Li Wu Fu

Bmi-1 Is a Novel Molecular Marker of Nasopharyngeal Carcinoma Progression and Immortalizes Primary Human Nasopharyngeal Epithelial Cells

The Bmi-1 oncoprotein regulates proliferation and oncogenesis in human cells. Its overexpression leads to senescence bypass in human fibroblasts and immortalization of human mammary epithelial cells. In this study, we report that compared with normal nasopharyngeal epithelial cells (NPEC), Bmi-1 is overexpressed in nasopharyngeal carcinoma cell lines. Importantly, Bmi-1 was also found to be overexpressed in 29 of 75 nasopharyngeal carcinoma tumors (38.7%) by immunohistochemical analysis. In contrast to nasopharyngeal carcinoma, there was no detectable expression of Bmi-1 in noncancerous nasopharyngeal epithelium. Moreover, high Bmi-1 expression positively correlated with poor prognosis of nasopharyngeal carcinoma patients. We also report that the overexpression of Bmi-1 leads to bypass of senescence and immortalization of NPECs, which normally express p16(INK4a) and exhibit finite replicative life span. Overexpression of Bmi-1 in NPECs led to the induction of human telomerase reverse transcriptase activity and reduction of p16(INK4a) expression. Mutational analysis of Bmi-1 showed that both RING finger and helix-turn-helix domains of it are required for immortalization of NPECs. Our findings suggest that Bmi-1 plays an important role in the development and progression of nasopharyngeal carcinoma, and that Bmi-1 is a valuable marker for assessing the prognosis of nasopharyngeal carcinoma patients. Furthermore, this study provides the first cellular proto-oncogene immortalized nasopharyngeal epithelial cell line, which may serve as a cell model system for studying the mechanisms involved in the tumorigenesis of nasopharyngeal carcinoma.

The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal epithelial cells

The polycomb group protein B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) is dysregulated in various cancers, and its upregulation strongly correlates with an invasive phenotype and poor prognosis in patients with nasopharyngeal carcinomas. However, the underlying mechanism of Bmi-1-mediated invasiveness remains unknown. In the current study, we found that upregulation of Bmi-1 induced epithelial-mesenchymal transition (EMT) and enhanced the motility and invasiveness of human nasopharyngeal epithelial cells, whereas silencing endogenous Bmi-1 expression reversed EMT and reduced motility. Furthermore, upregulation of Bmi-1 led to the stabilization of Snail, a transcriptional repressor associated with EMT, via modulation of PI3K/Akt/GSK-3beta signaling. Chromatin immunoprecipitation assays revealed that Bmi-1 transcriptionally downregulated expression of the tumor suppressor PTEN in tumor cells through direct association with the PTEN locus. This in vitro analysis was consistent with the statistical inverse correlation detected between Bmi-1 and PTEN expression in a cohort of human nasopharyngeal carcinoma biopsies. Moreover, ablation of PTEN expression partially rescued the migratory/invasive phenotype of Bmi-1-silenced cells, indicating that PTEN might be a major mediator of Bmi-1-induced EMT. Our results provide functional and mechanistic links between the oncoprotein Bmi-1 and the tumor suppressor PTEN in the development and progression of cancer.

Lapatinib (Tykerb, GW572016) reverses multidrug resistance in cancer cells by inhibiting the activity of ATP-binding cassette subfamily B member 1 and G member 2.

Lapatinib is active at the ATP-binding site of tyrosine kinases that are associated with the human epidermal growth factor receptor (Her-1 or ErbB1) and Her-2. It is conceivable that lapatinib may inhibit the function of ATP-binding cassette (ABC) transporters by binding to their ATP-binding sites. The aim of this study was to investigate the ability of lapatinib to reverse tumor multidrug resistance (MDR) due to overexpression of ABC subfamily B member 1 (ABCB1) and ABC subfamily G member 2 (ABCG2) transporters. Our results showed that lapatinib significantly enhanced the sensitivity to ABCB1 or ABCG2 substrates in cells expressing these transporters, although a small synergetic effect was observed in combining lapatinib and conventional chemotherapeutic agents in parental sensitive MCF-7 or S1 cells. Lapatinib alone, however, did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in sensitive and resistant cells. Additionally, lapatinib significantly increased the accumulation of doxorubicin or mitoxantrone in ABCB1- or ABCG2-overexpressing cells and inhibited the transport of methotrexate and E(2)17betaG by ABCG2. Furthermore, lapatinib stimulated the ATPase activity of both ABCB1 and ABCG2 and inhibited the photolabeling of ABCB1 or ABCG2 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner. However, lapatinib did not affect the expression of these transporters at mRNA or protein levels. Importantly, lapatinib also strongly enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBv200 cell xenografts in nude mice. Overall, we conclude that lapatinib reverses ABCB1- and ABCG2-mediated MDR by directly inhibiting their transport function. These findings may be useful for cancer combinational therapy with lapatinib in the clinic.

Erlotinib (Tarceva, OSI-774) Antagonizes ATP-Binding Cassette Subfamily B Member 1 and ATP-Binding Cassette Subfamily G Member 2-Mediated Drug Resistance

It has been reported that gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has the ability to modulate the function of certain ATP-binding cassette (ABC) transporters and to reverse ABC subfamily B member 1 (ABCB1; P-glycoprotein)- and ABC subfamily G member 2 (ABCG2; breast cancer resistance protein/mitoxantrone resistance protein)-mediated multidrug resistance (MDR) in cancer cells. However, it is unknown whether other EGFR TKIs have effects similar to that of gefitinib. In the present study, we have investigated the interaction of another EGFR TKI, erlotinib, with selected ABC drug transporters. Our findings show that erlotinib significantly potentiated the sensitivity of established ABCB1 or ABCG2 substrates and increased the accumulation of paclitaxel or mitoxantrone in ABCB1- or ABCG2-overexpressing cells. Furthermore, erlotinib did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in all cells and was unable to reverse MRP1-mediated MDR and had no effect on the parental cells. However, erlotinib remarkably inhibited the transport of E(2)17 beta G and methotrexate by ABCG2. In addition, the results of ATPase assays show that erlotinib stimulated the ATPase activity of both ABCB1 and ABCG2. Interestingly, erlotinib slightly inhibited the photolabeling of ABCB1 with [(125)I]iodoarylazidoprazosin (IAAP) at high concentration, but it did not inhibit the photolabeling of ABCG2 with IAAP. Overall, we conclude that erlotinib reverses ABCB1- and ABCG2-mediated MDR in cancer cells through direct inhibition of the drug efflux function of ABCB1 and ABCG2. These findings may be useful for cancer combinational therapy with erlotinib in the clinic.

Apatinib (YN968D1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters

Apatinib, a small-molecule multitargeted tyrosine kinase inhibitor, is in phase III clinical trial for the treatment of patients with non-small-cell lung cancer and gastric cancer in China. In this study, we determined the effect of apatinib on the interaction of specific antineoplastic compounds with P-glycoprotein (ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2). Our results showed that apatinib significantly enhanced the cytotoxicity of ABCB1 or ABCG2 substrate drugs in KBv200, MCF-7/adr, and HEK293/ABCB1 cells overexpressing ABCB1 and in S1-M1-80, MCF-7/FLV1000, and HEK293/ABCG2-R2 cells overexpressing ABCG2 (wild-type). In contrast, apatinib did not alter the cytotoxicity of specific substrates in the parental cells and cells overexpressing ABCC1. Apatinib significantly increased the intracellular accumulation of rhodamine 123 and doxorubicin in the multidrug resistance (MDR) cells. Furthermore, apatinib significantly inhibited the photoaffinity labeling of both ABCB1 and ABCG2 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner. The ATPase activity of both ABCB1 and ABCG2 was significantly increased by apatinib. However, apatinib, at a concentration that produced a reversal of MDR, did not significantly alter the ABCB1 or ABCG2 protein or mRNA expression levels or the phosphorylation of AKT and extracellular signal-regulated kinase 1/2 (ERK1/2). Importantly, apatinib significantly enhanced the effect of paclitaxel against the ABCB1-resistant KBv200 cancer cell xenografts in nude mice. In conclusion, apatinib reverses ABCB1- and ABCG2-mediated MDR by inhibiting their transport function, but not by blocking the AKT or ERK1/2 pathway or downregulating ABCB1 or ABCG2 expression. Apatinib may be useful in circumventing MDR to other conventional antineoplastic drugs.

Reversal of MDR1/P-glycoprotein-mediated multidrug resistance by vector-based RNA interference in vitro and in vivo.

Overexpression of P-glycoprotein (P-gp) encoded by MDR1 gene in cancer cells results in multidrug resistance (MDR) to structurally and mechanistically different chemotherapeutic drugs, which is a major cause for cancer chemotherapy failures to cancer patients. Recently, there were several reports showing that expression of siRNAs targeting MDR1 gene is able to reverse the P-gp mediated MDR, however, the in vivo reversal effects for MDR have still not been identified. We developed a novel MDR reversal system using RNA interference technique in human epidermoid carcinoma KBv200 cells. The stably expressing MDR1 shRNA cells (KBv200/MDR1sh) were established with transfection of vector pEGFPC2-H1-MDR1shDNA containing MDR1-V siRNA expression cassette, and we found that more than 90% of MDR1 mRNA and P-gp were reduced. KBv200/MDR1sh cells simultaneously showed stably expressing EGFP and kept low MDR1 expression beyond ten passages. Compared KBv200/MDR1sh cells with KBv200 cells, resistance to vincristine and doxorubicin decreased from 62.4-fold to 10.5-fold and from 74.5-fold to 9.5-fold respectively, and intracellular doxorubicin accumulation enhanced from 0.30°¿0.08 nmoles/106 cells to 0.86°¿0.16 nmoles/106 cells, and the fluorescence intensity of intracellular Rhodamine 123 accumulation increased from 3.58°¿1.63/106 cells to 13.96°¿3.07/106 cells. In the nude mice xenografts, vincristine (0.2 mg/kg of body weight) inhibited the growth of KBv200/MDR1sh solid tumors by 42.0%, but the same dose of vincristine didn’t inhibit the growth of KBv200 solid tumors significantly. These results suggest that administration of RNAi targeted MDR1 gene can effectively reverse MDR both in vitro and in vivo models.

Sensitization of ABCG2-overexpressing cells to conventional chemotherapeutic agent by sunitinib was associated with inhibiting the function of ABCG2

Sunitinib is an ATP-competitive multi-targeted tyrosine kinase inhibitor. In this study, we evaluated the possible interaction of sunitinib with P-glycoprotein (P-gp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), breast cancer resistance protein (BCRP, ABCG2) and lung-resistance protein (LRP) in vitro. Our results showed that sunitinib completely reverse drug resistance mediated by ABCG2 at a non-toxic concentration of 2.5muM and has no significant reversal effect on ABCB1-, ABCC1- and LRP-mediated drug resistance, although a small synergetic effect was observed in combining sunitinib and conventional chemotherapeutic agents in ABCB1 overexpressing MCF-7/adr and parental sensitive MCF-7 cells, ABCC1 overexpressing C-A120 and parental sensitive KB-3-1 cells. Sunitinib significantly increased intracellular accumulation of rhodamine 123 and doxorubicin and remarkably inhibited the efflux of rhodamine 123 and methotrexate by ABCG2 in ABCG2-overexpressing cells, and also profoundly inhibited the transport of [(3)H]-methotrexate by ABCG2. However, sunitinib did not affect the expression of ABCG2 at mRNA or protein levels. In addition, sunitinib did not block the phosphorylation of Akt and Erk1/2 in ABCG2-overexpressing or parental sensitive cells. Overall, we conclude that sunitinib reverses ABCG2-mediated MDR through inhibiting the drug efflux function of ABCG2. These findings may be useful for cancer combinational therapy with sunitinib in the clinic.

Vandetanib (Zactima, ZD6474) Antagonizes ABCC1- and ABCG2-Mediated Multidrug Resistance by Inhibition of Their Transport Function

Background ABCC1 and ABCG2 are ubiquitous ATP-binding cassette transmembrane proteins that play an important role in multidrug resistance (MDR). In this study, we evaluated the possible interaction of vandetanib, an orally administered drug inhibiting multiple receptor tyrosine kinases, with ABCC1 and ABCG2 in vitro. Methodology and Principal Findings MDR cancer cells overexpressing ABCC1 or ABCG2 and their sensitive parental cell lines were used. MTT assay showed that vandetanib had moderate and almost equal-potent anti-proliferative activity in both sensitive parental and MDR cancer cells. Concomitant treatment of MDR cells with vandetanib and specific inhibitors of ABCC1 or ABCG2 did not alter their sensitivity to the former drug. On the other hand, clinically attainable but non-toxic doses of vandetanib were found to significantly enhance the sensitivity of MDR cancer cells to ABCC1 or ABCG2 substrate antitumor drugs. Flow cytometric analysis showed that vandetanib treatment significantly increase the intracellular accumulation of doxorubicin and rhodamine 123, substrates of ABCC1 and ABCG2 respectively, in a dose-dependent manner (P<0.05). However, no significant effect was shown in sensitive parental cell lines. Reverse transcription-PCR and Western blot analysis showed that vandetanib did not change the expression of ABCC1 and ABCG2 at both mRNA and protein levels. Furthermore, total and phosphorylated forms of AKT and ERK1/2 remained unchanged after vandetanib treatment in both sensitive and MDR cancer cells. Conclusions Vandetanib is unlikely to be a substrate of ABCC1 or ABCG2. It overcomes ABCC1- and ABCG2-mediated drug resistance by inhibiting the transporter activity, independent of the blockade of AKT and ERK1/2 signal transduction pathways.

Anthracenedione derivative 1403P-3 induces apoptosis in KB and KBv200 cells via reactive oxygen species-independent mitochondrial pathway and death receptor pathway

Anthracenedione derivatives are potent cytotoxic agents to tumour cells. In this study, we investigated the anticancer activities of anthracenedione derivative 1403P-3 separated from the secondary metabolites of the mangrove endophytic fungus No. 1403. Our results demonatrated that 1403P-3 showed potent cytotoxicity not only to human epidermoid carcinoma drug-sensitive parental KB cells but also to multidrug resistant (MDR) KBv200 cells and the IC50 values were 19.66 and 19.27 μM, respectively. Further research indicated that 1403P-3 induced apoptosis in KB cells and KBv200 cells confirmed by Hoechst 33258 staining, detection of DNA fragmentation and cleavage of poly (ADP-ribose) polymerase (PARP). Furthermore, apoptosis triggered by 1403P-3 was characterized by the loss of mitochondrial membrane potential (ΔΨm), release of cytochrome c, cleavage of Bid, and activation of caspases-2, -3, -7, -8 and -9. Z-IETD-FMK, caspase-8 inhibitor could inhibit the activation of caspase-2 and cleavage of Bid induced by 1403P-3. However, activation of caspase-9 and cleavage of PARP caused by 1403P-3 were not inhibited by Z-IETD-FMK. Additionally, 1403P-3 did not influence the expression level of Bcl-2 and Bax. It is noteworthy that 1403P-3 decreased the generation of reactive oxygen species (ROS) in KB cells and KBv200 cells. DNA binding assay exhibited that apoptosis induced by 1403P-3 was not involved in intercalating to DNA. In summary, 1403P-3 induced apoptosis of KB cells and KBv200 cells through mitochondrial pathway and death receptor pathway. Furthermore, the mitochondrial pathway was independent of reactive oxygen species and activation of caspase-8.

Secalonic Acid D induced leukemia cell apoptosis and cell cycle arrest of G1 with involvement of GSK-3β/β-catenin/c-Myc pathway

The anticancer activities of secalonic acid D separated from the secondary metabolites of the mangrove endophytic fungus No. ZSU44 were investigated in this study. Secalonic acid D showed potent cytotoxicity to HL60 and K562 cells, and the IC50 values were 0.38 and 0.43 μmol/L, respectively. Annexin V-FITC/PI assay and western blot indicated that secalonic acid D induced apoptosis in HL60 and K562 cells.. In addition, secalonic acid D led to cell cycle arrest of G1 phase related to downregulation of c-Myc. Moreover, our data indicated that downregulation of c-Myc and cell cycle arrest of G1 phase were caused not by formation of G-quadruplex structures but by activation of GSK-3β followed by degradation of β-catenin.