| The American Cancer Society estimates228,190new lung cancer cases and159,480lung cancer deaths will occur in the United States in2013, accounting for13.7%and27.5%of the total cases worldwide. Lung cancers continue to be the most common causes of cancer death. Although chemotherapy has advanced considerably over last30years, the5-year relative survival rate for lung cancer remains only17%[1]. New therapeutic approaches to this highly invasive cancer are desperately needed.The cancer stem cell hypothesis was proposed many years ago. This hypothesis suggests that tumors originate from tissue stem cells that exhibit dysregulation of the normal self-renewal process. Some tumor cells retain stem cell properties, including the ability to self-renew and differentiate [2]. These stem-like tumor cells, which are termed cancer stem cells (CSCs), are referred to as tumor-initiating cells. CSCs within a tumor are thought to drive disease progression and metastasis [3]. Remarkably, compared with normal cancer cells, therapies against CSCs are less effective. CSCs exhibit significant resistance to conventional anti-tumor therapy. Many ATP-binding cassette (ABC) transporters are highly expressed by CSCs. These transporters, such as ABCG1, ABCB1and ABCC1, can actively efflux drugs from cells [4]. Therefore, although radiation and chemotherapy against tumors can cause complete regression, enough cancer stem cells could remain to cause tumor recurrence [5]. Thus, new treatments specifically targeting CSCs may significantly enhance the effect of chemotherapy. CSC markers such as CD133are widely accepted and used for the isolation of CSCs in many tumor types, including non-small cell lung cancer [6].If it is true that CSCs originate from normal stem cells, pathways regulating stem cell self-renewal and differentiation would also work on CSCs [5]. The Notch signaling pathway is considered to play an important role in the control of cell fates and developmental processes [7]. Several studies have reported the dysfunction of Notch pathways in the tumorigenesis of many cancers [8]. Furthermore, a high percentage of lung cancer lines express Notch receptors and their target genes, such as Jagged1, Hes1 and Heyl. The inhibition of Notch signaling can reduce tumor cell proliferation [9].ObjectiveTo explore the function of the Notch signaling pathway in the regulation of CSCs, we isolated CD133+cells from the human lung adenocarcinoma cell line A549. We compared the expression of Notch signaling between CD133+and CD133-cells and blocked Notch signaling using the y-secretase inhibitor DAPT. Furthermore, we try to examine the resistance of CSCs to drugs and the effect of GSI/CDDP combination therapy. To find weather Notch pathway inhibitors have an important application in CSC-targeting therapy of lung cancer.Methods1. We isolated CSCs from the human lung adenocarcinoma cell line A549. CD133was used as a stem cell marker for fluorescence-activated cell sorting (FACS) The cell line A549was purchased from the China Center for Type Culture Collection, Wuhan University. A549cells were cultured in RPMI-1640containing10%fetal bovine serum. We treated cancer cells with FcR Blocking reagent and CD133/2(293C3)-PE antibody and sorted CSCs using the high-speed sorting flow cytometer FACS. CD133+cells sorted from A549were maintained in serum-free stem cell media. Stem cell media was made in RPMI-1640, supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). We examined the biological characteristics of CSCs such as cell morphology, self-renewal and drug resistance.The Cell Counting Kit-8was used to quantify the proliferation of CD133+and CD133-cells. Growth curves were obtained by plotting the absorbance values over time.2. In order to compare the expression of Notch signaling in both CD133+and CD133-cells, we detected the expression of Notchl, Notch2and Hesl of cancer cells by real-time fluorescence quantitative polymerase chain reaction (RT-PCR) and western blotting. Total-RNA was extracted from CD133+and CD133-cells using TRIzol reagent. Gene expression was quantified using the comparative Ct method. Actin was used as an internal control. The protein expression was quantified using the Odyssey infrared imaging system. GAPDH was used for normalization. We blocked Notch signaling using the y-secretase inhibitor DAPT (GSI-Ⅸ) to determine the effect on CD133+and CD133-cells of A549.3. We studied the effect of Notch signaling on drug resistance in CD133+and CD133-cells. Cisplatin (CDDP) is a classic chemotherapy drug for lung cancer. Therefore, we used CDDP in our drug intervention. To further examine the effect of combining chemotherapy with GSI, we treated the two types of cells with2μM DAPT and6mg/L CDDP. Cell proliferation assays and Cell cycle analysis were used to evaluate the synergistic effect. The Cell Counting Kit-8was used to quantify the proliferation. Cancer cells were stained with propidium iodide (PI), then analyzed by flow cytometry after30min incubation at room temperature.Results1. We labeled A549cells with monoclonal CD133/2(293C3) antibodies conjugated to R-phycoerythrin (PE). These cells were separated into CD133+and CD133-cell fractions by FACS for the expression of CD133. The stem cell marker CD133was only expressed in0.5%±0.12%of all A549cells. After sorting, the percentage of CD133-expressing cells in the CD133+group was97.5%±1.72%. Cell proliferation assays over7days revealed that the difference between CD133+and CD133-cells in the rate of proliferation was not significant. CD133+cells appeared more resistant to chemotherapy compared with CD133-cells at the same CDDP concentration. The IC50was5.9±0.61mg/L in CD133-cells and21.1±0.97mg/L in CD133+cells, representing a significant difference between the two types of cells (P<0.01). We observed that5.75%±0.35%of CD133-cells and0.1%±0.02%of CD133+cells were in the G2/M phase. This finding remarkably demonstrated that in most CD133+cells, DNA replication was not active (P＜0.01). This inactivity of DNA replication may be the reason why cancer stem cells exhibit increased resistance to chemotherapy compared with normal cancer cells.2. RT-PCR revealed that both CD133+and CD133-cells express Notchl and Notch2, but CD133+cells expressed lower levels than CD133-cells (0.069±0.019and0.13±0.026). Hesl was expressed in CD133-cells but not in CD133+cells. Actin was used as an internal control. Western blotting analysis provided similar results to those obtained by RT-PCR (1.36±0.13vs0.40±0.08,1.01±0.20vs0.76±0.11, P＜0.05) DAPT has been shown to cause a reduction in Aβ40and A042levels and Notch signaling deficiencies at the morphological, molecular and biochemical levels in human cancer cells. We observed that DAPT could inhibit the growth of both CD133+and CD133-cells, but the effect was not strong. After being treated with2μM DAPT for48hours, CCK-8assay revealed that the cell viability of CD133-cells was79.2%±4.3%compared with control. In contrast, the cell viability of CD133+cells was68.2%±3.8%,(P<0.05) which suggested that the suppression of growth by DAPT was more effective in CD133+cells. DAPT seemed to have little effect on cell cycle changes in CD133-cells. After48h of DAPT treatment, the percentage of cells in the G2/M phase was5.65%±0.44%, which was not significantly different from the control group at5.75%±0.35%(P>0.05). The situation was similar for CD133+cells; the proportion of cells in the G2/M phase was0.00%in the DAPT group and0.1%±0.02%in control group. However, the proportion of cells in S phase increased from11.96%±0.89%to18.48%±0.64%(P<0.05) in CD133+cells.3. We observed that this combination enhanced the antitumor activity of the chemotherapy. When cells were only treated with6mg/L CDDP, the viability of the cells was43.5%±2.7%and66.7%±4.2%in CD133-and CD133+cells, respectively. However, when treated with GSI and CDDP combination therapy, the cell viability decreased to35.3%±1.8%and42.5%±2.1%, respectively. This observation supported our assumption that the Notch pathway blockade depresses the resistance of A549cells to chemotherapy. Moreover, we also observed that the increase in the inhibitory effect of combination treatment was14%in CD133-cells and55.6%in CD133+cells. It is particularly noteworthy that the synergistic effect of CDDP/GSI was especially significant for CD133+cells (P<0.01). CDDP can bind to DNA and interfere with the cell’s repair mechanisms, which eventually leads to cell death. In our study, CDDP arrested cells in the G2/M phase. When treated with6mg/L CDDP for48h, the proportion of cells in the G2/M phase increased to39.46%±1.84%in CD133-cells and17.50%±0.77%in CD133+cells. This result confirms that CD133+cells were not as sensitive as CD133-cells to chemotherapy (P＜0.05). We observed that the proportion of G2/M cells in CD133-cells exhibited no significant difference between CDDP and DAPT/CDDP combination therapy (39.46%±1.84%and39.53%±1.08%, P>0.05). However, we found that DAPT/CDDP combination therapy significantly potentiated the cell cycle arrest in the G2/M phase in CD133+cells (38.41%±0.93%vs0.1%±0.02%,38.41%±0.93%vs17.50%±0.77%, P＜0.05). Compared with the CDDP group, the percentage of G2/M cells in CD133+cells increased from17.50%±0.77%to38.41%±0.93%(P＜0.01), very close to the value of CD133-cells.ConclusionOur discovery demonstrated a depression of growth in CD133+A549cells caused by GSI. Blockade of Notch signaling pathway enhanced the effect of chemotherapy with CDDP. Furthermore, this DAPT/CDDP co-therapy caused a G2/M arrest and effectively eliminated both CD133-and CD133+cells. This synergetic effect was especially significant on CD133+cells. Further studies are needed to demonstrate the mechanisms of GSI-induced enhancement with CDDP. Nevertheless, targeting the Notch pathway has exhibited great potential to be an improved cancer treatment that could kill both replicating cancer cells and more quiescent cancer stem cells and reduce tumor relapse. |
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