The correlation of epithelial-mesenchymal transition-related gene expression and the clinicopathologic features of colorectal cancer patients in Taiwan.

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in the world. It has been the most prevalent malignancy in Taiwan for consecutive thirteen years. Despite the diversity of its etiologic and pathophysiologic factors, a biological process named as epithelial-mesenchymal transition (EMT) is indispensable in the progression of epithelial cancer. Our aim is to investigate the correlation between the expression of 8 EMT-related proteins (E-cadherin, ß-catenin, claudin-1, CD44, N-cadherin, fibronectin, vimentin, S100A4) and the clinicopathologic features of CRC in Taiwan, along with the DNA CpG epigenetic status of CD44 gene. In immunohistochemical assessment, decreased expression of E-cadherin is statistically associated with the progression of cancer stage, while decreased expression of claudin-1 as well as increased ß-catenin nuclear translocation and N-cadherin expression is statistically associated with the progression of histopathologic grade. E-cadherin, nuclear ß-catenin and claudin-1 are also associated with other important prognostic factors, including nodal metastasis, tumor deposits, and elevated serum CA 19-9 levels. In addition, the left-sided colon and rectal cancers show increased nuclear translocation of ß-catenin compared to the right-sided colon cancers, while the rectal cancers show increased fibronectin expression compared to the right-sided and left-sided colon cancers. Moreover, vimentin is aberrantly expressed in one case of signet-ring cell carcinoma. The DNA methylation levels of CD44 gene promoter between the tumoral and non-tumorous tissues by NGS comparison showed statistical difference on six CpG sites. However, such difference may not be sufficient because these DNA methylation proportions are too low to inactivate CD44 gene. Our results demonstrate the expression of E-cadherin, claudin-1, and nuclear ß-catenin is closely related to the clinicopathologic prognostic determinants of CRC in Taiwan. The DNA methylation level of CD44 gene and its protein expression, however, show no correlation with the clinicopathologic features in CRC.

Salinomycin acts through reducing AKT-dependent thymidylate synthase expression to enhance erlotinib-induced cytotoxicity in human lung cancer cells.

Erlotinib (TarcevaR) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor in the treatment of human non-small cell lung cancer (NSCLC). Salinomycin, a polyether antibiotic, has been promising a novel therapeutic agent for lung cancer, and down-regulated the expression of thymidylate synthase (TS) in NSCLC cell lines. Previous study showed that against EGFR and TS was strongly synergistic cytotoxicity in NSCLC cells. In this study, we showed that erlotinib (1.25-10µM) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung squamous cell carcinoma H1703 and adenocarcinoma H1975 cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting AKT activity with PI3K inhibitor LY294002 enhanced the cytotoxicity and cell growth inhibition of erlotinib. A combination of erlotinib and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced protein levels of phospho-AKT(Ser473), phospho-AKT(Thr308), and TS. Overexpression of a constitutive active AKT (AKT-CA) or Flag-TS expression vector reversed the salinomycin and erlotinib-induced synergistic cytotoxicity. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the erlotinib-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with erlotinib for lung cancer treatment.

Curcumin downregulates p38 MAPK-dependent X-ray repair cross-complement group 1 (XRCC1) expression to enhance cisplatin-induced cytotoxicity in human lung cancer cells.

Cisplatin is a well-studied and widely used chemotherapeutic agent and is effective in the treatment of the advanced human non-small cell lung cancer (NSCLC). Curcumin is a yellow pigment derived from the rhizome of Curcuma longa and has been proved to have antioxidant and antitumor properties. XRCC1 is an important scaffold protein involved in base excision repair and plays an important role in the development of lung cancer. In this study, we characterize the role of curcumin in the cytotoxicity, p38 MAPK activation, and XRCC1 expression affected by cisplatin in NSCLC cells. We show that curcumin enhanced the cytotoxicity induced by cisplatin in two NSCLC cells, A549 and H1703. Treatment with cisplatin alone increased XRCC1 mRNA and protein expression through p38 MAPK activation. Moreover, SB2023580 (p38 inhibitor) decreased the XRCC1 mRNA and protein stability upon cisplatin treatment. Knockdown of XRCC1 in NSCLC cells by transfection of XRCC1 siRNA or inactivation of p38 MAPK resulted in enhancing the cytotoxicity and cell growth inhibition induced by cisplatin. Curcumin inhibited the expression of XRCC1 in cisplatin-exposed NSCLC cells. Furthermore, transfection with constitutive active MKK6 or HA-p38 MAPK vectors rescued the XRCC1 protein level and also the cell survival suppressed by cisplatin and curcumin combination in A549 and H1703 cells. These findings suggested that the downregulation of XRCC1 expression by curcumin can enhance the chemosensitivity of cisplatin in NSCLC cells.

Minocycline enhances mitomycin C-induced cytotoxicity through down-regulating ERK1/2-mediated Rad51 expression in human non-small cell lung cancer cells.

Minocycline is a semisynthetic tetracycline derivative; it has anti-inflammatory and anti-cancer effects distinct from its antimicrobial function. However, the molecular mechanism of minocycline-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the MKK1/2-ERK1/2 signal pathway maintains the expression of Rad51 in NSCLC cells. In this study, minocycline treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1975. Treatment with minocycline decreased Rad51 mRNA and protein levels through MKK1/2-ERK1/2 inactivation. Furthermore, expression of constitutively active MKK1 (MKK1-CA) vectors significantly rescued the decreased Rad51 protein and mRNA levels in minocycline-treated NSCLC cells. However, combined treatment with MKK1/2 inhibitor U0126 and minocycline further decreased the Rad51 expression and cell viability of NSCLC cells. Knocking down Rad51 expression by transfection with small interfering RNA of Rad51 enhanced the cytotoxicity and cell growth inhibition of minocycline. Mitomycin C (MMC) is typically used as a first or second line regimen to treat NSCLC. Compared to a single agent alone, MMC combined with minocycline resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-ERK1/2, and reduced Rad51 protein levels. Overexpression of MKK1-CA or Flag-tagged Rad51 could reverse the minocycline and MMC-induced synergistic cytotoxicity. These findings may have implications for the rational design of future drug regimens incorporating minocycline and MMC for the treatment of NSCLC.

Resveratrol Enhances Etoposide-Induced Cytotoxicity through Down-Regulating ERK1/2 and AKT-Mediated X-ray Repair Cross-Complement Group 1 (XRCC1) Protein Expression in Human Non-Small-Cell Lung Cancer Cells.

Etoposide (VP-16), a topoisomerase II inhibitor, is an effective anti-cancer drug used for the treatment of non-small-cell lung cancer (NSCLC). Resveratrol is a naturally occurring polyphenolic compound that has been proved to have anti-cancer activity. XRCC1 is an important scaffold protein involved in base excision repair that is regulated by ERK1/2 and AKT signals and plays an important role in the development of lung cancer. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in etoposide treatment alone or combined with resveratrol-induced cytotoxicity in NSCLC cells has not been identified. In this study, etoposide treatment increased XRCC1 mRNA and protein expression through AKT and ERK1/2 activation in two NSCLC cells, H1703 and H1975. Knockdown of XRCC1 in NSCLC cells by transfection of XRCC1 siRNA or inactivation of ERK1/2 and AKT resulted in enhancing cytotoxicity and cell growth inhibition induced by etoposide. Resveratrol inhibited the expression of XRCC1 and enhanced the etoposide-induced cell death and anti-proliferation effect in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors could rescue the XRCC1 protein level and also the cell survival suppressed by co-treatment with etoposide and resveratrol. These findings suggested that down-regulation of XRCC1 expression by resveratrol can enhance the chemosensitivity of etoposide in NSCLC cells.

Down-regulation of ERK1/2 and AKT-mediated X-ray repair cross-complement group 1 protein (XRCC1) expression by Hsp90 inhibition enhances the gefitinib-induced cytotoxicity in human lung cancer cells.

Gefitinib (Iressa(R), ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT signaling activation. It has been shown that inhibition of Hsp90 function can enhance antitumor activity of EGFR-TKI. XRCC1 is an important scaffold protein in base excision repair, which could be regulated by ERK1/2 and AKT pathways. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in gefitinib alone or combination with an Hsp90 inhibitor-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. In this study, gefitinib treatment decreased XRCC1 mRNA and protein expression through ERK1/2 and AKT inactivation in two NSCLC cells, A549 and H1975. Knocking down XRCC1 expression by transfection with small interfering RNA of XRCC1 enhanced the cytotoxicity and cell growth inhibition of gefitinib. Combining treatment of gefitinib with an Hsp90 inhibitor resulted in enhancing the reduction of XRCC1 protein and mRNA levels in gefitinib-exposed A549 and H1975 cells. Compared to a single agent alone, gefitinib combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors rescued the XRCC1 protein level as well as the cell survival suppressed by an Hsp90 inhibitor and gefitinib. These findings suggested that down-regulation of XRCC1 can enhance the sensitivity of gefitinib for NSCLC cells.

Down-regulation of MSH2 expression by Hsp90 inhibition enhances cytotoxicity affected by tamoxifen in human lung cancer cells.

The anti-estrogen tamoxifen has been used worldwide as an adjuvant hormone therapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Human MutS homolog 2 (MSH2), a crucial element of the highly conserved DNA mismatch repair system, and expression of MSH2 have been down-regulated by Hsp90 function inhibition in human lung cancer. Therefore, in this study, we examined whether MSH2 plays a role in the tamoxifen and Hsp90 inhibitor-induced cytotoxic effect on NSCLC cells. The results showed that treatment with tamoxifen increased MSH2 mRNA and protein levels. The combination treatment with PI3K inhibitors (LY294002 or wortmannin) or knockdown AKT expression by specific small interfering RNA could decrease tamoxifen-induced MSH2 expression. Both knocking down MSH2 expression and co-treatment of PI3K inhibitors enhanced the cytotoxicity and cell growth inhibition of tamoxifen. Compared to a single agent alone, tamoxifen combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced MSH2 expression. These findings may have implications for the rational design of future drug regimens incorporating tamoxifen and Hsp90 inhibitors for the treatment of NSCLC.

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.

Tamoxifen enhances erlotinib-induced cytotoxicity through down-regulating AKT-mediated thymidine phosphorylase expression in human non-small-cell lung cancer cells.

Tamoxifen is a triphenylethylene nonsteroidal estrogen receptor (ER) antagonist used worldwide as an adjuvant hormone therapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Thymidine phosphorylase (TP) is an enzyme of the pyrimidine salvage pathway which is upregulated in cancers. In this study, tamoxifen treatment inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with tamoxifen decreased TP mRNA and protein levels through AKT inactivation. Furthermore, expression of constitutively active AKT (AKT-CA) vectors significantly rescued the decreased TP protein and mRNA levels in tamoxifen-treated NSCLC cells. In contrast, combination treatment with PI3K inhibitors (LY294002 or wortmannin) and tamoxifen further decreased the TP expression and cell viability of NSCLC cells. Knocking down TP expression by transfection with small interfering RNA of TP enhanced the cytotoxicity and cell growth inhibition of tamoxifen. Erlotinib (Tarceva, OSI-774), an orally available small molecular inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for clinical treatment of NSCLC. Compared to a single agent alone, tamoxifen combined with erlotinib resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-AKT and phospho-ERK1/2, and reduced TP protein levels. These findings may have implications for the rational design of future drug regimens incorporating tamoxifen and erlotinib for the treatment of NSCLC.

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.

Pemetrexed downregulates ERCC1 expression and enhances cytotoxicity effected by resveratrol in human nonsmall cell lung cancer cells.

The multitargeted antifolate pemetrexed has demonstrated certain clinical activities against nonsmall cell lung cancer (NSCLC). Resveratrol (3,5,4-trihydroxy-trans-stilbene) is a polyphenol found in grapes and other plants and has great potential as a preventative and therapeutic agent due to its anticarcinogenic activity. The efficacy of adding resveratrol to pemetrexed to prolong the survival of patients with NSCLC still remains unclear. The excision repair cross-complementation 1 (ERCC1) is a DNA repair gene coding 5' endonuclease in nucleotide excision repair and is overexpressed in chemo- or radioresistant carcinomas. In this study, resveratrol (10-50 µM) inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with resveratrol increased ERCC1 messenger RNA and protein levels in a MKK3/6-p38 MAPK signal activation-dependent manner. Furthermore, blocking p38 MAPK activation by SB202190 or knocking down ERCC1 expression by transfection with small interfering RNA of ERCC1 enhanced the cytotoxicity of resveratrol. Combining resveratrol with pemetrexed resulted in a synergistic cytotoxic effect, accompanied with the reduction of phospho-p38 MAPK and ERCC1 protein levels, and a DNA repair capacity. Expression of constitutively active MKK6 (MKK6E) or HA-p38 MAPK vectors significantly rescued the decreased p38 MAPK activity, and restored ERCC1 protein levels and cell survival in resveratrol and pemetrexed cotreated NSCLC cells. In this study, for the first time, we have demonstrated the synergistic effect of combined treatment with resveratrol and pemetrexed in human NSCLC cells through downregulation of the MKK3/6-p38 MAPK-ERCC1 signal, suggesting a potential benefit of combining resveratrol and pemetrexed to treat lung cancer in the future.