Expression of calcyclin-binding protein/Siah-1 interacting protein in normal and malignant human tissues: an immunohistochemical survey.

Calcyclin-binding protein (CacyBP)/Siah-1 interacting protein (SIP), a component of ubiquitin-mediated proteolysis, could bind the Skp1-Cul1-F box protein complex. Although CacyBP/SIP was implicated in p53-induced beta-catenin degradation, its exact function was still unknown. Our previous studies showed that CacyBP/SIP could modulate the multidrug-resistant phenotype of gastric cancer cells and was highly expressed in gastric cancer tissues compared with that in non-cancerous tissues. In this study, CacyBP/SIP protein expression profile in a broad range of human normal tissues and carcinomas was analyzed by immunohistochemistry staining with anti-CacyBP/SIP monoclonal antibody first produced in our laboratory. CacyBP/SIP was generally localized in the cytoplasm/nucleus. Positive staining of CacyBP/SIP was found in brain, heart, lymph node, and esophagus. Weak staining was shown in the rectum and kidney. No CacyBP/SIP was detected in other normal tissues. However, CacyBP/SIP was ubiquitously detected in all kinds of tumor tissues and was highly expressed in nasopharyngeal carcinoma, osteogenic sarcoma, and pancreatic cancer. To our knowledge, this is the first study on the CacyBP/SIP expression pattern in a broad range of human normal and tumor tissues. The data presented should serve as a useful reference for other investigators in future studies of CacyBP/SIP functions. Hopefully, this knowledge will lead to discovery of more roles of CacyBP/SIP in tumorigenesis.     

Clitocine Reversal of P-Glycoprotein Associated Multi-Drug Resistance through Down-Regulation of Transcription Factor NF-κB in R-HepG2 Cell Line

Multidrug resistance(MDR)is one of the major reasons for failure in cancer chemotherapy and its suppression may increase the efficacy of therapy. The human multidrug resistance 1 (MDR1) gene encodes the plasma membrane P-glycoprotein (P-gp) that pumps various anti-cancer agents out of the cancer cell. R-HepG2 and MES-SA/Dx5 cells are doxorubicin induced P-gp over-expressed MDR sublines of human hepatocellular carcinoma HepG2 cells and human uterine carcinoma MES-SA cells respectively. Herein, we observed that clitocine, a natural compound extracted from Leucopaxillus giganteus, presented similar cytotoxicity in multidrug resistant cell lines compared with their parental cell lines and significantly suppressed the expression of P-gp in R-HepG2 and MES-SA/Dx5 cells. Further study showed that the clitocine increased the sensitivity and intracellular accumulation of doxorubicin in R-HepG2 cells accompanying down-regulated MDR1 mRNA level and promoter activity, indicating the reversal effect of MDR by clitocine. A 5'-serial truncation analysis of the MDR1 promoter defined a region from position -450 to -193 to be critical for clitocine suppression of MDR1. Mutation of a consensus NF-κB binding site in the defined region and overexpression of NF-κB p65 could offset the suppression effect of clitocine on MDR1 promoter. By immunohistochemistry, clitocine was confirmed to suppress the protein levels of both P-gp and NF-κB p65 in R-HepG2 cells and tumors. Clitocine also inhibited the expression of NF-κB p65 in MES-SA/Dx5. More importantly, clitocine could suppress the NF-κB activation even in presence of doxorubicin. Taken together; our results suggested that clitocine could reverse P-gp associated MDR via down-regulation of NF-κB.     

Regulatory mechanism of ZNF139 in multi-drug resistance of gastric cancer cells

Our previous study found increased zinc finger protein 139 (ZNF139) expression in gastric cancer (GC) cells. Purpose of the study is to further clarify the role and mechanism of ZNF139 in multi-drug resistance (MDR) of GC cells. MTT assay, RT-PCR, Western blotting were employed to detect susceptibility of GC cells to chemotherapeutic agents (5-FU, L-OHP) in vitro, and expressions of ZNF139 and MDR associated genes MDR1/P-gp, MRP1, Bcl-2, Bax were also detected. siRNA specific to ZNF139 was transfected into MKN28 cells, then chemosensitivity of GC cells as well as changes of ZNF139 and MDR associated genes were detected. It’s found the inhibition rate of 5-FU, L-OHP to well-differentiated GC tissues and cell line was lower than that in the poorly differentiated tissues and cell line; expressions of ZNF139 and MDR1/P-gp, MRP1 and Bcl-2 in well-differentiated GC tissues and cell line MKN28 were higher, while Bax expression was lower. After ZNF139-siRNA was transfected into MKN28, ZNF139 expression in GC cells was inhibited by 90 %; inhibition rate of 5-FU, L-OHP to tumor cells increased, and expressions of MDR1/P-gp, MRP1 and Bcl-2 were down-regulated, while Bax was up-regulated. ZNF139 was involved in GC MDR by promoting expressions of MDR1/P-gp, MRP1 and Bcl-2 and inhibiting Bax simultaneously.     

Galectin-3 Silencing Inhibits Epirubicin-Induced ATP Binding Cassette Transporters and Activates the Mitochondrial Apoptosis Pathway via β-Catenin/GSK-3β Modulation in Colorectal Carcinoma

Multidrug resistance (MDR), an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC) transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi) on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp), MDR-associated protein (MRP) 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.     

Sunitinib Reverse Multidrug Resistance in Gastric Cancer Cells by Modulating Stat3 and Inhibiting P-gp Function

Sunitinib, a small-molecule multi-targeted tyrosine kinase inhibitor, has been applied in phase II clinical trial as second-line treatment for advanced gastric cancer. In this study, we determined the effect of Sunitinib on the multidrug resistance in gastric cancer cells selected by vincristine. Our results showed that Sunitinib significantly enhanced the cytotoxicity of adriamycin, vincristine, etoposide, 5-Fluorouracil, and cisplatin in multidrug-resistant gastric cancer cells (SGC7901/VCR). Sunitinib significantly increased the intracellular accumulation and retention of rhodamine 123 in the SGC7901/VCR cells. However, Sunitinib, at a concentration that reverses MDR, had no significant effect on P-gp protein or mRNA expression levels. In addition, the present study revealed that Sunitinib inhibited Stat3 and down-regulated Bcl-2 in SGC7901/VCR cells, which might also contribute to the reversal of MDR. In conclusion, Sunitinib reverses multidrug resistance in gastric cancer cells by inhibiting P-gp transporter function and modulating Stat3 and Bcl-2. Further study with Sunitinib may be helpful for developing combination therapeutic strategy or circumventing gastric cancer MDR to other conventional anti-cancer drugs.     

Suppression of multidrug resistance by treatment with TRAIL in human ovarian and breast cancer cells with high level of c-Myc

In this study, we investigated the role of c-Myc in overcoming multidrug resistance (MDR) in human ovarian and breast cancer cells by TRAIL. We showed that P-gp expressing MDR variants (Hey A8-MDR and MCF7-MDR cells) with high level of c-Myc were highly susceptible to TRAIL treatment when compared to their drug-sensitive parental human ovarian cancer Hey A8 and breast MCF-7 cells, respectively. Up-regulation of DR5 TRAIL receptor and down-regulation of c-FLIP and the promotion of caspase-dependent cell death, which contribute to TRAIL sensitization of MDR cells, were regulated by the over-expressed c-Myc in the MDR cells. After targeted inhibition of c-Myc with specific siRNA, these responses to TRAIL disappeared and TRAIL-induced apoptosis was also suppressed in MCF7-MDR cells. Treatment with TRAIL significantly reduced P-glycoprotein (P-gp)-mediated efflux of rhodamine123 in both Hey A8-MDR and MCF7-MDR cells. Furthermore, TRAIL significantly potentiated the cytotoxicity of vinblastine, vincristine, doxorubicin and VP-16 that are P-gp substrate anticancer drugs in both MDR cells, which resulted in the reversal effect of TRAIL on the MDR phenotype. The present study shows for the first time that elevated c-Myc expression in the MDR cells plays a critical role in overcoming MDR by TRAIL that can act as a specific sensitizer for P-gp substrate anticancer drug.     

Adrenomedullin prevents apoptosis in prostate cancer cells

The 52-aminoacid peptide adrenomedullin (AM) is expressed in the normal and malignant prostate. We have previously shown that prostate cancer cells produce and secrete AM, which acts as an autocrine growth inhibitory factor. We have evaluated in the present study the role of AM in prostate cancer cell apoptosis, induced either by serum deprivation or treatment with the chemotherapeutic agent etoposide (which acts as an inhibitor of topoisomerase II). For this purpose we over-expressed AM in PC-3, DU 145 and LNCaP cells, which were transfected with an expression vector carrying AM. We also treated the parental cell lines with synthetic AM in normal culture conditions and in conditions of induced-apoptosis. After serum removal, AM prevented apoptosis in DU 145 and PC-3 cells, but not in LNCaP cells. When treated with etoposide, AM prevented apoptosis in PC-3 and LNCaP cells, but not in DU 145 cells. Cell cycle analysis demonstrated a significant decrease in the percentage of AM-overexpressing PC-3 cells in the subG0/G1 phase after treatment with etoposide, as compared to the percentage of mock-transfected PC-3 treated cells. Western blot showed that protein levels of phosphorylated ERK1/2 increased in parental PC-3 cells after treatment with etoposide. In PC-3 cells overexpressing AM, phosphorylated ERK1/2 basal levels were lower than basal levels of parental PC-3 cells, and treatment with etoposide did not result in such an increase. Etoposide produced a significant increase in cleaved PARP in parental PC-3 cells. However, PC-3 clones overexpressing AM that were treated with etoposide only showed a mild increase in fragmented PARP. The ratio Bcl-2/Bax was reduced in parental or mock-transfected PC-3 cells after treatment with etoposide. On the contrary, this ratio was not reduced in PC-3 clones with AM overexpression that were treated with etoposide. All these data demonstrate that AM plays a protective role against induced apoptosis in prostate cancer cells. These results may have important implications in prostate cancer resistance to chemotherapeutic agents.     

CacyBP/SIP phosphatase activity in neuroblastoma NB2a and colon cancer HCT116 cells

Recently, we have reported that CacyBP/SIP could be a novel phosphatase for ERK1/2 kinase. In this work, we analyzed the CacyBP/SIP phosphatase activity toward ERK1/2 in 2 cell lines of different origin. We showed that overexpression of CacyBP/SIP in NB2a cells resulted in a lower level of phosphorylated ERK1/2 (P-ERK1/2) in the nuclear fraction while such overexpression in HCT116 cells had no effect on the level of P-ERK1/2. Moreover, we found that overexpression of CacyBP/SIP resulted in higher phosphatase activity in the nuclear fraction obtained from NB2a cells when compared with HCT116 cells. Using 2-D electrophoresis we showed that the pattern of spots representing CacyBP/SIP differed in these 2 cell lines and was probably due to a different phosphorylation state of this protein. We also established that after overexpression of CacyBP/SIP in NB2a cells, the amount of nuclear β-catenin was low, while it remained high in HCT116 cells. Since NB2a cells have differentiation potential and HCT116 cells do not, our data suggest that different activity of CacyBP/SIP in these 2 cell lines might affect the ERK1/2 pathway in the differentiation or proliferation processes.     

The CacyBP/SIP Protein is Sumoylated in Neuroblastoma NB2a Cells

The Calcyclin binding protein and Siah-1 interacting protein (CacyBP/SIP) protein is highly expressed in mammalian brain as well as in neuroblastoma NB2a cells and pheochromocytoma PC12 cells. This protein interacts with several targets such as cytoskeletal proteins or ERK1/2 kinase and seems to be involved in many cellular processes. In this work we examined a post-translational modification of CacyBP/SIP which might have an effect on its function. Since theoretical analysis of the amino acid sequence of CacyBP/SIP indicated several lysine residues which could potentially be sumoylated we checked experimentally whether this protein might be modified by SUMO attachment. We have shown that indeed CacyBP/SIP bound the E2 SUMO ligase, Ubc9, in neuroblastoma NB2a cell extract and was sumoylated in these cells. By fractionation of NB2a cell extract we have found that, contrary to the majority of SUMO-modified proteins, sumoylated CacyBP/SIP is present in the cytoplasmic and not in the nuclear fraction. We have also established that lysine 16 is the residue which undergoes sumoylation in the CacyBP/SIP protein.     

Calcyclin binding protein promotes DNA synthesis and differentiation in rat neonatal cardiomyocytes

During cardiac muscle development, most cardiomyocytes permanently withdraw from the cell cycle. Previously, by suppressive subtractive hybridization, we identified calcyclin-binding protein/Siah-interacting protein (CacyBP/SIP) as one of the candidates being upregulated in the hyperplastic to hypertrophic switch, suggesting an important role of CacyBP/SIP in cardiac development. To show the importance of CacyBP/SIP during myoblast differentiation, we report here that CacyBP/SIP is developmentally regulated in postnatal rat hearts. The overexpression of CacyBP/SIP promotes the differentiation and DNA synthesis of H9C2 cells and primary rat cardiomyocytes, as well as downregulates the expression of beta-catenin. Besides, CacyBP/SIP promotes the formation of myotubes and multinucleation upon differentiation. To investigate the cardioprotective role of CacyBP/SIP in cardiomyocytes, a hypoxia/reoxygenation model was employed. We found that CacyBP/SIP was upregulated during myocardial infarction (MI) and hypoxia/reoxygenation. As a conclusion, CacyBP/SIP may play a role in cardiomyogenic differentiation and possibly protection of cardiomyocytes during hypoxia/reoxygenation injury.     

A mechanism for P-glycoprotein-mediated apoptosis as revealed by verapamil hypersensitivity

Selection of tumor cell lines with anticancer drugs has led to the appearance of multidrug-resistant (MDR) subclones with P-glycoprotein 1 (P-gp1) expression. These cells are cross-resistant to several structurally and functionally dissimilar drugs. Interestingly, in the process of gaining resistance, MDR cells become hypersensitive or collaterally sensitive to membrane-active agents, such as calcium channel blockers, steroids, and local anaesthetics. In this report, hypersensitivity to the calcium channel blocker, verapamil, was analyzed in sensitive and resistant CHO cell lines. Our results show that treatment with verapamil preferentially induced apoptosis in MDR cells compared to drug-sensitive cells. This effect was independent of p53 activity and could be inhibited by overexpression of the Bcl-2 gene. The induction of apoptosis by verapamil had a biphasic trend in which maximum cell death occurred at 10 microM, followed by improved cell survival at higher concentrations (50 microM). We correlated this effect to a similar biphasic trend in P-gp1 ATPase activation by verapamil in which low concentrations of verapamil (10 microM) activated ATPase, followed by inhibition at higher concentrations. To confirm the relationship between apoptosis and ATPase activity, we used two inhibitors of P-gp1 ATPase, PSC 833 and ivermectin. These ATPase inhibitors reduced hypersensitivity to verapamil in MDR cells. In addition, low concentrations of verapamil resulted in the production of reactive oxygen species (ROS) in MDR cells. Taken together, these results show that apoptosis was preferentially induced by P-gp1 expressing cells exposed to verapamil, an effect that was mediated by ROS, produced in response the high ATP demand by P-gp1.     

Reversal of multidrug resistance of vincristine-resistant gastric adenocarcinoma cells through up-regulation of DARPP-32

Here we investigated the roles of DARPP-32 in multidrug resistance (MDR) of gastric cancer cells and the possible underlying mechanisms. We constructed the eukaryotic expression vector of DARPP-32 and transfected it into human vincristine-resistant gastric adenocarcinoma cell line SGC7901/VCR. Up-regulation of DARPP-32 could significantly enhance the sensitivity of SGC7901/VCR cells towards vincristine, adriamycin, 5-fluorouracil and cisplatin, and could decrease the capacity of cells to efflux adriamycin. What's more, the results of subrenal capsule assay confirmed that DARPP-32 might play a certain role in MDR of gastric cancer. DARPP-32 could significantly down-regulate the expression of P-gp and zinc ribbon domain-containing 1 (ZNRD1), but not alter the expression of multidrug resistance-associated protein (MRP) or the glutathione S-transferase (GST). DARPP-32 could also significantly decrease the anti-apoptotic activity of SGC7901/VCR cells. Further study of the biological functions of DARPP-32 might be helpful for understanding the mechanisms of MDR in gastric cancer.