Sheng-Chieh Tseng

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. Mol Cancer Ther; 11(3); 561–71. ©2011 AACR.

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.

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 metformins 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.

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.

Inhibition of p38 MAPK-dependent MutS homologue-2 (MSH2) expression by metformin enhances gefitinib-induced cytotoxicity in human squamous lung cancer cells

OBJECTIVES Gefitinib, a quinazoline-derived tyrosine kinase inhibitor, has anti-tumor activity in vivo and in vitro. Human MutS homologue-2 (MSH2) plays a central role in promoting genetic stability by correcting DNA replication errors. The present study investigated the effects of p38 mitogen-activated protein kinase (MAPK) signal on gefitinib-induced MSH2 expression in two human non-small cell lung squamous cancer cell lines. MATERIALS AND METHODS After the gefitinib treatment, the expressions of MSH2 mRNA were determined by real-time PCR and RT-PCR analysis. Protein levels of MSH2, phospho-MKK3/6, phospho-p38 MAPK were determined by Western blot analysis. We used specific MSH2, and p38 MAPK small interfering RNA to examine the role of p38 MAPK-MSH2 signal in regulating the chemosensitivity of gefitinib. Cell viability was assessed by MTS assay, trypan blue exclusion, and colony-forming ability assay. RESULTS Exposure of gefitinib increased MSH2 protein and mRNA levels, which was accompanied by MKK3/6-p38 MAPK activation in H520 and H1703 cells. Moreover, blocking p38 MAPK activation by SB202190 significantly decreased gefitinib-induced MSH2 expression by increasing mRNA and protein instability. In contrast, enhancing p38 activation using constitutively active MKK6 (MKK6E) increased MSH2 protein and mRNA levels. Specific inhibition of MSH2 expression by siRNA enhanced gefitinib-induced cytotoxicity. Metformin, an anti-diabetic drug, might reduce cancer risk. In human lung squamous cancer cells, metformin decreased gefitinib-induced MSH2 expression and augmented the cytotoxic effect and growth inhibition by gefitinib. Transient expression of MKK6E or HA-p38 MAPK vector could abrogate metformin and gefitinib-induced synergistic cytotoxic effect in H520 and H1703 cells. CONCLUSION Together, down-regulation of MSH2 expression can be a possible strategy to enhance the sensitivity of gefitinib to human lung squamous cancer cells.

Down-regulation of MSH2 expression by an Hsp90 inhibitor enhances pemetrexed-induced cytotoxicity in human non-small-cell lung cancer cells.

Elevated heat shock protein 90 (Hsp90) expression has been linked to poor prognosis in patients with non-small cell lung cancer (NSCLC). The multitargeted antifolate pemetrexed has demonstrated certain clinical activities against NSCLC. However, the efficacy of the combination of pemtrexed and Hsp90 inhibitor to prolong the survival of patients with NSCLC still remains unclear. Human MutS homolog 2 (MSH2), a crucial element of the highly conserved DNA mismatch repair system, and defects or polymorphisms of MSH2 have been found in lung cancer. In this study, we evaluated the effects of pemetrexed on NSCLC cell lines (H520 and H1703) and found that treatment with this drug at 20-50 µM increased the MSH2 mRNA and protein levels in a MKK3/6-p38 MAPK signal activation-dependent manner. Furthermore, the knockdown of MSH2 expression by transfection with small interfering RNA of MSH2 or the blockage of p38 MAPK activation by SB202190 enhanced the cytotoxicity of pemetrexed. Combining the drug treatment with an Hsp90 inhibitor resulted in an enhanced pemetrexed-induced cytotoxic effect, accompanied with the reduction of MSH2 protein and mRNA levels. The expression of constitutively active MKK6 (MKK6E) or HA-p38 MAPK vectors significantly rescued the decreased p38 MAPK activity, and restored the MSH2 protein levels and cell survival in NSCLC cells co-treated with pemetrexed and Hsp90 inhibitor. In this study, we have demonstrated that down-regulation of the MKK3/6-p38 MAPK signal with the subsequent reduction of MSH2 enhanced the cytotoxic effect of pemetrexed in H520 and H1703 cells. The results suggest a potential future benefit of combining pemetrexed and the Hsp90 inhibitor to treat lung cancer.

Abstract 1283: Role of ERCC1 in metformin enhancing paclitaxel-induced cytotoxicity.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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. Citation Format: Yun-Wei Lin, Sheng-Chieh Tseng, Yu-Ching Huang, Hsien-Chun Chiu, Huang-Jen Chen, Shao-Hsing Weng, Yi-Jhen Huang, Ting-Yu Wo. Role of ERCC1 in metformin enhancing paclitaxel-induced cytotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1283. doi:10.1158/1538-7445.AM2013-1283

Abstract 1301: Metformin induces cytotoxicity by TP and ERCC1 decrease.

Metformin is an anti-diabetic drug recently shown to inhibit cancer cell proliferation and growth, although the involved molecular mechanisms have not been elucidated. In many cancer cells, highly expression of thymidine phosphorylase (TP) and ERCC1 are associated with poor prognosis. We used A549 and H1975 huamn 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 cells viability. Specific inhibition of TP and ERCC1 expression by siRNA enhanced the metformin-induced cytotoxicity and growth inhibition. In conclusion, metformin induces cytotoxicity by down-regulating TP and ERCC1 expression in NSCLC cells. Citation Format: Yun-Wei Lin, Yu-Ching Huang, Sheng-Chieh Tseng, Hsien-Chun Chiu, Huang-Jen Chen, Yi-Jhen Huang, Ting-Yu Wo, Shao-Hsing Weng, Jen-Chung Ko. Metformin induces cytotoxicity by TP and ERCC1 decrease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1301. doi:10.1158/1538-7445.AM2013-1301