Jyh-g Chen

Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells

Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 μM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with 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.

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.

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

ABSTRACT 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 (1.25–10 &mgr;M) 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. Graphical abstract Figure. No caption available. HighlightsErlotinib decreases thymidylate synthase expression in human lung cancer cells.Downregulation of thymidylate synthase enhances the cytotoxicity of erlotinib.A combination of erlotinib and salinomycin shows a synergistic cytotoxicity.

Salinomycin enhances cisplatin-induced cytotoxicity in human lung cancer cells via down-regulation of AKT-dependent thymidylate synthase expression

Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore and has anticancer activity on various cancer cell lines. Cisplatin has been proved as chemotherapy drug for advanced human non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) is a key enzyme in the pyrimidine salvage pathway, and increased expression of TS is thought to be associated with resistance to cisplatin. In this study, we showed that salinomycin (0.5-2μg/mL) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung adenocarcinoma A549 and squamous cell carcinoma H1703 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 salinomycin. A combination of cisplatin and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-AKT, and TS expression. Overexpression of a constitutive active AKT (AKT-CA) expression vector reversed the salinomycin and cisplatin-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in salinomycin and cisplatin cotreated cells. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the cisplatin-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with cisplatin for lung cancer treatment.

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.

17-(Allylamino)-17-Demethoxygeldanamycin Enhances Etoposide-Induced Cytotoxicity via the Downregulation of Xeroderma Pigmentosum Complementation Group C Expression in Human Lung Squamous Cell Carcinoma Cells

Etoposide (VP16) is a topoisomerase II inhibitor and has been used for the treatment of non-small cell lung cancer (NSCLC). Xeroderma pigmentosum complementation group C (XPC) protein is a DNA damage recognition factor in nucleotide excision repair and involved in regulating NSCLC cell proliferation and viability. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is responsible for the stabilization and maturation of many oncogenic proteins. In this study, we report whether Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) enhanced etoposide-induced cytotoxicity in NSCLC cells through modulating the XPC expression. We found that etoposide increased XPC expression in an AKT activation manner in 2 squamous cell carcinoma H1703 and H520 cells. Knockdown of XPC using siRNA or inactivation of AKT by pharmacological inhibitor PI3K inhibitor (LY294002) enhanced the cytotoxic effects of etoposide. In contrast, enforced expression of XPC cDNA or AKT-CA (a constitutively active form of AKT) reduced the cytotoxicity and cell growth inhibition of etoposide. Hsp90 inhibitor 17-AAG enhanced cytotoxicity and cell growth inhibition of etoposide in NSCLC cells, which were associated with the downregulation of XPC expression and inactivation of AKT. Our findings suggested that the Hsp90 inhibition induced XPC downregulation involved in enhancing the etoposide-induced cytotoxicity in H1703 and H520 cells.

Down-Regulation of MutS Homolog 2 (MSH2) Expression by Curcumin Enhances Cytotoxicity Induced by Gemcitabine in Human Lung Adenocarcinoma Cells

Gemcitabine (2′,2′-difluorodeoxycytidine) is a difluorinated analog of deoxycytidine. It is used clinically to treat patients with non-small-cell lung cancer (NSCLC). Curcumin is a yellow pigment derived from the rhizome of Curcuma longa, and has been proven to have antioxidant and antitumor properties. Human MutS homolog 2 (MSH2) is a key DNA mismatch repair protein that plays an important role in maintaining genomic stability. Depletion of MSH2 from cells can reverse resistance to certain DNA-damaging agents. In this study, exposure of human lung adenocarcinoma A549 and H1975 cells to gemcitabine increased protein phosphorylation of MKK3/6 and p38 MAPK in a time- and dose-dependent manner; this was accompanied by increased expression of MSH2 mRNA and protein. Gemcitabine-induced cytotoxicity was significantly enhanced by MSH2 siRNA transfection or inactivation of p38 MAPK by SB202190 or p38 MAPK siRNA transfection. However, overexpression of MSH2 cDNA reduced gemcitabine-induced cytotoxicity. Furthermore, curcumin enhanced gemcitabine-induced cytotoxicity via inactivation of MKK3/6-p38 MAPK and downregulation of MSH2. Enforced expression of constitutively active MKK6 rescued cell viability and restored MSH2 protein levels that were suppressed by curcumin and gemcitabine. Suppression of MSH2 expression and a combination with curcumin may be considered as potential therapeutic modalities for gemcitabine-resistant NSCLC cells.