Biochemical characterization of resistance to mitoxantrone and adriamycin in Caco-2 human colon adenocarcinoma cells: a possible role for glutathione S-transferases.

Cytotoxicity of Adriamycin on human colon adenocarcinoma cell lines was investigated. Concentrations of Adriamycin producing 50% inhibition were very similar in HT29, Sw480, Sw620, and Sw1116 cells, whereas Caco-2 cells were relatively insensitive. As compared to the Sw1116 cell line, Caco-2 cells were also insensitive to mitoxantrone. Sensitivity to cisplatin, 5-fluorouracil, or ethacrynic acid was comparable in both cell lines. To find the mechanism for this mitoxantrone and Adriamycin resistance, several potential Adriamycin-detoxifying systems were characterized and quantified in both Sw1116 and Caco-2 cells. No dramatic differences in glutathione content and expression of both selenium dependent- and independent glutathione peroxidase, UDP-glucuronyltransferase, and cytochrome P-450 were found. However, highly significant differences in glutathione S-transferase activity were present, the expression of both class pi and class alpha glutathione S-transferases being much higher in the Caco-2 cell line. In addition, a slightly higher content of P-170 glycoprotein was present in the Caco-2 cells. These findings suggest that glutathione S-transferases, and to a lesser extent the P-170 glycoprotein, may be involved in mitoxantrone and Adriamycin resistance of Caco-2 colon carcinoma cells.     

Glutathione, glutathione-s-transferase and p-170 glycoprotein in metastases of malignant melanomas

P-170 glycoprotein, glutathione and glutathione S-transferases are important in in vitro drug resistance, but their clinical relevance is unclear. Therefore glutathione content, glutathione S-transferase enzyme activity, isoenzyme composition as well as P-170 glycoprotein level were studied in metastases of malignant melanomas of thirteen patients. P-170 glycoprotein and glutathione S-transferases were quantified by immunoblotting with monoclonal antibodies, glutathione S-transferase enzyme activity was measured with 1-chloro-2,4-dinitrobenzene as substrate, and glutathione was assayed by HPLC. Glutathione and glutathione S-transferase enzyme activity were measurable in all samples and mean values were 40+/-7 nmol/mg protein (mean+/-SEM; range: 13-98) and 310+/-72 nmol/min mg protein (range: 15-819), respectively. Glutathione S-transferases present were mainly of class pi (2817+/-402 ng/mg protein); class alpha enzymes were detectable only in one case in low amounts (71 ng/mg protein), and class mu transferases were present in 5 out of the 13 samples (38%; 391+/-206 ng/mg protein). The P-170 glycoprotein plasma membrane located drug efflux pump was found in 8 out of 12 samples (67%). In three samples values were much higher as compared to the other specimens. In the metastatic melanoma of one patient, both high levels of glutathione S-transferase and P-170 glycoprotein were found. Further studies are necessary to reveal whether melanoma tissues containing high levels of P-170 glycoprotein, glutathione S-transferases or a combination of both systems do respond differently towards anti-cancer drug treatment.     

Comparison of glutathione S-transferase activity between drug-resistant and-sensitive human tumor cells: Is glutathione S-transferase associated with multidrug resistance?

Summary We have studied the levels of glutathione S-transferase in drug-resistant and-sensitive human tumor cell lines to examine a possible involvement of glutathione S-transferase (GST) in multidrug resistance mechanisms. No increase in the activity of glutathione S-transferase was detected in myelogenous leukemia K562 resistant to adriamycin (K562/ADM), ovarian carcinoma cell line A2780 resistant to adriamycin (2780AD), or acute lymphoblastic leukemia cell line CCRF-CEM resistant to vinblastine (CEM-VLB100), compared with the drug-sensitive parent tumor cells. The human breast cancer cell lines Hattori and MCF-7 had a 12- to 63-fold lower level of glutathione S-transferase activity than K562, A2780, CCRF-CEM, and their drug-resistant sublines. Induction of ADM resistance in Hattori did not increase the activity of glutathione S-transferase. However, induction of colchicine resistance in MCF-7 resulted in a 70-fold increase in the activity of glutathione S-transferase. A revertant of the colchicine-resistant MCF-7 contained a level of glutathione S-transferase activity similar to that of the resistant subline. The increase of glutathione S-transferase activity did not alter the sensitivity of the cell to cytotoxic drugs. The increased activity was due to the appearance of glutathione S-transferase , as shown by enzyme inhibition using anti-glutathione S-transferase antibody. Our findings indicate that increased cellular glutathione S-transferase activity is not associated with the development of multidrug resistance.Abbreviations GST glutathione S-transferase - ADM adriamycin - VCR vincristine - VLB vinblastine     

阿霉素在小鼠体内诱导S－180细胞株抗药性的实验研究

利用ＢＡＢＬ／ｃ小鼠，腹腔接种Ｓ－１８０瘤细胞，阿霉素腹腔注射治疗１５个周期培育传代，得到抗阿霉素的Ｓ－１８０细胞株（Ｓ－１８０Ｒ）。此抗药细胞对阿霉素的抗药性比亲本细胞提高６６倍。对典型的ＤＮＡ拓扑异构酶Ⅱ抑制刻ＶＰ１６（Ｅｔｏｐｏｓｉｄｅ）抗药性增加９倍。经免疫组化进一步证实，抗药细胞显示多药抗药基因（ＭＤＲｇｅｎｅ）产物Ｐ－１７０糖蛋白过表达。流式萤光细胞仪测试结果也表明抗药细胞比亲本细胞排出药物能力提高８９倍。可以肯定这是ＭＤＲ基因过表达产物所致。本工作为筛选有效逆转抗药功能的药物及其他治疗手段提供了有用的动物模型。     

胃癌耐药细胞EXOs的提取鉴定及可能存在的耐药信息传递作用的初步探讨

目的:探讨胃癌耐药细胞（SGC7901/Adr）分泌的外泌体（exosomes,EXOs）在细胞间可能存在的耐药信息传递作用。方法:选用胃癌亲本细胞SGC7901及其阿霉素耐药细胞SGC7901/Adr为模型,用PEG方法提取EXOs;透射电子显微镜观察鉴定细胞EXOs形态;Western Blot检测CD9、CD63、TSG101、Calreticulin以及P-糖蛋白（P-gp）的表达;运用激光共聚焦显微镜观察胃癌SGC7901和SGC7901/Adr细胞及加入阿霉素耐药细胞SGC7901/Adr外泌体处理后的敏感细胞内阿霉素药物浓度变化。结果:透射电子显微镜下观察到,胃癌SGC7901和SGC7901/Adr细胞来源的EXOs为直径在30~100 nm之间的囊性小泡,呈圆形或椭圆形,大小均匀一致。Western Blot检测胃癌SGC7901和SGC7901/Adr细胞来源的EXOs,携带有外泌体生物标记分子CD9（四次跨膜分子）、CD63（四次跨膜分子）以及TSG101表达,Calreticulin为阴性表达。进一步检测发现在SGC7901/Adr细胞来源的EXOs中存在P-gp蛋白表达。经过阿霉素耐药细胞SGC7901/Adr外泌体处理后的敏感细胞,胞内可见阿霉素聚集减少,核区分布减少,荧光强度减弱。结论:胃癌耐药细胞分泌的EXOs可能通过传递P-gp蛋白的形式,具有传递耐药信息的作用。     

人结肠癌LoVo/Adr细胞耐药性与细胞内Ca2+浓度的关系

背景及目的：基于在耐药细胞中Ca^2 增高,且钙通道拮抗剂维拉帕米可逆转多药耐药性,推测Ca^2 可能在耐药性中起一定的作用。本文的目的是探讨人结肠癌LoVo/Adr细胞耐药性与细胞内Ca^2 浓度之间的关系。方法：以Fluo-3/AM标记LoVo和LoVo/Adr细胞内Ca^2 ,用共聚焦显微镜观察其浓度变化;采用MTT法检测维拉帕米逆转细胞耐药性的伤脑筋和流式细胞仪检测细胞内阿霉素的浓度。结果：LoVo细胞和LoVo/Adr细胞Fluo-3/AM的荧光强度分别为850.45和1495.88;加用5mg/L维拉帕米后,LoVo细胞和LoVo/Adr细胞荧光强度分别为813.25和1284.14。说明耐药株LoVo/Adr细胞内Ca^2 浓度显著高于敏感株LoVo细胞,维拉帕米不能明显显著LoVo/Adr细胞内Ca^2 浓度,但5mg/L维拉帕米可显著增加LoVo/Adr细胞内阿霉素的浓度,对阿霉素和长春新碱的增敏倍数分别是8.85倍和5.31倍。结论：细胞内Ca^2 浓度增高可能是耐药株LoVo/Adr的表型特征之一,但与该细胞株耐药性无直接关系。     

Energy metabolism of human LoVo colon carcinoma cells: correlation to drug resistance and influence of lonidamine.

The relationship between modification of energy metabolism and extent of drug resistance was investigated in two sublines (LoVoDX and LoVoDX10) from human LoVo colon carcinoma cells that exhibit different degrees of resistance to doxorubicin. Results indicated that the extent of alteration in energy metabolism strictly correlated with degree of resistance. In LoVoDX cells, only 14CO2 production was enhanced, whereas in the more resistant LoVoDX10 cells, both 14CO2 and aerobic lactate production were stimulated. The basal and glucose-supported efflux rate and the amount of drug extruded by LoVoDX10 cells were significantly higher than in the resistant LoVoDX cells. Because the expression of surface P-170 glycoprotein was similar in both cell lines, this phenomenon was attributed to increased efflux pump activity resulting from greater ATP availability. Inhibition of 14CO2 production, aerobic glycolysis, and clonogenic activity by lonidamine (LND) increased with enhancement of the energy metabolism. Moreover, LND, by affecting energy-yielding processes, reduced intracellular ATP content, lowered the energy supply to the ATP-driven efflux pump, and inhibited, almost completely, doxorubicin extrusion by resistant LoVo cells. These findings strongly suggest that LND, currently used in tumor therapy, reduces drug resistance by restoring the capacity to accumulate and retain drug of cells with the MDR phenotype that overexpress P-170.     

Bap29varP, a variant of Bap29, influences the cell surface expression of the human P-glycoprotein

P-glycoprotein (Pgp), a plasma membrane (PM) glycoprotein, is responsible for the development of multidrug resistance. The mechanism by which Pgp is targeted to the PM is not defined. To identify proteins that influence Pgp trafficking, we utilized the yeast two-hybrid analysis procedure, which identified a new isoform of endoplasmic reticulum (ER)-bound Bap29, termed Bap29varP, as an interacting protein with the N-terminus of Pgp. The drug-resistant human breast cancer MCF-7 (MCF-7/Adr(R)) cells express both Bap29varP and approximately 170 kDa Pgp, which are however absent in the drug-sensitive MCF-7 cells. When Bap29varP was overexpressed in MCF-7/Adr(R) cells, Pgp was predominantly localized in the ER and intracellular vesicles, suggesting Bap29varP influences Pgp trafficking. When Pgp was expressed in MCF-7 cells, it was exclusively found in the ER with a molecular mass of approximately 160 kDa slightly smaller than that of the molecular mass of Pgp expressed in MCF-7/Adr(R) cells. On the other hand, when Pgp was expressed in Bap29varP-containing human colon adenocarcinoma HT-29 cells, it was localized at the PM. These findings together suggest that Bap29varP acts as an essential chaperone, influencing the processing and trafficking of Pgp to the cell surface.     

Endogenous tumor necrosis factor functions as a resistant factor against adriamycin

One of the mechanisms of cytotoxicity by tumor necrosis factor (TNF) and heat is the induction of reactive oxygen molecules. Cells producing endogenous tumor necrosis factor (enTNF) show resistance to the cytotoxicity of exogenous TNF and heat by inducing manganous superoxide dismutase (MnSOD) to scavenge the reactive oxygen molecules. Intracellular hydroxyl radical production is also involved in adriamycin-induced cytotoxicity. In this study, we therefore examined the possibility that enTNF may act as a protective protein against adriamycin-induced cytotoxicity in a manner similar to that in which it protects against exogenous TNF and heat. Adriamycin-sensitive L-M (mouse tumorigenic fibroblast) cells, originally expressing no enTNF, were transfected with an expression vector which directs the synthesis of non-secretory-type human TNF (enTNF). The stable transformants became resistant to adriamycin with increased levels of MnSOD. Conversely, when HeLa (human uterine cervical cancer) cells, which originally produce an appreciable amount of enTNF, were transfected with an anti-sense TNF mRNA expression vector to inhibit enTNF synthesis, their intracellular MnSOD activity was suppressed and adriamycin sensitivity was enhanced. However, no alterations in expression of multidrug-resistant gene products—P-170 glycoprotein, glutathione S-transferase π (GST-π) and the intracellular concentrations of glutathione (GSH)-were observed in these transfectants as compared to their parent cells. These results indicate that enTNF exerts its intracellular protective effect against adriamycin-induced cytotoxicity by the same mechanism as that against exogenous TNF and heat, namely scavenging reactive oxygen with induced MnSOD.     

GCS在人乳腺癌细胞多药耐药中的作用及与P-gP的关系

目的探讨葡萄糖神经酰胺合成酶（GCS）在人乳腺癌细胞多药耐药中的作用及其与P-糖蛋白（P-gP）的关系。方法采用MTT法检测多柔比星（阿霉素）对人乳腺癌耐药细胞株MCF-7／Adr和敏感株MCF-7的抑制率和IC50。以GCS抑制剂D，L-threo-1-phenyl-2-decanoyl—amino-3-morpholino-1-propanol（PDMP）预处理MCF-7／Adr后检测抑制率和IC50。运用流式细胞术（FCM）检测人MCF-7及MCF-7／Adr中GCS、P-gp的表达，以PDMP预处理细胞后检测GCS、P-gP的表达。FCM法检测细胞中ADM的荧光强度。结果MCF-7／Adr对MCF-7的耐药倍数为22．7倍，PDMP作用后阿霉素对MCF-7／Adr的抑制率升高，IC50下降（P〈0．05）。MCF-7／Adr中GCS和P-gp的表达均高于MCF-7，PDMP使MCF-7／Adr中GCS表达下降（P〈0．05），对P—gp表达无明显影响（P〉0．05）。FCM检测显示PDMP可使阿霉素在MCF-7内潴留增多。结论GCS在MCF-7／Adr多药耐药中起重要作用，PDMP能影响P-gP功能，GCS与P-gP有-定关系。     

LoVo/Adr细胞中sorcin基因的表达与细胞Ca2+浓度的关系

目的：探讨在人结肠癌耐药株LoVo／Adr细胞中可溶性耐药相关钙结合蛋白（soluble resistance related calcium binding protein，sorcin）与Ca^2＋浓度的关系。方法：以Fluo-3／AM标记细胞内Ca^2＋，用共聚焦显微镜观察其浓度变化；采用RT-PCR和免疫组化的方法检测细胞中sorcin基因的mRNA水平和蛋白水平。结果：与亲本LoVo细胞相比，耐药LoVo／Adr细胞中Ca^2＋浓度显著增高（P〈0．01）；sorcin基因在LoVo细胞中几乎不表达，而在耐药LoVo／Adr细胞中，无论mRNA水平还是蛋白水平均显著高表达（P〈0．01）。结论：耐药LoVo／Adr细胞中sorcin高表达没有引起细胞内Ca^2＋浓度的降低。     

Role of free radicals in an adriamycin-resistant human small cell lung cancer cell line

In two Adriamycin (Adr) resistant sublines (GLC4-Adr1 and GLC4-Adr2) of a human small cell lung carcinoma cell line, GLC4, cross-resistance for radiation was found. GLC4-Adr1 has an acquired Adr resistance factor of 44 after culturing without Adr for 20 days and GLC4-Adr2, the same subline cultured without Adr for 3 months, has a decreased but stable resistance factor of 8. One of the assumed mechanisms of Adr is that the effect is mediated through the formation of free radicals. Therefore free radical scavenging might play a role in these Adr resistant cell lines. Adr, H2O2, and X-ray induced cytotoxicity were evaluated. Glutathione (GSH) levels and activities of associated enzymes were determined as well as Adr, H2O2, and X-ray induced DNA breaks and repair. GSH level was decreased in GLC4-Adr1, but restored to the normal level in GLC4-Adr2. Superoxide dismutase, catalase, glutathione-peroxidase, and glutathione S-transferase were not elevated in the resistant sublines. Adr induced a decreased amount of DNA breaks in GLC4-Adr1 compared to GLC4. For X-ray and H2O2 a comparable amount of DNA damage was found. GLC4-Adr1 was able to repair DNA breaks induced by Adr, X-ray, and H2O2 better than GLC4. In conclusion, no increased enzyme capacity for detoxification of free radicals could be detected in the cytosol of the resistant cells. The resistance against free radicals in the GLC4-Adr1 line may at least in part be a result of increased DNA repair.     

Modulation of multidrug resistance by verapamil or mdr1 anti-sense oligodeoxynucleotide does not change the high susceptibility to lymphokine-activated killers in mdr-resistant human carcinoma (LoVo) line

Two sublines were derived from the colon adenocarcinoma line LoVo, the first one was sensitive (LoVo/H) and the second one was made resistant to doxorubicin (LoVo/Dx). When tested for susceptibility to lysis by different types of immune effectors, LoVo/Dx appeared more sensitive than LoVo/H to the killing of CD3+CD5+CD16-, CD3- CD16+)-enriched lymphokine activated killers (LAK) or activated macrophages. In order to check whether this effect was due to different expression of glycoprotein P170 between the two LoVo sublines (30% vs. 90% of positive cells), a pharmacological and genetic modulation of P170 was carried out in LoVo cells. Treatment of LoVo/Dx with the calcium channel blocker verpamil (VRP), strongly impaired P170 function as evaluated by reduced Dx resistance, without affecting the lysability of LoVo/Dx cells by LAKs. Moreover, the significant inhibition of P170 expression resulting from the treatment of LoVo/Dx with mdr1 anti-sense olideoxynucleotide also failed to change the high lysability of LoVo/Dx by LAKs. These results, therefore, indicate that molecules other than P170 are involved in the increased lysis of LoVo/Dx subline by immune effectors and that down-regulation of the P170 expression or function will not reduce the potential effectiveness of cancer chemo-immunotherapy.     

多药耐药乳腺癌细胞MCF-7/Adr中体外转录的小分子干扰RNA(siRNA)对mdr1基因的干扰作用

目的 探讨多药耐药乳腺癌细胞MCF-7／Adr中体外转录的小分子干扰RNA（siRNA）对mdr1基因的干扰作用。方法将siRNA转染多药耐药乳腺癌细胞MCF-7／Adr后，采用共聚焦荧光显微镜、Northernblot和Westernblot分析siRNA在蛋白和mRNA水平对mdr1基因的静止作用；并用MTT法测定siRNA处理后阿霉素对MCF-7／Adr细胞的杀伤作用。结果 特异性siRNA能明显抑制P170蛋白表达及其mRNA，并能明显提高阿霉素对MCF-7／Adr细胞的杀伤作用。结论 siRNA能逆转细胞耐药性，可能将为肿瘤耐药的治疗提供又一新技术。     

In vitro and in vivo modulation of multi-drug resistance with amiodarone.

The modulating effect on drug resistance of amiodarone (AM) and its metabolite desethylamiodarone (DEA) was studied in a P-glycoprotein-positive human colon carcinoma cell line COLO 320, and a human small-cell lung carcinoma cell line GLC4 and its adriamycin (Adr)-resistant subline GLC4-Adr (both P-glycoprotein-negative). AM, DEA and verapamil induced an increase in cytotoxicity of Adr, vincristine and etoposide (VP16) in COLO 320 cells, while in the GLC4 and GLC4-Adr cell line no effect was seen. In the COLO 320 cell line, AM caused more intracellular, and especially intranuclear, fluorescence of Adr and more Adr-induced DNA strand breaks as compared to Adr alone. Moreover, an increase in VP16-induced topoisomerase II-DNA complexes was observed when AM was added. Competition between AM and Adr for the same efflux pump was suggested in efflux studies. The colony-forming unit granulocyte macrophage (CFU-GM) assay showed no increase in cytotoxicity of Adr when AM was added. Fourteen patients with Adr-resistant tumors were treated with Adr and AM. In these patients, peak serum levels of AM plus DEA of 10 microM were reached. Patient serum (20%) obtained after the first i.v. AM infusion induced in vitro significantly more cell kill of Adr in COLO 320 cells. Apart from a transient first-degree AV block in one patient, no cardiac toxicity was observed with the combination of Adr and AM. Bone-marrow toxicity was the same as expected from Adr alone in these patients. One of the 13 evaluable patients obtained a partial remission.     

Leuprorelin acetate affects adhesion molecule expression in human prostate cancer cells

Multidrug resistance is the most predominant phenomenon leading to chemotherapy treatment failure in breast cancer patients. Despite many studies having suggested that overexpression of epidermal growth factor receptor (EGFR) is a potent predictor of malignancy in cancers, systematic research of EGFR in multidrug resistant (MDR) breast cancer cells is lacking. In order to clarify the role of EGFR in MDR breast cancer cells, MCF7/Adr expressing relatively higher EGFR, and its parental cell line MCF7 expressing relatively lower EGFR, were chosen for this study. Knockdown of EGFR by siRNA in MCF7/Adr cells showed that EGFR siRNA inhibits cell migration, invasion and proliferation in vitro; converse effects were observed in MCF7 cells transfected with pcDNA3.0-EGFR plasmid. Moreover, we found that EGFR upregulated migration and invasion via EMMPRIN, MMP2 and MMP9 in addition to promoting cell cycle passage via elevation of cyclin D1 and CDK4 in MDR breast cancer cells. Interestingly, MCF7/Adr cells not expressing EGFR showed significant decrease of P-glycoprotein (P-gp) and ABCG2 expression levels, and became more sensitive to treatment of adriamycin (ADR) and paclitaxel (Taxol); the above results indicated that MDR of cancer cells is related to S-phase arrest. In conclusion, EGFR is an important factor enhancing the malignancy of MDR breast cancer cells, partially, inducing MDR. Anti-EGFR therapy may improve outcome in chemorefractory breast cancer patients.     

The role of the MDR-1/P-170 mechanism in the development of multidrug resistance in chronic myeloid leukemia

We studied blood and bone marrow cells from 42 patients with Ph-chromosome positive chronic myeloid leukemia (CML) and 20 normal subjects for amplification of the multidrug resistance gene (MDR-1) by Southern blotting and for overexpression of P-glycoprotein (P-170) by immunocytochemistry on intact cells with the monoclonal antibody C219. No P-170 could be detected in normal bone marrow or buffy coat. Overexpression of P-170 without amplification of MDR-1 was found in four of 11 patients with chronic phase CML at diagnosis, seven of 16 patients treated with busulfan or hydroxyurea in chronic phase and four of 15 patients in blast crisis. The P-170 overexpression involved only cells of the granulocyte lineage and varied from weak to strong in individual patients. It did not correlate with duration of or response to treatment during chronic phase. In transformation P-170 expression was seen in differentiated cells of the granulocyte lineage but not in blast cells, although three patients had been treated intensively with lipophilic and other cytotoxic drugs to which they had become resistant. We conclude that resistance to busulfan and hydroxyurea in chronic phase and resistance of blast cells to other cytotoxic drugs in transformation are not mediated primarily through the MDR-1/P-170 pathway.     

Isolation by flow cytometry of a human ovarian tumor cell subpopulation exhibiting a high glutathione content phenotype and increased resistance to adriamycin

We have used monochlorobimane as a quantitative marker by which cells of naturally high or low GSH contents were purified by fluorescence-activated cell sorting (FACS). The cell line chosen for this purpose, MLS, was a human ovarian tumor cell line established from a patient who had received extensive chemotherapy and showed evidence of 'multidrug' resistance. Cells of a specified volume were sorted into subpopulations containing the 1% most dim (low GSH) and 1% most bright (high GSH) cells. With an increasing number of sortings, cell subpopulations emerged with progressively lower (dim) and higher (bright) GSH content as compared to the parent population. After 4 sortings, GSH contents were 10.6 +/- 0.8, 5.1 +/- 0.4, and 7.2 +/- 0.7 X 10(-18) moles/micron3 for MLS/bright, MLS/dim and MLS/parent respectively. The high and low GSH phenotypes were of limited stability reverting to the parent phenotype by the sixth week following the last FACS. Cells with high GSH content were more resistant to adriamycin than cells with low GSH content, for example, at 1 log cell kill MLS/bright was 1.6 fold more resistant than MLS/dim. An ADR resistant variant of the MLS line, designated MLS/ADRR/2, established by twice treating MLS cells with 1 microgram/ml ADR for 2 hr, also showed increased GSH content (1.3-fold) and ADR resistance as compared with the parent line. These results illustrate the possible importance of tumor cell GSH status in determining the response to chemotherapy of a heterogenous population of tumor cells with diverse GSH contents.     

The leukocyte protein L-plastin induces proliferation, invasion and loss of E-cadherin expression in colon cancer cells

L-plastin, a gene that codes for an actin-bundling protein, is upregulated in the metastatic colon cancer cell line SW620, when compared to its premetastatic counterpart SW480. The aim of our study was to characterise the effect of L-plastin overexpression on SW480 cells in the context of the acquisition of a metastatic phenotype. SW480 cell lines overexpressing L-plastin were established (SW480-LPL). Analysis of these cell lines revealed significantly higher rates of proliferation and invasion than the control cell line (SW480-Ctrl). In addition, the expression of E-cadherin was lost from SW480-LPL cells. Treatment of SW480-LPL cells with cytochalasin B, an inhibitor of endocytosis, attenuated the loss of E-cadherin expression in these cells. The association of L-plastin overexpression with an increased rate of proliferation and invasion, and loss of E-cadherin expression in the SW480 colon cancer cell line indicates that L-plastin plays an important mechanistic role in colorectal cancer metastasis (supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html).     

Bcl-2 has differing effects on the sensitivity of breast cancer cells depending on the antineoplastic drug used

The aim of this paper was to evaluate the role of bcl-2 in the susceptibility of the MCF7 ADR human breast carcinoma line overexpressing the P-170 glycoprotein (P-170) to various drugs. The sensitivity to four multidrug resistance (MDR)-related drugs (doxorubicin (ADR), vincristine (VCR), vinblastine (VBL), actinomycin D (ACTD)) and three MDR-non-related drugs (cisplatin (DDP), bischloroethylnitrosourea (BCNU), 5-fluorouracil (5-FU)) was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay in three bcl-2-overexpressing clones obtained from the MCF7 ADR line. We found that the bcl-2-overexpressing clones show increased resistance to DDP and BCNU, while no difference to 5-FU were observed between the control cells and bcl-2 transfectants. Surprisingly, bcl-2-overexpressing clones displayed an increased sensitivity compared with the control cells to the MDR-related drugs ADR, VCR, VBL and ACTD. Focusing on DDP and ADR, we found that the increased resistance of the bcl-2 transfectants to DDP was correlated to their ability to prevent apoptosis, while the enhanced sensitivity to ADR was associated with an increased ADR accumulation and a decreased ADR efflux. Moreover, while bcl-2 overexpression does not induce changes in P-170 glycoprotein expression, it did induce a reduction of the adenosine triphosphate (ATP) levels and basal protein kinase C (PKC) activity, both of which have a crucial role in the regulation of the MDR phenotype. In conclusion, the effect of bcl-2 on antineoplastic sensitivity observed in this study underscores the idea that bcl-2 may have distinct biological effects depending on the anticancer drug used.