Precision targeting tumor cells using cancer-specific InDel mutations with CRISPR-Cas9.

An ideal cancer therapeutic strategy involves the selective killing of cancer cells without affecting the surrounding normal cells. However, researchers have failed to develop such methods for achieving selective cancer cell death because of shared features between cancerous and normal cells. In this study, we have developed a therapeutic strategy called the cancer-specific insertions-deletions (InDels) attacker (CINDELA) to selectively induce cancer cell death using the CRISPR-Cas system. CINDELA utilizes a previously unexplored idea of introducing CRISPR-mediated DNA double-strand breaks (DSBs) in a cancer-specific fashion to facilitate specific cell death. In particular, CINDELA targets multiple InDels with CRISPR-Cas9 to produce many DNA DSBs that result in cancer-specific cell death. As a proof of concept, we demonstrate here that CINDELA selectively kills human cancer cell lines, xenograft human tumors in mice, patient-derived glioblastoma, and lung patient-driven xenograft tumors without affecting healthy human cells or altering mouse growth.

Augmented ERAD (ER-associated degradation) activity in chondrocytes is necessary for cartilage development and maintenance.

Chondrocytes secrete massive extracellular matrix (ECM) molecules that are produced, folded, and modified in the endoplasmic reticulum (ER). Thus, the ER-associated degradation (ERAD) complex-which removes misfolded and unfolded proteins to maintain proteostasis in the ER- plays an indispensable role in building and maintaining cartilage. Here, we examined the necessity of the ERAD complex in chondrocytes for cartilage formation and maintenance. We show that ERAD gene expression is exponentially increased during chondrogenesis, and disruption of ERAD function causes severe chondrodysplasia in developing embryos and loss of adult articular cartilage. ERAD complex malfunction also causes abnormal accumulation of cartilage ECM molecules and subsequent chondrodysplasia. ERAD gene expression is decreased in damaged cartilage from patients with osteoarthritis (OA), and disruption of ERAD function in articular cartilage leads to cartilage destruction in a mouse OA model.

Integrin ß-like 1 protein (ITGBL1) promotes cell migration by preferentially inhibiting integrin-ECM binding at the trailing edge.

BACKGROUND: Cell migration is a basic cellular behavior involved in multiple phenomena in the human body such as embryonic development, wound healing, immune reactions, and cancer metastasis. For proper cell migration, integrin and the ECM binding complex must be disassembled for the retraction of trailing edges. OBJECTIVE: Integrin must be differentially regulated at leading edges or trailing edges during cell migration. Previously, we showed that ITGBL1 was a secreted protein and inhibits integrin activity. Therefore, we examined the function of ITGBL1 on the retraction of trailing edges during cell migration. METHODS: To examined the function of ITGBL1 on cell migration, we knocked-down or overexpressed ITGBL1 by using ITGBL1 siRNA or ITGBL1 plasmid DNA in human chondrocytes or ATDC5 cells. We then characterized cellular migration and directionality by performing wound healing assays. Also, to analyze leading-edge formation and trailing-edge retraction, we labeled cell membranes with membrane-GFP and performed live imaging of migrating cells and. Finally, we specifically detected active forms of integrin, FAK and Vinculin using specific antibodies upon ITGBL1 depletion or overexpression. RESULT: In this study, ITGBL1 preferentially inhibited integrin activity at the trailing edges to promote cell migration. ITGBL1-depleted cells showed increased focal adhesions at the membranous traces of trailing edges to prevent the retraction of trailing edges. In contrast, overexpression of ITGBL1 upregulated directional cell migration by promoting focal adhesion disassembly at the trailing edges. CONCLUSION: ITGBL1 facilitates directional cell migration by promoting disassembly of the trailing edge focal adhesion complex.

Extracellular matrixes and neuroinflammation.

The extracellular matrix is a critical component of every human tissue. ECM not only functions as a structural component but also regulates a variety of cellular processes such as cell migration, differentiation, proliferation, and cell death. In addition, current studies suggest that ECM is critical for the pathophysiology of various human diseases. ECM is composed of diverse components including several proteins and polysaccharide chains such as chondroitin sulfate, heparan sulfate, and hyaluronic acid. Each component of ECM exerts its own functions in cellular and pathophysiological processes. One of the interesting recent findings is that ECM is involved in inflammatory responses in various human tissues. In this review, we summarized the known functions of ECM in neuroinflammation after acute injury and chronic inflammatory diseases of the central nerve systems. [BMB Reports 2020; 53(10): 491-499].

Additive effect of walnut and chokeberry on regulation of antioxidant enzyme gene expression and attenuation of lipid peroxidation in d-galactose-induced aging-mouse model.

Studies have highlighted the association between the cellular damage caused by reactive oxygen species and aging. The reducing sugar d-galactose causes aging-related changes and oxidative stress. Lipids are the first target of free radicals, and lipid peroxidation is related to aging. Walnut (Juglans regia Chandler) kernel contains antioxidant phenolic compounds, and chokeberry (Aronia melanocarpa) is one of the richest sources of polyphenols, including anthocyanins, among other fruits. Polyphenols from chokeberry exhibit antioxidant and anti-inflammatory activities. In this study, the additive antioxidative effect of walnut and chokeberry mixture was evaluated by oxidative stress index in d-galactose-induced aging model. Thirty-five Balb/c mice (8 weeks old) were divided into following five groups (n = 7 in each group): normal control (C), d-galactose control (D), d-galactose with chokeberry diet (CH), d-galactose with walnut diet (W), and d-galactose with walnut and chokeberry mixture diet (WCH). In all treatment diets groups, the levels of serum, hepatic, and kidney malonaldehyde were significantly lower than D group and the levels were approaching to control level. Moreover, the kidney malondialdehyde levels were significantly lower in WCH group compared with the control group. This study also confirmed the activities of antioxidant enzymes in liver, as the levels of superoxide dismutase, and glutathione peroxidase were significantly increased in CH group compared to in W or CH groups. The results of this study supported the additive effect of walnut and chokeberry on increment of antioxidant enzyme gene expression in liver and consequently the attenuation of lipid peroxidation in serum, liver, and kidney in d-galactose-induced aging-mouse model. Further studies are needed to investigate the detailed mechanism underlying the additive antioxidative effects in various tissues.

ITGBL1 modulates integrin activity to promote cartilage formation and protect against arthritis.

Developing and mature chondrocytes constantly interact with and remodel the surrounding extracellular matrix (ECM). Recent research indicates that integrin-ECM interaction is differentially regulated during cartilage formation (chondrogenesis). Integrin signaling is also a key source of the catabolic reactions responsible for joint destruction in both rheumatoid arthritis and osteoarthritis. However, we do not understand how chondrocytes dynamically regulate integrin signaling in such an ECM-rich environment. Here, we found that developing chondrocytes express integrin-ß-like 1 (Itgbl1) at specific stages, inhibiting integrin signaling and promoting chondrogenesis. Unlike cytosolic integrin inhibitors, ITGBL1 is secreted and physically interacts with integrins to down-regulate activity. We observed that Itgbl1 expression was strongly reduced in the damaged articular cartilage of patients with osteoarthritis (OA). Ectopic expression of Itgbl1 protected joint cartilage against OA development in the destabilization of the medial meniscus-induced OA mouse model. Our results reveal ITGBL1 signaling as an underlying mechanism of protection against destructive cartilage disorders and suggest the potential therapeutic utility of targeting ITGBL1 to modulate integrin signaling in human disease.

3'-Sialyllactose protects against osteoarthritic development by facilitating cartilage homeostasis.

3'-Sialyllactose has specific physiological functions in a variety of tissues; however, its effects on osteoarthritic development remain unknown. Here, we demonstrated the function of 3'-sialyllactose on osteoarthritic cartilage destruction. In vitro and ex vivo, biochemical and histological analysis demonstrated that 3'-sialyllactose was sufficient to restore the synthesis of Col2a1 and accumulation of sulphated proteoglycan, a critical factor for cartilage regeneration in osteoarthritic development, and blocked the expression of Mmp3, Mmp13 and Cox2 induced by IL-1ß, IL-6, IL-17 and TNF-α, which mediates cartilage degradation. Further, reporter gene assays revealed that the activity of Sox9 as a transcription factor for Col2a1 expression was accelerated by 3'-sialyllactose, whereas the direct binding of NF-κB to the Mmp3, Mmp13 and Cox2 promoters was reduced by 3'-sialyllactose in IL-1ß-treated chondrocytes. Additionally, IL-1ß induction of Erk phosphorylation and IκB degradation, representing a critical signal pathway for osteoarthritic development, was totally blocked by 3'-sialyllactose in a dose-dependent manner. In vivo, 3'-sialyllactose protected against osteoarthritic cartilage destruction in an osteoarthritis mouse model induced by destabilization of the medial meniscus, as demonstrated by histopathological analysis. Our results strongly suggest that 3'-sialyllactose may ameliorate osteoarthritic cartilage destruction by cartilage regeneration via promoting Col2a1 production and may inhibit cartilage degradation and inflammation by suppressing Mmp3, Mmp13 and Cox2 expression. The effects of 3'-sialyllactose could be attributed in part to its regulation of Sox9 or NF-κB and inhibition of Erk phosphorylation and IκB degradation. Taken together, these effects indicate that 3'-sialyllactose merits consideration as a natural therapeutic agent for protecting against osteoarthritis.

A Recombinant Secondary Antibody Mimic as a Target-specific Signal Amplifier and an Antibody Immobilizer in Immunoassays.

We construct a novel recombinant secondary antibody mimic, GST-ABD, which can bind to the Fc regions of target-bound primary antibodies and acquire multiple HRPs simultaneously. We produce it in tenth of mg quantities with a bacterial overexpression system and simple purification procedures, significantly reducing the manufacturing cost and time without the use of animals. GST-ABD is effectively conjugated with 3 HRPs per molecule on an average and selectively bind to the Fc region of primary antibodies derived from three different species (mouse, rabbit, and rat). HRP-conjugated GST-ABD (HRP-GST-ABD) is successfully used as an alternative to secondary antibodies to amplify target-specific signals in both ELISA and immunohistochemistry regardless of the target molecules and origin of primary antibodies used. GST-ABD also successfully serves as an anchoring adaptor on the surface of GSH-coated plates for immobilizing antigen-capturing antibodies in an orientation-controlled manner for sandwich-type indirect ELISA through simple molecular recognition without any complicated chemical modification.

Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway.

Terminally misfolded proteins are selectively recognized and cleared by the endoplasmic reticulum-associated degradation (ERAD) pathway. SEL1L, a component of the ERAD machinery, plays an important role in selecting and transporting ERAD substrates for degradation. We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1L(cent)). Strikingly, SEL1L(cent) forms a homodimer with two-fold symmetry in a head-to-tail manner. Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. We identified that the full-length SEL1L forms a self-oligomer through the SEL1L(cent) domain in mammalian cells. Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. Therefore, we propose that certain SLR motifs of SEL1L play a unique role in membrane bound ERAD machinery.

An enhanced ascorbate peroxidase 2/antibody-binding domain fusion protein (APEX2-ABD) as a recombinant target-specific signal amplifier.

A recombinant target-specific signal amplifier was constructed by genetically fusing the enhanced ascorbate peroxidase 2 (APEX2) and an antibody-binding domain (ABD). The fusion protein APEX2-ABD possessed the peroxidase activity and the antibody-binding capability simultaneously and replaced the conventional HRP-conjugated secondary antibodies in a TSA assay for amplifying fluorescence signals.

A thioredoxin fold protein Sh3bgr regulates Enah and is necessary for proper sarcomere formation.

The sh3bgr (SH3 domain binding glutamate-rich) gene encodes a small protein containing a thioredoxin-like fold, SH3 binding domain, and glutamate-rich domain. Originally, it was suggested that increased expression of Sh3bgr may cause the cardiac phenotypes in Down's syndrome. However, it was recently reported that the overexpression of Sh3bgr did not cause any disease phenotypes in mice. In this study, we have discovered that Sh3bgr is critical for sarcomere formation in striated muscle tissues and also for heart development. Sh3bgr is strongly expressed in the developing somites and heart in Xenopus. Morpholino mediated-knockdown of sh3bgr caused severe malformation of heart tissue and disrupted segmentation of the somites. Further analysis revealed that Sh3bgr specifically localized to the Z-line in mature sarcomeres and that knockdown of Sh3bgr completely disrupted sarcomere formation in the somites. Moreover, overexpression of Sh3bgr resulted in abnormally discontinues thick firmaments in the somitic sarcomeres. We suggest that Sh3bgr does its function at least partly by regulating localization of Enah for the sarcomere formation. In addition, we provide the data supporting Sh3bgr is also necessary for proper heart development in part by affecting the Enah protein level.

Interferon ß protects against lethal endotoxic and septic shock through SIRT1 upregulation.

Lipopolysaccharide (LPS), an endotoxin derived from gram-negative bacteria, promotes the secretion of proinflammatory cytokines and mediates endotoxemia through activation of mitogen activated protein kinases, NF-κB, and interferon regulatory factor-3. Silent information regulator transcript-1 (SIRT1), an NAD-dependent deacetylase, mediates NF-κB deacetylation, and inhibits its function. SIRT1 may affect LPS-mediated signaling pathways and endotoxemia. Here we demonstrate that SIRT1 blocks LPS-induced secretion of interleukin 6 and tumor necrosis factor α in murine macrophages, and protects against lethal endotoxic and septic shock in mice. We also demonstrate that interferon ß increases SIRT1 expression by activating the Janus kinase--signal transducer and activator of transcription (JAK-STAT) pathway in mouse bone marrow derived macrophages. In vivo treatment of interferon ß protects against lethal endotoxic and septic shock, which is abrogated by infection with dominant negative SIRT1-expressing adenovirus. Our work suggests that both SIRT1 and SIRT1-inducing cytokines are useful targets for treating patients with sepsis.

Interplay between estrogen receptor and AKT in estradiol-induced alternative splicing.

BACKGROUND: Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. METHODS: MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. RESULTS: We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF. CONCLUSION: E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.

Lipopolysaccharide induces CD38 expression and solubilization in J774 macrophage cells.

CD38, an ADP ribosyl cyclase, is a 45 kDa type II transmembrane protein having a short N-terminal cytoplasmic domain and a long C-terminal extracellular domain, expressed on the surface of various cells including macrophages, lymphocytes, and pancreatic ß cells. It is known to be involved in cell adhesion, signal transduction and calcium signaling. In addition to its transmembrane form, CD38 is detectable in biological fluids in soluble forms. The mechanism by which CD38 is solubilized from the plasma membrane is not yet clarified. In this study, we found that lipopolysaccharide (LPS) induced CD38 upregulation and its extracellular release in J774 macrophage cells. Furthermore, it also increased CD38 expression at the mRNA level by activating the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway. However, LPS decreased the levels of CD38 in the plasma membrane by releasing CD38 into the culture supernatant. LPS-induced CD38 release was blocked by the metalloproteinase-9 inhibitor indicating that MMP-9 solubilizes CD38. In conclusion, the present findings demonstrate a potential mechanism by which C38 is solubilized from the plasma membrane.

NAADP mediates insulin-stimulated glucose uptake and insulin sensitization by PPARγ in adipocytes.

Insulin stimulates glucose uptake through the membrane translocation of GLUT4 and GLUT1. Peroxisome proliferator-activated receptor γ (PPARγ) enhances insulin sensitivity. Here, we demonstrate that insulin stimulates GLUT4 and GLUT1 translocation, and glucose uptake, by activating the signaling pathway involving nicotinic acid adenine dinucleotide phosphate (NAADP), a calcium mobilizer, in adipocytes. We also demonstrate that PPARγ mediates insulin sensitization by enhancing NAADP production through upregulation of CD38, the only enzyme identified for NAADP synthesis. Insulin produced NAADP by both CD38-dependent and -independent pathways, whereas PPARγ produced NAADP by CD38-dependent pathway. Blocking the NAADP signaling pathway abrogated both insulin-stimulated and PPARγ-induced GLUT4 and GLUT1 translocation, thereby inhibiting glucose uptake. CD38 knockout partially inhibited insulin-stimulated glucose uptake. However, CD38 knockout completely blocked PPARγ-induced glucose uptake in adipocytes and PPARγ-mediated amelioration of glucose tolerance in diabetic mice. These results demonstrated that the NAADP signaling pathway is a critical molecular target for PPARγ-mediated insulin sensitization.

SIRT1 Inhibits p53 but not NF-κB Transcriptional Activity during Differentiation of Mouse Embryonic Stem Cells into Embryoid Bodies.

BACKGROUND AND OBJECTIVES: SIRT1, a histone diacetylase, modify transactivation function of various transcription factor including p53 and NF-κB. p53 and NF-κB is involved in in vitro differentiation of mouse embryonic stem cells (mESC) into mouse embryoid body (mEB). These suggest that SIRT1 might affect in vitro differentiation of mESC into mEB by regulation of p53 and NF-κB. METHODS AND RESULTS: In this study we analyzed the effect of SIRT1 in in vitro differentiation of mESC into mEB using wild and SIRT1 knockout mESC. To examine SIRT1-specific gene in mESC, this study conducted microarray-based differential gene expression analysis between wild and SIRT1 knockout mESC. Comparing their gene expression patterns, this study determined a list of genes regulated by SIRT1. cDNA microarray data-set analysis revealed that genes associated with transcription and signal transduction are significantly modified in SIRT1 knockout mESC. cDNA microarray data-set analysis between mESC and EB in wild and SIRT1 showed that SIRT1 inhibits p53 signaling pathway but not affect NF-κB signaling pathway. CONCLUSIONS: This study suggests that SIRT1 modify mESC differentiation by regulation of p53 transcriptional activity.

Connexin-43 hemichannels mediate cyclic ADP-ribose generation and its Ca2+-mobilizing activity by NAD+/cyclic ADP-ribose transport.

The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD(+). Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fcγ receptor (FcγR) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD(+) export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR stimulation and NAD(+)/cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization.

Overexpression of SIRT1 protects pancreatic beta-cells against cytokine toxicity by suppressing the nuclear factor-kappaB signaling pathway.

OBJECTIVE: SIRT1, a class III histone/protein deacetylase, is known to interfere with the nuclear factor-kappaB (NF-kappaB) signaling pathway and thereby has an anti-inflammatory function. Because of the central role of NF-kappaB in cytokine-mediated pancreatic beta-cell damage, we postulated that SIRT1 might work in pancreatic beta-cell damage models. RESEARCH DESIGN AND METHODS: RINm5F (RIN) cells or isolated rat islets were treated with interleukin-1beta and interferon-gamma. SIRT1 was activated by resveratrol, a pharmacological activator, or ectopic overexpression. The underlying mechanisms of SIRT1 against cytokine toxicity were further explored. RESULTS: Treatment of RIN cells with cytokines induced cell damage, and this damage was well correlated with the expression of the inducible form of nitric oxide (NO) synthase (iNOS) and NO production. However, SIRT1 overexpression completely prevented cytokine-mediated cytotoxicity, NO production, and iNOS expression. The molecular mechanism by which SIRT1 inhibits iNOS expression appeared to involve the inhibition of the NF-kappaB signaling pathway through deacetylation of p65. In addition, SIRT1 activation by either resveratrol or adenoviral-directed overexpression of SIRT1 could prevent cytokine toxicity and maintain normal insulin-secreting responses to glucose in isolated rat islets. CONCLUSIONS: This study will provide valuable information not only into the mechanisms underlying beta-cell destruction but also into the regulation of SIRT1 as a possible target to attenuate cytokine-induced beta-cell damage.

Troglitazone enhances tamoxifen-induced growth inhibitory activity of MCF-7 cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been identified as a potential source of therapy for human cancers. However, PPARgamma ligands have a limitation for breast cancer therapy, since estrogen receptor alpha (ER(alpha)) negatively interferes with PPARgamma signaling in breast cancer cells. Here we show that ER(alpha) inhihits PPARgamma transactivity and ER(alpha)-mediated inhibition of PPARgamma transactivity is blocked by tamoxifen, an estrogen receptor blocker. The activation of ER(alpha) with 17-beta-estradiol blocked PPRE transactivity induced by troglitazone, a PPARgamma ligand, indicating the resistance of ER(alpha)-positive breast cancer cells to troglitazone. Indeed, troglitazone inhibited the growth of ER(alpha)-negative MDA-MB-231 cells more than that of ER(alpha)-positive MCF-7 cells. Combination of troglitazone with tamoxifen led to a marked increase in growth inhibition of ER(alpha)-positive MCF-7 cells compared to either agent alone. Our data indicates that troglitazone enhances the growth inhibitory activity of tamoxifen in ER(alpha)-positive MCF-7 cells.

Induction of G1 phase arrest and apoptosis in MDA-MB-231 breast cancer cells by troglitazone, a synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) ligand.

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands inhibit cell proliferation and induce apoptosis in cancer cells. Here we wished to determine whether the PPARgamma ligand induces apoptosis and cell cycle arrest of the MDA-MB-231 cell, an estrogen receptor alpha negative breast cancer cell line. The treatment of MDA-MB-231 cell with PPARgamma ligands was shown to induce inhibition of cell growth in a dose-dependent manner as determined by MTT assay. Cell cycle analysis showed a G1 arrest in MDA-MB-231 cells exposed to troglitazone. An apoptotic effect by troglitazone demonstrated that apoptotic cells elevated by 2.5-fold from the control level at 10 microM, to 3.1-fold at 50 microM and to 3.5-fold at 75 microM. Moreover, troglitazone treatment, applied in a dose-dependent manner, caused a marked decrease in pRb, cyclin D1, cyclin D2, cyclin D3, Cdk2, Cdk4 and Cdk6 expression as well as a significant increase in p21 and p27 expression. These results indicate that troglitazone causes growth inhibition, G1 arrest and apoptotic death of MDA-MB-231 cells.