Identification of novel potential PI3Kα inhibitors for cancer therapy.

Phosphatidylinositol 3-kinase alpha (PI3Kα) is among the most important PI3K isoforms and has been associated with multiple human cancers. Therefore, PI3Kα has garnered considerable attention as a viable target for anticancer drug discovery, and thus the identification and development of highly potent inhibitors of this isoform has become an important line of research. Here, structure-based virtual screening, bioassays, and molecular dynamics simulations were performed to discover novel potential PI3Kα inhibitors. TCM-N1 (ZINC13382850) was identified as a possible PI3Kα inhibitor. Particularly, fluorescence quenching assays determined that the binding affinity of the aforementioned compound was superior to that of a reference ligand (BYL719; i.e. a known PI3Kα inhibitor). Moreover, enzymatic activity and cell proliferation inhibition assays indicated that TCM-N1 possessed a moderate inhibition activity against PI3Kα and a relatively high anti-tumor proliferation ability in gastric, colorectal, and cervical cancer cells. The binding model and related thermodynamic parameters further demonstrated that TCM-N1 was tightly embedded into the ATP-binding pocket via hydrogen bonds, van der Waals interactions, and hydrophobic interactions. Therefore, this study provides promising insights into the development and design of more potent PI3Kα-inhibiting analogs. Communicated by Ramaswamy H. Sarma.

The microRNA miR-19a-3p suppresses cell growth, migration, and invasion in multiple myeloma via the Wnt/ß-catenin pathway.

BACKGROUND: MicroRNAs have been suggested as potential regulators in the development of multiple myeloma (MM) through affecting the expression of their target genes. This study aimed to investigate the effects of miR-19a-3p in MM, and its underlying mechanisms in regulating cell proliferation and invasion. METHODS: Bone marrow samples from 25 MM patients and 12 healthy donors were collected and miR-19a-3p and Wnt1 mRNA expression was assessed. The effects of miR-19a-3p on cell proliferation, migration, and invasion in U226 and RPMI-8226 MM cells were evaluated by miR-19a-3p overexpression. Luciferase assays were performed to explore the potential target genes. Knock down or overexpression of Wnt1 was used to explore the effects of miR-19a-3p on cell growth, migration, and invasion. RESULTS: The expression of miR-19a-3p was downregulated in MM and cell lines, while Wnt1 mRNA levels were increased. Overexpression of miR-19a-3p inhibited cell proliferation, migration, and invasion in U226 and RPMI-8226 cells. Additionally, western blot assays revealed that miR-19a-3p could suppress Wnt1, ß-catenin, cyclin D1, and c-Myc expression. Knockdown of Wnt1 also inhibited cell growth, migration, and invasion. Moreover, luciferase reporter assay revealed direct binding between Wnt1 and miR-19a-3p. Wnt1 overexpression partially reversed the suppressive effects of miR-19a-3p on cell proliferation, migration, and invasion in U266 cells. CONCLUSIONS: The expression of miR-19a-3p was downregulated in MM patients and MM cell lines. Overexpression of miR-19a-3p inhibited proliferation, migration, and invasion by targeting Wnt1 via the Wnt/ß-catenin signaling pathway.

mTORC1-Sch9 regulates hydrogen sulfide production through the transsulfuration pathway.

Endogenous hydrogen sulfide mediates anti-aging benefits of dietary restriction (DR). However, it is unclear how H2S production is regulated by pathways related to DR. Due to the importance of mTORC1 pathway in DR, we investigated the effects of Sch9, a yeast homolog of mammalian S6K1 and a major substrate of mTORC1 on H2S production in yeast Saccharomyces cerevisiae. We found that inhibition of the mTORC1-Sch9 pathway by SCH9 deletion, rapamycin or myriocin treatment resulted in a dramatic decrease in H2S production. Although deficiency of SCH9 did not alter the intracellular level of methionine, the intracellular level of cysteine increased in Δsch9 cells. The expression of CYS3 and CYS4, two transsulfuration pathway genes encoding cystathionine gamma-lyase (CGL) and cystathionine beta-synthase (CBS), were also decreased under mTORC1-Sch9 inhibition. Overexpression of CYS3 or CYS4 in Δsch9 cells or WT cells treated with rapamycin rescued the deficiency of H2S production. Finally, we also observed a reduction in H2S production and lowering of both mRNA and protein levels of CGL and CBS in cultured human cells treated with rapamycin to reduce mTORC1 pathway activity. Thus, our findings reveal a probably conserved mechanism in which H2S production by the transsulfuration pathway is regulated by mTORC1-Sch9 signaling.

Insight into the structural stability of wild-type and histidine mutants in Pin1 by experimental and computational methods.

Pin1, a polypeptide proline isomerase parvulin, plays a key role in Alzheimer's disease (AD), common tumors and cancers. Two conservative histidine residues, His59 and His157, are important for maintaining the stability of the PPIase domain. Hence multiple spectral and computational techniques were performed to investigate the potential mechanism of two histidine residues. Thermal denaturation indicated that both residues His59 and His157 are not sensitive to the lower temperatures, while residue His59 is more sensitive to the higher temperatures than residue His157. Acidic denaturation suggested that influences of both residues His59 and His157 to acidic stability were the difference from Pin1-WT. ANS and RLS spectra hinted that there was no significant effect on hydrophobic change and aggregation by histidine mutations. The GndHCl-induced denaturation implied that residues His59 and His157 contributed the most to the chemical stability. MD simulations revealed that residues His59 and His157 mutations resulted in that the hydrogen bond network of the dual histidine motif was destroyed wholly. In summary, these histidine residues play an important role in maintaining the structural stability of the PPIase domain.

In Silico Investigation of the Anti-Tumor Mechanisms of Epigallocatechin-3-Gallate.

The EGCG, an important component of polyphenol in green tea, is well known due to its numerous health benefits. We employed the reverse docking method for the identification of the putative targets of EGCG in the anti-tumor target protein database and these targets were further uploaded to public databases in order to understand the underlying pharmacological mechanisms and search for novel EGCG-associated targets. Similarly, the pharmacological linkage between tumor-related proteins and EGCG was manually constructed in order to provide greater insight into the molecular mechanisms through a systematic integration with applicable bioinformatics. The results indicated that the anti-tumor mechanisms of EGCG may involve 12 signaling transduction pathways and 33 vital target proteins. Moreover, we also discovered four novel putative target proteins of EGCG, including IKBKB, KRAS, WEE1 and NTRK1, which are significantly related to tumorigenesis. In conclusion, this work may provide a useful perspective that will improve our understanding of the pharmacological mechanism of EGCG and identify novel potential therapeutic targets.

Mitochondria-mediated apoptosis was induced by oleuropein in H1299 cells involving activation of p38 MAP kinase.

Lung cancer is the main health threat in the world. Recently, oleuropein has been reported to have potent antioxidant and anticancer activities. However, the antitumor effects of oleuropein on H1299 cells are not well understood. Therefore, the purpose of this paper is tantamount to explore the effects of oleuropein on H1299 cells and its underlying mechanism that may be involved. Oleuropein treatment in H1299 cells resulted in cell cycle distribution at G2 /M arrest and apoptosis in a dose-dependent manner. Mitochondria-mediated apoptosis was verified by the increase in Bax/Bcl-2 ratio, release of cytochrome c, and activation of caspase-3 on oleuropein-induced H1299 cells. In addition, our data also demonstrated that the p38 mitogen-activated protein kinase (MAPK) signaling pathway has a critical role in oleuropein-induced apoptosis. Moreover, we used transcriptome analysis to identify differentially expressed genes (DEGs) in H1299 cells by oleuropein and SB203580 treatment. Many DEGs were annotated to metabolic pathways, cell cycle, pathways in cancer, MAPK signaling pathway by Kyoto Encyclopedia of Genes and Genomes and Gene ontology enrichment methods. Network and expression analysis found that DEGs, including RPS6A5, GADD45A, and MKP, play a key role in the p38 MAPK signaling pathway. In H1299 cells, oleuropein resulted in the expression of numerous genes related to cell signaling, metabolism pathway and directly associated with apoptosis. These results illustrated that oleuropein-induced apoptosis via mitochondrial apoptotic cascade was activated by the p38 MAPK signaling pathway in H1299 cells. Thus, oleuropein as a natural compound and therapeutic drug has potential application value in the treatment of lung cancer.

Silencing Ras-Related C3 Botulinum Toxin Substrate 1 Inhibits Growth and Migration of Hypopharyngeal Squamous Cell Carcinoma via the P38 Mitogen-Activated Protein Kinase Signaling Pathway.

BACKGROUND Ras-related C3 botulinum toxin substrate 1 (Rac1) is implicated in a variety of cellular functions and is related to tumor growth and metastasis. This study aimed to explore the role of Rac1 in hypopharyngeal squamous cell carcinoma (HSCC). MATERIAL AND METHODS The Rac1 expression in HSCC tissues was determined by quantitative real-time polymerase chain reaction and Western blot analysis. The level of Rac1 in HSCC cells was downregulated by a Rac1-specific shRNA. Then, the growth and metastasis of HSCC cells were assessed in vitro by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, flow cytometry, Hoechst staining, and Transwell assay. Moreover, cells transfected with Rac1 shRNA or negative control were injected subcutaneously into the right axilla of mice, and then the effects of Rac1 silencing on the growth of HSCC were also explored in vivo. Additionally, activation of the P38 mitogen-activated protein kinase (MAPK) signaling pathway was assessed by Western blot. RESULTS Rac1 was highly expressed in HSCC tissues. Silencing Rac1 inhibited the proliferation and cell cycle progress of HSCC cells, and induced their apoptosis. Rac1 silencing also suppressed the migration and invasion of HSCC cells. In vivo study showed that silencing Rac1 suppressed the growth of tumor bodies. Moreover, the P38 MAPK signaling pathway was implicated in the tumor-suppressing effect of Rac1 silencing in vitro and in vivo. CONCLUSIONS Silencing Rac1 suppressed the growth and migration of HSCC through the P38 MAPK signaling pathway. Due to its contribution in HSCC, Rac1 has the potential to become a promising antitumor therapeutic target for HSCC.

P38 MAP kinase is involved in oleuropein-induced apoptosis in A549 cells by a mitochondrial apoptotic cascade.

Lung cancer is one of malignant tumors that cause great threats to human health, which causes the fastest growing morbidity and mortality. Oleuropein as natural production exerts anticancer effects in several cancer cells. In the study, we investigated apoptotic effect of oleuropein on A549 cells and the underlying mechanisms. Oleuropein markedly decreased cell viability in A549 cells by resulting in G2/M phase arrest, but failed to decreased cell viability in BEAS-2B cells significantly. Apoptosis by oleuropein was confirmed by apoptotic morphology, accumulation in a sub-G1 peak, nucleus fragmentation and cleavage of PARP. Dose-dependent elevation in p-p38MAPK and p-ATF-2 was observed whereas apparent changes could not be observed in p-JNK and p-c-Jun, showing activation of p38MAPK but not JNK. Interestingly, ERK1/2 appeared to be constant while p-ERK1/2 was reduced dose-dependently. Oleuropein caused decrease in mitochondrial membrane potential, increase in Bax/Bcl- 2 ratio, release of mitochondrial cytochrome c and activation of caspase-9 and caspase-3, implying that mitochondrial apoptotic pathway was activated. Additionally, oleuropein-induced apoptosis was dramatically attenuated by Z-VAD-FMK (caspase inhibitor). The p38MAPK inhibitor prevented production of apoptotic bodies and reduced expressions of cleaved-PARP, p-P38, p-ATF-2 and release of cytochrome c. Taken together, these results demonstrated p38MAPK signaling pathway mediated oleuropein-induced apoptosis via mitochondrial apoptotic cascade in A549 cells. Oleuropein has the potential to be a therapeutic drug for lung cancer treatment.

Interaction between Pin1 and its natural product inhibitor epigallocatechin-3-gallate by spectroscopy and molecular dynamics simulations.

The binding of epigallocatechin-3-gallate (EGCG) to wild type Pin1 in solution was studied by spectroscopic methods and molecular dynamics simulations in this research to explore the binding mode and inhibition mechanism. The binding constants and number of binding sites per Pin1 for EGCG were calculated through the Stern-Volmer equation. The values of binding free energy and thermodynamic parameters were calculated and indicated that hydrogen bonds, electrostatic interaction and Van der Waals interaction played the major role in the binding process. The alterations of Pin1 secondary structure in the presence of EGCG were confirmed by far-UV circular dichroism spectra. The binding model at atomic-level revealed that EGCG was bound to the Glu12, Lys13, Arg14, Met15 and Arg17 in WW domain. Furthermore, EGCG could also interact with Arg69, Asp112, Cys113 and Ser114 in PPIase domain.

Unfolded-protein response-associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract.

Failure of lens fiber cell denucleation (LFCD) is associated with congenital cataracts, but the pathobiology awaits elucidation. Recent work has suggested that mechanisms that direct the unidirectional process of LFCD are analogous to the cyclic processes associated with mitosis. We found that lens-specific mutations that elicit an unfolded-protein response (UPR) in vivo accumulate p27(Cdkn1b), show cyclin-dependent kinase (Cdk)-1 inhibition, retain their LFC nuclei, and are cataractous. Although a UPR was not detected in lenses expressing K6W-Ub, they also accumulated p27 and showed failed LFCD. Induction of a UPR in human lens epithelial cells (HLECs) also induced accumulation of p27 associated with decreased levels of S-phase kinase-associated protein (Skp)-2, a ubiquitin ligase that regulates mitosis. These cells also showed decreased lamin A/C phosphorylation and metaphase arrest. The suppression of lamin A/C phosphorylation and metaphase transition induced by the UPR was rescued by knockdown of p27. Taken together, these data indicate that accumulation of p27, whether related to the UPR or not, prevents the phosphorylation of lamin A/C and LFCD in maturing LFCs in vivo, as well as in dividing HLECs. The former leads to cataract and the latter to metaphase arrest. These results suggest that accumulation of p27 is a common mechanism underlying retention of LFC nuclei.

The structural and functional role of the three tryptophan residues in Pin1.

Pin1, the only known isomerase catalyzing phosphorylated pSer/pThr-Pro motifs in proteins, plays unique roles in human diseases notably cancers and Alzheimer's disease. Herein, site-directed mutagenesis was employed to construct the tryptophan mutants of Pin1, including W11L, W34L, and W73L. Spectral methodologies, activity measurement, and proteinase resistance analysis were used to investigate the structural and functional role of the tryptophan residues in Pin1. In general, W11 and W34 are essential to the structure and the function of Pin1, because their mutations influence the structure of WW domain of Pin1, potentially attenuate the binding affinity of Pin1 to substrates, and thus inhibit the enzymatic activity of Pin1. Particularly, W11 mutation results in significantly varied structural features of Pin1 as revealed by fluorescence and circular dichroism (CD) spectroscopies, and decreases the enzymatic activity, thermal stability, and proteinase resistance of Pin1, all of which give an explanation for the high conservation of W11 in vivo. The synchronous fluorescence spectra indicate that W11 and W34 mutations possibly block their energy transfer to Y23 or Y24, suggesting the structural rearrangement in WW domain. By contrast, W73 is of minor importance for the structure and the function of Pin1, because the parameters of W73L observed in several experiments are very similar to wild-type Pin1. These observations are very beneficial for further understanding the structural and functional characteristics of Pin1 and for unveiling the pathogenesis of Pin1-related diseases especially those caused by tryptophan mutations.

Role of pH in structural changes for Pin1 protein: an insight from molecular dynamics study.

Pin1 protein is closely associated with the pathogenesis of cancers and Alzheimer's disease (AD). Previously, we have shown the acid-induced denaturation of Pin1 was determined by means of fluorescence emission, synchronous fluorescence etc., indicating an intermediate state around chromophores in Pin1 at about 4.0. Molecular dynamics simulations for the wild type Pin1 and its mutants were performed to explore the role of pH in the conformation changes of Pin1 protein. Our present study shows that one protein domain (PPIase domain) is more sensitive than the other one (WW domain) in Pin1 protein, and also our study shows that the integrality of the two conserve tryptophan in one domain (WW) is important in response to low pH. We arrive at the last result with the analysis of the protein root mean square distance and the analysis of the radius of gyration. The analysis of protein solvent accessible surface area values have proven our previous experiment result that there is an intermediate state around tryptophan residues at about pH 4.0. Moreover, acidic states of the protein can break the alpha-helixes in Pin1, especially the alpha-helix α3 close to active sites; as a result, Pin1 loses most of its activity at low pH. The results help us to understand the role of pH in Pin1, provide us insights into the conformation change at the atomic-level and emphasize the important role of decreased pH in the pathogenesis of some Pin1-related diseases, and support the therapeutic approach for the related Pin1 diseases by targeting acidosis and modifying the intracellular pH gradients.

Oleuropein induced apoptosis in HeLa cells via a mitochondrial apoptotic cascade associated with activation of the c-Jun NH2-terminal kinase.

Oleuropein could inhibit growth and/or induce apoptosis in several cancer cell lines. In this study, we investigate how oleuropein strongly induces apoptotic cell death in HeLa human cervical carcinoma cells. Oleuropein induced HeLa cells apoptosis as demonstrated by induction of a sub-G(1) peak in flow cytometry and apoptosis-related morphological changes observed by fluorescence microscopy after being stained by Hoechst 33324. The results also showed that 150 - 200 µM oleuropein–treated HeLa cells were arrested at the G(2)/M phase. Western blot analysis revealed that the phosphorylated ATF-2, c-Jun NH(2)-terminal kinase (JNK) protein, p53, p21, Bax, and cytochrome c protein in the cytoplasm significantly increased in a dose-dependent manner after treatment of oleuropein for 24 h. Additionally, increasing levels of Bax in response to JNK/SPAK signaling, which formed mitochondrial membrane channels, accounted for releasing of cytochrome c and activation of caspase-9 and -3. SP600125 (20 µM), a JNK(1/2) inhibitor, markedly suppressed the formation of apoptotic bodies and JNK activation induced by oleuropein at 200 µM. Thus, oleuropein-induced apoptosis was activated by the JNK/SPAK signal pathway. The result shows that oleuropein holds promise as a potential chemotherapeutic agent for the treatment of HeLa cells.

Induction of systemic resistance against tobacco mosaic virus by Ningnanmycin in tobacco.

Ningnanmycin (NNM) is an antiviral agent firstly isolated from Strepcomces noursei var·xichangensisn. Studies have shown that NNM promotes PAL, POD and SOD activity and possesses antiviral activity against tobacco mosaic virus (TMV). In this study, our results demonstrated that NNM inhibited the polymerization process of TMV coat protein (TMV-CP) in vitro and promoted the systemic accumulation of pathogenesis-related proteins (PRs), which are the markers of systemic acquired resistance (SAR). An non-expressor, pathogenesis-related genes 1 (NPR1) that regulates SAR and induces systemic resistance (ISR), increased. In addition, the Jaz3 expression increase showed that NNM also induced ISR. Based on the results of this work and earlier reports, it is suggesting that NNM induces tobacco systemic resistance against TMV via activating multiple plant defense signaling pathways.

Insight into the structural stability of wild type and mutants of the tobacco etch virus protease with molecular dynamics simulations.

The efficiency and high specificity of tobacco etch virus protease (TEVp) has made it widely used for cleavage of recombinant fusion proteins. However, TEVp suffers from a few intrinsic defects such as self-cleavage, poorly expressed in E. coli and less soluble. So some mutants were designed to improve it, such as S219V, T17S/N68D/I77V and L56V/S135G etc. MD simulations for the WT TEVp and its mutants were performed to explore the underlying dynamic effects of mutations on TEVp. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of TEVp, including the elongation of ß-sheet, conversion of loop to helix and the flexibility of active core. Our present study indicates that the three mutants for TEVp can change their secondary structure and tend to form more helixes and sheets to improve stability. The study also helps us to understand the effects of some mutations on TEVp, provides us insights into the change of them at the atomic level and gives a potential rational method to design an improved protein.

Aluminum(III) interferes with the structure and the activity of the peptidyl-prolyl cis-trans isomerase (Pin1): a new mechanism contributing to the pathogenesis of Alzheimer's disease and cancers?

The enzyme peptidyl-prolyl cis-trans isomerase (Pin1) may play an important role in preventing the development of Alzheimer's disease (AD). The structural and functional stability of Pin1 is extremely important. Previously, we have determined the stability of Pin1 under stressed conditions, such as thermal treatment and acidic-pH. Considering that aluminum (Al(III)) is well known for its potential neurotoxicity in the pathogenesis of AD, we examined whether Al(III) affects the structure and function of Pin1, by means of a PPIase activity assay, intrinsic fluorescence, circular dichroism (CD) spectroscopy, FTIR, and differential scanning calorimetry (DSC). The intrinsic tryptophan fluorescence measurements mainly show that Al(III) may bind to the clusters nearby W11 and W34 in the WW domain of Pin1, quenching the intrinsic fluorescence of the two tryptophan residues, which possibly results in the decreased binding affinity of Pin1 to substrates. The secondary structural analysis as revealed by FTIR and CD measurements indicate that Al(III) induces the increase in ß-sheet and the decrease in α-helix in Pin1. Furthermore, the changes of the thermodynamic parameters for Pin1 as monitored by DSC confirm that the thermal stability of Pin1 significantly increases in the presence of Al(III). The Al(III)-induced structural changes of Pin1 result in a sharp decrease of the PPIase activity of Pin1. To some extent, our research is suggestive that Al(III) may inhibit the isomerization activity of Pin1 in vivo, which may contribute to the pathogenesis of AD.

P38 MAP kinase mediates apoptosis after genipin treatment in non-small-cell lung cancer H1299 cells via a mitochondrial apoptotic cascade.

Genipin, an active constituent of Gardenia fruit, has been reported to show an anti-tumor effect in several cancer cell systems. Here, we demonstrate how genipin exhibits a strong apoptotic cell death effect in human non-small-cell lung cancer H1299 cells. Genipin-mediated decrease in cell viability was observed through apoptosis as demonstrated by induction of a sub-G1 peak through flow cytometry, DNA fragmentation measured by TUNEL assay, and cleavage of poly ADP-ribose-polymerase. During genipin-induced apoptosis, the mitochondrial execution pathway was activated by caspase-9 and -3 activation as examined by a kinetic study, cytochrome c release, and a dose-dependent increase in Bax/Bcl-2 ratio. A search for the downstream pathway reveals that genipin-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2. SB203580, a p38MAPK inhibitor, markedly blocked the formation of TUNEL-positive apoptotic cells in genipin-treated cells. Besides, the interference of p38MAPK inhibited Bax expression and cytochrome c release. Altogether, our observations imply that genipin causes increased levels of Bax in response to p38MAPK signaling, which results in the initiation of mitochondrial death cascade, and therefore it holds promise as a potential chemotherapeutic agent for the treatment of H1299 cells.

The acidic pH-induced structural changes in Pin1 as revealed by spectral methodologies.

Pin1 is closely associated with the pathogenesis of cancers and Alzheimer's disease (AD). Previously, we have shown the characteristics of the thermal denaturation of Pin1. Herein, the acid-induced denaturation of Pin1 was determined by means of fluorescence emission, synchronous fluorescence, far-UV CD, ANS fluorescence and RLS spectroscopies. The fluorescence emission spectra and the synchronous fluorescence spectra suggested the partially reversible unfolding (approximately from pH 7.0 to 4.0) and refolding (approximately from pH 4.0 to 1.0) of the structures around the chromophores in Pin1, apparently with an intermediate state at about pH 4.0-4.5. The far-UV CD spectra indicated that acidic pH (below pH 4.0) induced the structural transition from α-helix and random coils to ß-sheet in Pin1. The ANS fluorescence and the RLS spectra further suggested the exposure of the hydrophobic side-chains of Pin1 and the aggregation of it especially below pH 2.3, and the aggregation possibly resulted in the formation of extra intermolecular ß-sheet. The present work primarily shows that acidic pH can induce kinds of irreversible structural changes in Pin1, such as the exposure of the hydrophobic side-chains, the transition from α-helix to ß-sheet and the aggregation of Pin1, and also explains why Pin1 loses most of its activity below pH 5.0. The results emphasize the important role of decreased pH in the pathogenesis of some Pin1-related diseases, and support the therapeutic approach for them by targeting acidosis and modifying the intracellular pH gradients.

Apoptosis induced by genipin in human leukemia K562 cells: involvement of c-Jun N-terminal kinase in G2/M arrest.

AIM: To investigate the effect of genipin on apoptosis in human leukemia K562 cells in vitro and elucidate the underlying mechanisms. METHODS: The effect of genipin on K562 cell viability was measured using trypan blue dye exclusion and cell counting. Morphological changes were detected using phase-contrast microscopy. Apoptosis was analyzed using DNA ladder, propidium iodide (PI)-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of genipin on cell cycle distribution was determined using PI staining. Caspase 3 activity was analyzed to detect apoptosis at different time points. Protein levels of phospho-c-Jun, phosphor-c-Jun N-terminal kinase (p-JNK), phosphor-p38, Fas-L, p63, and Bax and the release of cytochrome c were detected using Western blot analysis. RESULTS: Genipin reduced the viability of K562 cells with an IC(50) value of approximately 250 µmol/L. Genipin 200-400 µmol/L induced formation of typical apoptotic bodies and DNA fragmentation. Additionally, genipin 400 µmol/L significantly increased the caspase 3 activity from 8-24 h and arrested the cells in the G2/M phase. After stimulation with genipin 500 µmol/L, the levels of p-JNK, p-c-Jun, Fas-L, Bax, and cytochrome c were remarkably upregulated, but there were no obvious changes of p-p38. Genipin 200-500 µmol/L significantly upregulated the Fas-L expression and downregulated p63 expression. Dicoumarol 100 µmol/L, a JNK1/2 inhibitor, markedly suppressed the formation of apoptotic bodies and JNK activation induced by genipin 400 µmol/L. CONCLUSION: These results suggest that genipin inhibits the proliferation of K562 cells and induces apoptosis through the activation of JNK and induction of the Fas ligand.

Genipin induced apoptosis associated with activation of the c-Jun NH2-terminal kinase and p53 protein in HeLa cells.

Genipin is a metabolite of geniposide isolated from an extract of Gardenia fructus. Some observations suggested that genipin could induce cell apoptosis in hepatoma cells and PC3 human prostate cancer cells. However, the effects of genipin on HeLa human cervical carcinoma cells are still unknown. In this study, we provided evidences that genipin induced the death of HeLa cells through apoptotic pathway in a dose-dependent manner. Genipin could remarkably induce cytotoxicity in HeLa cells and inhibit its proliferation. Induction of the apoptosis by genipin was confirmed by analysis of DNA fragmentation and induction of sub-G(1) peak through flow cytometry. The results also showed that genipin-treated HeLa cells cycle was arrested at G(1) phase. Western blot analysis revealed that the phosphorylated c-Jun NH(2)-terminal kinase (JNK) protein, phospho-Jun protein, p53 protein and bax protein significantly increased in a dose-dependent manner after treatment of genipin for 24 h, and to our knowledge, the activation of JNK maybe result in the increase of the p53 protein level, and the increase of the p53 protein led to the accumulation of bax protein, bax protein further induced cell apoptotic death eventually. Taken all these together, it is possible to develop genipin as an anti-cancer drug.