In vitro and ex vivo anti­tumor effect and mechanism of Tucatinib in leukemia stem cells and ABCG2­overexpressing leukemia cells.

Leukemia stem cells (LSCs), which evade standard chemotherapy, may lead to chemoresistance and disease relapse. The overexpression of ATP­binding cassette subfamily G member 2 (ABCG2) is an important determinant of drug resistance in LSCs and it can serve as a marker for LSCs. Targeting ABCG2 is a potential strategy to selectively treat and eradicate LSCs, and, hence, improve leukemia therapy. Tucatinib (Irbinitinib) is a novel tyrosine kinase inhibitor, targeting ErbB family member HER2, and was approved by the Food and Drug Administration in April 2020, and in Switzerland in May 2020 for the treatment of HER2­positive breast cancer. In the present study, the results demonstrated that tucatinib significantly improved the efficacy of conventional chemotherapeutic agents in ABCG2­overexpressing leukemia cells and primary leukemia blast cells, derived from patients with leukemia. In addition, tucatinib markedly decreased the proportion of leukemia stem cell­like side population (SP) cells. In SP cells, isolated from leukemia cells, the intracellular accumulation of Hoechst 33342, which is an ABCG2 substrate, was significantly elevated by tucatinib. Furthermore, tucatinib notably inhibited the efflux of [3H]­mitoxantrone and, hence, there was a higher level of [3H]­mitoxantrone in the HL60/ABCG2 cell line. The result from the ATPase assay revealed that tucatinib may interact with the drug substrate­binding site and stimulated ATPase activity of ABCG2. However, the protein expression level and cellular location of ABCG2 were not affected by tucatinib treatment. Taken together, these data suggested that tucatinib could sensitize conventional chemotherapeutic agents, in ABCG2­overexpressing leukemia cells and LSCs, by blocking the pump function of the ABCG2 protein. The present study revealed that combined treatment with tucatinib and conventional cytotoxic agents could be a potential therapeutic strategy in ABCG2­positive leukemia.

Diallyl thiosulfinate enhanced the anti-cancer activity of dexamethasone in the side population cells of multiple myeloma by promoting miR-127-3p and deactivating the PI3K/AKT signaling pathway.

BACKGROUND: Side population (SP) cells, which have similar features to those of cancer stem cells, show resistance to dexamethasone (Dex) treatment. Thus, new drugs that can be used in combination with Dex to reduce the population of SP cells in multiple myeloma (MM) are required. Diallyl thiosulfinate (DATS, allicin), a natural organosulfur compound derived from garlic, has been shown to inhibit the proliferation of SP cells in MM cell lines. Therefore, we investigated the effect of a combination of DATS and Dex (DAT + Dex) on MM SP cells. METHODS: SP cells were sorted from MM RPMI-8226 and NCI-H929 cell lines using Hoechst 33342-labeled fluorescence-activated cell sorting. The growth of SP cells was evaluated using the cell counting kit-8 assay. Cell cycle and apoptosis assays were conducted using a BD Calibur flow cytometer. miRNA expression was measured using quantitative reverse transcription-polymerase chain reaction. Phosphoinositide 3-kinase (PI3K), phosphorylated AKT (p-AKT), AKT, p-mechanistic target of rapamycin (mTOR), and mTOR levels were measured using western blot analysis. RESULTS: Our results showed that the combination of DATS+Dex inhibited sphere formation, colony formation, and proliferation of MM SP cells by inducing apoptosis and cell cycle arrest in the G1/S phase. In addition, the combination of DATS+Dex promoted miR-127-3p expression and inhibited PI3K, p-AKT, and p-mTOR expression in SP cells. Knockdown of miR-127-3p expression weakened the effect of DATS+Dex on cell proliferation, colony formation, apoptosis, and cell cycle of MM SP cells. Additionally, knockdown of miR-127-3p activated the PI3K/AKT/mTOR signaling pathway in MM SP cells cotreated with DATS+Dex. CONCLUSION: We demonstrated that cotreatment with DATS+Dex reduced cell proliferation, promoted apoptosis, and caused cell cycle arrest of MM SP cells by promoting miR-127-3p expression and deactivating the PI3K/AKT/mTOR signaling pathway.

Verteporfin induces apoptosis and reduces the stem cell-like properties in Neuroblastoma tumour-initiating cells through inhibition of the YAP/TAZ pathway.

Neuroblastoma is an embryonal malignancy of early childhood arising from the embryonic sympatho-adrenal lineage of the neural crest. About half of all cases are currently classified as high-risk of disease recurrence, with an overall survival rate of less than 40% at 5 years despite intensive therapy. Recent studies on matched primary tumours and at the relapse revealed downregulation of genes transcriptionally silenced by YAP as significant association with neuroblastoma relapse. Here, we evaluated the pharmacological targeting of YAP/TAZ with the YAP/TAZ-TEAD inhibitor Verteporfin (VP) in Tumour Initiating Cells (TICs) derived from High-Risk Neuroblastoma patients. VP treatment suppresses YAP/TAZ expression, induces apoptosis and causes the re-organization of the cytoskeleton reducing cells migration and clonogenic ability. Moreover, VP reduces the percentage of side population cells and ABC transporters involved in drug resistance, and the percentage of stem cell subpopulations CD133+ and CD44+ of TICs. Finally, we demonstrated that VP sensitizes TICs to the standard drugs used for neuroblastoma therapy etoposide and cis-platin opening the way to use VP as drug repositioning candidate for recurrent neuroblastoma.

4,4'­Bond secalonic acid D targets SP cells and inhibits metastasis in hepatocellular carcinoma.

The existence of cancer stem cells (CSCs) is considered to be the main reason for chemoresistance, metastasis and the ultimate failure of treatment in hepatocellular carcinoma (HCC). However, there are a few chemical agents that may inhibit CSCs. The present study identified that 4,4'­bond secalonic acid D (4,4'­SAD), a compound isolated from the marine­derived fungus Penicillium oxalicum, inhibited the growth of side population (SP) cells isolated from human liver cancer cell lines PLC/PRF/5 and HuH­7 by attenuating the expression of ATP­binding cassette superfamily G member 2. Furthermore, the results of wound healing, Transwell, western blotting and reverse transcription­quantitative PCR assays demonstrated that 4,4'­SAD suppressed the invasion and migration of SP cells by downregulating matrix metallopeptidase 9 (MMP­9) and upregulating the antagonist tissue inhibitor of metalloproteinases 1 in vitro. Moreover, in vivo study results found that 4,4'­SAD had anti­lung metastasis efficacy via the decrease of MMP­9 expression in the H22 HCC model of Kunming mice. Therefore, the present study identified the potential of 4,4'­SAD as a promising candidate for the treatment of advanced liver cancer.

Overexpressing microRNA-34a overcomes ABCG2-mediated drug resistance to 5-FU in side population cells from colon cancer via suppressing DLL1.

Colon cancer side population (SP) cells are a small subset of cancer cells that have cancer stemness capacity and enhanced drug resistance. ABCG2 is a multidrug resistance-related protein in SP cells and has been demonstrated to be regulated by Notch signalling pathway. Recently, microRNAs are reported to play a critical role in SP cell fate. However, their role in ABCG2-mediated drug resistance in colon cancer SP cells remains unclear. In the current study, the different expressions of miR-552, miR-611, miR-34a and miR-5000-3p were compared within SP and non-SP cells, which were separated from human colon cancer cell lines (SW480 and LoVo). We found that miR-34a was significantly down-regulated in SP cells and that overexpressing miR-34a overcame drug resistance to 5-fluorouracil (5-FU). The luciferase reporter assay indicated that miR-34a negatively regulated DLL1, a ligand of Notch signalling pathway, via binding with 3'-untranslated region of its messenger RNA. In addition, overexpressing miR-34a overcame ABCG2-mediated resistance to 5-FU via DLL1/Notch pathway in vitro, and suppressed tumour growth under 5-FU treatment in vivo. In conclusion, our findings suggest that miR-34a acts as a tumour suppressor via enhancing chemosensitivity to 5-FU in SP cells, which provides a novel therapeutic target in chemotherapy-resistant colon cancer.

SOX2 upregulates side population cells and enhances their chemoresistant ability by transactivating ABCC1 expression contributing to intrinsic resistance to paclitaxel in melanoma.

Paclitaxel is the last choice for the treatment of advanced melanoma as a second-line chemotherapeutic agent, but there are still many cases of intrinsic resistance to paclitaxel in melanoma and the reasons that cause paclitaxel resistance remain unclear. Here, we identified that high expression of SRY-box transcription factor 2 (SOX2) and high ratio of side population (SP) cells reduced the sensitivity to paclitaxel in melanoma cells. The knockout and the induction of SOX2 completely depleted and significantly upregulated the ratios of melanoma SP cells, respectively. These data suggest that SOX2, a pluripotent transcription factor for inducing cancer stem cells in melanoma, is also sufficient and necessary for the induction of melanoma SP cells. ATP-binding cassette (ABC) subfamily C member 1 (ABCC1) is one of ABC transporters which causes SP cells to be resistance to chemotherapeutic agents by efficiently pumping drugs out of cells. The knockout and the induction of ABCC1 significantly increased and decreased the sensitivity of melanoma cells to paclitaxel. High expression of ABCC1 was identified in melanoma cell lines with high expression of SOX2 and in their SP cells. SOX2 was identified to induce ABCC1 transcription. Taken together, SOX2 upregulates SP cells and enhances their chemoresistant ability by increasing ABCC1 expression, which contributes to intrinsic resistance to paclitaxel in melanoma. Our findings will lead to new insights into melanoma biology and therapy resistance, and eventually to new therapeutic targets.

Limonin enhances the radiosensitivity of nasopharyngeal carcinoma cells via attenuating Stat3-induced cell stemness.

The inhibitory effects of limonin have been disclosed in various tumors, however, its roles in nasopharyngeal carcinoma (NPC) progression are never been revealed. In the current work, we collected NPC cells with a higher stemness compared with bulk cells through isolating the side population (SP) cells. It was found that limonin exhibited a stronger inhibitory effect on SP cells than that in bulk cells, which was evident by a lower IC50 value. Additionally, limonin attenuated the stemness and migration ability of SP cells with the higher stemness, characterized as decreasing the spheroid formation ability, expression of stemness markers and migration ability. Moreover, the proportion of SP cells in G0 phase was remarkably higher than that in bulk cells. Notably, upon limonin treatment, the proportion of SP cells in G0 was decreased and S/G2/M increased. Furthermore, limonin enhanced the radiosensitivity of NPC cells. The mechanistic studies based on RNA-sequencing analysis revealed that limonin inhibited the gene transcription driven by Stat3 (signal transducer and activator of transcription 3) and an activator of Stat3 (Colivelin or IL-6) rescued the inhibitory effects of limonin. Therefore, these results demonstrate that limonin could reduce the stemness of NPC cells and thus the radiosensitivity through suppressing Stat3 transcriptional activity.

The PI3K/AKT signaling pathway regulates ABCG2 expression and confers resistance to chemotherapy in human multiple myeloma.

Side population (SP) cells are involved in the development of multidrug resistance (MDR) in human multiple myeloma (MM), due to their cancer stem cell (CSC)­like phenotypes. ATP­binding cassette (ABC) drug transporter proteins have been reported to be closely associated with MDR in leukemia; however, the correlation between ABC proteins and the progression of MM remains unclear. The present study used MM cell lines and clinical samples to determine the role of ABC subfamily G member 2 (ABCG2) in MM via flow cytometry, reverse transcription­quantitative polymerase chain reaction and western blotting. SP cells sorted from MM cell lines, including NCI­H929 cells, via fluorescence­activated cell sorting, exhibited CSC­like phenotypes and expressed high levels of ABCG2. Expression of ABCG2 and activation of the phosphatidylinositol 3­kinase (PI3K)/AKT serine/threonine kinase (AKT) signaling pathway was positively associated with the proportion of SP cells in the NCI­H929 cell line. In addition, suppression of the PI3K/AKT pathway using LY294002 or rapamycin counteracted the protective effects of ABCG2 against chemotherapeutic drug treatment. Mechanistically, PI3K/AKT signaling may regulate ABCG2 expression, and ABCG2 may regulate phosphatase and tensin homolog expression via a potential negative feedback loop. Furthermore, SP cell proportion, ABCG2 expression and PI3K/AKT pathway activation were associated with disease progression in patients with MM. These findings indicated the critical roles of ABCG2 and PI3K/AKT signaling in controlling stemness of MM cells, and suggested a novel strategy for targeting ABCG2 and PI3K/AKT signaling to treat MM with MDR.

Bortezomib Is More Effective to Side Population of RPMI8226 Myeloma Cells than Classical Anti-myeloma Agents.

AIM: Cytotoxic chemotherapy-based treatment of multiple myeloma (MM) is not curative, and the disease eventually recurs. This is partially because although currently available anti-MM strategies are effective in targeting the bulk of tumor cells, they do not target the tumor-initiating subpopulation of cancer stem cells. This study investigated the prevalence and biological functions of side population (SP) cells in MM cell lines including RPMI8226, ARH77, MM.1R and IM 9. MATERIALS AND METHODS: Flow cytometry-based Hoechst 33342 staining was used to evaluate the existence of SP cells. In addition, the ability of SP cells to regenerate the original population was determined. RESULTS: The frequency of SP cells was heterogeneous. Most cell lines (ARH77, IM9, and MM.1R) contained fewer than 1% SP cells; however, RPMI8226 contained approximately 10% SP cells. Sorted SP cells showed a higher proliferative ability and clonogenicity than the MP in the RPMI8226 myeloma cell line. The activity of ATP-binding cassette subfamily G member 2 (ABCG2), which is associated with high rates of proliferation, was higher in SP cells. However, the expression of specific surface markers such as cluster of differentiation (CD)138, CD34, CD38, CD19, CD20, and CD27 did not differ between SP and MP cells. Bortezomib was the only agent that significantly affected proliferation of both SP and MP cells. CONCLUSION: Our studies demonstrated that the SP fraction of myeloma cells possessed clonogenic tumor-initiating potential and revealed new mechanisms of action for bortezomib on SP cells.

Side population cells in anaplastic thyroid cancer and normal thyroid.

Comparison of studies of cells derived from normal and pathological tissues of the same organ can be fraught with difficulties, particular with cancer where a number of different diseases are considered cancer within the same tissue. In the thyroid, there are 4 main types of cancer, three of which arise from follicular epithelial cells; papillary and follicular which are classified as differentiated, and anaplastic which is classified as undifferentiated. One assay that can be utilised for isolation of cancer stem cells is the side population (SP) assay. However, SP studies have been limited in part due to lack of optimal isolation strategies and in the case of anaplastic thyroid cancer (ATC) are further compounded by lack of access to ATC tumors. We have used thyroid cell lines to determine the optimal conditions to isolate viable SP cells. We then compared SP cells and NSP cells (bulk tumour cells without the SP) of a normal thyroid cell line N-thy ori-3-1 and an anaplastic thyroid cancer cell line SW1736 and showed that both SP cell populations displayed higher levels of stem cell characteristics than the NSP. When we compared SP cells of the N-thy ori-3-1 and the SW1736, the SW1736 SP had a higher colony forming potential, expressed higher levels of stem cell markers and CXCR4 and where more migratory and invasive, invasiveness increasing in response to CXCL12. This is the first report showing functional differences between ATC SP and normal thyroid SP and could lead to the identification of new therapeutic targets to treat ATC.

Dual inhibition of enhancer of zeste homolog 1/2 overactivates WNT signaling to deplete cancer stem cells in multiple myeloma.

Multiple myeloma (MM) is an incurable hematological malignancy caused by accumulation of abnormal clonal plasma cells. Despite the recent development of novel therapies, relapse of MM eventually occurs as a result of a remaining population of drug-resistant myeloma stem cells. Side population (SP) cells show cancer stem cell-like characteristics in MM; thus, targeting these cells is a promising strategy to completely cure this malignancy. Herein, we showed that SP cells expressed higher levels of enhancer of zeste homolog (EZH) 1 and EZH2, which encode the catalytic subunits of Polycomb repressive complex 2 (PRC2), than non-SP cells, suggesting that EZH1 as well as EZH2 contributes to the stemness maintenance of the MM cells and that targeting both EZH1/2 is potentially a significant therapeutic approach for eradicating myeloma stem cells. A novel orally bioavailable EZH1/2 dual inhibitor, OR-S1, effectively eradicated SP cells and had a greater antitumor effect than a selective EZH2 inhibitor in vitro and in vivo, including a unique patient-derived xenograft model. Moreover, long-term continuous dosing of OR-S1 completely cured mice bearing orthotopic xenografts. Additionally, PRC2 directly regulated WNT signaling in MM, and overactivation of this signaling induced by dual inhibition of EZH1/2 eradicated myeloma stem cells and negatively affected tumorigenesis, suggesting that repression of WNT signaling by PRC2 plays an important role in stemness maintenance of MM cells. Our results show the role of EZH1/2 in the maintenance of myeloma stem cells and provide a preclinical rationale for therapeutic application of OR-S1, leading to significant advances in the treatment of MM.

Hypericin affects cancer side populations via competitive inhibition of BCRP.

OBJECTIVE: Cancer stem-like cells (CSLCs) are considered a root of tumorigenicity and resistance. However, their identification remains challenging. The use of the side population (SP) assay as a credible marker of CSLCs remains controversial. The SP assay relies on the elevated activity of ABC transporters that, in turn, can be modulated by hypericin (HYP), a photosensitizer and bioactive compound of St. John's Wort (Hypericum perforatum), a popular over-the-counter antidepressant. Here we aimed to comprehensively characterize the SP phenotype of cancer cells and to determine the impact of HYP on these cells. METHODS: Flow cytometry and sorting-based assays were employed, including CD24-, CD44-, CD133-, and ALDH-positivity, clonogenicity, 3D-forming ability, ABC transporter expression and activity, and intracellular accumulation of HYP/Hoechst 33342. The tumorigenic ability of SP, nonSP, and HYP-treated cells was verified by xenotransplantation into immunodeficient mice. RESULTS: The SP phenotype was associated with elevated expression of several investigated transporters and more intensive growth in non-adherent conditions but not with higher clonogenicity, tumorigenicity or ALDH-positivity. Despite stimulated BCRP level and MRP1 activity, HYP reversibly decreased the SP proportion, presumably via competitive inhibition of BCRP. HYP-selected SP cells acquired additional traits of resistance and extensively eliminated HYP. CONCLUSIONS: Our results suggest that SP is not an unequivocal CSLC-marker. However, SP could play an important role in modulating HYP-treatment and serve as a negative predictive tool for HYP-based therapies. Moreover, the use of supplements containing HYP by cancer patients should be carefully considered, due to its proposed effect on drug efflux and complex impact on tumor cells, which have not yet been sufficiently characterized.

Elimination of stem-like cancer cell side-population by auranofin through modulation of ROS and glycolysis.

Cancer side-population (SP) represents a sub-population of stem-like cancer cells that have an important role in drug resistance due to their high expression of the ATP-binding cassette transporter ABCG2 involved in drug export. Auranofin (AF), a clinical drug of gold complex that is used in treatment of rheumatoid arthritis, has been reported inducing tumor antiproliferation. However, whether AF can impact SP cells remains unclear. Our study showed that AF caused a depletion of SP cells and a downregulation of stem cell markers, and impaired their ability to form tumor colonies in vitro and incidence to develop tumors in vivo of lung cancer cells. Reactive oxygen species (ROS) had an important role in mediating AF-induced depletion of SP cells, which could be reversed by antioxidant NAC. Further study revealed that AF could also cause ATP depletion by inhibition of glycolysis. The depletion of cellular ATP might impair the function of ABCG2 pump, leading to increased drug accumulation within the cells and thus enhancing anticancer activity of chemotherapeutic agents such as adriamycin. Synergistic effect of AF and adriamycin was demonstrated both in vitro and in vivo. Simultaneous increase of ROS and inhibition of glycolysis is a novel strategy to eliminate stem-like cancer cells. Combination of AF with adriamycin seems to be promising to enhance therapeutic effectiveness.

Tonsil-derived mesenchymal stem cells (T-MSCs) prevent Th17-mediated autoimmune response via regulation of the programmed death-1/programmed death ligand-1 (PD-1/PD-L1) pathway.

Our knowledge of the immunomodulatory role of mesenchymal stem cells (MSCs) in both the innate and adaptive immune systems has dramatically expanded, providing great promise for treating various autoimmune diseases. However, the contribution of MSCs to Th17-dominant immune disease, such as psoriasis and its underlying mechanism remains elusive. In this study, we demonstrated that human palatine tonsil-derived MSCs (T-MSCs) constitutively express both the membrane-bound and soluble forms of programmed death-ligand 1 (PD-L1), which enables T-MSCs to be distinguished from MSCs originating from other organs (i.e. bone marrow or adipose tissue). We also found that T-MSC-derived PD-L1 effectively represses Th17 differentiation via both cell-to-cell contact and a paracrine effect. Further, T-MSCs increase programmed death-1 (PD-1) expression on T-cells by secreting IFN-ß, which may enhance engagement with PD-L1. Finally, transplantation of T-MSCs into imiquimod-induced psoriatic skin inflammation in mice significantly abrogated disease symptoms, mainly by blunting the Th17 response in a PD-L1-dependent manner. This study suggests that T-MSCs might be a promising cell source to treat autoimmune diseases such as psoriasis, via its unique immunoregulatory features. Copyright © 2017 John Wiley & Sons, Ltd.

Dexamethasone reduces side population fraction through downregulation of ABCG2 transporter in MCF-7 breast cancer cells.

Side population (SP) cells represent a rare population among breast cancer cells. SP cells have been reported to act as cancer stem­like cells, and to participate in the development of multidrug resistance via modulating the expression of ATP-binding cassette subfamily G member 2 (ABCG2). Dexamethasone is a corticosteroid drug that has been used as an adjuvant treatment to enhance the efficacy of chemotherapeutic agents; however, its effects in breast cancer have yet to be thoroughly investigated. In the present study, the effects of dexamethasone were investigated using the human MCF­7 breast cancer cell line, and SPs were examined in detail. Cellular proliferation, SP fractions and ABCG2 expression were examined following treatment of MCF­7 cells with dexamethasone. Dexamethasone was revealed to cause a dose­ and time­dependent decrease in cancer cell proliferation, and it also decreased the size of the SP fraction of MCF­7 cells and the expression of the ABCG2 transporter. The effects of dexamethasone on cellular proliferation, SP fraction and ABCG2 expression were abolished following the administration of the glucocorticoid antagonist RU486. These results suggested that dexamethasone may target breast cancer cell SPs and thus increase the sensitivity of tumor cells to chemotherapy. Therefore, it may be hypothesized that dexamethasone can be used as a chemosensitizer in the adjuvant treatment of patients with breast cancer.

Targeting BCRP/ABCG2 by RNA interference enhances the chemotherapy sensitivity of human colon cancer side population cells.

Relapse and metastasis are frequent in colon cancer and may be linked to stem cell characteristics. This study isolated side population (SP) cells from a colon cancer cell line (Colo-320) and examined their self-renewal and differentiation abilities. Compared to non-SP (NSP) cells, SP colon cancer cells were more tumorigenic in vivo and exhibited more invasive characteristics and a greater ability to form colonies. Additionally, more cells were in G0/G1 phase and more highly expressed the multidrug resistance protein BCRP/ABCG2. We achieved enhanced chemotherapy sensitivity by transfecting SP cells with a hairpin-like, small interfering RNA (siRNA) eukaryotic expression plasmid targeting BCRP/ABCG2.

Regulation of stem-like cancer cells by glutamine through ß-catenin pathway mediated by redox signaling.

BACKGROUND: Cancer stem cells (CSCs) are thought to play an important role in tumor recurrence and drug resistance, and present a major challenge in cancer therapy. The tumor microenvironment such as growth factors, nutrients and oxygen affect CSC generation and proliferation by providing the necessary energy sources and growth signals. The side population (SP) analysis has been used to detect the stem-like cancer cell populations based on their high expression of ABCG2 that exports Hoechst-33342 and certain cytotoxic drugs from the cells. The purpose of this research is to investigate the effect of a main nutrient molecule, glutamine, on SP cells and the possible underlying mechanism(s). METHODS: Biochemical assays and flow cytometric analysis were used to evaluate the effect of glutamine on stem-like side population cells in vitro. Molecular analyses including RNAi interfering, qRT-PCR, and immunoblotting were employed to investigate the molecular signaling in response to glutamine deprivation and its influence on tumor formation capacity in vivo. RESULTS: We show that glutamine supports the maintenance of the stem cell phenotype by promoting glutathione synthesis and thus maintaining redox balance for SP cells. A deprivation of glutamine in the culture medium significantly reduced the proportion of SP cells. L-asparaginase, an enzyme that catalyzes the hydrolysis of asparagine and glutamine to aspartic acid and glutamate, respectively, mimics the effect of glutamine withdrawal and also diminished the proportion of SP cells. Mechanistically, glutamine deprivation increases intracellular ROS levels, leading to down-regulation of the ß-catenin pathway. CONCLUSION: Glutamine plays a significant role in maintaining the stemness of cancer cells by a redox-mediated mechanism mediated by ß-catenin. Inhibition of glutamine metabolism or deprivation of glutamine by L-asparaginase may be a new strategy to eliminate CSCs and overcome drug resistance.

Regulation of voltage-gated potassium channels attenuates resistance of side-population cells to gefitinib in the human lung cancer cell line NCI-H460.

BACKGROUND: Side-population (SP) cells that exclude anti-cancer drugs have been found in various tumor cell lines. Moreover, SP cells have a higher proliferative potential and drug resistance than main population cells (Non-SP cells). Also, several ion channels are responsible for the drug resistance and proliferation of SP cells in cancer. METHODS: To confirm the expression and function of voltage-gated potassium (Kv) channels of SP cells, these cells, as well as highly expressed ATP-binding cassette (ABC) transporters and stemness genes, were isolated from a gefitinib-resistant human lung adenocarcinoma cell line (NCI-H460), using Hoechst 33342 efflux. RESULTS: In the present study, we found that mRNA expression of Kv channels in SP cells was different compared to Non-SP cells, and the resistance of SP cells to gefitinib was weakened with a combination treatment of gefitinib and Kv channel blockers or a Kv7 opener, compared to single-treatment gefitinib, through inhibition of the Ras-Raf signaling pathway. CONCLUSIONS: The findings indicate that Kv channels in SP cells could be new targets for reducing the resistance to gefitinib.

Bafilomycin A1 triggers proliferative potential of senescent cancer cells in vitro and in NOD/SCID mice.

Anticancer therapies that induce DNA damage tend to trigger senescence in cancer cells, a process known as therapy-induced senescence (TIS). Such cells may undergo atypical divisions, thus contributing to tumor re-growth. Accumulation of senescent cancer cells reduces survival of patients after chemotherapy. As senescence interplays with autophagy, a dynamic recycling process, we sought to study whether inhibition of autophagy interferes with divisions of TIS cells. We exposed human colon cancer HCT116 cells to repeated cycles of a chemotherapeutic agent - doxorubicin (doxo) and demonstrated induction of hallmarks of TIS (e.g. growth arrest, hypertrophy, poliploidization and secretory phenotype) and certain properties of cancer stem cells (increased NANOG expression, percentages of CD24+ cells and side population). Colonies of small and highly proliferative progeny appeared shortly after drug removal. Treatment with bafilomycin A1 (BAF A1), an autophagy inhibitor, postponed short term in vitro cell re-population. It was associated with reduction in the number of diploid and increase in the number of poliploid cells. In a long term, a pulse of BAF A1 resulted in reactivation of autophagy in a subpopulation of HCT116 cells and increased proliferation. Accordingly, the senescent HCT116 cells treated with BAF A1 when injected into NOD/SCID mice formed tumors, in contrast to the controls.Our results suggest that senescent cancer cells that appear during therapy, can be considered as dormant cells that contribute to cancer re-growth, when chemotherapeutic treatment is stopped. These data unveil new mechanisms of TIS-related cancer maintenance and re-population, triggered by a single pulse of BAF A1 treatment.

Isolation, identification, and characterization of cancer stem cells: A review.

Cancer stem cells (CSCs) or tumor-initiating cells (TICs) as a small subset of neoplastic cells are able to produce a tumor (tumorigenesis), maintain the population of tumorigenic cells (self-renewal), and generate the heterogeneous cells constructing the entire tumor (pluripotency). The research on stationary and circulating CSCs due to resistance to conventional therapies and inability in complete eradication of cancer is critical for developing novel therapeutic strategies for a more effective reduction in the risk of tumor metastasis and cancer recurrence. This review compiles information about different methods of detection and dissociation, side population, cellular markers, and establishment culture of CSCs, as well as characteristics of CSCs such as tumorigenicity, and signaling pathways associated with self-renewal and the capability of the same histological tumor regeneration in various cancers.