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。     

Preferential radiosensitization of human prostatic carcinoma cells by mild hyperthermia

PURPOSE: Recent cell culture studies by us and others suggest that some human carcinoma cells are more sensitive to heat than are rodent cells following mild hyperthermia. In studying the cellular mechanism of enhanced thermosensitivity of human tumor cells to hyperthermia, prostatic carcinoma cells of human origin were found to be more sensitive to mild hyperthermia than other human cancer cells. The present study was designed to determine the magnitude of radiosensitization of human prostatic carcinoma cells by mild hyperthermia and to examine whether the thermal radiosensitization is related to the intrinsic thermosensitivity of cancer cells. METHODS AND MATERIALS: Two human prostatic carcinoma cell lines (DU-145 and PC-3) and other carcinoma cells of human origin, in particular, colon (HT-29), breast (MCF-7), lung (A-549), and brain (U-251) were exposed to temperatures of 40-41 degrees C. Single acute dose rate radiation and fractionated radiation were combined with mild hyperthermia to determine thermal radiosensitization. The end point of the study was the colony-forming ability of single-plated cells. RESULTS: DU-145 and PC-3 cells were found to be exceedingly thermosensitive to 41 degrees C for 24 h, relative to other cancer cell lines. Ninety percent of the prostatic cancer cells were killed by a 24 h heat exposure. Prostatic carcinoma cells exposed to a short duration of heating at 41 degrees C for 2 h resulted in a substantial enhancement of radiation-induced cytotoxicity. The thermal enhancement ratios (TERs) of single acute dose radiation following heat treatment 41 C for 2 h were 2.0 in DU-145 cells and 1.4 in PC-3 cells. The TERs of fractionated irradiation combined with continuous heating at 40 degrees C were similarly in the range of 2.1 to 1.4 in prostate carcinoma cells. No significant radiosensitization was observed in MCF-7 and HT-29 cells under the same conditions. CONCLUSION: The present data suggest that a significant radiosensitization of prostatic cancer cells could be obtained by the combined treatment of radiation and mild hyperthermia. Future clinical trials should be aimed at achieving mild heating and fractionated radiation therapy.     

RNAi沉默RON基因对人结肠癌HT-29细胞侵袭和耐药性的抑制作用

目的:探讨RNAi沉默酪氨酸激酶受体RON(recepteur d'origine nantais)基因对人结肠癌HT-29细胞侵袭和对抗肿瘤药物敏感性的影响.方法:构建RON基因的RNAi慢病毒载体Lv-RON-siRNA.Real-time PCR和Western blotting检测RON基因的沉默效率及RON蛋白表达水平;Transwell侵袭实验和ATP-TCA(ATP-tumor chemosensitivity assay)检测RON基因对HT-29细胞侵袭和对药物敏感性的影响.结果:慢病毒载体Lv-RON-siRNA感染HT-29细胞对RON基因的沉默效果达到70％.Lv-RON-siRNA感染后,HT-29细胞侵袭力较对照组明显降低(0.97±0.072 vs 1.29±0.076,P＜0.05).Lv-RON-siRNA感染后,HT-29细胞对5-氟尿嘧啶(5-fluorouraci,5-FU)的IC90值和IC50值分别为(14.28 ±1.34)、(8.93±1.20) μg/ml,顺铂(cisplatin,DDP)的IC90值和IC50值分别为(1.91±0.22)、(0.64±0.07) μg/ml,均明显低于对照组(P＜0.01).结论:沉默RON基因表达能抑制HT-29细胞的侵袭力,提高细胞对5-FU和DDP的敏感性.     

Sensitivity of soft tissue sarcoma cell lines to chemotherapeutic agents: identification of ecteinascidin-743 as a potent cytotoxic agent.

The cytotoxic effects of ecteinascidin-743(ET-743), a novel marine natural product, were evaluated and compared with that of clinically used anticancer agents methotrexate, doxorubicin, etoposide, and paclitaxel in eight human soft tissue sarcoma (STS) cell lines. HT-1080, a fibrosarcoma cell line, and HS-42, a malignant mesodermal cell line, were the most sensitive of the cell lines to methotrexate, doxorubicin, etoposide, and paclitaxel. Other cell lines (IC50s) varied considerably and were more resistant to these agents. ET-743 was more potent than any of these agents, with IC50s in the pM range in all of the cell lines. Cytotoxicity of ET-743 was dose- and time-related (4-72 h exposure). Cytotoxic concentrations of ET-743 produced a S/G2 block in all of the cell lines tested. Three colon adenocarcinoma cell lines, HCT-8, HT-29, and HCT-116, and one breast cancer cell line, MCF-7, were 1-2 logs less sensitive to ET-743 than the STS cell lines. Cell lines were also characterized as to expression of oncogenes and tumor suppressor genes to attempt to correlate sensitivity of these cell lines to ET-743 and other chemotherapeutic agents. All of the cell lines except M8805, a malignant fibrous histiocytoma cell line, had mutations in p53 and/or overexpressed the MDM2 protein. Only HS-18, a liposarcoma cell line, lacked expression of the retinoblastoma protein. None of the cell lines had detectable expression of P-glycoprotein as measured by immunohistochemistry. ET-743 is an extremely potent cytotoxic agent against human STS cell lines and is being evaluated as an antitumor agent in this disease.     

HERG K+ channel expression-related chemosensitivity in cancer cells and its modulation by erythromycin

Purpose Previous studies have found that the HERG K⁺ channel is highly expressed in some cancers. In the study reported here, we investigated HERG expression in various cancer cell lines, its correlation with chemosensitivity to vincristine, paclitaxel, and hydroxy-camptothecin, and its biochemical modulation.Methods The MTT assay and clonogenic assay were used to detect the cytotoxicity of anticancer drugs in vitro. HERG expression was analyzed by Western blotting or immunocytochemistry. Gene transfection was used to examine the changes in HERG-related chemosensitivity. Cell cycle phase distribution was detected by flow cytometry and drug combinations were evaluated by the MTT assay.Results HERG expression levels differed widely between various human cancer cell lines and HT-29 cells expressing high levels of HERG were more sensitive than A549 cells expressing low levels of HERG to vincristine, paclitaxel, and hydroxy-camptothecin. In terms of IC₅₀, the chemosensitivities of herg-transfected A549 cells to vincristine, paclitaxel and hydroxy-camptothecin were significantly increased. However, for cisplatin and 5-fluorouracil, no significant difference between herg-transfected A549 cells and parent A549 cells was detected. Erythromycin, a HERG K⁺ channel blocker, suppressed the growth of various cancer cells and the potency was correlated with HERG expression levels. Combinations of erythromycin and vincristine, paclitaxel or hydroxy-camptothecin showed synergy in cytotoxicity to HT-29 cells. Erythromycin also enhanced the G₂/M arrest induced by vincristine in HT-29 cells. There were synergistic effects between erythromycin and vincristine, paclitaxel, and hydroxy-camptothecin, and chemosensitivity was correlated with HERG expression level.Conclusions HERG expression levels and chemosensitivity were positively correlated for vincristine, paclitaxel, and hydroxy-camptothecin. Erythromycin was active as a modulator. These results suggest that HERG may serve as a molecular marker and modulating target for individualized cancer therapy.     

黄芪多糖通过调控miR-20a/TGFBR2 分子轴降低结直肠癌HT-29/DDP细胞的顺铂耐药性

[摘要] 目的：探讨黄芪多糖（APS）通过调控miR-20a/TGFBR2 分子轴对结直肠癌（CRC）顺铂耐药细胞HT-29/DDP增殖、侵袭、凋亡和耐药性的影响及其机制。方法：以人CRC HT-29 细胞、HT-29/DDP细胞为亲本和耐药细胞模型,将HT-29/DDP细胞随机分为4 组：对照组、APS 处理组、过表达miR-20a + APS 组、沉默TGFBR2 + APS 组。用不同质量浓度的APS（0、0.5、1.0、1.5 和2.0 mg/ml）处理HT-29/DDP细胞后,以qPCR和Wb实验检测细胞中miR-20a 和TGFBR2 的表达水平;用CCK-8、Transwell 和Annexin V-FITC/PI 染色流式细胞术检测对HT-29/DDP细胞增殖、侵袭和凋亡的影响;用双荧光素酶报告基因验证miR-20a 与TGFBR2的靶向作用关系。构建裸鼠皮下CRC HT-29/DDP细胞移植瘤模型,观察APS对移植瘤生长的影响及其机制。结果：APS显著抑制HT-29/DDP 细胞的增殖（P<0.01）,且呈剂量依赖性。miR-20a 在经APS 处理后的HT-29/DDP 细胞中低表达（P<0.01）、TGFBR2 的表达水平显著上调（P<0.01）。双荧光素酶报告基因证实miR-20a 靶向作用TGFBR2 并下调其表达水平。体内外实验证实APS通过靶向下调miR-20a 对TGFBR2 的抑制作用,提高HT-29/DDP细胞的药物敏感性,进而抑制该细胞增殖、侵袭和促进凋亡（均P<0.01);同时发现,该作用与抑制PCNA、Bcl-2 蛋白而促进Bax、Caspase-3 蛋白表达有关联。结论：APS通过下调miR-20a对TGFBR2 表达的抑制作用,从而逆转HT-29/DDP细胞对顺铂的耐药性。     

Increased expression of the major heat shock protein Hsp72 in human prostate carcinoma cells is dispensable for their viability but confers resistance to a variety of anticancer agents

The major heat shock protein Hsp72 is expressed at high levels in various types of cancer. Here we attempt to clarify the role of Hsp72 in prostate cancer cells by studying the effects of specific downregulation of this protein using siRNA and antisense RNA approaches. Contrary to previous reports, specific depletion of Hsp72 did not reduce viability of the prostate carcinoma cell lines PC-3 and DU-145. However, even short-term downregulation of Hsp72 in these cells made them more sensitive to hyperthermia, inhibitors of proteasome and Hsp90, and tumor necrosis factor. Interestingly, prolonged downregulation of Hsp72 in PC-3 cells over 3 weeks aggravated these effects, as well as enhanced the sensitivity of cells to oxidative stress, radiation, cis-platinum, vinblastin and taxol. The increased sensitivity to the anticancer agents was due to increased apoptosis, as well as other types of cell death, which resulted in the loss of clonogenic survival. Prolonged downregulation of Hsp72 led to severe suppression of the major survival pathways, ERK and NF-kappaB, which may be responsible for enhanced sensitivity of prostate carcinoma cells to a variety of anticancer treatments, as well as reduction of the cell's capability of forming colonies in soft agar.     

Chemotherapy enhances TNF-related apoptosis-inducing ligand DISC assembly in HT29 human colon cancer cells

Cytokines such as Fas-ligand (Fas-L) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) can induce human colon cancer cell apoptosis through engagement of their death domain receptors. All the cancer cells are not sensitive to these cytokines. We have shown recently that low doses of cytotoxic drugs could restore TRAIL-induced cell death in resistant colon cancer cell lines. The present work further explores the death pathway triggered by the cytotoxic drug/TRAIL combination in HT-29 colon cancer cells (www.alexis-corp.com). Clinically relevant concentrations of cisplatin, doxorubicin and 5-fluorouracil synergize with TRAIL to trigger HT-29 cell death. Activation of this pathway leads to apoptosis that involves both caspases and the mitochondria. An increased recruitment of Fas-associated death domain (FADD) and procaspase-8 to the TRAIL-induced death-inducing signaling complex (DISC) was shown in cells exposed to anticancer drugs. Following caspase-8 activation at the DISC level, the mitochondria-dependent death pathway is activated, as demonstrated by the cleavage of Bid, the dissipation of DeltaPsi(m), the release of mitochondrial proteins in the cytosol and the inhibitory effect of Bcl-2 expression. Importantly, besides mitochondrial potentiation, we show here that cytotoxic drugs sensitize HT-29 colon cancer cells to TRAIL-induced cell death by enhancing FADD and procaspase-8 recruitment to the DISC, a novel mechanism whose efficacy could depend partly on Bcl-2 expression level.     

Prediction of chemosensitivity of colorectal cancer to 5-fluorouracil by gene expression profiling with cDNA microarrays

5-Fluorouracil (5-FU) is the most widely used anticancer agent for gastrointestinal cancers. Because many tumors show primary resistance, it is clinically meaningful to predict tumor sensitivity to the drug before treatment. cDNA microarrays containing 21,168 clones were used to identify genes associated with sensitivity to 5-FU. Gene expression profiling of 3 colorectal cancer cell lines (DLD-1, HT-29 and NUGC-3) and the corresponding 5-FU-resistant sublines (DLD-1/FU, HT-29/FU and NUGC-3/5FU/L) showed 81 genes that were differentially expressed. The gene set thus identified successfully predicted the sensitivities of 5 other colorectal cancer cell lines and could also separate 5-FU resistant clinical samples from sensitive ones.     

Re-evaluating gadolinium(III) texaphyrin as a radiosensitizing agent

Gadolinium(III) texaphyrin (Gd-tex) was recently proposed as a radiosensitizing agent that combines preferential tumor uptake with detection of drug localization by magnetic resonance imaging (S. W. Young et al., Proc. Natl. Acad. Sci. USA, 93: 6610-6615, 1996). In view of the initial report on this compound, four radiobiology laboratories undertook independent efforts to further study radiosensitization by Gd-tex. In addition to repeating the previously reported studies on Gd-tex in HT-29 cells, we tested five other human tumor cell lines (U-87 MG, U251-NCI, SW480, A549, and MCF-7). These studies included a Gd-tex treatment period of 24 h before irradiation (as in the original publication), with concentrations of Gd-tex ranging from 20-500 microM. In neither the HT-29 cells nor any of the other five human cell lines did we see radiation sensitization by Gd-tex. Two cell lines (MCF-7 and U-87 MG) were further tested for radiosensitization by Gd-tex under hypoxic conditions. No radiosensitization was observed in either case. Finally, the radiation response of two tumor lines were assessed in vivo. Neither HT-29 xenografts in severe combined immunodeficient (SCID) mice nor RIF-1 tumors growing in C3H mice demonstrated radiosensitization after Gd-tex treatment before single or fractionated doses of radiation. Our results raise questions about the efficacy of Gd-tex as a radiosensitizing agent.     

Anti-Proliferative Effects of Hesa-A on Human Cancer Cells with Different Metastatic Potential

Background: During the past few years, Hesa-A, a herbal-marine mixture, has been used to treat cancer as analternative medicine in Iran. Based on a series of studies, it is speculated that Hesa-A possesses special cytotoxiceffects on invasive tumors. To test this hypothesis, we investigated the selective anticancer effects of Hesa-A onseveral cancer cell lines with different metastatic potential. Materials and Methods: Hesa-A was prepared innormal saline as a stock solution of 10 mg/ml and further diluted to final concentrations of 100μg/ml, 200μg/ml, 300μg/ml and 400μg/ml. MTT-based cytotoxicity assays were performed with A549 (lung non small cancer),MCF-7 (breast adenocarcinoma), SKOV3 (ovarian cancer), and PC-3 (prostate adenocarcinoma) cells. Results:All treated cancer cells showed significant (P<0.01) or very significant (P<0.0001) differences in comparison tonegative control at almost all of the tested doses (100-400 μg/ml). At the lower dose (100 μg/ml), Hesa-A reducedcell viability to 66%, 45.3%, 35.5%, 33.2% in SKOV3, A549, PC-3 and MCF-7 cells, respectively. Moreover, atthe highest dose (400μg/ml), Hesa-A resulted in 88.5%, 86.6% , 84.9% and 79.3% growth inhibition in A549,MCF-7, PC-3 and SKOV3 cells, respectively. Conclusions: Hesa-A exert potent cytotoxic effects on differenthuman cancer cells, especially those with a high metastatic potential.     

Effects of physiological oxygen environment on drug-induced cell lethality of multicellular tumor spheroids from human lung cancer

Advanced malignant tumors of certain histological types contain a hypoxic and necrotic core. Multicellular tumor spheroids (MTS) have the characteristics of chronically hypoxic cells in the center. We studied the effects of physiological oxygen environment on MTS growth and the cell lethality produced by doxorubicin (DXR) and cisplatin (DDP). MTS were made from 2 human lung cancer cell lines; PC-6 small cell and PC-10 squamous cell carcinoma, and grown for 2, 3 or 4 weeks; either in 5% CO2/air or 5% 02/5% CO2/90% N2. They were exposed to graded concentrations of DXR for 1 hr and cell lethality was determined by clonogenic assay. In the physiological oxygen environment MTS growth was retarded for both cell lines. PC-6 MTS grown in physiological oxygen environment were more sensitive to DXR than those developed in air. The differential sensitivity was most pronounced with the 2 week old MTS and gradually narrowed with increasing MTS size. In contrast, PC-10 MTS developed in the physiological oxygen environment were more resistant to DXR than those in air; the differences were again most pronounced in 2 week old MTS. There were little differences in cell kill effects of DDP, irrespective of cells being in monolayer or in MTS and growing in air or in physiological oxygen environment. These observations are consistent with the interpretation that cells in PC-6 MTS are scarcely affected by the physiological oxygen environment but easily affected by DXR, whereas cells in PC-10 MTS responded vice versa.     

Epigenetics and miRNA as predictive markers and targets for lung cancer chemotherapy

Lung cancer cells show inherent and acquired resistance to chemotherapy. The lack of good predictive markers/novel targets and the incomplete understanding of the mechanisms of resistance limit the success of lung cancer response to chemotherapy. In the present study, we used an isogenic pair of lung adenocarcinoma cell lines; A549 (wild-type) and A549DOX11 (doxorubicin resistant) to study the role of epigenetics and miRNA in resistance/response of non-small cell lung cancer (NSCLC) cells to doxorubicin. Our results demonstrate differential expression of epigenetic markers whereby the level of HDACs 1, 2, 3 and4, DNA methyltransferase, acetylated H2B and acetylated H3 were lower in A549DOX11 compared to A549 cells. Fourteen miRNAs were dys-regulated in A549DOX11 cells compared to A549 cells, of these 14 miRNAs, 4 (has-mir-1973, 494, 4286 and 29b-3p) have shown 2.99 – 4.44 fold increase in their expression. This was associated with reduced apoptosis and higher resistance of A549DOX11cells to doxorubicin and etoposide. Sequential treatment with the epigenetic modifiers trichostatin A or 5-aza-2''-deoxycytidine followed by doxorubicin resulted in: (i) enhanced sensitivity of both cell lines to doxorubicin especially at low concentrations, (ii) enhanced doxorubicin-induced DNA damage in both cell lines, (iii) dysregulation of some miRNAs in A549 cells. In conclusion, A549DOX11 cells resistant to DNA damaging drugs have epigenetic profile and miRNA expression different from the sensitive cells. Moreover, epigenetic modifiers may reverse the resistance of certain NSCLC cells to DNA damaging agents by enhancing induction of DNA damage. This may open the door for using epigenetic profile/miRNA expression of some cancer cells as resistance markers/targets to improve response of resistant cells to doxorubicin and for the use of combination doxorubicin/epigenetic modifiers to reduce doxorubicin toxicity.     

Cerivastatin demonstrates enhanced antitumor activity against human breast cancer cell lines when used in combination with doxorubicin or cisplatin

Competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase are commonly used in the clinic to treat hypercholesterolemia and have been reported to exert antitumor effects. Cerivastatin is a novel, synthetic and the most pharmacologically potent inhibitor of HMG-CoA reductase. We decided to examine the cytostatic/cytotoxic activity of cerivastatin against human breast cancer cell lines and to test whether the effects of cerivastatin could be potentiated by doxorubicin and cisplatin. Cytostatic/cytotoxic effects of cerivastatin used alone or in the combination with chemotherapeutics were measured with MTT assay. The cell cycle distribution and apoptosis induction were evaluated with flow cytometer. The expression of p21 and p27 cyclin-dependent kinase inhibitors was measured with Western blotting. Isobologram analysis was performed to study the drug interactions. We observed that cerivastatin exerts cytostatic/cytotoxic effects against four human tumor cell lines (T-47D, T4-2, MDA-MB-231, MCF-7). We also demonstrated that cerivastatin exerts growth inhibitory effect through induction of p21 cyclin-dependent kinase inhibitor and inhibition of cell cycle progression. In the two tumor cell lines studied, one sensitive (MDA-MB-231) and one moderately resistant (T4-2) to the cytostatic/cytotoxic effects of cerivastatin we examined the effects of combined treatment with cerivastatin and either doxorubicin or cisplatin. Cerivastatin potentiated cytostatic/cytotoxic effects of cisplatin against T4-2 cells and those of doxorubicin against both cell lines. In T4-2 cells the interaction between doxorubicin and cerivastatin and between cisplatin and cerivastatin was found to be synergistic. Altogether, these studies indicate that cerivastatin is another HMG-CoA reductase inhibitor with potent antitumor effects.     

光甘草定对肺腺癌A549细胞恶性生物学行为的影响及其分子机制

目的：探讨光甘草定对肺腺癌细胞A549 恶性生物学行的影响及其分子机制。方法：常规方法培养A549 细胞和人正常肺上皮细胞BEAS-2B,用不同浓度的光甘草定和或顺铂对其进行处理,通过结晶紫染色、CCK-8 法检测光甘草定、顺铂对A549、BEAS-2B细胞增殖活力的影响,Transwell 小室实验、细胞划痕实验检测光甘草定对A549 细胞侵袭、迁移能力的影响,流式细胞术检测光甘草定对A549 细胞凋亡的影响,3D超低黏附板培养法培养A549 细胞后采用CCK-8法检测光甘草定对A549 细胞增殖的影响,WB法检测光甘草定对A549 细胞中上皮间质转化（EMT）相关蛋白表达的影响;构建A549 细胞移植瘤模型后检测光甘草定、顺铂对移植瘤的生长以及移植瘤组织中EMT相关蛋白表达的影响。结果：光甘草定、顺铂呈剂量依赖性地显著抑制A549 细胞的增殖（P<0.05 或P<0.01）、细胞迁移（P<0.05 或P<0.01）和侵袭能力（P<0.05 或P<0.01）,光甘草定能诱导A549 细胞凋亡（P<0.01）,抑制A549 细胞中N-cadherin、snail 和vimentin 蛋白的表达,促进E-cadherin 蛋白表达;光甘草定、顺铂均能抑制A549移植瘤的生长,抑制移植瘤组织中Ki67、N-cadherin、snail 和vimentin 蛋白的表达、促进E-cadherin 蛋白的表达。结论：光甘草定可通过抑制A549细胞的增殖、迁移和侵袭,诱导A549细胞凋亡,其机制可能与其抑制细胞的EMT进程而产生抑癌作用有关。     

Widdrol induces apoptosis via activation of AMP-activated protein kinase in colon cancer cells

Widdrol, a natural sesquiterpene present in Juniperus sp., has been shown to exert anticancer and antifungal effects. Emerging evidence has suggested that AMP-activated protein kinase (AMPK), which functions as a cellular energy sensor, is a potential therapeutic target for human cancers. In this study, we found that AMPK mediates the anticancer effects of widdrol through induction of apoptosis in HT-29 colon cancer cells. We showed that widdrol induced the phosphorylation of AMPK in a dose- and time-dependent manner. The selective AMPK inhibitor compound C abrogated the inhibitory effect of widdrol on HT-29 cell growth. In addition, we demonstrated that widdrol induced apoptosis and this was associated with the activation of caspases, including caspase?3/7 and caspase-9, in HT-29 cells. We also demonstrated that transfection of HT-29 cells with AMPK siRNAs significantly suppressed the widdrol-mediated apoptosis and the activation of caspases. However, cell cycle arrest induced by widdrol was not affected by transfection of HT-29 cells with AMPK siRNAs. Furthermore, widdrol inhibited HT-29 tumor growth in a human tumor xenograft model. Taken together, our results suggest that the anticancer effect of widdrol may be mediated, at least in part, by induction of apoptosis via AMPK activation.     

Role of phloretin as a sensitizer to TRAIL-induced apoptosis in colon cancer

Phloretin is one of the apple polyphenols with anticancer activities. Since tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) serves important roles in inducing apoptosis, the present study examined the effect of phloretin on TRAIL-induced apoptosis in colon cancer cells. Treatment with both phloretin and TRAIL markedly suppressed the survival of cancer cells from several colon cancer cell lines compared with that of cells treated with either TRAIL or phloretin. Additionally, decreased numbers of colonies were observed following addition of phloretin and TRAIL. Furthermore, TRAIL- and phloretin-treated HT-29-Luc cells exhibited decreased luciferase activity. Increased apoptosis was observed in phloretin- and TRAIL-treated HT-29-Luc colon cancer cells, accompanying elevated levels of cleaved poly(ADP-ribose) polymerase, and caspase-3, −8 and −9. The expression levels of MCL1 apoptosis regulator BCL2 family member (Mcl-1) were decreased following addition of phloretin in colon cancer cells. In addition, overexpression of Mcl-1 in phloretin- and TRAIL-treated HT-29-Luc cells resulted in increased cell survival. Treatment of HT-29-Luc cells with a combination of cycloheximide (CHX) and phloretin led to a more prominent decrease in Mcl-1 expression compared with that in cells treated with CHX alone, while Mcl-1 expression was recovered by treatment with MG132. Binding of ubiquitin with Mcl-1 was verified using immunoprecipitation. Intraperitoneal injection of both TRAIL and phloretin into tumor xenografts was associated with a decreased tumor volume compared with that following injection with either TRAIL or phloretin. Overall, the present results suggest a synergistic effect of phloretin on TRAIL-induced apoptosis in colon cancer cells.     

Isolation and characterization of a mitomycin C-resistant variant of human colon carcinoma HT-29 cells

To investigate the resistant mechanisms against MMC in human tumor cells, we isolated an MMC-resistant variant (HT-29/MMC) of HT-29 human colon carcinoma cells. HT-29/MMC cells showed 5-fold resistance to MMC as compared with the parental cell line but did not show cross-resistance to Adriamycin, vincristine, ACNU, bleomycin, or cisplatin. Treatment of the cells with dicoumarol, an inhibitor of DT-diaphorase, reduced the cytotoxicity of MMC in DT-diaphorase proficient HT-29 cells but not in HT-29/MMC cells. HT-29/MMC cells were 5 times more sensitive than HT-29 cells to menadione, which is detoxified by DT-diaphorase. DT-diaphorase was deficient in HT-29/MMC cells as determined by the enzyme activity and immunoblot analysis of the cytoplasmic proteins. Levels of cytochrome P-450 reductase and glutathione S-transferase, however, were comparable in both cell lines. The amount of [3H]-MMC found covalently bound to chromosomal DNA in HT-29/MMC cells was one-fourth that detected in HT-29 cells. Treatment with dicoumarol reduced the DNA-bound MMC in HT-29 cells but not in HT-29/MMC cells. These results indicate that the deficiency in DT-diaphorase, an activating enzyme of MMC, is one of the mechanisms of resistance in HT-29/MMC cells.Abbreviations MMC mitomycin C - DCPIP 2,6-dichlorophenolindophenol - TEMPOL 4-hydroxytetramethyl piperidine-1-oxyl - ACNU 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - PMSF phenyl methyl sulfonyl fluoride - PBS.(–) phosphatebuffered saline without calcium or magnesium This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan Correspondence to: Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan     

In vitro antitumour activity of the novel imidazoisoquinoline SDZ 62-434.

The novel imidazoisoquinoline SDZ 62-434, originally identified as a platelet-activating factor (PAF) antagonist, has antiproliferative activity in a range of cell lines from human solid and haematological malignancies. Using an MTT cytotoxicity assay, IC50 values of 5 microM - 111 microM were observed following a 24 h exposure. Similar results were obtained using a clonogenic assay. The HT29 colon adenocarcinoma was particularly sensitive while the MCF-7 breast carcinoma was the most resistant in our panel. Only a 2-3 fold cross-resistance was seen in the doxorubicin and cisplatin resistant variants of the A2780 ovarian carcinoma; the drug did not modulate sensitivity to doxorubicin in either parent or resistant lines. No cross-resistance to SDZ 62-434 was seen in a doxorubicin-resistant MCF-7 variant. Cytotoxicity was not due to non-specific membrane lysis. The potent PAF antagonist WEB 2086 did not modulate SDZ 62-434 cytotoxicity, indicating no role for PAF receptors. Precursor incorporation studies in A2780 cells showed that DNA synthesis was inhibited more effectively than protein synthesis while RNA synthesis was unaffected. SDZ 62-434 inhibited both bombesin and platelet-derived growth factor-induced DNA synthesis in quiescent Swiss 3T3 cells. This suggests a possible role for SDZ 62-434 as an inhibitor of signal transduction in cancer cells.     

Determinants of response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in human lung cancer cell lines.

BACKGROUND: Small-cell lung cancer (SCLC) is more sensitive to anticancer agents than non-small-cell lung cancer (NSCLC), but few studies have analyzed the mechanisms of natural drug resistance responsible for this difference. PURPOSE: To elucidate these mechanisms, we determined drug sensitivity and evaluated the biochemical parameters affecting response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in both types of cancer cell lines, in particular the activity and content of DNA topoisomerase II, as well as etoposide uptake and cell doubling time. METHODS: Drug sensitivity and cellular uptake of etoposide were determined by clonogenic assay and accumulation of radiolabeled drug, respectively. The topoisomerase II activity was assayed by decatenation of kinetoplast DNA to minicircle DNA using nuclear protein, and the content was determined by immunoblot analysis of nuclear extracts. We also compared the topoisomerase II content in parent cell lines with that in lines with cisplatin resistance acquired in vitro. RESULTS: Sensitivities to doxorubicin and etoposide were higher in SCLC cell lines than in NSCLC lines, and the difference was statistically significant. Etoposide uptake in SCLC cells was higher than in NSCLC cells; the difference was statistically significant, but this difference may not be sufficient to account for the variation in sensitivities of the cell lines. Topoisomerase II activities of nuclear protein from SCLC cell lines were reproducibly twofold higher than those for NSCLC cell lines. The topoisomerase II content in nuclear protein appeared to be higher in SCLC cell lines than in NSCLC cell lines and corresponded to the sensitivities to doxorubicin and etoposide. In the cisplatin-resistant NSCLC cell lines PC-7/CDDP and PC-14/CDDP, the topoisomerase II content was increased compared with that in the parent lines, but the topoisomerase II content in other cisplatin-sensitive parent lines was similar to that in resistant sublines. CONCLUSIONS: These findings suggest that the topoisomerase II activity and content may be major factors in determining sensitivity to topoisomerase II inhibitors.