Cancer Stem-like Side Population Research Articles

Omega-3 Fatty Acid-Enriched Fish Oil and Selenium Combination Modulates Endoplasmic Reticulum Stress Response Elements and Reverses Acquired Gefitinib Resistance in HCC827 Lung Adenocarcinoma Cells

Non-small cell lung cancer (NSCLC)-carrying specific epidermal growth factor receptor (EGFR) mutations can be effectively treated by a tyrosine kinase inhibitor such as gefitinib. However, the inevitable development of acquired resistance leads to the eventual failure of therapy. In this study, we show the combination effect of omega-3 fatty acid-enriched fish oil (FO) and selenium (Se) on reversing the acquired gefitinib-resistance of HCC827 NSCLC cells. The gefitinib-resistant subline HCC827GR possesses lowered proapoptotic CHOP (CCAAT/enhancer-binding protein homologous protein) and elevated cytoprotective GRP78 (glucose regulated protein of a 78 kDa molecular weight) endoplasmic reticulum (ER) stress response elements, and it has elevated β-catenin and cyclooxygenase-2 (COX-2) levels. Combining FO and Se counteracts the above features of HCC827GR cells, accompanied by the suppression of their raised epithelial-to-mesenchymal transition (EMT) and cancer stem markers, such as vimentin, AXL, N-cadherin, CD133, CD44, and ABCG2. Accordingly, an FO and Se combination augments the gefitinib-mediated growth inhibition and apoptosis of HCC827GR cells, along with the enhanced activation of caspase -3, -9, and ER stress-related caspase-4. Intriguingly, gefitinib further increases the elevated ABCG2 and cancer stem-like side population in HCC827GR cells, which can also be diminished by the FO and Se combination. The results suggest the potential of combining FO and Se in relieving the acquired resistance of NSCLC patients to targeted therapy.

Identification, characterization and microRNA expression profiling of side population cells in human oral squamous cell carcinoma Tca8113 cell lines.

The present study aimed to evaluate the stem cell markers, characteristics and biological functions of cancer stem-like side population (SP) cells in human oral cancer. SP cells were isolated from the human oral squamous cell carcinoma Tca8113 cell line by Hoechst 33342 fluorescence dye and flow cytometry. The colony forming and proliferative capability of SP and non-SP cells were detected using a live-cell analysis system in vitro. The number of cells expressing stem cell markers was compared between SP cells and non-SP cells by flow cytometry. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to detect the mRNA and protein expression levels of stem cell genes, respectively. Differential expression of microRNAs (miRNAs) in SP and non-SP cells was determined by microarray hybridization and an miRNA regulation network was produced. With regard to the proliferation capability, SP cells reached 60.0% confluence after 40 h of growth compared with 35.1% confluence for non-SP cells (P<0.05). The number of colonies in SP cells was 43.1±9.2 compared with 33.0±8.2 of non-SP cells (P<0.05). The aldehyde dehydrogenase-1 (ALDH1)-positive cell number in the SP cells was increased by 10 times compared with the non-SP cells (P<0.01). The mRNA and protein expression levels of ALDH1, SRY-box 2, POU class 5 homeobox 1 and Nanog homeobox in SP cells were significantly higher compared with non-SP cells (P<0.05). Microarray hybridization demonstrated that 21 miRNAs were upregulated and 13 miRNAs were downregulated in SP cells compared with non-SP cells. SP cells in Tca8113 demonstrated greater capability of proliferation and colony formation compared with non-SP cells in vitro. Stem cell markers were overexpressed in SP cells compared with non-SP cells.

Ovarian cancer stem cells promote tumour immune privilege and invasion via CCL5 and regulatory T cells.

Emerging evidence indicates a link between the increased proportion of regulatory T cells (Tregs ) and reduced survival in patients who have been diagnosed with cancer. Cancer stem cells (CSCs) have been indicated to play a vital role in tumour initiation, drug resistance and recurrence. However, the relationship between Tregs and CSCs remains largely unknown. Here, we sorted out ovarian cancer stem-like side population (SP) cells and CD133+ cells to investigate the influence of ovarian CSCs on Tregs . Among the various immune-related molecules that we assessed, C-C motif chemokine ligand 5 (CCL5) was the most elevated in ovarian CSCs relative to that in the non-CSCs. The expression of its receptor, C-C motif chemokine receptor 5 (CCR5), was also increased on the surface of Tregs in ovarian cancer patients. This receptor-ligand expression profile indicated that ovarian CSCs recruit Tregs via CCL5-CCR5 interactions. We further assessed the expression of interleukin (IL)-10 in Tregs cultured with different cancer cells. Tregs cultured in conditioned medium (CM) from ovarian CD133+ cells expressed a higher level of IL-10 than Tregs cultured in CM from CD133- cells, indicating that Tregs exert pronounced immune-inhibitory functions in CSC-rich environments. Furthermore, co-culture with ovarian cancer cell lines induced the expression of matrix metalloproteinase-9 (MMP9) in Tregs which, in turn, enhanced the degradation of the extracellular matrix and enabled the invasion of tumour cells, thereby facilitating tumour metastasis. For the first time, to our knowledge, our findings describe the relationship between ovarian CSCs and Tregs , and demonstrated that these two cell populations co-operate to promote tumour immune tolerance and enhance tumour progression.

Isolation and characterization of multidrug-resistant side population cells in prostate carcinoma

Purpose: To isolate and characterize cancer stem-like side population (SP) cells from prostate cancer tissues using Hoechst 33342 dye exclusion.Methods: The presence of SP cells was analyzed in tumor samples by fluorescence activated cell sorting. The cell survival rate and ability for cell self-renewal using the sphere formation assay were evaluated after treatment with multiple drugs.Results: SP cells in the prostate cancer samples constituted 2.8 %, but fell to 0.6 % after treatment with verapamil. The SP cells showed high resistance to drugs such as 5-fluorouracil, cisplatin, paclitaxel (2 μmol/L) and oxaliplatin. The survival rate of SP cells after treatment with these drugs was significantly higher (p &lt; 0.01) than that of non-SP cells. Furthermore, the number of spheres generated in serumfree medium was significantly higher in prostate cancer SP cells than in non-SP cells.Conclusion: The presence of SP cells is responsible for prostate treatment failure and tumor recurrence. Therefore, isolation and characterization of SP cells may provide new insights into the development of novel therapeutic agents targeting cancer stem cells for complete eradication of the tumor.Keywords: Side population cells, ABC transporters, Cancer stem cells, Chemotherapy, Prostate treatment failure, Tumor recurrence, Drug resistance

Increased multi-drug resistance and reduced apoptosis in osteosarcoma side population cells are crucial factors for tumor recurrence.

The present study investigated the characteristic features of cancer stem cells (CSCs) using an aggressive human osteosarcoma cell line OS-65. Hoechst 33342 dye exclusion was used to distinguish the cancer stem-like side population (SP) cells from OS-65 cells. Furthermore, the SP cells were characterized via chemoresistance and cell death assays, reverse transcription-quantitative polymerase chain reaction and immunofluorescence. The present study identified ~3.3% of cancer stem-like SP cells from OS-65 cells whose prevalence is reduced significantly (0.9%) following treatment with verapamil. It was demonstrated that osteosarcoma SP cells are highly efficient at generating additional sarcospheres as transcriptional regulation of stemness genes, including SOX2, OCT-4 and NANOG, is highly upregulated. Notably, these SP cells demonstrated high resistance against chemotherapeutic drugs and apoptosis via elevated transcriptional regulation of several ATPase binding cassette (ABC) transporter and anti-apoptotic proteins, including ABCG2, ABCB1/MDR1 ABCB5, B cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein, respectively. The results of the present study suggested that CSCs may be a novel therapeutic target for the prevention of tumor relapse.

Phenylethyl isothiocyanate reverses cisplatin resistance in biliary tract cancer cells via glutathionylation-dependent degradation of Mcl-1.

Biliary tract cancer (BTC) is a highly malignant cancer. BTC exhibits a low response rate to cisplatin (CDDP) treatment, and therefore, an understanding of the mechanism of CDDP resistance is urgently needed. Here, we show that BTC cells develop CDDP resistance due, in part, to upregulation of myeloid cell leukemia 1 (Mcl-1). Phenylethyl isothiocyanate (PEITC), a natural compound found in watercress, could enhance the efficacy of CDDP by degrading Mcl-1. PEITC-CDDP co-treatment also increased the rate of apoptosis of cancer stem-like side population (SP) cells and inhibited xenograft tumor growth without obvious toxic effects. In vitro, PEITC decreased reduced glutathione (GSH), which resulted in decreased GSH/oxidized glutathione (GSSG) ratio and increased glutathionylation of Mcl-1, leading to rapid proteasomal degradation of Mcl-1. Furthermore, we identified Cys16 and Cys286 as Mcl-1 glutathionylation sites, and mutating them resulted in PEITC-mediated degradation resistant Mcl-1 protein. In conclusion, we demonstrate for the first time that CDDP resistance is partially associated with Mcl-1 in BTC cells and we identify a novel mechanism that PEITC can enhance CDDP-induced apoptosis via glutathionylation-dependent degradation of Mcl-1. Hence, our results provide support that dietary intake of watercress may help reverse CDDP resistance in BTC patients.

Abnormal Wnt signaling and overexpression of ABCG2 contributes to drug efflux properties of side population cells in nasopharyngeal carcinoma.

The presence of cancer stem cells (CSCs) has major implications in the choice of cancer treatment strategy and is responsible for tumor relapse. CSCs have been isolated and characterized in several types of cancer; however, studies concerning the CSCs from nasopharyngeal carcinoma (NPC) are limited. Thus, the present study was designed to isolate and characterize the cancer stem-like side population (SP) cells from NPC samples. The fluorescence-activated cell sorting (FACS)-based Hoechst 33342 dye exclusion technique identified that 3.9% of cells from NPC samples were cancer stem-like SP cells. Upon treatment with verapamil (ABC transporter inhibitor), the percentage of SP cells was significantly reduced to 0.7%, which confirms that the ABC transporter protein exhibits a significant role in drug exclusion. Fluorescence microscopy analysis revealed that the FACS purified SP cells showed increased expression of ABCG2 (ATP transporter protein), Oct-4 and CD44 (stem cell surface protein). Furthermore, these SP cells exhibited increased mRNA expression of ABCG2 and anti-apoptotic factor Bmi-1, which contribute to multi-drug resistance and increased cell survival rate. Notably, the Wnt/β-catenin signaling pathways are altered in SP cells. In addition, using reverse transcription‑quantitative polymerase chain reaction analysis it was observed that the cells exhibited increased expression of DKK1 and AXIN2. In conclusion, data from the present study clearly demonstrated that the presence of cancer stem-like SP cells from NPC may be responsible for chemotherapeutic drug resistance, tumor recurrence and invasion.

Isolation and phenotypic characterization of cancer stem-like side population cells in colon cancer.

Previous studies in cancer biology suggest that chemotherapeutic drug resistance and tumor relapse are driven by cells within a tumor termed 'cancer stem cells'. In the present study, a Hoechst 33342 dye exclusion technique was used to identify cancer stem‑like side population (SP) cells in colon carcinoma, which accounted for 3.4% of the total cell population. Following treatment with verapamil, the population of SP cells was reduced to 0.6%. In addition, the sorted SP cells exhibited marked multidrug resistance and enhanced cell survival rates compared with non‑SP cells. The SP cells were able to generate more tumor spheres and were CD133 positive. Subsequent biochemical analysis revealed that the levels of the adenosine triphosphate‑binding cassette sub‑family G member 2 transporter protein, B‑cell lymphoma anti‑apoptotic factor and autocrine production of interleukin‑4 were significantly enhanced in the colon cancer SP cells, which contributed to drug resistance, protection of the cells from apoptosis and tumor recurrence. Therefore, the findings suggested that treatment failure and colon tumorigenesis is dictated by a small population of SP cells, which indicate a potential target in future therapies.

Cluster of differentiation 44- and octamer-binding transcription factor-4-positive stem-like osteosarcoma cells involved in tumor development.

Osteosarcoma (OS) is an aggressive primary bone cancer that usually affects children and young adolescents. Previous studies have demonstrated the implications of a small sub-population of cancer stem cells on treatment failure and tumor recurrence. The present study analyzed the characteristic features of the stem-like cells within the human OS-55 cell line. It was identified that 2.3% of the OS-55 cells were cancer stem-like side population (SP) cells. Following treatment with verapamil, the population of SP cells was reduced to 0.7%. The sphere formation assay revealed that the OS cells were able to rapidly form tumor spheres (also known as sarcospheres). Immunofluorescence analysis identified that the OS-55 cells expressed the cluster of differentiation 44, octamer-binding transcription factor-3/4A and Nanog stem cell surface markers. The results of the present study suggest that, as with other tumors, OS also contains a sub-population of cancer stem-like cells, which may have important implications in cancer diagnosis and treatment.

Elimination of cancer stem-like cells and potentiation of temozolomide sensitivity by Honokiol in glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) is the most common adult malignant glioma with poor prognosis due to the resistance to radiotherapy and chemotherapy, which might be critically involved in the repopulation of cancer stem cells (CSCs) after treatment. We had investigated the characteristics of cancer stem-like side population (SP) cells sorted from GBM cells, and studied the effect of Honokiol targeting on CSCs. GBM8401 SP cells possessed the stem cell markers, such as nestin, CD133 and Oct4, and the expressions of self-renewal related stemness genes, such as SMO, Notch3 and IHH (Indian Hedgehog). Honokiol inhibited the proliferation of both GBM8401 parental cells and SP cells in a dose-dependent manner, the IC50 were 5.3±0.72 and 11±1.1 μM, respectively. The proportions of SP in GBM8401 cells were diminished by Honokiol from 1.5±0.22% down to 0.3±0.02% and 0.2±0.01% at doses of 2.5 μM and 5 μM, respectively. The SP cells appeared to have higher expression of O 6-methylguanine-DNA methyltransferase (MGMT) and be more resistant to Temozolomide (TMZ). The resistance to TMZ could be only slightly reversed by MGMT inhibitor O 6-benzylguanine (O 6-BG), but markedly further enhanced by Honokiol addition. Such significant enhancement was accompanied with the higher induction of apoptosis, greater down-regulation of Notch3 as well as its downstream Hes1 expressions in SP cells. Our data indicate that Honokiol might have clinical benefits for the GBM patients who are refractory to TMZ treatment.

Isolation, culture and identification of cancer stem-like cells from human thyroid carcinoma tissues

Objective To isolate and culture human thyroid cancer stem-like cells side population(SP) from human thyroid carcinoma specimens and compare the differences in octamer-binding transcription factor 4(Oct-4)and adenosine triphosphate binding cassette transporter G2(ABCG2)expression between SP cells and non-SP cells. Methods SP and non-SP cells were sorted from human thyroid carcinoma cells by fluorescence-activated cell sorting(FACS). The expression of Oct-4 and ABCG2 in SP cells and non-SP cells was detected by Western blotting. Results SP cells with stem cell potency were isolated from human thyroid carcinoma cells. The content of sorted SP cells in experimental group and control group was 1.40% and 0.155% respectively(P< 0.05). The expression of Oct-4 and ABCG2 was significantly higher in SP cells than in non-SP cells. Conclusion SP cells, which have the potency of stem cells, exist in human thyroid carcinoma cells. Key words: Thyroid carcinoma; Side population cells; Cancer stem cells

Isolation and characterization of cancer stem cells from medulloblastoma.

Brain cancer stem cells are a subset of tumor cells present in several types of brain tumor that evade treatment regimens and are responsible for tumor recurrence. Recent reports on several tumors have suggested that Hoechst 33342 dye exclusion is a powerful technique for isolating cancer stem cell-like side population (SP) cells. In the present study, we attempted to isolate the SP cells from medulloblastoma, a malignant brain tumor in children. The tumor samples obtained were subjected to fluorescence-activated cell sorting analysis for SP cell isolation. Further, the SP cells were characterized by a sphere-formation assay and analyzed for expression of stem cell genes by reverse transcription-polymerase chain reaction (RT-PCR). Using flow cytometry, we isolated 2.9% of cancer stem-like SP cells from malignant medulloblastoma, which was reduced to 0.4% in the presence of verapamil, an inhibitor of ABC transporter. These SP cells undergo rapid proliferation and have a high tendency to form tumor spheres when compared with non-SP cells. Further, RT-PCR analysis revealed that the isolated SP cells have increased expression of neural stem cell markers such as nestin, Notch1, and the ABC transporter protein ABCG2. Therefore, our findings suggest that SP cells of medulloblastoma share the characteristics of cancer stem cells. The increased expression of stem cell markers and ABCG2 protein may function collectively and be responsible for drug and apoptosis resistance, as well as tumor recurrence and invasion.

Parthenolide inhibits cancer stem-like side population of nasopharyngeal carcinoma cells via suppression of the NF-κB/COX-2 pathway.

Cancer stem cells play a central role in the pathogenesis of nasopharyngeal carcinoma and contribute to both disease initiation and relapse. In this study, cyclooxygenase-2 (COX-2) was found to regulate cancer stem-like side population cells of nasopharyngeal carcinoma cells and enhance cancer stem-like cells' characteristics such as higher colony formation efficiency and overexpression of stemness-associated genes. The regulatory effect of COX-2 on cancer stem-like characteristics may be mediated by ABCG2. COX-2 overexpression by a gain-of-function experiment increased the proportion of side population cells and their cancer stemness properties. The present study also demonstrated that in contrast to the classical chemotherapy drug 5-fluorouracil, which increased the proportion of side population cells and upregulated the expression of COX-2, parthenolide, a naturally occurring small molecule, preferentially targeted the side population cells of nasopharyngeal carcinoma cells and downregulated COX-2. Moreover, we found that the cancer stem-like cells' phenotype was suppressed by using COX-2 inhibitors NS-398 and CAY10404 or knocking down COX-2 with siRNA and shRNA. These findings suggest that COX-2 inhibition is the mechanism by which parthenolide induces cell death in the cancer stem-like cells of nasopharyngeal carcinoma. In addition, parthenolide exhibited an inhibitory effect on nuclear factor-kappa B (NF-κB) nucler translocation by suppressing both the phosphorylation of IκB kinase complex and IκBα degradation. Taken together, these results suggest that parthenolide may exert its cancer stem cell-targeted chemotherapy through the NF-κB/COX-2 pathway.

Effects of doxorubicin and gemcitabine on the induction of apoptosis in breast cancer cells.

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related mortality in females worldwide. The efficacy of chemotherapeutic drugs on breast cancer is hindered by many factors, including that cancer stem-like side population (SP) cells may contribute to tumorigenesis and resistance to chemotherapy. Doxorubicin and gemcitabine are two of the most effective and widely used chemotherapeutic agents for the treatment of various human malignancies. To the best of our knowledge, the effects of doxorubicin and gemcitabine on breast cancer SP cells are poorly understood. In the present study, we examined the potential roles of doxorubicin and gemcitabine in breast cancer main population (MP) and SP cells, and the mechanisms of doxorubicin and gemcitabine. The results showed that doxorubicin and gemcitabine decreasede proliferation, and increased apoptosis in the MCF7 MP and SP cells in vitro. Furthermore, doxorubicin and gemcitabine inhibited tumor growth and improved the survival rate of mice in vivo. Additionally, the mechanisms of doxorubicin and gemcitabine in MCF7 MP and SP cells were determined.

Differentiation therapy: sesamin as an effective agent in targeting cancer stem-like side population cells of human gallbladder carcinoma

BackgroundRecent studies have demonstrated that side population (SP) cells isolated from various cancer cell lines and primary tumors possess stem cell-like properties. Sesamin, a food-derived agent, possesses anti-cancer activities both in vitro and in vivo. The present study was designed to determine whether sesamin also have effects on cancer stem-like SP cells from gallbladder cancer (GBC).MethodsIn this study, we sorted SP cells by flow cytometry. SP cells were cultured and treated with sesamin. Tumor-sphere formation, colony formation, Matrigel invasion and tumorigenic potential were determined. Expression of nuclear NF-κB, IL-6, p-Stat3, Twist, E-cadherin and Vimentin was measured by Western blot, immunofluorescence staining or RT-PCR analysis. Nuclear NF-κB activity and IL-6 protein level were assessed with ELISA. Xenograft tumors were generated in nude mice.ResultsAfter treated with sesamin, SP cells differentiated into cells expressing the epithelial marker (E-cadherin). Sesamin effectively affected SP cells stem cell-like characteristics (i.e., tumor-sphere formation, colony-formation, Matrigel invasion), weakened the drug-resistance of SP cells and inhibited tumor growth both in vitro and in vivo. Treatment with sesamin significantly reduced the expression of nuclear NF-κB, IL-6, p-Stat3, Twist and Vimentin (a mesenchymal marker) in SP cells. Nuclear NF-κB activity and IL-6 level were also decreased after treatment with sesamin.ConclusionFood-derived sesamin directs the epithelial differentiation of cancer stem-like SP cells from GBC, which is associated with attenuation of NF-κB-IL-6-Stat3-Twist signal pathway.

Pien Tze Huang suppresses the stem-like side population in colorectal cancer cells

Accumulating evidence suggests that a small population of cells termed cancer stem cells (CSCs) are crucial in tumor development and drug resistance, leading to cancer relapse and metastasis and eventually the failure of clinical cancer treatment. Therefore, targeting CSCs is a promising approach for anticancer therapies. Due to the drug resistance and adverse effects of currently used chemotherapies, traditional Chinese medicines (TCM) have recently received attention due to the relatively few side-effects. Thus, they have been used as important alternative remedies for various diseases, including cancer. Pien Tze Huang (PZH), a well-known TCM formula that was first prescribed more than 450years ago in the Ming Dynasty, has been used in China and Southeast Asia for centuries as a folk remedy for various types of cancer. Previously, it was reported that PZH inhibits colon cancer growth via the promotion of cancer cell apoptosis and inhibition of cell proliferation and tumor angiogenesis, which is probably mediated by its regulatory effect on multiple intracellular signaling pathways. To elucidate the mechanism of the tumoricidal activity of PZH, the aim of the present study was to investigate the effect of PZH on CSCs that were isolated as the side population (SP) from the HT-29 colorectal cancer cell line. The results demonstrated that PZH significantly and dose-dependently reduced the percentage of the colorectal cancer stem-like SP cells, decreased the viability and sphere-forming capacity of HT-29 SP cells, indicating that PZH is potent in suppressing the growth of colorectal cancer stem cells. Moreover, PZH treatment in HT-29 SP cells markedly inhibited the mRNA levels of ABCB1 and ABCG2, which are members of the ABC transporter superfamily, thereby contributing to the SP phenotype and multi-drug resistance. Findings of the present study suggest that inhibiting the growth of CSCs is a potential mechanism by which PZH can be used in cancer treatment.

Vitamin D Analog EB1089 Induces Apoptosis in a Subpopulation of SGC-7901 Gastric Cancer Cells Through a Mitochondrial-Dependent Apoptotic Pathway

Gastric cancer is the second leading cause of cancer death worldwide. Cancer stem-like side population (SP) cells may be important factors that hinder efficacy of chemopreventative and chemotherapeutic approaches in gastric cancer. EB1089 is an antitumor agent that has been used in many cancers; however, no reports to date have determined the effects of EB1089 in gastric cancer. In our study, SP and main population (MP) cells were isolated from 4 gastric cancer cell lines in different stages of differentiation by flow cytometry (FCM) and confirmed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. EB1089 decreased the proliferation, increased apoptosis, and induced mitochondrial damage in the SP cells isolated from 1 cell type (SGC-7901), but not MP cells, through increased Bax and decreased Bcl-2 and Bcl-xL protein expression. This protein expression pattern induced the activation of caspase-3 and caspase-9. The effects of EB1089 on SGC-7901 SP cells were blocked by treating cells with vitamin D receceptor (VDR) siRNA or butin (an inhibitor of the mitochondrial apoptosis pathway). Our results suggest that EB1089 targets SGC-7901 SP cells through a mitochondrial apoptosis pathway. However, further studies are needed to elucidate the signal transduction between VDR and the mitochondrial apoptosis pathway.

MiR-125b regulates side population in breast cancer and confers a chemoresistant phenotype

Resistance to chemotherapy is a major obstacle for the effective treatment of breast cancer and is partially due to the presence of drug resistant stem cell-like side population (SP). Previous studies have shown elevated miR-125b is associated with chemoresistance and metastasis; however, the relationship between miR-125b and SP cells remains unknown. In this study, we isolated and characterized SP cells in a panel of breast cancer cell lines and primary cancer cells from breast cancer patients. SP cells showed cancer stem cells (CSCs) properties, including self-renewal, resistance to chemotherapy and high expression of stem cell markers. The percentage of SP cells was higher in chemotherapy resistant patients compared to that in chemotherapy responsive patients (5.8 ± 2.4% in non-responsive patients vs. 1.2 ± 0.5% in responsive patients, P = 0.012). Importantly, SP cells had higher level of miR-125b than NSP cells and the elevated miR-125b expression in chemoresistant cancer cells were due to high percentage of SP cells. Overexpression of miR-125b correlated with an increase in tumor SP and CSC property, whereas knockdown of miR-125b correlated with decreased incidence of SP. In addition, miR-125b overexpression in breast cancer cells induced epithelial-mesenchymal transition (EMT)-like cellular marker alteration, suggesting a potential mechanism of miR-125b in the regulation of cancer stem-like SP cells. Taken together, these results suggest an important role for miR-125b in breast cancer chemoresistance by maintaining cancer stem-like SP fraction, and raise the possibility that miR-125b may be a significant prognostic response marker for cancer therapy.

Identification of Cancer Stem-Like Side Population Cells in Purified Primary Cultured Human Laryngeal Squamous Cell Carcinoma Epithelia

Cancer stem-like side population (SP) cells have been identified in many solid tumors; however, most of these investigations are performed using established cancer cell lines. Cancer cells in tumor tissue containing fibroblasts and many other types of cells are much more complex than any cancer cell line. Although SP cells were identified in the laryngeal squamous cell carcinoma (LSCC) cell line Hep-2 in our pilot study, it is unknown whether the LSCC tissue contains SP cells. In this study, LSCC cells (LSCCs) were primary cultured and purified from a surgically resected LSCC specimen derived from a well-differentiated epiglottic neoplasm of a Chinese male. This was followed by the verification of epithelium-specific characteristics, such as ultrastructure and biomarkers. A distinct SP subpopulation (4.45±1.07%) was isolated by Hoechst 33342 efflux analysis from cultured LSCCs by using a flow cytometer. Cancer stem cell (CSC)-associated assays, including expression of self-renewal and CSC marker genes, proliferation, differentiation, spheroid formation, chemotherapy resistance, and tumorigenicity were then conducted between SP and non-SP (NSP) LSCCs. In vitro and in vivo assays revealed that SP cells manifested preferential expression of self-renewal and CSC marker genes, higher capacity for proliferation, differentiation, and spheroid formation; enhanced resistance to chemotherapy; and greater xenograft tumorigenicity in immunodeficient mice compared with NSP cells. These findings suggest that the primary cultured and purified LSCCs contain cancer stem-like SP cells, which may serve as a valuable model for CSC research in LSCC.

Distinct Radiosensitivity of Lung Carcinoma Stem-Like Side Population and Main Population Cells

Lung cancer is a leading cause of cancer death worldwide. Efficacy of radiation therapy on lung cancer is hindered by many factors. Among these, both cancer stem-like side population (SP) and main population (MP) cells may contribute to tumorigenesis and resistance to radiation therapy. However, the detailed mechanism responsible for this effect remains unknown. The SP and MP cells were obtained from lewis lung carcinoma cells and analyzed the DNA dye (Hoechst 33342) method and flow cytometry. The levels of ABCG2 and CD133 markers were examined by reverse transcription polymerase chain reaction, Western blot, and immunofluorescence. The effects of ionizing radiation (IR) on the growth and apoptosis of SP and MP cells were determined by 3-(4, 5-dimethylthiazol-2-y)-2, 5-diphenylterazolium bromide (MTT), colony formation, and apoptosis assays. Mitochondrial membrane potential and intracellular reactive oxygen species production were measured by flow cytometry. Finally, the expression of Bax, Bcl-xL, Bcl-2, activated caspase-3 and caspase-9 proteins were examined by Western Blot. IR decreased proliferation, increased apoptosis and mitochondria damage in MP, but not in SP cells. Protein levels of Bcl-2 and Bcl-xl were decreased, while Bax expression was increased in MP cells following IR exposure. In addition, increased activation of caspase-3 and caspase-9 were detected after IR exposure in MP cells, but not in SP cells. Our results show that IR decreases proliferation, increases apoptosis, and induces mitochondria damage in MP cells, but not in SP cells, through increased Bax and decreased Bcl-2 and Bcl-xl protein expression. This protein expression pattern induces activation of caspase-3 and caspase-9. This study suggests that IR exposure targets MP cells through a mitochondrial apoptosis pathway. However, more work is required to further confirm these results using in vivo xenograft models. More importantly, further studies are warranted to elucidate the radioresistant mechanisms of SP cells.