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Cantharidin (CTD), a component of natural mylabris (Mylabris phalerata Pallas), has been shown to have biological activities and induce cell death in many human cancer cells. In the present study, we investigated the effect of CTD on cell migration and invasion of NCI-H460 human lung cancer cells. Cell viability was examined and results indicated that CTD decreased the percentage of viable cells in dose-dependent manners. CTD inhibited cell migration and invasion in dose-dependent manners. Gelatin zymography analysis was used to measure the activities of matrix metalloproteinases (MMP-2/-9) and the results indicated that CTD inhibited the enzymatic activities of MMP-2/-9 of NCI-H460 cells. Western blotting was used to examine the protein expression of NCI-H460 cells after incubation with CTD and the results showed that CTD decreased the expression of MMP-2/-9, focal adhesion kinase (FAK), Ras homolog gene family, member A (Rho A), phospho-protein kinase B (AKT) (Thr308)(p-AKT(308)), phospho-extracellular signal-regulated kinase1/2 (p-ERK1/2), phospho-p38 mitogen-activated protein (MAP) kinase (p-p38), phospho c-Jun N-terminal kinase 1/2 (p-JNK1/2), nuclear factor-κB (NF-κB) and urokinase plasminogen activator (UPA). Furthermore, confocal laser microscopy was used to confirm that CTD suppressed the expression of NF-κB p65, but did not significantly affect protein kinase C (PKC) translocation in NCI-H460 cells. Based on those observations, we suggest that CTD may be used as a novel anticancer metastasis agent for lung cancer in the future., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Cancer metastasis is the major cause of cancer patient death. Melanoma is a highly important metastasis in human cancer. Cantharidin (CTD), identified as an active component of natural mylabris (Mylabris phalerata Pallas), induces apoptosis in many human cancer cells. In the present study, we investigated the anti-metastasis effects of CTD in human melanoma cancer A375.S2 cells. Flow cytometry was used to measure CTD-induced cytotoxic effects in A375.S2 cells. Wound healing assay indicated that CTD suppressed the migration of A375.S2 cells in a dose-dependent manner. The Matrigel Transwell Assay was used for cell migration and invasion examination and the results showed that CTD inhibited both. Gelatin zymography was used to investigate the activities of MMP-2/9 and the results indicated that CTD inhibited the enzymatic activities of MMP-2/9 in A375.S2 cells. The protein expression of A375.S2 cells following incubation with CTD was examined by western blotting and the results showed that CTD decreased the expression of ERK1/2, PI3K, FAK, MMP-2, -9, COX-2, NF-κB p65, TIMP 1, TIMP 2, VEFG, uPA, Rho A, GRB2, ROCK-1 and Ras, but increased the expressions of p38, JNK, p-c-jun and PKC. Based on those observations, we suggest that CTD may be used as a novel anti-cancer metastasis agent of human melanoma cancer in the future., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

α-phellandrene (α-PA) is a cyclic monoterpene, present in natural plants such as Schinus molle L. α-PA promotes immune responses in mice in vivo. However, there is no available information on whether α-PA affects gene expression in leukemia cells. The present study determined effects of α-PA on expression levels of genes associated with DNA damage, cell cycle and apoptotic cell death in mouse leukemia WEHI-3 cells. WEHI-3 cells were treated with 10 μM α-PA for 24 h, cells were harvested and total RNA was extracted, and gene expression was analyzed by cDNA microarray. Results indicated that α-PA up-regulated 10 genes 4-fold, 13 by over 3-fold and 175 by over 2-fold; 21 genes were down-regulated by over 4-fold, 26 genes by over 3-fold and expression of 204 genes was altered by at leas 2-fold compared with the untreated control cells. DNA damage-associated genes such as DNA damage-inducer transcript 4 and DNA fragmentation factor were up-regulated by 4-fold and over 2-fold, respectively; cell-cycle check point genes such as cyclin G2 and cyclin-dependent kinases inhibitor 2D and IA (p21) were up-regulated by over 3-fold and over 2-fold, respectively; apoptosis-associated genes such as BCL2/adenovirus EIB interacting protein 3, XIAP-associated factor 1, BCL2 modifying factor, caspase-8 and FADD-like apoptosis regulator were over 2-fold up-regulated. Furthermore, DNA damage-associated gene TATA box binding protein was over 4-fold down-regulated, and D19Ertd652c (DNA segment) over 2-fold down-regulated; cell cycle-associated gene cyclin E2 was over 2-fold down-regulated; apoptosis associated gene growth arrest-specific 5 was over 9-fold down-regulated, Gm5426 (ATP synthase) was over 3-fold down-regulated, and death box polypeptide 33 was over 2-fold down-regulated. Based on these observations, α-PA altered gene expression in WEHI-3 cells in vitro., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

This study was conducted to evaluate the hepatoprotective effect of Agaricus blazei Murrill extract (ABM) against experimentally induced carbon tetrachloride (CCl(4)) toxicity in male BALB/c mice. The experiments included a normal group (no induction by CCl(4)), CCl(4-)induction group (with hepatotoxicity by CCl(4) and without treatment) and experimental groups with low dose (200 mg) or high dose (2,000 mg) of ABM extract (per kilogram mouse weight). All groups other than the normal group were treated with intraperitoneal injections of CCl(4) twice a week. Mice were tube-fed with experimental ABM extracts or double-distilled water, accordingly, on the remaining four days each week. The whole experimental protocol lasted 8 weeks; blood and liver samples were collected for biochemical and tissue histochemical analysis. Only administration of a high dose of ABM to treatment groups resulted in a significant abrogation of CCL(4)-induced increase of serum aspartate aminotransferase (AST) and alanine transaminase (ALT). Post-treatment with ABM also did not significantly reverse the alterations of glutathione peroxidase (GSHPx) and catalase. Both high- and low-dose ABM treatment reduced hepatic necrosis and fibrosis caused by CCl(4) in comparison with the CCl(4) control group in the histochemical analyses. Our results suggest that the ABM extract affects the levels of ALT and AST in mice.

Gallic acid (GA) induces apoptosis in different types of cancer cell lines. In this study, we investigate the apoptotic effects induced by GA in human promyelocytic leukemia HL-60 cells, and clarify the underlying mechanism. Our results showed that GA reduced the viability of HL-60 cells in a dose- and time-dependent manner. GA led to G(0)/G(1) phase arrest in HL-60 cells through promoting p21 and p27 and inhibiting the levels of cyclin D and cyclin E. GA caused DNA damage and fragmentation in HL-60 cells as assayed using DAPI staining and Comet assay. Flow cytometric analysis revealed that GA increased Ca(2+) levels and reduced the mitochondrial membrane potential (ΔΨ(m)) in HL-60 cells. Apoptotic protein expressions were determined by Western blotting. The results indicated that GA-mediated apoptosis of HL-60 cells mainly depended on mitochondrial pathway, by promoting the release of cytochrome c, apoptosis-inducing factor (AIF) and endonuclease G (Endo G) and by up-regulating the protein expression of Bcl-2-associated X protein (BAX), caspase-4, caspase-9 and caspase-3. In addition, GA also activated the death receptor-dependent pathway by enhancing the protein expressions of fatty acid synthase (FAS), FAS ligand (FASL), caspase-8 and BCL-2 interacting domain (BID). We determined the mRNA expression of the gene levels of these proteins by real-time PCR. The results showed that GA-mediated apoptosis of HL-60 cells mainly depended on up-regulation of the mRNA of caspase-8, caspase-9, caspase-3, AIF and Endo G. In conclusion, GA-induced apoptosis occurs through the death receptor and mitochondria-mediated pathways. The evaluation of GA as a potential therapeutic agent for treatment of leukemia seems warranted.

Etomidate is an important tool in the arsenal of the emergency physician, and it has been used in a variety of scenarios for both intubation and procedural sedation. In the present study, we investigated the cytotoxicity of etomidate including induction of apoptosis, and levels of protein and gene expressions associated with apoptotic cell death in murine leukemia RAW264.7 cells in vitro. Cytotoxic and apoptotic responses to etomidate of RAW264.7 cells, including cell morphological changes and cell viability were examined and measured by phase-contrast microscopy and flow cytometric assay, respectively. Results indicated that etomidate increased apoptotic cell morphological changes and reduced cell viability in RAW264.7 cells. 4',6-Diamidino-2-phenylindole (DAPI) staining also showed that etomidate induced the formation of apoptotic bodies, a characteristic of apoptosis. Results from Western blotting indicated that etomidate enhanced the levels of cytochrome c, apoptosis-inducing factor (AIF), endonuclease G (Endo G), caspase-9, caspase-3 active form and Bax proteins, but it inhibited the expression of Bcl-xl, leading to apoptosis. DNA microarray assay indicated that etomidate increased the expression of 17 genes (LOC676175; Gm14636; 2810021G02Rik; Iltifb; Olfr1167; Ttc30b; Olfr766; Gas5; Rgs1; LOC280487; V1rd4; Hist1h2bc; V1rj3; Gm10366; Olfr192; Gm10002 and Cspp1) and reduced the expression of 15 genes: (Gm10152; Gm5334; Olfr216; Lcn9; Gm10683; Gm5100; Tdgf1; Cypt2; Gm5595; 1700018F24Rik; Gm10417; Maml2; Olfr591; Trdn and Apol7c). In conclusion, etomidate induced cytotoxic and apoptotic effects the in murine leukemia RAW264.7 cells in vitro.

Enhanced fruit and vegetable consumption is closely related to reduced cancer incidence as shown in epidemiological studies. Ganoderma lucidum, one of the most well-known traditional Chinese medicines, has been demonstrated to have pharmacological activities and antitumor effects, in Asian populations. However, the promotion of immune responses in normal BALB/c mice is unclear. In the present study, we investigated the immune responses of BALB/c mice after treatment with G. lucidum extract in vivo. The results demonstrated that G. lucidum extract was able to promote the proliferation of splenocytes under Concanavalin A or lipopolysaccharide stimulation. Compared with the control group, phagocytosis of macrophage was significantly enhanced by intraperitoneal administration of G. lucidum extract at both 3 and 6 mg/kg. Compared with the control group, natural killer cell activity was significantly enhanced by intraperitoneal administration of G. lucidum extract (6 mg/kg). Results of cytometric bead array and flow cytometry indicated that the expressions of interleukin-6 and interferon-gamma also increased (p<0.001) by treatment with G. lucidum extract (3 and 6 mg/kg). In conclusion, the findings of this study implied that G. lucidum extract was able to effectively promote immune responses in BALB/c mice.

The ethanol extract of Dunaliella salina (EDS) on proliferation and apoptosis in the A549 human lung cancer cell line and their associated protein expressions were investigated. After 24 and 48 h treatment, MTT assay showed that 25 microg/ml of EDS significantly reduced A549 cell proliferation by 25.2% (p<0.05) and 48.3% (p<0.01), respectively. To explore its molecular mechanisms in regulating cell proliferation, we first showed that EDS markedly reduced A549 proliferation via inhibition of BrdU incorporation at 25 microg/ml by 65.8% (p<0.001). By cytometric analysis, EDS was found to induce apoptosis and cell cycle arrest in the G0/G1 phase. In the DNA gel electrophoresis assay, EDS (25, 50 and 100 microg/ml) induced significant apoptosis at 48 h. Annexin V/Propodium iodide double staining demonstrated that administration of EDS (25 microg/ml) in 12, 24 and 48 h induces apoptosis of 27.7%, 30.7%, and 38.7%. Western blotting assay demonstrated that EDS significantly increased the expression of cyclin-dependent kinase (CDK) inhibitors p53 and p21 and death-receptor proteins Fas and FasL. Bax expression was also elevated by treatment with EDS. Our data suggested that EDS could influence the antiproliferative effects and induce cell cycle G0/G1 arrest and apoptosis of A549 lung cancer cells.

Curcumin (1, 7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5- dione), a natural polyphenol product of the plant Curcuma longa, exhibited potent inhibitory activities against proliferation, induced cell cycle arrest and exhibited the induction of apoptosis in several tumor cell lines. In our previous studies, we have shown that curcumin induced cell cycle arrest and apoptosis on human leukemia HL-60 and mouse leukemia WEHI-3 cells; there are no reports regarding whether or not it affects leukemia cells in vivo. In the present study, we investigated the effects of curcumin on WEHI-3 in BALB/c mice and the results indicated that curcumin reduces the percentage of Mac-3 marker, which is the precursor of macrophage. Curcumin induced significant effects on the population of B cells from murine leukemia in vivo. We also investigated the weights of spleen and liver from murine leukemia and the results showed that curcumin reduced the weight of the liver and spleen. From the pathological examinations, the effects of curcumin on the liver and spleen from mice after being injected with WEHI-3 cells were apparent. Both organs were enlarged. In conclusion, curcumin affect WEHI-3 cells in vivo.