ATP-binding Cassette Sub-family G Member 2 Expression Research Articles

Investigation of MRP1 and ABCG2 Gene Expression in Chronic Myeloid Leukemia (CML) Patients

Background: This study evaluated and compared the quantitative expression of multidrug resistance-associated protein 1 (MRP1) and ATP-binding cassette subfamily G member 2 (ABCG2), two Multidrug Resistance (MDR) related genes, in 30 CML patients and 27 normal subjects. Methods: Total RNA was isolated from peripheral blood mononuclear cells (MNCs) using the Trizol reagent. Then cDNAs were synthesized. Gene expression was quantified using Real-Time PCR System. The relative expression of target genes was calculated using the 2-ΔΔCt method. Results: High expression of MRP1 and ABCG2 mRNAs were detected in the patient group. Intra-group comparisons also revealed increased expression of ABCG2 in Accelerated Phase (AP)-Blastic Crisis (BC) patients compared to Chronic Phase (CP) patients. At the same time, the increased expression of MRP1 in AP-BC patients was not statistically significant. Conclusion: Considering the broad spectrum of ATP Binding Cassette (ABC) transporter superfamily substrates, they can play an essential role in cell fate determination. High expression of MRP1 and ABCG2 genes can result in the efflux of therapeutic agents and subsequent reduction in their intracellular concentration. This mechanism finally protects cells from the therapeutic effects of medications. On the other hand, these transporters can export growth factors out of the cell. Such exported molecules may have a growth-inducing effect on adjacent cells. These are the possible mechanisms for the participation of MRP1 and ABCG2 genes in conferring drug resistance to CML cells.

Elevated Production of Mitochondrial Reactive Oxygen Species via Hyperthermia Enhanced Cytotoxic Effect of Doxorubicin in Human Breast Cancer Cell Lines MDA-MB-453 and MCF-7.

Hyperthermia (HT) treatment is a noninvasive cancer therapy, often used with radiation therapy and chemotherapy. Compared with 37 °C, 42 °C is mild heat stress for cells and produces reactive oxygen species (ROS) from mitochondria. To involve subsequent intracellular accumulation of DOX, we have previously reported that the expression of ATP-binding cassette sub-family G member 2 (ABCG2), an exporter of doxorubicin (DOX), was suppressed by a larger amount of intracellular mitochondrial ROS. We then hypothesized that the additive effect of HT and chemotherapy would be induced by the downregulation of ABCG2 expression via intracellular ROS increase. We used human breast cancer cell lines, MCF-7 and MDA-MB-453, incubated at 37 °C or 42 °C for 1 h to clarify this hypothesis. Intracellular ROS production after HT was detected via electron spin resonance (ESR), and DOX cytotoxicity was calculated. Additionally, ABCG2 expression in whole cells was analyzed using Western blotting. We confirmed that the ESR signal peak with HT became higher than that without HT, indicating that the intracellular ROS level was increased by HT. ABCG2 expression was downregulated by HT, and cells were injured after DOX treatment. DOX cytotoxicity enhancement with HT was considered a result of ABCG2 expression downregulation via the increase of ROS production. HT increased intracellular ROS production and downregulated ABCG2 protein expression, leading to cell damage enhancement via DOX.

Regulation of ABCG2 expression by Wnt5a through FZD7 in human pancreatic cancer cells.

ATP-binding cassette subfamily G member 2 (ABCG2), a member of the ABC transporter superfamily, has been implicated in the development of chemotherapeutic drug resistance in cancer cells. However, the regulators of ABCG2 expression and their roles in anticancer drug resistance have not been fully characterized, especially in the context of pancreatic cancer. The aim of the present study was to investigate whether ABCG2 contributed to drug resistance in pancreatic cancer and to elucidate its regulatory molecular pathways. Using immunohistochemical analysis of pancreatic ductal adenocarcinoma and adjacent healthy tissue samples, the present study identified a positive correlation between ABCG2 and Wnt5a, a member of the Wnt family of secreted proteins. It was also determined that treatment with recombinant human Wnt5a protein could upregulate the expression of ABCG2 in the Capan-2 human pancreatic cancer cell line and enhance its resistance to gemcitabine. The upregulation of ABCG2 by Wnt5a was inhibited by small interfering RNA silencing of Frizzled class receptor 7 (FZD7) or by FZD7 inhibitors. Moreover, both FZD7 silencing or inhibition of its function attenuated gemcitabine resistance induced by Wnt5a in Capan-2 cells. Therefore, the present findings suggested that Wnt5a and FZD7 acted as upstream regulators of ABCG2 expression and that FZD7 may be an essential factor for Wnt5a-induced gemcitabine resistance in pancreatic cancer cells.

SATP‑binding cassette subfamilyG member2 enhances the multidrug resistance properties of human nasal natural killer/Tcell lymphoma side population cells.

Extranodal natural killer (NK)/T cell lymphoma, nasal type (ENKL) is a rare type of non-Hodgkin's lymphoma that is associated with limited effective treatment options and unfavorable survival rate, which is partly the result of multidrug resistance (MDR). The presence of side population (SP) cells-SNK-6/ADM-SP (SSP) cells has been previously used to explore mechanisms of drug resistance. ATP-binding cassette subfamily G member 2 (ABCG2) is a gene involved in MDR and is closely associated with SPs. However, the function of ABCG2 in SSP cells is unclear. The present study verified the high expression of ABCG2 in SSP cells. The IC50 values of doxorubicin, cytarabine, cisplatin, gemcitabine and l-asparaginase were tested to evaluate drug sensitivity in SSP cells with different levels of ABCG2 expression. ABCG2 was identified as a gene promoting in MDR. ABCG2 upregulated cell proliferation, increased clonogenicity, increased invasive ability and decreased apoptosis, in vivo and in vitro, when cells were treated with gemcitabine. To conclude, ABCG2 enhanced MDR and increased the typical biological characteristics associated with cancer cells in SP cells. With further investigation of the ABCG2 gene could have the potential to reverse MDR in ENKL.

SOX9 enhances sorafenib resistance through upregulating ABCG2 expression in hepatocellular carcinoma

Sorafenib is a multi-kinase blocker and one of the few suggested drug treatments for aggressive hepatocellular carcinoma (HCC) patients. However, drug resistance to sorafenib may often occur over time and cause further tumor aggression. Recently, cancer stem cells were found in HCC and were speculated to be involved in tumor progression. SOX9 is highly expressed in HCC cancer stem cells and promotes cell proliferation and self-renewal. Meanwhile, HCC patients with higher SOX9 expression show poorer prognosis. Whether SOX9 is involved in sorafenib resistance in HCC is still unclear. Here, we found that sorafenib treatment increased the proportion of SOX9 positive cells in HCC cell lines. Overexpression of exogenous SOX9 in HCC increased sorafenib resistance both in vitro and in vivo, whereas down-regulation led to inhibition of sorafenib resistance. Knock-down of SOX9 by RNA interference caused down-regulation of downstream genes, including ATP binding cassette subfamily G member 2 (ABCG2). The drug resistance to sorafenib caused by SOX9 overexpression could be ameliorated by ABCG2 inhibition in HCC cell lines. In the cohort of patients taken sorafenib, we found that patients with lower SOX9 expression had more prolonged overall survival (OS) and progression-free survival (PFS). Univariate and multivariate Cox analysis shows that SOX9 expression exerts as an independent risk factor for the OS and PFS of HCC patients with sorafenib treatment. These findings demonstrate that SOX9 enhances sorafenib resistance and may regulate this process by modulating ABCG2 expression.

GPER mediates decreased chemosensitivity via regulation of ABCG2 expression and localization in tamoxifen-resistant breast cancer cells

Rescue chemotherapy is usually the preferred treatment for patients with advanced estrogen receptor-positive (ER+) breast cancer with endocrinotherapy resistance. However, these patients often simultaneously show a poor response to cytotoxic drugs, and thus the detailed mechanism of this resistance needs to be further investigated. Our previous research indicated that the G-protein-coupled estrogen receptor (GPER) is a novel mediator of the development of multidrug resistance, including resistance to both endocrinotherapy and chemotherapy, and ATP binding cassette subfamily G member 2 (ABCG2) has been identified as an engine that confers cancer cells with chemoresistance by expelling xenobiotics and chemotherapeutics. Here, we are the first to show that the expression levels of GPER and ABCG2 are markedly increased in tamoxifen-resistant ER + metastases compared to the corresponding primary tumors. A plasma membrane expression pattern of GPER and ABCG2 was observed in patients with metastases. Furthermore, both ER modulator tamoxifen, GPER-specific agonist G1 and pure ER antagonist ICI 182,780 significantly enhanced ABCG2 expression in tamoxifen-resistant breast cancer cells (MCF-7R) but not in tamoxifen-sensitive cells (MCF-7). The activated downstream GPER/EGFR/ERK and GPER/EGFR/AKT signaling pathways were responsible for regulating the expression and cell membrane localization of ABCG2, respectively, in MCF-7R cells. Interestingly, the above phenomenon could be alleviated by inhibitors of both the indicated signaling pathways and by knockdown of GPER in MCF-7R cells. More importantly, the tamoxifen-induced GPER/ABCG2 signaling axis was shown to play a pivotal role in the development of chemotherapy (doxorubicin) resistance both in vitro and in vivo. The clinical data further revealed that tamoxifen-resistant patients with high GPER/ABCG2 signaling activation had poor progression-free survival (PFS) when given rescue anthracycline chemotherapy. Therefore, our data provide novel insights into GPER-mediated chemoresistance and provide a rationale for the GPER/ABCG2 signaling axis being a promising target for reversing chemoresistance in patients with advanced ER + tamoxifen-resistant breast cancer.

Itraconazole Alters the Stem Cell Characteristics of A549 and NCI-H460 Human Lung Cancer Cells by Suppressing Wnt Signaling.

BackgroundCancer stem cells (CSCs) behave as their malignant counterparts, but persist after treatment, and possess properties that allow them to interact with their environment. Itraconazole, an antifungal agent, also has a role in suppressing tumor progression, but its effects in regulating tumor cell stemness remain unclear. This study aimed to evaluate the effects of itraconazole on A549 and NCI-H460 human lung cancer cell stemness in vitro.Material/MethodsA549 and NCI-H460 human lung cancer cells and BEAS-2B normal bronchial epithelial cells were cultured with and without itraconazole. Cell viability was evaluated. The expression of stem cell markers, CD133, ATP binding cassette subfamily G member 2 (ABCG2), and aldehyde dehydrogenase 1 (ALDH1), were measured by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Sphere-forming cells were evaluated in vitro.ResultsItraconazole reduced the expression of stemness molecules CD133, ABCG2, and ALDH1 in A549 and NCI-H460 human lung cancer cells, and the numbers of sphere-forming cells were reduced. However, itraconazole had little effect on cell viability but enhanced the chemosensitivity of A549 and NCI-H460 cells. Itraconazole inhibited Wnt signaling. Re-activation of Wnt signaling restored itraconazole-mediated inhibition on A549 and NCI-H460 cell stemness.ConclusionsItraconazole altered the stemness characteristics of A549 and NCI-H460 human lung cancer cells by suppressing Wnt signaling but did not affect cell viability.

Single-nucleotide polymorphisms in a short basic motif in the ABC transporter ABCG2 disable its trafficking out of endoplasmic reticulum and reduce cell resistance to anticancer drugs

ATP-binding cassette (ABC) subfamily G member 2 (ABCG2) belongs to the ABC transporter superfamily and has been implicated in multidrug resistance of cancers. Although the structure and function of ABCG2 have been extensively studied, little is known about its biogenesis and the regulation thereof. In this study, using mutagenesis and several biochemical analyses, we show that the positive charges in the vicinity of the RKR motif downstream of the ABC signature drive trafficking of nascent ABCG2 out of the endoplasmic reticulum (ER) onto plasma membranes. Substitutions of and naturally occurring single-nucleotide polymorphisms within these positively charged residues disabled the trafficking of ABCG2 out of the ER. A representative ABCG2 variant in which the RKR motif had been altered underwent increased ER stress-associated degradation. We also found that unlike WT ABCG2, genetic ABCG2 RKR variants have disrupted normal maturation and do not reduce accumulation of the anticancer drug mitoxantrone and no longer confer resistance to the drug. We conclude that the positive charges downstream of the ABC signature motif critically regulate ABCG2 trafficking and maturation. We propose that single-nucleotide polymorphisms of these residues reduce ABCG2 expression via ER stress-associated degradation pathway and may contribute to reduced cancer drug resistance, improving the success of cancer chemotherapy.

Moderate hypoxia modulates ABCG2 to promote the proliferation of mouse spermatogonial stem cells by maintaining mild ROS levels

The aim of this study was to investigate the effects of different oxygen (O2) concentrations on the growth of mouse spermatogonial stem cells (SSCs) and the possible mechanisms of cell proliferation in vitro. The SSCs from testicular cells were cultured in various O2 concentrations (1%, 2.5%, 5%, and 20% O2) for 7 days. Colonies of SSCs were identified morphologically and by immunofluorescence. The number of mouse SSC colonies and the area covered by them were measured. Cell cycle progression of the SSCs was analyzed to identify the state of cell proliferation. The effects of O2 concentrations on the levels of intracellular reactive oxygen species (ROS) and expression of ATP binding cassette subfamily G member 2 (ABCG2) were also analyzed in the SSCs. Following culturing for 7 days, the SSCs were treated with Ko143 (a specific inhibitor of ABCG2) for 1 h, and the ROS level and expression of bcl-2, bax, and p53 were analyzed. The results showed that mouse SSCs formed compact colonies and had unclear borders in different O2 concentrations for 7 days, and there were no major morphologic differences between the O2 treatment groups. The expression of the SSC marker, GFR α1 was studied in each O2 treatment group. The number and area of SSC colonies, and the number of GFR α1 positive cells were the highest in the 2.5% O2 treatment group. Compared with other O2 concentrations, the number of cells in G0 cycle was significantly higher, while the level of intracellular ROS was lower at 1% O2. Moreover, the intracellular ROS levels gradually increased with increasing O2 concentration from 1% to 20%. The expression of ABCG2 in the SSCs cultured at 2.5% O2 was higher than in the other O2 groups. Inhibition of ABCG2 increased intracellular ROS generation, and the expression of the pro-apoptotic genes bax and p53, and decreased the expression of the anti-apoptotic gene bcl-2. In conclusion, moderate to low O2 tension increases ABCG2 expression to maintain mild ROS levels, triggers the expression of the anti-apoptotic genes, suppresses the proapoptotic gene pathway, and further promotes the proliferation of mouse SSCs in vitro.

Sox9 Enhances Sorafenib Resistance Through Upregulating ABCG2 Expression in Hepatocellular Carcinoma

Sorafenib is a multikinase blocker and the only suggested drug treatment for aggressive hepatocellular carcinoma (HCC) patients. However, drug resistance to sorafenib often occurs after a period of time and cause further tumor aggression. Cancer stem cells are recently found in HCC and regarded to be involved in this process. Sox9 is highly expressed in HCC cancer stem cells and promotes cell proliferation and self-renewal. Meanwhile, HCC patients with higher Sox9 expression show poorer prognosis. Whether Sox9 is involved in sorafenib resistance in HCC is still unclear. Here, we found that sorafenib treatment increased Sox9 expression in HCC cell lines. Overexpression of exogenous Sox9 in HCC increased sorafenib resistance both in vitro and in vivo, and such phenomenon could be inhibited when Sox9 was down-regulated. We checked the down-stream gene expression profile through Sox9 RNA interference and found ATP binding cassette subfamily G member 2 (ABCG2) expression was closely affected by Sox9. Furthermore, we overexpressed Sox9 in combination with ABCG2 inhibition in HCC cell lines, and confirmed that the drug resistance to sorafenib could be ameliorated. In the cohort of patients who took sorafenib, we found those with lower Sox9 expression had longer OS and PFS. Cox analysis shows that Sox9 expression is an independent risk factor of HCC and logistic regression analysis reveals that Sox9 expression, tumor capsule deficiency, tumor diameters and microvascular invasion are risk factors of prognosis of HCC patients. Conclusion: These findings demonstrate that Sox9 enhances sorafenib resistance of HCC and meanwhile Sox9 may regulate such process through modulating ABCG2 expression. Funding: Natural Science Foundation of China. Declaration of Interest: We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled ”Sox9 enhances sorafenib resistance through upregulating ABCG2 expression in hepatocellular carcinoma”. Ethical Approval: The animal experiments in this study conformed to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and were approved by the Institutional Animal Care and Use Committee of Second Military Medical University (Shanghai,China).

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. ATP-binding 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 transcription-quantitative polymerase chain reaction and western blotting. SP cells sorted from MM cell lines, including NCI-H929 cells, via fluorescence-activated cell sorting, exhibited CSC-like phenotypes and expressed high levels of ABCG2. Expression of ABCG2 and activation of the phosphatidylinositol 3-kinase (PI3K)/AKT serine/threonine kinase (AKT) signaling pathway was positively associated with the proportion of SP cells in the NCI-H929 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.

Silencing growth hormone receptor inhibits estrogen receptor negative breast cancer through ATP-binding cassette sub-family G member 2

Growth hormone receptor (GHR) plays a vital role in breast cancer chemoresistance and metastasis but the mechanism is not fully understood. We determined if GHR could be a potential therapeutic target for estrogen receptor negative (ER−ve) breast cancer, which are highly chemoresistant and metastatic. GHR was stably knocked down in ER-ve breast cancer cells and its effect on cell proliferation, metastatic behavior, and chemosensitivity to docetaxel (DT) was assessed. Microarray analysis was performed to identify potential GHR downstream targets involved in chemoresistance. GHR and ATP-binding cassette sub-family G member 2 (ABCG2) overexpression and knockdown studies were performed to investigate the mechanism of GHR-induced chemoresistance. Patient-derived xenografts was used to study the effect of GHR and ABCG2. Immunohistochemical data was used to determine the correlation between GHR, pAKT, pmTOR, and ABCG2 expressions. GHR silencing drastically reduced the chemoresistant and metastatic behavior of ER-ve breast cancer cells and also inhibited AKT/mTOR pathway. In contrast, activation, or overexpression of GHR increased chemoresistance and metastasis by increasing the expression and promoter activity, of ABCG2. Inhibition of JAK2/STAT5 signaling repressed GHR-induced ABCG2 promoter activity and expression. Further, ABCG2 knockdown significantly increased the chemosensitivity. Finally, patient-derived xenograft studies revealed the role of GHR in chemoresistance. Overall, these findings demonstrate that targeting GHR could be a novel therapeutic approach to overcome chemoresistance and associated metastasis in aggressive ER-ve breast cancers.

Protein Arginine Methyltransferase 3 Enhances Chemoresistance in Pancreatic Cancer by Methylating hnRNPA1 to Increase ABCG2 Expression.

Pancreatic cancer is poorly responsive to chemotherapy due to intrinsic or acquired resistance. Our previous study showed that epigenetic modifying enzymes including protein arginine methyltransferase 3 (PRMT3) are dysregulated in gemcitabine (GEM)-resistant pancreatic cancer cells. Here, we attempt to elucidate the role of PRMT3 in chemoresistance. Overexpression of PRMT3 led to increased resistance to GEM in pancreatic cancer cells, whereas reduction of PRMT3 restored GEM sensitivity in resistant cells. We identified a novel PRMT3 target, ATP-binding cassette subfamily G member 2 (ABCG2), which is known to play a critical role in drug resistance. PRMT3 overexpression upregulated ABCG2 expression by increasing its mRNA stability. Mass spectrometric analysis identified hnRNPA1 as a PRMT3 interacting protein, and methylation of hnRNPA1 at R31 by PRMT3 in vivo and in vitro. The expression of methylation-deficient hnRNPA1-R31K mutant reduced the RNA binding activity of hnRNPA1 and the expression of ABCG2 mRNA. Taken together, this provides the first evidence that PRMT3 methylates the RNA recognition motif (RRM) of hnRNPA1 and promotes the binding between hnRNPA1 and ABCG2 to enhance drug resistance. Inhibition of PRMT3 could be a novel strategy for the treatment of GEM-resistant pancreatic cancer.

Aspirin enhances the sensitivity of hepatocellular carcinoma side population cells to doxorubicin via miR-491/ABCG2.

Objective: To explore whether aspirin (ASA) enhances the sensitivity of hepatocellular carcinoma (HCC) side population (SP) cells to doxorubicin (Doxo) via miR-491/ATP-binding cassette sub-family G member 2 (ABCG2).Methods: Non-SP and SP cells were isolated from MHCC-97L cell line using flow cytometry analysis and fluorescence-activated cell sorting. Colony formation assay was performed to determine the colony-formation ability of cells. Cell viability of SP cells was determined with the MTT assay. Luciferase reporter assay was applied in confirming the binding between miR-491 and ABCG2.Results: Although the Doxo treatment lowered the colony-formation ability of both non-SP and SP cells, the colony-formation ability of SP cells was 2-fold higher than that of non-SP cells (P<0.05). Doxo slightly inhibited the cell viability of SP cells in a concentration-dependent manner; the addition of ASA dramatically enhanced the inhibitory effect of Doxo on SP cell viability in a concentration-dependent manner (P<0.05). Compared with non-SP cells, the miR-491 expression was significantly decreased in SP cells, which was significantly reversed by ASA (P<0.05). miR-491 directly controlled the ABCG2 expression. In the presence of Doxo, miR-491 inhibitor reduced the inhibitory effect of ASA on the cell viability of SP cells, which was significantly reversed by knockdown of ABCG2 (P<0.05).Conclusion: ASA enhanced the sensitivity of SP cells to Doxo via regulating the miR-491/ABCG2 signaling pathway.

SDF-1/CXCR4 axis promotes the growth and sphere formation of hypoxic breast cancer SP cells by c-Jun/ABCG2 pathway

ATP-binding cassette sub-family G member 2 (ABCG2) confers to the major phenotypes of side population (SP) cells, the cancer stem-like cells. In this study, the SP cells displayed a distinctly higher ABCG2 expression level, sphere formation efficiency (SFE) and growth rate even under hypoxia condition. CXCR4 overexpression by pcDNA-CXCR4 transfection robustly increased ABCG2 expression, and promoted SFE and growth of hypoxic SP cells, while CXCR4 inhibitor AMD3100 could suppress the promotion. Additionally, we found that CXCR4 promoted the expression of c-Jun, a major gene in the oncogenic JNK/c-Jun pathway. Our data on electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays both showed that c-Jun directly bound with the ABCG2 promoter sequence. Moreover, overexpression of JNK/c-Jun promoted ABCG2 expression, SFE, and growth of hypoxic SP cells and the promotion could be rescued by c-Jun inhibitor SP600125. In conclusion, CXCR4 increases the growth and SFE of breast cancer SP cells under hypoxia through c-Jun-mediated transcriptional activation of ABCG2.

Protective role of ABCG2 against oxidative stress in colorectal cancer and its potential underlying mechanism.

Colorectal cancer (CRC), one of the most common cancer types worldwide, is associated with a high mortality rate, and oxidative stress and inflammation play a vital role in this malignancy. A previous study by our group indicated that ATP binding cassette subfamily G member 2 (ABCG2) was capable of protecting cells from reactive oxygen species (ROS)‑mediated cell damage and death. In the present study, the role of ABCG2 in CRC was investigated by using clinical samples and cell lines. The levels of oxidative stress markers and inflammatory factors were increased, while the levels of antioxidants were decreased in CRC tissues or the serum of patients. Notably, high expression of ABCG2 in CRC tissues which may be the feedback of over-oxidative reaction, was associated with a poor prognosis. Further invitro study indicated that the downregulation of ABCG2 induced ROS generation and inflammatory reactions, and inhibited the production of antioxidants. The nuclear factor (NF)-κB signaling pathway was activated under oxidative stress induced by ABCG2 knockdown. In conclusion, the present results indicated that ABCG2 may relieve oxidative stress and inflammatory response by inhibiting the NF-κB signaling pathway in cell models, and may thus play a potential protective role in CRC. This information may provide novel theoretical mechanisms and future targets for CRC therapy.

Combined effects of octreotide and cisplatin on the proliferation of side population cells from anaplastic thyroid cancer cell lines.

Anaplastic thyroid cancer (ATC) represents the most aggressive subtype of thyroid cancer and has a poor prognosis. In addition to surgery, chemotherapy is an important treatment for ATC; however, the therapeutic effects of current chemotherapies for ATC are not particularly promising. There is a high proportion of side population (SP) cells in ATC, which may be a reason for its drug resistance. In the present study, the antitumor activities of combined octreotide (OCT) and cisplatin (DDP) on the proliferation and apoptosis of ATC SP cells were evaluated. First, SP cells from 8305C and BHT101 cell lines were detected and sorted. Following in vitro culture for 1 week, cluster of differentiation (CD)44, CD133, ATP-binding cassette (ABC) subfamily B member 1 (ABCB1), ABC subfamily G member 2 (ABCG2) and somatostatin receptor expression was detected to characterize the SP cells. An MTT assay was performed to investigate the combined effects on 8305C-SP cell proliferation in vitro, and a mouse model was used to investigate the combined effects on 8305C-SP cell proliferation in vivo. Annexin V/propidium iodide staining was used to investigate the combined effects on 8305C-SP cell apoptosis. Chemotherapeutic drug resistance-associated protein expression and apoptosis-associated protein expression were also detected following combined treatment. As a result, SP cells were identified in 8305C and BHT101 cells, and the proportion of 8305C-SP cells was increased compared with that of BTH101-SP cells. SP cells have enhanced proliferation, tumorigenicity and drug resistance compared with main population cells. The combined treatment of OCT with DDP suppressed the proliferation of 8305C-SP cells in vitro and in vivo, and induced 8305C-SP cell apoptosis. Combined treatment decreased the ABCB1 and ABCG2 expression by SP cells and activated mitochondrial apoptotic signaling, resulting in cell apoptosis. In conclusion, these data support the hypothesis that combined treatment with OCT and DDP induces ATC cell apoptosis and suppresses cell proliferation. These data provide a theoretical basis for further combined chemotherapy clinical applications.

Abstract 893: Hyperthermia regulates both SLC22A16 expression and ABCG2 expression via ROS production to enhance the cytotoxicity of doxorubicin

Abstract Introduction: Hyperthermia (HT) is a non-invasive cancer therapy. Treatment temperature between 41°C to 44°C has no cytotoxic damage in normal cells, however shows cytotoxicity in cancer cells because of the underdeveloped vascular system. HT often used with other cancer therapy such as radiation therapy and chemotherapy. However mechanism of synergistic effect using these therapies remains unclear. Compared to 37°C, 42°C is mild heat stress for cells, thus superoxide anion is released from tissue. Superoxide anion is produced by mitochondrial electron transport chain. Reactive oxygen species (ROS), produced by mild heat stress, can be released from mitochondria. We have previously reported that ATP-binding cassette sub-family G member 2 (ABCG2) expression was suppressed by increasing mitochondrial ROS, andinduction of the cancer specific porphyrin accumulation. ABCG2 is a transporter of doxorubicin (DOX), therefore we hypothesized that synergistic effect of HT and chemotherapy would be induced by down-regulation of ABCG2 expression via intracellular ROS increase. In this study, we investigated if cytotoxic effect of breast cancer cell using DOX can be enhance by HT via intracellular ROS increase. Materials and methods: The murine breast cancer cell line, 4T1E was incubated at 37°C or 42°C for 1h. Intracellular ROS generation after HT treatment was detected by electron spin resonance (ESR). Twenty four hours after HT treatment, cells were incubated in medium containing 0, 0.1 and 1 μM DOX for 24 h. Cell viability was measured using the Cell Counting Kit 8, a water-soluble tetrazolium-8 based colorimetric assay. ABCG2 expression in whole cells was analyzed by Western blotting. Results and discussion: ESR signal peak with HT treatment became high as compared to without HT treatment, indicating intracellular ROS level was increased by HT treatment. Cell viability and ABCG2 expression were decreased by DOX exposure and by HT treatment. The enhancement of HT treatment effect by DOX is considered to be result of down-regulation of ABCG2 expression by ROS. Conclusion: HT treatment involved intracellular ROS production and down-regulated the expression of ABCG2 protein. HT treatment also enhanced the cell damage by DOX. Citation Format: Hirofumi Matsui. Hyperthermia regulates both SLC22A16 expression and ABCG2 expression via ROS production to enhance the cytotoxicity of doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 893.

Expression of CD44, CD44v9, ABCG2, CD24, Bmi-1 and ALDH1 in stage I and II oral squamous cell carcinoma and their association with clinicopathological factors.

Cancer stem cells (CSCs) exhibit self-replication, self-differentiation, drug resistance and immune evasion activities. In recent years CSCs have become increasingly important for the treatment of malignant tumors. CSCs express specific markers, including cluster of differentiation (CD)44, CD44 variant 9 (CD44v9), ATP-binding cassette sub-family G member 2 (ABCG2), CD24, B lymphoma Mo-MLV insertion region 1 homolog (BMI-1) and aldehyde dehydrogenase 1 (ALDH1). However, the prognostic value of their expression in patients with oral squamous cell carcinoma (OSCC) are not well known. The present study evaluated these markers in stage I and II patients with OSCC and examined the association between T classification, histological differentiation, classification of invasion mode, lymph node metastasis and disease-free survival rate. Tissue specimens were obtained from 70 patients with stage I or II OSCC following either surgery or biopsy. Immunohistochemistry was performed and positive staining was defiend as 10% positive cells. CD44 and CD44v9 expressions were strongly detected in all OSCC tissues compared with normal epithelial cells. A total of 22 (31.4%) cases expressed ABCG2 and there was a significant association between ABCG2 expression and invasion. A total of 41 cases (59.0%) expressed CD24 and there was a significant association between CD24 expression and invasion. A total of 33 cases (47.1%) expressed BMI-1 and there was a significant association between BMI-1 expression and the disease-free survival rate. A total of 18 cases (25.7%) expressed ALDH1. Although there was no association between ALDH1 expression and T classification, there were significant associations between ALDH1 expression and histological differentiation, invasion mode, metastasis and the disease-free survival rate. Multivariate analysis revealed that ALDH1 expression was the only prognostic factor for disease-free survival rate. The results of the present study suggest that the positivity of ALDH1 detected in patients with OSCC correlates with the number of cells undergoing epithelial mesenchymal transition and metastasis. These findings indicated that the expression of ALDH1 may be an effective prognostic marker indicating the survival of patients with stage I and II OSCC.

AB108. P082. Role of epigenetic modifying enzymes in the chemoresistance of pancreatic cancer

: Gemcitabine (GEM) is a chemotherapeutic drug and the standard treatment option for pancreatic cancer patients. Although some patients respond well in the treatment firstly, development of resistance is commonly observed in clinic. To dissect the mechanism underlying GEM resistance, we screened for the epigenetic modifying enzymes that are increased in GEM-resistant pancreatic cancer cells and identified 21 upregulated enzymes in resistant cells. We first addressed the functional role of a H3K9 methyltransferase EHMT2 and found that ectopic expression of EHMT2 in pancreatic cancer cells increased GEM resistance while inactivation of EHMT2 in resistant cancer cells reduced it. Mechanistically, we showed that interleukin-8 (IL-8) is one of the downstream effectors of EHMT2 to increase GEM resistance. EHMT2-overexpressing cancer cells exhibited autocrine IL-8/CXCR1/2 stimulation to prevent GEM cytotoxicity which could be attenuated by anti-IL-8 antibody or chemical inhibitor. IL-8 released by cancer cells also activated pancreatic stellate cell (PSC) to increase GEM resistance. Combination of EHMT2 inhibitor and GEM significantly decreased tumor growth, metastasis, IL-8 expression and PSC activation in animals. Expanding from our previous study, we also found the upregulation of protein arginine methyltransferase 3 (PRMT3) in GEM-resistant pancreatic cancer cells. By using proteomics approach, we identified a number of PRMT3-interacting proteins. One candidate gene possibly involves in the chemoresistance is the transporter ATP binding cassette subfamily G member 2 (ABCG2). In this study, we will introduce how PRMT3 modulates the expression of ABCG2 via a novel post-transcriptional mechanism. Our results suggest that PRMT3 could be a potential target to overcome GEM resistance.