The riminophenazine agents clofazimine and B669 inhibit the proliferation of intrinsically multidrug resistant carcinoma cell lines

The sensitivity to clofazimine, B669 and eight standard anti-tumour chemotherapeutic agents of four intrinsically multidrug resistant (MDR) cell lines [three human hepatocellular carcinoma cell lines (HepG2, PLC and Mahlavu) and a human colorectal carcinoma cell line (CaCo2)] were compared with that of a human cervix epithelioid carcinoma cell line (HeLa). The MDR cell lines and HeLa cells showed equivalent sensitivity to the riminophenazines, methotrexate and 5-fluorouracil, but exhibited ranging levels of resistance to vinblastine, doxorubicin, mitomycin C, daunorubicin, etoposide and vincristine. Riminophenazines may be useful agents in the chemotherapy of tumours with intrinsic MDR.     

Three-dimensional culture system as a model for studying cancer cell invasion capacity and anticancer drug sensitivity

BACKGROUND: Three-dimensional (3-D) culture systems that simulate the tumor extracellular microenvironment may be appropriate to test cancer cell potential for invasion and tumor cell sensitivity to anticancer drugs. MATERIALS AND METHODS: Human PC-3 prostate, A549 colon, HT-29 lung and MCF-7 and MDA-MB231 breast cancer cells were embedded and grown in collagen gel surrounded by a fibrin clot. Increasing concentrations of cisplatin, doxorubicin, paclitaxel and 5-fluorouracil were comparatively evaluated for their ability to inhibit tumor cell proliferation and colony formation in vitro. RESULTS: All cells, except MDA, formed colonies in collagen. PC-3, A549 and HT-29 cells massively invaded fibrin forming migratory fronts. Cell colonies were also formed in fibrin (secondary tumor-like structures) apart from migratory fronts; HT-29 cells were the most aggressive in this regard MDA cells were particularly sensitive to doxorubicin, while MCF-7 cells showed sensitivity to all anticancer regimens tested. A549 cells were the tumor cell type with greatest potential for invasion and were sensitive mostly to cisplatin. PC-3 cells were primarily sensitive to cisplatin and doxorubicin, while HT-29 cells were sensitive to fluorouracil and doxorubicin. CONCLUSION: 3-D collagen cell culture systems can be used to study cancer cell potential for invasion and their relative sensitivity/resistance to anticancer drugs.     

Rapid up-regulation of mdr1 expression by anthracyclines in a classical multidrug-resistant cell line.

Studies were carried out in a variant human multidrug-resistant (MDR) cell line CEM/A7R, which expresses very low levels of mdr1 mRNA and P-glycoprotein (P-gp). The induction of mdr1 RNA expression by three anthracyclines, (doxorubicin, daunorubicin, epirubicin), VP-16 and two vinca alkaloids (vincristine, vinblastine) was semiquantitatively assessed by scanning Northern blots on a phosphorimager. The relative level of mdr1 expression was expressed as ratio of mdr1 to the internal RNA (actin). A significant increase (P < 0.02) in expression of mdr1 was noted within 4 hrs of exposure to 1.5 micrograms ml-1 daunorubicin or epirubicin. Neither vinblastine nor vincristine had any effect on mdr1 levels after an 8 h exposure. With increasing concentrations of daunorubicin or epirubicin in a fixed 24 h time period, mdr1 expression increased, although a biphasic response was seen. Based on MRK 16 binding, an increase in P-gp levels was seen in the CEM/A7R line after a 24 h exposure to 1 microgram ml-1 daunorubicin or epirubicin. The rapid increase in mdr1 expression after a short period of exposure to doxorubicin, daunorubicin or epirubicin suggests that induction of mdr1 expression may have an important role in the development of drug-resistant tumours.     

Lack of a role for MRP1 in platinum drug resistance in human ovarian cancer cell lines.

The level of expression of the multidrug resistance-associated protein (MRP1) in a panel of human ovarian carcinoma cell lines and their variants with acquired cisplatin resistance was determined using Western blotting. No overexpression of MRP1 was detected in any of the cell lines. In addition, we have transfected the MRP1 gene into an intrinsically cisplatin-resistant cell line SKOV3, previously shown to have elevated levels of glutathione (GSH). The MRP1-transfected line SKOV3-S2 was shown to be cross-resistant to doxorubicin, vincristine and etoposide but not to paclitaxel, vinblastine and platinum agents, such as cisplatin, JM216 [bis-acetato-ammine-dichloro-cyclohexylamine platinum (IV)] and AMD473 [cis-ammine dichloro (2-methyl-pyridine) platinum (II)]. No cross-resistance to any of the platinum agents was observed in a MRP1-overexpressing human lung cancer cell line with acquired doxorubicin resistance. Reduction of GSH levels (80-90%) by buthionine sulphoximine (BSO) produced significant potentiation in cisplatin sensitivity in the parental SKOV3, the vector-alone control SKOV3-puro and the MRP1-transfected line SKOV3-S2. The degree of sensitization was similar in all cell lines (1.6-fold). However, selective sensitization by BSO to vincristine was observed in the MRP1-transfected line (4.1-fold) but not in the vector control. No significant differences were observed in cisplatin accumulation in the SKOV3-puro and the SKOV3-S2 cells, although both these transfected lines accumulated significantly more than the parental line. Our results suggest that MRP1 does not play a significant role in platinum resistance in the human tumour cell lines investigated in this study.     

The acridonecarboxamide GF120918 potently reverses P-glycoprotein-mediated resistance in human sarcoma MES-Dx5 cells

The doxorubicin-selected, P-glycoprotein (P-gp)-expressing human sarcoma cell line MES-Dx5 showed the following levels of resistance relative to the non-P-gp-expressing parental MES-SA cells in a 72 h exposure to cytotoxic drugs: etoposide twofold, doxorubicin ninefold, vinblastine tenfold, taxotere 19-fold and taxol 94-fold. GF120918 potently reversed resistance completely for all drugs. The EC50s of GF120918 to reverse resistance of MES-Dx5 cells were: etoposide 7+/-2 nM, vinblastine 19+/-3 nM, doxorubicin 21+/-6 nM, taxotere 57+/-14 nM and taxol 91+/-23 nM. MES-Dx5 cells exhibited an accumulation deficit relative to the parental MES-SA cells of 35% for [3H]-vinblastine, 20% for [3H]-taxol and [14C]-doxorubicin. The EC50 of GF120918, to reverse the accumulation deficit in MES-Dx5 cells, ranged from 37 to 64 nM for all three radiolabelled cytotoxics. [3H]-vinblastine bound saturably to membranes from MES-Dx5 cells with a KD of 7.8+/-1.4 nM and a Bmax of 5.2+/-1.6 pmol mg(-1) protein. Binding of [3H]-vinblastine to P-gp in MES-Dx5 membranes was inhibited by GF120918 (K = 5+/-1 nM), verapamil (Ki = 660+/-350 nM) and doxorubicin (Ki = 6940+/-2100 nM). Taxol, an allosteric inhibitor of [3H]-vinblastine binding to P-gp, could only displace 40% of [3H]-vinblastine (Ki = 400+/-140 nM). The novel acridonecarboxamide derivative GF120918 potently overcomes P-gp-mediated multidrug resistance in the human sarcoma cell line MES-Dx5. Detailed analysis revealed that five times higher GF120918 concentrations were needed to reverse drug resistance to taxol in the cytotoxicity assay compared to doxorubicin, vinblastine and etoposide. An explanation for this phenomenon had not been found.     

Liver metastasis models of colon cancer for evaluation of drug efficacy using NOD/Shi-scid IL2Rgammanull (NOG) mice

To examine the drug efficacy of a novel farnesyltransferase inhibitor (FTI), CH4512600, in vivo, we developed a reliable liver metastasis model of human colon cancer using NOD/Shi-scid IL2Rgamma(null) (NOG) mice. Eleven human colon cancer cell lines were examined for their ability to form diverse metastatic foci in the livers of NOG mice. When inoculated with 10(4) COLO320DM, HCT 116, HT-29, WiDr, LoVo and LS174T cells, liver metastasis was evident in 100% (6/6), 100% (6/6), 88.9% (8/9), 87.5% (7/8), 83.3% (5/6) and 50.0% (3/6) of the NOG mice, respectively. CaCo2, COLO201, LS123, SW48 and SW1417 showed no metastasis when seeded at 10(4) cells even in NOG mice. The mRNA expression levels and genetic mutations of N, H and K-RAS genes, which directly affect the levels of cellular RAS protein that would be molecular target for FTI, were also examined in these six metastatic human colon cancer cell lines for molecular biological and genotypic characteristics. Only three cell lines had a point mutation in the RAS oncogene. LS174T cell line had a point mutation of the K-RAS gene at codon 12 (gly12 --> asp; G12D), and HCT 116 and LoVo cell lines had a point mutation of the K-RAS gene at codon 13 (gly13 --> asp; G13D). Relative gene expression levels of N, H and K-RAS genes in the HCT 116 cell line were 2.6-5.0-fold lower than that of LS174T and LoVo cell lines. We selected HCT 116 cell line from our liver metastasis model for evaluation of FTI CH4512600 efficacy in vivo. Using the NOG mouse liver metastasis model, we demonstrated the effectiveness of FTI CH4512600 to suppress tumor growth in vivo and to prolong mouse survival significantly from 36.9+/-2.9 to 50.3+/-9.4 days.     

AQP5 silencing suppresses p38 MAPK signaling and improves drug resistance in colon cancer cells

It is known that aquaporin 5 (AQP5) may represent a novel therapeutic target for treating colon cancer (CC), but whether AQP5 plays a role in the regulation of multidrug resistance (MDR) of colon cancer still remains unclear. In the present study, AQP5 and P-glycoprotein (P-gp), glutathione S-transferase-π (GST-π), topoisomerase II (TOPO II), and thymidylate synthase (TS) were checked in CC and adjacent cancer tissues; AQP5-siRNA was applied to silencing AQP5 in CC cell line HT-29, 5-fluorouracil (5-FU), and cisplatin (DDP) added on cells, and sulforhodamine B (SRB) was used; fluorescence real-time quantitative RT-PCR and Western blot were employed to detect changes in multidrug resistance factor and expression mitogen-activated protein kinase (MAPK) signaling pathway in HT-29. The results showed that AQP5 is significantly induced in cancer tissues than that in adjacent cancer tissues. The expression of AQP5 is positively correlated with drug resistance factors, as demonstrated by the increased expressions of P-gp, GST-π, and TOPO II in CC tissues compared to that in adjacent cancer tissues. Conversely, knockdown of AQP5 in HT-29 human colon cancer cells increased inhibition rates of cancer chemotherapeutic drugs such as 5-FU and DDP. The improved efficacies of chemotherapeutic drugs are associated with the decreased expression of P-gp, GST-π, and TOPO II. In addition, phosphorylation of p38 MAPK was increased by knockdown of AQP5 in HT-29 cells while phosphorylation and expression of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and Protein kinase B (AKT) were not affected. P38 MAPK inhibitor increased the drug sensitivity of HT-29 cells in a similar way as AQP5-siRNAs do. So these results indicate that AQP5 is associated with drug resistance of colon cancer, and that the AQP5-P38 MAPK pathway may represent a potential drug target to improve drug resistance of colon cancer cells.     

Treatment of human colon cancer xenografts with TRA-8 anti-death receptor 5 antibody alone or in combination with CPT-11.

PURPOSE: This study was designed to evaluate the in vitro cytotoxicity and in vivo efficacy of TRA-8, a mouse monoclonal antibody that binds to the DR5 death receptor for tumor necrosis factor-related apoptosis-inducing ligand (also called Apo2L), alone and in combination with CPT-11, against human colon cancer cells and xenografts. EXPERIMENTAL DESIGN: DR5 expression was assessed on human colon cancer cell lines using flow cytometry, and cellular cytotoxicity after TRA-8 treatment, alone and in combination with SN-38, was determined by measuring cellular ATP levels. Tumor growth inhibition and regression rates of well-established subcutaneous COLO 205, SW948, HCT116, and HT-29 colon cancer xenografts in athymic nude mice treated with TRA-8 or CPT-11 alone and in combination were determined. (99m)Tc-TRA-8 was used to examine tumor localization of TRA-8 in animals bearing each of the four xenografts. In addition, whole-body biodistribution and imaging was carried out in COLO 205-bearing animals using in vivo single-photon emission computed tomography imaging and tissue counting. RESULTS: DR5 expression was highest on HCT116, intermediate on SW948 and COLO 205 cells, and lowest on HT-29 cells. COLO 205 cells were the most sensitive to TRA-8-induced cytotoxicity in vitro, SW948 and HCT116 cell lines were moderately sensitive, and HT-29 cells were resistant. Combination treatment with TRA-8 and SN-38 produced additive to synergistic cytotoxicity against all cell lines compared with either single agent. The levels of apoptosis in all cell lines, including HT-29, were increased by combination treatment with SN-38. In vivo, combination therapy with TRA-8 and CPT-11 was superior to either single-agent regimen for three of the xenografts: COLO 205, SW948, and HCT116. COLO 205 tumors were most responsive to therapy with 73% complete regressions after combination therapy. HT-29 cells derived no antitumor efficacy from TRA-8 therapy. Tumor xenografts established from the four colon cancer cell lines had comparable specific localization of (99m)Tc-TRA-8. CONCLUSIONS: In vitro and in vivo effects of TRA-8 anti-DR5 monoclonal antibody on four different colon cancer cell lines and xenografts were quite variable. The HT-29 cell line had low surface DR5 expression and was resistant to TRA-8 both in vitro and in vivo. Three cell lines (COLO 205, SW948, and HCT116) exhibited moderate to high sensitivity to TRA-8-mediated cytotoxicity which was further enhanced by the addition of SN-38, the active metabolite of CPT-11. In vivo, the combination of TRA-8 and CPT-11 treatment produced the highest antitumor efficacy against xenografts established from the three TRA-8-sensitive tumor cell lines. All four colon cancer xenografts had comparable localization of (99m)Tc-TRA-8. These studies support the strategy of TRA-8/CPT-11 combined treatment in human colon cancer clinical trials.     

Pharmacokinetic profile of the microtubule stabilizer patupilone in tumor-bearing rodents and comparison of anti-cancer activity with other MTS in vitro and in vivo

Introduction  Patupilone is a microtubule stabilizer (MTS) currently in clinical development. Here, we evaluate the anti-cancer activity in vitro and in vivo in comparison to paclitaxel and describe the pharmacokinetics (PK) of patupilone in tumor-bearing nude mice and rats. Methods  The potency in vitro of patupilone and two other MTS, paclitaxel and ixabepilone, was determined using human colon carcinoma cell lines with low (HCT-116, HT-29, RKO) and high (HCT-15) P-glycoprotein expression (P-gp), as well as two multi-drug resistance (MDR) model cell pairs, MCF7/ADR and KB-8511 cells and their respective drug-sensitive parental counterparts. The PK of patupilone was investigated in nude mice bearing HCT-15 or HT-29 xenografts and in rats bearing s.c. pancreatic CA20498 tumors or A15 glioma tumors. Anti-cancer activity in vivo was compared to that of paclitaxel using three different human tumor colon models. The retention and efficacy of patupilone was compared in small and large HT-29 xenografts whose vascularity was determined by non-invasive magnetic resonance imaging. Results  Patupilone was highly potent in vitro against four different colon carcinoma cell lines including those showing multi-drug-resistance. In contrast, paclitaxel and ixabepilone displayed significantly reduced activity with markedly increased resistance factors. In both rats and mice, a single i.v. bolus injection of patupilone (1.5–4 mg/kg) rapidly distributed from plasma to all tissues and was slowly eliminated from muscle, liver and small intestine, but showed longer retention in tumor and brain with no apparent elimination over 24 h. Patupilone showed significant activity against three human colon tumor models in vivo, unlike paclitaxel, which only had activity against low P-gp expressing tumors. In HT-29 tumors, patupilone activity and retention were independent of tumor size, blood volume and flow. Conclusions  The high potency of patupilone, which is not affected by P-gp expression either in vitro or in vivo, and favorable PK, independent of tumor vascularity, suggest that it should show significant activity in colorectal cancer and in other indications where high P-gp expression may compromise taxane activity.     

Effects of carvedilol on MDR1-mediated multidrug resistance: comparison with verapamil

The reversing effects of carvedilol and other β-adrenoceptor antagonists on multidrug resistance (MDR) were assessed in HeLa cells and the MDR1-overexpressing derivative Hvr100–6 cells, established by stepwise increases of vinblastine concentration in the culture medium. The inhibitory effects on the transcellular transport and intracellular accumulation of [³H]vinblastine and [³H]daunorubicin were also assessed using LLC-GA5-COL150 cell monolayers, established by transfection of human MDR1 cDNA into porcine kidney epithelial LLC-PK, cells. The cytotoxic effects of vinblastine, paclitaxel, doxorubicin and daunorubicin in Hvr100–6 were reversed 1.4- to 7.1-fold by carvedilol at the realistic clinical concentration of 1 μM , whereas other β-adrenoceptor antagonists had weaker or no such effects. Transport experiments using LLC-GA5-COL150 cell monolayers demonstrated that this effect of carvedilol was due to the inhibition of MDR1-mediated transport of vinblastine, paclitaxel, doxorubicin and daunorubicin. These MDR1-mediated reversing effects of carvedilol were similar to those of 1 μM verapamil, suggesting that carvedilol could be a candidate modulator of MDR in clinical use. Since other β-adrenoceptor antagonists had no inhibitory effect on transport, the effects of carvedilol were not related to β-adrenoceptors and might have been due to antioxidant activity. (Cancer Sci 2003; 94: 81–86)     

Enhanced in vitro invasiveness and drug resistance with altered gene expression patterns in a human lung carcinoma cell line after pulse selection with anticancer drugs

The human lung carcinoma cell line DLKP was exposed to sequential pulses of 10 commonly used chemotherapeutic drugs (VP-16, vincristine, taxotere, mitoxantrone, 5-fluorouracil, methotrexate, CCNU, BCNU, cisplatin and chlorambucil); resulting cell lines exhibited resistance to the selecting agents (ranging approx. 1.5- to 36-fold) and, in some cases, cross-resistance to methotrexate (approx. 1.4- to 22-fold), vincristine (1.6- to 262-fold), doxorubicin (Adriamycin, approx. 1.1- to 33-fold) and taxotere (approx. 1.1- to 36-fold). Several of the variants displayed collateral sensitivity to cisplatin. A marked increase in in vitro invasiveness and motility was observed with variants pulsed with mitoxantrone, 5-fluorouracil, methotrexate, BCNU, cisplatin and chlorambucil. There was no significant change in invasiveness of cells pulsed with VP-16, vincristine, taxotere or CCNU. All of the pulse-selected variants showed elevated levels of MDR-1/P-gp protein by Western blot analysis, although mdr-1 mRNA levels were not increased (except for DLKP-taxotere). In DLKP-taxotere, MRP1 protein levels were also greatly elevated, but mrp1 mRNA levels remained unchanged. BCRP was upregulated in DLKP-mitoxantrone at both the mRNA and protein levels. Gelatin zymography, Western blot and RT-PCR showed that DLKP and its variants secreted MMPs 2, 9 and 13. MMP inhibition assays suggested that MMP-2 plays a more important role than MMPs 9 and 13 in cell invasion of these DLKP drug-resistant variants in vitro. These results indicate that drug exposure may induce not only resistance but also invasiveness in cancer cells.     

In-vitro cytotoxicity in ovarian-cancer of low-dose continuous exposure to microtubule inhibitors (Paclitaxel taxotere) combined with Cisplatin

We present our data on the cytotoxicity of microtubule inhibitors (paclitaxel and taxotere) and their mode of action against cisplatin-sensitive or -resistant ovarian cancer cell lines in vitro. To try to establish an optimal administration schedule in clinical use, the cytotoxicity of microtubule inhibitors alone and in sequential combination with cisplatin was investigated. With microtubules alone, a marked dose- and schedule-dependent growth inhibition was demonstrated in lower concentrations and only schedule-dependency in higher concentrations. The cytotoxicity of taxotere was approximately 0.8- to 132-fold that of paclitaxel. No cross resistance to cisplatin was observed. In the relation between assay AUC (area under the concentration curve) and growth inhibition, increasing AUC by dose escalation seemed not to be advantageous. In a combination with cisplatin, the treatment of microtubules over 48 h followed by cisplatin administration demonstrated the most effective cytotoxicity in cisplatin-resistant cell line.     

HERG钾通道在肿瘤细胞的表达与阿霉素化疗敏感性的关系及红霉素的调节作用

背景与目的:HERG钾通道在许多肿瘤组织中表达,而在肿瘤起源的相应正常组织中却是低表达或不表达。本研究探讨HERG钾通道蛋白在肿瘤细胞的表达及其与阿霉素化疗敏感性的关系,同时观察红霉素的生化调节作用。方法:采用Westernblot法检测HERG钾通道蛋白在肿瘤细胞的表达情况,经过质粒的分离和纯化、基因转染技术、MTT法研究HERG钾通道蛋白的表达与阿霉素的抗肿瘤作用的关系,同时采用MTT法检测红霉素的抗肿瘤作用及其与阿霉素联用时的协同作用,荧光显微镜观察阿霉素进入肿瘤细胞的情况。结果:HERG在不同肿瘤细胞的表达量不同,表达较高的HT鄄29细胞对阿霉素敏感性比表达较低的A549细胞弱。将herg基因转染入表达较低的A549细胞后,从IC50来看,阿霉素的增殖抑制作用明显降低。红霉素能明显抑制HT鄄29细胞的增殖,与阿霉素联用能协同抑制HT鄄29细胞的增殖。herg转染入A549细胞后,阿霉素在肿瘤细胞内的含量基本没有改变。结论:HERG钾通道蛋白的表达与阿霉素的化疗敏感性可能呈一种负相关。红霉素对HERG高表达的HT鄄29细胞增殖具有明显的抑制作用,而对HERG低表达的A549细胞基本无作用,且与阿霉素联用存在明显的协同作用。     

RBM8A在人结肠癌细胞株中的表达及其意义

目的 检测RBM8A在不同结肠癌细胞株中的表达,为进一步体外实验筛选靶细胞。方法 采用实时定量反转录聚合酶连锁反应（qRT-PCR）法以及蛋白免疫印迹（Western-blot）法检测RBM8A在结肠癌细胞株SW480、SW620、HT29、LOVO、COLO320DM、T84及人正常结肠上皮细胞株NCM460中的表达。结果 RBM8A在7株细胞株中均有表达,其中在结肠癌细胞株SW620和COLO320DM中的表达高于正常细胞株,差异有统计学意义（P＜0.05）,而其余细胞株中RBM8A的表达与正常细胞株差异无统计学意义（P＞0.05）。结论 RBM8A在结肠癌细胞株SW620和COLO320DM中高表达,SW620和COLO320DM细胞株或可作为靶细胞。     

结肠癌 COLO 耐药株的构建及其与肿瘤干细胞的关系

目的：构建结肠癌 COLO 耐药株,探索其功能特性及其与肿瘤干细胞的关系。方法通过逐渐递增化疗药5-氟尿嘧啶（5-FU）浓度的方法,构建了耐0．10μmol／ml 和0．20μmol／ml 5-FU 的结肠癌 COLO 耐药株各1株,分别命名为 COLO／5-FU-1、COLO／5-FU-2。从增殖能力、克隆形成能力、迁移和侵袭能力、微球体形成能力、干性基因的表达、交叉耐药等方面与亲本结肠癌细胞株 COLO 进行对比。结果在存活检测中,常规培养后第4天,结肠癌耐药株 COLO／5-FU-2、COLO／5-FU-1、亲本 COLO 细胞的 A 值分别为0．61±0．13、0．54±0．07、0．41±0．09,差异有统计学意义（F ＝63．43,P ＝0．033）。随着耐5-FU 药物浓度的增加,COLO 耐药株的克隆形成率逐渐增强,COLO／5-FU-2、COLO／5-FU-1、亲本 COLO的克隆形成率依次为（87．6±12．7）％、（65．3±9．7）％、（38．5±7．6）％,差异有统计学意义（F ＝33．64, P ＝0．017）。COLO／5-FU-2、COLO／5-FU-1、亲本 COLO 每高倍视野中迁移穿过基底膜的细胞数分别为（482±39）个、（434±45）个、（373±38）个,侵袭穿过基底膜的细胞数分别为（174±42）个、（112±31）个、（87±29）个,差异均有统计学意义（F ＝109．61,P ＝0．009;F ＝67．31,P ＝0．032）。与亲本结肠癌细胞株COLO 比较,耐药株表达更高的干性基因,差异有统计学意义（F ＝47．31,P ＝0．042）;且耐药株对其他化疗药如米托蒽醌存在交叉耐药[如给药96 h,米拖蒽醌对亲本 COLO 的抑制率高于 COLO／5-FU-1、COLO／5-FU-2（0．749±0．042、0．423±0．024、0．342±0．018）,差异有统计学意义（F ＝12．61,P ＝0．028）]。COLO／5-FU-2、COLO／5-FU-1、亲本 COLO 细胞的微球体形成率依次为（8．90±0．97）％、（6．20±0．75）％、（3．90±0．32）％,差异有统计学意义（F ＝164．32,P ＝0．006）。结论结肠癌 COLO耐药株有肿瘤干细胞样细胞的特点,是富集了的肿瘤干细胞。     

Discovery and characterization of OC144-093, a novel inhibitor of P-glycoprotein-mediated multidrug resistance

OC144-093 is a novel substituted diarylimidazole (Mr 495) generated using the OntoBLOCK system, a solid-phase combinatorial chemistry technology, in combination with high-throughput cell-based screening. OC144-093 reversed multidrug resistance (MDR) to doxorubicin, paclitaxel, and vinblastine in human lymphoma, breast, ovarian, uterine, and colorectal carcinoma cell lines expressing P-glycoprotein (P-gp) with an average EC50 of 0.032 microM. Inhibition of MDR by OC144-093 was reversible, but the effect persisted for at least 12 h after removal of compound from the culture medium. OC144-093 had no effect on the response to cytotoxic agents by cells in vitro lacking P-gp expression or expressing a multidrug resistance-associated protein (MRP-1). OC144-093 was not cytotoxic by itself against 15 normal, nontransformed, or tumor cell lines, regardless of P-gp status, with an average cytostatic IC50 of >60 microM. OC144-093 blocked the binding of [3H]azidopine to P-gp and inhibited P-gp ATPase activity. The compound was >50% p.o. bioavailable in rodents and dogs and did not alter the plasma pharmacokinetics of i.v.-administered paclitaxel. OC144-093 increased the life span of doxorubicin-treated mice engrafted with MDR P388 leukemia cells by >100% and significantly enhanced the in vivo antitumor activity of paclitaxel in MDR human breast and colon carcinoma xenograft models, without a significant increase in doxorubicin or paclitaxel toxicity. The results demonstrate that OC144-093 is an orally active, potent, and nontoxic inhibitor of P-gp-mediated multidrug resistance that exhibits all of the desired properties for treatment of P-gp-mediated MDR, as well as for prevention of MDR prior to selection and/or induction of refractory disease.     

Induced resistance in the human non small cell lung carcinoma (NCI-H460) cell line in vitro by anticancer drugs

Exposure of human non-small cell lung cancer cells (NCI-H460) to gradually increasing concentrations of doxorubicin resulted in the appearance of a new cell line (NCI-H460/R) that was resistant to doxorubicin (96.2-fold) and cross-resistant to etoposide, paclitaxel, vinblastine and epirubicin. Slight cross-resistance to two MDR-unrelated drugs 8-Cl-cAMP and sulfinosine was observed. Flow cytometry analysis showed that the accumulation of doxorubicin in the resistant cells was 88.4% lower than in the parental cells. Also, verapamil significantly decreased the efflux rate in NCI-H460 and NCI-H460/R cells, whereas curcumin inhibited the efflux in NCI-H460 cells only. Gene expression data confirmed the induction of mdr1 (P-gp), as judged by the observed 15-fold increase in its mRNA concentration in doxorubicin-resistant NCI-H460/R cells. In contrast, mrp1 and lrp expression was unaffected by the doxorubicin resistance. Further work should develop a rationale for a novel treatment of NSCLC with appropriate modulators of resistance aimed at improving the outcome of the acquired drug resistance.     

Expression of protein kinase C beta1 confers resistance to TNFalpha- and paclitaxel-induced apoptosis in HT-29 colon carcinoma cells.

The expression of different protein kinase C (PKC) isoenzymes has been shown to vary with proliferation rates, differentiation or apoptosis in normal colon crypts. In addition, the activity of some PKC isoenzymes appears to be reduced in colorectal cancer. The aim of the present work was to determine whether modulation of PKC expression would affect the susceptibility of a p53-defective colon carcinoma cell line to different apoptotic treatments. HT-29 cells exhibited sensitivity to paclitaxel (Taxol) and tumor necrosis factor alpha (TNFalpha) in a dose- and time-dependent manner but were relatively resistant to etoposide. Inhibition of PKC activity augmented the susceptibility of HT-29 cells to apoptosis, and phorbol ester induction of PKC reduced such susceptibility. Transfected HT-29(PKC) cells, hyper-expressing the beta1 isoform of PKC, were less sensitive to TNFalpha and paclitaxel than the normal counterpart. The present data 1) indicate that the expression of PKC influences the susceptibility of HT-29 colon cancer cells to apoptotic drugs apparently regardless of their mechanism of action, and 2) suggest paclitaxel as a potential candidate for the treatment of colon cancer, possibly in association with inhibitors of PKC (alpha and beta) at doses not cytotoxic per se.