Disulfiram/copper Complex Inhibits ALDH-positive Non-Small Cell Lung Cancer Stem Cells

The cancer stem cell model proposes that tumor progression,drug resistance,metastasis,and relapse after therapy may be driven by a subset of cells within a tumor.Therefore,the identification of CSCs has important implications for future therapeutic approaches.During the past few years,cancer stem cells(CSCs)have been identified in,and isolated from,solid tumors such as breast,brain,colon,pancreatic,and prostate tumors.Recent evidence suggests that like other tumors,human lung cancers may also harbor CSC populations.However,identification and targeting of human lung CSCs has been hampered by the lack of reliable lung cancer stem cell markers.The aldehyde dehydrogenase(ALDH)family comprises cytosolic isoenzymes responsible for oxidizing intracellular aldehydes.Recent evidence suggests that ALDH activity has also identified CSCs in a variety of tumor types.Disulfiram(DSF)is an aldehyde dehydrogenase inhibitor that was used for alcohol aversion therapy since the 1940s.Accumulating evidence demonstrates that DSF has strong anticancer activity against lung cancer and other certain types of cancers both in several preclinical studies and clinical trails,and the cytotoxicity of DSF is copper(Cu)dependent.Previous studies in our lab showed that DSF/Cu could inhibit tumor angiogenesis and invasion.These findings led us to investigate the effect of DSF/Cu complex treatment on self-renewal,proliferation,tumor forming ability and tumor recurrence driven by ALDH-positive NSCLC stem cells in vitro and in vivo.1.ALDH-positive cells represent cancer stem cells in NSCLC cell lines.Aldeflor assays followed by FACS analysis were used to assess the presence of a cell population with ALDH activity,and then colony forming assays were used to compare the colony forming capacity ofALDH-positive and ALDH-negative cells in NSCLC cell lines.The results showed that,in H1299 and H460 cell lines,the ALDH-positive cells show a signifiantly higher colony-forming effiiency than the ALDH-negative cells.Nanog,Oct-4 and Sox2 were expressed at higher levels in ALDH-positive H1299 cells than in ALDH-negative H1299 cells,suggesting that in the H1299 cell line,ALDH expression may be essential for maintaining self-renewal and tumorigenesis.The data of animal experiments showed that,the ALDH-positive H1299 cells generated much larger tumors than the ALDH-negative cells in all the NOD/SCID mice.Furthermore,the secondary xenograft tumors that developed from the ALDH-positive cells were significantly larger than the tumors from ALDH-negative cells.Taken together,the results of the in vivo and in vitro assays indicated that ALDH could be a single CSCs marker in some NSCLC cell lines.2.DSF/Cu inhibits ALDH-positive NSCLC stem cells in vitro and in vivo.We investigated whether DSF/Cu could inhibit ALDH-positive NSCLC stem cells in vitro and tumors derived from sorted ALDH-positive CSCs in vivo.DSF/Cu(0.5/1 μmol/1)significantly inhibited the expression of stem cell transcription factors(Sox2,Oct-4 and Nanog)and reduced the capacities of NSCLC stem cells for self-renewal,proliferation and invasion in vitro.DSF/Cu(60/2.4 mg/kg,twice a week)reduced the size of tumors derived from sorted ALDH-positive stem cells.And DSF/Cu treatment could also inhibit the expression of Nanog and Oct-4 in tumor tissues in a dose-dependent manner.Taken together,these results suggested that DSF/Cu was able to eliminate NSCLC stem cells both in vivo and in vivo.3.Disulfiam/copper complex inhibits non-small cell lung cancer recurrence driven by ALDH-positive cancer stem cells.Two other NOD/SCID xenograft models were used to determine whether DSF/Cu could target NSCLC stem cells and inhibit tumor recurrence in vivo.To determine whether DSF/Cu can inhibit tumor regrowth in vivo,we assessed the functional presence of CSCs by assaying for in vivo tumor-seeding ability after drug treatment in vitro.The results confim that ALDH-positive CSCs within NSCLCs are resistant to paclitaxel and DSF alone,but are sensitive to DSF/Cu.Furthermore,we used a secondary xenograft model to test whether DSF/Cu treatment could eliminate ALDH-positive cells and inhibit tumor recurrence,which was reflected by reduced tumor growth in recipient mice that were inoculated with tumor cells derived from primary xenografts.The results suggest that DSF/Cu was able to eliminate NSCLC stem cells in primary xenografts,thereby abrogating the regrowth of tumors in secondary mice.4.ALDH1A1 plays a key role in the DSF/Cu induced elimination of cancer stem cells.Recent evidence suggests that ALDH1 or ALDH3A1,which may be lung tumor stem cell markers or therapeutic targets,are highly expressed in some NSCLC cell lines as well as in patient lung cancer samples.However,the stem cell-related function and signifiance of the ALDHs have not yet been thoroughly investigated in NSCLCs.RNA interference and overexpression of ALDH isozymes suggested that ALDH1A1,which plays a key role in ALDH-positive NSCLC stem cells,might be the target of the DSF/Cu complex.Collectively,our data demonstrate that DSF/Cu targets ALDH1A1 to inhibit NSCLC recurrence driven by ALDH-positive CSCs.Thus,the DSF/Cu complex may represent a potential therapeutic strategy for NSCLC patients.We examined the efficacy of the DSF/Cu complex against ALDH-positive NSCLC stem cells in cell lines and lung cancer xenografts.Our in vivo data showed that the DSF/Cu complex was more effective than DSF alone at eliminating ALDH-positive cells and inhibiting tumor recurrence,as reflected by the inhibition of tumor growth in recipient mice that were inoculated with tumor cells derived from DSF/Cu-treated cell lines or primary xenografts.Furthermore,we investigated the stem cell-related function and significance of ALDH isozymes in NSCLC cell lines.Our data showed that ALDH1A1,which plays a key role in ALDH-positive NSCLC stem cells,is the target of the DSF/Cu complex.Collectively,our data demonstrate that DSF/Cu targets ALDH1A1 to inhibit NSCLC recurrence driven by ALDH-positive CSCs.Thus,the DSF/Cu complex may represent a potential therapeutic strategy for NSCLC patients.