Attenuation of PI3K-Akt-mTOR Pathway to Reduce Cancer Stemness on Chemoresistant Lung Cancer Cells by Shikonin and Synergy with BEZ235 Inhibitor.

Lung cancer is considered the number one cause of cancer-related deaths worldwide. Although current treatments initially reduce the lung cancer burden, relapse occurs in most cases; the major causes of mortality are drug resistance and cancer stemness. Recent investigations have provided evidence that shikonin generates various bioactivities related to the treatment of cancer. We used shikonin to treat multi-resistant non-small lung cancer cells (DOC-resistant A549/D16, VCR-resistant A549/V16 cells) and defined the anti-cancer efficacy of shikonin. Our results showed shikonin induces apoptosis in these ABCB1-dependent and independent chemoresistance cancer sublines. Furthermore, we found that low doses of shikonin inhibit the proliferation of lung cancer stem-like cells by inhibiting spheroid formation. Concomitantly, the mRNA level and protein of stemness genes (Nanog and Oct4) were repressed significantly on both sublines. Shikonin reduces the phosphorylated Akt and p70s6k levels, indicating that the PI3K/Akt/mTOR signaling pathway is downregulated by shikonin. We further applied several signaling pathway inhibitors that have been used in anti-cancer clinical trials to test whether shikonin is suitable as a sensitizer for various signaling pathway inhibitors. In these experiments, we found that low doses shikonin and dual PI3K-mTOR inhibitor (BEZ235) have a synergistic effect that inhibits the spheroid formation from chemoresistant lung cancer sublines. Inhibiting the proliferation of lung cancer stem cells is believed to reduce the recurrence of lung cancer; therefore, shikonin's anti-drug resistance and anti-cancer stem cell activities make it a highly interesting molecule for future combined lung cancer therapy.

Relationship between cancer stem cell-related SNPs and survival outcomes in patients with primary lung cancer.

BACKGROUND: Cancer stem cells may be the source of cancer-causing mutant cells and are closely related to the prognosis of cancer. Our study aimed to investigate the potential association between single-nucleotide polymorphisms (SNPs) of cancer stem cell-related genes and the prognosis of lung cancer patients. METHODS: The SNP loci were genotyped by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), and the overall survival of subjects was analyzed by log-rank test after stratifying and adjusting their demographic data, clinical data, and genotypes. The correlation between survival time and quality of life of lung cancer under codominant, dominant, recessive, and additive genetic models was analyzed by the Cox regression model. The association between SNP polymorphism and the prognosis of lung cancer was analyzed by Stata16.0 software, and their heterogeneity was tested. Interaction analysis was performed using R software (version 4.2.0). RESULTS: Stratified analysis unveiled that rs3740535 had recessive AA genotype and additive GG genotype; Rs3130932 dominant GT + GG genotype, additive TT genotype; Rs13409 additive TT genotype; Rs6815391 recessive CC genotype and additional TT genotype were associated with increased risk of lung cancer death. Rs3130932 recessive GG genotype was associated with a reduced risk of lung cancer death. CONCLUSION: Rs3740535, rs3130932, rs13409, and rs6815391 are associated with the overall survival of lung cancer patients and may be valuable for the prognosis of lung cancer patients.

Curcumin Induces Ferroptosis in A549 CD133+ Cells through the GSH-GPX4 and FSP1-CoQ10-NAPH Pathways.

BACKGROUND: Cancer stem cells (CSCs) are characterized by an ability for unlimited proliferation and efficiency of self-renewal. The targeting of lung CSCs (LCSCs)-related signaling pathways represent a promising therapeutic strategy for treatment of lung cancer. Ferroptosis a potential strategy for LCSCs treatment, and curcumin cloud induce ferroptosis. In this study, we aimed to observe the effects of curcumin on LCSCs via ferroptosis-related pathways. METHODS: In this study, A549 cluster of differentiation (CD)133+ and A549 CD133- cells were isolated using magnetic bead-based separation. Colony formation and sphere formation assays, as well as cells injection in non-obese diabetes/severe combined immune deficiency (NOD/SCID) mice, were used to analyze the tumorigenic ability of cells differentially expressing CD133. A549 CD133+ cells were treated with different doses of curcumin (0, 10, 20, 40, 80 µM). Cell viability, glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1) expressions were measured. The 50% inhibitory concentration (IC50) of curcumin, two ferroptosis inducers, inhibitor of GPX4 (RSL3) and inhibitor of FSP1 (iFSP1), and a ferroptosis inhibitor, ferrostatin-1 (Fer-1), were used to investigate the mechanism underlying the effect of curcumin on ferroptosis in A549 CD133+ cells. RESULTS: A549 CD133+ cells had greater tumorigenic ability than A549 cells. Curcumin treatment suppressed the expressions of GPX4 (glutathione peroxidase 4) and FSP1 in A549 CD133+ cells, thereby inducing ferroptosis. RSL3 and iFSP1 respectively suppressed the GSH (glutathione)-GPX4 and FSP1 (ferroptosis suppressor protein 1)-CoQ10 (coenzyme Q10)-nicotinamide adenine dinucleotide (NADH) pathways in A549 CD133+ cells. However, the roles of curcumin were blocked by Fer-1 treatment. CONCLUSIONS: In this study, curcumin induced ferroptosis through inhibiting the GSH-GPX4 and FSP1-CoQ10-NADH pathways in A549 CD133+ cells, resulting in the inhibition of their self-renewal potential.

S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) inhibits lung cancer tumorigenesis by regulating cell plasticity.

BACKGROUND: Lung cancer is one of the most frequently diagnosed cancers characterized by high mortality, metastatic potential, and recurrence. Deregulated gene expression of lung cancer, likewise in many other solid tumors, accounts for their cell heterogeneity and plasticity. S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as Inositol triphosphate (IP(3)) receptor-binding protein released with IP(3) (IRBIT), plays roles in many cellular functions, including autophagy and apoptosis but AHCYL1 role in lung cancer is largely unknown. RESULTS: Here, we analyzed the expression of AHCYL1 in Non-Small Cell Lung Cancer (NSCLC) cells from RNA-seq public data and surgical specimens, which revealed that AHCYL1 expression is downregulated in tumors and inverse correlated to proliferation marker Ki67 and the stemness signature expression. AHCYL1-silenced NSCLC cells showed enhanced stem-like properties in vitro, which correlated with higher expression levels of stem markers POU5F1 and CD133. Also, the lack of AHCYL1 enhanced tumorigenicity and angiogenesis in mouse xenograft models highlighting stemness features. CONCLUSIONS: These findings indicate that AHCYL1 is a negative regulator in NSCLC tumorigenesis by modulating cell differentiation state and highlighting AHCYL1 as a potential prognostic biomarker for lung cancer.

HIF1α and HIF2α regulate non-small-cell lung cancer dedifferentiation via expression of Sox2 and Oct4 under hypoxic conditions.

OBJECTIVE: To explore HIF1α and HIF2α regulate the dedifferentiation of lung cancer cells under hypoxic conditions through Sox2 and Oct4. MATERIALS AND METHODS: HIF1α, HIF2α, Sox2 and Oct4 expression was analysed in lung cancer tissues. We analysed sphere formation by single-cell of differentiated lung cancer under hypoxia, and detected the expression of CD133, CD44, Sox2, Oct4, HIF1α and HIF2α. We knocked out HIF1α, HIF2α, Sox2 or Oct4 in cells, cultured the cells under hypoxic conditions and detected CD133 and CD44 using western blotting. We also detected the apoptosis rate of cells with HIF1α, HIF2α, Sox2 or Oct4 knockout. RESULTS: There was more sphere formation of differentiated lung cancer cells under hypoxic conditions than of control cells under normoxic conditions. These newly formed spheres highly expressed CD133 and CD44. TCGA database showed high expression of HIF1α and HIF2α in lung cancer tissues. After knocking out HIF1α and HIF2α, the expression of Sox2, Oct4, CD133 and CD44 decreased significantly, and after knocking out Sox2 or Oct4, the expression of CD133 and CD44 decreased. CONCLUSION: HIF1α and HIF2α regulate non-small-cell lung cancer dedifferentiation through Sox2 and Oct4 under hypoxic conditions.

Research progress of Chinese materia medica in inhibiting malignant biological behavior of lung cancer stem cells / 国际中医中药杂志

Chinese materia medica can inhibit the proliferation, invasion, migration and expression of drug-resistant proteins of lung cancer stem cells (LCSCs), and induce apoptosis and delay self-renewal, as well as exert anti-tumor effects by interfering with their ecological niche, immune microenvironment and aerobic glycolysis, etc. The biomarkers involved mainly include CD133, CD44, ALDH and ABCG2, while the related signaling pathways are Wnt/β-catenin, Hedgehog, and Notch. The research on the intervention of LCSCs by Traditional Chinese Medicine (TCM) is generally few, mostly concentrated in basic research, and the selected experimental indicators have a high repetition rate, involving fewer cell types and signaling pathways; there is a relative lack of clinical trials, which lack an organic connection with basic experiments. In the future, the quality of research is expected to be improved, and in-depth study of TCM with anti-lung cancer stem cell effect should be carried out, with the purpose to promote the precise treatment of lung cancer.

On the mechanisms of photodynamic action of photosensitizers based on polycationic derivatives of synthetic bacteriochlorin against human lung cancer cells A549 (in vitro study).

BACKGROUND: One of the tasks of anticancer photodynamic therapy is increasing the efficacy of treatment of cancer nodes with large (clinically relevant) sizes using near-infrared photosensitizers (PS). We study the photodynamic action against A549 human lung cancer cells using PS based on polycationic derivatives of synthetic bacteriochlorin. METHODS: The efficacy and mechanisms of the photodynamic action of PS based on polycationic derivatives of synthetic bacteriochlorin against A549 lung cancer cells were studied in vitro using immunocytochemical and morphological methods. RESULTS: It was found that PS based on tetracationic and octacationic derivatives of synthetic bacteriochlorin induce necrosis, apoptosis, decreasing of proliferative and mitotic activity, as well as reducing the number of ALDH1-positive cancer cells with signs of stem cells in A549 human lung cancer cell culture. The IC50 values (concentration of a PS that reduces cells survival by 50%) were about 0.69 µM for tetracationic PS and 0.57 µM for octacationic PS under irradiation at 30 J/cm2 while in the "dark" control they were higher than 100 µM for both PSs. CONCLUSIONS: Photosensitizers based on polycationic derivatives of synthetic bacteriochlorin have high phototoxicity against A549 cancer cells caused by the induction of necrosis and apoptosis of cancer cells, including cells with signs of stemness, and a sharp decrease of mitotic and proliferative activity.

Effect of chronic intermittent hypoxia-induced HIF-1α/ATAD2 expression on lung cancer stemness.

BACKGROUND: Obstructive sleep apnea is associated with increased lung cancer incidence and mortality. Cancer stem cells (CSCs) are characterized by their self-renewing ability, which contributes to metastasis, recurrence, and drug resistance. ATPase family AAA domain-containing protein 2 (ATAD2) induces malignancy in different types of tumors. However, a correlation between ATAD2 expression and CSCs in lung cancer has not yet been reported. METHODS: The relative messenger RNA (mRNA) levels of ATAD2, CD44, CD133, and hypoxia-inducible factor (HIF)-1α were determined using reverse-transcription quantitative polymerase chain reaction. ATAD2 protein levels were determined using Western blotting. Cell counting kit-8, 5-ethynyl-2'-deoxyuridine (EdU), and colony formation assays were performed to analyze the proliferation of lung cancer cells. Transwell migration and invasion assays were performed to evaluate cell migration and invasion, respectively. Tumor sphere formation analysis was used to determine tumor spheroid capacity. The link between ATAD2 and HIF-1α was verified using a dual-luciferase reporter assay. Immunofluorescence staining was performed to assess mitochondrial reactive oxygen species (mtROS) production. Flow cytometry analysis was conducted to determine the CD133 and CD44 positive cell ratio. RESULTS: We evaluated the relative expression of ATAD2 in four lung cancer cell lines (A549, SPC-A1, H460, and H1299 cells) and found increased mRNA and protein levels of ATAD2 in lung cancer samples. ATAD2 overexpression was a poor prognostic factor for lung cancer patients. Loss of ATAD2 reduced lung cancer cell viability and proliferation. Additionally, ATAD2 knockdown repressed lung cancer cell migration, invasion, stem-cell-like properties, and mtROS production. Chronic intermittent hypoxia (CIH)-induced HIF-1α expression significantly activated ATAD2 during lung cancer progression. CONCLUSIONS: This study found that CIH induced HIF-1α expression, which acts as a transcriptional activator of ATAD2. The present study also suggests a novel mechanism by which the integrity of CIH-triggered HIF-1α/ATAD2 may determine lung cancer aggressiveness via the interplay of mtROS and stemness in lung cancer cells.

BTB and CNC homology 1 (Bach1) induces lung cancer stem cell phenotypes by stimulating CD44 expression.

BACKGROUND: Growing evidence suggests that cancer stem cells (CSCs) are responsible for cancer initiation in tumors. Bach1 has been identified to contribute to several tumor progression, including lung cancer. The role of Bach1 in CSCs remains poorly known. Therefore, the function of Bach1 on lung CSCs was focused currently. METHODS: The expression of Bach1, CD133, CD44, Sox2, Nanog and Oct4 mRNA was assessed using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). Protein expression of Bach1, CD133, CD44, Sox2, Nanog, Oct4, p53, BCL2, BAX, p-p38, p-AKT1, c-Fos and c-Jun protein was analyzed by western blotting. 5-ethynyl-29-deoxyuridine (EdU), colony formation, Flow cytometry analysis and transwell invasion assay were carried out to analyze lung cancer cell proliferation, apoptosis and invasion respectively. Tumor sphere formation assay was utilized to evaluate spheroid capacity. Flow cytometry analysis was carried out to isolate CD133 or CD44 positive lung cancer cells. The relationship between Bach1 and CD44 was verified using ChIP-qPCR and dual-luciferase reporter assay. Xenograft tumor tissues were collected for hematoxylin and eosin (HE) staining and IHC analysis to evaluate histology and Ki-67. RESULTS: The ratio of CD44 + CSCs from A549 and SPC-A1 cells were significantly enriched. Tumor growth of CD44 + CSCs was obviously suppressed in vivo compared to CD44- CSCs. Bach1 expression was obviously increased in CD44 + CSCs. Then, via using the in vitro experiment, it was observed that CSCs proliferation and invasion were greatly reduced by the down-regulation of Bach1 while cell apoptosis was triggered by knockdown of Bach1. Loss of Bach1 was able to repress tumor-sphere formation and tumor-initiating CSC markers. A repression of CSCs growth and metastasis of shRNA-Bach1 was confirmed using xenograft models and caudal vein injection. The direct interaction between Bach1 and CD44 was confirmed by ChIP-qPCR and dual-luciferase reporter assay. Furthermore, mitogen-activated protein kinases (MAPK) signaling pathway was selected and we proved the effects of Bach1 on lung CSCs were associated with the activation of the MAPK pathway. As manifested, loss of Bach1 was able to repress p-p38, p-AKT1, c-Fos, c-Jun protein levels in lung CSCs. Inhibition of MAPK signaling remarkably restrained lung CSCs growth and CSCs properties induced by Bach1 overexpression. CONCLUSION: In summary, we imply that Bach1 demonstrates great potential for the treatment of lung cancer metastasis and recurrence via activating CD44 and MPAK signaling.

Epithelial membrane protein 3 regulates lung cancer stem cells via the TGF­ß signaling pathway.

Epithelial membrane protein 3 (EMP3) is a transmembrane glycoprotein that contains a peripheral myelin protein 22 domain. EMP3 first received attention as a tumor suppressor, but accumulating evidence has since suggested that it may exhibit a tumor­promoting function. Nonetheless, the biological function of EMP3 remains largely unclear with regards to its role in cancer. Herein, it was shown that EMP3 expression is upregulated in non­small cell lung cancer (NSCLC) cells overexpressing aldehyde dehydrogenase 1 (ALDH1). EMP3 was shown to be involved in cell proliferation, the formation of cancer stem cells (CSCs) and in epithelial­mesenchymal transition (EMT). The ability to resist irradiation, one of the characteristics of CSCs, decreased when the EMP3 mRNA expression was knocked down using small interfering RNA. In addition, when EMP3 knockdown reduced the migratory ability of cells, a characteristic of EMT. Additionally, it was shown that the TGF­ß/Smad signaling axis was a target of EMP3. EMP3 was found to interact with TGF­ß receptor type 2 (TGFBR2) upon TGF­ß stimulation in lung CSCs (LCSC). As a result, binding of EMP3­TGFBR2 regulates TGF­ß/Smad signaling activation and consequently affects CSCs and EMT. Kaplan­Meier analysis results confirmed that patients with high expression of EMP3 had poor survival rates. Taken together, these findings showed that EMP3 may be a potential target for management of LCSCs with high expression of ALDH1, and that EMP3 is involved in TGF­ß/Smad signaling activation where it promotes acquisition of cancerous properties in tumors.

Aluminium (III) phthalocyanine chloride tetrasulphonate is an effective photosensitizer for the eradication of lung cancer stem cells.

Cancer stem cells (CSCs) are considered to contribute to the recurrence of lung cancer due to their stem-like nature and the involvement of genetic markers associated with drug efflux, regeneration and metastases. Photodynamic therapy (PDT) is a cost-effective and non-invasive therapeutic application that can act as an alternative therapy for lung cancer when considering CSC involvement. Stem-like cells derived from the A549 lung cancer cell line, positive for CD133, CD56 and CD44 antigen markers, were characterized, intracellular localization of aluminium (III) phthalocyanine chloride tetrasulphonate (AlPcS4Cl) determined and its anti-cancer PDT effects were evaluated. Results confirmed that isolated cells were stem cell-like and subcellular localization of AlPcS4Cl in integral organelles involved in cell homeostasis supported the destruction of CSC. AlPcS4Cl's effectivity was demonstrated with CSC eradication showing a significant increase in cytotoxicity and cell death via apoptosis, caused by a decrease in mitochondrial membrane potential. PDT could serve as a palliative treatment for lung cancer and improve prognosis by elimination of lung CSCs.

A novel CXCR4 antagonist counteracts paradoxical generation of cisplatin-induced pro-metastatic niches in lung cancer.

Platinum-based chemotherapy remains widely used in advanced non-small cell lung cancer (NSCLC) despite experimental evidence of its potential to induce long-term detrimental effects, including the promotion of pro-metastatic microenvironments. In this study, we investigated the interconnected pathways underlying the promotion of cisplatin-induced metastases. In tumor-free mice, cisplatin treatment resulted in an expansion in the bone marrow of CCR2+CXCR4+Ly6Chigh inflammatory monocytes (IMs) and an increase in lung levels of stromal SDF-1, the CXCR4 ligand. In experimental lung metastasis assays, cisplatin-induced IMs promoted the extravasation of tumor cells and the expansion of CD133+CXCR4+ metastasis-initiating cells (MICs). Peptide R, a novel CXCR4 inhibitor designed as an SDF-1 mimetic peptide, prevented cisplatin-induced IM expansion, the recruitment of IMs into the lungs, and the promotion of metastasis. At the primary tumor site, cisplatin treatment reduced tumor size while simultaneously inducing tumor release of SDF-1, MIC expansion, and recruitment of pro-invasive CXCR4+ macrophages. Co-recruitment of MICs and CCR2+CXCR4+ IMs to distant SDF-1-enriched sites also promoted spontaneous metastases that were prevented by CXCR4 blockade. In clinical specimens from NSCLC patients SDF-1 levels were found to be higher in platinum-treated samples and related to a worse clinical outcome. Our findings reveal that activation of the CXCR4/SDF-1 axis specifically mediates the pro-metastatic effects of cisplatin and suggest CXCR4 blockade as a possible novel combination strategy to control metastatic disease.

A high-throughput screen identifies inhibitors of lung cancer stem cells.

Metastasis is the main cause of cancer morbidity and mortality. Cancer stem cells (CSCs) are a rare subpopulation of cancer cells that can drive metastasis. The identification of CSC inhibitors and CSC-related genes is an alluring strategy for suppressing metastasis. Here, we established a simple and repeatable high-throughput CSC inhibitor screening platform that combined tumor sphere formation assays and cell viability assays. Human lung cancer cells were cocultured with 1280 pharmacologically active compounds (FDA-approved). Fifty-four candidate compounds obtained from our screening system completely or partially inhibited tumor sphere formation. A total of 5 of these 54 compounds (prochlorperazine dimaleate, thioridazine hydrochloride, ciproxifan hydrochloride, Ro 25-6981 hydrochloride, and AMN 082) completely inhibited the self-renewal of CSCs without cytotoxicity in vitro via their targets and suppressed lung cancer metastasis in vivo, suggesting that our screening platform is selective and reliable. DRD2, HRH3, and GRIN2B exhibited potent genes promoting CSCs in vitro experiments and clinical datasets. Further validation of the top hit (DRD2) and previously published studies demonstrate that our screening platform is a useful tool for CSC inhibitor and CSC-related gene screening.

Synergistic Roles of Curcumin in Sensitising the Cisplatin Effect on a Cancer Stem Cell-Like Population Derived from Non-Small Cell Lung Cancer Cell Lines.

Cancer stem cells (CSCs) represent a small subpopulation within a tumour. These cells possess stem cell-like properties but also initiate resistance to cytotoxic agents, which contributes to cancer relapse. Natural compounds such as curcumin that contain high amounts of polyphenols can have a chemosensitivity effect that sensitises CSCs to cytotoxic agents such as cisplatin. This study was designed to investigate the efficacy of curcumin as a chemo-sensitiser in CSCs subpopulation of non-small cell lung cancer (NSCLC) using the lung cancer adenocarcinoma human alveolar basal epithelial cells A549 and H2170. The ability of curcumin to sensitise lung CSCs to cisplatin was determined by evaluating stemness characteristics, including proliferation activity, colony formation, and spheroid formation of cells treated with curcumin alone, cisplatin alone, or the combination of both at 24, 48, and 72 h. The mRNA level of genes involved in stemness was analysed using quantitative real-time polymerase chain reaction. Liquid chromatography-mass spectrometry was used to evaluate the effect of curcumin on the CSC niche. A combined treatment of A549 subpopulations with curcumin reduced cellular proliferation activity at all time points. Curcumin significantly (p < 0.001) suppressed colonies formation by 50% and shrank the spheroids in CSC subpopulations, indicating inhibition of their self-renewal capability. This effect also was manifested by the down-regulation of SOX2, NANOG, and KLF4. Curcumin also regulated the niche of CSCs by inhibiting chemoresistance proteins, aldehyde dehydrogenase, metastasis, angiogenesis, and proliferation of cancer-related proteins. These results show the potential of using curcumin as a therapeutic approach for targeting CSC subpopulations in non-small cell lung cancer.

Sulforaphane Suppressed Stemness Characters of Lung Adenocarcinoma Stem Cells by Down-regulating Methylation of miR-200c Promoter / 肿瘤防治研究

Objective To investigate the effect of sulforaphane (SFN) on the proliferation and self-renewal of lung cancer stem cells and its regulatory mechanism. Methods MTT method was used to detect the effect of SFN on the proliferation of lung adenocarcinoma cell lines H460 and A549; tumor sphere formation experiment was used to detect the ability of tumor sphere formation; Western blot was applied to explore the expression of stemness-related proteins (such as β-catenin, Klf4, c-myc) in lung adenocarcinoma cells before and after SFN treatment; NGS sequencing was used to analyze the effect of SFN on the expression profile of tumor cell miRNAs. qRT-PCR verified the changes in the transcription level of key miRNAs by SFN. Western blot was used to detect the effect of SFN on the expression of DNMTs in tumor cells. We constructed miR-200c promoter-GFP plasmid, and applied IF, methylation PCR and DNA sequencing methods to detect the effect of SFN on the methylation level of tumor spheres and miRNA promoter. Results The miRNAs expression profile of lung adenocarcinoma tumor spheres changed significantly after SFN (5.0μmol/L) treatment, and miRNA-200c increased the most. Compared with the control group, the expression of β-catenin, Klf4, c-myc and Vimentin genes in H460 and A549 cells of SFN-S group decreased, and the protein expression levels of DNMT1 and DNMT3a were also significantly decreased. Compared with the control group, H460 and A549 cells stably expressing pEGFP-R200c plasmid in SFN-S group significantly reduced tumor sphere diameter, while tumor sphere fluorescence intensity increased, and GFP protein expression was up-regulated. There were 9 CpG-rich regions in the miR-200c promoter region in the above-mentioned pEGFP-R200c plasmid cell line, and the methylation levels were 88.9%, 44.4% and 38.8% in the control group, SFN-S group and 5-Aza-dC group, respectively. Conclusion SFN may downregulate the expression of stem-related genes in lung cancer stem cells by epigenetically decreasing the methylation level of miR-200c promoter and promoting the transcription of miR-200c.

Effective Gold Nanoparticle-Antibody-Mediated Drug Delivery for Photodynamic Therapy of Lung Cancer Stem Cells.

Cancer stem cells (CSCs) are a leading contributor to lung cancer mortality rates. CSCs are responsible for tumor growth and recurrence through inhibition of drug-induced cell death, decreasing the effect of traditional cancer therapy and photodynamic therapy (PDT). PDT can be improved to successfully treat lung cancer by using gold nanoparticles (AuNPs), due to their size and shape, which have been shown to facilitate drug delivery and retention, along with the targeted antibody (Ab) mediated selection of CSCs. In this study, a nanobioconjugate (NBC) was constructed, using a photosensitizer (PS) (AlPcS4Cl), AuNPs and Abs. The NBC was characterized, using spectroscopy techniques. Photodynamic effects of the NBC on lung CSCs was evaluated, using biochemical assays 24 h post-irradiation, in order to establish its anticancer effect. Results showed successful conjugation of the nanocomposite. Localization of the NBC was seen to be in integral organelles involved in cell homeostasis. Biochemical responses of lung CSCs treated using AlPcS4Cl -AuNP and AlPcS4Cl-AuNP-Ab showed significant cell toxicity and cell death, compared to free AlPcS4Cl. The PDT effects were enhanced when using the NBC, showing significant lung CSC destruction to the point of eradication.

[Isolation and Biological Characterization of Lung Cancer Stem Cells].

Objective To explore the methods of screening and biological characteristics of lung cancer stem cells. Methods We selected the ABCG2 +and ABCG2 -cells from SPC-A-1/adriamycin(ADM)cell line induced by ADM and analyzed the tumorigenicity of ABCG2 +and ABCG2 -cells in vivo by flow cytometry and transplantation in nude mice. Results The average fluorescence intensity of SPC-A-1 cells was(1.001±0.014)×10 2,which was significantly lower than that of SPC-A-1/ADM cells [(10.257±0.023) ×10 2 ](t=17.320,P=0.001);the difference was also statistically significant between the ABCG2/BCRP-FITC treatment group and the SPC-A-1 control group(t=5.269,P=0.021) and the SPC-A-1 control group(t=6.869, P=0.012) and between the SPC-A-1/ADM cell control group and the SPC-A-1/ADM cell homotype control group(t=8.112,P=0.015).The positive rate of SPC-A-1/ADM cells treated with ABCG2/BCRP-FITC was 9.8%,39.84 times higher than that of SPC-A-1 cells;it showed significant difference between the ABCG2/BCRP-FITC group and the SPC-A-1/ADM group(t=9.120,P=0.005) and the SPC-A-1/ADM group(t=8.257,P= 0.006).The positive rate of group B cells was 684 times that of group A cells,and the difference was statistically significant(t=11.235,P=0.001),and the fluorescence intensity of group B cells was strong.The average tumorigenic volume of the mice inoculated with SPC-A-1 cells,group A cells,and group B cells was(6.96±1.82),(6.70±2.55),and(9.17±2.41) mm 3,respectively.Among them,group B was the highest,but there was no significant difference among these three groups(F=2.362,P=0.086).The tumorigenic rate of group B cells was 75.00%,which was significantly higher than that of SPC-A-1 cells and group A cells(F=19.780,P=0.002). Conclusion ABCG2 cells from human lung adenocarcinoma SPC-A-1/ADM cell line can be isolated by ABCG2 antibody combined with immunomagnetic beads sorting method,and the tumor formation rate in nude mice can be observed to explore the identification and biological characterization of lung cancer stem cells.

The FDA-Approved Anti-Asthma Medicine Ciclesonide Inhibits Lung Cancer Stem Cells through Hedgehog Signaling-Mediated SOX2 Regulation.

Ciclesonide is an FDA-approved glucocorticoid (GC) used to treat asthma and allergic rhinitis. However, its effects on cancer and cancer stem cells (CSCs) are unknown. Our study focuses on investigating the inhibitory effect of ciclesonide on lung cancer and CSCs and its underlying mechanism. In this study, we showed that ciclesonide inhibits the proliferation of lung cancer cells and the growth of CSCs. Similar glucocorticoids, such as dexamethasone and prednisone, do not inhibit CSC formation. We show that ciclesonide is important for CSC formation through the Hedgehog signaling pathway. Ciclesonide reduces the protein levels of GL1, GL2, and Smoothened (SMO), and a small interfering RNA (siRNA) targeting SMO inhibits tumorsphere formation. Additionally, ciclesonide reduces the transcript and protein levels of SOX2, and an siRNA targeting SOX2 inhibits tumorsphere formation. To regulate breast CSC formation, ciclesonide regulates GL1, GL2, SMO, and SOX2. Our results unveil a novel mechanism involving Hedgehog signaling and SOX2 regulated by ciclesonide in lung CSCs, and also open up the possibility of targeting Hedgehog signaling and SOX2 to prevent lung CSC formation.

Involvement of collagen XVII in pluripotency gene expression and metabolic reprogramming of lung cancer stem cells.

BACKGROUND: Recent advancements in cancer biology field suggest that glucose metabolism is a potential target for cancer treatment. However, little if anything is known about the metabolic profile of cancer stem cells (CSCs) and the related underlying mechanisms. METHODS: The metabolic phenotype in lung CSC was first investigated. The role of collagen XVII, a putative stem cell or CSC candidate marker, in regulating metabolic reprogramming in lung CSC was subsequently studied. Through screening the genes involved in glycolysis, we identified the downstream targets of collagen XVII that were involved in metabolic reprogramming of lung CSCs. Collagen XVII and its downstream targets were then used to predict the prognosis of lung cancer patients. RESULTS: We showed that an aberrant upregulation of glycolysis and oxidative phosphorylation in lung CSCs is associated with the maintenance of CSC-like features, since blocking glycolysis and oxidative phosphorylation reduces sphere formation, chemoresistance, and tumorigenicity. We also showed that the Oct4-hexokinase 2 (HK2) pathway activated by collagen XVII-laminin-332 through FAK-PI3K/AKT-GSB3ß/ß-catenin activation induced the upregulation of glycolysis and maintenance of CSC-like features. Finally, we showed that collagen XVII, Oct4, and HK2 could be valuable markers to predict the prognosis of lung cancer patients. CONCULSIONS: These data suggest the Oct4-HK2 pathway regulated by collagen XVII plays an important role in metabolic reprogramming and maintenance of CSC-like features in lung CSCs, which may aid in the development of new strategies in cancer treatment.

Isolation and Biological Characterization of Lung Cancer Stem Cells / 中国医学科学院学报

To explore the methods of screening and biological characteristics of lung cancer stem cells. We selected the ABCG2 and ABCG2 cells from SPC-A-1/adriamycin(ADM)cell line induced by ADM and analyzed the tumorigenicity of ABCG2 and ABCG2 cells by flow cytometry and transplantation in nude mice. The average fluorescence intensity of SPC-A-1 cells was(1.001±0.014)×10 ,which was significantly lower than that of SPC-A-1/ADM cells [(10.257±0.023) ×10 ](=17.320,=0.001);the difference was also statistically significant between the ABCG2/BCRP-FITC treatment group and the SPC-A-1 control group(=5.269,=0.021) and the SPC-A-1 control group(=6.869, =0.012) and between the SPC-A-1/ADM cell control group and the SPC-A-1/ADM cell homotype control group(=8.112,=0.015).The positive rate of SPC-A-1/ADM cells treated with ABCG2/BCRP-FITC was 9.8%,39.84 times higher than that of SPC-A-1 cells;it showed significant difference between the ABCG2/BCRP-FITC group and the SPC-A-1/ADM group(=9.120,=0.005) and the SPC-A-1/ADM group(=8.257,= 0.006).The positive rate of group B cells was 684 times that of group A cells,and the difference was statistically significant(=11.235,=0.001),and the fluorescence intensity of group B cells was strong.The average tumorigenic volume of the mice inoculated with SPC-A-1 cells,group A cells,and group B cells was(6.96±1.82),(6.70±2.55),and(9.17±2.41) mm ,respectively.Among them,group B was the highest,but there was no significant difference among these three groups(=2.362,=0.086).The tumorigenic rate of group B cells was 75.00%,which was significantly higher than that of SPC-A-1 cells and group A cells(=19.780,=0.002). ABCG2 cells from human lung adenocarcinoma SPC-A-1/ADM cell line can be isolated by ABCG2 antibody combined with immunomagnetic beads sorting method,and the tumor formation rate in nude mice can be observed to explore the identification and biological characterization of lung cancer stem cells.