The Effects Of Arsenic Trioxide On Cancer Stem-like Cells In Lung Cancer And The Possible Mechanism

?Background and Objective?Lung cancer is the most frequently diagnosed malignant tumor and has the highest cancer mortality rate throughout the world [1].Radiotherapy,chemotherapy and targeted therapy are used in the treatment of advanced lung cancer,but the prognosis has not been significantly improved[2].Therefore,the clinicians need to find a more effective strategy.Recently,researchers found that the tumor tissues are composed of heterogeneous cancer cell populations.Cancer stem cells?CSCs?,also known as cancer initiating cells,are small subpopulations of cancer cells,which exhibit the characteristics of normal stem cells,such as self renewal capability and multipotency.They can drive the initiation,development,invasion and metastasis of tumors,and are resistant to radiotherapy and chemotherapy[3,4].They play key roles in the differentiation,proliferation,self-renewal and angiogenesis of tumors.If we can effectively inhibit the cancer stem cells,it may be possible to block the development of tumor from the source.Arsenic trioxide?As₂O₃?,the effective component of arsenic,has been used as a traditional remedy in China for thousands of years.In recent decades,As₂O₃ has been proved to promote differentiation of the APL-derived stem cells and thus induce complete remission in patients with acute promyelocytic leukemia?APL?with minimal toxicity [5-7].As₂O₃ has been approved by FDA for treatment of APL.The combination of As₂O₃ and PI3 K inhibitor strongly diminishes acute myeloid leukemia stem cells[8].Furthermore,Studies have shown that As₂O₃ inhibits the cancer stem-like cells in gliomas[9,10],hepatocellular carcinoma[11] and pancrease cancer[12] via downregulation of Notch or Hedgehog activation.In lung cancer,we have previously demonstrated that As₂O₃ can induce cell apoptosis and arrest cell cycle [13].As₂O₃ can inhibit angiogenesis in lung cancer [14,15].Moreover,in animal models,As₂O₃ can induce the formation of specific glandular cavity in tumor tissue and decrease the expression of Ki-67,suggesting that As₂O₃ may induce the differentiation of lung cancer.However,we don't know whether As₂O₃ inhibit lung cancer stem cells.In this study,we evaluated whether As₂O₃ has inhibitory effects on cancer stem-like cells in lung cancer,and further explored the possible mechanism.?Methods?Part 1 Effects of As₂O₃ on cancer stem-like cells in lung cancer1.Effects of As₂O₃ on cell proliferation of lung cancer cellsLung cancer cells were treated with 0,1,2,4,8 and 16?mol/L As₂O₃,respectively.The cells were incubated for another 24,48 or 72 hours.Cell viability was determined by the CCK8 assay.2.Effects of As₂O₃ on clonogenic capacity of lung cancer cellsLung cancer cells were treated with 0,1,2 and 4?mol/L As₂O₃ for 72 hours,respectively.Subsequently,2×103 cells per well were seeded in 6-well culture dishes.After for 2 weeks,macroscopic colonies were counted and photographed.3.Effects of As₂O₃ on tumor sphere formation of lung cancer cellsLung cancer cells were treated with 0,1,2 and 4?mol/L As₂O₃ for 72 hours,respectively.Subsequently,1×104 cells per well were cultured in serum-free conditions.After 4-7 days,pictures were taken under a microscope and the number of tumor spheres was counted.4.Effects of As₂O₃ on the expression of stem cell biomarker CD133Lung cancer cells were treated with different concentrations of As₂O₃ for 72 hours.q PCR,Western Blot and flow cytometry analysis were performed to determine the expression of stem cell biomarker CD133.5.Effects of As₂O₃ on the expression of stem cell transcription factors such as Sox2,Oct4,C-myc,nanog,Klf4 and ?-cateninLung cancer cells were treated with 0,1,2 and 4?mol/L As₂O₃ for 72 hours.q PCR and Western Blot analysis were performed to determine the expression levels of stem cell transcription factors such as Sox2,Oct4,C-myc,nanog,Klf4 and ?-catenin.Part 2 The mechanism of As₂O₃ on lung cancer stem-like cellsLung cancer cells were treated with 0,1,2 and 4?mol/L As₂O₃ for 72 hours.Then we used q PCR and Western Blot analysis to measure the expression of Hedgehog signaling molecules such as Ptch,Smo,Gli1,Gli2,N-myc and GAS1.?Results?Part 1 Effects of As₂O₃ on cancer stem-like cells in lung cancer1.Effects of As₂O₃ on cell proliferation of lung cancer cells?1?Effects of As₂O₃ on cell proliferation of human lung adenocarcinoma cell line A549As₂O₃ could inhibit the proliferation of A549 cells in a concentration-and timedependent manner.IC50 was 10.860 ?M after treatment with As₂O₃ for 72 hours.?2?Effects of As₂O₃ on cell proliferation of human large cell lung cancer cell line NCI-H460As₂O₃ could inhibit the proliferation of NCI-H460 cells in concentration-and time-dependent manner.IC₅₀ was 5.610?M after treatment with As₂O₃ for 72 hours.?3?Effects of As₂O₃ on cell proliferation of human lung squamous cell line SK-MES-10,1,2,4,8 and 16 ?M As₂O₃ could not inhibit the proliferation of SK-MES-1?P>0.05?.?4?Effects of As₂O₃ on cell proliferation of human small cell lung cancer cell line NCI-H446As₂O₃ could inhibit the proliferation of NCI-H446 cells in a concentration-and timedependent manner.IC50 was 4.252 ?M after treatment with As₂O₃ for 72 hours.2.Effects of As₂O₃ on clonogenic capacity of lung cancer cells?1?Effects of As₂O₃ on clonogenic capacity of human lung adenocarcinoma cell line A549CCK8 assay indicated that As₂O₃ could inhibit cell viability of A549,NCI-H460 and NCI-H446 cells.However,SK-MES-1 cells were not sensitive to As₂O₃ treatment.Therefore,we employed A549,NCI-H460 and NCI-H446 cells as subjects of our follow-up studies.In A549 cells,the differences of colonies between the As₂O₃-treated group and the control group were not statistically significant?P>0.05?.?2?Effects of As₂O₃ on clonogenic capacity of human large cell lung cancer cell line NCI-H460Clonogenic capacity of NCI-H460 cells could be remarkably inhibited by 2 and 4 ?M As₂O₃?P<0.001?.However,1 ?M As₂O₃ could not inhibit the clonogenic capacity of NCI-H460 cells?P>0.05?.?3?Effects of As₂O₃ on clonogenic capacity of human small cell lung cancer cell line NCI-H446The clonogenic capacity of NCI-H446 cells could be remarkably inhibited by 1,2 and 4 ?M As₂O₃?P<0.01?.3.Effects of As₂O₃ on tumor sphere formation of lung cancer cells?1?Effects of As₂O₃ on tumor sphere formation of human lung adenocarcinoma cell line A549There were no significant differences of tumor sphere numbers between the As₂O₃-treated group and the control group?P>0.05?.Besides,the volume of tumor spheres could not be influenced by As₂O₃ treatment.?2?Effects of As₂O₃ on tumor sphere formation of human large cell lung cancer cell line NCI-H4601,2 and 4 ?M As₂O₃ was able to significantly decrease the number of the tumor spheres?P<0.001?.?3?Effects of As₂O₃ on tumor sphere formation of human small cell lung cancer cell line NCI-H4461,2 and 4 ?M As₂O₃ was able to remarkably reduce the number of the tumor spheres in a dose dependent manner?P<0.001?.4.Effects of As₂O₃ on the expression of stem cell biomarker CD133?1?Effects of As₂O₃ on the expression of stem cell biomarker CD133 in human large cell lung cancer cell line NCI-H460These results showed that As₂O₃ had significant inhibitory effects on cancer stem-like cells in NCI-H460 and NCI-H446.Therefore,we employed NCI-H460 and NCI-H446 as subjects of our follow-up studies.We found that As₂O₃ significantly suppressed the expression of stem cell biomarker CD133 in NCI-H460 cells at m RNA and protein levels in a dose-dependent manner.?2?Effects of As₂O₃ on the expression levels of stem cell biomarker CD133 in human small cell lung cancer cell line NCI-H446We found that As₂O₃ significantly suppressed the expression of stem cell biomarker CD133 in NCI-H446 cells at m RNA and protein levels in a dose-dependent manner.5.Effects of As₂O₃ on the expression of stem cell transcription factors such as Sox2,Oct4,C-myc,nanog,Klf4 and ?-catenin?1?Effects of As₂O₃ on the expression of stem cell transcription factors such as Sox2,Oct4,C-myc,nanog,Klf4 and ?-catenin in human large cell lung cancer cell line NCI-H460The expression of Sox2 and Oct4 at mRNA and protein levels in As₂O₃-treated groups was lower than that in control group.However,As₂O₃ has no significant effects on the expression of C-myc,nanog,Klf4 and ?-catenin.?2?Effects of As₂O₃ on the expression of stem cell transcription factors such as Sox2,Oct4,C-myc,nanog,Klf4 and ?-catenin in human small cell lung cancer cell line NCI-H446The expression of Sox2 and Oct4 at m RNA and protein levels in As₂O₃-treated groups was significantly lower than that in control group.However,there were no significant differences of expression levels of C-myc,nanog,Klf4 and ?-catenin between the As₂O₃-treated groups and the vehicle-treated group.Part 2 The mechanism of As₂O₃ on lung cancer stem-like cells1.As₂O₃ down-regulated Hedgehog pathway in human large cell lung cancer cell line NCI-H460As₂O₃ could significantly decrease the expression of Smo,Gli1,Gli2,GAS1 and N-myc at m RNA and protein levels in a concentration-dependent manner in NCI-H460 cells.However,As₂O₃ had no significant effect on the expression level of Ptch.2.As₂O₃ down-regulated hedgehog signaling in human small cell lung cancer cell line NCI-H446As₂O₃ could significantly decrease the expression of Smo,Gli1,Gli2,GAS1 and N-myc at m RNA and protein levels in a concentration dependent manner in NCI-H446 cells.However,As₂O₃ had no significant effect on the expression of Ptch.?Conclusion?1.As₂O₃ could inhibit cancer stem-like cells in human large cell lung cancer cell line NCI-H460 and human small cell lung cancer cell line NCI-H446.2.As₂O₃ exerted its effects by down-regulation of Hedgehog signaling pathway.