ABCG2 Regulates The Pattern Of Self-renewing Divisions In Cisplatin-resistant Non-small Cell Lung Cancer Cell Lines

IntroductionChemotherapy is a key method of treatment in the primary and palliative care of patients with lung cancer, of which non-small cell lung cancer(NSCLC) accounts for the majority of cases. Cisplatin is one of the most commonly used chemotherapeutic drugs in the treatment of lung cancer, particularly for NSCLC. However, there are still some patients who exhibit drug resistance to cisplatin-based chemotherapy or responds well initially and then gradually relapses. To overcome drug resistance, patients with NSCLC are administered large doses of drugs, which generally increases the adverse effects, yet fails to improve the clinical prognosis or outcome. Therefore, a better understanding of the molecular mechanisms underlying cisplatin resistance is warranted in order to further understand the exact mechanisms of chemo-resistance and make more individual chemotherapy strategies for NSCLC patients in the future.There is increasing evidence that the active export of chemotherapeutic drugs from cancer cells is one of the main mechanisms of drug resistance. Cancer cells often gain drug resistance through the overexpression of membrane transport proteins that effectively efflux of antitumor drugs. The ATP-binding cassette(ABC) multidrug transporters such as ABCG2(BCRP/MXR/ABCP) are considered responsible for the majority of drug efflux in human cancer in vitro. The overexpression of ABCG2 has been reported to confer drug resistance upon NSCLC to various chemotherapeutic drugs. Furthermore, previous study also showed ABCG2 cloud impact clinical outcome in platinum-based chemotherapy for advanced NSCLC patients. For example, the ABCG2-positive patients had lower response rate to chemotherapy and shorter progression-free survival and overall survival than ABCG2-negative patients. Despite these advances, the potential function of ABCG2 remains incompletely understood and may be more complicated than we would like to think.Recent evidence have demonstrated that ABCG2 is involved in the proliferation of cancer cells and suppression of ABCG2 could inhibit cancer cell proliferation, which may propose a novel function of ABCG2 in cancer cell proliferation. Meanwhile, a recent study demonstrated that ABCG2 could directly regulate the switch between symmetric and asymmetric cell division in cardiac side population, determining progenitor cell fate decisions. Traditionally, there were two basic models of stem cell divisions, including asymmetric and symmetric cell divisions. With asymmetric cell division model, two daughter cells with divergent fates are generated, one capable of self-renewal and the other committed to differentiation. With symmetric division model, two identical daughter cells are formed that retain stem cell properties or become early committed cells. From the engineering prospective, the asymmetric model has the advantage of keeping the stem cell population level steady. However, an obvious disadvantage is its inability to replenish the stem cell pool in case of injury. This problem is naturally solved by the symmetric model. Several studies showed that progenitor/stem cells divide asymmetrically during physiological tissue homeostasis, however, these progenitor/stem cells revert to symmetric division and rapid proliferation after tissue injury. In clinical, chemotherapy is typically administered in cycles that are separated by 3-week intervals to allow restoration of hematopoietic and other normal cells damaged by the anti-tumor drugs, we ofen see the tumor cells in some patients can aggressively repopulate during these intervals and restore the tumor to its pretreatment size. Taken together with previous studies, we speculate that the tumor chemotherapy is similar to tissue damage, the "surviving" drug-resistant tumor cells will changes its division parttern from asymmetric division to symmetrical division, which will prompt the drug-resistant tumor cells grow rapidly and lead to tumor recurrence. Given the recurrent cells are mainly composed of symmetrical division, this will cause recurrent tumor is drug-resistant, and the chemotherapy drugs couldn’t narrow the mass of tumor, and finally lead to chemotherapy failure.Above all, we speculate that the ABCG2 may not only act as a multi drug-resistant protein which mediated chemotherapy tolerance, but also against chemotherapy drugs through promoting the tumor cells proliferate rapidly by the way of symmetrical division. To test our hypothesis, this research intends to establish stable resistance cell lines from lung cancer A549 cells and H460 cells through cisplatin cyclical induced in vitro culture. And then we will explore whether the over expression of ABCG2 is seen in drug resistance in non-small cell lung cancer cells through the Real- time PCR and Western Blot. At the same time, we will observe whether there is difference of the cell civision patterns between the parental cells and resistant cells using PKH26 dye marking observation of a single cell division. Furthermore, we will establish stable ABCG2-overexpressing and stable shRNA-ABCG2-knock-down cell lines to evaluate the potential role of ABCG2 in cancer cell division. The successful implementation of this study, it will further deepen the understanding of the role of ABCG2 on drug resistance in non small cell lung cancer, and provide some theoretical basis to prevent lung cancer recurrence.Methods1. Isolation and identification of cisplatin-resistant A549/CisR cells and H460/CisR cells separately from parental A549 cells and parental H460 cells: Cisplatin-resistant(CisR) variants of each cell line were derived from each original parental(PT) cell line by continuous exposure to cisplatin(Sigma-Aldrich, UK) following initial dose-response studies of cisplatin(0.1 mM–100 mM) over 72 h from which IC50 values were obtained. Initially, each CisR subline was treated with cisplatin(IC50) for 72 h. The media was removed and cells were allowed to recover for a further 72 h. This development period was carried out for approximately 6 months, after which time IC50 concentrations were re-assessed in each resistant cell line. A significant fold increase of IC50 was observed between parental cells and cisplatin-resistant cells, this means we established the cisplatin-resistant non-small cell lung cancer cell successfully.2. Investigate the expression of ABCG2 and cell division pattern among A549 cells, A549/CisR cells, H460 cells and H460/CisR cells: Real-time PCR and Western-blot were carried out to detect the expression pattern of ABCG2 among A549 cells, A549/CisR cells, H460 cells and H460/CisR cells. Then PKH-26 staining was used to deterimine the cell division pattern of A549 cells, A549/CisR cells, H460 cells and H460/CisR cells.3. Establishment of stable ABCG2-overexpressing or shRNA-ABCG2-knockdown cell lines:A549 cells were transfected with the recombinant lentivirus of ABCG2 or control lenti-virus(GFP-lentivirus), which was constructed by GeneChem Co. Ltd.(Shanghai, China). To establish A549/CisRshABCG2 cells, A549/CisR cells were transfected with SMART vector shRNA lentiviral particles targeted against the ABCG2 genewith 5 μg/ml of Polybrene(Sigma-Aldrich) to inhibit ABCG2 gene expression. An empty SMART vector expressing Turbo GFP was used as control. The stable shRNA-ABCG2 cell line, A549/CisRshABCG2, was established and the expression of ABCG2 was evaluated by Western-blot and PCR.4. Explore the role of ABCG2 in regulating the pattern of self-renewing divisions in cisplatin-resistant non-small cell lung cancer cell lines: PKH-26 staining and was used to deterimine the cell division pattern of A549 cells、A549/ABCG2 cells、A549/GFP cells、A54/CisR cells、A549/CisRshABCG2 cells and A549/CisRshGFP cells.Results1. In A549 cells, the IC50 concentration of cisplatin-resistant cells was determined to be 43.01 μM compared to 6.14 μM in the original parental cell line; a 7-fold increase in the concentration of cisplatin was required to obtain a 50% inhibition in cell growth. A significant increase in IC50 concentrations was also observed in H460/CisR cells(29.19 vs. 4.01 μM), indicating a 7.28-fold increase in the H460/CisR cell lines compared to the parental cells. Taken together, the results demonstrated a cisplatin-resistant phenotype in two NSCLC cell lines following continuous exposure to cisplatin in vitro.2. The level of expression of ABCG2 in the A549/CisR cells was higher than in the parental cells. Similar results were also observed in H460/CisR cells. Moreover, a higher level of expression of ABCG2 mRNA was observed in A549/CisR cells and H460/CisR cells as compared to the parental cells. Moreover, the pattern of cell division in A549 parental cells was 73.9% asymmetric, 16.7% symmetric, and 9.4% undefined(according to PHK-26 staining which could not be classified by PKH-26 dye). The pattern of cell division in H460 parental cells was similar to the pattern of A549 parental cells, including 82.2% asymmetric, 9.5% symmetric and 8.3% undefined.The pattern of cell division in A549/CisR cells was 77.2% symmetric, 10.1% asymmetric and 12.7% undefined. The pattern of cell division in H460/CisR cells comprised of 73.9% symmetric, 13.3% asymmetric and 12.8% undefined. In summary, these results suggest that symmetric and asymmetric divisions co-exist in cisplatin-resistant NSCLC cells and the parental cells, but with different proportions. Thus, parental NSCLC cells mainly divide asymmetrically, whereas cisplatin-resistant NSCLC cells mainly divide symmetrically.3. To determine the relationship between ABCG2 and cell division in cisplatin-resistant NSCLC cells, we transfected the recombinant lentivirus of ABCG2 into A549 cells to establish a stable ABCG2-overexpressing cell line(A549/ABCG2). The results of Western-blot and real-time quantification PCR showed that the expression of ABCG2 in A549/ABCG2 cells was clearly increased. Moreover, PKH-26 staining showed that a significantly greater proportion of A549/ABCG2 cells(81.7%) divided symmetrically than A549 parental cells(16.7%). These data indicate that ABCG2 overexpression increases the potential for symmetric division in A549 cells.4. To further confirm that the characteristics of ABCG2 described above could regulate cell division in NSCLC cells, we transfected A549/CisR cells with SMART vector shRNA lentiviral particles targeted against ABCG2 to inhibit expression of the gene. The level of expression of ABCG2 in A549/CisRshABCG2 cells was significantly lower than A549/CisR cells. Furthermore, PKH-26 staining showed that a significantly lower percentage of A549/CisRshABCG2 cells(11.1%) divided symmetrically than A549/CisR cells(77.2%). These findings, in addition to the observation that ABCG2 overexpression increased the percentage of symmetric division, indicate that ABCG2 regulates the pattern of cell self-renewing divisions in cispl-atin-resistant NSCLC cell lines.Conclusions1. We established a cisplatin-resistant phenotype in two NSCLC cell lines following continuous exposure to cisplatin in vitro. A significant fold increase of IC50 was observed between parental cells and cisplatin-resistant cells.2. The level of expression of ABCG2 in the cisplatin-resistant NSCLC cells was higher than in the parental NSCLC cells. and parental NSCLC cells mainly divide asym-metrically, whereas cisplatin-resistant NSCLC cells mainly divide symmetrically.3. ABCG2 could regulate the pattern of cell self-renewing divisions in cisplatin-resistant NSCLC cell line. Overexpression of ABCG2 in A549 parental cells significantly increased the proportion of symmetric division, whereas knockdown of ABCG2 in drug-resistant A549 cells significantly increased the proportion of asymmetric division.