Ectopic production of heparin-binding growth factors and receptors for basic fibroblast growth factor by rat mammary epithelial cell lines derived from malignant metastatic tumours

A rat mammary (Rama) epithelial cell line, Rama 704, derived from normal rat mammary gland does not possess any detectable cell-surface receptors for basic fibroblast growth factor (bFGF), produces a barely detectable level of bFGF mRNA and does not contain detectable levels of bFGF-like activity. Similar results have been obtained with the Rama 37 epithelial cells derived from benign tumours. However, 4 independently isolated epithelial cell lines derived from malignant rat mammary tumours and their metastases possess receptors for bFGF and contain between 2 ng and 9 ng heparin-binding, growth-stimulatory activity per 10⁶ cells. The weakly metastatic Rama 600 cells possess high- and low-affinity receptors for bFGF, (K_D 20 pM and 8 nM, respectively), while the moderately metastatic Rama 800 cells possess only high-affinity receptors (K_D 40 pM). The moderately metastatic C18PLN and 267LU cells, derived from metastases arising from benign Rama 37 cells which had been transfected with DNA from the malignant Rama 800 cells, also possess only high-affinity receptors (K_D 36 pM and 80 pM, respectively). Our results show that within the Rama system there is a correlation between the appearance of heparin-binding growth factors and of high-affinity but not low-affinity receptors for bFGF with the malignant phenotype.     

The role of NAD(P)H: quinone oxidoreductase in mitomycin C- and porfiromycin-resistant HCT 116 human colon-cancer cells

Summary A mitomycin C (MMC)- and porfiromycin (PFM)-resistant subline of the HCT 116 human coloncancer cell line was isolated after repeated exposure of HCT 116 cells to increasing concentrations of MMC under aerobic conditions. The MMC-resistant subline (designated HCT 116-R30A) was 5 times more resistant than the parent cells to MMC and PFM under aerobic conditions. Both the MMC-resistant cells and the parent HCT 116 cells accumulated similar amounts of PFM by passive diffusion, but levels of macromolecule-bound PFM were about 50% lower in the resistant cell line, implying a decrease in PFM reductive activation in the resistant cells. The finding that microsomes from either sensitive or resistant cells showed an equal ability to reduce MMC and PFM indicated that the activity of NADPH cytochrome P-450 reductase (EC 1.6.2.4) was not changed in the resistant subline. Soluble extracts of HCT 116 cells reduced MMC and PFM more effectively at pH 6.1, and NADH and NADPH were utilized equally well as electron donors under both aerobic and anaerobic conditions. These data suggest that quinone reductase (EC 1.6.99.2; DT-diaphorase) in soluble extracts is responsible for the reduction of MMC. Quinone reductase activities in soluble extracts of HCT 116-R30A cells for the reduction of dichlorophenol indophenol (DCPIP) and menadione-cytochrome c at optimal pHs were decreased by 95% as compared with those obtained in parent cells. However, the MMC-reducing activity of HCT 116-R30A soluble extracts was only 50% lower than that of the parent cell extracts. The kinetic constants (K _m, V_max) found for quinone reductase in the two cell lines with respect to the substrates DCPIP and menadione differed. Two species of mRNA for quinone reductase (2.7 and 1.2 kb) were detected in both cell lines, and there was no detectable difference between parent and resistant cells in the steady-state level of either of these mRNA species. Furthermore, incubation with the quinone reductase inhibitor dicoumarol rendered HCT 116 cells more resistant to MMC. Alteration of the quinone reductase activity in HCT 116-R30A cells appears to be the mechanism responsible for their resistance to MMC and PFM.Abbreviations NAD(P)H reduced nicotinamide adenosine dinucleotide (phosphate) - MMC mitomycin C - PFM porfiromycin - PBS phosphate-buffered saline - DCPIP 2,6-dichlorophenol indophenol - DIC dicoumarol - c- and t-AHME cis- andtrans-7-amino-1-hydroxyl-2-methoxyaminomitosene - EDTA ethylenediaminetetraacetic acid - AME 7-aminio-2-methoxyaminomitosene - DMSO dimethylsulfoxide - SSC 20xSSC contains 3m NaCl and 0.3m sodium citrat - SSPE: 20xSSPE contains 3m NaCl, 0.2m monosodium phosphate, and 20mm EDTA - SDS sodium dodecyl sulfate This work was supported by grant CH-412 from the American Cancer Society and by an award from the Bressler Fund of the University of Maryland School of Medicine (to S. P.)     

Up-regulation of GPR48 induced by down-regulation of p27Kip1 enhances carcinoma cell invasiveness and metastasis

A reduced expression level of the cyclin-dependent kinase inhibitor p27(Kip1) is associated with increased tumor malignancy and poor prognosis in individuals with various types of cancer. To investigate the basis for this relation, we applied microarray analysis to screen for genes differentially expressed between p27(+/-) and parental (p27(+/+)) HCT116 human colon carcinoma cells. Expression of the gene for G protein-coupled receptor 48 (GPR48) was increased in the p27(+/-) cells. Forced expression of GPR48 increased both in vitro invasive activity and lung metastasis potency of HCT116 cells. In contrast, depletion of endogenous GPR48 by RNA interference reduced the invasive potential of HeLa and Lewis lung carcinoma cells not only in vitro but also in vivo. Moreover, GPR48 expression was significantly associated with lymph node metastasis and inversely correlated with p27 expression in human colon carcinomas. GPR48 may thus play an important role in invasiveness and metastasis of carcinoma and might therefore represent a potential prognostic marker or therapeutic target.     

Production of basic fibroblast growth factor-like factor by cultured human cholangiocellular carcinoma cells

An extract of cultured human cholangiocellular carcinoma cells (HuCC-T1) was found to contain high mitogenic activity for BALB/c3T3 cells. The growth factor eliciting most of the mitogenic activity was purified and concluded to be identical with basic fibroblast growth factor (bFGF)-like factor on the basis of its molecular weight and heparin-Sepharose elution profile, and the results of immunoblotting and radioimmunoassay. HuCC-T1 cells also secreted bFGF-like factor into serum-free medium. A combination of insulin and transferrin or bovine serum albumin stimulated the growth of HuCC-T1 cells in serum-free medium. However, bFGF did not stimulate their growth in the presence and absence of these supplements. Neutralizing monoclonal antibody against bFGF did not inhibit growth. These results indicate that bFGF-like factor is not a growth factor for this cell line.     

Production of Basic Fibroblast Growth Factor-like Factor by Cultured Human Cholangiocellular Carcinoma Cells

An extract of cultured human cholangiocellular carcinoma cells (HuCC-T1) was found to contain high mitogenic activity for BALB/c3T3 cells. The growth factor eliciting most of the mitogenic activity was purified and concluded to be identical with basic fibroblast growth factor (bFGF)-like factor on the basis of its molecular weight and heparin-Sepharose elution profile, and the results of immunoblotting and radioimmunoassay. HuCC-T1 cells also secreted bFGF-like factor into serum-free medium. A combination of insulin and transferrin or bovine serum albumin stimulated the growth of HuCC-T1 cells in serum-free medium. However, bFGF did not stimulate their growth in the presence and absence of these supplements. Neutralizing monoclonal antibody against bFGF did not inhibit growth. These results indicate that bFGF-like factor is not a growth factor for this cell line.     

FOXJ1调控AKT信号通路对结直肠癌细胞凋亡和侵袭能力的影响

目的 探讨叉头框转录因子J1(FOXJ1)对人结直肠癌细胞凋亡和侵袭能力的影响及机制.方法 应用Western Blot方法检测人结直肠癌细胞HCT116、HT29、SW480及人正常肠上皮细胞FHC中FOXJ1的表达水平.细胞中转染pEGFP-N1-FOXJ1和pEGFP-N1,Western Blot检测细胞中FOXJ1、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、基质金属蛋白酶-2(MMP-2)、蛋白激酶B(AKT)、磷酸化的蛋白激酶B(p-AKT)的表达水平,流式细胞仪检测细胞的凋亡情况,Transwell小室检测细胞的侵袭能力.结果 人结直肠癌细胞HCT116、HT29、SW480中FOXJ1的表达水平均明显低于人正常肠上皮细胞FHC(P﹤0.01).人结直肠癌细胞HCT116中FOXJ1的表达水平下降最多,后续选用HCT116细胞继续研究.转染pEGFP-N1后的细胞凋亡率、侵袭细胞数目及细胞中FOXJ1、Cleaved Caspase-3、MMP-2、AKT、p-AKT的表达水平与对照组比较,差异均无统计学意义(P﹥0.05).转染pEGFP-N1-FOXJ1后的细胞凋亡率及细胞中FOXJ1、Cleaved Caspase-3的表达水平明显高于对照组(P﹤0.01);转染pEGFP-N1-FOXJ1后的侵袭细胞数目及细胞中MMP-2、p-AKT的表达水平明显低于对照组(P﹤0.01).结论 FOXJ1在人结直肠癌细胞中过表达.FOXJ1能够促进结直肠癌细胞凋亡,抑制结直肠癌细胞侵袭,其作用机制可能与AKT信号通路有关.     

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.     

Basic fibroblast growth factor-like activity and receptors are expressed in a human glioma cell line

Basic fibroblast growth factor (bFGF), a potent mitogen and angiogenic peptide, has been examined as an autocrine regulator of glioma cell growth. The addition of purified bovine pituitary bFGF to an established human glioma cell line, SNB-19, doubled the density of these cells in chemically defined medium. Half-maximal stimulation occurred at 8.2 ng/ml (480 pM). Also, human recombinant bFGF (hr-bFGF) significantly enhanced the growth of SNB-19 cells in soft agar. SNB-19 cells expressed both high and low affinity binding sites for hr-bFGF. These cells expressed approximately 13,000 high affinity sites/cell (Kd = 16.6 +/- 1.7 pM) and 9.5 x 10(6) low affinity sites/cell (Kd = 61.2 +/- 4.1 nM). The results of cross-linking experiments with iodinated hr-bFGF demonstrated the presence of two bands with molecular masses of 145 and 130 kDa. High affinity receptors were also demonstrated in SNB-19 tumors grown in nude mice. SNB-19 cell extracts contained mitogenic activity that eluted from heparin-agarose with high salt (1.2-2 M NaCl) and exhibited many properties normally associated with authentic bFGF. This material cross-reacted with a monoclonal antibody to hr-bFGF, comigrated with hr-bFGF by Western blot analysis, competed with 125I-hr-bFGF in a radioreceptor assay, and stimulated SNB-19 cell growth. These results indicate that a human glioma cell line both expresses and utilizes a bFGF-like growth factor. Such a factor may be an important autocrine regulator of glioma cell growth and may also facilitate its neoplastic progression.     

Retinoic acid modulates both invasion and plasma membrane ruffling of MCF-7 human mammary carcinoma cells in vitro.

The invasiveness of MCF-7 human mammary carcinoma cells was tested in vitro via confronting cultures with embryonic chick heart fragments. Invasive (e.g. MCF-7/6) and non-invasive (e.g. MCF-7/AZ) variants were detected. Automated image analysis of time-lapse video-microscopy recordings showed that the plasma membrane ruffling activity of the invasive MCF-7/6 variant was higher than the ruffling activity of the non-invasive MCF-7/AZ variant. Addition of all-trans-retinoic acid to the culture medium (10(-6) M) inhibited both invasion and ruffling of MCF-7/6 cells, while MCF-7/AZ cells became invasive and acquired an increased ruffling by the same type of treatment. A similar opposite effect on MCF-7 cells was not found after treatment with other ligands of the nuclear steroid/thyroid receptor superfamily. Triiodo-l-thyronine (up to 10(-5) M) and beta-oestradiol (up to 10(-6) M) did not alter the invasiveness of the cells, while dexamethasone (10(-6) M) and the pure anti-oestrogen ICI 164,384 inhibited both invasion and ruffling. Our data show that retinoic acid can modulate invasiveness in opposite directions.     

Urokinase-type plasminogen activator and its receptor in colorectal cancer: independent prognostic factors of metastasis and cancer-specific survival and potential therapeutic targets

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR), plasminogen (Plg), and plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) have been observed in many cancers and may contribute to progression and metastasis. In our study, we examined the expression of the 5 proteins by immunohistochemistry in 59 consecutive primary colorectal cancers (CRC) and correlated the protein expression with patient outcome. In addition, we determined the effect of down-regulation of uPAR on the invasive/metastatic capability of CRC cells, by measuring antisense-uPAR transfected HCT116 and control cell lines, in terms of uPAR expression, uPA-binding activity, invasiveness through Matrigel in vitro and metastasis after cecal orthotopic implantation in nude mice in vivo. We found that higher expression of uPA or uPAR in primary tumor tissues was positively correlated with distant metastasis of CRC (Mann-Whitney, p < 0.02) and negatively correlated with both patient overall survival (OS) and cancer-specific survival (CSS; Cox model, p < 0.04). The prognostic value of uPA and uPAR for both OS and CSS was independent of other variables (multivariate Cox model, p < 0. 007). Antisense-uPAR transfected HCT116 cells, which expressed significantly lower levels of total cellular and cell surface uPAR proteins and uPA-binding activity compared with either wild-type or cells transfected with vector alone (Bonferroni, p < 0.05/3), consistently showed decreased invasiveness through Matrigel (Bonferroni, p < 0.05/3) and decreased metastasis formation in nude mice (Fisher, p < 0.05). Our data suggest that uPAR and uPA are independent prognostic factors in CRC; anti-uPAR treatment, which affects both uPAR and uPA levels, may have potential for new treatment of the disease.     

TIP30基因对大肠癌细胞HCT116生物学特性的影响

目的通过构建基因真核表达载体分析TIP30对大肠癌细胞HCT116生物学特性的影响,为TIP30在大肠癌基因治疗中的应用提供依据。方法构建pCMV4-flag-TIP30真核表达载体并转染HCT116细胞,RT-PCR和Western blot检测TIP30基因表达,体外侵袭实验检测细胞侵袭能力,软琼脂实验检测细胞成瘤性。结果成功构建稳定表达TIP30的HCT116细胞模型,转染TIP30 的HCT116细胞增殖受抑,侵袭能力及克隆形成能力均减弱。结论大肠癌HCT116细胞TIP30过表达不仅能抑制其生长、诱导其凋亡,并能降低其侵袭、迁移能力,为TIP30基因治疗提供依据。     

hPTTG1基因沉默下调bFGF表达并抑制A2780细胞侵袭的研究

目的：探讨RNAi技术沉默卵巢癌细胞A2780细胞内hPTFG1表达对其侵袭性的影响及调控机制。方法：利用阳离子转染试剂将hPTTG1siRNA转染A2780细胞．荧光实时定量PCR检测hPTTG1及bFGF表达水平;MTT基质黏附实验检测细胞黏附能力;细胞侵袭重建基底膜实验测定细胞体外侵袭能力;划痕实验检测细胞迁移能力;Western blot检测bFGF蛋白表达水平。结果：hPTTG1siRNA转染组hPTTG1mRNA表达下降,抑制率为（76．8±4．1）％;转染hPTTG1siRNA后细胞黏附、侵袭和迁移能力降低;hPTTG1干扰后bFGFmRNA及蛋白表达下降。结论：hPTTG1siRNA能够抑制A2780细胞侵袭,可能是通过下调bFGF表达实现的。     

The mismatch repair protein, hMLH1, mediates 5-substituted halogenated thymidine analogue cytotoxicity, DNA incorporation, and radiosensitization in human colon cancer cells

Deficiency in DNA mismatch repair (MMR) is found in some hereditary (hereditary nonpolyposis colorectal cancer) and sporadic colon cancers as well as other common solid cancers. MMR deficiency has recently been shown to impart cellular resistance to multiple chemical agents, many of which are commonly used in cancer chemotherapy. It is therefore of interest to find an approach that selectively targets cells that have lost the ability to perform MMR. In this study, we examine the response of MMR-proficient (hMLH1+) and MMR-deficient (hMLH1-) colon carcinoma cell lines to the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) before and after irradiation. These dThd analogues are used clinically as experimental sensitizing agents in radioresistant human cancers, and there is a direct correlation between the levels of dThd analogue DNA incorporation and tumor radiosensitization. In contrast to the well-characterized, marked increase in cytotoxicity (> 1 log cell kill) found with 6-thioguanine exposures in HCT116/3-6 (hMLH1+) cells compared to HCT116 (hMLH1-) cells, we found only modest cytotoxicity (10-20% cell kill) in both cell lines when treated with IdUrd or BrdUrd for 1 population doubling. Upon further analysis, the levels of halogenated dThd analogues in DNA were significantly lower (two to three times lower) in HCT116/3-6 cells than in HCT116 cells, and similar results were found in Mlh1+/+ spontaneously immortalized murine embryonic fibroblasts and fibroblasts from Mlh1 knockout mice. As a result of the higher levels of the dThd analogue in DNA, there was an increase in radiation sensitivity in HCT116 cells but not in HCT116/3-6 cells after pretreatment with IdUrd or BrdUrd when compared to treatment with radiation alone. Additionally, we found no differences in the cellular metabolic pathways for dThd analogue DNA incorporation because the enzyme activities of dThd kinase and thymidylate synthase, as well as the levels of triphosphate pools, were similar in HCT116 and HCT116/3-6 cells. These data suggest that the hMLH1 protein may participate in the recognition and subsequent removal of halogenated dThd analogues from DNA. Consequently, whereas MMR-deficient cells and tumor xenografts have shown intrinsic resistance to a large number of chemotherapeutic agents, the 5-halogenated dThd analogues appear to selectively target such cells for potential enhanced radiation sensitivity.     

新合成吩嗪衍生物体外诱导癌细胞周期阻滞和凋亡的实验

目的 探讨新合成吩嗪衍生物抗癌活性及其作用机制。方法 MTT法检测细胞增殖,吖啶橙/溴乙锭（AO/EB）染色荧光显微镜观察细胞形态,流式细胞仪检测细胞周期,Annexin V-FITC/7-AAD双染检测凋亡,Western blot检测p53和caspase-3蛋白的表达。结果 pn18、pn23、pc27和pc28四种化合物在体外抑制癌细胞增殖,尤其对人结直肠癌细胞HCT116作用明显,且呈时间和剂量依赖性。pc28在四种化合物中作用最强,48 h对HepG2、HCT116和A549细胞半数抑制浓度（IC₅₀）分别为（6.62±2.69）、（10.83±1.41）和（22.39±4.31）μmol。20 μmol化合物作用于HCT116细胞24 h后,细胞密度降低,形态变圆,细胞内明亮绿色荧光与染色质浓缩相关,死亡细胞呈橙黄色和红色与细胞膜通透性增加有关;pc28诱导的细胞晚期凋亡比例由0.345%升高至19.7%。20 μmol pn18和pc27诱导G₀/G₁期细胞分别升高17.5%和25.0%,pn23和pc28诱导S期细胞分别升高18.4%和11.0%。pn23和pc28诱导p53表达升高,pc28也能上调并激活caspase-3。结论 四种合成的新型吩嗪衍生物在体外能有效抑制多种癌细胞增殖,其作用机制可能与诱导细胞周期阻滞和凋亡相关。     

Design of a selectable reporter for the detection of mutations in mammalian simple repeat sequences

To study the mutator phenotype characteristic of tumors showingwidespread replication errors at simple DNA repeat sequences(RER+), we designed a selectable reporter system for the detectionof such mutations in mammalian cells. A hygromycin B phosphotransferasegene was rendered out-of-frame by the insertion of a (CA)₁₃dinucleotide repeat tract immediately following the ATG startcodon, and subcloned into a retroviral expression vector containinga G418 (neo) selectable marker. Following transduction of thisconstruct into cultured cells, clonal neo+ cell lines were establishedand then tested for their ability to form colonies in hygromycinB-containing medium. Using this system, we found that the HCT116,LS174T and LS180 human colon carcinoma cell lines acquire hygromycinresistance (hyg^r) at a 100-fold higher frequency than the HT29,SW480, DLD-1 and HCT15 human colon carcinoma and NIH3T3 fibroblastcell lines, and at a 25-fold higher rate than the Rat 6 embyrofibroblast cell line. DNA sequence analysis indicated that frameshiftmutations had occurred within the CA dinucleotide repeat tractin HCT116 cells that became hyg^r. Thus, the mutation rates atsimple repeated sequences in mammalian cell lines can be readilydetermined and studied using this system.     

Activation of p21-Dependent G1/G2 Arrest in the Absence of DNA Damage as an Antiapoptotic Response to Metabolic Stress

The folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase, ALDH1L1), a metabolic regulator of proliferation, activates p53-dependent G1 arrest and apoptosis in A549 cells. In the present study, we have demonstrated that FDH-induced apoptosis is abrogated upon siRNA knockdown of the p53 downstream target PUMA. Conversely, siRNA knockdown of p21 eliminated FDH-dependent G1 arrest and resulted in an early apoptosis onset. The acceleration of FDH-dependent apoptosis was even more profound in another cell line, HCT116, in which the p21 gene was silenced through homologous recombination (p21(-/-) cells). In contrast to A549 cells, FDH caused G2 instead of G1 arrest in HCT116 p21(+/+) cells; such an arrest was not seen in p21-deficient (HCT116 p21(-/-)) cells. In agreement with the cell cycle regulatory function of p21, its strong accumulation in nuclei was seen upon FDH expression. Interestingly, our study did not reveal DNA damage upon FDH elevation in either cell line, as judged by comet assay and the evaluation of histone H2AX phosphorylation. In both A549 and HCT116 cell lines, FDH induced a strong decrease in the intracellular ATP pool (2-fold and 30-fold, respectively), an indication of a decrease in de novo purine biosynthesis as we previously reported. The underlying mechanism for the drop in ATP was the strong decrease in intracellular 10-formyltetrahydrofolate, a substrate in two reactions of the de novo purine pathway. Overall, we have demonstrated that p21 can activate G1 or G2 arrest in the absence of DNA damage as a response to metabolite deprivation. In the case of FDH-related metabolic alterations, this response delays apoptosis but is not sufficient to prevent cell death.     

Induction of a less aggressive phenotype in human colon carcinoma HCT116 cells by chronic exposure to HDAC inhibitor SAHA

Histone deacetylase inhibitors (HDACis) are anticancer molecules that epigenetically modulate cell functions. Chronic exposure of HCT116 colon cancer cells to SAHA has been investigated for a better understanding of resistance mechanisms but, surprisingly, a less aggressive tumor phenotype both in vitro and in vivo was obtained after exposure to increasing concentrations of SAHA. Indeed, HCT116/SAHA cells when injected into nude mice showed a reduced engraftment and growth with respect to HCT116 cells. This difference was not observed inoculating the cells into NOD/SCID mice that, differently from nude mice, lack NK activity, thus suggesting the involvement of the native immune response in impairment of HCT116/SAHA cell growth. In agreement with this result, a growing induction of NKG2D ligand expression, MICA and MICB, that are molecular mediators of NK cell killing, was confirmed in HCT116/SAHA chronically exposed to SAHA. A reduced clonogenic efficiency was also observed in HCT116/SAHA with respect to HCT116 cells. Interestingly, even after chronic exposure to SAHA, HCT116/SAHA cells developed only a moderate resistance to SAHA both in vitro and in vivo and they acquired a collateral sensitivity to anthracyclines. These results are of note and probably rely on the fact that, having simultaneously many different targets, HDACis would require many different mutations to display high resistance index. Moreover, to understand the molecular basis of HCT116/SAHA cell phenotype a gene expression profile of cancer genes was evaluated in HCT116 incubated with SAHA for 24 h and in HCT116/SAHA cells to identify selectively regulated genes.     

4'-O-Alkyaloenin derivatives and their sulfates directed toward overcoming multidrug resistance in tumor cells

The cytotoxic effects on HCT 116, Hep G2 and HCT 116/VCR 100-1-1 cell lines of synthetic 4'-O-alkylaloenins (2-17), 4'-O-benzylaloenin (18) and 4'-O-allylaloenin (19) were examined by MTT assay, and compared with that of aloenin (1) isolated from Aloe arborescens Mill. Var. natalensis Berger which showed no marked effect (IC50 value: > 100 microM). The cytotoxic effects of 4'-O-alkylaloenin sulfates (21-29) were also examined on the same cell lines. The introduction of a longer alkyl group at the O-4' position of 1 resulted in a higher cytotoxic action on HCT 116 and Hep G2 cells. Among 4'-O-alkylaloenins 2-17, 4'-O-tetradecylaloenin 14 was the most cytotoxic to both on HCT 116 cells (IC50 value: 5.3+/-2.3 microM) and Hep G2 cells (IC50 value: 4.0+/-0.6 microM). Also among 4'-O-alkylaloenin sulfates 21-29, 4'-O-dodecylaloenin sulfate 29 was the most cytotoxic to both on HCT 116 (IC50 value: 4.8+/-0.2 microM) and Hep G2 cells (IC50 value: 4.0+/-0.5 microM). 4'-O-Alkylaloenins 7-14 and 4'-O-alkylaloenin sulfates 24-29 were also cytotoxic to Hep G2 and HCT 116/VCR 100-1-1 cell lines, which overexpress P-glycoprotein, as well as HCT 116 cell lines which scarcely express it.     

Influence of p53 and p21Waf1 expression on G2/M phase arrest of colorectal carcinoma HCT116 cells to proteasome inhibitors

Ubiquitin-mediated protein degradation in vertebrates has been implicated in cell cycle control. In this report we explored the effects of proteasome inhibitors (MG132, lactacystin and ALLN) on cell cycle distribution. Colorectal carcinoma HCT116 cells were treated with proteasome inhibitor MG132. The results showed that MG132 inhibited cell proliferation in a dose-dependent manner. MG132 arrested HCT116 cells at G2/M phase, which was associated with drug-induced blockade of p53 degradation and/or induction of p53-related gene expression along with the accumulation of cyclin B, cyclin A and p21. MG132 treated HCT116 (wild-type) had a similar cell cycle distribution as the MG132 treated HCT116 (p53-/-) and HCT116 (p21-/-) cells, suggesting that p53 and p21 may not be essential for MG132-induced G2/M phase arrest. The release experiments from nocodazole-induced mitotic phase cells indicated that MG132 inhibits the proliferation of HCT116 cells via arrest in the G2 phase. In addition, when HCT116 cells were exposed to combination of sodium butyrate and MG132 enhanced cell growth inhibition and induction of apoptosis were observed.