Novel plant triterpenoid drug amooranin overcomes multidrug resistance in human leukemia and colon carcinoma cell lines

Amooranin (AMR), a plant terpenoid, isolated from Amoora rohituka, was investigated for its ability to overcome multidrug resistance in human leukemia and colon carcinoma cell lines. AMR IC(50) values of multidrug-resistant leukemia (CEM/VLB) and colon carcinoma (SW620/Ad-300) cell lines were higher (1.9- and 6-fold) than parental sensitive cell lines (CEM and SW620). AMR induced G(2)+M phase-arrest during cell cycle traverse in leukemia and colon carcinoma cell lines and the percentage of cells in G(2)+M phase increased in a dose-dependent manner. Coincubation of tumor cells with both DOX and AMR reversed DOX resistance in 104-fold DOX-resistant CEM/VLB and 111-fold DOX-resistant SW620/Ad-300 cell lines with a dose modification factor of 50.9 and 99.6, respectively. Flow cytometric assay showed that AMR causes enhanced cellular DOX accumulation in a dose-dependent manner. AMR inhibits photolabeling of P-glycoprotein (P-gp) with [(3)H]-azidopine and the blocking effect enhanced with increasing concentrations of AMR. Our results show that AMR competitively inhibits P-gp-mediated DOX efflux, suggestive of a mechanism underlying the enhanced DOX accumulation and reversal of multidrug resistance by AMR.     

A novel 7-modified camptothecin analog overcomes breast cancer resistance protein-associated resistance in a mitoxantrone-selected colon carcinoma cell line

We selected a mitoxantrone-resistant HT29 colon carcinoma cell line (HT29/MIT) that exhibited a very high degree of resistance to the selecting agent and marked resistance to topotecan and SN38, but limited resistance to doxorubicin. The development of drug resistance was independent of expression of P-glycoprotein or multidrug resistance-associated protein but was associated with high up-regulation of the breast carcinoma resistance protein (BCRP) as shown by Western blot analysis. BCRP overexpression was associated with a reduced intracellular accumulation of topotecan, a known substrate for BCRP. Conversely, a lipophilic 7-modified camptothecin analogue (ST1481) displayed a complete lack of cross-resistance in HT29/MIT cells, suggesting that the drug was not a substrate for BCRP because no defects in intracellular accumulation were found. This conclusion is consistent with the antitumor efficacy of ST1481 against a BCRP-expressing tumor. These results may have therapeutic implications because the antitumor efficacy of ST1481 is in part related to a good bioavailability after oral administration, and the drug is currently under Phase I clinical evaluation.     

Camptothecin overcomes MDR1-mediated resistance in human KB carcinoma cells.

In order to understand the high efficacy of camptothecin derivatives against human colon tumor xenografts in nude mice, we have studied the transport properties of camptothecin derivatives across cellular membranes of MDR1-overexpressing cells. MDR1 overexpression was shown to have little effect on camptothecin cytotoxicity; camptothecin was equally cytotoxic to both the drug-sensitive parental cell line, KB 3-1, and its multidrug-resistant derivative, KB V1. The ability of camptothecin to overcome MDR1-mediated resistance is most likely due to unimpaired accumulation of camptothecin in MDR1 cells as suggested from the following experiments: (a) cytotoxicity of camptothecin against KB V1 cells was not altered by the known MDR1-reversing agent, verapamil; (b) camptothecin was ineffective as compared with vinblastine in competing with [3H]azidopine for photoaffinity labeling of MDR1; (c) camptothecin was equally efficient in trapping cellular topoisomerase I molecules on chromosomal DNA in the form of cleavable complexes in both KB 3-1 and KB V1 cells. The mechanism by which camptothecin overcomes MDR1-mediated resistance has been further studied using a number of uncharged and charged camptothecin derivatives. In contrast to the uncharged camptothecin derivatives, such as 9-amino-camptothecin and 10,11-methylenedioxy-camptothecin, the charged camptothecin derivative, topotecan, showed reduced cytotoxicity against MDR1-overexpressing KB V1 cells. The reduced cytotoxicity of topotecan in KB V1 cells was due to the overexpression of MDR1 in KB V1 cells since verapamil restored both topotecan accumulation and cytotoxicity. These results suggest that the charge on camptothecin can affect the drug's sensitivity to MDR1. The possible effect of membrane permeability in determining drug selectivity of MDR1 is discussed.     

By inhibiting PFKFB3, aspirin overcomes sorafenib resistance in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the few cancers with a continuous increase in incidence and mortality. Drug resistance is a major problem in the treatment of HCC. In this study, two sorafenib‐resistant HCC cell lines and a nude mouse subcutaneously tumor model were used to explore the possible mechanisms leading to sorafenib resistance, and to investigate whether aspirin could increase the sensitivity of hepatoma cells to sorafenib. The combination of aspirin and sorafenib resulted in a synergistic antitumor effect against liver tumors both in vitro and in vivo. High glycolysis and PFKFB3 overexpression occupied a dominant position in sorafenib resistance, and can be targeted and overcome by aspirin. Aspirin plus sorafenib induced apoptosis in tumors without inducing weight loss, hepatotoxicity or inflammation. Our results suggest that aspirin overcomes sorafenib resistance and their combination may be an effective treatment approach for HCC.     

A recombinant IL-4-Pseudomonas exotoxin inhibits protein synthesis and overcomes apoptosis resistance in human CLL B cells

We have determined that CLL B cells consistently express type 3 membrane receptors for the Th2-derived cytokine IL-4 (IL-4R). Furthermore, when added to CLL B cells, IL-4 induces increased apoptosis resistance, increased protein synthesis in CLL B cells and rapid onset activation of STAT1, STAT5 and STAT6. Since the IL-4-IL-4R pathway is intact in CLL B cells and is related to apoptosis resistance, we considered whether we could target this pathway. A recombinant IL-4 Pseudomonas exotoxin fusion protein (IL-4 PE), known to bind to IL-4R, was incubated with CLL B cells. IL-4 PE (10 ng/ml) cultured with CLL B cells resulted in an increase of apoptosis/death from mean levels of 46.6+/-7.0 of non-exposed cells to 69+/-8.6 (n=6). By measuring in vitro protein synthesis, two predominant patterns of sensitivity were observed. In one, CLL B cell clones (n=4) were found to be extremely sensitive to IL-4 PE (IC50's range=6-25 ng/ml). In the second, low concentrations of IL-4 PE induced agonist activity while increasing concentrations induced cytotoxicity in 6 of 21 patient-derived cells. These studies suggest that the IL-4R, on B-CLL cells, can serve as a unique molecular target for directing cytotoxic agents in the therapy of B-CLL.     

Inhibition of BMK1 pathway suppresses cancer stem cells through BNIP3 and BNIP3L

Cancer stem cells (CSCs) possess many characteristics associated with stem cells and are believed to drive tumor initiation. Although targeting of CSCs offers great promise for the new generation of therapeutics, lack of the effective drugable target and appropriate pharmacological reagents significantly impedes the development of chemotherapies. Here, we show that the phosphorylation of BMK1 was significantly correlated with not only embryonic and induced pluripotent stem (iPS) cells, but also the CSCs. It was showed that activation of BMK1 by the expression of MEK5D enhanced the self-renew (sphere formation), proliferation (clone formation) and tumorigenic capacity of CSCs. While BMK1 inhibitor, XMD8-92, suppressed these capacities. RNA-seq and microarray analysis revealed that inhibition of BMK1 significantly enhanced the expression of BNIP3 and BNIP3L, which play important roles in cell death. Further study indicated that shRNA-mediated knock down of BNIP3 and BNIP3L impairs the BMK1 inhibitor, XMD8-92-induced suppression of sphere formation and clone formation of CSC. Collectively, these results not only indicate that BMK1 plays an important role in maintaining “stemness” of CSCs, but also implicate that BMK1 might be a potential drug target for CSCs.     

5, 7-Dimethoxyflavone sensitizes TRAIL-induced apoptosis through DR5 upregulation in hepatocellular carcinoma cells

Purpose  

5, 7-dimethoxyflavone (DMF) has been reported to induce apoptosis in various cancer cells. The aim of this study was to examine whether DMF sensitizes human hepatocellular carcinoma (HCC) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and its mechanism.     

Esculetin enhances TRAIL-induced apoptosis through DR5 upregulation in human oral cancer SAS cells

Esculetin has been shown to selectively induce tumor apoptosis in several types of cancers and is regarded as a promising chemotherapeutic agent. In this study, we showed that esculetin significantly suppressed the growth of oral cancer SAS cells in a dose-dependent manner. DNA content flow cytometry and TUNEL assay revealed that esculetin induced cell cycle arrest and apoptosis. Western blotting showed esculetin increased DR5 protein expression and activated caspase-8, which differed from previous studies conducted in other cell types. Furthermore, treatment with esculetin significantly increased TRAIL-induced apoptosis in SAS cells and the TRAIL-sensitizing effect was blocked by DR5/Fc chimera protein. Our results indicate that esculetin enhances TRAIL-induced apoptosis primarily through upregulation of DR5. Combination of esculetin and TRAIL may be a novel treatment strategy for oral cancers.     

PSK induces apoptosis through the inhibition of activated STAT3 in human esophageal carcinoma cells

PSK, a protein-bound polysaccharide, is widely used in Japan as an immunopotentiating biological response modifier for cancer patients. PSK exerts antitumor activities through stimulation of the host's immune response; however, few studies have addressed the direct actions of PSK on tumor cells. Recently, it has been found that STAT3 is aberrantly activated in various types of malignancies, and plays a crucial role in tumor cell proliferation and survival. In the present study, STAT3 was constitutively activated in KYSE170 and TE13 esophageal carcinoma cells, and PSK inhibited proliferation and induced apoptosis in these cells in a dose-dependent manner. Based on these findings, the relationship between STAT3 and apoptosis in these cells was investigated. Results showed that PSK inhibited the expression of activated STAT3 and stimulated the expression of pro-apoptotic Bax in a dose-dependent manner, without affecting the expression of anti-apoptotic Bcl-xL and Mcl-1. These results indicate that PSK may induce apoptosis in esophageal carcinoma cells by inhibiting the expression of activated STAT3.     

Galectin-3 leads to attenuation of apoptosis through Bax heterodimerization in human thyroid carcinoma cells

Cancer cells survive escaping normal apoptosis and the blocks in apoptosis that keep cancer cells alive are promising candidates for targeted therapy. Galectin-3 (Gal-3) is, a member of the lectin family, which is involved in cell growth, adhesion, proliferation and apoptosis. It remains elusive to understand the role of Gal-3 on apoptosis in thyroid carcinoma cells. Here, we report that Gal-3 heterodimerizes Bax, mediated by the carbohydrate recognition domain (CRD) of Gal-3, leading to anti-apoptotic characteristic. Gal-3/Bax interaction was suppressed by an antagonist of Gal-3, in which in turn cells became sensitive to apoptosis. The data presented here highlight that Gal-3 is involved in the anti-apoptosis of thyroid carcinoma cells. Thus, it suggests that targeting Gal-3 may lead to an improved therapeutic modality for thyroid cancer.     

地塞米松诱导结肠癌细胞凋亡的机制研究

目的：探讨地塞米松（Ｄｅｘ）体外抑制结肠癌细胞株的增殖并诱导其凋亡的作用机制。方法：采用ＭＴＴ及流式细胞仪检测细胞增殖抑制及凋亡,ＲＴ-ＰＣＲ检测ＧＲ、ＩκＢ及Ｂcl-２的表达改变,凝胶迁移或电泳迁移率实验（ＥＭＳＡ）检测Ｄｅｘ作用过程中的ＮＦ-κＢ活性改变。结果：Ｄｅｘ体外作用后,对４种结肠癌细胞株ＬｏＶｏ、ＨＣＴ-１１６、ＨＴ-２９及ＳＷ-４８０均有抑制作用,ＬｏＶｏ和ＨＣＴ-１１６细胞作用最显著。流式细胞仪检测显示,Ｄｅｘ（１×１０^-4mol/L）诱导７２ｈ后,在ＬｏＶｏ和ＨＣＴ-１１６细胞检测到细胞凋亡和坏死峰,明显高于未加Ｄｅｘ的对照组,具有显著性差异。ＲＴ ＰＣＲ检测显示Ｄｅｘ作用后ＧＲα、ＩκＢ表达升高,Ｂｃｌ-２无明显改变,ＥＭＳＡ结果也显示ＮＦ-κＢ活性明显降低。结论：地塞米松可能通过上调ＧＲα、ＩκＢ表达,抑制ＮＦ-κＢ活性来抑制ＬｏＶｏ细胞的增殖,并诱导其凋亡。     

Establishment of drug resistance in human gastric and colon carcinoma xenograft lines

We established multidrug-resistant human gastric and colon xenograft lines by means of intratumoral injections of four agents, doxorubicin (DXR), cisplatin (CDDP), 5-fluorouracil (5-FU) and mitomycin C (MMC), into subcutaneous SC1NU and SW480 tumors once a week or less. Such intermittent drug exposure is commonly used in clinical chemotherapeutic protocols. All xenograft lines acquired resistance to the injected drugs as evaluated by in vivo drug-resistance tests. Many of the drug-resistant lines showed various patterns of cross resistance to other drugs. In order to analyze the mechanism of resistance in vivo, we investigated the expression of drug resistance gene, which has been extensively studied in vitro. We used four complementary DNAs (cDNAs) for multidrug resistance (MDR1), glutathione S-transferase-pi (GST-pi), thymidylate synthase (TS) and dehydrofolate reductase (DHFR), as probes. We observed GST-pi, DHFR and TS mRNA expression at various levels, but MDR1 mRNA expression was found only in SW480/DXR by the method of poly (A+) RNA selection. Four resistant SW480 lines had higher TS mRNA expressions. Six resistant lines had stronger GST-pi mRNA expression. Five resistant lines had higher DHFR mRNA expression. Drug resistance genes related to the treated drug were also expressed in this in vivo model; MDR1 in SW480/DXR, GST-pi in SW480/CDDP and in SC1NU/CDDP and TS in SW480/5-FU. In contrast to in vitro resistant lines which have been reported as models of drug resistance, the expression of drug resistance genes in vivo was not always correlated to the acquisition of cross resistance. These resistant xenograft lines and the methods developed to induce drug resistance in vivo should be useful for studies on the mechanism of drug resistance in the clinical setting.     

In vivo circumvention of human colon carcinoma resistance to bleomycin.

Metabolic inactivation of bleomycin (BLM) by cysteine proteinase-like enzymes is thought to be a major mechanism of BLM tumor resistance. We now report that the human colon carcinoma COLO-205 is highly resistant to BLM and that E-64, a cysteine proteinase inhibitor, sensitizes COLO-205 to BLM. Treatment of COLO-205-bearing nude mice with either E-64 (40 mg/kg) or BLM (10 mg/kg) alone did not inhibit COLO-205 growth. However, pretreatment with E-64 prior to BLM prevented these xenografts from growing. Analysis by high performance liquid chromatography of in vivo BLM metabolism following [3H]BLM A2 treatment of COLO-205-bearing nude mice showed a different metabolic profile among the various organs and the tumor. Whereas [3H]BLM A2 was the only major radioactive peak detected in sera and tumors, several metabolites, including deamido-BLM A2, were found in kidney, liver, and lung as early as 15 min. Pretreatment of mice with E-64 inhibited tumor, kidney, and lung BLM A2 metabolism. Furthermore, pretreatment with E-64 increased BLM A2 accumulation in tumors (6.1-fold), kidney (4.0-fold), lung (2.8-fold), liver (1.8-fold), and serum (1.7-fold). E-64 pretreatment did not enhance the major toxicity of BLM, pulmonary fibrosis, as determined by both lung hydroxyproline levels and histopathology. Thus, the cysteine proteinase inhibitor E-64 affects the metabolic fate and the levels of accumulation of BLM in vivo. These results demonstrate that resistance of human COLO-205 tumors to BLM can be circumvented by E-64 without enhancement of the major side effect of BLM, suggesting a possible clinical use of this combination therapy.     

Camptothecin acts synergistically with imatinib and overcomes imatinib resistance through Bcr-Abl independence in human K562 cells

In this study, we have tried to find new targets and effective drugs for imatinib-resistant chronic myelogenous leukemia (CML) cells displaying loss of Bcr-Abl kinase target dependence. The imatinib-resistant K562/R1, -R2 and -R3 cells showed profound declines of Bcr-Abl level and concurrently exhibited up-regulation of Bcl-2 and Ku70/80, and down-regulation of Bax, DNA-PKcs and BRCA1, suggesting that loss of Bcr-Abl after exposure to imatinib might be accompanied by other cell survival mechanism. K562/R3 cells were more sensitive to camptothecin (CPT)- and radiation-induced apoptosis than K562 cells, indicating hypersensitivity of imatinib-resistant cells to DNA damaging agents. Moreover, when K562 cells were treated with the combination of imatinib with CPT, the level of Bax and the cleavage of PARP-1 and DNA-PK were significantly increased in comparison with the effects of each drug. Therefore, our study suggests that CPT can be used to treat CML with loss of Bcr-Abl expression.