Sulforaphane attenuates EGFR signaling in NSCLC cells.

BACKGROUND: EGFR, a receptor tyrosine kinase (RTK), is frequently overexpressed and mutated in non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitors (TKIs) have been widely used in the treatment of many cancers, including NSCLC. However, intrinsic and acquired resistance to TKI remains a common obstacle. One strategy that may help overcome EGFR-TKI resistance is to target EGFR for degradation. As EGFR is a client protein of heat-shock protein 90 (HSP90) and sulforaphane is known to functionally regulate HSP90, we hypothesized that sulforaphane could attenuate EGFR-related signaling and potentially be used to treat NSCLC. RESULTS: Our study revealed that sulforaphane displayed antitumor activity against NSCLC cells both in vitro and in vivo. The sensitivity of NSCLC cells to sulforaphane appeared to positively correlate with the inhibition of EGFR-related signaling, which was attributed to the increased proteasomal degradation of EGFR. Combined treatment of NSCLC cells with sulforaphane plus another HSP90 inhibitor (17-AAG) enhanced the inhibition of EGFR-related signaling both in vitro and in vivo. CONCLUSIONS: We have shown that sulforaphane is a novel inhibitory modulator of EGFR expression and is effective in inhibiting the tumor growth of EGFR-TKI-resistant NSCLC cells. Our findings suggest that sulforaphane should be further explored for its potential clinical applications against NSCLC.

Proteomic analyses of genes regulated by heterogeneous nuclear ribonucleoproteins A/B in Jurkat cells.

Several lines of evidence suggest that hnRNPs A/B (hnRNPs A1, A2/B1, and A3) play an important role in proliferation, although the functional overlap among members of hnRNPs A/B remains largely unknown. In this study, we have employed RNAi knockdown and proteomic approaches to investigate the biological functions of hnRNPs A/B. Depletion of hnRNP A2, but not A1 or A3, produced a significant inhibition of cellular proliferation in Jurkat cells. Analysis of the proteomes in the cells depleted for hnRNP A1, A2, or A3 has identified a total of 167 differentially expressed proteins in the depleted cells. Network analysis of the proteins altered in the cells depleted for hnRNP A2 revealed that the biological processes likely affected by these proteins are related to cell cycle, cytoskeleton rearrangement, and transcription regulation. Indeed, we have confirmed that the level of RhoA and CrkL was selectively reduced in the cells depleted of hnRNP A2, but not in the cells depleted for hnRNP A1 or A3. Therefore, we suggest that the reduced proliferation observed in the cells depleted of hnRNP A2 may result from its effects on cell adhesion processes in the Jurkat cells.

Site-specific separation and detection of phosphopeptide isomers with pH-mediated stacking capillary electrophoresis-electrospray ionization-tandem mass spectrometry.

This study reported a pH-mediated stacking CE coupled with ESI MS/MS method to determine the phosphorylation sites of three synthetic phosphopeptides containing structural isomers. These phosphopeptides mimic the phosphopeptides (amino acid residues 12-25) derived from the trypsin-digested products of human lamin A/C protein. The LODs were determined to be 118, 132 and 1240 fmol for SGAQASS(19)TpPL(22)SPTR, SGAQASS(19)TPL(22)SpPTR, and SGAQASS(19)TpPL(22)SpPTR, respectively. The established method was employed to analyze the phosphorylation sites of the trypsin-digested products of glutathione S-transferase-lamin A/C (1-57) fusion protein that had been phosphorylated in vitro by cyclin-dependent kinase 1. The results indicated that this method is feasible to specifically determine the phosphorylation site from phosphopeptide isomers in the trypsin-digested products of a kinase-catalyzed phosphoprotein, which should benefit the investigation of protein kinase-mediated cellular signal transduction.

N-(1-Pyrenyl) maleimide inhibits telomerase activity in a cell free system and induces apoptosis in Jurkat cells.

Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. Agents that interact selectively with telomerase are anticipated to exert specific action on cancer cells. In this study, we evaluated maleimide derivatives for their potency and selectivity of telomerase inhibition. Among the several N-substituted derivatives of maleimide tested, N-(1-Pyrenyl) maleimide was shown to exert the greatest inhibition of telomerase in a cell free system, with an IC50 value of 0.25 µM. Importantly, we demonstrated that N-(1-pyrenyl) maleimide induces apoptosis in Jurkat T cells and displays the greatest differential cytotoxicity against hematopoietic cancer cells. These results suggest that N-(1-pyrenyl) maleimide is an attractive maleimide to be tested and developed as anti-cancer drug.

Telomeric DNA-binding activities of heterogeneous nuclear ribonucleoprotein A3 in vitro and in vivo.

Telomeres are dynamic DNA-protein complexes that protect the ends of linear chromosome. Telomere-binding proteins play crucial role in the maintenance of telomeres. HnRNP A3 has been shown recently to bind specifically to single-stranded telomeric DNA in vitro, although its in vivo telomere function remains unknown. In this study, the DNA-binding properties of hnRNP A3 in vitro as well as its putative role of telomere maintenance in vivo were investigated. The minimal sequence for hnRNP A3 binding to DNA was determined as an undecamer with the following consensus sequence 5'-[T/C]AG[G/T]NN[T/C]AG[G/T]N-3'. Confocal microscopy and chromatin-immunoprecipitation (ChIP) analyses showed that hnRNP A3 is associated with telomere in vivo. Knocking-down the expression of hnRNP A3 had no effect on telomere length maintenance and did not affect cell proliferation. In contrast, overexpression of hnRNP A3 resulted in the production of steady-state short telomeres in OECM1 cells. These results suggest that hnRNP A3 is associated with telomere in vivo and acts as a negative regulator of telomere length maintenance.

PriA participates in nascent DNA synthesis in Escherichia coli.

The question of whether discontinuous DNA replication operates only for the lagging strand or for both strands in E. coli remains unresolved. In this study, the participation of priA, B, C and rep genes in discontinuous DNA replication was examined by analyzing the size distribution of nascent DNA synthesized in wild-type, lig-7 and polA4113 genetic backgrounds. Inactivation of priA, but not priB, priC or rep, resulted in a significant increase of high molecular weight (HMW) DNA in the short pulse-labeled DNA in the wild-type lig(+) polA(+) strains. Inactivation of priA also produced a significant increase of HMW DNA in the nascent DNA synthesized in lig-7 and polA4113 strains. These results indicate that PriA is involved in the discontinuous synthesis of nascent DNA.

Heterogeneous nuclear ribonucleoprotein A3 binds single-stranded telomeric DNA and inhibits telomerase extension in vitro.

Telomeres are dynamic DNA-protein complexes at the end of linear chromosomes. Maintenance of functional telomeres is required for chromosome stability, and to avoid the activation of DNA damage response pathway and cell cycle arrest. Telomere-binding proteins play crucial roles in the maintenance of functional telomeres. In this study, we employed affinity pull-down and proteomic approach to search for novel proteins that interact with the single-stranded telomeric DNA. The proteins identified by two-dimensional gel electrophoresis were further characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF-TOF tandem MS. Among the five identified proteins, we report here the biochemical properties of a novel protein, hnRNP A3. The purified hnRNP A3 bound specifically to G-rich strand, but not to C-rich strand or double-stranded telomeric DNA. The RRM1 (RNA recognition motif 1) domain, but not RRM2, of hnRNP A3 is sufficient to confer specific binding to the telomeric sequence. In addition, we present evidence that hnRNP A3 can inhibit telomerase extension in vitro. These biochemical properties of hnRNP A3 suggest that hnRNP A3 can participate in telomere regulation in vivo.

Telomerase prevents accelerated senescence in glucose-6-phosphate dehydrogenase (G6PD)-deficient human fibroblasts.

Fibroblasts derived from glucose-6-phosphate dehydrogenase (G6PD)-deficient patients display retarded growth and accelerated cellular senescence that is attributable to increased accumulation of oxidative DNA damage and increased sensitivity to oxidant-induced senescence, but not to accelerated telomere attrition. Here, we show that ectopic expression of hTERT stimulates telomerase activity and prevents accelerated senescence in G6PD-deficient cells. Stable clones derived from hTERT-expressing normal and G6PD-deficient fibroblasts have normal karyotypes, and display no sign of senescence beyond 145 and 105 passages, respectively. Activation of telomerase, however, does not prevent telomere attrition in earlier-passage cells, but does stabilize telomere lengths at later passages. In addition, we provide evidence that ectopic expression of hTERT attenuates the increased sensitivity of G6PD-deficient fibroblasts to oxidant-induced senescence. These results suggest that ectopic expression of hTERT, in addition to acting in telomere length maintenance by activating telomerase, also functions in regulating senescence induction.

Heterogeneous Nuclear Ribonucleoproteins A1 and A2 Function in Telomerase-Dependent Maintenance of Telomeres.

The A/B subfamily of heterogeneous nuclear ribonucleoproteins (hnRNPs A/B), which includes hnRNP A1, A2/B1, and A3, plays an important role in cell proliferation. The simultaneous suppression of hnRNP A1/A2, but not the suppression of hnRNP A1 or A2 alone, has been shown to inhibit cell proliferation and induce apoptosis in cancer cells, but not in mortal normal cells. However, the molecular basis for such a differential inhibition of cell proliferation remains unknown. Here, we show that the simultaneous suppression of hnRNP A1 and hnRNP A2 resulted in dysfunctional telomeres and induced DNA damage responses in cancer cells. The inhibition of apoptosis did not alleviate the inhibition of cell proliferation nor the formation of dysfunctional telomeres in cancer cells depleted of hnRNP A1/A2. Moreover, while proliferation of mortal normal fibroblasts was not sensitive to the depletion of hnRNP A1/A2, the ectopic expression of hTERT in normal fibroblasts rendered these cells sensitive to proliferation inhibition, which was associated with the production of dysfunctional telomeres. Our study demonstrates that hnRNP A1 and A2 function to maintain telomeres in telomerase-expressing cells only, suggesting that the maintenance of functional telomeres in telomerase-expressing cancer cells employs factors that differ from those used in the telomerase-negative normal cells.

The in vivo anti-leukemia activity of N-(1-Pyrenlyl) maleimide in a bioluminescent mouse model.

In a search for anticancer drugs by screening for inhibitors of telomerase, we have identified several small-molecule inhibitors that selectively inhibit telomerase in a cell-free system. Among these inhibitors, N-(1-pyrenyl) maleimide (NPM) induced apoptosis and displayed the greatest differential cytotoxicity against acute T cell leukemia-derived Jurkat cells cultured in vitro. In this work, the in vivo anti-leukemia activity of NPM was investigated using a bioluminescent mouse model. The luciferase-expressing Jurkat cells (Jurkat-Luc) were mixed with matrigel and injected subcutaneously into the nude mice. Drug treatment was commenced on day 7 after tumor implantation. The growth of xenografted tumors was significantly inhibited in the mice treated with NPM, which is comparable to the inhibitory effect of a classical anti-leukemia drug, cyclophosphamide. Combined treatment with NPM and cyclophosphamide further enhanced the growth inhibition of xenografted Jurkat-Luc cells. Immunohistochemistry staining with cleaved caspase 3 (cl-caspase 3) indicated a very heavy staining of cl-caspase 3 only in the tumor implants excised from the NPM-treated mice. We conclude that NPM induced apoptosis and inhibited the growth of xenografted Jurkat-Luc cells in nude mice, demonstrating that NPM displays anti-leukemia activity in vivo.

2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells.

Magnolol, a hydroxylated biphenol compound isolated from the bark of Magnolia officinalis, has been shown to exhibit anti-proliferative effect in various cancer cells, including skin cancer cells. Methoxylation of magnolol appears to improve its anti-inflammatory activity, yet the effect of this modification on the agent's antitumor activity remains unknown. In this work, we report that 2-O-methylmagnolol (MM1) displays improved antitumor activity against skin cancer cells compared to magnolol both in vitro and in vivo. The increased antitumor activity of MM1 appears to correlate with its increased ability to induce apoptosis. DNA microarray and network pathway analyses suggest that MM1 affects certain key factors involved in regulating apoptosis and programmed cell death. Interestingly, the level of the long non-coding (lnc) RNA of growth arrest-specific 5 (GAS5) was increased in MM1-treated cells, and inhibition of lncRNA GAS5 inhibited MM1-induced apoptosis. Conversely, overexpression of lncRNA GAS5 inhibited cell proliferation and promoted cell apoptosis in skin cancer cells. The expression of lncRNA GAS5 in the skin cancer tissues was found to be lower than that in the adjacent normal tissues in a majority of patients. Taken together, our findings suggest that MM1 has improved antitumor activity in skin cancer cells, and that this is due, at least in part, to the upregulation of lncRNA GAS5 and the enhancement of apoptosis.

A major role of PKC theta and NFkappaB in the regulation of hTERT in human T lymphocytes.

Expression of the telomerase catalytic subunit (TERT) is the rate-limiting determinant of telomerase activity in most human cells. In this work, we examined the participation of protein kinase C (PKC) in the regulation of hTERT expression in human T lymphocytes. Transient expression assays using luciferase reporter plasmids containing hTERT promoter showed that overexpression of PKC theta, but not the other PKC isoforms, could activate the promoter activity of hTERT in resting T lymphocytes. Among the PKC theta-activated signalings, we presented evidence that the expression of hTERT is mediated through NFkappaB but not through MEK or c-Jun N-terminal kinase pathways. Analysis of the hTERT promoter occupancy in vivo using chromatin immunoprecipitation assays, however, did not detect an increased binding of NFkappaB to the hTERT promoter in the activated T cells, although an increased binding of cMyc and Sp1 was detected. Together with the observation that inhibition of NFkappaB eliminated the induction of cMyc in activated T cells, these results suggest that PKC theta-activated NFkappaB signaling regulates the expression of hTERT via cMyc in human T lymphocytes.

Heterogeneous nuclear ribonucleoproteins A1 and A2 modulate expression of Tid1 isoforms and EGFR signaling in non-small cell lung cancer.

The Tid1 protein is a DnaJ co-chaperone that has two alternative splicing isoforms: Tid1 long form (Tid1-L) and Tid1 short form (Tid1-S). Recent studies have shown that Tid1-L functions as a tumor suppressor by decreasing EGFR signaling in various cancers, including head and neck cancer and non-small cell lung cancer (NSCLC). However, the molecular mechanism responsible for regulating the alternative splicing of Tid1 is not yet known. Two splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNP) A1 and A2, participate in alternative splicing and are known to be overexpressed in lung cancers. In this work, we examined if hnRNP A1 and A2 could regulate the alternative splicing of Tid1 to modulate tumorigenesis in NSCLC. We report that RNAi-mediated depletion of both hnRNP A1/A2 (but not single depletion of either) increased Tid1-L expression, inhibited cell proliferation and attenuated EGFR signaling. Analyses of the expression levels of hnRNP A1, hnRNP A2, EGFR and Tid1-L in NSCLC tissues revealed that hnRNP A1 and A2 are positively correlated with EGFR, but negatively correlated with Tid1-L. NSCLC patients with high-level expression of hnRNP A1, hnRNP A2 and EGFR combined with low-level expression of Tid1-L were associated with poor overall survival. Taken together, our results suggest that hnRNP A1 or A2 are both capable of facilitating the alternative splicing of exon 11 in the Tid1 pre-mRNA, thereby suppressing the expression of Tid1-L and allowing EGFR-related signaling to facilitate NSCLC tumorigenesis.

Potent inhibition of human telomerase by helenalin.

Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. Inhibition of telomerase in cancer cells has been shown to limit the growth of human cancer cells in culture. In this study, we report that helenalin, a natural sesquiterpene lactone, is a potent and selective inhibitor for human telomerase. In vitro studies indicate that this drug can inactivate telomerase directly in a manner that is dependent on concentration and time. The inhibitory action of this drug on telomerase is selective since the presence of excessive externally added proteins did not protect the inhibition and all of the other enzymes tested in this study were not inhibited by this drug. Furthermore, we demonstrated that helenalin can inhibit the expression of hTERT and telomerase in hematopoietic cancer cells. Therefore, the anti-tumor activity of helenalin is attributed, at least in part, to the inhibition of telomerase.

N-(1-pyrenyl) maleimide induces bak oligomerization and mitochondrial dysfunction in Jurkat Cells.

N-(1-pyrenyl) maleimide (NPM) is a fluorescent reagent that is frequently used as a derivatization agent for the detection of thio-containing compounds. NPM has been shown to display a great differential cytotoxicity against hematopoietic cancer cells. In this study, the molecular mechanism by which NPM induces apoptosis was examined. Here, we show that treatment of Jurkat cells with NPM leads to Bak oligomerization, loss of mitochondrial membrane potential (Δψm), and release of cytochrome C from mitochondria to cytosol. Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis. Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak. Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

The dual role of protein kinase C in the regulation of telomerase activity in human lymphocytes.

Protein kinase C (PKC) has been implicated to play an essential function in the upregulation of telomerase activity in activated T cells, yet its role in the regulation of telomerase activity remains largely unknown. In this work, we present evidence that PKC activity is required both for the induction of hTERT expression and for the post-transcriptional control of telomerase enzyme activity in T lymphocytes. Of the several PKC isoforms present in lymphocytes, only the level of PKC-zeta was greatly increased during T cell activation, implicating that PKC-zeta may be required for the post-transcriptional control of telomerase enzyme activity in T lymphocytes.

The antitumor agent PBT-1 directly targets HSP90 and hnRNP A2/B1 and inhibits lung adenocarcinoma growth and metastasis.

Natural products are the major sources of currently available anticancer drugs. We recently reported that phenanthrene-based tylophorine derivative-1 (PBT-1) may be a potential antitumor agent for lung adenocarcinoma. We therefore examined the direct targets of PBT-1 and their effects in inhibiting lung adenocarcinoma. We found that PBT-1 reduced the level of Slug and inhibits the migration, invasion, and filopodia formation of lung adenocarcinoma CL1-5 cells in vitro. In addition, PBT-1 displayed in vivo antitumor and antimetastasis activities against subcutaneous and orthotopic xenografts of CL1-5 cells in nude mice. Chemical proteomics showed that heat shock protein 90 (HSP90) and heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) bound PBT-1 in CL1-5 cells. Inhibition of HSP90 and hnRNP A2/B1 reduced the activation of AKT and Slug expression. Taken together, these findings suggest that PBT-1 binds to HSP90 and/or hnRNP A2/B1 and initiates antitumor activities by affecting Slug- and AKT-mediated metastasis and tumorigenesis.

Phosphoproteome profiling of the sexually transmitted pathogen Trichomonas vaginalis.

BACKGROUND/PURPOSE(S): Trichomoniasis caused by Trichomonas vaginalis is the most common non-viral sexually transmitted infection. Morphological transformation from the trophozoite stage to the amoeboid or pseudocyst stage is crucial for T. vaginalis infection and survival. Protein phosphorylation is a key post-translational modification involved in the regulation of several biological processes in various prokaryotes and eukaryotes. More than 880 protein kinases have been identified in the T. vaginalis genome. However, little is known about the phosphorylation of specific proteins and the distribution of phosphorylated proteins in different stages of the morphological transformation of T. vaginalis. METHODS: To obtain a more comprehensive understanding of the T. vaginalis phosphoproteome, we analyzed phosphorylated proteins in the three morphological stages using titanium dioxide combined with LC-MS/MS. RESULTS: A total of 93 phosphopeptides originating from 82 unique proteins were identified. Among these proteins, 21 were detected in all stages, 29 were identified in two different stages, and 32 were stage specific. CONCLUSION: Identification of stage-specific phosphorylated proteins indicates that phosphorylation of these proteins may play a key role in the morphological transformation of T. vaginalis.

Proteomic analysis of the differential protein expression reveals nuclear GAPDH in activated T lymphocytes.

Despite the important role of T cell activation in the adaptive immunity, very little is known about the functions of proteins that are differentially expressed in the activated T cells. In this study, we have employed proteomic approach to study the differentially expressed proteins in activated T cells. A total of 25 proteins was characterized that displayed a decreased expression, while a total of 20 proteins was characterized that displayed an increased expression in the activated T cells. Among them, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified unexpectedly as one of the up-regulated proteins. Western blot analysis of proteins separated by 2-dimensional gel electrophoresis had identified several modified GAPDHs which were detectable only in the activated T cells, but not in resting T cells. These modified GAPDHs had higher molecular mass and more basic PI, and were present in the nucleus of activated T cells. Promoter occupancy studies by chromatin immunoprecipitation assay revealed that nuclear GAPDH could be detected in the promoter of genes that were up-regulated during T cell activation, but not in the promoter of genes that were not unaffected or down-regulated. Our results suggest that nuclear GAPDH may function as transcriptional regulator in activated T cells.

Potent inhibition of human telomerase by U-73122.

Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. In this study, we report that U-73122, an amphiphilic alkylating agent that is commonly used as an inhibitor for phospholipase C, is also a potent and selective inhibitor of human telomerase. The inhibition of telomerase by U-73122 was attributed primarily to the pyrrole-2,5-dione group, since its structural analog U-73343 did not inhibit telomerase. In confirmation, we observed that telomerase was inhibited by N-ethylmaleimide, but not N-ethylsuccinimide. The IC(50) value of U-73122 for the in vitro inhibition of telomerase activity is 0.2 microM, which is comparable to or slightly more sensitive than that for phospholipase C. The inhibitory action of U-73122 on telomerase appears to be rather selective since the presence of externally added proteins did not protect the inhibition and the IC(50) values for the other enzymes tested in this study were at least an order of magnitude higher than that for telomerase. Furthermore, we demonstrate that U-73122 can inhibit telomerase in hematopoietic cancer cells. The potent and selective inhibition of telomerase by U-73122 raises the potential exploitation of this drug and other alkylating agents as telomerase inhibitor.