Heat shock protein 90 inhibitor 17-AAG down-regulates thymidine phosphorylase expression and potentiates the cytotoxic effect of tamoxifen and erlotinib in human lung squamous carcinoma cells.

Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, in cancer cells, are related to a poor prognosis in a variety of cancers. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is involved in the stabilization and maturation of many oncogenic proteins. The aim of this study is to elucidate whether Hsp90 inhibitor 17-AAG could enhance tamoxifen- and erlotinib-induced cytotoxicity in nonsmall cell lung cancer (NSCLC) cells via modulating TP expression in two squamous NSCLC cell lines, H520 and H1703. We found that 17-AAG reduced TP expression via inactivating the MKK1/2-ERK1/2-mitogen-activated protein kinase (MAPK) pathway. TP knockdown with siRNA or ERK1/2 MAPK inactivation with the pharmacological inhibitor U0126 could enhance the cytotoxic and growth inhibitory effects of 17-AAG. In contrast, MKK1-CA or MKK2-CA (a constitutively active form of MKK1/2) vector-enforced expression could reduce the cytotoxic and cell growth inhibitory effects of 17-AAG. Furthermore, 17-AAG enhanced the cytotoxic and cell growth inhibitory effects of tamoxifen and erlotinib in NSCLC cells, which were associated with TP expression downregulation and MKK1/2-ERK1/2 signal inactivation. Taken together, Hsp90 inhibition downregulates TP, enhancing the tamoxifen- and erlotinib-induced cytotoxicity in H520 and H1703 cells.

Nitroglycerin-induced downregulation of AKT- and ERK1/2-mediated radiation-sensitive 52 expression to enhance pemetrexed-induced cytotoxicity in human lung cancer cells.

Nitroglycerin (NTG)-a nitric oxide-donating drug-is traditionally administered via the sublingual route to treat acute myocardial angina attacks. NTG also increases tumor blood flow and, consequently, cancer drug delivery to tumor cells. In the homologous recombination pathway, radiation-sensitive 52 (Rad52) plays a crucial role in DNA repair by promoting the annealing of complementary single-stranded DNA and stimulating radiation-sensitive 51 (Rad51) recombinase activity. Pemetrexed-a multitargeted antifolate agent-exhibits satisfactory clinical activity in wild-type nonsquamous non-small-cell lung cancer (NSCLC) cells. However, the synergistic activity of combination therapy with NTG and pemetrexed against NSCLC cells has not yet been clarified. In 2 NSCLC cell lines (i.e. lung squamous cell carcinoma H520 and lung adenocarcinoma H1975 cells), NTG reduced Rad52 expression; in addition, decreased phospho-AKT and phospho-ERK1/2 protein levels were observed. Enhancement of AKT or ERK1/2 activity through transfection with a constitutively active AKT (AKT-CA) vector or constitutively active mitogen-activated protein kinase kinase 1 (MKK1-CA) vector increased the Rad52 protein level and cell survival, which were suppressed by NTG. The knockdown of Rad52 expression by using small interfering RNA or by inhibiting AKT and ERK1/2 activity enhanced the cytotoxicity and cell growth inhibition induced by NTG. Moreover, NTG synergistically enhanced the cytotoxicity and cell growth inhibition induced by pemetrexed in NSCLC cells; these effects were associated with AKT and ERK1/2 inactivation and, consequently, Rad52 downregulation in H520 and H1975 cells. The results provide a rationale for combining NTG and pemetrexed in lung cancer treatment to improve lung cancer control.

Downregulation of p38 MAPK Activation and Radiation-Sensitive 52 Expression Enhances 5-Fluorouracil and Erlotinib-Induced Cytotoxicity in Human Lung Squamous Cells.

INTRODUCTION: 5-Fluorouracil (5-FU) is used to treat various cancers, including non-small-cell lung cancer (NSCLC). It inhibits nucleotide synthesis and induces single- and double-strand DNA breaks. In the homologous recombination pathway, radiation-sensitive 52 (Rad52) plays a crucial role in DNA repair by promoting the annealing of complementary single-stranded DNA and stimulating Rad51 recombinase activity. Erlotinib (Tarceva) is a selective epidermal growth factor receptor tyrosine kinase inhibitor with clinical activity against NSCLC cells. However, whether the combination of 5-FU and erlotinib has synergistic activity against NSCLC cells is unknown. METHODS: After the 5-FU and/or erlotinib treatment, the expressions of Rad52 mRNA were determined by quantitative real-time polymerase chain reaction analysis. Protein levels of Rad52 and phospho-p38 MAPK were determined by Western blot analysis. We used specific Rad52 or p38 MAPK small interfering RNA and p38 MAPK inhibitor (SB2023580) to examine the role of p38 MAPK-Rad52 signal in regulating the chemosensitivity of 5-FU and/or erlotinib. Cell viability was assessed by MTS assay and trypan blue exclusion assay. RESULTS: In 2 squamous cell carcinoma cell lines, namely, H520 and H1703, 5-FU reduced Rad52 expression in a p38 MAPK inactivation-dependent manner. Enhancement of p38 MAPK activity by transfection with MKK6E (a constitutively active form of MKK6) vector increased the Rad52 protein level and cell survival by 5-FU. However, in human lung bronchioloalveolar cell adenocarcinoma A549 cells, 5-FU reduced Rad52 expression and induced cytotoxicity independent of p38 MAPK. Moreover, 5-FU synergistically enhanced the cytotoxicity and cell growth inhibition of erlotinib in NSCLC cells; these effects were associated with Rad52 downregulation and p38 MAPK inactivation in H520 and H1703 cells. CONCLUSION: The results provide a rationale for combining 5-FU and erlotinib in lung cancer treatment.

Downregulation of Xeroderma Pigmentosum Complementation Group C Expression by 17-Allylamino-17-Demethoxygeldanamycin Enhances Bevacizumab-Induced Cytotoxicity in Human Lung Cancer Cells.

INTRODUCTION: Xeroderma pigmentosum complementation group C (XPC) protein is an important DNA damage recognition factor involved in nucleotide excision repair and regulation of non-small-cell lung cancer (NSCLC) cell proliferation and viability. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) blocks ATP binding to heat shock protein 90 (Hsp90), resulting in destabilization of Hsp90-client protein complexes. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor expressed by many types of tumors. Bevacizumab (Avastin) is a humanized monoclonal antibody against human VEGF used as an antiangiogenesis agent in the therapy of many cancers, proving successful in increasing objective tumor response rate and prolonging overall survival in NSCLC patients. METHODS: After the bevacizumab and/or 17-AAG treatment, the expressions of XPC mRNA were determined by quantitative real-time PCR analysis. Protein levels of XPC and phospho-AKT were determined by Western blot analysis. We used specific XPC small interfering RNA and PI3K inhibitor (LY294002) to examine the role of the AKT-XPC signal in regulating the chemosensitivity of bevacizumab and 17-AAG. Cell viability was assessed by the MTS assay and trypan blue exclusion assay. RESULTS: In this study, bevacizumab decreased XPC expression in human lung squamous cell carcinoma H520 and H1703 cells via AKT inactivation. Enhancement of AKT activity by transfection with constitutively active AKT vectors increased XPC expression and cell survival after treatment with bevacizumab. In addition, 17-AAG synergistically enhanced bevacizumab-induced cytotoxicity and cell growth inhibition in H520 and H1703 cells, associated with downregulation of XPC expression and inactivation of AKT. DISCUSSION/CONCLUSION: Together, these results may provide a rationale to combine bevacizumab with Hsp90 inhibitors in future to enhance therapeutic effects for lung cancer.

Nitroglycerin Enhances Cisplatin-Induced Cytotoxicity via AKT Inactivation and Thymidylate Synthase Downregulation in Human Lung Cancer Cells.

Nitroglycerin (NTG), a nitric oxide-donating drug, may increase tumor blood flow and consequently increase cancer drug delivery to tumor cells. Thymidylate synthase (TS) is an essential enzyme for the de novo synthesis of deoxythymidine monophosphate; we had found that knocking down the expression of TS sensitizes lung cancer cells to cisplatin-induced cytotoxicity. However, whether NTG and cisplatin could induce synergistic cytotoxicity in nonsmall cell lung cancer (NSCLC) cells through modulating TS expression is unknown. In this study, NTG decreased TS expression in an AKT, also known as Protein kinase B (PKB) inactivation dependent manner in human lung adenocarcinoma A549 and squamous cell carcinoma H1703 cells. Enhancement of AKT activity by transfection with constitutive active AKT vectors increased the TS expression level as well as the cell survival pretreated by NTG. Moreover, NTG synergistically enhanced cytotoxicity and cell growth inhibition by cisplatin treatment in NSCLC cells, which were associated with downregulation of TS expression and inactivation of AKT in A549 and H1703 cells. Together, these results may provide a rationale to combine NTG with cisplatin-based chemotherapy to enhance the therapeutic effect for lung cancer in the future.

Capsaicin enhances erlotinib-induced cytotoxicity via AKT inactivation and excision repair cross-complementary 1 (ERCC1) down-regulation in human lung cancer cells.

Capsaicin, a natural active ingredient of green and red peppers, has been demonstrated to exhibit anti-cancer properties in several malignant cell lines. Excision repair cross-complementary 1 (ERCC1) has a leading role in the nucleotide excision repair (NER) process because of its involvement in the excision of DNA adducts. Erlotinib (TarcevaR) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has demonstrated clinical activity in non-small cell lung cancer (NSCLC) cells. However, whether capsaicin and erlotinib could induce synergistic cytotoxicity in NSCLC cells through modulating ERCC1 expression is unknown. In this study, capsaicin decreased the ERCC1 expression in an AKT inactivation dependent manner in two human lung adenocarcinoma cells, namely, A549 and H1975. Enhancement of AKT activity by transfection with constitutive active AKT vectors increased the ERCC1 protein level as well as the cell survival by capsaicin. Moreover, capsaicin synergistically enhanced the cytotoxicity and cell growth inhibition of erlotinib in NSCLC cells, which were associated with the down-regulation of ERCC1 expression and inactivation of AKT in A549 and H1975 cells. Together, these results may provide a rationale to combine capsaicin with erlotinib for lung cancer treatment.

Inhibition of thymidine phosphorylase expression by Hsp90 inhibitor potentiates the cytotoxic effect of salinomycin in human non-small-cell lung cancer cells.

Salinomycin is a polyether ionophore antibiotic having anti-tumorigenic property in various types of cancer. Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. 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-AAG could enhance salinomycin-induced cytotoxicity in NSCLC cells through modulating TP expression in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1975. We found that salinomycin increased TP expression in a MKK3/6-p38 MAPK activation manner. Knockdown of TP using siRNA or inactivation of p38 MAPK by pharmacological inhibitor SB203580 enhanced the cytotoxic and growth inhibition effects of salinomycin. In contrast, enforced expression of MKK6E (a constitutively active form of MKK6) reduced the cytotoxicity and cell growth inhibition of salinomycin. Moreover, Hsp90 inhibitor 17-AAG enhanced cytotoxicity and cell growth inhibition of salinomycin in NSCLC cells, which were associated with down-regulation of TP expression and inactivation of p38 MAPK. Together, the Hsp90 inhibition induced TP down-regulation involved in enhancing the salinomycin-induced cytotoxicity in A549 and H1975 cells.

Astaxanthin enhances erlotinib-induced cytotoxicity by p38 MAPK mediated xeroderma pigmentosum complementation group C (XPC) down-regulation in human lung cancer cells.

Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects that include anti-cancer and anti-inflammatory properties. Xeroderma pigmentosum complementation group C (XPC) protein is an important DNA damage recognition factor in nucleotide excision repair and is involved in regulating non-small cell lung cancer (NSCLC) cell proliferation and viability. Erlotinib (TarcevaR) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has demonstrated clinical activity in NSCLC cells. However, whether astaxanthin and erlotinib could induce synergistic cytotoxicity in NSCLC cells through modulating XPC expression is unknown. In this study, we found that p38 MAPK activation by astaxanthin decreased XPC expression in two human lung adenocarcinoma A549 and H1975 cells. Inactivation of p38 MAPK by pharmacological inhibitor SB203580 or the specific small interfering RNA (siRNA) rescued the astaxanthin-reduced XPC mRNA and protein levels. Enforced expression of XPC cDNA or inhibiting the p38 MAPK activity reduced the cytotoxicity and cell growth inhibition of astaxanthin. In contrast, knockdown of XPC using siRNA enhanced the cytotoxic effects of astaxanthin. Moreover, astaxanthin synergistically enhanced cytotoxicity and cell growth inhibition of erlotinib in NSCLC cells, which were associated with the down-regulation of XPC expression and activation of p38 MAPK. Our findings suggested that the astaxanthin induced p38 MAPK mediated XPC down-regulation enhanced the erlotinib-induced cytotoxicity in A549 and H1975 cells.

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.

Yellow and green pigments from Calophyllum inophyllum L. seed oil induce cell death in colon and lung cancer cells.

Natural compounds have been candidates for anticancer medicine over the last 20 years. During the process of isolating seed oil from Calophyllum inophyllum L., yellow and green pigments containing multiple compounds with an aromatic structure were identified. High-performance liquid chromatography and nuclear magnetic resonance analysis of these pigments revealed that the compounds present were identical, but the concentration of the compounds was different. Treatment with the pigments was able to induce the death of DLD-1 human colon cancer cells and increase the percentage of the cells in the sub-G1 and sub-G2/M phases in a dose-dependent manner. Additionally, the pigments were able to exhibit cytotoxic activity on A549 and H1975 human non-small cell lung cancer (NSCLC) cell lines at 24 h, with half-maximal inhibitory concentrations (IC50) values of 0.1206 and 0.0676%, respectively for green pigments, and 0.0434 and 0.0501%, respectively for yellow pigments. Furthermore, a decrease in IC50 value was associated with an increase in the duration of treatment. However, a sharp decrease in IC50 value of the yellow pigment was observed for H1975 cells at 48 h and for A549 cells at 72 h compared with no change in IC50 value for the green pigment with time, suggesting that the pigments function and induce cell death differently in the two cell lines. An investigation was performed into the synergistic effect of the green pigment and gefitinib (Iressa®, ZD1839), which is a selective epidermal growth factor receptor-tyrosine kinase inhibitor to block growth factor-mediated cell proliferation. The combination of the green pigment and gefitinib resulted in an enhancement of the decrease in viability of A549 and H1975 cells compared with treatment with gefitinib alone, which suggested that treatment with the green pigments was able to enhance the sensitivity of NSCLC cells to gefitinib. In conclusion, these pigments may be considered for development as anti-colon cancer agents.

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.

Astaxanthin enhances pemetrexed-induced cytotoxicity by downregulation of thymidylate synthase expression in human lung cancer cells.

Pemetrexed, a multitargeted antifolate agent, has demonstrated clinical activity in non-small cell lung cancer (NSCLC) cells. Increased expression of thymidylate synthase (TS) is thought to be associated with resistance to pemetrexed. Astaxanthin exhibits a wide range of beneficial effects including anti-cancer and anti-inflammatory properties. In this study, we showed that down-regulating of TS expression in two NSCLC cell lines, human lung adenocarcinoma H1650 and squamous cell carcinoma H1703 cells, with astaxanthin were associated with decreased MKK1/2-ERK1/2 activity. Enforced expression of constitutively active MKK1 (MKK1-CA) vector significantly rescued the decreased TS mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with a MKK1/2 inhibitor (U0126 or PD98059) further decreased the TS expression in astaxanthin-exposed NSCLC cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting ERK1/2 activity enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Combination of pemetrexed and astaxanthin resulted in synergistic enhancing cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-MKK1/2, phopho-ERK1/2, and TS expression. Overexpression of MKK1/2-CA reversed the astaxanthin and pemetrexed-induced synergistic cytotoxicity. Our findings suggested that the down-regulation of MKK1/2-ERK1/2-mediated TS expression by astaxanthin is an important regulator of enhancing the pemetrexed-induced cytotoxicity in NSCLC cells.

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.

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.

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.

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.

Lipopeptides extract from Bacillus amyloliquefaciens induce human oral squamous cancer cell death.

A lipopeptide extract of Bacillus amyloliquefaciens BACY1 (BLE) was found to induce cell death in human oral squamous cell carcinoma (OSCC) cell lines, SCC4 and SCC25, in this study. The results of MTT assay showed that BLE inhibited OSCC cell proliferation in a dose-dependent manner. BLE was also effective in increasing the sub-G1 phases. Furthermore, when membrane damage in SCC4 cells treated with BLE was monitored by LDH assay, release of LDH was significantly increased. The protein and mRNA levels of pro-apoptotic Bax, and caspase-3 were up-regulated by BLE. Taken together, these results suggest that BLE induces apoptosis and then inhibits the cell proliferation of human OSCC 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.