Host immune defense peptide LL-37 activates caspase-independent apoptosis and suppresses colon cancer.

Cathelicidins are a family of bacteriocidal polypeptides secreted by macrophages and polymorphonuclear leukocytes (PMN). LL-37, the only human cathelicidin, has been implicated in tumorigenesis, but there has been limited investigation of its expression and function in cancer. Here, we report that LL-37 activates a p53-mediated, caspase-independent apoptotic cascade that contributes to suppression of colon cancer. LL-37 was expressed strongly in normal colon mucosa but downregulated in colon cancer tissues, where in both settings its expression correlated with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells. Exposure of colon cancer cells to LL-37 induced phosphatidylserine externalization and DNA fragmentation in a manner independent of caspase activation. Apoptogenic function was mediated by nuclear translocation of the proapoptotic factors, apoptosis-inducing factor (AIF) and endonuclease G (EndoG), through p53-dependent upregulation of Bax and Bak and downregulation of Bcl-2 via a pertussis toxin-sensitive G-protein-coupled receptor (GPCR) pathway. Correspondingly, colonic mucosa of cathelicidin-deficient mice exhibited reduced expression of p53, Bax, and Bak and increased expression of Bcl-2 together with a lower basal level of apoptosis. Cathelicidin-deficient mice exhibited an increased susceptibility to azoxymethane-induced colon tumorigenesis, establishing pathophysiologic relevance in colon cancer. Collectively, our findings show that LL-37 activates a GPCR-p53-Bax/Bak/Bcl-2 signaling cascade that triggers AIF/EndoG-mediated apoptosis in colon cancer cells.

A rice bran polyphenol, cycloartenyl ferulate, elicits apoptosis in human colorectal adenocarcinoma SW480 and sensitizes metastatic SW620 cells to TRAIL-induced apoptosis.

High intake of whole grain food has been suggested as an important factor for reducing the risk of colon cancer, owing to the abundance of indigestible fibers. Our findings demonstrated that, among various rice bran phenolic compounds tested, cycloartenyl ferulate (CF) showed the most prominent in vitro growth inhibition on human colorectal adenocarcinoma SW480, but had low toxicity on normal colon CCD-18-Co cells. The anticancer activity of CF was further illustrated by its ability to induce significant regression of SW480 xenograft in nude mice. CF elevated the death receptors DR4 and DR5 and triggered both the death receptor and the mitochondrial apoptosis pathways. Depletion of anti-apoptotic Bcl-2 and up-regulation of pro-apoptotic Bak were observed, accompanied by dissipation of the mitochondrial membrane potential and release of cyto c and SMAC/DIABLO from mitochondria into the cytosol. Bid was found to be cleaved by caspase-8, so that the death receptor pathway might be exaggerated by the mitochondrial pathway. Strikingly, we showed for the first time that CF also sensitized the metastatic and resistant colon cancer SW620 to TRAIL-induced apoptosis and the mechanisms involved at least enhanced activation of caspase-8 and -3. This study provides a clear evidence that the health-beneficial properties of whole grain consumption are not only limited by the presence of dietary fibers but also other molecules that can either act as a chemopreventive agent to directly induce tumor regression or as a sensitizer to enhance TRAIL-induced apoptosis in metastatic cancer cells.

Chemical composition and antiproliferative activity of essential oil from the leaves of a medicinal herb, Schefflera heptaphylla.

Schefflera heptaphylla (L.) Frodin is a medicinal herb widely used as a main ingredient of the popular health tea formulation against infections in Southern China. Twenty-seven volatile compounds were identified by GC-MS analysis from the essential oil obtained from the leaves of S. heptaphylla, and 17 of them belonged to monoterpenes or sesquiterpenes. The main volatile constituent in S. heptaphylla was found to be a monoterpene, beta-pinene, comprising about 22% of the total volatile components. The essential oil showed significant antiproliferative activity against three cancer cell lines, MCF-7, A375 and HepG2 cells, with IC50 values of 7.3 microg/mL, 7.5 microg/mL and 6.9 microg/mL, respectively. The result of the cytotoxicity assay indicates that (-)-beta-pinene and (+)-beta-pinene (commercially available from Sigma) also possessed antiproliferative activity against the cancer cells MCF-7, A375 and HepG2 with IC50 values ranging from 147.1 to 264.7 microm.

Antiviral and anti-proliferative glycoproteins from the rhizome of Smilax glabra Roxb (Liliaceae).

The glycoproteins possessing antiviral and anti-proliferative activities were isolated from the Chinese medicinal herb Smilax glabra (known as tufuling), by extraction with 0.2 M NaCl, ammonium sulfate precipitation, fetuin-agarose affinity chromatography and gel filtration. The molecular mass of the fetuin-binding glycoprotein (designated SGPF2) was estimated to be about 58 kDa, with a major protein subunit of 26 kDa. The non-fetuin binding glycoproteins (in the unadsorbed fraction) were further separated into 5 different subfractions (SGPF1a-SGPF1e) with anion-exchange chromatography, all of which also contained the major band at 26 kDa. All the isolated proteins of 26 kDa had similar N-terminal amino acid sequences, implying that they were probably the isoforms originated putatively from a multigene family with different binding affinity and ionic strength. The glycoprotein SGPF2 exhibited antiviral activity against respiratory syncytial virus (RSV) with a median inhibitory concentration (IC(50)) of 62.5 microg/ml and Herpes simplex virus type 1 (HSV-1) had an IC(50) of 31.3 microg/ml. The glycoprotein potencies for antiviral activity appeared to depend on the molecules' binding affinity for fetuin, that is, the fetuin-binding protein was more potent than the non-fetuin binding proteins. Further examination revealed that these glycoproteins also had the ability to suppress the proliferation of MCF-7 cells. The possible mechanism of anti-proliferative action as analyzed by DNA flow cytometry indicated that they could induce apoptosis mediated via sub-G(1) phase of the MCF-7 cell cycle. For example, there was an increase by 75.8% of the control level of apoptosis after incubation with SGPF1a.

Avian influenza virus A/HK/483/97(H5N1) NS1 protein induces apoptosis in human airway epithelial cells.

Avian H5N1 influenza virus causes a remarkably severe disease in humans, with an overall case fatality rate of greater than 50%. Human influenza A viruses induce apoptosis in infected cells, which can lead to organ dysfunction. To verify the role of H5N1-encoded NS1 in inducing apoptosis, the NS1 gene was cloned and expressed in human airway epithelial cells (NCI-H292 cells). The apoptotic events posttransfection were examined by a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling assay, flow cytometric measurement of propidium iodide, annexin V staining, and Western blot analyses with antibodies specific for proapoptotic and antiapoptotic proteins. We demonstrated that the expression of H5N1 NS1 protein in NCI-H292 cells was sufficient to induce apoptotic cell death. Western blot analyses also showed that there was prominent cleavage of poly(ADP-ribose) polymerase and activation of caspase-3, caspase-7, and caspase-8 during the NS1-induced apoptosis. The results of caspase inhibitor assays further confirmed the involvement of caspase-dependent pathways in the NS1-induced apoptosis. Interestingly, the ability of H5N1 NS1 protein to induce apoptosis was much enhanced in cells pretreated with Fas ligand (the time posttransfection required to reach >30% apoptosis was reduced from 24 to 6 h). Furthermore, 24 h posttransfection, an increase in Fas ligand mRNA expression of about 5.6-fold was detected in cells transfected with H5N1 NS1. In conclusion, we demonstrated that the NS1 protein encoded by avian influenza A virus H5N1 induced apoptosis in human lung epithelial cells, mainly via the caspase-dependent pathway, which encourages further investigation into the potential for the NS1 protein to be a novel therapeutic target.

Ethyl acetate extract of Chinese medicinal herb Sarcandra glabra induces growth inhibition on human leukemic HL-60 cells, associated with cell cycle arrest and up-regulation of pro-apoptotic Bax/Bcl-2 ratio.

Sarcandra glabra (Thunb.) Nakai, colloquially known as Caoshanhu, is a Chinese medicinal herb with reported anti-tumor, anti-inflammatory, anti-viral and non-specific immunoenhancing properties. Although the plant has been clinically used for treating a variety of diseases, its bioactive ingredients are largely unknown and its mode of action has never been investigated. In this study, the anti-tumor property of ethyl acetate (EA) extract of S. glabra was investigated by determining its in vitro growth-inhibitory effects on a panel of human cancer cell lines of different histotypes. Growth inhibition of the EA extract on the cancer cells seemed to be selective, and the leukemic HL-60 was found to be the most responsive after 48 h of treatment (IC50=58 microg/ml). Flow cytometric studies further illustrated that the extract might interfere with DNA replication and thus arrested the cell cycle at S phase in the leukemic cells, followed by DNA fragmentation and loss of phospholipid asymmetry in the plasma membrane after 72 h of treatment. Concurrently, the pro-apoptotic Bax/Bcl-2 ratio was also up-regulated by more than 178% of the control level. All these findings suggested that the extract had initiated apoptosis to kill the leukemic cells. Results from this pioneer study help to establish a scientific foundation for future research and development of the bioactive ingredients in EA extract of S. glabra as efficacious anti-cancer agents.

Quantitative analysis of anticancer 3-deoxyanthocyanidins in infected sorghum seedlings.

3-Deoxyanthocyanidins are structurally related to the anthocyanin pigments, which are popular as health-promoting phytochemicals. Here, it is demonstrated that the 3-deoxyanthocyanidins are more cytotoxic on human cancer cells than the 3-hydroxylated anthocyanidin analogues. At 200 microM concentration, luteolinidin reduced the viability of HL-60 and HepG2 cells by 90 and 50%, respectively. Sorghum is a major source of 3-deoxyanthocyanidins, which are present as seed pigments and as phytoalexins responding to pathogen attack. On the basis of the collision-induced dissociation spectra of luteolinidin and apigeninidin, an LC-MS/MS method, operating in multiple-reaction monitoring mode, was developed for the specific detection and accurate quantification of these compounds in complex mixtures, which may be difficult to analyze using absorbance measurements. The results demonstrated that inoculated sorghum seedlings could be utilized for convenient and large-scale production of 3-deoxyanthocyanidins. A quantity of almost 270 microg/g (fresh weight) of luteolinidin was produced 72 h after fungal inoculation of 1-week-old seedlings.

In vitro antiviral activities of Chinese medicinal herbs against duck hepatitis B virus.

Fifty-six different Chinese medicinal herbs from 29 families were evaluated for their antiviral activities against duck hepatitis B virus (DHBV) in vitro. The DHBV DNA level in primary duck hepatocyte cultures was monitored by dot blot hybridization and the cytotoxicity was evaluated by MTT assay. Anti-DHBV activities were found more strongly in the aqueous extracts of Ardisia chinensis and Pithecellobium clypearia with selective indices of 2.6 and >2.7, respectively, which were comparable to that of 2',3'-dideoxycytidine. Further research on the isolation of the active antiviral phytochemicals from these herbs may provide alternative options for the treatment of chronic hepatitis B.

Growth-inhibitory effects of a beta-glucan from the mycelium of Poria cocos on human breast carcinoma MCF-7 cells: cell-cycle arrest and apoptosis induction.

Because of the reported immune-enhancing and anti-tumor activities of some mushroom polysaccharides, their applications as biological response modifiers have attracted significant attention. We have purified a water-soluble beta-glucan PCM3-II, comprising mainly 1right curved arrow 3 and 1right curved arrow 4 linkages, from the mycelia of Poria cocos (Schw.) Wolf (Fu-ling). In this study, the growth-inhibitory effect of PCM3-II was further explored on the human breast carcinoma MCF-7 cells in vitro. The dose effect of PCM3-II was studied by incubating the breast cancer cells with 12.5-400 microg/ml of the glucan for 72 h. The MTT study showed that PCM3-II reduced proliferation and viability of the MCF-7 cells dose-dependently, so that the cancer-cell growth was decreased by 50% of the control level at 400 microg/ml of the glucan. The time effect of PCM3-II was then investigated by treating the breast cancer cells with 400 microg/ml of the glucan for 24, 48 and 72 h, respectively. Results from the flow cytometry study demonstrated that PCM3-II induced cell-cycle G1 arrest time-dependently and about 90% of the cells in cell cycle were accumulated at G1 phase after 72 h of treatment. The G1 arrest was associated with downregulations of the unscheduled cyclin D1 and cyclin E expressions in the breast cancer cells. Apoptosis was also induced by PCM3-II in the MCF-7 cells, so that the subG1 cells in DNA histogram of the flow cytometry were elevated by 5-fold of the control level at 48 h and by 24-fold at 72 h of treatment. The immunoblot study also showed that the glucan induced depletion of the antiapoptotic Bcl-2 protein, but not the proapoptotic Bax protein, so that the Bax/Bcl-2 ratio was elevated in the breast cancer cells at the time when the most prominent apoptosis was also observed. In conclusion, although the detailed mechanism for the anti-tumor activity of the P. cocos beta-glucan still needs further investigation, this study provides preliminary insights into its mode of action and perspectives of its development as a water-soluble anti-tumor agent.

Ethyl acetate extract of Patrinia scabiosaefolia downregulates anti-apoptotic Bcl-2/Bcl-X(L) expression, and induces apoptosis in human breast carcinoma MCF-7 cells independent of caspase-9 activation.

Patrinia scabiosaefolia Fisch. is a Chinese medicinal herb used traditionally for treating intestinal carbuncle. Although Patrinia scabiosaefolia has also been suggested for cancer therapy, there has not been any scientific evidence supporting this application. In this study, a panel of human cancer cells, including breast carcinoma MCF-7; hepatocellular carcinoma HepG2; skin melanoma A375; lung carcinoma A549 and prostate adenocarcinoma PC-3, were treated in vitro with ethyl acetate extract of Patrinia scabiosaefolia (EAE-PS) for 48 h. Results from MTT study showed that MCF-7 was the most responsive (IC50 = 112.3 microg/ml) while PC-3 was the most resistant (IC50 = 348.7 microg/ml) one to cell growth inhibition. DNA flow cytometry demonstrated that EAE-PS induced apoptosis in the resistant MCF-7 cells by 14.5-fold of the control level after 36 h of treatment. Immunoblot studies further illustrated that although EAE-PS downregulated the anti-apoptotic Bcl-2/Bcl-X(L) expression in breast cancer cells, the induced apoptosis could not be prevented by the caspase-9 inhibitor (Z-LEHD-FMK). All these results suggest that EAE-PS retards MCF-7 cell growth by activating the caspase-independent mitochondrial cell death pathway. Results from this study support future research and development of the bioactive ingredients from Patrinia scabiosaefolia as anticancer agents, especially against those apoptosis-resistant cancers with deregulated Bcl-2/Bcl-X(L) expression.

The chlorophyllin-induced cell cycle arrest and apoptosis in human breast cancer MCF-7 cells is associated with ERK deactivation and Cyclin D1 depletion.

Targeting the mitogen-activated protein kinases (MAPKs) has been suggested as a novel strategy to treat cancer. Chlorophyllin (CHL) is the sodium-copper salt of chlorophyll derivative and is a commonly used food dye for green coloration; CHL was found previously to retard growth of the human breast carcinoma MCF-7 cells. Extracellular signal-regulated kinases (ERKs) constitute a subfamily of MAPKs, participating in cell survival, proliferation and differentiation. We report here the first evidence that CHL deactivates ERKs to inhibit the breast cancer cell proliferation. The results from flow cytometry showed that 200 microg/ml CHL reduced the phosphorylated and activated ERK-positive cells in different cell cycle phases from the control of >96 to <38% at 24 h of incubation; the ERK deactivations occurred in both dose- and time-dependent manner, so that nearly all ERKs were de-activated by 400 microg/ml CHL at 72 h of treatment. Immunoblot studies, however, illustrated that the levels of total ERKs were not significantly affected by the CHL treatments, suggesting that the phytochemical retards the enzyme activation rather than its expression. Cyclin D1, but not its enzyme Cdk6, was also depleted after the CHL treatments; the depletions were associated with elevations of G0/G1 cells. Apoptosis occurred time-dependently with the ERK deactivations by 400 microg/ml CHL; the apoptotic cells elevated from 2.7-fold of the control level at 24 h, to 4.7-fold at 48 h and to 16.6-fold at 72 h of treatment. Bcl-2 was also depleted at 72 h when there was the most prominent elevation of the apoptotic cells, suggesting that it participates during the exacerbation rather than the initiation phases of the CHL-induced apoptosis. Results from this study support further research on CHL for preventing and treating those tumors with deregulated ERK activations.

Docosahexaenoic acid modulates different genes in cell cycle and apoptosis to control growth of human leukemia HL-60 cells.

Although previous studies have shown that docosahexaenoic acid (DHA; 22:6 omega 3) from fish oils inhibits growth of different cancers, safety issues have been raised repeatedly about contaminations of toxins in these oils. Cultured microalgae are suggested recently as an alternative cleaner and safer source of the fatty acid. We investigated in this study the function of DHA from the enriched microalga Crypthecodinium cohnii (ADHA) in cell-growth control and its mechanism in human leukemia HL-60 cells. ADHA retarded proliferation of the leukemia cells dose-dependently by 4-93% of the control level, after 72-h incubations with 10-160 micro M of the fatty acid; and the 50% inhibitory concentration (IC50) was estimated as 74 micro M. DNA-flow cytometry study showed that ADHA arrested G0/G1 cells by 12-22% and induced apoptotic cells by 569-906% of their controls, after incubation with the IC50 of ADHA for 24, 48 and 72 h. The modes of cell-cycle arrest and pro-apoptotic actions of ADHA were further elucidated. Gene-array analysis illustrated that ADHA modulated a number of cell-cycle and apoptosis genes to control the cell growth; in particular, the fatty acid up-regulated the transcriptional repressor E2F-6 and pro-apoptotic Bax by 1435 and 4172% respectively, after 24 h of incubation. Semi-quantitative RT-PCR study further showed that ADHA induced elevation of the Bax mRNA transcript time-dependently. In meanwhile, ADHA also induced phosphorylation and thus inactivation of Rb protein in the leukemia cells. All these results suggest that ADHA up-regulates Bax and inactivates Rb protein to induce the cell-growth control and apoptosis in human leukemia HL-60 cells.

Synergistic action of piroxicam on the eicosapentaenoic acid-induced apoptosis is associated with enhanced down-regulation of anti-apoptotic Bcl-2 expression but not promoted activation of pro-apoptotic Bid protein.

Eicosapentaenoic acid (EPA) is a dietary polyunsaturated fatty acid (PUFA) that is found abundantly in fish oils and induces apoptosis in human promyelocytic leukemia HL-60 cells. Cyclooxygenase (COX) converts EPA intracellularly into various inflammatory mediators that may affect the bioavailability of this fatty acid for inducing apoptosis in the cancer cells. In this study, effect of piroxicam (PRX), a COX inhibitor, on the EPA-induced apoptosis in HL-60 cells was investigated. EPA arrested cell cycle of the leukemic cells at G0/G1 phase after 8-h incubation and induced apoptosis after 24-h incubation. PRX induced neither cell-cycle arrest nor apoptosis significantly in HL-60 cells even after 48-h incubation. However, 24-h incubation with PRX followed by 24-h incubation with EPA significantly elevated both early-phase and late-phase apoptotic cells by 35% and 166% respectively, when compared to those induced by the fatty acid only. This synergistic action of PRX on the EPA-induced apoptosis was associated with enhanced down-regulation of anti-apoptotic Bcl-2 protein expression, but not promoted activation of pro-apoptotic Bid protein.

Docosahexaenoic acid from a cultured microalga inhibits cell growth and induces apoptosis by upregulating Bax/Bcl-2 ratio in human breast carcinoma MCF-7 cells.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid that comprises 22 carbons and 6 alternative double bonds in its hydrocarbon chain (22:6omega3). Previous studies have shown that DHA from fish oil controls the growth and development of different cancers; however, safety issues have been raised repeatedly about contamination of toxins in fish oil that makes it no longer a clean and safe source of the fatty acid. We investigated the cell growth inhibition of DHA from the cultured microalga Crypthecodinium cohnii (algal DHA [aDHA]) in human breast carcinoma MCF-7 cells. aDHA exhibited growth inhibition on breast cancer cells dose-dependently by 16.0% to 59.0% of the control level after 72-h incubations with 40 to 160 microM of the fatty acid. DNA flow cytometry shows that aDHA induced sub-G(1) cells, or apoptotic cells, by 64.4% to 171.3% of the control levels after incubations with 80 mM of the fatty acid for 24, 48, and 72 h. Western blot studies further show that aDHA did not modulate the expression of proapoptotic Bax protein but induced the downregulation of anti-apoptotic Bcl-2 expression time-dependently, causing increases of Bax/Bcl-2 ratio by 303.4% and 386.5% after 48- and 72-h incubations respectively with the fatty acid. Results from this study suggest that DHA from the cultured microalga is also effective in controlling cancer cell growth and that downregulation of antiapoptotic Bcl-2 is an important step in the induced apoptosis.