Jo Hua Chiang

Danthron, an anthraquinone derivative, induces DNA damage and caspase cascades-mediated apoptosis in SNU-1 human gastric cancer cells through mitochondrial permeability transition pores and Bax-triggered pathways

Anthraquinones have been shown to induce apoptosis in different types of tumor cells, but the mechanisms of danthron-induced cytotoxicity and apoptosis in human gastric cancer cells have not been adequately explored. This study investigated the roles of caspase cascades, ROS, DNA damage, mitochondrial disruption, and Bax and Bcl-2 proteins in danthron-induced apoptosis of SNU-1 human gastric cancer cells, a commonly used cell culture system for in vitro studies. Cells were incubated with different concentrations of danthron in a time- and/or dose-dependent manner. Cell morphological changes (shrinkage and rounding) were examined by a phase-contrast microscope, whereas cell viability and apoptotic populations were determined by flow cytometric analysis using propidium iodide (PI) and annexin V-FITC staining. The fluorescent DAPI nucleic acid stain and Comet assay were applied to detect danthron-induced chromatin condensation (an apoptotic characteristic) and DNA damage. Increasing the levels of caspase-3, -8, and -9 activities was involved in danthron-induced apoptosis, and they could be attenuated by inhibitors of specific caspases, indicating that danthron triggered the caspase-dependent apoptotic pathway. Further studies with flow cytometric analyses indicated that cellular levels of ROS, cytosolic Ca(2+), and mitochondrial permeability transition (MPT) pore opening were increased, but the level of mitochondrial membrane potential (ΔΨ(m)) was decreased. Also, the ratio of Bax/Bcl-2 levels and other proapoptotic proteins associated with modulating the ΔΨ(m) were up-regulated. Apoptotic signaling was also stimulated after exposure to danthron and determined by Western blotting and real-time PCR analyses. In summary, it is suggested that danthron-induced apoptotic cell death was involved in mitochondrial depolarization, which led to release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G) and caused the activation of caspase-9 and -3 in SNU-1 human gastric cancer cells.

Cantharidin induces G2/M phase arrest and apoptosis in human colorectal cancer colo 205 cells through inhibition of CDK1 activity and caspase-dependent signaling pathways

Cantharidin (CTD) is a traditional Chinese medicine and an effective component isolated from blister beetle, and it has been demonstrated to have anticancer, antibiotic, antivirus activities and immune-regulated functions. It has been reported that CTD induces cell cycle arrest and apoptosis in many cancer cell types. However, there are no reports showing that CTD would induce cell cycle arrest and apoptosis in human colorectal cancer colo 205 cells. In this study, we studied colo 205 cells which were treated with CTD and demonstrated its molecular mechanisms in apoptosis. CTD induced growth inhibition, G2/M phase arrest and apoptosis in colo 205 cells. The IC50 is 20.53 µM in CTD-treated colo 205 cells. DAPI/TUNEL double staining and Annexin V assays were used to confirm the apoptotic cell death in colo 205 cells after CTD exposure. CTD caused G2/M arrest, down-regulated CDK1 activity, decreased Cyclin A, Cyclin B, CDK1 and increased CHK1 and p21 protein levels. Colorimetric assays also indicated that CTD triggered activities of casapse-8, -9 and -3 in colo 205 cells. Moreover, CTD increased ROS production and decreased the level of mitochondrial membrane potential (ΔΨm) in colo 205 cells. Consequently, CTD-induced growth inhibition was significantly attenuated by N-acetylcysteine (NAC, a scavenger). CTD stimulated the protein levels of Fas/CD95, the caspase-3 active form, cytochrome c and Bax, but suppressed the protein levels of pro-caspase-8, pro-caspase-9 and Bcl-2, determined by Western blot analysis. Based on our observations, we suggest that CTD is able to induce G2/M phase arrest and apoptosis in colo 205 cells through inhibition of CDK1 activity and caspase-dependent signaling pathways.

Antitumor effects of emodin on LS1034 human colon cancer cells in vitro and in vivo: Roles of apoptotic cell death and LS1034 tumor xenografts model

Emodin, an active natural anthraquinone derivative, is found in the roots and rhizomes of numerous Chinese medicinal herbs and exhibits anticancer effects on many types of human cancer cell lines. The aim of this study investigated that emodin induced apoptosis of human colon cancer cells (LS1034) in vitro and inhibited tumor nude mice xenografts bearing LS1034 in vivo. In in vitro study, emodin induced cell morphological changes, decreased the percentage of viability, induced G2/M phase arrest and increased ROS and Ca(2+) productions as well as loss of mitochondrial membrane potential (ΔΨ(m)) in LS1034 cells. Emodin-triggered apoptosis was also confirmed by DAPI staining and these effects are concentration-dependent. Western blot analysis indicated that the protein levels of cytochrome c, caspase-9 and the ratio of Bax/Bcl-2 were increased in LS1034 cells after emodin exposure. Emodin induced the productions of ROS and Ca(2+) release, and altered anti- and pro-apoptotic proteins, leading to mitochondrial dysfunction and activations of caspase-9 and caspase-3 for causing cell apoptosis. In in vivo study, emodin effectively suppressed tumor growth in tumor nude mice xenografts bearing LS1034. Overall, the potent in vitro and in vivo antitumor activities of emodin suggest that it might be developed for treatment of colon cancer in the future.

Rutin inhibits the proliferation of murine leukemia WEHI-3 cells in vivo and promotes immune response in vivo.

Flavonoids are polyphenolic compounds found in various foods of plants. Rutin, one of the flavonoids, had been showed induced apoptosis in cancer cells. There is no available information to address rutin affects murine leukemia cells in vivo. In the present study, we are focused on the in vivo effects of rutin on leukemia WEHI-3 cells. The effects of rutin on WEHI-3 in BALB/c mice in vivo were also examined and the results indicated that rutin decreased the percentage of Mac-3 marker, indicating that the differentiation of the precursor of macrophage and T cells was inhibited. The weights of liver and spleen were decreased from rutin-treated groups compared to the control groups and the results indicated that rutin decreased the weight of these organs. One of the major characteristic of WEHI-3 leukemia is the enlarged spleen in murine after i.p. injection of WEHI-3 cells. After the pathological examination, the function of rutin was observed in the liver and spleen in the mice previously injected with WEHI-3 cells. Rutin promoted the activity of macrophage phagocytosis in cells which isolated from peritoneal (i.p.). Taken together, rutin can affect WEHI-3 cells in vivo.

Berberine inhibits human tongue squamous carcinoma cancer tumor growth in a murine xenograft model.

Our primary studies showed that berberine induced apoptosis in human tongue cancer SCC-4 cells in vitro. But there is no report to show berberine inhibited SCC-4 cancer cells in vivo on a murine xenograft animal model. SCC-4 tumor cells were implanted into mice and groups of mice were treated with vehicle, berberine (10mg/kg of body weight) and doxorubicin (4mg/kg of body weight). The tested agents were injected once per four days intraperitoneally (i.p.), with treatment starting 4 weeks prior to cells inoculation. Treatment with 4mg/kg of doxorubicin or with 10mg/kg of berberine resulted in a reduction in tumor incidence. Tumor size in xenograft mice treated with 10mg/kg berberine was significantly smaller than that in the control group. Our findings indicated that berbeirne inhibits tumor growth in a xenograft animal model. Therefore, berberine may represent a tongue cancer preventive agent and can be used in clinic.

Novel quinazolinone MJ-29 triggers endoplasmic reticulum stress and intrinsic apoptosis in murine leukemia WEHI-3 cells and inhibits Leukemic mice

The present study was to explore the biological responses of the newly compound, MJ-29 in murine myelomonocytic leukemia WEHI-3 cells in vitro and in vivo fates. We focused on the in vitro effects of MJ-29 on ER stress and mitochondria-dependent apoptotic death in WEHI-3 cells, and to hypothesize that MJ-29 might fully impair the orthotopic leukemic mice. Our results indicated that a concentration-dependent decrease of cell viability was shown in MJ-29-treated cells. DNA content was examined utilizing flow cytometry, whereas apoptotic populations were determined using annexin V/PI, DAPI staining and TUNEL assay. Increasing vital factors of mitochondrial dysfunction by MJ-29 were further investigated. Thus, MJ-29-provaked apoptosis of WEHI-3 cells is mediated through the intrinsic pathway. Importantly, intracellular Ca2+ release and ER stress-associated signaling also contributed to MJ-29-triggered cell apoptosis. We found that MJ-29 stimulated the protein levels of calpain 1, CHOP and p-eIF2α pathways in WEHI-3 cells. In in vivo experiments, intraperitoneal administration of MJ-29 significantly improved the total survival rate, enhanced body weight and attenuated enlarged spleen and liver tissues in leukemic mice. The infiltration of immature myeloblastic cells into splenic red pulp was reduced in MJ-29-treated leukemic mice. Moreover, MJ-29 increased the differentiations of T and B cells but decreased that of macrophages and monocytes. Additionally, MJ-29-stimulated immune responses might be involved in anti-leukemic activity in vivo. Based on these observations, MJ-29 suppresses WEHI-3 cells in vitro and in vivo, and it is proposed that this potent and selective agent could be a new chemotherapeutic candidate for anti-leukemia in the future.

Bufalin increases sensitivity to AKT/mTOR-induced autophagic cell death in SK-HEP-1 human hepatocellular carcinoma cells

Bufalin is the major component of Chan-Su (a traditional Chinese medicine, TCM) extracts from the venom of Bufo bufo gargarizan. In the present study, we investigated the pharmacological mechanisms of cell cycle arrest and autophagic cell death induced by bufalin in SK-HEP-1 human hepatocellular carcinoma cells in vitro. Bufalin inhibited cell survival by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. In addition, bufalin induced G2/M phase arrest by reducing CDK1 activity. Bufalin triggered DNA fragmentation and apoptotic cell death in SK-HEP-1 cells by DNA gel electrophoresis, TUNEL and caspase-3 activity assay, while bufalin induced autophagic cell death by double-membrane vacuoles (transmission electron microscopy, TEM), acidic vesicular organelles (acridine orange staining) and cleavage of microtubule-associated protein 1 light chain 3 (LC3). Protein expression levels of cyclin A and B, CDK1, phospho-CDK1 (Thr161), Cdc25c, phospho-Cdc25c (Ser198), phospho-AKT (Thr308), phospho-AKT (Ser473), phospho‑mTOR (Ser2481) were downregulated. In contrast, protein expression levels of the Chk1, Wee1, LC3-II, Beclin-1, Atg 5, Atg 7 and Atg 12 were upregulated in SK-HEP-1 cells after bufalin treatment. Inhibition of autophagy by 3-methyladenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) or bafilomycin A1 (an inhibitor of the vacuolar proton pump of lysosomes and endosomes) reduced the effect of bufalin on cell viability and enhanced the effect of bufalin on apoptosis. In conclusion, bufalin triggered autophagic cell death and G2/M phase arrest through the AKT/mTOR signaling pathway in SK-HEP-1 cells. Our findings showed that bufalin may be potentially efficacious in the treatment of human hepatocellular carcinoma.

Benzyl isothiocyanate inhibits murine WEHI-3 leukemia cells in vitro and promotes phagocytosis in BALB/c mice in vivo

Many evidences have shown that dietary intake of cruciferous vegetables could protect against the risk of various types of malignancies. Benzyl isothiocyanate (BITC), one of the compounds from cruciferous vegetables, had shown induced cell cycle arrest and apoptosis in cancer cells. However, there is no available information to address that BITC affects murine leukemia cells in vitro and in vivo. Here, we investigated in vitro effects of BITC on murine leukemia WEHI-3 cells. BITC decreased the percentage of viable cells via G0/G1 arrest and apoptosis in WEHI-3 cells. BITC induced apoptosis through the dysfunction of mitochondria (decreased the levels of mitochondria membrane potential) and activation of caspase-3. Then we investigated in vivo effects of BITC on murine leukemia WEHI-3 cells and the results indicated that BITC decreased the weights of liver and spleen and it also decreased the percentage of CD11b and Mac-3 markers, indicating that the differentiation of the precursor of macrophage and B cells was inhibited. BITC promoted the activity of macrophage phagocytosis in cells which are isolated from PBMC and peritoneal (i.p.). Taken together, BITC can affect WEHI-3 cells in vitro and in vivo.

Curcumin inhibits human lung large cell carcinoma cancer tumour growth in a murine xenograft model

Curcumin can decrease viable cells through the induction of apoptosis in human lung cancer NCI‐H460 cells in vitro. However, there are no reports that curcumin can inhibit cancer cells in vivo. In this study, NCI‐H460 lung tumour cells were implanted directly into nude mice and divided randomly into four groups to be treated with vehicle, curcumin (30 mg/kg of body weight), curcumin (45 mg/kg of body weight) and doxorubicin (8 mg/kg of body weight). Each agent was injected once every 4 days intraperitoneally (i.p.), with treatment starting 4 weeks after inoculation with the NCI‐H460 cells. Treatment with 30 mg/kg and 45 mg/kg of curcumin or with 8 mg/kg of doxorubicin resulted in a reduction in tumour incidence, size and weight compared with the control group. The findings indicate that curcumin can inhibit tumour growth in a NCI‐H460 xenograft animal model in vivo. Copyright

Quercetin Inhibited Murine Leukemia WEHI-3 Cells In Vivo and Promoted Immune Response

Enhanced flavonoid consumption is closely related with a reduced cancer incidence as shown in epidemiological studies. Quercetin (3,5,7,3′,4′‐pentahydroxylflavone) is one of the active components of flavonoids which exist in natural plants, particularly in onions and fruits. It was reported that quercetin induced apoptosis in human cancer cell lines, including human leukemia HL‐60 cells, but there is no available information as to its effects on leukemia cells in vivo. The purpose of the present studies was to focus on the in vivo effects of quercetin on leukemia WEHI‐3 cells. The effects of quercetin on WEHI‐3 cells injected into BALB/c mice were examined. Quercetin decreased the percentage of Mac‐3 and CD11b markers, suggesting that the differentiation of the precursors of macrophages and T cells was inhibited. There was no effect on CD3 levels but increased CD19 levels. Quercetin decreased the weight of the spleen and liver compared with the olive oil treated animals. Quercetin stimulated macrophage phagocytosis of cells isolated from peritoneum. Quercetin also promoted natural killer cell activity. Based on pathological examination, an effect of quercetin was observed in the spleen of mice previously injected with WEHI‐3 cells. Apparently, quercetin affects WEHI‐3 cells in vivo. Copyright