoline in drinking water was established to discover stage-associated biomarkers. A proteomics approach, immunoblot and immunohistochemical analysis were used to validate the expressed biomarkers in mice with OSCC. Human plasma samples were also collected and candidate biomarkers were evaluated using enzyme-linked immunosorbent assay. RESULTS Proteomic profiling of mouse plasma samples indicated that haptoglobin and apolipoprotein A1 precursor were up-regulated in the mice with OSCC. Immunoblotting of plasma samples and immunohistochemical analysis of oral tissues showed a significantly higher level of haptoglobin in the OSCC mice than in the control mice. The expression of haptoglobin in human plasma samples from 52 patients with OSCC indicated a strong correlation between the increasing levels of haptoglobin and the clinical stages of OSCC (P<0.01). CONCLUSIONS These results suggest that haptoglobin has a great potential as a sensitive plasma biomarker for early detection of patients with OSCC.

Cantharidin induces apoptosis of H460 human lung cancer cells through mitochondria-dependent pathways

Lung cancer is one of the leading causes of death in cancer-related diseases. Cantharidin (CTD) is one of the components of natural mylabris (Mylabris phalerata Pallas). Numerous studies have shown that CTD induced cytotoxic effects on cancer cells. However, there is no report to demonstrate that CTD induced apoptosis in human lung cancer cells. Herein, we investigated the effect of CTD on the cell death via the induction of apoptosis in H460 human lung cancer cells. Flow cytometry assay was used for examining the percentage of cell viability, sub-G1 phase of the cell cycle, reactive oxygen species (ROS) and Ca²⁺ productions and the levels of mitochondrial membrane potential (∆Ψm). Annexin V/PI staining and DNA gel electrophoresis were also used for examining cell apoptosis. Western blot analysis was used to examine the changes of apoptosis associated protein expression and confocal microscopy for examining the translocation apoptosis associated protein. Results indicated that CTD significantly induced cell morphological changes and decreased the percentage of viable H460 cells. CTD induced apoptosis based on the occurrence of sub-G1 phase and DNA fragmentation. We found that CTD increased gene expression (mRNA) of caspase-3 and -8. Moreover, CTD increased ROS and Ca2+ production and decreased the levels of ∆Ψm. Western blot analysis results showed that CTD increased the expression of cleavage caspase-3 and -8, cytochrome c, Bax and AIF but inhibited the levels of Bcl-xL. CTD promoted ER stress associated protein expression such as GRP78, IRE1α, IRE1β, ATF6α and caspase-4 and it also promoted the expression of calpain 2 and XBP-1, but inhibited calpain 1 that is associated with apoptosis pathways. Based on those observations, we suggest that CTD may be used as a novel anticancer agent for the treatment of lung cancer in the future.

Suppressions of Migration and Invasion by Cantharidin in TSGH-8301 Human Bladder Carcinoma Cells through the Inhibitions of Matrix Metalloproteinase-2/-9 Signaling

Cancer metastasis becomes an initial cause of cancer death in human population. In many cancers, it has been shown that the high levels of matrix metalloproteinase (MMP)-2 and/or MMP-9 are associated with the invasive phenotypes of cancer cells. In this study, we investigated the effects of cantharidin, a derivative of blister beetles which is one of the traditional Chinese medicines, on the adhesion, migration, and invasion of human bladder cancer TSGH-8301 cells. Cantharidin effectively suppressed TSGH-8301 cell adhesion, migration, and invasion in a concentration-dependent manner. Results from Western blotting, RT-PCR, and gelatin zymography assays indicated that cantharidin blocked the protein levels, gene expression (mRNA), and activities of MMP-2 and -9 in TSGH-8301 cells. Cantharidin also significantly suppressed the protein expressions of p-p38 and p-JNK1/2 in TSGH-8301 cells. Taken together, cantharidin was suggested to present antimetastatic potential via suppressing the levels of MMP-2 and MMP-9 expression that might be mediated by targeting the p38 and JNK1/2 MAPKs pathway in TSGH-8301 human bladder cancer cells.

Norcantharidin triggers cell death and DNA damage through S-phase arrest and ROS-modulated apoptotic pathways in TSGH 8301 human urinary bladder carcinoma cells

Norcantharidin (NCTD) is one of the ingredients of blister beetles which have been used in Chinese medicine for a long time. The purpose of this study was to investigate the inhibitory effects of NCTD on TSGH 8301 human bladder cancer cells in vitro and the mechanisms through which it exerts its anticancer action. Cell morphological analysis was performed using a phase-contrast microscope. The percentage of viable cells, cell cycle distribution, sub-G1 phase (apoptosis), reactive oxygen species (ROS) production and the levels of mitochondrial membrane potential (∆Ψ(m)) were analyzed by flow cytometry. DNA condensation and damage were determined by DAPI staining and comet assay. Apoptosis-associated protein level changes in TSGH 8301 cells following exposure to NCTD were examined, measured and determined by western blotting. Analysis of protein translocation was conducted by immunostaining and confocal laser microscopy. The results indicated that NCTD promoted cytotoxic effects, including the induction of cell morphological changes and the decrease in the percentage of viability, the induction of S-phase arrest as well as sub-G1 phase (apoptosis) in TSGH 8301 cells. The activities of caspase-3 and -9 were upregulated following NCTD treatment. Western blotting indicated that NCTD upregulated Fas, FasL, Bax, Bid, cytochrome c, caspase-3, -8 and -9 that led to the induction of apoptosis through the Fas extrinsic pathway. Furthermore, NCTD induced AIF and Endo G that were released from mitochondria to induce apoptosis through the mitochondrial-independent pathway. NCTD upregulated ROS production, downregulated ∆Ψ(m) and ERK, JNK, p38 protein kinases in TSGH 8301 cells. These findings suggest that NCTD triggers apoptosis in TSGH 8301 human bladder cancer cells via the Fas receptor, activation of the caspse-8, -9 and -3, mitochondrial-dependent and -independent pathways. NCTD may be useful for developing new therapeutic regimens for the treatment of bladder cancer.

The crude extract of Corni Fructus inhibits the migration and invasion of U‐2 OS human osteosarcoma cells through the inhibition of matrix metalloproteinase‐2/‐9 by MAPK signaling

Osteosarcoma is the most common primary malignancy of the bone cancers. In the Chinese population, the crude extract of Corni Fructus (CECF) has been used as Traditional Chinese medicine to treat several different diseases for hundreds of years. In the present study, effects of CECF on inhibition of migration and invasion in U‐2 OS human osteosarcoma cells were examined. CECF significantly inhibited migration and invasion of U‐2 OS human osteosarcoma cells. We also found that CECF inhibited activities of matrix metalloproteinases‐2 (MMP‐2) and matrix metalloproteinases‐9 (MMP‐9). CECF decreased protein levels of FAK, PKC, SOS1, MKK7, MEKK3, GRB2, NF‐κB p65, COX‐2, HIF‐1α, PI3K, Rho A, ROCK‐1, IRE‐1α, p‐JNK1/2, p‐ERK1/2, p‐p38, Ras, p‐PERK, MMP‐2, MMP‐9, and VEGF in U‐2 OS cells. Results of this study indicate that CECF may have potential as a novel anticancer agent for the treatment of osteosarcoma by inhibiting migration and invasion of cancer cells

Induction of apoptosis by curcumin in murine myelomonocytic leukemia WEHI‐3 cells is mediated via endoplasmic reticulum stress and mitochondria‐dependent pathways

Curcumin, derived from the food flavoring spice turmeric (Curcuma longa), has been shown to exhibit anticancer activities and induce apoptosis in many types of cancer cell lines. In our previous study, curcumin was able to inhibit murine myelomonocytic leukemia WEHI‐3 cells in vivo. However, there is no report addressing the cytotoxic responses and the mechanisms underlying curcumin‐induced apoptotic cell death in WEHI‐3 cells. Therefore, we hypothesized that that curcumin affected WEHI‐3 cells and triggered cell death through apoptotic signaling pathways. The effects of curcumin on WEHI‐3 cells were investigated by using flow cytometric analysis, comet assay, confocal laser microscopy and Western blotting. In this study, we found that curcumin induced apoptosis in WEHI‐3 cells in a dose‐dependent (5–20 μM) manner. Interestingly, curcumin enhanced the level of the antiapoptotic protein Bcl‐2 which might show that curcumin‐induced apoptosis is done through the ER stress signaling pathways based on the increase of CIEBP homologous protein (CHOP), activating transcription factor 6 (ATF‐6), inositol‐requiring enzyme 1 (IRE1), and caspase‐12 in WEHI‐3 cells. Moreover, curcumin increased the reactive oxygen species (ROS) production and cytosolic Ca2+ release, and induced DNA damage, but decreased the level of mitochondrial membrane potential (ΔΨm) in WEHI‐3 cells. In conclusion, curcumin‐induced apoptosis occurs through the ROS‐affected, mitochondria‐mediated and ER stress‐dependent pathways. The evaluation of curcumin as a potential therapeutic agent for treatment of leukemia seems warranted.

Sulforaphane Induces Cell Death Through G2/M Phase Arrest and Triggers Apoptosis in HCT 116 Human Colon Cancer Cells

Sulforaphane (SFN), an isothiocyanate, exists exclusively in cruciferous vegetables, and has been shown to possess potent antitumor and chemopreventive activity. However, there is no available information that shows SFN affecting human colon cancer HCT 116 cells. In the present study, we found that SFN induced cell morphological changes, which were photographed by contrast-phase microscopy, and decreased viability. SFN also induced G2/M phase arrest and cell apoptosis in HCT 116 cells, which were measured with flow cytometric assays. Western blotting indicated that SFN increased Cyclin A, cdk 2, Cyclin B and WEE1, but decreased Cdc 25C, cdk1 protein expressions that led to G2/M phase arrest. Apoptotic cell death was also confirmed by Annexin V/PI and DAPI staining and DNA gel electrophoresis in HCT 116 cells after exposure to SFN. The flow cytometric assay also showed that SFN induced the generation of reactive oxygen species (ROS) and Ca[Formula: see text] and decreased mitochondria membrane potential and increased caspase-8, -9 and -3 activities in HCT 116 cell. Western blotting also showed that SFN induced the release of cytochrome c, and AIF, which was confirmed by confocal microscopy examination. SFN induced ER stress-associated protein expression. Based on those observations, we suggest that SFN may be used as a novel anticancer agent for the treatment of human colon cancer in the future.

Bufalin induces apoptosis in vitro and has Antitumor activity against human lung cancer xenografts in vivo

Bufalin has been shown to be effective against a variety of cancer cells, but its role in lung cancer has never been studied in an animal model. In this study, we evaluated bufalin effects in a human lung cancer cell line NCI‐H460 both in vitro and in vivo. Bufalin caused significant cytotoxicity in NCI‐H460 cells at a concentration as low as 1 μM. DNA condensation was observed in bufalin‐treated cells in a dose‐dependent manner. Mitochondrial membrane potential (ΔΨm) was reduced and reactive oxygen species (ROS) were increased in bufalin‐treated NCI‐H460 cells. Levels of several proapoptotic proteins such as Fas, Fas‐ligand, cytochrome c, apoptosis protease activating factor‐1, endonuclease G, caspase‐3 and caspase‐9 were increased after bufalin treatment. At the same time, anti‐apoptotic B‐cell lymphoma 2 protein levels were reduced. Bufalin decreased glucose regulated protein‐78 gene expression but increased growth arrest‐ and DNA damage‐inducible 153 gene expression. Bufalin injected intraperitoneally in a dose‐dependent manner reduced tumor size in BALB/C nu/nu mice implanted with NCI‐H460 cells. Bufalin injection did not produce significant drug‐related toxicity in experimental animals except at a high dose (0.4 mg kg−1). In conclusion, low concentrations of bufalin can induce apoptosis in the human lung cancer cell line NCI‐H460 in vitro. Bufalin also reduced tumor size in mice injected with NCI‐H460 cells without significant drug‐related toxicity. These results indicate that bufalin may have potential to be developed as an agent for treating human non‐small cell lung cancer.