Estrogenic effects of extracts from cabbage, fermented cabbage, and acidified brussels sprouts on growth and gene expression of estrogen-dependent human breast cancer (MCF-7) cells

Cruciferous vegetable extracts from freeze-dried cabbage (FDC), freeze-dried fermented cabbage (FDS), and acidified Brussels sprouts (ABS) were prepared by exhaustive extraction with ethyl acetate. Estrogenic and antiestrogenic effects of these extracts were analyzed. To identify whether the extracts are potential estrogen receptor (ER) ligands that can act as agonists or antagonists, the binding affinity of extracts for the ER was measured using a competitive radiometric binding assay. The extracts bound with low affinity to the ER, and the relative binding affinity is estradiol > FDS > FDC > ABS. These extracts were evaluated for their estrogenic and antiestrogenic activities in estrogen-dependent human breast cancer (MCF-7) cells using as endpoints proliferation and induction of estrogen-responsive pS2 gene expression, which was analyzed using Northern blot assay. At low concentrations (5-25 ng/mL) all of the extracts reduced 1 nM estradiol-induced MCF-7 cell proliferation. Extracts at 25 ng/mL also inhibited estradiol-induced pS2 mRNA expression. At higher extract concentrations (50 ng/mL-25 microg/mL), however, increased proliferation in MCF-7 cells was observed. Similarly, expression of the pS2 gene was induced by higher extract concentrations (0.25-25 microg/mL). The pure estrogen antagonist, ICI 182,780, suppressed the cell proliferation induced by the extracts as well as by estradiol and also the induction of pS2 expression by the extracts. The ER subtype-selective activities of FDC and FDS were analyzed using a transfection assay in human endometrial adenocarcinoma (HEC-1) cells. FDS acted as an ERalpha-selective agonist while FDC fully activated both ER-alpha and ER-beta. Growth of the ER-negative MDA-231 cells was not affected by the extracts or by estradiol. This study demonstrates that cruciferous vegetable extracts act bifunctionally, like an antiestrogen at low concentrations and an estrogen agonist at high concentrations.     

Potential chemopreventive properties of anthocyanin-rich aqueous extracts from in vitro produced tissue of sweetpotato (Ipomoea batatas L.)

Anthocyanin-rich aqueous extracts from cell suspension cultures of a high anthocyanin-producing sweetpotato PL (purple line) cell line grown under two different media conditions, MM (multiplication medium) and APM (high anthocyanin-producing medium) and from the cell line's donor tissue, field-grown storage root (SR) of sweetpotato, cv. Ayamurasaki, were evaluated for antioxidative (DPPH test), antimutagenic (Salmonella/reversion assay; mutagen, Trp-P-1), and antiproliferative (human promyelocytic leukaemia cells HL-60) activities. Both cell line extracts MM and APM exhibited higher radical scavenging activities (RSA), 3.8- and 1.4-fold, respectively, than the SR extract. The antimutagenic activity of all extracts was found to be dose-dependent. At a dose of 1 mg/plate, the highest activity exhibited APM (73% inhibition of Trp-P-1-induced reverse mutation of Salmonella typhimurium TA98), followed by MM (54% inhibition) and SR (36% inhibition). The MM extract was the strongest inhibitor of the proliferation of human promyelocytic leukemia cells. At a concentration of 1.6 mg/mL medium during 24 h, it suppressed the growth of 47% of HL-60 cells. A significantly lower growth suppression effect displayed APM and SR extracts (21 and 25%, respectively). Total anthocyanin levels and anthocyanin composition in evaluated samples seem to be related to their activities. The MM extract, which exhibited the highest RSA and antiproliferation activities, contained the highest level of anthocyanins. Among them, nonacylated cyanidin 3-sophoroside-5-glucoside dominated. It is speculated that the presence of this anthocyanin contributed toward enhanced activities of MM extract.     

Andrographolide inhibits ICAM-1 expression and NF-κB activation in TNF-α-treated EA.hy926 cells

Several lines of evidence indicate that inflammation and endothelial cell dysfunction are important initiating events in atherosclerosis. Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, induces the expression of cell adhesion molecules and results in monocyte adherence and atheromatous plaque formation. Andrographolide (AP) is a major bioactive diterpene lactone in Andrographis paniculata that has anti-inflammatory activity. A previous study demonstrated the role of heme oxygenase 1 (HO-1) in the inhibition of TNF-α-induced ICAM-1 expression by AP. The present study investigated the effect of AP on the IKK/NF-κB signaling pathway, which mediates TNF-α-induced ICAM-1 expression in EA.hy926 cells. Similar to the previous study, AP inhibited TNF-α-induced ICAM-1 mRNA and protein levels, its expression on the cell surface, and subsequent adhesion of HL-60 cells to EA.hy926 cells. AP inhibited TNF-α-induced κB inhibitor (IκB) kinase (IKK) and IκBα activation, p65 nuclear translocation, NF-κB and DNA binding activity, and promoter activity of ICAM-1. Although AP increased the intracellular cAMP concentration and induced the phosphorylation of cAMP response element-binding protein (CREB), knocking down CREB protein expression by transfecting the cells with CREB-specific small interfering RNA did not relieve the inhibition of ICAM-1 expression by AP. Taken together, these results suggest that AP down-regulates TNF-α-induced ICAM-1 expression at least in part via attenuation of activation of NF-κB in EA.hy926 cells rather than through activation of CREB. The results suggest that AP may have potential as a cardiovascular-protective agent.     

Berry phenolic extracts modulate the expression of p21(WAF1) and Bax but not Bcl-2 in HT-29 colon cancer cells

Previous studies have shown that anthocyanin-rich berry extracts inhibit the growth of cancer cells in vitro. The objective of this study was to compare the effects of berry extracts containing different phenolic profiles on cell viability and expression of markers of cell proliferation and apoptosis in human colon cancer HT-29 cells. Berry extracts were prepared with methanol extraction, and contents of the main phenolic compounds were analyzed using HPLC. Anthocyanins were the predominant phenolic compounds in bilberry, black currant, and lingonberry extracts and ellagitannins in cloudberry extract, whereas both were present in raspberry and strawberry extracts. Cells were exposed to 0-60 mg/mL of extracts, and the cell growth inhibition was determined after 24 h. The degree of cell growth inhibition was as follows: bilberry > black currant > cloudberry > lingonberry > raspberry > strawberry. A 14-fold increase in the expression of p21WAF1, an inhibitor of cell proliferation and a member of the cyclin kinase inhibitors, was seen in cells exposed to cloudberry extract compared to other berry treatments (2.7-7-fold increase). The pro-apoptosis marker, Bax, was increased 1.3-fold only in cloudberry- and bilberry-treated cells, whereas the pro-survival marker, Bcl-2, was detected only in control cells. The results demonstrate that berry extracts inhibit cancer cell proliferation mainly via the p21WAF1 pathway. Cloudberry, despite its very low anthocyanin content, was a potent inhibitor of cell proliferation. Therefore, it is concluded that, in addition to anthocyanins, also other phenolic or nonphenolic phytochemicals are responsible for the antiproliferative activity of berries.     

In Vitro Study on Effect of Germinated Wheat on Human Breast Cancer Cells

This research investigated the possible anticancer effects of germinated wheat flour (GWF) on growth and apoptosis of human breast cancer cells. In a series of in vitro experiments, estrogen‐receptor‐positive (MCF‐7) and ‐negative (MDA‐MB‐231) cells were cultured and treated with GWF that was obtained after a three‐day germination. The GWF significantly reduced the growth of both of MCF‐7 and MDA‐MB‐231 cells. In analysis with a flow cytometer, the GWF significantly up‐regulated apoptosis of both cell lines. These results indicate that GWF induced apoptosis and inhibited growth of the human breast cell lines, thereby demonstrating its potential inhibition of breast cancer cell proliferation.     

Antioxidant and antiproliferative activities of Spirulina and Chlorella water extracts

Liver fibrosis is a chronic liver disease that will further develop to cirrhosis if severe damage continues to form. A potential treatment for liver fibrosis is to inhibit activated hepatic stellate cell (HSC) proliferation and, subsequently, to induce HSC apoptosis. It has been reported that antioxidants are able to inhibit the proliferation of HSCs. In this study, the aqueous extract of spirulina was chosen as the source of antioxidant to investigate the inhibitory effect on the proliferation of HSC. The growth inhibitory effects of aqueous spirulina and chlorella extract on human liver cancer cells, HepG2, were also studied and compared in pairs. Results indicated that the total phenol content of spirulina was almost five times greater than that of chlorella (6.86 +/- 0.58 vs 1.44 +/- 0.04 mg tannic acid equivalent/g of algae powder, respectively). The antioxidant activity of spirulina determined by the ABTS*+ method was higher than chlorella (EC50: 72.44 +/- 0.24 micromol of trolox equivalent/g of spirulina extract vs 56.09 +/- 1.99 micromol of trolox equivalent/g of chlorella extract). Results of DPPH* assay also showed a similar trend as the ABTS*+ assay (EC50: 19.39 +/- 0.65 micromol of ascorbic acid equivalent/g of spirulina extract vs 14.04 +/- 1.06 micromol of ascorbic acid equivalent/g of chlorella extract). The aqueous extracts of these two algae both showed antiproliferative effects on HSC and HepG2, but spirulina was a stronger inhibitor than chlorella. Annexin-V staining showed that aqueous extract of spirulina induced apoptosis of HSC after 12 h of treatment. In addition, the aqueous extract of spirulina triggered a cell cycle arrest of HSC at the G2/M phase.     

Selenocystine induces S-phase arrest and apoptosis in human breast adenocarcinoma MCF-7 cells by modulating ERK and Akt phosphorylation

Selenocystine (SeC) is a nutritionally available selenoamino acid with selective anticancer effects on a number of human cancer cell lines. The present study shows that SeC inhibited the proliferation of human breast adenocarcinoma MCF-7 cells in a time- and dose-dependent manner, through the induction of cell cycle arrest and apoptotic cell death. SeC-induced S-phase arrest was associated with a marked decrease in the protein expression of cyclins A, D1, and D3 and cyclin-dependent kinases (CDKs) 4 and 6, with concomitant induction of p21waf1/Cip1, p27Kip1, and p53. Exposure of MCF-7 cells to SeC resulted in apoptosis as evidenced by caspase activation, PARP cleavage, and DNA fragmentation. SeC treatment also triggered the activation of JNK, p38 MAPK, ERK, and Akt. Inhibitors of ERK (U0126) and Akt (LY294002), but not JNK (SP600125) and p38 MAPK (SB203580), suppressed SeC-induced S-phase arrest and apoptosis in MCF-7 cells. The findings establish a mechanistic link between the PI3K/Akt pathway, MAPK pathway, and SeC-induced cell cycle arrest and apoptosis in MCF-7 cells.     

Topoisomerase I and II enzyme inhibitory aqueous extract of Ardisia compressa and ardisin protect against benomyl oxidation of hepatocytes

Tea preparations of Ardisia compressa (AC) have been used in folk medicine against liver disorders. The objective of this study was to evaluate the in vitro topoisomerase I and II enzyme inhibition and the antioxidant effect of an aqueous extract from dry leaves of AC and a pure component (ardisin) purified from AC on benomyl (Be)-induced cytotoxicity in primary culture rat hepatocytes. Lipid peroxidation (malondialdehyde), antioxidant enzyme activities of glutathione reductase, glutathione peroxidase, and superoxide dismutase, and glutathione levels were studied. Topoisomerase I and II enzyme inhibition was used to guide purification of ardisin, which was purified using TLC, MPLC, and preparative and analytical HPLC methods. Benomyl increased malondialdehyde (58% change in comparison to the control) and glutathione peroxidase (10%), producing a significant consumption of endogenous antioxidant glutathione (65%, P < 0.05). A 94% hepatocyte protection was observed when cells were first exposed to ardisin (0.27 microg/mL), followed by Be (35 microg/mL). Cell protection by the tea extract of AC (AE) was greater than that by (-)-epigallocatechin 3-gallate (EGCG). Ardisin showed a clear inhibition of topoisomerases I and II catalytic activity in Saccharomyces cerevisiae mutant cells JN 394, JN394t(-)(1), and JN394t-(2)(-)(5). The potency of ardisin was superior to that of AE and EGCG as an antioxidant, protecting rat hepatocytes when exposed to Be. On the basis of the effective concentrations of equivalents to [+]catechin found in the present study, it can be estimated that, in order to gain antioxidative protection, a person would need to ingest approximately 1 L of AC tea per day, with a total content of 10.8 g of plant material.     

STAT3基因转染牛乳腺上皮细胞及生物学特性观察

应用PCR技术从含有人STAT3基因cDNA的质粒pOTB7中扩增STAT3基因片段。将其克隆入真核表达载体pEGFP-C1中,构建重组表达质粒。利用脂质体介导法将重组表达质粒转染原代牛乳腺上皮细胞(Bovine Mammary Epithelial cells)。G418筛选阳性克隆,RT-PCR检测STAT3基因在牛乳腺上皮细胞中的表达情况,并通过流式细胞术(Flow cytometry)检测转染细胞的增殖能力、周期以及DNA含量。结果表明,转染24h后大约16%的细胞被导入含STAT3基因的重组表达质粒。在mRNA水平证实细胞内有STAT3基因的高表达。导入STAT3基因后,细胞增殖能力增强,染色体倍数发生变化,细胞的增殖寿命较对照细胞延长了13代,表明导入STAT3基因后,能延长体外培养的牛乳腺上皮细胞的寿命。     

Growth suppression of human cancer cells by polyphenolics from sweetpotato (Ipomoea batatas L.) leaves

Sweetpotato leaves (Ipomoea batatas L.) contain a high content of polyphenolics that consist of caffeic acid, chlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and 3,4,5-tri-O-caffeoylquinic acid. We investigated the suppression of the proliferation of selected human cancer cells by phenolic compounds isolated from sweetpotato leaf. The human cancer cells used in this research included a stomach cancer (Kato III), a colon cancer (DLD-1), and a promyelocytic leukemia cell (HL-60). Caffeic acid and di- and tricaffeoylquinic acids dose-dependently depressed cancer cell proliferation, and the difference in sensitivity between caffeoylquinic acid derivatives and each kind of cancer cell was observed. Specifically, 3,4,5-tri-O-caffeoylquinic acid effectively depressed the growth of three kinds of cancer cells, and caffeic acid had an exceptionally higher effect against HL-60 cells than other di- and tricaffeoylquinic acids. In attempting to clarify the mechanism of growth suppression with the addition of the apoptotic inhibitor N-ethylmaleimide, we observed that the nuclear granulation in 3,4,5-tri-O-caffeoylquinic acid-treated HL-60 cells suggested apoptosis induction. This effect was confirmed by DNA fragmentation, an increase of caspase-3 activity, and expression of c-Jun. Growth suppression of HL-60 cells by 3,4,5-tri-O-caffeoylquinic acid was determined to be the result of apoptotic death of the cells. These results indicate that 3,4,5-tri-O-caffeoylquinic acid may have potential for cancer prevention.     

Total cranberry extract versus its phytochemical constituents: antiproliferative and synergistic effects against human tumor cell lines

Cranberries (Vaccinium macrocarpon Ait.) are an excellent dietary source of phytochemicals that include flavonol glycosides, anthocyanins, proanthocyanidins (condensed tannins), and organic and phenolic acids. Using C-18 and Sephadex Lipophilic LH-20 column chromatography, HPLC, and tandem LC-ES/MS, the total cranberry extract (TCE) has been analyzed, quantified, and separated into fractions enriched in sugars, organic acids, total polyphenols, proanthocyanidins, and anthocyanins (39.4, 30.0, 10.6, 5.5, and 1.2% composition, respectively). Using a luminescent ATP cell viability assay, the antiproliferative effects of TCE (200 microg/mL) versus all fractions were evaluated against human oral (KB, CAL27), colon (HT-29, HCT116, SW480, SW620), and prostate (RWPE-1, RWPE-2, 22Rv1) cancer cell lines. The total polyphenol fraction was the most active fraction against all cell lines with 96.1 and 95% inhibition of KB and CAL27 oral cancer cells, respectively. For the colon cancer cells, the antiproliferative activity of this fraction was greater against HCT116 (92.1%) than against HT-29 (61.1%), SW480 (60%), and SW620 (63%). TCE and all fractions showed >/=50% antiproliferative activity against prostate cancer cells with total polyphenols being the most active fraction (RWPE-1, 95%; RWPE-2, 95%; 22Rv1, 99.6%). Cranberry sugars (78.8 microg/mL) did not inhibit the proliferation of any cancer cell lines. The enhanced antiproliferative activity of total polyphenols compared to TCE and its individual phytochemicals suggests synergistic or additive antiproliferative interactions of the anthocyanins, proanthocyanidins, and flavonol glycosides within the cranberry extract.     

Aqueous extract from Spanish black radish (Raphanus sativus L. Var. niger) induces detoxification enzymes in the HepG2 human hepatoma cell line

Spanish black radish (Raphanus sativus L. var. niger) is a member of the Cruciferae family that also contains broccoli and Brussels sprouts, well-known to contain health-promoting constituents. Spanish black radishes (SBR) contain high concentrations of a glucosinolate unique to the radish family, glucoraphasatin, which represents >65% of the total glucosinolates present in SBR. The metabolites of glucosinolates, such as isothiocyanates, are implicated in health promotion, although it is unclear whether glucosinolates themselves elicit a similar response. The crude aqueous extract from 0.3 to 3 mg of dry SBR material increased the activity of the phase II detoxification enzyme quinone reductase in the human hepatoma HepG2 cell line with a maximal effect at a concentration of 1 mg/mL. Treatment of HepG2 cells with the crude aqueous extract of 1 mg of SBR per mL also significantly induced the expression of mRNA corresponding to the phase I detoxification enzymes: cytochrome P450 (CYP) 1A1, CYP1A2, and CYP1B1 as well as the phase II detoxification enzymes: quinone reductase, heme oxygenase 1, and thioredoxin reductase 1. Previous studies have shown that the myrosinase metabolites of different glucosinolates vary in their ability to induce detoxification enzymes. Here, we show that while glucoraphasatin addition was ineffective, the isothiocyanate metabolite of glucoraphasatin, 4-methylthio-3-butenyl isothiocyanate (MIBITC), significantly induced phase II detoxification enzymes at a concentration of 10 microM. These data demonstrate that the crude aqueous extract of SBR and the isothiocyanate metabolite of glucoraphasatin, MIBITC, are potent inducers of detoxification enzymes in the HepG2 cell line.     

Ginkgo biloba extract inhibits endotoxin-induced human aortic smooth muscle cell proliferation via suppression of toll-like receptor 4 expression and NADPH oxidase activation

Toll-like receptor 4 (TLR4) initiates the inflammatory response in blood vessels in reaction to immune stimuli such as lipopolysaccharide (LPS) produced by gram-negative bacteria. LPS-induced proliferation and functional perturbation in vascular smooth muscle cells play important roles during atherogenesis. Ginkgo biloba extract is an antiatherothrombotic Chinese herbal medicine with anti-inflammatory properties. The effects of G. biloba extract on LPS-induced proliferation and TLR4 expression and the underlying mechanisms for these actions, in human aortic smooth muscle cells (HASMCs), were examined in vitro. LPS-induced proliferation was mediated by the expression of TLR4 in HASMCs. LPS increased the expression of TLR4 in HASMCs, and this effect was mediated by the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, phosphorylation of intracellular mitogen-activated protein kinases (MAPKs), and increases in the cytoplasmic level of HuR and TLR4 mRNA stability. G. biloba extract inhibited LPS-induced HASMC proliferation and decreased the expression of TLR4 by inhibiting LPS-induced NADPH oxidase activation, mRNA stabilization, and MAPK signaling pathways. These results suggest that LPS-induced TLR4 expression contributes to HASMC proliferation and that G. biloba inhibits LPS-stimulated proliferation of HASMCs by decreasing TLR4 expression.     

Cancer chemopreventive effects of lycopene: suppression of MMP-7 expression and cell invasion in human colon cancer cells

Clinical studies indicate that high blood levels of leptin or matrix metalloproteinase-7 (MMP-7; matrilysin) proteins are associated with tumor progression of human colorectal cancer (CRC). Leptin could play an important role in cell migration and invasion of cancer cells. Our previous study indicated that lycopene could inhibit the proliferation of human colon cancer cells in vitro. However, the inhibitory effects of lycopene on the progression of human colon cancer cells have not been demonstrated yet. In this study, we investigated the inhibitory effects of lycopene on tumor progression including cell invasion and MMP-7 expression in leptin-stimulated human colon cancer cells in vitro. Our results demonstrated that lycopene significantly inhibited leptin-mediated cell invasion and MMP-7 expression in human colon cancer HT-29 cells. Lycopene could augment the expression and stability of E-cadherin proteins. Our results showed that MAPK/ERK and PI3K/Akt signaling pathways played important roles in leptin-mediated MMP-7 expression and cell invasion. Lycopene could effectively inhibit the phosphorylation of Akt, glycogen synthase kinase-3β (GSK-3β) and ERK 1/2 proteins. The molecular mechanisms of lycopene were in part through decreases in nuclear levels of AP-1 and β-catenin proteins. These novel findings suggested that lycopene could act as a chemopreventive agent to suppress MMP-7 expression and leptin-mediated cell invasion in human colon cancer HT-29 cells.     

Apple polyphenols affect protein kinase C activity and the onset of apoptosis in human colon carcinoma cells

Polyphenol-rich apple extracts have been reported to suppress human colon cancer cell growth in vitro. The protein kinase C (PKC) is among the signaling elements known to play an important role in colon carcinogenesis. In the present study, we investigated whether apple polyphenols affect PKC activity and induce apoptosis in the human colon carcinoma cell line HT29. A polyphenol-rich apple juice extract (AE02) was shown to inhibit cytosolic PKC activity in a cell-free system. In contrast, incubation of HT29 cells for 1 or 3 h with AE02 up to 2 mg/mL did not affect the cytosolic PKC activity. After prolonged incubation (24 h), cytosolic PKC activity was modulated, albeit a u-shaped curve of effectiveness was observed, with an initial inhibitory effect followed by the recurrence and even induction of enzyme activity. Concomitantly, in the cytosol, a significant decrease of the protein levels of PKCalpha, PKCbetaII, and PKCgamma together with a significant increase of a proapoptotic PKCdelta fragment was observed. However, the effects on the protein levels of these PKC isoforms in the cytosol were not associated with translocation between the different cellular compartments but might instead result from the onset of apoptosis. Indeed, the treatment with AE02 was shown to induce apoptosis by the activation of caspase-3, DNA fragmentation, and cleavage of poly(ADP ribose) polymerase. So far, identified and available constituents of the apple extract did not contribute substantially to the observed effects on PKC and apoptosis induction. In summary, apple polyphenols were found to inhibit PKC activity in a cell-free system. However, our results indicate that within intact cells PKC does not represent the primary target of apple polyphenols but appears to be affected in the course of apoptosis induction.     

Cordycepin stimulated steroidogenesis in MA-10 mouse Leydig tumor cells through the protein kinase C Pathway

Cordycepin (3'-deoxyadenosine) is an adenosine analogue isolated from Cordyceps sinensis , which is a Chinese herbal medicine known to have many benefits, including adjustment of the physical condition, an anticancer effect, and enhancement of sexual performance. It was previously demonstrated that cordycepin could simultaneously activate steroidogenesis and apoptosis in MA-10 mouse Leydig tumor cells. However, the mechanism remains elusive. Thus, aim of the present study was to investigate the steroidogenic and apoptotic mechanism of cordycepin in MA-10 cells. MA-10 cells were treated with cordycepin at various dosages and time courses plus different protein kinase inhibitors. Steroid production, protein expression, and cell viability were then determined. Results illustrated that cordycepin stimulated MA-10 cell steroidogenesis in dose- and time-dependent relationships. However, cordycepin could not induce steroidogenic acute regulatory (StAR) protein expression. However, cordycepin did activate the phospholipase C/protein kinase C (PLC/PKC), but not PKA and PI3K, pathway to induce MA-10 cell steroidogenesis. Moreover, cordycepin could stimulate the phosphorylation of PKC, extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun N-terminal kinase (c-JNK), but not p38, in MA-10 cells. In addition, cordycepin could activate the PKC pathway to induce MA-10 cell death, and this death effect was not caused by cordycepin-stimulated progesterone from MA-10 cells. In conclusion, cordycepin stimulated intracellular PLC/PKC and MAPK signal transduction pathways to induce steroidogenesis and cell death in MA-10 mouse Leydig tumor cells.     

A synthetic naringenin derivative, 5-hydroxy-7,4'-diacetyloxyflavanone-N-phenyl hydrazone (N101-43), induces apoptosis through up-regulation of Fas/FasL expression and inhibition of PI3K/Akt signaling pathways in non-small-cell lung cancer cells

Naringenin, a well-known naturally occurring flavonone, demonstrates cytotoxicity in a variety of human cancer cell lines; its inhibitory effects on tumor growth have spurred interest in its therapeutic application. In this study, naringenin was derivatized to produce more effective small-molecule inhibitors of cancer cell proliferation, and the anticancer effects of its derivative, 5-hydroxy-7,4'-diacetyloxyflavanone-N-phenyl hydrazone (N101-43), in non-small-cell lung cancer (NSCLC) cell lines NCI-H460, A549, and NCI-H1299 were investigated. Naringenin itself possesses no cytotoxicity against lung cancer cells. In contrast, N101-43 inhibits proliferation of both NCI-H460 and A549 cell lines; this capacity is lost in p53-lacking NCI-H1299 cells. N101-43 induces apoptosis via sub-G1 cell-cycle arrest in NCI-H460 and via G0/G1 arrest in A549 cells. Expression of apoptosis and cell-cycle regulatory factors is altered: Cyclins A and D1 and phospho-pRb are down-regulated, but expression of CDK inhibitors such as p21, p27, and p53 is enhanced by N101-43 treatment; N101-43 also increases expression levels of the extrinsic death receptor Fas and its binding partner FasL. Furthermore, N101-43 treatment diminishes levels of cell survival factors such as PI3K and p-Akt dose-dependently, and N101-43 additionally induces cleavage of the pro-apoptotic factors caspase-3, caspase-8, and poly ADP-ribose polymerase (PARP). Cumulatively, these investigations show that the naringenin derivative N101-43 induces apoptosis via up-regulation of Fas/FasL expression, activation of caspase cascades, and inhibition of PI3K/Akt survival signaling pathways in NCI-H460 and A549 cells. In conclusion, these data indicate that N101-43 may have potential as an anticancer agent in NSCLC.