Phosphoproteomics Reveals the Role of Constitutive KAP1 Phosphorylation by B-cell Receptor Signaling in Chronic Lymphocytic Leukemia.

Application of B-cell receptor (BCR) pathway inhibitor ibrutinib for chronic lymphocytic leukemia (CLL) is a major breakthrough, yet the downstream effects following inhibition of BCR signaling and during relapse await further clarification. By comparative phosphoproteomic profiling of B cells from patients with CLL and healthy donors, as well as CLL B cells collected at multiple time points during the course of ibrutinib treatment, we provided the landscape of dysregulated phosphoproteome in CLL and its dynamic alterations associated with ibrutinib treatment. Particularly, differential phosphorylation events associated with several signaling pathways, including BCR pathway, were enriched in patient CLL cells. A constitutively elevated phosphorylation level of KAP1 at serine 473 (S473) was found in the majority of CLL samples prior to treatment. Further verification showed that BCR activation promoted KAP1 S473 phosphorylation, whereas ibrutinib treatment abolished it. Depletion of KAP1 in primary CLL cells decelerated cell-cycle progression and ectopic expression of a KAP1 S473 phospho-mimicking mutant accelerated G2-M cell-cycle transition of CLL cells. Moreover, temporal phosphoproteomic profiles using a series of CLL cells isolated from one patient during the ibrutinib treatment revealed the dynamic changes of several molecules associated with BCR signaling in the ibrutinib responsive and recurrent stages. IMPLICATIONS: This phosphoproteomic analysis and functional validation illuminated the phosphorylation of KAP1 at S473 as an important downstream BCR signaling event and a potential indicator for the success of ibrutinib treatment in CLL.

An Enzyme-Mediated Aza-Michael Addition Is Involved in the Biosynthesis of an Imidazoyl Hybrid Product of Conidiogenone B.

Meleagrin B is a terpene-alkaloid hybrid natural product that contains both the conidiogenone and meleagrin scaffold. Their derivatives show diverse biological activities. We characterized the biosynthesis of (-)-conidiogenone B (1), which involves a diterpene synthase and a P450 monooxygenase. In addition, an α,ß-hydrolase (Con-ABH) was shown to catalyze an aza-Michael addition between 1 and imidazole to give 3S-imidazolyl conidiogenone B (6). Compound 6 was more potent than 1 against Staphylococcus aureus strains.

Proteogenomics of Non-smoking Lung Cancer in East Asia Delineates Molecular Signatures of Pathogenesis and Progression.

Lung cancer in East Asia is characterized by a high percentage of never-smokers, early onset and predominant EGFR mutations. To illuminate the molecular phenotype of this demographically distinct disease, we performed a deep comprehensive proteogenomic study on a prospectively collected cohort in Taiwan, representing early stage, predominantly female, non-smoking lung adenocarcinoma. Integrated genomic, proteomic, and phosphoproteomic analysis delineated the demographically distinct molecular attributes and hallmarks of tumor progression. Mutational signature analysis revealed age- and gender-related mutagenesis mechanisms, characterized by high prevalence of APOBEC mutational signature in younger females and over-representation of environmental carcinogen-like mutational signatures in older females. A proteomics-informed classification distinguished the clinical characteristics of early stage patients with EGFR mutations. Furthermore, integrated protein network analysis revealed the cellular remodeling underpinning clinical trajectories and nominated candidate biomarkers for patient stratification and therapeutic intervention. This multi-omic molecular architecture may help develop strategies for management of early stage never-smoker lung adenocarcinoma.

Glycoproteomic Alterations in Drug-Resistant Nonsmall Cell Lung Cancer Cells Revealed by Lectin Magnetic Nanoprobe-Based Mass Spectrometry.

Understanding the functional role of glycosylation-mediated pathogenesis requires deep characterization of glycoproteome, which remains extremely challenging due to the inherently complex nature of glycoproteins. We demonstrate the utility of lectin-magnetic nanoprobe (MNP@lectin) coupled to Orbitrap HCD-CID-MS/MS for complementary glycotope-specific enrichment and site-specific glycosylation analysis of the glycoproteome. By three nanoprobes, MNP@ConA, MNP@AAL, and MNP@SNA, our results revealed the first large-scale glycoproteome of nonsmall cell lung cancer (NSCLC) with 2290 and 2767 nonredundant glycopeptides confidently identified (Byonic score ≥100) in EGFR-TKI-sensitive PC9 and -resistant PC9-IR cells, respectively, especially with more fucosylated and sialylated glycopeptides in PC9-IR cells. The complementary enrichment was demonstrated with only five glycopeptides commonly enriched in three MNP@lectins. Glycotope specificity of 79 and 62% for enrichment was achieved using MNP@AAL and MNP@SNA, respectively. Label-free quantitation revealed predominant fucosylation in PC9-IR cells, suggesting its potential role associated with NSCLC resistance. Moreover, without immunoprecipitation, this multilectin nanoprobe allows the sensitive identification of 51 glycopeptides from 10 of 12 reported sites from onco-protein EGFR. Our results not only demonstrated a sensitive approach to study the vastly under-represented N-glycoprotome but also may pave the way for a glycoproteomic atlas to further explore the site-specific function of glycoproteins associated with drug resistance in NSCLC.

Rapid High-pH Reverse Phase StageTip for Sensitive Small-Scale Membrane Proteomic Profiling.

Membrane proteins are crucial targets for cancer biomarker discovery and drug development. However, in addition to the inherent challenges of hydrophobicity and low abundance, complete membrane proteome coverage of clinical specimen is usually hindered by the requirement of large amount of starting materials. Toward comprehensive membrane proteomic profiling for small amounts of samples (10 µg), we developed high-pH reverse phase (Hp-RP) combined with stop-and-go extraction tip (StageTip) technique, as a fast (∼15 min.), sensitive, reproducible, high-resolution and multiplexed fractionation method suitable for accurate quantification of the membrane proteome. This approach provided almost 2-fold enhanced detection of peptides encompassing transmembrane helix (TMH) domain, as compared with strong anion exchange (SAX) and strong cation exchange (SCX) StageTip techniques. Almost 5000 proteins (∼60% membrane proteins) can be identified in only 10 µg of membrane protein digests, showing the superior sensitivity of the Hp-RP StageTip approach. The method allowed up to 9- and 6-fold increase in the identification of unique hydrophobic and hydrophilic peptides, respectively. The Hp-RP StageTip method enabled in-depth membrane proteome profiling of 11 lung cancer cell lines harboring different EGFR mutation status, which resulted in the identification of 3983 annotated membrane proteins. This provides the largest collection of reference peptide spectral data for lung cancer membrane subproteome. Finally, relative quantification of membrane proteins between Gefitinib-resistant and -sensitive lung cancer cell lines revealed several up-regulated membrane proteins with key roles in lung cancer progression.

Mining Missing Membrane Proteins by High-pH Reverse-Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry.

Despite significant efforts in the past decade toward complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still "missing proteins". Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using nonsmall cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. To increase identification coverage, we employed Hp-RP StageTip prefractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue was incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet, and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring using synthetic peptides, we provided additional evidence of eight missing proteins including seven with transmembrane helix domains. This study demonstrates that mining missing proteins focused on cancer membrane subproteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224.

Metformin induces cytotoxicity by down-regulating thymidine phosphorylase and excision repair cross-complementation 1 expression in non-small cell lung cancer cells.

Metformin is an antidiabetic drug recently shown to inhibit cancer cell proliferation and growth, although the involved molecular mechanisms have not been elucidated. In many cancer cells, high expression of thymidine phosphorylase (TP) and Excision repair cross-complementation 1 (ERCC1) is associated with poor prognosis. We used A549 and H1975 human non-small cell lung cancer (NSCLC) cell lines to investigate the role of TP and ERCC1 expression in metformin-induced cytotoxicity. Metformin treatment decreased cellular TP and ERCC1 protein and mRNA levels by down-regulating phosphorylated MEK1/2-ERK1/2 protein levels in a dose- and time-dependent manner. The enforced expression of the constitutively active MEK1 (MEK1-CA) vectors significantly restored cellular TP and ERCC1 protein levels and cell viability. Specific inhibition of TP and ERCC1 expression by siRNA enhanced the metformin-induced cytotoxicity and growth inhibition. Arachidin-1, an antioxidant stilbenoid, further decreased TP and ERCC1 expression and augmented metformin's cytotoxic effect, which was abrogated in lung cancer cells transfected with MEK1/2-CA expression vector. In conclusion, metformin induces cytotoxicity by down-regulating TP and ERCC1 expression in NSCLC cells.

Metformin-mediated downregulation of p38 mitogen-activated protein kinase-dependent excision repair cross-complementing 1 decreases DNA repair capacity and sensitizes human lung cancer cells to paclitaxel.

Metformin, an extensively used and well-tolerated drug for treating individuals with type 2 diabetes, has recently gained significant attention as an anticancer drug. On the other hand, paclitaxel (Taxol) is a new antineoplastic drug that has shown promise in the treatment of non-small cell lung cancer (NSCLC). High expression levels of excision repair cross-complementary 1 (ERCC1) in cancers have been positively associated with the DNA repair capacity and a poor prognosis in NSCLC patients treated with platinum-containing chemotherapy. In this current study, paclitaxel was found to increase phosphorylation of mitogen-activated protein kinase (MAPK) kinase 3/6 (MKK3/6)-p38 MAPK as well as protein and mRNA levels of ERCC1 in H1650 and H1703 cells. Moreover, paclitaxel-induced ERCC1 protein and mRNA levels significantly decreased via the downregulation of p38 activity by either a p38 MAPK inhibitor SB202190 or p38 knockdown with specific small interfering RNA (siRNA). Specific inhibition of ERCC1 with siRNA was found to enhance the paclitaxel-induced cytotoxic effect and growth inhibition. Furthermore, metformin was able to not only decrease the paclitaxel-induced p38 MAPK-mediated ERCC1 expression, but also augment the cytotoxic effect induced by paclitaxel. Finally, expression of constitutive activate MKK6 or HA-p38 MAPK vectors in lung cancer cells was able to abrogate ERCC1 downregulation by metformin and paclitaxel as well as cell viability and DNA repair capacity. Overall, our results suggest that inhibition of the p38 MAPK signaling by metformin coupled with paclitaxel therapy in human NSCLC cells may be a clinically useful combination, which however will require further validation.

HSP90 inhibition induces cytotoxicity via down-regulation of Rad51 expression and DNA repair capacity in non-small cell lung cancer cells.

Heat shock protein 90 (HSP90) is an exciting new target in cancer therapy. Repair protein Rad51 is involved in protecting non-small cell lung cancer (NSCLC) cell lines against chemotherapeutic agent-induced cytotoxicity. This study investigated the role of Rad51 expression in HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced cytotoxicity in two NSCLC cell lines, A549 and H1975. The 17-AAG treatment decreased cellular Rad51 protein and mRNA levels and phosphorylated MKK1/2-ERK1/2 protein levels, and disrupted the HSP90 and Rad51 interaction. This triggered Rad51 protein degradation through the 26S proteasome pathway. The 17-AAG treatment also decreased the NSCLC cells' DNA repair capacity, which was restored by the forced expression of the Flag-Rad51 vector. Specific inhibition of Rad51 expression by siRNA further enhanced 17-AAG-induced cytotoxicity. In contrast, enhanced ERK1/2 activation by the constitutively active MKK1/2 (MKK1/2-CA) vector significantly restored the 17-AAG-reduced Rad51 protein levels and cell viability. Arachidin-1, an antioxidant stilbenoid, further decreased Rad51 expression and augmented the cytotoxic effect and growth inhibition of 17-AAG. The 17-AAG and arachidin-1-induced synergistic cytotoxic effects and decreased DNA repair capacity were abrogated in lung cancer cells with MKK1/2-CA or Flag-Rad51 expression vector transfection. In conclusion, HSP90 inhibition induces cytotoxicity by down-regulating Rad51 expression and DNA repair capacity in NSCLC cells.

Inhibition of thymidine phosphorylase expression by using an HSP90 inhibitor potentiates the cytotoxic effect of cisplatin in non-small-cell lung cancer cells.

Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. In this study, we examined the role of TP expression in relation to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced cytotoxicity in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1650. Treatment with 17-AAG (0.1-1 µM) resulted in a decrease in cellular TP protein and mRNA levels, which was accompanied by a downregulation of phosphorylated MKK1/2-ERK1/2 and AKT protein levels. The 17-AAG treatment disrupted the interaction between HSP90 and TP and triggered TP protein degradation through the ubiquitin-26S proteasome pathway. Specific inhibition of TP expression by siRNA further enhanced the cell death and growth inhibition that had been induced by 17-AAG. An enhancement of ERK1/2 or AKT activation by transfecting the cancer cells with constitutively active MKK1/2 or AKT expression vectors significantly restored the 17-AAG-reduced TP protein levels as well as cell viability. In contrast, a combination of U0126 (MKK1/2 inhibitors) or LY294002 (PI3K inhibitor) further decreased the TP expression and cell viability induced by 17-AAG. Moreover, 17-AAG enhanced the cisplatin-induced cytotoxic effect through downregulation of the cisplatin-induced TP expression and ERK1/2 and AKT activation. Taken together, our results suggest that the down-modulation of TP protein induced by 17-AAG represents a key factor in enhancing the cytotoxic effects of cisplatin in NSCLC cells.

Enhancement of mitomycin C-induced cytotoxicity by curcumin results from down-regulation of MKK1/2-ERK1/2-mediated thymidine phosphorylase expression.

Curcumin (diferuloylmethane), a phenolic compound obtained from the rhizome of Curcuma longa, has been found to inhibit cell proliferation in various human cancer cell lines, including non-small cell lung cancer (NSCLC). Thymidine phosphorylase (TP) is considered an attractive therapeutic target, because increased TP expression can suppress cancer cell death induced by DNA-damaging agents. Mitomycin C (MMC), a chemotherapeutic agent used to treat NSCLC, inhibits tumour growth through DNA cross-linking and breaking. Whether MMC can affect TP expression in NSCLC is unknown. Therefore, in this study, we suggested that curcumin enhances the effects of MMC-mediated cytotoxicity by decreasing TP expression and ERK1/2 activation. Exposure of human NSCLC cell lines H1975 and H1650 to curcumin decreased MMC-elicited phosphorylated MKK1/2-ERK1/2 protein levels. Moreover, curcumin significantly decreased MMC-induced TP protein levels by increasing TP mRNA and protein instability. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased TP protein levels and cell viability in curcumin- and MMC-co-treated cells. In contrast, U0126, a MKK1/2 inhibitor, augmented the cytotoxic effect and the down-regulation of TP by curcumin and MMC. Specific inhibition of TP by siRNA significantly enhanced MMC-induced cell death and cell growth inhibition. Our results suggest that suppression of TP expression or administration of curcumin along with MMC may be a novel lung cancer therapeutic modality in the future.

Inhibition of p38 MAPK-dependent excision repair cross-complementing 1 expression decreases the DNA repair capacity to sensitize lung cancer cells to etoposide.

Etoposide (VP-16), a topoisomerase II inhibitor, is an effective anticancer drug currently used for the treatment of a wide range of cancers. Excision repair cross-complementary 1 (ERCC1) is a key protein involved in the process of nucleotide excision repair. High level of ERCC1 expression in cancers is associated with resistance to DNA damage-based chemotherapy. In this study, the effects of p38 mitogen-activated protein kinase (MAPK) signal on the ERCC1 expression induced by etoposide in non-small cell lung cancer (NSCLC) cell lines was investigated. Etoposide increased phosphorylated MAPK kinase 3/6 (MKK3/6)-p38 MAPK and ERCC1 protein and mRNA levels in A549 and H1975 cells. Moreover, SB202190, a p38 inhibitor, or knockdown of p38 expression by specific short interfering RNA (siRNA) significantly decreased the etoposide-induced ERCC1 protein levels and DNA repair capacity in etoposide-exposed NSCLC cells. Enhancement of p38 activation by constitutively active MKK6 (MKK6E) increased ERCC1 protein levels. Specific inhibition of ERCC1 by siRNA significantly enhanced the etoposide-induced cytotoxicity and hypoxanthine guanine phosphoribosyltransferase (hprt) gene mutation rate. Moreover, the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) could decrease the etoposide-induced p38 MAPK-mediated ERCC1 expression and augment the cytotoxic effect and growth inhibition by etopsoside. 17-AAG and etoposide-induced synergistic cytotoxic effect and DNA repair capacity decrease could be abrogated in lung cancer cells with MKK6E or HA-p38 MAPK expression vector transfection. Our results suggest that in human NSCLC cells, ERCC1 is induced by etoposide through the p38 MAPK pathway, and this phenomenon is required for NSCLC survival and resistant DNA damage.

Curcumin enhances the mitomycin C-induced cytotoxicity via downregulation of MKK1/2-ERK1/2-mediated Rad51 expression in non-small cell lung cancer cells.

Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), has been reported to suppress the proliferation of a wide variety of tumor cells. Rad51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. A high expression of Rad51 has been reported in chemo- or radio-resistant carcinomas. Therefore, in the current study, we will examine whether curcumin could enhance the effects of mitomycin C (MMC), a DNA interstrand cross-linking agent, to induce cytotoxicity by decreasing Rad51 expression. Exposure of two human non-small lung cancer (NSCLC) cell lines (A549 and H1975) to curcumin could suppress MMC-induced MKK1/2-ERK1/2 signal activation and Rad51 protein expression. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased Rad51 protein levels in curcumin and MMC co-treated human lung cells. Moreover, the synergistic cytotoxic effect induced by curcumin combined with MMC was decreased by MKK1-CA-mediated enhancement of ERK1/2 activation by a significant degree. In contrast, MKK1/2 inhibitor, U0126 was shown to augment the cytotoxicity of curcumin and MMC through downregulation of ERK1/2 activation and Rad51 expression. Depletion of endogenous Rad51 expression by siRad51 RNA transfection significantly enhanced MMC and/or curcumin induced cell death and cell growth inhibition. In contrast, an overexpression of Rad51 protected lung cancer cells from synergistic cytotoxic effects induced by curcumin and MMC. We concluded that Rad51 inhibition may be an additional action mechanism for enhancing the chemosensitization of MMC by curcumin in NSCLC.

Synergistic effect of curcumin and cisplatin via down-regulation of thymidine phosphorylase and excision repair cross-complementary 1 (ERCC1).

Curcumin (diferuloylmethane), a phenolic compound obtained from the rhizome of Curcuma longa, is known to have antiproliferative and antitumor properties. Thymidine phosphorylase (TP), an enzyme of the pyrimidine salvage pathway, is considered an attractive therapeutic target, and its expression could suppress cancer cell death induced by DNA damage agents. Excision repair cross-complementary 1 (ERCC1) is a protein involved the process of nucleotide excision repair. The ERCC1 gene is expressed at high levels in cancers and has been associated with resistance to platinum-based chemotherapy. In this study, the effects of curcumin on TP and ERCC1 expression induced by cisplatin in non-small-cell lung cancer (NSCLC) cell lines was investigated. Exposure of the NSCLC cells to various concentrations of curcumin (5-40 µM) down-regulates the mRNA and protein levels of TP and ERCC1 through destabilization of the mRNA and proteins via a mechanism involving inactivation of MKK1/2-extracellular signal-regulated kinase (ERK1/2). Depletion of endogenous TP or ERCC1 expression by transfection with specific small interfering RNAs significantly decreases cell viability in curcumin-exposed NSCLC cells. Curcumin enhances the sensitivity of cisplatin treatment for NSCLC through inactivation of ERK1/2 and by decreasing the TP and ERCC1 protein levels. Enhancement of ERK1/2 signaling by constitutively active MKK1/2 causes an increase in TP and ERCC1 protein levels and promotes cell viability after cotreatment with curcumin and cisplatin. Enhancement of the cytotoxicity to cisplatin by administration of curcumin is mediated by down-regulation of the expression levels of TP and ERCC1 and by inactivation of ERK1/2.

Up-regulation of extracellular signal-regulated kinase 1/2-dependent thymidylate synthase and thymidine phosphorylase contributes to cisplatin resistance in human non-small-cell lung cancer cells.

Chemotherapy for advanced human non-small-cell lung cancer (NSCLC) includes platinum-containing compound such as cisplatin in combination with a second- or third-generation cytotoxic agent. 5-Fluorouracil (5-FU) belongs to antimetabolite chemotherapeutics, and its mechanism of cytotoxicity is involved in the inhibition of thymidylate synthase (TS). TS and thymidine phosphorylase (TP) are key enzymes of the pyrimidine salvage pathway. In this study, we have examined the molecular mechanism of TS and TP in regulating drug sensitivity to cisplatin in NSCLC cell lines. Cisplatin could increase the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MKK1/2)-extracellular signal-regulated kinase 1/2 (ERK1/2) and the protein levels of TS and TP through enhancing the protein stability in A549 and H1975 cells. Blocking ERK1/2 activation by MKK1/2 inhibitor [U0126; 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene)] decreased TS and TP protein levels in both cell lines treated with cisplatin. Depletion of endogenous TS or TP expression by specific small interfering RNA transfection significantly increased cisplatin-induced cell death and growth inhibition. Combined treatment with 5-FU could decrease cisplatin-induced ERK1/2 activation and the induction of TS and TP, which subsequently resulted in synergistic cytotoxic effects. Enforced expression of constitutive active MKK1/2 vectors rescued the protein levels of phospho-ERK1/2, TS, and TP, and the cell viability that were decreased by cisplatin and 5-FU combination. In contrast, U0126 enhanced drug sensitivity to cisplatin and/or 5-FU in lung cancer cells. In conclusion, the up-regulation of ERK1/2-dependent TS and TP can protect human lung cancer cells from cisplatin-induced cytotoxicity.

Modulation of Rad51, ERCC1, and thymidine phosphorylase by emodin result in synergistic cytotoxic effect in combination with capecitabine.

Thymidine phosphorylase (TP) is the rate-limiting enzyme for the activation of capecitabine (pro-drug of fluorouracil), and as a useful predictor of tumor response to capecitabine-based chemotherapy. Overexpression of Rad51 and ERCC1 induce resistance to chemotherapeutic agents. Emodin, one of the main bioactive anthraquinone derivatives in the roots and rhizomes of numerous plants, possesses potent antitumor effects. Accordingly, we aimed to explore the molecular mechanism of emodin enhances the capecitabine-induced cytotoxicity through controlling Rad51, ERCC1, and TP expression in human non-small cell lung cancer (NSCLC). The results show that capecitabine increases the phosphorylation of MKK1/2-ERK1/2 and protein levels of Rad51 and ERCC1 through enhancing the protein stability. Depletion of endogenous Rad51 or ERCC1 expression by specific small interfering RNA transfection significantly increases capecitabine-induced cell death and growth inhibition. Emodin enhances the capecitabine-induced cytotoxic effects through ERK1/2 inactivation and decreasing the Rad51 and ERCC1 protein levels induced by capecitabine. Enhancement of ERK1/2 signaling by constitutively active MKK1/2 (MKK1/2-CA) results in increasing Rad51 and ERCC1 protein levels and cell viability in NSCLC cell lines treated with emodin and capecitabine. Interestingly, emodin enhances TP mRNA and protein expression in capecitabine treated NSCLC cell lines, and depletion of the TP expression decreases the cytotoxic effects induced by capecitabine and emodin. We conclude that enhancing the cytotoxicity to capecitabine by emodin is mediated by down-regulation the expression of Rad51 and ERCC1 and up-regulation TP expression.