salinomycin and Lung-Neoplasms

Salinomycin (Sal) is a recently identified anti-tumor drug for treating several types of solid tumor; however, its effects on the migratory and invasive properties of non-small cell lung cancer (NSCLC) remain unclear. This study investigated the inhibitory effect underlying mechanisms of Salon transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) and cell migration. Sal solidly blocked cell migration and invasion enhancement by TGF-β1-induced EMT, through recovering E-cadherin loss and suppressing mesenchymal markers induction, as well as TGF-β1-mediated AMPK/SIRT signaling activity upregulation. The pharmacologic inhibition or knockdown of AMPK or SIRT1 can act synergistically with Sal to inhibit TGF-β1-induced MMP-2 and MMP-9. In contrast, AMPK or SIRT1 upregulation can protect against TGF-β1-induced MMP-2 and MMP-9 inhibition by Sal. Next we demonstrated that the MMP-2 and MMP-9 knockdown can act synergistically with Sal to inhibit TGF-β1-induced EMT. Moreover, treatment of PMA of MMP activator increased TGF-β1-induced MMP-2 and MMP-9, even with Sal. Our results demonstrate that Sal suppresses TGF-β1-induced EMT by downregulating MMP-2 and MMP-9 through the AMPK/SIRT pathway, thereby inhibiting lung cancer cell migration and invasion.

Topics: AMP-Activated Protein Kinases; Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Neoplasm Invasiveness; Pyrans; Signal Transduction; Sirtuin 1; Transforming Growth Factor beta1; Xenograft Model Antitumor Assays

Tumor dissemination and recurrence is attributed to highly resistant cancer stem cells (CSCs) which may constitute a fraction of circulating tumor cells (CTCs). Small cell lung cancer (SCLC) constitutes a suitable model to investigate the relation of CTCs and CSCs due to rapid tumor spread and a high number of CTCs. Expansion of five SCLC CTC lines (BHGc7, 10, 16, 26 and UHGc5) in vitro at our institution allowed for the analysis of CSC markers and cytotoxicity of the CSC-selective drugs salinomycin and niclosamide against CTC single cell suspensions or CTC spheroids/ tumorospheres (TOS). Salinomycin exerted dose-dependent cytotoxicity against the SCLC lines but, with exception of BHGc7 TOS, there was no markedly enhanced activity against TOS. Similarly, niclosamide exhibits high activity against BHGc7 TOS and UHGc5 TOS but not against the other CTC spheroids. High expression of the CSC marker CD133 was restricted to three SCLC tumor lines and the BHGc10 CTC line. All SCLC CTCs are CD24-positive but lack expression of CD44 and ABCG2 in contrast to the SCLC tumor lines which show a phenotype more similar to that of CSCs. The stem cell marker SOX2 was found in all CTC lines and SCLC GLC14/16, whereas elevated expression of Oct-3/4 and Nanog was restricted to BHGc26 and UHGc5. In conclusion, the SCLC CTCs established from patients with relapsed disease lack a typical CSC phenotype in respect to chemosensitivity to CSC-selective drugs, surface markers, expression of pluripotent stem cell and transcription factors.

Topics: Antigens, CD; Antineoplastic Agents; Cell Line; Cell Proliferation; Cell Survival; Humans; Lung Neoplasms; Nanog Homeobox Protein; Neoplastic Cells, Circulating; Neoplastic Stem Cells; Niclosamide; Octamer Transcription Factor-3; Pyrans; Small Cell Lung Carcinoma; SOXB1 Transcription Factors

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.

Topics: A549 Cells; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cytotoxins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; HSP90 Heat-Shock Proteins; Humans; Lung Neoplasms; Pyrans; Thymidine Phosphorylase

The existing of avidity cancer stem cells (CSCs) made it an optical strategy to kill cancer cells and CSCs at the same time. Here, we constructed a CSCs specific nanocarrier naming T-S-NLC using the CD133+ targeting peptide TISWPPR (TR) as the targeting moiety attached to the distal end of PEG on salinomycin (Sal) loaded nanostructured lipid carriers (NLC), its pharmaceutical characteristics proved it 128.73 ± 2.09 nm, anionic spheroid with sustained release profile. It's in vitro targeting effect in CD133+ CSCs indicated that it exhibited superior CSCs internalization over non-modified NLC or free drug. Afterwards, it was used in combination with previously designed EGFR specific A-P-NLC (AEYLR peptide-PEG-modified paclitaxel loaded NLC) to achieve the goal to kill the cancer cells and CSCs, simultaneously. The in vitro tumor targeting effect of T-S-NLC + A-P-NLC was affirmed by cellular uptake and proliferation inhibition effect in NCI-H1299 and S180 cell lines showing advanced results over single preparation groups. In vivo tumor targeting effect in S180 tumor-bearing mice also validated the better tumor accumulative effect of the combined group. Last but not least, the in vivo antitumor effect strongly identified the greater tumor suppression effect of T-S-NLC + A-P-NLC than single preparation groups or combined use of free drugs while maintaining a good living state of the mice. To sum up, the combined usage of PTX and Sal active targeting NLC naming A-P-NLC + T-S-NLC which killed cancer cells and CSCs at the same time was a promising drug delivery system.

Topics: Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Humans; Lipids; Lung Neoplasms; Mice; Nanostructures; Neoplastic Stem Cells; Paclitaxel; Particle Size; Peptides; Polyethylene Glycols; Pyrans

Initiation and recurrence of lung cancer, the most fatal cancer worldwide, are attributed to lung cancer stem cells (CSCs). Evidence suggests that cancer cells can be turned into CSCs in a spontaneous way, and therefore simultaneous elimination of lung CSCs and cancer cells is crucial to achieve effective therapy of lung cancer. In lung cancer, epidermal growth factor receptor (EGFR) is overexpressed in both CSCs and cancer cells. The present study developed salinomycin poly(ethylene glycol) 2000-distearoylphosphatidylethanolamine nanomicelles conjugated with EGFR aptamers (M-SAL-EGFR) to kill lung CSCs and cancer cells. The 24 nm sized M-SAL-EGFR was prepared by a lipid film based method. The EGFR was overexpressed in lung CSCs and cancer cells. Results revealed that the M-SAL-EGFR could efficiently bind to EGFR-overexpressing lung CSCs and cancer cells, and induced enhanced cyotoxic effect than non-targeted M-SAL and salinomycin. Administration of M-SAL-EGFR in mice with lung cancer xenograft inhibited tumor growth more effectively compared with M-SAL and salinomycin. The EGFR aptamers were thus able to promote effective salinomycin delivery to lung cancer. Our results also suggest that the M-SAL-EGFR represents a promising approach for targeting both lung CSCs and cancer cells.

Topics: Animals; Cell Line, Tumor; Drug Delivery Systems; ErbB Receptors; Lung Neoplasms; Mice; Micelles; Nanocomposites; Neoplastic Stem Cells; Phosphatidylethanolamines; Polyethylene Glycols; Pyrans

As the leading cause of cancer-associated mortality worldwide, lung cancer is often associated with therapy failure and decreases in survival; these factors are often attributed to lung cancer-initiating cells (CICs). In addition, sufficient evidence has suggested that simultaneous targeting of CICs, together with cancer cells, is critical for the achievement of preferable therapeutic efficacy, due to the spontaneous conversion between CICs and cancer cells. Salinomycin sodium (SS) is an antibacterial therapeutic agent that exerts potent activity against CICs in various types of cancer, including lung cancer. The present study generated SS lipid-polymer hybrid nanoparticles (NPs) with cluster of differentiation (CD)133 and epidermal growth factor receptor (EGFR) antibodies (CD133/EGFR SS NPs) for the simultaneous treatment of lung CICs and cancer cells. The activity of CD133/EGFR SS NPs was analyzed using cytotoxicity and tumorsphere formation assays, flow cytometry, and an in vivo anticancer assay in mice bearing lung cancer xenografts. The results revealed that CD133/EGFR SS NPs effectively promoted SS delivery to lung CICs and cancer cells, achieving superior therapeutic effects compared with non-targeted NPs or NPs with a single antibody. Furthermore, CD133/EGFR SS NPs exhibited the best efficacy in inhibiting tumor growth compared with the control agents in lung cancer-bearing mice. In conclusion, CD133/EGFR SS NPs may be capable of efficiently targeting and treating lung CICs together with cancer cells, and may represent an effective treatment for lung cancer.

Topics: AC133 Antigen; Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; Drug Delivery Systems; ErbB Receptors; Humans; Lactic Acid; Lung Neoplasms; Male; Mice; Mice, Nude; Nanoparticles; Neoplastic Stem Cells; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Pyrans; Xenograft Model Antitumor Assays

Erlotinib (Tarceva

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; ErbB Receptors; Erlotinib Hydrochloride; Humans; Lung Neoplasms; Proto-Oncogene Proteins c-akt; Pyrans; Thymidylate Synthase

Treatment resistance significantly inhibits the efficiency of targeted cancer therapies in drug-sensitive genotypes. In the current work, we studied mechanisms for rapidly occurring, adaptive resistance in targeted therapy-sensitive lung, breast, and melanoma cancer cell lines. The results show that in ALK translocated lung cancer lines H3122 and H2228, cells with cancer stem-like cell features characterized by high expression of cancer stem cell markers and/or in vivo tumorigenesis can mediate adaptive resistance to oncogene ablative therapy. When pharmacological ablation of ALK oncogene was accompanied with PI3K inhibitor or salinomycin therapy, cancer stem-like cell features were reversed which was accompanied with decreased colony formation. Furthermore, co-targeting was able to block the formation of acquired resistance in H3122 line. The results suggest that cells with cancer stem-like cell features can mediate adaptive resistance to targeted therapies. Since these cells follow the stochastic model, concurrent therapy with an oncogene ablating agent and a stem-like cell-targeting drug is needed for maximal therapeutic efficiency.

Topics: Aldehyde Dehydrogenase; Anaplastic Lymphoma Kinase; Animals; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Lung Neoplasms; MCF-7 Cells; Melanoma; Mice; Mice, Inbred NOD; Mice, SCID; Molecular Targeted Therapy; Neoplasms; Oxidoreductases Acting on CH-NH Group Donors; Phosphoinositide-3 Kinase Inhibitors; Pyrans; Receptor Protein-Tyrosine Kinases; Xenograft Model Antitumor Assays

The presence of cancer stem cells (CSCs) is linked to preexisting or acquired drug resistance and tumor relapse. Therefore, targeting both differentiated tumor cells and CSCs was suggested as an effective approach for non-small cell lung cancer (NSCLC) treatment. After screening of chemotherapeutic agents, tyrosine kinase inhibitors (TKIs) or monoclonal antibody in combination with the putative stem cell killer Salinomycin (SAL), we found Metformin (METF), which modestly exerted a growth inhibitory effect on monolayer cells and alveospheres/CSCs of 5 NSCLC cell lines regardless of their EGFR, KRAS, EML4/ALK and LKB1 status, interacted synergistically with SAL to effectively promote cell death. Inhibition of EGFR (AKT, ERK1/2) and mTOR (p70 s6k) signaling with the combination of METF and SAL can be augmented beyond that achieved using each agent individually. Phospho-kinase assay further suggested the multiple roles of this combination in reducing oncogenic effects of modules, such as ß-catenin, Src family kinases (Src, Lyn, Yes), Chk-2 and FAK. Remarkably, significant reduction of sphere formation was seen under combinatorial treatment in all investigated NSCLC cell lines. In conclusion, METF in combination with SAL could be a promising treatment option for patients with advanced NSCLC irrespective of their EGFR, KRAS, EML4/ALK and LKB1 status.

Topics: AMP-Activated Protein Kinase Kinases; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Line, Tumor; ErbB Receptors; Humans; Lung Neoplasms; Metformin; Microtubule-Associated Proteins; Neoplastic Stem Cells; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Pyrans; ras Proteins; Serine Endopeptidases; Signal Transduction

Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. In this study, we found that salinomycin induced autophagy in human non-small cell lung cancer (NSCLC) cells. Furthermore, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. We also showed that salinomycin triggered more apoptosis and less autophagy in A549 cells in which CDH1 expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.

Topics: Activating Transcription Factor 4; Antigens, CD; Apoptosis; Autophagy; Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Line, Tumor; Cytoprotection; Drug Screening Assays, Antitumor; Endoplasmic Reticulum Stress; Humans; Lung Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyrans; Repressor Proteins; Signal Transduction; TOR Serine-Threonine Kinases; Transcription Factor CHOP; Up-Regulation

A major challenge for oncologists and pharmacologists is to develop more potent and less toxic drugs that will decrease the tumor growth and improve the survival of lung cancer patients. Salinomycin is a polyether antibiotic used to kill gram-positive bacteria including mycobacteria, protozoans such as plasmodium falciparum, and the parasites responsible for the poultry disease coccidiosis. This old agent is now a serious anti-cancer drug candidate that selectively inhibits the growth of cancer stem cells. We investigated the impact of salinomycin on survival, colony growth, migration and invasion of the differentiated human non-small cell lung cancer lines LNM35 and A549. Salinomycin caused concentration- and time-dependent reduction in viability of LNM35 and A549 cells through a caspase 3/7-associated cell death pathway. Similarly, salinomycin (2.5-5 µM for 7 days) significantly decreased the growth of LNM35 and A549 colonies in soft agar. Metastasis is the main cause of death related to lung cancer. In this context, salinomycin induced a time- and concentration-dependent inhibition of cell migration and invasion. We also demonstrated for the first time that salinomycin induced a marked increase in the expression of the pro-apoptotic protein NAG-1 leading to the inhibition of lung cancer cell invasion but not cell survival. These findings identify salinomycin as a promising novel therapeutic agent for lung cancer.

Topics: A549 Cells; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Growth Differentiation Factor 15; Humans; Lung Neoplasms; Pyrans; RNA Interference; RNA, Small Interfering

Cancer stem cells (CSCs) are thought to be responsible for tumor initiation and recurrence after chemotherapy. Targeting CSCs and non-CSCs with specific compounds may be an effective approach to reduce lung cancer growth and metastasis. The aim of this study was to investigate the effect of salinomycin, a selective inhibitor of CSCs, with or without combination with paclitaxel, in a metastatic model. To evaluate the effect of these drugs in metastasis and tumor microenvironment we took advantage of the immunocompetent and highly metastatic LLC mouse model. Aldefluor assays were used to analyze the ALDH+/- populations in murine LLC and human H460 and H1299 lung cancer cells. Salinomycin reduced the proportion of ALDH+ CSCs in LLC cells, whereas paclitaxel increased such population. The same effect was observed for the H460 and H1299 cell lines. Salinomycin reduced the tumorsphere formation capacity of LLC by more than 7-fold, but paclitaxel showed no effect. In in vivo experiments, paclitaxel reduced primary tumor volume but increased the number of metastatic nodules (p<0.05), whereas salinomycin had no effect on primary tumors but reduced lung metastasis (p<0.05). Combination of both drugs did not improve the effect of single therapies. ALDH1A1, SOX2, CXCR4 and SDF-1 mRNA levels were higher in metastatic lesions than in primary tumors, and were significantly elevated in both locations by paclitaxel treatment. On the contrary, such levels were reduced (or in some cases did not change) when mice were administered with salinomycin. The number of F4/80+ and CD11b+ cells was also reduced upon administration of both drugs, but particularly in metastasis. These results show that salinomycin targets ALDH+ lung CSCs, which has important therapeutic effects in vivo by reducing metastatic lesions. In contrast, paclitaxel (although reducing primary tumor growth) promotes the selection of ALDH+ cells that likely modify the lung microenvironment to foster metastasis.

Topics: Aldehyde Dehydrogenase; Animals; Cell Line, Tumor; Cell Proliferation; Chemokine CXCL12; Flow Cytometry; Humans; Immunohistochemistry; Lung Neoplasms; Mice; Neoplasm Recurrence, Local; Neoplastic Stem Cells; Paclitaxel; Pyrans; Real-Time Polymerase Chain Reaction; Receptors, CXCR4; SOXB1 Transcription Factors

Lung cancer is a leading cause of death in human. Cancer stem cells have been regarded as basis for failure of current therapeutic options. Salinomycin was shown to kill these cancer stem cells in some types of cancer such as breast cancer and leukemia. The in vitro anticancer activities of salinomycin have been validated against the lung cancer cell line A549 via sulforhodamine B and colony formation assay. Salinomycin has been demonstrated to significantly rupture the in vitro lung cancer tumorospheres from ALDH positive A549 lung cells using flow cytometry. Expression of stem cell markers OCT-4, NANOG and SOX2 in ALDH positive A549 lung cells was decreased significantly by real-time RT-PCR analysis after 24 hour salinomycin treatment. Taken together, salinomycin may provide a promising approach for lung cancer chemotherapy.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents; Biomarkers; Cell Line, Tumor; Humans; Lung Neoplasms; Neoplastic Stem Cells; Pyrans; Structure-Activity Relationship