Protective Autophagy in 5-Fluorouracil-Resistant Colorectal Cancer Cells and ERK-RSK-ABCG2 Linkage / Autofagia Protectora en Células de Cáncer Colorrectal Resistentes al 5-Fluorouracilo y Enlace ERK-RSK-ABCG2

SUMMARY: To evaluate the anti-cancer effects of yeast extract on resistant cells, autophagy and necroptosis were investigated in 5-fluorouracil (5-FU)-resistant colorectal cancer cells. Further underlying characteristics on drug resistance were evaluated, focused on ERK-RSK-ABCG2 linkage. SNU-C5 and 5-FU resistant SNU-C5 (SNU-C5/5-FUR) colorectal cancer cells were adopted for cell viability assay and Western blotting to examine the anti-cancer effects of yeast extract. Yeast extract induced autophagy in SNU-C5 cells with increased Atg7, Atg12-5 complex, Atg16L1, and LC3 activation (LC3-II/LC3-I), but little effects in SNU-C5/5-FUR cells with increased Atg12-5 complex and Atg16L1. Both colorectal cancer cells did not show necroptosis after yeast extract treatment. Based on increased ABCG2 and RSK expression after yeast extract treatment, drug resistance mechanisms were further evaluated. As compared to wild type, SNU-C5/5-FUR cells showed more ABCG2 expression, less RSK expression, and less phosphorylation of ERK. ABCG2 inhibitor, Ko143, treatment induces following changes: 1) more sensitivity at 500 mM 5-FU, 2) augmented proliferation, and 3) less phosphorylation of ERK. These results suggest that protective autophagy in SNU-C5/5-FUR cells with increased ABCG2 expression might be candidate mechanisms for drug resistance. As the ERK responses were different from each stimulus, the feasible mechanisms among ERK-RSK-ABCG2 should be further investigated in 5-FU-resistant CRC cells.

Para evaluar los efectos anticancerígenos del extracto de levadura en células resistentes, se investigaron la autofagia y la necroptosis en células de cáncer colorrectal resistentes al 5-fluorouracilo (5-FU). Además se evaluaron otras características subyacentes de la resistencia a los medicamentos centrándose en el enlace ERK-RSK-ABCG2. Se usaron células de cáncer colorrectal SNU-C5 (SNU-C5/5-FUR) resistentes a SNU-C5 y 5- FU para el ensayo de viabilidad celular y la transferencia Western para examinar los efectos anticancerígenos del extracto de levadura. El extracto de levadura indujo autofagia en células SNU-C5 con mayor activación de Atg7, complejo Atg12-5, Atg16L1 y LC3 (LC3-II/LC3-I), pero pocos efectos en células SNU-C5/5-FUR con aumento de Atg12-5 complejo y Atg16L1. Ambas células de cáncer colorrectal no mostraron necroptosis después del tratamiento con extracto de levadura. Se evaluaron los mecanismos de resistencia a los medicamentos. en base al aumento de la expresión de ABCG2 y RSK después del tratamiento con extracto de levadura.En comparación con las de tipo salvaje, las células SNU-C5/5-FUR mostraron más expresión de ABCG2, menos expresión de RSK y menos fosforilación de ERK. El tratamiento con inhibidor de ABCG2, Ko143, induce los siguientes cambios: 1) más sensibilidad a 5-FU 500 mM, 2) proliferación aumentada y 3) menos fosforilación de ERK. Estos resultados sugieren que la autofagia protectora en células SNU-C5/5-FUR con mayor expresión de ABCG2 podría ser un mecanismo candidato para la resistencia a los medicamentos. Como las respuestas de ERK fueron diferentes de cada estímulo, los mecanismos factibles entre ERK-RSK- ABCG2 deberían investigarse más a fondo en células CCR resistentes a 5-FU.

Correlation between the mRNA levels of BCRP and LUNX genes and pathological types and stages of patients with non-small cell lung cancer / 中华医学遗传学杂志

OBJECTIVE@#To analyze the correlation between the mRNA levels of breast cancer resistance protein (BCRP) and lung-specific X protein (LUNX) genes with pathological types and stages of patients with non-small cell lung cancer (NSCLC) and their significance for prognosis.@*METHODS@#Eighty nine patients with NSCLC admitted to Huaihe Hospital of Henan University between June 2015 and June 2018 were recruited, with 55 patients with benign lung lesions admitted during the same period of time selected as the control group. The mRNA levels of BCRP and LUNX genes were detected in the peripheral blood samples from the two groups, and their correlation with the clinicopathological characteristics and prognosis of the patients was analyzed.@*RESULTS@#The expression rates of BCRP and LUNX mRNA in the NSCLC group were significantly higher compared with the control group (P < 0.05). The level of BCRP mRNA of the NSCLC patients has correlated with the degree of differentiation and TNM staging (P < 0.05), but not with gender, age, smoking, pathological types and lymph node metastasis (P > 0.05). The level of LUNX mRNA of them has correlated with the degree of differentiation, TNM staging and lymph node metastasis (P < 0.05), but not with gender, age, smoking, and pathological types (P > 0.05). Compared with those with no expression, the overall survival rate of patients with BCRP and LUNX expression was significantly lower (P < 0.05). The degree of differentiation, TNM staging, lymph node metastasis, and expression of the BCRP and LUNX mRNA may all affect the prognosis of the patients.@*CONCLUSION@#The levels of BCRP and LUNX mRNA in the peripheral blood of patients with NSCLC are significantly increased. The expression of BCRP mRNA is correlated with the degree of differentiation and TNM staging, whilst the expression of LUNX mRNA is correlated with the differentiation degree, TNM staging and lymph node metastasis. Both may be used as independent predictors for the prognosis of patients with NSCLC.

A review: drug-drug interactions of epithelial growth factor receptor-tyrosine kinase inhibitors / 中华肿瘤杂志

Mutations in the epithelial growth factor receptor (EGFR) is a driving factor that causes non-small cell lung carcinoma (NSCLC). The epithelial growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is a crucial discovery in the treatment of lung cancer, particularly the efficacy of EGFR-TKIs is superior to that of the standard chemotherapy for patients with EGFR mutation-positive advanced NSCLC. Patients with NSCLC use EGFR-TKIs and other medications simultaneously is commonly seen, especially among those with comorbidities, which increases the risk of drug-drug interactions (DDIs) of EGFR-TKIs. The most common mechanisms underlying the DDIs of EGFR-TKIs are modulations of cytochrome P450 (CYP) and drug transporters [including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP)], as well as gastrointestinal acid-inhibitory drugs [proton pump inhibitors (PPIs) and H(2) receptor antagonists (H(2)RA)]. Inhibitors or inducers of CYP enzymes and drug transporters can inhibit or accelerate the metabolism of EGFR-TKIs, which increase or reduce the exposure of EGFR-TKIs, thereby affect the efficacy and safety of EGFR-TKIs. In addition, PPIs or H(2)RA can decrease the solubility, bioavailability and efficacy of EGFR-TKIs. This review summarizes the mechanisms of DDIs of gefitinib, erlotinib, icotinib, afatinib, dacomitinib and osimertinib; the management recommendations for DDIs of those EGFR-TKIs from the Chinese and global guideline, as well as from the recent pre-clinical and clinical studies, which provide the reference and evidence for managing the combination therapies of EGFR-TKIs and other medications in clinics.

ABCG2 polymorphism, age and leukocyte count may contribute to oral mucositis in oncopediatric patients

Abstract The study investigated the relationship between genetic polymorphisms and the development of oral mucositis in pediatric patients undergoing chemotherapy involving methotrexate. A longitudinal study was conducted with 64 patients, and oral mucositis was evaluated by the modified Oral Assessment Guide, which aims to diagnose and classify oral mucositis. Epithelial cells were obtained by mouthwash and DNA was extracted. The polymorphisms MTHFR (rs1801133), DNMT3B (rs2424913), ABCC2 (rs717620), ABCG2 (rs2231137) and ABCG2 (rs2231142) were analyzed by PCR-RFLP method. Demographic, hematological and biochemical data were collected from medical records. Statistical analysis was performed using the SPSS software adopting a p-value of 0.05. Male sex predominated (56.2%), and the mean age was 10.8 years (± 4.9). Oral mucositis affected 65.6% of the patients, of which 61.9% developed the severe form of the disease. For the ABCG2 gene (rs2231142), the rare A allele and CA genotype were more frequent in individuals with mucositis (p= 0.02; RR = 0.60; CI = 0.387 - 0.813). The severity of the disease was mainly observed in younger patients (median = 9 years; p=0.02). Patients with severe oral mucositis presented lower leukocytes count (median = 2.150 mm3) compared to patients with the mild/moderate form (median = 4.200 mm3; p=0.03). Female patients and each 10,000-platelet increase were protective factors against the onset of oral mucositis (p=0.02). It is concluded that rs2231142 polymorphism increases the likelihood of oral mucositis and younger patients and patients with low leukocytes counts are more likely to develop severe form.

Resumo O presente estudo investigou a relação entre cinco polimorfismos genéticos e o desenvolvimento de mucosite oral em pacientes pediátricos recebendo quimioterapia com metrotexato. O estudo longitudinal foi conduzido com 64 pacientes e a mucosite oral avaliada pelo Oral Assessment Guide modificado, que tem como objetivo diagnosticar e classificar a mucosite oral. Células epiteliais bucais foram obtidas por bochecho e o DNA foi extraído. Os polimorfismos MTHFR (rs1801133), DNMT3B (rs2424913), ABCC2 (rs717620), ABCG2 (rs2231137) e ABCG2 (rs2231142), foram analisados pela técnica de PCR-RFLP. Dados demográficos, hematológicos e bioquímicos foram coletados a partir de registros médicos. Análise estatística foi realizada utilizando o software SPSS adotando um valor de p=0,05. Observou-se que, o sexo masculino foi predominante (56,2%), e a idade média foi de 10,8 anos (± 4.9). A mucosite oral acometeu 65,6% dos pacientes, dos quais, 61,9% desenvolveram a forma grave da doença. Para o gene ABCG2 (rs2231142), o alelo raro A e o genótipo CA foram mais frequentes em indivíduos com mucosite (p= 0.02; RR = 0.60; CI = 0.387 - 0.813). A gravidade da doença foi observada principalmente em pacientes mais jovens (mediana = 9 anos; p=0.02). Além disso, os pacientes com mucosite oral grave apresentaram menor contagem de leucócitos (mediana = 2150 mm3) em comparação aos pacientes com a forma leve/moderada (mediana = 4200 mm3; p=0.03). Pacientes do sexo feminino e aumento a cada 10.000 plaquetas foram fatores de proteção contra o aparecimento de mucosite oral (p=0.02). Concluiu-se que a presença do polimorfismo rs2231142 aumenta o risco de o paciente desenvolver a mucosite oral, bem como pacientes mais jovens e menor contagem de leucócitos contribui com a severidade.

Establishment of Cytarabine-resistant Acute Lymphoblastic Leukemia Cell Lines and Its Resistance Mechanism / 中国实验血液学杂志

OBJECTIVE@#To establish cytarabine-resistant acute lymphoblastic leukemia (ALL) cell lines and investigate its possible resistant mechanism.@*METHODS@#Low-concentration cytarabine (Ara-C) continuously induced and cultured Jurkat and Nalm-6 cells to construct cytarabine-resistant cell lines Jurkat/Ara-C and Nalm-6/Ara-C. The cell viability was detected by CCK-8 assay, and the distribution of cell cycle was detected by flow cytometry. Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of multidrug resistant gene and Ara-C metabolic enzymes. The expression levels of cyclin were detected by Western blot.@*RESULTS@#Jurkat/Ara-C and Nalm-6/Ara-C drug-resistant cell lines were successfully established, the resistance index of which was 1 973.908±161.163 and 7 231.643± 1 190.624, respectively. Drug-resistant cell lines had no cross-resistance to commonly used chemotherapeutic drugs, such as doxorubicin. Flow cytometry showed that the ratio of G@*CONCLUSION@#Cytarabine-resistant ALL cell lines are successfully established by using low concentration continuous induction method, and its drug-resistant mechanism may be related to the deficiencies of DCK and cyclinB1.

Effect of ABCG2 on Side Population Cells and Related Pathways in Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To investigate the biological characteristics of ABCG2 and its effect on side population cells (SP cells) of multiple myeloma (MM) so as to find the way for reversing drug resistance.@*METHODS@#The silence of ABCG2 expression was performed throngh interfering the MM cells by using siRNA. then the ratio of SP cell of MM, speed of cell proliferation and sensitivity of cells to chermotherapentic drugs before and after interference were compared, the biological functions of ABCG2 and its role in regulation of SP cells and PTEN/PI3K/AKT signaling pathway was clarified.@*RESULTS@#siRNA interference could down regulate the expression of ABCG2 at both mRNA and protein level. After siRNA interference, the proliferation of multiple myeloma cells was decreased slightly, the expression of PTEN was increased, the activity of PI3K/AKT pathway was inhibited, and the ratio of SP cells was decreased.@*CONCLUSION@#In multiple myeloma, down-regulation of expression ABCG2 can negatively regulate the expression of PTEN, the PI3K/AKT signaling pathway is inhibited,the ratio of SP decreased,and the response to drug is increased.

Evidence of progenitor cells in the adult human cochlea: sphere formation and identification of ABCG2

OBJECTIVES: The aim of this study was to search for evidence of stem or progenitor cells in the adult human cochlea by testing for sphere formation capacity and the presence of the stem cell marker ABCG2. METHODS: Cochleas removed from patients undergoing vestibular schwannoma resection (n=2) and from brain-dead organ donors (n=4) were dissociated for either flow cytometry analysis for the stem cell marker ABCG2 or a sphere formation assay that is widely used to test the sphere-forming capacity of cells from mouse inner ear tissue. RESULTS: Spheres were identified after 2-5 days in vitro, and the stem cell marker ABCG2 was detected using flow cytometric analysis after cochlear dissociation. CONCLUSIONS: Evidence suggests that there may be progenitor cells in the adult human cochlea, although further studies are required.

Silencing of ABCG2 by MicroRNA-3163 Inhibits Multidrug Resistance in Retinoblastoma Cancer Stem Cells

To investigate the function and regulation mechanism of ATP-binding cassette, subfamily G, member 2 (ABCG2) in retinoblastoma cancer stem cells (RCSCs), a long-term culture of RCSCs from WERI-Rb1 cell line was successfully established based on the high expression level of ABCG2 on the surface of RCSCs. To further explore the molecular mechanism of ABCG2 on RCSCs, a microRNA that specifically targets ABCG2 was predicted. Subsequently, miR-3163 was selected and confirmed as the ABCG2-regulating microRNA. Overexpression of miR-3163 led to a significant decrease in ABCG2 expression. Additionally, ABCG2 loss-of-function induced anti-proliferation and apoptosis-promoting functions in RCSCs, and multidrug resistance to cisplatin, carboplatin, vincristine, doxorubicin, and etoposide was greatly improved in these cells. Our data suggest that miR-3163 has a significant impact on ABCG2 expression and can influence proliferation, apoptosis, and drug resistance in RCSCs. This work may provide new therapeutic targets for retinoblastoma.

The effect of MDR1 (P-gp) and ABCG2 on the drug resistance in Hep 2 cells / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To study the effect of MDR1 (P-gp) and ABCG2 on the drug resistance in Hep 2 cells.@*METHOD@#Flow cytometry was used to detect the variations of the antitumor drugs accumulation and discharging, and activity variations when MDR1 and ABCG2 inhibitors were used in Hep-2.@*RESULT@#The accumulation and discharging of mitoxantrone was significantly higher than the control group when ABCG2 inhibitor FTC was used in Hep-2 (P<0. 05). In contrast, P-gp did not appear similar case; To the mitoxantrone and cisplatin, there was no statistical correlation about activity of Hep-2 between P-gp or ABCG2 antagonist and the control; To the doxorubicin, combining FTC and P-gp, the activity of Hep-2 was higher than the control and difference was significant (P<. 05), In contrast, FTC and P-gp did not appear similar case when used alone; To the 5-FU, when PGP used, the activity of Hep-2 was higher than that in the control and difference was significant (P<0. 05), In con- trast, FTC and FTC+P-gp did not appear similar case; To the paclitaxel, when P-gp or FTC+P-gp used, the activity of Hep-2 was higher than that in the control and difference was significant(P<0. 05).@*CONCLUSION@#ABCG2 may lead to drug resistance mainly by changing the ability of cell in accumulating and discharging chemotherapy drugs. P-gp has other way. P-gp and ABCG2 play different roles in different drug resistance.

Effect of Ursolic Acid on Breast Cancer Resistance Protein-mediated Transport of Rosuvastatin In Vivo and Vitro / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To evaluate whether ursolic acid can inhibit breast cancer resistance protein (BCRP)-mediated transport of rosuvastatin in vivo and in vitro.</p><p><b>METHODS</b>Firstly, we explored the pharmacokinetics of 5-fluorouracil (5-FU, a substrate of BCRP) in rats in the presence or absence of ursolic acid. Secondly, we studied the pharmacokinetics of rosuvastatin in rats in the presence or absence of ursolic acid or Ko143 (inhibitor of BCRP). Finially, the concentration-dependent transport of rosuvastatin and the inhibitory effects of ursolic acid and Ko143 were examined in Madin-Darby Canine Kidney (MDCK) 2-BCRP421CC (wild type) cells and MDCK2-BCRP421AA (mutant type) cells.</p><p><b>RESULTS</b>As a result, significant changes in pharmacokinetics parameters of 5-FU were observed in rats following pretreatment with ursolic acid. Both ursolic acid and Ko143 could significantly affect the pharmacokinetics of rosuvastatin. The rosuvastatin transport in the BCRP overexpressing system was increased in a concentration-dependent manner. However, there was no statistical difference in BCRP-mediated transport of rosuvastatin betweent the wild type cells and mutant cells. The same as Ko143, ursolic acid inhibited BCRP-mediated transport of rosuvastatin in vitro.</p><p><b>CONCLUSION</b>Ursolic acid appears to be a potent modulator of BCRP that affects the pharmacokinetic of rosuvastatin in vivo and inhibits the transport of rosuvastatin in vitro.</p>

Expression of ABCG2 and p-glycoprotein in residual breast cancer tissue after chemotherapy and their correlation with epithelial-mesenchymal transition / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the expression of breast cancer resistance protein (ABCG2), p-glycoprotein (P-gp) in residual breast cancer tissue after chemotherapy and their correlation with epithelial mesenchymal transition (EMT).</p><p><b>METHODS</b>Seventy-six cases of breast cancer were collected. The expression of ABCG2, P-gp and EMT markers E-cadherin and vimentin in residual breast cancer tissue after chemotherapy was detected by immunohistochemistry (EnVision method). MCF7 cells were divided into three group:untreated control group, positive control (TGF-β1 induced) group and drug surviving cells (DSC) group (selected viable MCF7 cells after docetaxel and epirubicin treatment). The expression of EMT markers E-cadherin and vimentin was detected by immunofluorescence. The mRNA and protein expression of ABCG2, P-gp and EMT markers were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively.</p><p><b>RESULTS</b>Compared with breast cancer tissue before chemotherapy, ABCG2, P-gp and vimentin protein were highly expressed in residual breast cancer tissue after chemotherapy. The expression of ABCG2 and P-gp correlated positively with vimentin protein (r1=0.97, P1=0.000; r2=0.83, P2=0.001) and negatively with E-cadherin protein (r3=-0.55, P3=0.010; r4=-0.43, P4=0.020) expression. RT-PCR results showed that ABCG2, P-gp and vimentin mRNA were highly expressed in residual breast cancer tissue after chemotherapy. The expression of ABCG2 and P-gp mRNA correlated positively with vimentin mRNA (r1=0.99, r2=0.96, P<0.05) but negatively with E-cadherin protein (r3=-0.99, r4=-0.98, P<0.05); Western blot showed that ABCG2, P-gp and vimentin protein were highly expressed in residual breast cancer tissue after chemotherapy. The expression of ABCG2 and P-gp protein correlated positively with vimentin protein (r1=0.98, r2=0.89, P<0.05) and negatively with E-cadherin protein (r3=-0.47, r4=-0.33, P<0.05).</p><p><b>CONCLUSIONS</b>The expression of resistance-associated proteins in the residual breast cancer tissue after chemotherapy is significantly correlated with EMT. The expression of EMT profile may be one of important mechanisms for multidrug resistance in breast cancer.</p>

The drug-drug interaction mediated by efflux transporters and CYP450 enzymes / 药学学报

Multidrug regimens and corresponding drug interactions cause many adverse reactions and treatment failures. Drug efflux transporters: P-glycoprotein (P-gp), multidrug resistance associated protein (MRP) and breast cancer resistance protein (BCRP) in conjunction with metabolizing enzymes (cytochrome P450, CYP450) are major factors in such interaction. In recent years, a large number of studies have shown that P-gp plays a role in the oxidative metabolism of its substrates that are also substrates of CYP3A4. Combined actions of P-gp and CYP3A could account in some part for the low oral bioavailability determined for many of these dual substrates. P-gp along with efflux transporters (MRP and BCRP) having overlapping substrate specificity plays critical role in drug disposition. The relationship between MRP or BCRP and CYP3A is similar to that between P-gp and CYP3A. In this paper, we summarize the classification of efflux transporters, the main metabolizing enzymes CYP3A, clinical significance interactions mediated by efflux transporters and CYP450 enzymes and in vitro studies.

Correlation of MDR1 and ABCG2 genetic polymorphisms with the efficacy and adverse events of irinotecan chemotherapy in patients with colorectal cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the correlation of MDR1 and ABCG2 genetic polymorphisms with the efficacy and adverse events of irinotecan chemotherapy in patients with colorectal cancer (CRC).</p><p><b>METHODS</b>Clinical data of CRC patients treated with irinotecan-based chemotherapy in the Peking University Cancer Hospital between January 1996 and December 2011 were collected, and their blood samples were collected accordingly. Genomic DNA was extracted from blood samples. The following SNP detection of MDR1 and ABCG2 genes was conducted by direct sequencing method. The correlation of genetic SNPs with efficacy and toxicity of irinotecan treatment was further analyzed.</p><p><b>RESULTS</b>Allele frequencies of MDR1 2677 G>T/A, ABCG2 421 C>A, 34 G>A, 376 C>T were comparable with previous studies. Genetic SNPs results from peripheral blood samples and tumor tissues were highly consistent. Patients carrying MDR1 2677 wild type had higher clinical benefit than those carrying mutant genotype, while the differences were not significant. The progression-free survival (PFS) was longer in wild-type patients as compared to mutant-type patients in second-line chemotherapy (P=0.012). There were no significant correlations between ABCG2 421 C>A, 34 G>A, 376 C>T and chemotherapy efficacy. No significant correlations were observed between MDR1 2677 G>T/A, ABCG2 421 C>A, ABCG2 34 G>A, ABCG2 376 C>T and irinotecan-related grade 3 and 4 neutropenia or diarrhea.</p><p><b>CONCLUSION</b>MDR1 2677 G>T/A may be served as a biomarker in predicting the efficacy of irinotecan chemotherapy in patients with colorectal cancer.</p>

Metformin induces apoptosis in hepatocellular carcinoma Huh-7 cells in vitro and its mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>to investigate the effects of antidiabetic drug metformin on proliferation and apoptosis in human hepatocellular carcinoma cell line Huh-7 cells.</p><p><b>METHODS</b>Huh-7 cells were treated with metformin at different concentrations. Cell viability was determined by MTT assay. Cell apoptosis and CD133(+) expression rate were detected by flow cytometery (FCM). Expressions of PTEN, Akt, p-Akt, Bcl-2, Bax proteins in the cells were measured by Western blot. The effect of metformin on the hepatosphere formation was observed in the serum-free suspension culture. Reverse transcription-polymerase chain reaction (RT-PCR) was used to validate the expression levels of stemness marker genes CD133, β-catenin, and ABCG2 mRNA in the hepatospheres.</p><p><b>RESULTS</b>The proliferation of Huh-7 cells was inhibited by metformin in a dose- and time-dependent manner. The early and late cell apoptosis rates induced by metformin at dose of 10 mmol/L for 48 hrs were (22.29 ± 0.8)% and (13.87 ± 1.2)%, respectively, and 25 mmol/L for 48 hrs (15.28 ± 2.1)% and (25.89 ± 2.3)%, respectively. Western blotting results revealed that the expression of CD133, phosphorylated Akt and the Bcl-2/Bax ratio were downregulated, and PTEN was upregulated in the Huh-7 cells after treated with 25 mmol/L metformin for 48 hrs. Metformin inhibited the formation of hepatospheres. Metformin also downregulated the expression of several cancer stem cells (CSCs)-related genes which are involved in the signaling pathways governing the self-renewal, proliferation and differentiation of CSCs in the hepatospheres.</p><p><b>CONCLUSIONS</b>Metformin inhibits the proliferation of human hepatocellular carcinoma Huh-7 cells and enhances their apoptosis in vitro. It may be related to the downregulation of PI3K/Akt signal pathway and selectively targeting CD133(+) cells.</p>

Effect of 5-fluorouracil on the expression of ATP-binding cassette superfamily G member 2 in human colon cancer cell SW480 / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of 5-fluorouracil (5-FU) on the expression of ATP-binding cassette superfamily G member 2 (ABCG2) in human colon cancer cell SW480.</p><p><b>METHODS</b>SW480 cells were treated with various concentrations of 5-FU. CCK8 assay was utilized to detect the 5-FU IC50 to SW480 cells. Positive expression of ABCG2 was detected by flow cytometry, and mRNA expression of ABCG2 was detected by real time polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The 5-FU IC50 to SW480 cells increased as the drug concentration increased (P<0.05). Flow cytometry revealed that positive expression rate of ABCG2 in normal SW480 cells (group A) was (6.26±0.86)%. Immediately after treatment with 5-FU for 48 hours, the positive expression rate of ABCG2 (group B) was (3.43±1.18)% (P<0.05). In the second passage of cells after treatment with 5-FU for 48 hours, the positive expression rate of ABCG2 (group C) was (12.91±3.42)% (P<0.05). The mRNA expression of ABCG2 detected by RT-PCR was in accordance with the results from flow cytometry.</p><p><b>CONCLUSION</b>Expression of ABCG2 in SW480 cells can be affected by various concentrations of 5-FU.</p>

Expression of breast cancer resistance protein and p-glycoprotein in residual breast cancer tissue after chemotherapy and its correlation with cancer stem cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To compare the expression differences of breast cancer resistance protein(BCRP/ABCG2) and P-glycoprotein(P-gp) in breast cancer tissue before chemotherapy and in residual breast cancer tissue, and to explore its correlation with breast cancer stem cells.</p><p><b>METHODS</b>Immunohistochemistry was used to detect the expression of ABCG2, P-gp, and breast cancer stem cells(BCSCs) markers(CD44 and CD24) in breast cancer tissue before chemotherapy and residual breast cancer tissue after chemotherapy. Immunofluorescence was applied for determination of the CD44 and CD24 protein expressions of BCSCs microspheres cells. The monoclone-forming ability of BCSCs microspheres cells was detected by limited dilution assay. The expressions of ABCG2, P-gp, CD44, and CD24 proteins were detected by Western blot.</p><p><b>RESULTS</b>Compared with those in breast cancer tissue before chemotherapy, the expression levels of ABCG2 and P-gp were positively correlated with the expression level of CD44 protein(Χ(2)=41.34, r=0.83;Χ(2)=22.81, r=0.61) in residual breast cancer tissue after chemotherapy;meanwhile, they were negatively correlated with the expression of CD24 protein(Χ(2)=-21.25, r=0.72;Χ(2)=-17.26, r=0.65) (all P<0.05) .The diameter of BCSCs microspheres were increased significantly after chemotherapy.The content of BCSCs increased by about 2.5 times after chemotherapy.The expressions of ABCG2, P-gp and CD44 proteins significantly increased and that of CD24 protein significantly declined(P<0.05) .</p><p><b>CONCLUSION</b>Chemotherapy endows residual breast cancer tissue with cancer stem cells-like features, leading to multidrug resistance of breast cancer.</p>

Preparation and characterization of irinotecan hydrochloride loaded PEO-PPO-PEO micelles and its mechanism of decreasing drug intestinal toxicity / 药学学报

In this work, we developed PEO-PPO-PEO micelles loaded with irinotecan hydrochloride (CPT-11) using breast cancer resistance protein (BCRP) inhibitory material PEO20-PPO70-PEO20, and studied its mechanism of decreasing CPT-11 induced delayed diarrhea and intestinal toxicity. BCRP-overexpressing MDCKII (MDCKII/BCRP) cells were used to evaluate the effect of PEO20-PPO70-PEO20 and PEO-PPO-PEO micelles on transmembrane transport of CPT-11 in vitro. The biliary excretion, delayed diarrhea and intestinal damage of CPT-11 loaded PEO-PPO-PEO micelles of rats were investigated. The results showed that the obtained micelles could decrease the biliary excretion of CPT-11, ameliorate delayed diarrhea and intestinal toxicity of rats through inhibiting BCRP-mediated CPT-11 efflux. PEO-PPO-PEO micelles were promising carriers to reduce intestinal toxicity of CPTs.

Establishment of BCRP expressed pig kidney cell line LLC-PK1/BCRP and its biological profile / 药学学报

To establish a pig kidney cell line LLC-PK1/BCRP in which human breast cancer resistance protein was highly expressed, the expression vector pcDNA3.1(+)-BCRP which contained BCRP gene was constructed and transfected into LLC-PKI cells via liposomes. After selecting with G418, population doubling time, flow cytometry and Western blotting analysis were used to evaluate the cell line. MTT assays were employed to determine the drug resistance index of mitoxantrone and doxorubicin. Invert fluorescent microscope was used to observe the efflux of fluorescence dye Hoechst 33342 by BCRP, furthermore, the BCRP's inhibitor GF120918 was applied to reverse the efflux of Hoechst 33342. The experiment results showed that the expression of BCRP protein increased in LLC-PK1/BCRP cell. The population doubling time of LLC-PK1/BCRP cell was a little longer than that of the parental cell LLC-PK1. The resistance indexes to mitoxantrone and doxorubicin were 51.95 and 6.09 times, respectively, higher than LLC-PK1 cell. The efflux of Hoechst 33342 was significantly enhanced and could be reversed by GF120918. So a LLC-PK1/BCRP cell line was established, which highly expressed BCRP protein successfully. This cell line could be a valuable model to further investigate the biological profile of BCRP and select the substrate and inhibitor of BCRP.

Role of transporters in hepatic drug disposition / 药学学报

Liver is regarded as one of the most important organs for drug clearance in the body, which mediates both the metabolism and biliary excretion of drugs. Transporters are a class of functional membrane proteins and control the movement of substances into or out of cells. Transporters, which are extensively expressed in the liver, play important roles in the drug hepatic disposition by regulating the uptake of drugs from blood into hepatocytes or the efflux of drugs and their metabolites into bile. In this review, the localization, functions and substrate selectivity of the major transporters in the liver will be summarized, and the impacts of these transporters on drug hepatic disposition, the potential drug-drug interactions as well as their genetic polymorphisms will also be reviewed.

Breast cancer resistance protein (BCRP/ABCG2): its role in multidrug resistance and regulation of its gene expression / 癌症

Breast cancer resistance protein (BCRP)/ATP-binding cassette subfamily G member 2 (ABCG2) is an ATP-binding cassette (ABC) transporter identified as a molecular cause of multidrug resistance (MDR) in diverse cancer cells. BCRP physiologically functions as a part of a self-defense mechanism for the organism; it enhances elimination of toxic xenobiotic substances and harmful agents in the gut and biliary tract, as well as through the blood-brain, placental, and possibly blood-testis barriers. BCRP recognizes and transports numerous anticancer drugs including conventional chemotherapeutic and targeted small therapeutic molecules relatively new in clinical use. Thus, BCRP expression in cancer cells directly causes MDR by active efflux of anticancer drugs. Because BCRP is also known to be a stem cell marker, its expression in cancer cells could be a manifestation of metabolic and signaling pathways that confer multiple mechanisms of drug resistance, self-renewal (stemness), and invasiveness (aggressiveness), and thereby impart a poor prognosis. Therefore, blocking BCRP-mediated active efflux may provide a therapeutic benefit for cancers. Delineating the precise molecular mechanisms for BCRP gene expression may lead to identification of a novel molecular target to modulate BCRP-mediated MDR. Current evidence suggests that BCRP gene transcription is regulated by a number of trans-acting elements including hypoxia inducible factor 1α, estrogen receptor, and peroxisome proliferator-activated receptor. Furthermore, alternative promoter usage, demethylation of the BCRP promoter, and histone modification are likely associated with drug-induced BCRP overexpression in cancer cells. Finally, PI3K/AKT signaling may play a critical role in modulating BCRP function under a variety of conditions. These biological events seem involved in a complicated manner. Untangling the events would be an essential first step to developing a method to modulate BCRP function to aid patients with cancer. This review will present a synopsis of the impact of BCRP-mediated MDR in cancer cells, and the molecular mechanisms of acquired MDR currently postulated in a variety of human cancers.