Expression of P-gp, MRP, LRP, GST-π and TopoIIα and intrinsic resistance in human lung cancer cell lines

This study aimed to determine the relationship between the endogenous levels of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathione-s-transferase-π (GST?π) and topoisomerase IIα (TopoIIα) and intrinsic drug resistance in four human lung cancer cell lines, SK-MES-1, SPCA-1, NCI-H-460 and NCI-H-446, of different histological types. The expression of P-gp, MRP, LRP, GST-π and TopoIIα was measured by immunofluorescence, Western blotting and RT-PCR. Drug resistance to cisplatin, doxorubicin and VP-16 was determined using MTT assays. The correlation between expression of the resistance-related proteins and their roles in the resistance to drugs in these cancer cell lines was analyzed. We found that the endogenous levels of P-gp, MRP, LRP, GST-π and TopoIIα in the four cell lines varied. The level of GST-π in the SK-MES-1 cells was the highest, whereas the level of P-gp in the SPCA-1 cells was the lowest. The chemoresistance to cisplatin, doxorubicin and VP-16 in the four cell lines was different. The SPCA-1 cell line was most resistance to cisplatin; SK-MES-1 was most resistance to VP-16; whereas SK-MES-1 was most sensitive to doxorubicin. There was a positive correlation between GST-π expression and resistance to cisplatin, between TopoIIα expression and resistance to VP-16; and a negative correlation was noted between TopoIIα expression and resistance to doxorubicin. In summary, the endogenous expression of P-gp, MRP, LRP, GST-π and TopoIIα was different in the four human lung cancer cell lines of different histological types, and this variance may be associated with the variation in chemosensitivity to cisplatin, doxorubicin and VP-16. Among the related proteins, GST-π may be useful for the prediction of the intrinsic resistance to cisplatin, whereas TopoIIα may be useful to predict resistance to doxorubicin and VP-16 in human lung cancer cell lines.     

Expression of HIF-1α and PTEN in bnman multiple myeloma bone marrow

Objective To investigate HIF-1α and PTEN expression in multiple myeloma(MM).Methods The expression of HIF-1α and PTEN was studied in 28 cases of MM with immunohistochemistry.Results Over-expression of HIF-1α was frequently(20 of 28 cases,71.4%)detected in MM bone marrow.Expression of HIF-1α between MM and control group bone marrow has significant differences(P＜0.01).The positive expression rate of PTEN in MM bone marrow was 42.9%(12 0f 28 cases).Expression of PTEN between MM and control group bone marrow has significant difierences(P＜0.01).There was negative correlation between the expression of HIF-1α protein and the PTEN protein(P＜0.05,r=-0.542).Conclusion The absence or low expression of PTEN and the increased levels of HIF-1α may play a critical role in the formation.development and invasion of MM.HIF-1α and PTEN may be used to estimate the progress of MM.     

Inhibitory effects of epigallocatechin-3-gallate on cell proliferation and the expression of HIF-1α and P-gp in the human pancreatic carcinoma cell line PANC-1

The aim of this study was to verify the inhibitory effects of epigallocatechin-3-gallate (EGCG) on cell proliferation and the expression of hypoxia-inducible factor 1 (HIF-1α) and multidrug resistance protein 1 (MDR1/P-gp) in the human pancreatic carcinoma cell line PANC-1, thereby, reversing drug resistance of pancreatic carcinoma and improving its sensitivity to cancer chemotherapy. The human pancreatic carcinoma cell line PANC-1 was incubated under hypoxic conditions with different concentrations of epigallocatechin-3-gallate (EGCG) for indicated hours. The effects of EGCG on the mRNA or protein expression of HIF-1α and MDR1 were determined by RT-PCR or western blotting. Cellular proliferation and viability assays were measured using Cell Counting Kit-8. Western blotting revealed that EGCG inhibits the expression of the HIF-1α protein in a dose-dependent manner, while RT-PCR showed that it does not have any effects on HIF-1α mRNA. In addition, EGCG attenuated the mRNA and protein levels of P-gp in a dose-dependent manner, reaching a peak at the highest concentration. Furthermore, EGCG inhibited the proliferation of PANC-1 cells in a concentration- and time-dependent manner. The attenuation of HIF-1α and the consequently reduced P-gp could contribute to the inhibitory effects of EGCG on the proliferation of PANC-1 cells.     

Expression of HIF-1α in hepatocellular carcinoma and its relationship with vasculogenic mimicry and clinical pathology

Objective:The aim of this study was to discuss HIF-1αexpression and vasculogenic mimicry (VM) in hepatocel-lular carcinoma (HCC) and their relationship with the clinical pathological features and clinical significance. Methods:Two hundred and seven specimens from patients in The Af iliated Hospital of North Sichuan Medical Col ege who received hepatic cellcarcinoma resection were tested by immunohistochemistry and double staining of CD31 and PSA. Then detected the expression of HIF-1α, VM, and analysed the relationship between clinical pathology. Results:The HIF-1αpositive rate was 71.01%and its expression was associated with liver cirrhosis, tumor size and TNM stage (P<0.05). HIF-1αprotein expres-sion was positively associated with the VM (γ=0.1988, P=0.0041). Conclusion:Hypoxia may be the reason for VM in high invasive HCC, regulating the tumor microenvironment may have great significance in inhibiting invasion and metastasis of HCC.     

Expression and clinical implication of HIF-1α mRNA in mononuclear cells of bone marrow of multiple myeloma

Objective To analyze the correlation between the expression of HIF-1α in multiple myeloma (MM) and clinical indexes in order to illustrate the expression and implication of HIF-1α gene in MM. Methods RQ-PCR method was used to amplify HIF-1α mRNA of bone marrow mononuclear cells isolated from 28 cases of MM patients and the control group. β-actin was used as internal standard. HIF-1α mRNA expression was analyzed by SDS software and the ratios of HIF-1α/ β-actin were calculated. Results The relative expression level of HIF-1α mRNA in MM bone marrow mononuclear cells was 12.68 times as that in control group. HIF-1α mRNA was positively correlated with β2-MG (r =0.575, P =0.000), ESR (r =0.522,P =0.000), LDH (r=0.286, P=0.044) and CRP (r =0.356, P =0.011). There was a negative correlation between HIF-1α mRNA and Hb (r =-0.556, P =0.000). Conclusion The expression of HIF-1α mRNA was up-regulated and HIF-1α was related to a number of clinical indexes. HIF-1α may be used to estimate the progress of MM and hopeful to be a new molecular target in cancer therapy.     

Expression and clinical implication of HIF-1α mRNA in mononuclear cells of bone marrow of multiple myeloma

Objective To analyze the correlation between the expression of HIF-1α in multiple myeloma (MM) and clinical indexes in order to illustrate the expression and implication of HIF-1α gene in MM. Methods RQ-PCR method was used to amplify HIF-1α mRNA of bone marrow mononuclear cells isolated from 28 cases of MM patients and the control group. β-actin was used as internal standard. HIF-1α mRNA expression was analyzed by SDS software and the ratios of HIF-1α/ β-actin were calculated. Results The relative expression level of HIF-1α mRNA in MM bone marrow mononuclear cells was 12.68 times as that in control group. HIF-1α mRNA was positively correlated with β2-MG (r =0.575, P =0.000), ESR (r =0.522,P =0.000), LDH (r=0.286, P=0.044) and CRP (r =0.356, P =0.011). There was a negative correlation between HIF-1α mRNA and Hb (r =-0.556, P =0.000). Conclusion The expression of HIF-1α mRNA was up-regulated and HIF-1α was related to a number of clinical indexes. HIF-1α may be used to estimate the progress of MM and hopeful to be a new molecular target in cancer therapy.     

Expression of Drug Resistance Proteins Pgp, MRP1, MRP3, MRP5 AND GST-π in Human Glioma

Summary Chemotherapy in glioma is poorly effective: the blood–brain barrier and intrinsic and/or acquired drug resistance of tumor cells could partly explain this lack of major effect. We investigated expression of P-glycoprotein (Pgp), multidrug resistance protein (MRP) 1, MRP3, MRP5 and glutathione-S-transferase π (GST-π) in malignant glioma patients. Cytofluorimetric analysis of 48 glioma specimens and 21 primary cultures showed high levels of MRP1, moderate levels of MRP5 and low levels of Pgp, GST-π and MRP3. Immunohistochemistry (25 glioma specimens) showed expression of GST-π (66.7% of cases), MRP1 (51.3%), MRP5 (45.8%), Pgp (34.8%) and MRP3 (29.9%) in tumor cells. Moreover, analysis of tumor samples by real time quantitative PCR showed mRNA expression of all investigated genes. Tumor vasculature, analyzed in glioma specimens and in tumor derived endothelial cells, showed expression of all investigated proteins. Non-tumor brain samples (from a patient with arteriovenous malformation and from one with epilepsy), normal human astrocytes and cultured endothelial cells were also analyzed: astrocytes and endothelial cells expressed the highest levels of the investigated proteins, mainly MRP1 and MRP5. No significant differences in proteins expression were detected between primary or recurrent gliomas, suggesting that glioma chemoresistance is mostly intrinsic. Therefore, we detected, for the first time, the presence of MRP3 and MRP5 on glioma specimens – both in tumor and endothelial cells – and we delineated an expression profile of chemoresistance proteins in glioma. The possible association of inhibitors of drug efflux pumps with chemotherapy could be investigated to improve drugs delivery into the tumor and their cytotoxic effects.     

HIF-1α and GLUT1 Gene Expression is Associated with Chemoresistance of Acute Myeloid Leukemia

Aims: Much evidence suggests that increased glucose metabolism in tumor cells might contribute to thedevelopment of acquired chemoresistance. However, the molecular mechanisms are not fully clear. Therefore,we investigated a possible correlation of mRNA expression of HIF-1α and GLUT1 with chemoresistance in acutemyeloid leukemia (AML). Methods: Bone marrow samples were obtained from newly diagnosed and relapsedAML (M3 exclusion) cases. RNA interference with short hairpin RNA (shRNA) was used to stably silence GLUT1or HIF-1α gene expression in an AML cell line and HIF-1α and GLUT1 mRNA expression was measured byreal-time quantitative polymerase chain reaction assay (qPCR). Results: High levels of HIF-1α and GLUT1 wereassociated with poor responsiveness to chemotherapy in AML. Down-regulation of the expression of GLUT1 byRNA interference obviously sensitized drug-resistant HL-60/ADR cells to adriamycin (ADR) in vitro, comparablewith RNA interference for the HIF-1α gene. Conclusions: Our data revealed that over-expression of HIF-1α andGLUT1 might play a role in the chemoresistance of AML. GLUT1 might be a potential target to reverse suchdrug resistance.     

Prostate cancer cell malignancy via modulation of HIF-1α pathway with isoflurane and propofol alone and in combination

Background:

Surgery is considered to be the first line treatment for solid tumours. Recently, retrospective studies reported that general anaesthesia was associated with worse long-term cancer-free survival when compared with regional anaesthesia. This has important clinical implications; however, the mechanisms underlying those observations remain unclear. We aim to investigate the effect of anaesthetics isoflurane and propofol on prostate cancer malignancy.

Methods:

Prostate cancer (PC3) cell line was exposed to commonly used anaesthetic isoflurane and propofol. Malignant potential was assessed through evaluation of expression level of hypoxia-inducible factor-1α (HIF-1α) and its downstream effectors, cell proliferation and migration as well as development of chemoresistance.

Results:

We demonstrated that isoflurane, at a clinically relevant concentration induced upregulation of HIF-1α and its downstream effectors in PC3 cell line. Consequently, cancer cell characteristics associated with malignancy were enhanced, with an increase of proliferation and migration, as well as development of chemoresistance. Inhibition of HIF-1α neosynthesis through upper pathway blocking by a PI-3K-Akt inhibitor or HIF-1α siRNA abolished isoflurane-induced effects. In contrast, the intravenous anaesthetic propofol inhibited HIF-1α activation induced by hypoxia or CoCl₂. Propofol also prevented isoflurane-induced HIF-1α activation, and partially reduced cancer cell malignant activities.

Conclusions:

Our findings suggest that modulation of HIF-1α activity by anaesthetics may affect cancer recurrence following surgery. If our data were to be extrapolated to the clinical setting, isoflurane but not propofol should be avoided for use in cancer surgery. Further work involving in vivo models and clinical trials is urgently needed to determine the optimal anaesthetic regimen for cancer patients.     

HIF-1α and HSP90: target molecules selected from a tumorigenic papillary thyroid carcinoma cell line

It is important to properly identify aggressive tumors among differentiated thyroid cancers that are most often indolent. By comparison of a tumorigenic clone with an originally less tumorigenic papillary thyroid carcinoma (PTC) cell line, we looked for markers involved in the aggressive biology of thyroid cancer. Human PTC cell lines BHP10-3 and its tumorigenic subclone BHP10-3SC(mice) were compared using microarray analysis. Upregulated genes in the tumorigenic clone were selected for RT-PCR, immunoblot analysis and immunohistochemistry in human tissue. Hypoxia-inducible factor (HIF)-1α and its chaperone protein heat shock protein (HSP)90 showed significantly increased expression in BHP10-3SC(mice) and human PTC tissue. These two genes, HIF-1α and HSP90, were further validated using siRNA gene knockdown, pharmacological inhibition using 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of both HSP90 and HIF-1α and in vivo orthotopic animal model. Invasiveness of BHP10-3SC(mice) was abrogated by blockade of HIF-1αin vitro by both siRNA and 17-AAG. The same finding was demonstrated in the orthotopic animal model. These findings support that HIF-1α is important in tumorigenesis of PTC and that it may serve to be an important target for identification and treatment of aggressive tumors.     

Reduced levels of topoisomerase IIα and IIβ in a multidrug-resistant lung-cancer cell line

We have previously shown that the doxorubicinselected multidrug-resistant small-cell lung-cancer cell line H69AR is resistant to VP-16-induced single-strand DNA breaks as compared with its parental H69 cell line. Levels of immunoreactive topoisomerase II are also reduced in H69AR cells. In the present study, we found that cleaved complex formation in the presence of VP-16 was decreased in H69AR cells as compared with H69 cells. In addition, the resistant cells contained lower levels of both topoisomerase II and topoisomerase II protein and mRNA. However, these changes were not accompanied by a decrease in the P4-unknotting (strand-passing) activity of 0.67M NaCl nuclear extracts of H69AR cells, nor was there any difference in VP-16 inhibition of unknotting activity in the H69 and H69AR nuclear extracts. These data suggest that reduced levels of topoisomerase II and II may contribute to the resistance of H69AR cells to VP-16 and other drugs that target these isoenzymes.This work was supported by a grant from the National Cancer Institute of Canada (to S. P. C. C.). One of the authors (C. D. E.) was supported in part by a Queen's University graduate fellowship, and another (S. P. C. C.) is a Career Scientist of the Ontario Cancer Treatment and Research Foundation     

Expression of E-cadherin, β-catenin and topoisomerase IIα in leiomyosarcomas

Introduction  

The expression of E-cadherin, β-catenin and topoisomerase II has been associated with clinical outcome of several cancers including sarcomas. We aimed to evaluate the expression of these markers in leiomyosarcomas (LMS).