Immunohistochemical analysis of nm23 protein expression in malignant bone tumors

Expression levels of nm23 protein in 72 malignant bone tumors comprising 41 osteosarcomas, 22 chondrosarcomas, 6 Ewing's sarcomas, and 2 malignant fibrous histiocytomas were examined immunohistochemically, using anti-nm23 protein polyclonal antibody, and compared with 51 cases of benign bone tumors or tumor-like lesions. Malignant bone tumors showed significantly higher nm23 protein expression than benign bone tumors or tumor-like lesions (P<0.0001). In chondrosarcoma, nm23 expression increased in high-grade tumors (grade I versus grade II and III:P=0.0229). In the cases of osteosarcoma, however, grade IV osteosarcomas showed decreased expression of nm23 compared with grade III tumors (P=0.0122). There was no significant relationship between nm23 expression and histological type. nm23 expression had no correlation with metastatic potential in osteosarcoma, although the therapy was not uniform in our cases. Furthermore, in 6 cases of osteosarcoma and 1 case of Ewing's sarcoma, there was no clear tendency for a decrease of nm23 in the metastatic sites compared with primary sites, as reported in breast cancer. These results showed that, in contrast to reports on breast cancer and experimental models, nm23 protein expression in human bone tumors may be associated with malignant potentiality, except in cases of osteosarcoma.     

Expression and significance of hypoxia-inducible factor-1 alpha and MDR1/P-glycoprotein in human colon carcinoma tissue and cells

Purpose  

Hypoxia in tumors is generally associated with chemoresistance and radioresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1) and the multidrug resistance (MDR1) gene/transporter P-glycoprotein (P-gp) has not been clearly investigated. This study aims at examining the expression levels of HIF-1α and MDR1/P-gp in human colon carcinoma tissues and cell lines (HCT-116, HT-29, LoVo, and SW480) and ascertaining whether HIF-1α plays an important role in tumor multidrug resistance with MDR1/P-gp.     

Expression of multidrug resistance-associated protein gene in Ewing's sarcoma and malignant peripheral neuroectodermal tumor of bone

The expression of multidrug resistance-associated protein (MRP) mRNA was examined in ten samples of Ewing's sarcoma of bone (ES) and in one nude mice transplantable ES and two malignant peripheral neuroectodermal tumor (MPNT) cell lines using an RT-PCR assay. MRP mRNA expression was recognized in eight of the ten clinical specimen and in all three cell lines. On the other hand, the expression of multidrug resistance gene (MDR1) was demonstrated in three of the ten clinical samples and all three cell lines. Our results may contribute to elucidation of the mechanism of anti-cancer-drug resistance in this tumor.     

MBR rearrangement and P-glycoprotein expression are not independent prognostic factors like p53 protein in malignant lymphoma

Non-Hodgkin‘s lymphomas (NHL) are B-cell malignancies which generally present molecular abnormalities, such as bcl-2 translocation t(14; 18) predominantly in the follicular subgroup. Other molecular events have been described in NHL, including p53 gene mutation and overexpression of one chemoresistance mechanism, the multidrug resistance system, P-glycoprotein (MDR1/P-gp). In this study, we analysed samples from 44 NHL patients with the presence of the bcl-2 major breakpoint region (MBR) rearrangement in 29 and without in 15. Immunochemical analysis revealed that 39 samples were positive for bcl-2 protein expression in tumoral cells (88.6%). Seventeen (38.6%) patients expressed P-gp and 9 (20.5%) expressed p53 proteins. Eleven patients expressed both bcl-2 and P-gp proteins, four expressed bcl-2 and p53 proteins whereas four expressed bcl-2, p53 and P-gp proteins. Our results confirm the importance of p53 expression as a key prognostic factor, and no objective response (OR) was found in patients with p53 positivity. MBR rearrangement was not associated with poor response to chemotherapy (62.1% OR in MBR positive patients v. 60% OR in MBR negative patients). The clinical impact of P-gp cannot be identified because no relationship was observed between P-gp expression and prognosis (58.8% OR in P-gp positive patients v. 63% OR in P-gp negative patients).     

MDR1 and MRP expression in chronic B-cell lymphoproliferative disorders

The role of the MDR1 and MRP genes in drug resistance in patients with chronic lymphocytic leukaemia (CLL)/non-Hodgkin's lymphoma (NHL) is unclear. We hypothesized that any relationship between levels of expression and exposure to P-glycoprotein (P-gp) transportable drugs may become evident by using a measure of gene expression that combined the number of positive cells and the degree of positivity. 68 CLL/NHL patients were analysed using flow cytometry with MDR1 and MRP specific antibodies and were divided into subgroups, untreated ( n  = 31), treated with non P-gp transportable drugs ( n  = 26), those treated with low total doses of P-gp transportable drugs ( n  = 6) and patients treated with high total doses of P-gp transportable drugs ( n  = 5). The group exposed to high doses of P-gp transportable drugs had higher levels of MDR1 expression when compared to all other groups ( P  < 0.05, ANOVA). A positive correlation between the level of MDR1 expression and the cumulative dose of P-gp transportable drugs was demonstrated ( P  = 0.02). MRP expression was higher in those patients exposed to high doses of P-gp transportable drugs when compared to all other groups ( P  < 0.05, ANOVA), although only a trend towards a linear dose correlation effect could be established ( P  = 0.08). We concluded that MDR1 and MRP are involved in drug resistance but only in patients treated with P-gp transportable drugs.     

Drug-induced changes in the expression of MDR-associated genes: Investigations on cultured cell lines and chemotherapeutically treated leukemias

Summary The induced expression of multiple drug resistance (MDR)-associated genes as a direct response of tumor cells to antineoplastic drugs could be an important factor influencing the success of cancer chemotherapy. We investigated the effects of such compounds on mdr1/P-glycoprotein (P-gp) gene expression and drug sensitivities in the T-lymphoblastoid human cell line CCRF-CEM and MDR sublines. Thereby, we observed that actinomycin D or adriamycin administered at sublethal concentrations induced increases of mdr1 mRNA levels and resistance within 72 h. Furthermore, on leukemia cell samples collected before and after chemotherapy we checked by a complementary DNA polymerase chain reaction (cDNA-PCR) approach for similar alterations in the relative expression levels of the MDR-associated genes (a) mdr1/P-gp (b) mrp (MDR related protein), and (c) the topoisomerase II isoforms and. We found a concomitant increase in mdr1 and mrp gene expression combined with a decreased expression of topoisomerase II in the course of the second relapse of an acute lymphoblastic leukemia (ALL). This points to the emergence of at least three different MDR mechanisms in this type of leukemia unresponsive to chemotherapy. A chronic myeloid leukemia (CML) in blast crisis, however, showed combined increases in mdr1 (about 20-fold) and mrp (about four fold) gene expression after intense but unsuccessful chemotherapy over a 6-month period. Our results indicate the occurrence of induced resistance in vitro and in vivo and suggest a contribution of the newly identified ATP-binding cassette (ABC) transporter MRP in MDR. CSG-CR Cooperative Study Group — Cellular Resistance We dedicate the work to: G. Frese     

Effect of the cyclin-dependent kinases inhibitor p27 on resistance of ovarian cancer multicellular spheroids to anticancer chemotherapy

Purpose: A low proliferating fraction in solid tumors limits the effectiveness of cell-cycle-dependent chemotherapeutic agents. To understand the molecular basis of such resistance, we examined the expression of the cyclin-dependent kinases inhibitor p27, and relationship with drug resistance and P-gp expression in ovarian cancer multicellular spheroids. Methods: We cultured ovarian cancer cells (A2780 and CAOV3) as multicellular spheroids and examined the expression of p27 and P-glycoprotein (P-gp) by western blot, flow cytometry and confocal. We also analyzed the cell-cycle distribution by flow cytometry. In addition, trypan blue exclusion testing and cell apoptosis analysis were used to detect the sensitivity to Taxol. Results: When transferred from monolayer to three-dimensional culture, a consistent upregulation of p27 protein and P-gp protein was observed in ovarian cancer cell lines. Compared with monolayer cells, there was a significant increase of G0-G1 phase cells and decrease of S and G2-M phase cells in spheroid cells. Aggregates of cells showed higher cell viability than monolayer cells. Antisense oligodeoxynucleotide (ASON) -mediated downregulation of p27 reduced intercellular adhesion, increased cell proliferation, downregulated P-gp expression and sensitized cells to Taxol. Conclusions: Our results implicate that p27 serves as a regulator of drug resistance in ovarian tumors. ASON-mediated alteration of p27 reverses resistance of ovarian cancer to anticancer agents that are associated with increased sensitivity of ovarian cancer cells to chemotherapeutic agents.Hui Xing and Shixuan Wang have contributed equally in this work     

Immunohistochemical studies on the expression of P-glycoprotein and p53 in relation to histological differentiation and cell proliferation in hepatocellular carcinoma

Localization of P-glycoprotein (P-gp) and p53 was immunohistochemically examined in 41 patients with hepatocellular carcinoma (HCC) in order to determine the relationship between the expression of P-gp and p53 and the degree of histological differentiation or cell proliferation in HCC. P-gp showed different patterns of expression between cancerous and cirrhotic liver hepatocytes, and the expression in cancerous tissue also varied according to the degree of histological differentiation. In cirrhotic liver hepatocytes, expression of P-gp was found on bile canalicular membranes. In the case of cancerous tissue, P-gp was localized on the canalicular membranes in well-differentiated HCC showing a trabecular pattern, as recognized cirrhotic liver hepatocytes. In moderately differentiated HCC showing pseudo-glandular patterns, predominant expression of P-gp was found on the luminal side of cell membranes of the glandular ducts. The P-gp expression rate was 87.5% in well-differentiated HCC, 84% in moderately differentiated HCC, and 37.5% in poorly differentiated HCC, indicating a marked decrease with decreasing degree of differentiation. On the other hand, the rate of mutation of p53, a tumor suppressor gene, was 12.5% in well-differentiated HCC, 52.0% in moderately differentiated HCC, and 85.5% in poorly differentiated HCC, showing a significant increase with decreasing degree of differentiation (P<0.005). The labeling index (LI) of proliferating cell nuclear antigen (PCNA) tended to increase with the progression of chronic liver disease, with a markedly high value of 24.0±1.5% in cases of HCC. The PCNA LI was 15.6±11.9% in well-differentiated HCC, 23.1±15.1% in moderately differentiated HCC, and 50.1±13.3% in poorly differentiated HCC, which indicated a significantly increase in poorly differentiated HCC (P<0.001). Thus, it became apparent that abnormal expressions of P-gp and p53 and the cell proliferation in HCC vary according to the degree of histological differentiation of the malignancy. This suggests that more effective chemotherapy for HCC can be potentially developed by considering the pattern and level of expression of P-gp as a mechanism of drug resistance and the extent of histological differentiation.     

P-glycoprotein expression in adult acute myeloid leukemia: correlation with induction treatment outcome

Drug resistance has become a major cause of the treatment failure in patients with acute leukemia. P-glycoprotein (P-gp), which is associated with multidrug resistance (MDR) phenotype, has been reported to be an important predictor of the treatment outcome. The aim of this study was to analyze the value of P-gp expression in bone marrow cells as a predictor of the response to remission induction chemotherapy, as well as duration of remission in adult patients with newly diagnosed acute myeloid leukemia (AML). We examined the expression of P-gp in 31 patients using the monoclonal antibody UIC2. Direct immunofluorescent labeling was performed and samples were analyzed by flow cytomery. Kolmogorov-Smirnov test (D-value) was used to estimate UIC2 staining. A D > or = 0.3 for labeling of gated leukaemic blasts as compared to that of the isotypic control was defined positive (+) and compared to clinical data. P-gp expression was found in 14/31 (45.6%) patients, 17/31 (54.8%) of the samples were found P-gp negative(-). No correlation was found regarding age, sex and FAB subtype, altough 6/14 (43%) cases with more than 50% of cells having P-gp expression, were CD34+/CD7+. Complete remission rates were significantly lower in UIC2+ patients than in UIC2- cases (70% vs 35%, p < 0.01). Complete remission duration was also shorter in UIC2+ patients (6 vs 12.4 months). Our data indicate, that P-gp expression is a reliable marker of resistance to induction treatment in patients with de novo AML and can help to identify patients who may require alternative regimens designed to overcome therapy resistance.     

The effect of glucosylceramide synthase on P-glycoprotein function in K562/AO2 leukemia drug-resistance cell line

Previous work from our laboratory demonstrated that glucosylceramide synthase (GCS) and multidrug resistance 1 gene (MDR1) are co-overexpressed in drug-resistant leukemia cells. We hypothesized that GCS and MDR1 may interact. In this study, we used RNA interference (RNAi) to silence the GCS or MDR1 gene in K562/AO2 drug-resistant cells. The sensitivity of cells to different treatments with doxorubicin was evaluated. We used Taqman probe fluorescence real-time quantitative PCR, and detected expression of GCS and MDR1 mRNAs in different interfering groups. Intracellular mean fluorescence intensity (MFI), which represents rhodamine123 (rh123) retention, was determined by flow cytometry (FCM). An MTT cytotoxicity assay showed that the 50% inhibition concentration (IC50) of doxorubicin of K562/AO2 cells (138.25 ± 3.75 μg/ml) was significantly higher than that of K562 drug-sensitive cells (2.125 ± 0.125 μg/ml), and that IC50 was evidently lower in K562/AO2 cells, whether it was transfected with a small interfering RNA (siRNA) targeting GCS (GCSsiRNA) or one targeting MDR1 (MDR1siRNA). Compared with untreated K562/AO2 cells, the inhibition rates of GCS mRNA in the cells transfected with GCSsiRNA for 9 and 36 h were 56.67 ± 9.29% (p < 0.05) and 74 ± 6.38% (p < 0.05), respectively. Interestingly, the expression of MDR1 mRNA was also inhibited to 51.7 ± 4.5% (p < 0.05) 36 h after transfection with GCSsiRNA, but there was no significant difference in MDR1 expression at 9 h post-transfection in cells treated with GCSsiRNA and a negative control. It is well known that rh123 retention in cells results from an efflux function of P-glycoprotein (P-gp). In K562 cells, rh123 retention was much higher than in K562/AO2 cells (p < 0.01). We also noted that rh123 retention in the K562/AO2 cells transfected with GCSsiRNA for 48 h was significantly higher than in the negative control group. In conclusion, we show in the present study that inhibition of the GCS gene affects the expression of MDR1 mRNA and P-gp function.     

Expression of P-glycoprotein and p53 in advanced hepatocellular carcinoma treated by single agent chemotherapy: Clinical correlation

Hepatocellular carcinoma (HCC), a chemoresistant tumour, is the most common fatal cancer in Taiwan. Hepatocellular carcinoma frequently expresses a high level of P-glycoprotein (P-gp), which is a specific phenotype of a multidrug-resistance gene, and harbours mutations of the tumour suppressor gene p53. A modulatory relationship between p53 and P-gp has been reported. In this study, we analysed the expression of P-gp in relation to chemotherapeutic response and p53 protein expression in advanced HCC. Prechemotherapeutic tumour samples were obtained from 25 patients with HCC which had been treated with either etoposide (VP-16) or doxorubicin. P-glycoprotein and p53 in HCC were visualized by immunohistochemical staining using the monoclonal antibodies JSB-1 and DO1, respectively. We investigated the correlation of P-gp expression with chemotherapeutic responses, clinicopathological features and p53 protein expression. In our study, seven cases achieved partial remission, and the remaining 18 cases had a poor response to chemotherapy. Expression of P-gp was observed in 13 tumours (52%). Positive P-gp protein expression was significantly associated with non-responders (8% or 1/13 vs 50% or 6/12, P= 0.03). Thus, P-gp expression inversely correlated with chemotherapeutic response. Expression of p53 protein was seen in 12 cases and did not correlate with chemosensitivity or P-gp expression. In summary, P-gp expression correlates with the chemosensitivity of HCC that has been treated with VP-16 or doxorubicin and p53 mutations do not appear to be a major determinant of P-gp expression in advanced HCC.     

Up‐regulation of breast cancer resistance protein expression in hepatoblastoma following chemotherapy: A study in patients and in vitro

Aim: Hepatoblastoma (HB), the most common pediatric malignant liver tumor, is treated with chemotherapy to facilitate surgical resection. Previous studies suggest that HB acquires chemoresistance via increased expression of multidrug resistance protein 1 (MDR1, ABCC1). There is no well established evidence that this also occurs in the clinical setting and little is known about the effects of chemotherapeutic treatments on HB in situ. Methods: Clinical and histopathological features and expression patterns of ABC transporters in diagnostic needle biopsies from 7 HBs taken before chemotherapy were compared with those in surgically resected tumors. To understand the mechanism s leading to chemoresistance we also investigated the involvement of hypoxia on protein expression and functional activity of drug transporters (BCRP and MDR1) in cultures of HepG2 human HB cells. Results: We found that chemotherapeutical treatment of HBs led to an increased expression of the breast cancer resistance protein (BCRP, ABCG2) in all patients studied. There was no change in the expression pattern of MDR1 or other ABC transporters. Chemotherapy‐induced specific vascular abnormalities associated with areas of necrosis and fibrosis were seen in all cases, suggesting tumor hypoxia. The observations of increased BCRP expression in hypoxic areas of three‐dimensional HepG2 aggregates and the enhanced BCRP function in monolayer cultures of HepG2 cells under hypoxic conditions, support a role for hypoxia in enhanced BCRP expression. Conclusions: Chemotherapeutical treatment of HB leads to vascular alterations that modify the tumor microenvironment, and increased BCRP expression in which hypoxia might play a role. No evidence was found for upregulation of MDR1 in HBs as suggested from previous experimental studies.     

P-glycoprotein (P-170) expression in acute leukemias

Multidrug resistance (MDR) is still a major obstacle to chemotherapy success in acute myeloid leukemia (AML) and to a less extent acute lymphoblastic leukemia (ALL). Recent studies have shown that the expression of certain gene products mediate the development of resistance to chemotherapeutic agents. The most well characterized of these genes is the multidrug resistance gene MDR-1. This study was planned to study the expression of P-glycoprotein/170 in patients with acute leukemia and the effect of Cyclosporin A (CSA) as a modulator of P-glycoprotein functional activity. The study was carried out on 20 patients with acute leukemia (14 AML cases and 6 ALL cases). In addition, 6 normal individuals served as a reference group. Flow cytometric analysis of P-gp/170 surface expression was performed using UIC-2 MoAb together with the functional assay using Rhodamine 123 (Rh 123) and Cyclosporin A as a modulator.P-gp/170 was expressed on the leukemic cells of 37.5% of relapsed patients (40.0% of AML and 33.3% of ALL cases), whereas 27.2% of de novo patients expressed P-gp/170 (33.3% of AML cases and 0% of ALL cases). The functional activity of MDR-1 gp was 71.4% in AML and 33.3% in ALL patients compared with16.6% in normal lymphocytes. From this study, it is clear that P-gp/170 is expressed to a higher degree in leukemic cells and this is greater in relapsed compared to de novo cases and more in AML than ALL blasts. Functional activity is a more sensitive predictor of chemoresistance than P-gp/170 surface expression.     

Pharmacogenomics of MDR1/ABCB1 gene: the influence on risk and clinical outcome of haematological malignancies

Pharmacogenomics is a rapidly developing field of biomedical research, which investigates phenotypic and pharmacodynamic consequences of the genetic variations among individuals. The multi-drug resistance-1, MDR1 (ABCB1) gene belongs to ATP-binding cassette (ABC) family and encodes for membrane transporter P-glycoprotein (P-gp). A wide array of P-gp substrates comprises toxic xenobiotics and numerous commonly used medications including anti-cancer drugs. Under physiological conditions P-gp protects cells against toxins, whereas in malignant cells P-gp confers multi-drug resistance phenotype. Moreover, characteristic tissue localisation enables P-gp to influence the uptake, tissue distribution and elimination of P-gp transported drugs. A number of recent studies identified variety of single nucleotide polymorphisms (SNPs) in the MDR1 gene and demonstrated significant ethnic differences in their allelic frequency distribution. Furthermore, it was shown that some of these SNPs, especially silent C3435T polymorphism in exon 26, may alter P-gp expression and transport activity. Consequently, it is likely that specific functional MDR1 haplotypes may result with altered exposure to toxins and drugs, thus influencing predisposition to certain diseases as well as efficacy or toxicity of pharmacotherapy. In this paper, we focus on the available data concerning the impact of MDR1 polymorphism on the risk and clinical outcome of haematological malignancies. The structure and function of P-gp as well as results of studies addressing the relevance of MDR1 polymorphism in non-haematological disorders are also briefly discussed.     

Multidrug resistance after retroviral transfer of the human MDR1 gene correlates with P-glycoprotein density in the plasma membrane and is not affected by cytotoxic selection.

Multidrug resistance (MDR) in mammalian cells is associated with the expression of the MDR1 gene encoding P-glycoprotein (P-gp), an and active efflux pump for various lipophilic compounds. MDR transfectants can be isolated after MDR1 gene transfer and selection with cytotoxic drugs; low levels of drug resistance have also been observed in unselected NIH 3T3 mouse cells after retrovirus-mediated transfer of mouse mdr1 cDNA. MDR cell lines possess multiple phenotypic changes, suggesting that P-gp function could be complemented by some additional mechanisms associated with cytotoxic selection. To determine whether cytotoxic selection contributes to the MDR phenotype of MDR1-expressing cells, NIH 3T3 cells infected with a recombinant retrovirus carrying the human MDR1 gene were selected by two different procedures: (i) noncytotoxic selection for increased P-gp expression on the cell surface by multiple rounds of immunofluorescence labeling and flow sorting or (ii) one or more steps of selection with a cytotoxic drug. The levels of MDR in both types of infectants showed an excellent correlation with the P-gp density in the plasma membrane, expressed as immunoreactivity with a P-gp-specific antibody normalized by reactivity with an antibody against an unrelated antigen. Cytotoxic selection conferred no additional increase in resistance relative to P-gp density. These results indicate that P-gp density in the plasma membrane may be sufficient to determine the level of MDR.     

高温对SGC7901/ADM细胞多药耐药蛋白表达的影响和意义

目的:研究高温43℃对多药耐药基因表达产物P-gp、MRP、LRP在蛋白水平上的影响及其生物学意义,更深入地了解热效应逆转耐药的机制.方法:采用免疫细胞化学染色法、RT-PCR以及Western blot方法,检测在不同温度和不同药物作用下,人胃癌耐药细胞株SGC7901/ADM和对照的人胃癌敏感细胞SGC7901株中,与人胃癌多药耐药相关的分子MDR1、P-gp、MRP、LRP在蛋白水平上的表达差异.结果:采用免疫细胞化学染色法检测人胃癌耐药株SGC7901/ADM细胞,发现高温43℃60 min处理可使P-gp蛋白表达下调率(down-regulated rate,DRR)31.78%(P=0.016),而MRP表达DRR为20.22%(P=0.037),差异有统计学意义,LRP未表达.采用Western blot法在SGC7901细胞中未检测出P-gp蛋白表达,而SGC7901/ADM细胞中P-gp高表达;在ADM和CDDP处理组细胞中,高温43℃时P-gp的表达量较37℃时DRR分别为45.65%(P=0.007)、17.95%(P=0.021),差异有显著学意义;TAX处理组DRR为11.90%,差异无显著学意义(P=0.065).结论:高温43℃的短期处理对人胃癌SGC7901/ADM细胞中P-gp和MRP多药耐药蛋白的表达有一定的抑制作用,可能是逆转耐药的机制之一.