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Biomedical subjects

Ching-Shih Chen

Publications and source records attributed to Ching-Shih Chen.

At least 19 recordsLinked to original sources

Antitumor effects of a novel phenylbutyrate-based histone deacetylase inhibitor, (S)-HDAC-42, in prostate cancer.

PURPOSE: To assess the antitumor effects of a novel phenylbutyrate-derived histone deacetylase (HDAC) inhibitor, (S)-HDAC-42, vis-à-vis suberoylanilide hydroxamic acid (SAHA) in in vitro and in vivo models of human prostate cancer. EXPERIMENTAL DESIGN: The in vitro effects of (S)-HDAC-42 and SAHA were evaluated in PC-3, DU-145, or LNCaP human prostate cancer cell lines. Cell viability, apoptosis, and indicators of HDAC inhibition were assessed. Effects on Akt and members of the Bcl-2 and inhibitor of apoptosis protein families were determined by immunoblotting. Immunocompromised mice bearing established s.c. PC-3 xenograft tumors were treated orally with (S)-HDAC-42 (50 mg/kg q.o.d. or 25 mg/kg q.d.) or SAHA (50 mg/kg q.d.) for 28 days. In vivo end points included tumor volumes and intratumoral changes in histone acetylation, phospho-Akt status, and protein levels of Bcl-xL and survivin. RESULTS: (S)-HDAC-42 was more potent than SAHA in suppressing the viability of all cell lines evaluated with submicromolar IC50 values. Relative to SAHA, (S)-HDAC-42 exhibited distinctly superior apoptogenic potency, and caused markedly greater decreases in phospho-Akt, Bcl-xL, and survivin in PC-3 cells. The growth of PC-3 tumor xenografts was suppressed by 52% and 67% after treatment with (S)-HDAC-42 at 25 and 50 mg/kg, respectively, whereas SAHA at 50 mg/kg suppressed growth by 31%. Intratumoral levels of phospho-Akt and Bcl-xL were markedly reduced in (S)-HDAC-42-treated mice, in contrast to mice treated with SAHA. CONCLUSIONS: (S)-HDAC-42 is a potent orally bioavailable inhibitor of HDAC, as well as targets regulating multiple aspects of cancer cell survival, which might have clinical value in prostate cancer chemotherapy and warrants further investigation in this regard.

Administration, Oral↗

Overcoming trastuzumab resistance in HER2-overexpressing breast cancer cells by using a novel celecoxib-derived phosphoinositide-dependent kinase-1 inhibitor.

Although trastuzumab has been successfully used in patients with HER2-overexpressing metastatic breast cancer, resistance is a common problem that ultimately culminates in treatment failure. In light of the importance of Akt signaling in trastuzumab's antitumor action, we hypothesized that concurrent inhibition of Akt could enhance trastuzumab sensitivity and moreover reverse the resistant phenotype in HER2-positive breast cancer cells. Based on our finding that celecoxib mediates antitumor effects through the inhibition of phosphoinositide-dependent kinase-1 (PDK-1)/Akt signaling independently of cyclooxygenase-2 (COX-2), we used celecoxib as a scaffold to develop a COX-2-inactive PDK-1 inhibitor, 2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012). Here, we investigated the effect of OSU-03012 on trastuzumab-mediated apoptosis in four breast cancer cell lines with different HER2 expression and trastuzumab-resistance status, including MDA-MB-231, BT474, SKBR3, and insulin-like growth factor-I receptor-overexpressing SKBR3 (SKBR3/IGF-IR). Effects of trastuzumab and OSU-03012, individually or in combination, on cell viability and changes in pertinent biomarkers including HER2 expression, phosphorylation of Akt, p27(kip1), and the PDK-1 substrate p70(S6K) were assessed. OSU-03012 alone was able to trigger apoptosis in all cell lines with equal potency (IC(50) = 3-4 microM), suggesting no cross-resistance with trastuzumab. Medium dose-effect analysis indicates that OSU-03012 potentiated trastuzumab's antiproliferative effect in HER2-positive cells, especially in SKBR3/IGF-IR cells, through the down-regulation of PDK-1/Akt signaling. This synergy, however, was not observed in HER2-negative MDA-MB-231 cells. This combination treatment represents a novel strategy to increase the efficacy of trastuzumab and to overcome trastuzumab resistance in the treatment of HER2-positive breast cancer.

3-Phosphoinositide-Dependent Protein Kinases↗

Development of small-molecule cyclin D1-ablative agents.

Previously, we demonstrated that the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist troglitazone mediated the repression of cyclin D1 in MCF-7 breast cancer cells by facilitating proteasome-facilitated proteolysis. This PPARgamma-independent mechanism provided a molecular basis for using troglitazone as scaffold to develop a novel class of cyclin D1-ablative agents. The proof of principle of this premise is provided by Delta2TG, in which the introduction of a double bond adjacent to the thiazolidinedione ring abrogated the PPARgamma activity while retaining the activity in cyclin D1 repression. Structural optimization of Delta2TG led to STG28 [(S)-5-(4-{[6-(allyloxy)-2,5,7,8-tetramethylchroman-2-yl]methoxy}-3-methoxybenzylidene)thiazolidine-2,4-dione], which exhibited low micromolar potency in ablating cyclin D1 and inhibiting MCF-7 cell proliferation. It is noteworthy that STG28 mediated the proteasomal degradation of cyclin D1 with a high degree of specificity. Exposure to STG28 did not cause any appreciable change in the expression levels of a series of other cyclins and CDK-dependent kinases. In light of the pivotal role of cyclin D1 in promoting tumorigenesis and drug resistance, this novel cyclin D1-ablating agent may have therapeutic relevance in cancer therapy.

Antineoplastic Agents↗

Downregulation of survivin expression and concomitant induction of apoptosis by celecoxib and its non-cyclooxygenase-2-inhibitory analog, dimethyl-celecoxib (DMC), in tumor cells in vitro and in vivo.

BACKGROUND: 2,5-Dimethyl-celecoxib (DMC) is a close structural analog of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib (Celebrex) that lacks COX-2-inhibitory function. However, despite its inability to block COX-2 activity, DMC is able to potently mimic the anti-tumor effects of celecoxib in vitro and in vivo, indicating that both of these drugs are able to involve targets other than COX-2 to exert their recognized cytotoxic effects. However, the molecular components that are involved in mediating these drugs' apoptosis-stimulatory consequences are incompletely understood. RESULTS: We present evidence that celecoxib and DMC are able to down-regulate the expression of survivin, an anti-apoptotic protein that is highly expressed in tumor cells and known to confer resistance of such cells to anti-cancer treatments. Suppression of survivin is specific to these two drugs, as other coxibs (valdecoxib, rofecoxib) or traditional NSAIDs (flurbiprofen, indomethacin, sulindac) do not affect survivin expression at similar concentrations. The extent of survivin down-regulation by celecoxib and DMC in different tumor cell lines is somewhat variable, but closely correlates with the degree of drug-induced growth inhibition and apoptosis. When combined with irinotecan, a widely used anticancer drug, celecoxib and DMC greatly enhance the cytotoxic effects of this drug, in keeping with a model that suppression of survivin may be beneficial to sensitize cancer cells to chemotherapy. Remarkably, these effects are not restricted to in vitro conditions, but also take place in tumors from drug-treated animals, where both drugs similarly repress survivin, induce apoptosis, and inhibit tumor growth in vivo. CONCLUSION: In consideration of survivin's recognized role as a custodian of tumor cell survival, our results suggest that celecoxib and DMC might exert their cytotoxic anti-tumor effects at least in part via the down-regulation of survivin - in a manner that does not require the inhibition of cyclooxygenase-2. Because inhibition of COX-2 appears to be negligible, it might be worthwhile to further evaluate DMC's potential as a non-coxib alternative to celecoxib for anti-cancer purposes.

Animals↗

Increased PTEN expression due to transcriptional activation of PPARgamma by Lovastatin and Rosiglitazone.

Germline mutations in the tumor suppressor gene PTEN (protein phosphatase and tensin homolog located on chromosome ten) predispose to heritable breast cancer. The transcription factor PPARgamma has also been implicated as a tumor suppressor pertinent to a range of neoplasias, including breast cancer. A putative PPARgamma binding site in the PTEN promoter indicates that PPARgamma may regulate PTEN expression. We show here that the PPARgamma agonist Rosiglitazone, along with Lovastatin, induce PTEN in a dose- and time-dependent manner. Lovastatin- or Rosiglitazone-induced PTEN expression was accompanied by a decrease in phosphorylated-AKT and phosphorylated-MAPK and an increase in G1 arrest. We demonstrate that the mechanism of Lovastatin- and Rosiglitazone-associated PTEN expression was a result of an increase in PTEN mRNA, suggesting that this increase was transcriptionally-mediated. Compound-66, an inactive form of Rosiglitazone, which is incapable of activating PPARgamma, was unable to elicit the same response as Rosiglitazone, signifying that the Rosiglitazone response is PPARgamma-mediated. To support this, we show, using reporter assays including dominant-negative constructs of PPARgamma, that both Lovastatin and Rosiglitazone specifically mediate PPARgamma activation. Additionally, we demonstrated that cells lacking PTEN or PPARgamma were unable to induce PTEN mediated cellular events in the presence of Lovastatin or Rosiglitazone. These data are the first to demonstrate that Lovastatin can signal through PPARgamma and directly demonstrate that PPARgamma can upregulate PTEN at the transcriptional level. Since PTEN is constitutively active, our data indicates it may be worthwhile to examine Rosiglitazone and Lovastatin stimulation as mechanisms to increase PTEN expression for therapeutic and preventative strategies including cancer, diabetes mellitus and cardiovascular disease.

Antineoplastic Agents↗

OSU-03012 promotes caspase-independent but PERK-, cathepsin B-, BID-, and AIF-dependent killing of transformed cells.

We determined one mechanism by which the putative phosphoinositide-dependent kinase (PDK)-1 inhibitor 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}acetamide (OSU-03012) killed primary human glioma and other transformed cells. OSU-03012 caused a dose-dependent induction of cell death that was not altered by p53 mutation, expression of ERBB1 vIII, or loss of phosphatase and tensin homolog deleted on chromosome 10 function. OSU-03012 promoted cell killing to a greater extent in glioma cells than in nontransformed astrocytes. OSU-03012 and ionizing radiation caused an additive, caspase-independent elevation in cell killing in 96-h viability assays and true radiosensitization in colony formation assays. In a cell type-specific manner, combined exposure to OSU-03012 with a mitogen-activated protein kinase kinase 1/2 inhibitor, phosphoinositide 3-kinase/AKT inhibitors, or parallel molecular interventions resulted in a greater than additive induction of cell killing that was independent of AKT activity and caspase function. OSU-03012 lethality as a single agent or when combined with signaling modulators was not modified in cells lacking expression of BIM or of BAX/BAK. OSU-03012 promoted the release of cathepsin B from the lysosomal compartment and release of AIF from mitochondria. Loss of BH3-interacting domain (BID) function, overexpression of BCL(XL), and inhibition of cathepsin B function suppressed cell killing and apoptosis-inducing factor (AIF) release from mitochondria. In protein kinase R-like endoplasmic reticulum kinase-/- cells, the lethality of OSU-03012 was attenuated which correlated with reduced cleavage of BID and with suppression of cathepsin B and AIF release into the cytosol. Our data demonstrate that OSU-03012 promotes glioma cell killing that is dependent on endoplasmic reticulum stress, lysosomal dysfunction, and BID-dependent release of AIF from mitochondria, and whose lethality is enhanced by irradiation or by inhibition of protective signaling pathways.

3-Phosphoinositide-Dependent Protein Kinases↗

alpha-Tocopheryl succinate induces apoptosis in prostate cancer cells in part through inhibition of Bcl-xL/Bcl-2 function.

Although the antitumor effect of alpha-tocopheryl succinate (vitamin E succinate) has been well demonstrated, its underlying mechanism remains elusive. This study provides evidence that inhibition of Bcl-xL/Bcl-2 function represents a major pathway whereby alpha-tocopheryl succinate mediates apoptosis induction in prostate cancer cells. In vitro data indicate that alpha-tocopheryl succinate was able to disrupt the binding of Bak BH3 peptide to Bcl-xL and Bcl-2 with IC50 of 26 microm, in line with its potency in antiproliferation. Treatment of PC-3 cells with this agent led to reduced association of Bcl-2 and Bcl-xL with Bak, leading to caspase-dependent apoptosis. Moreover, overexpression of Bcl-xL protected LNCaP cells from the apoptosis induction. This mechanistic finding provided a basis to develop potent Bcl-xL/Bcl-2 inhibitors. Docking of alpha-tocopheryl succinate into the Bak peptide-binding site indicates that it adopted a unique hairpin-shaped conformation for protein interactions. We rationalized that the hemisuccinate and the two proximal isopranyl units of the side chain played a crucial role in ligand anchoring and protein-ligand complex stabilization, respectively. However, exposure of the distal isopranyl unit to a polar environment might diminish the binding affinity of alpha-tocopheryl succinate. This premise was corroborated by a structure-activity analysis of a series of derivatives with truncated side chains and/or altered carboxyl terminus. This computer model predicted that the removal of the distal isopranyl unit from the side chain would improve binding affinity, leading to two agents with significantly higher potency in inhibiting Bak peptide binding and in suppressing prostate cancer cell proliferation.

Apoptosis↗

Cytotoxicity of a non-cyclooxygenase-2 inhibitory derivative of celecoxib in non-small-cell lung cancer A549 cells.

Lung cancer is one of the most common causes of cancer death worldwide. Although recent advances in chemotherapy and radiation therapy have yielded modest improvements in patient outcomes, overall survival remains poor. Therefore, new therapeutic targets are needed. Phosphoinositide-dependent kinase-1 (PDK1) is one potential target. The aim of the present studies was to investigate the potential of a celecoxib-derived PDK1 inhibitor (OSU03013), that does not inhibit cyclooxygenase-2, to kill lung cancer cells in vitro. Using human non-small-cell lung cancer A549 cells, OSU03013 dose-dependently induced apoptosis. After 6 h of treatment with 7.5 microM OSU03013, 26% of the cells were apoptotic, compared to 4% of the control cells as determined by measuring the sub-G1 peak of propidium iodide stained cells with flow cytometry. A similar increase in apoptosis was evident using the Cell Death ELISA assay. OSU03013-induced apoptosis was accompanied by a reduction in the mitochondrial membrane potential, the release of cytochrome c and the cleavage of caspase-3. Surprisingly, the phosphorylation of Akt at serine 473 was increased in A549 cells treated with 7.5 microM OSU03013. However, the toxicity of OSU03013 was reduced in A549 cells expressing a constitutively active form of Akt. These data demonstrate that OSU03013 induces apoptosis in A549 cells via the mitochondrial pathway. Inhibition of the Akt pathway appears uninvolved in this toxicity, although Akt can provide protection. These results also suggest the potential of celecoxib-derived agents to treat some forms of lung cancer.

Apoptosis↗

Peroxisome proliferator-activated receptor gamma-independent repression of prostate-specific antigen expression by thiazolidinediones in prostate cancer cells.

In light of the potential use of the thiazolidinedione family of peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists in prostate cancer treatment, this study assessed the mechanism by which these agents suppress prostate-specific antigen (PSA) secretion in prostate cancer cells. Two lines of evidence indicate that the effect of thiazolidinediones on PSA down-regulation is independent of PPARgamma activation. First, this thiazolidinedione-mediated PSA down-regulation is structure-specific irrespective of the relative PPARgamma agonist potency. Second, the PPARgamma-inactive analogs of troglitazone and ciglitazone [Delta2TG (5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl-methoxy)-benzylidene]-thiazolidine-2,4-dione) and Delta2CG (5-[4-(1-methyl-cyclohexylmethoxy)-benzylidene]-thiazolidine-2,4-dione), respectively] exhibit higher potency than the parent compound in inhibiting dihydrotestosterone (DHT)-stimulated PSA secretion. Although 10 microM troglitazone and Delta2TG significantly inhibit PSA secretion, they do not alter the expression level of androgen receptor (AR) or interfere with DHT-activated nuclear translocation of AR. However, reporter gene and chromatin immunoprecipitation studies indicate that troglitazone and Delta2TG block AR recruitment to the androgen response elements within the PSA promoter. Thus, this study raises the question of whether the ability of oral troglitazone to reduce PSA levels in prostate cancer patients is therapeutically relevant. A major concern is that the concentration for troglitazone to mediate antitumor effects is severalfold higher than that of PSA down-regulation, which is difficult to attain at therapeutic doses. Nevertheless, it is noteworthy that troglitazone and Delta2TG at high doses were able to inhibit AR expression. From a translational perspective, separation of PPARgamma agonist activity from AR down-regulation provides a molecular basis to use troglitazone as a platform to design AR-ablative agents.

Animals↗

Targeting histone deacetylase in cancer therapy.

Histone deacetylase (HDAC) is recognized as one of the promising targets for cancer treatment as many HDAC inhibitors have entered clinical trials for both solid and liquid tumors. Nevertheless, the mechanisms underlying the antiproliferative effects of HDAC inhibitors remain elusive. Although they have been shown to regulate the transcription of a defined set of genes through chromatin remodeling, increasing evidence suggests that modifications of the epigenetic histone code may not be the primary mechanism for HDAC inhibitor-mediated growth inhibition and apoptosis in cancer cells. While histones still represent a primary target for the physiological function of HDACs, the antitumor effect of HDAC inhibitors might also be attributed to transcription-independent mechanisms by modulating the acetylation status of a series of nonhistone targets. Also noteworthy is the effect of HDAC inhibitors on Akt downregulation through the alteration of protein phosphatase 1 (PP1) complex formation. To provide an overview of the use of HDAC inhibitors in cancer treatment, this review addresses the following subjects: (1) the physiological relevance of HDAC-mediated acetylation of histone and nonhistone substrates, (2) the chemical biology of HDACs and development of a novel class of HDAC inhibitors, and (3) the protein acetylation-independent effect of HDAC inhibitors on the activation status of signaling kinases.

Antineoplastic Agents↗

A structurally optimized celecoxib derivative inhibits human pancreatic cancer cell growth.

Deregulation of the phosphatidylinositol 3-kinase (PI-3K)/PDK-l/Akt signaling cascade is associated with pancreatic cancer tumor invasion, angiogenesis, and tumor progression. As such, it has been postulated that PDK-1/Akt signaling inhibitors may hold promise as novel therapeutic agents for pancreatic cancer. Disadvantages of currently available Akt inhibitors include tumor resistance, poor specificity, potential toxicity, and poor bioavailability. Previous studies have demonstrated that OSU-03012, a celecoxib derivative, specifically inhibits PDK-1 mediated phosphorylation of Akt with IC(50) values in the low muM range. Human pancreatic cancer cell lines AsPC-1, BxPC-3, Mia-PaCa 2, and PANC-1 were cultured in media containing varying concentrations of OSU-03012, 5-fluorouracil (5-FU), and gemcitabine, and changes in Akt phosphorylation and cell viability were evaluated using western blotting and a 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, respectively. Treatment with OSU-03012 resulted in decreased PDK-1-mediated Akt phosphorylation and cell growth inhibition for all cell lines with IC(50) values ranging between 1.0 and 2.5 muM. Resistance to 5-FU and gemcitabine was observed in cell lines AsPC-1 and BxPC-3. Further analyses indicate that OSU-03012 induces both proapoptotic and antiproliferative effects in these cells. Taken together, these data suggest that OSU-03012 has potential value as a novel therapy for pancreatic cancer.

3-Phosphoinositide-Dependent Protein Kinases↗

Neutrophil gelatinase-associated lipocalin as a survival factor.

NGAL (human neutrophil gelatinase-associated lipocalin) and its mouse analogue 24p3 are members of the lipocalin family of small secreted proteins. These proteins are up-regulated in a number of pathological conditions, including cancers, and may function as transporters of essential factors. Although previous publications have suggested that 24p3 has pro-apoptotic functions, other data are more suggestive of a survival function. The current study was designed to determine whether NGAL is pro- or anti-apoptotic. Apoptosis induced in human adenocarcinoma A549 cells by the 5-lipoxygenase-activating-protein inhibitor MK886, or several celecoxib-derived PDK1 (phosphoinositide-dependent kinase 1) inhibitors that are devoid of cyclo-oxygenase-2 inhibitory activity, was accompanied by a dose- and time-dependent increase of NGAL mRNA levels, as was reported previously with 24p3. A similar induction of NGAL mRNA was observed in human breast cancer MCF7 cells treated with MK886, indicating this was not a cell-specific effect. Treatment of A549 cells with up to 150 mug/10(6) cells of purified recombinant NGAL protein had no effect on viability, whereas antisera against the full-length NGAL protein induced apoptosis in these cells. The stable overexpression of NGAL in A549 cells had no effect on proliferation or viability. However, the cell death induced by a PDK1 inhibitor was reduced by 50% in NGAL-overexpressing cells. Decreasing NGAL mRNA and protein expression with siRNA (small interfering RNA) in A549 cells increased the toxicity of a PDK1 inhibitor by approx. 45%. These data indicate that, although the induction of NGAL correlates with apoptosis, this induction represents a survival response. Because NGAL is a secreted protein, it may play an extracellular role in cell defence against toxicants and/or facilitate the survival of the remaining cells.

3-Phosphoinositide-Dependent Protein Kinases↗

Histone acetylation-independent effect of histone deacetylase inhibitors on Akt through the reshuffling of protein phosphatase 1 complexes.

Despite advances in understanding the role of histone deacetylases (HDACs) in tumorigenesis, the mechanism by which HDAC inhibitors mediate antineoplastic effects remains elusive. Modifications of the histone code alone are not sufficient to account for the antitumor effect of HDAC inhibitors. The present study demonstrates a novel histone acetylation-independent mechanism by which HDAC inhibitors cause Akt dephosphorylation in U87MG glioblastoma and PC-3 prostate cancer cells by disrupting HDAC-protein phosphatase 1 (PP1) complexes. Of four HDAC inhibitors examined, trichostatin A (TSA) and HDAC42 exhibit the highest activity in down-regulating phospho-Akt, followed by suberoylanilide hydroxamic acid, whereas MS-275 shows only a marginal effect at 5 microm. This differential potency parallels the respective activities in inducing tubulin acetylation, a non-histone substrate for HDAC6. Evidence indicates that this Akt dephosphorylation is not mediated through deactivation of upstream kinases or activation of downstream phosphatases. However, the effect of TSA on phospho-Akt can be rescued by PP1 inhibition but not that of protein phosphatase 2A. Immunochemical analyses reveal that TSA blocks specific interactions of PP1 with HDACs 1 and 6, resulting in increased PP1-Akt association. Moreover, we used isozyme-specific small interfering RNAs to confirm the role of HDACs 1 and 6 as key mediators in facilitating Akt dephosphorylation. The selective action of HDAC inhibitors on HDAC-PP1 complexes represents the first example of modulating specific PP1 interactions by small molecule agents. From a clinical perspective, identification of this PP1-facilitated dephosphorylation mechanism underscores the potential use of HDAC inhibitors in lowering the apoptosis threshold for other therapeutic agents through Akt down-regulation.

Apoptosis↗

Structure-based optimization of phenylbutyrate-derived histone deacetylase inhibitors.

Previously, we developed a strategy to develop a novel class of histone deacetylase (HDAC) inhibitors by tethering short-chain fatty acids with Zn(2+)-chelating motifs, which led to N-hydroxy-4-(4-phenylbutyryl-amino)benzamide (HTPB), a hydroxamate-tethered phenylbutyrate derivative with sub-micromolar potency in inhibiting HDAC activity and cancer cell proliferation. In this study, we carried out structure-based optimization of HTPB by using the framework generated by the structure of histone deacetylase-like protein (HDLP)-trichostatin A (TSA) complexes. Docking of HTPB into the HDLP binding domain suggested that the hydrophobic microenvironment encompassed by Phe-198 and Phe-200 could be exploited for structural optimization. This premise was corroborated by the greater potency of (S)-(+)-N-hydroxy-4-(3-methyl-2-phenylbutyrylamino)-benzamide [(S)-11] (IC(50) in HDAC inhibition, 16 nM), of which the isopropyl moiety was favorable in interacting with this hydrophobic motif. (S)-11 at concentrations as low as 0.1 microM was effective in causing histone hyperacetylation and p21(WAF/CIP1) overexpression and suppressing proliferation in cancer cells.

Acetylation↗

Celecoxib analogues disrupt Akt signaling, which is commonly activated in primary breast tumours.

INTRODUCTION: Phosphorylated Akt (P-Akt) is an attractive molecular target because it contributes to the development of breast cancer and confers resistance to conventional therapies. Akt also serves as a signalling intermediate for receptors such as human epidermal growth factor receptor (HER)-2, which is overexpressed in 30% of breast cancers; therefore, inhibitors to this pathway are being sought. New celecoxib analogues reportedly inhibit P-Akt in prostate cancer cells. We therefore examined the potential of these compounds in the treatment of breast cancer. The analogues were characterized in MDA-MB-453 cells because they overexpress HER-2 and have very high levels of P-Akt. METHODS: To evaluate the effect of the celecoxib analogues, immunoblotting was used to identify changes in the phosphorylation of Akt and its downstream substrates glycogen synthase kinase (GSK) and 4E binding protein (4EBP-1). In vitro kinase assays were then used to assess the effect of the drugs on Akt activity. Cell death was evaluated by poly(ADP-ribose) polymerase cleavage, nucleosomal fragmentation and MTS assays. Finally, tumour tissue microarrays were screened for P-Akt and HER-2 expression. RESULTS: OSU-03012 and OSU-O3013 inhibited P-Akt and its downstream signalling through 4EBP-1 and GSK at concentrations well below that of celecoxib. Disruption of P-Akt was followed by induction of apoptosis and more than 90% cell death. We also noted that the cytotoxicity of the celecoxib analogues was not significantly affected by serum. In contrast, the presence of 5% serum protected cells from celecoxib induced death. Thus, the structural modification of the celecoxib analogues increased P-Akt inhibition and enhanced the bioavailability of the drugs in vitro. To assess how many patients may potentially benefit from such drugs we screened tumour tissue microarrays. P-Akt was highly activated in 58% (225/390) of cases, whereas it was only similarly expressed in 35% (9/26) of normal breast tissues. Furthermore, HER-2 positive tumours expressed high levels of P-Akt (P < 0.01), supporting in vitro signal transduction. CONCLUSION: We determined that Celecoxib analogues are potent inhibitors of P-Akt signalling and kill breast cancer cells that overexpress HER-2. We also defined an association between HER-2 and P-Akt in primary breast tissues, suggesting that these inhibitors may benefit patients in need of new treatment options.

Anti-Inflammatory Agents, Non-Steroidal↗

Celecoxib derivatives induce apoptosis via the disruption of mitochondrial membrane potential and activation of caspase 9.

Celecoxib is a potent nonsteroid antiinflammatory drug (NSAID) that has shown great promise in cancer chemoprevention and treatment. The tumor suppression activity of celecoxib and other NSAIDs have been related to the induction of apoptosis in many cancer cell lines and animal models. While celecoxib is a specific inhibitor of cyclooxygenase (COX)-2, recent data indicate that its apoptotic properties may also be mediated through COX-independent pathways. In our study, we evaluated second generation celecoxib derivatives, lacking COX-2 inhibitory activity, in a premalignant and malignant human oral cell culture model to determine their potential anticancer effect and mechanisms responsible for the COX-independent apoptotic activity. Celecoxib and its derivatives delayed the progression of cells through the G(2)/M phase and induced apoptosis. The derivatives with apolar substituents at the terminal phenyl moiety of celecoxib greatly enhanced apoptosis and cell cycle delay. Apoptosis and cell cycle arrest appeared to be independent of derivative induced inhibition of PDK1 and phosphorylation of Akt and Erk1/2. Derivatives induced apoptosis was mediated by the cleavage and activation of caspase-9 and caspase-3, but not caspase 8, implicating the mitochondrial pathway for apoptosis induction. Inhibitors of caspase-3 and caspase-9 and cyclosporin A, a mitochondrial membrane potential stabilizer, attenuated derivative induced apoptosis. Inhibition of caspase-3 prevented the activation of caspase 8, while the inhibition of caspase-9 inhibitor blocked activation of both caspase 3 and 8 by the derivatives. Apoptosis was independent of Bcl-2. These results indicate that the second generation celecoxib derivatives induce apoptosis in human oral cancer lines by the disruption of mitochondrial membrane potential activating caspase 9 and downstream caspase 3 and 8. This suggests that the modification of the celecoxib structure can lead to highly effective COX-independent growth inhibitory and apoptotic agents in chemoprevention and therapy.

3-Phosphoinositide-Dependent Protein Kinases↗

Thiazolidenediones mediate apoptosis in prostate cancer cells in part through inhibition of Bcl-xL/Bcl-2 functions independently of PPARgamma.

Certain members of the thiazolidenedione family of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, such as troglitazone and ciglitazone, exhibit antitumor effects; however, the underlying mechanism remains inconclusive. This study shows that the effect of these thiazolidenedione members on apoptosis in prostate cancer cells is independent of PPARgamma activation. First, close structural analogues of thiazolidenediones, whereas devoid of PPARgamma activity, retain the ability to induce apoptosis with equal potency. Second, both PC-3 (PPARgamma-expressing) and LNCaP (PPARgamma-deficient) cells are sensitive to apoptosis induction by troglitazone and its PPARgamma-inactive analogue irrespective of their PPARgamma expression status. Third, rosiglitazone and pioglitazone, potent PPARgamma agonists, show marginal effects on apoptosis even at high concentrations. Evidence indicates that the apoptotic effect of troglitazone, ciglitazone, and their PPARgamma-inactive analogues 5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-benzylidene]-2,4-thiazolidine-dione (Delta2-TG) and 5-[4-(1-methyl-cyclohexylmethoxy)-benzylidene]-thiazolidine-2,4-dione, respectively, is in part attributable to their ability to inhibit the anti-apoptotic functions of Bcl-xL and Bcl-2. Treatment of PC-3 cells with troglitazone or Delta2-TG led to reduced association of Bcl-2 and Bcl-xL with Bak, leading to caspase-dependent apoptosis. Bcl-xL overexpression protects LNCaP cells from apoptosis induction by troglitazone and Delta2-TG in an expression level-dependent manner. Considering the pivotal role of Bcl-xL/Bcl-2 in regulating mitochondrial integrity, this new mode of mechanism provides a framework to account for the PPARgamma-independent action of thiazolidenediones in inducing apoptosis in cancer cells. Moreover, dissociation of these two pharmacologic activities provides a molecular basis to develop novel Bcl-xL/Bcl-2 inhibitors, of which the proof of principle is illustrated by a Delta2-TG analogue with potent in vivo antitumor activities.

Animals↗

Synergistic interactions between imatinib mesylate and the novel phosphoinositide-dependent kinase-1 inhibitor OSU-03012 in overcoming imatinib mesylate resistance.

Resistance to the Ableson protein tyrosine (Abl) kinase inhibitor imatinib mesylate has become a critical issue for patients in advanced phases of chronic myelogenous leukemia. Imatinib-resistant tumor cells develop, in part, as a result of point mutations within the Abl kinase domain. As protein kinase B (Akt) plays a pivotal role in Abl oncogene-mediated cell survival, we hypothesize that concurrent inhibition of Akt will sensitize resistant cells to the residual apoptotic activity of imatinib mesylate, thereby overcoming the resistance. Here, we examined the effect of OSU-03012, a celecoxib-derived phosphoinositide-dependent kinase-1 (PDK-1) inhibitor, on imatinib mesylate-induced apoptosis in 2 clinically relevant breakpoint cluster region (Bcr)-Abl mutant cell lines, Ba/F3p210(E255K) and Ba/F3p210(T315I). The 50% inhibitory concentration (IC50) values of imatinib mesylate to inhibit the proliferation of Ba/F3p210(E255K) and Ba/F3p210(T315I) were 14 +/- 4 and 30 +/- 2 microM, respectively. There was no cross-resistance to OSU-03012 in these mutant cells with an IC50 of 5 microM irrespective of mutations. Nevertheless, in the presence of OSU-03012 the susceptibility of these mutant cells to imatinib-induced apoptosis was significantly enhanced. This synergistic action was, at least in part, mediated through the concerted effect on phospho-Akt. Together these data provide a novel therapeutic strategy to overcome imatinib mesylate resistance, especially with the Abl mutant T315I.

3-Phosphoinositide-Dependent Protein Kinases↗